Device, maker skill evaluation method, program, and device, and test device performance evaluation method, program, and device
阅读说明：本技术 装置，制备者技能评估方法、程序和装置，以及测试装置性能评估方法、程序和装置 (Device, maker skill evaluation method, program, and device, and test device performance evaluation method, program, and device ) 是由 铃木幸荣 海野洋敬 濑尾学 和泉贤 大崎优介 川岛优大 桥本路缘 于 2018-11-22 设计创作，主要内容包括：提供一种装置,包括：多个孔；布置在各孔中并含有特定拷贝数的可扩增试剂的试剂组合物；以及孔划分成的两组或更多组,所述两组或更多组具有以相同特定拷贝数布置的可扩增试剂但除特定拷贝数以外的试剂组合物的组成不同。优选地,除可扩增试剂的特定拷贝数以外的组成包括引物和扩增剂中的至少任一者。更优选地,装置包括特定拷贝数不同的两组或更多组。还更优选地,各孔组至少包括其中可扩增试剂的特定拷贝数为0的阴性对照组和其中可扩增试剂的特定拷贝数接近检测极限的组。(Providing an apparatus comprising: a plurality of holes; a reagent composition disposed in each well and containing a specific copy number of amplifiable reagents; and two or more groups into which the wells are divided, the two or more groups having amplifiable reagents arranged at the same specific copy number but differing in composition of reagent compositions other than the specific copy number. Preferably, the composition other than the specific copy number of the amplifiable agent comprises at least any one of a primer and an amplifiable agent. More preferably, the apparatus comprises two or more sets differing in specific copy numbers. Still more preferably, each well group includes at least a negative control group in which the specific copy number of the amplifiable reagent is 0 and a group in which the specific copy number of the amplifiable reagent is close to the detection limit.)
1. An apparatus, comprising:
a plurality of holes; and
the holes are divided into two or more groups, the two or more groups having different compositions of holes.
2. An apparatus, comprising:
a plurality of holes;
a reagent composition disposed in each well and comprising a specific copy number of amplifiable reagents; and
the wells are divided into two or more groups having amplifiable reagents arranged at the same particular copy number, but the reagent compositions differ in composition except for the particular copy number.
3. The apparatus of claim 2, comprising
The specific copy number is different for the groups.
4. An apparatus, comprising:
a plurality of holes;
an amplifiable reagent disposed in each well at a particular copy number; and
the wells are divided into two or more groups that differ in their specific copy number of amplifiable reagents.
5. The apparatus of any one of claims 2-4,
wherein at least one of the groups into which the wells are divided is a group in which the specific copy number of the amplifiable reagent is near the detection limit.
6. The apparatus of any one of claims 2-4,
wherein at least one of the groups into which the well is divided is a group in which the specific copy number of the amplifiable reagent is a copy number greater than a quantification limit.
7. The apparatus of any one of claims 2-6,
wherein at least one of the groups into which the well is divided is a negative control group in which the specific copy number of the amplifiable reagent is 0.
8. The apparatus of any one of claims 2-7,
wherein at least one of the groups into which the well is divided is a positive control group in which the specific copy number of the amplifiable reagent is 100 or more.
9. The apparatus of any one of claims 2-8,
wherein at least one of the groups into which the wells are divided is the group with the least copy number except for the negative control group, the at least one group being located at least at wells near the periphery of the device.
10. The apparatus of any one of claims 2-9,
wherein each well comprises at least any one of a primer and an amplification agent.
11. The apparatus of any of claims 2-10, comprising
An identification unit configured to be able to identify information about a specific copy number of the amplifiable reagent in the well.
12. The apparatus of any one of claims 2-11,
wherein the amplifiable reagent is a nucleic acid.
13. The apparatus as set forth in claim 12, wherein,
wherein the nucleic acid is introduced into the nucleic acid of the nucleus.
14. The apparatus of claim 13, wherein the first and second electrodes are disposed in a substantially cylindrical configuration,
wherein the cell is a yeast cell.
15. The apparatus of any one of claims 2-14,
wherein the specific copy number is a counted copy number of the amplifiable reagent.
16. The apparatus of any one of claims 13-15,
wherein the cells are discharged by an inkjet method.
17. A manufacturer skill assessment method for assessing the skill of a manufacturer preparing a reagent composition, the manufacturer skill assessment method comprising:
obtaining value information on Ct values in an apparatus according to any one of claims 1-16; and
evaluating the skill of the preparation based on the obtained information on the Ct value.
18. A manufacturer skill assessment program for assessing the skill of a manufacturer preparing a reagent composition, the manufacturer skill assessment program causing a computer to perform a process comprising:
obtaining value information on Ct values in an apparatus according to any one of claims 1-16; and
evaluating the skill of the preparation based on the obtained information on the Ct value.
19. A maker skill evaluation device configured to evaluate a skill of a maker preparing a reagent composition, the maker skill evaluation device comprising:
a Ct-value-information obtaining unit configured to obtain information on a Ct value in the apparatus according to any one of claims 1 to 16; and
a skill evaluation unit configured to evaluate a skill of the preparation based on the obtained information on the Ct value.
20. A test apparatus performance evaluation method for evaluating performance of a test apparatus configured to test a test target, the test apparatus performance evaluation method comprising:
obtaining value information on Ct values in an apparatus according to any one of claims 1-16; and
evaluating the performance of the test device based on the information about the Ct value.
21. A test apparatus performance evaluation program for evaluating performance of a test apparatus configured to test a test target, the test apparatus performance evaluation program causing a computer to execute a process comprising:
obtaining value information on Ct values in an apparatus according to any one of claims 1-16; and
evaluating the performance of the test device based on the information about the Ct value.
22. A test device performance evaluation device configured to evaluate a performance of a test device configured to test a test target, the test device performance evaluation device comprising:
a Ct-value-information obtaining unit configured to obtain value information on a Ct value in the apparatus according to any one of claims 1 to 16; and
a performance evaluation unit configured to evaluate performance of the test device based on information on a Ct value.
Technical Field
The present disclosure relates to an apparatus, a maker skill evaluation method, a program, and an apparatus, and a test apparatus performance evaluation method, a program, and an apparatus
Background
Polymerase Chain Reaction (PCR) and quantitative polymerase chain reaction (qPCR) are used, for example, for qualitative and quantitative evaluation of nucleic acids. Recently, PCR has also been used for negative tests, for example, of genetically modified crops or food (GMO) and of viral contamination. Therefore, it is necessary to ensure the reliability of the result.
In order to ensure the results, the guarantee of the device performance and the management of the measurement system are performed based on the device temperature control management and the user maintenance management.
A series of nucleic acid samples for qualitative and quantitative evaluation of nucleic acids is generated by serial dilution of nucleic acid samples with known concentrations. For example, a method of diluting a DNA fragment having a specific base sequence by a limiting dilution method and selecting a dilution solution including a target copy number based on a real-time PCR result of the obtained dilution solution is proposed (for example, see PTL 1).
A standard nucleic acid kit obtained by sealing a standard nucleic acid solution serially diluted into different concentrations in a plurality of sample-filled portions has also been proposed (for example, see PTL 2).
Further, for management of the measurement system, it is proposed to ensure the measurement result based on temperature management during the PCR reaction (for example, see NPL 1).
In recent years, real-time polymerase chain reaction (real-time PCR) has become increasingly important in qualitative and quantitative gene testing. The performance evaluation of real-time polymerase chain reactions, especially the performance evaluation between apparatuses or facilities, is important. Currently, a calibration curve based on external standards using absorbance is used. However, these calibration curves do not accurately define the absolute number of nucleic acids. In particular, the inability to accurately define an absolute number has a large impact on low copy numbers.
A series of nucleic acid samples for such a calibration curve was generated by serial dilution of nucleic acid samples with known concentrations. For example, a method of diluting a DNA fragment having a specific base sequence by a limiting dilution method and selecting a dilution solution including a target copy number based on a real-time PCR result of the obtained dilution solution has been proposed (for example, see PTL 1).
Recently, the following methods have also been proposed: a specific number (copy number) of molecules of DNA fragments are introduced into cells by a gene recombination technique, the cells are cultured, and the cultured cells are isolated by manipulation, thereby preparing a sample containing one copy of DNA having a target base sequence (for example, see PTL 3).
Reference list
Patent document
PTL1 Japanese unexamined patent application publication No. 2014-33658
PTL 3 Japanese unexamined patent application publication No. 2015-
Non-patent document
NPL 1:ISO/TS 20836:2005。
Disclosure of Invention
Technical problem
It is an object of the present disclosure to provide a device for appropriately evaluating the skills of a maker preparing a reagent composition, or a device capable of appropriately evaluating the in-plane uniformity of a test device, the performance between test devices, and the performance between test facilities.
Problem solving scheme
According to an aspect of the disclosure, a device comprises a plurality of wells, and two or more groups into which the wells are divided, the two or more groups having different compositions of the wells.
Advantageous effects of the invention
The present disclosure may provide a device for appropriately evaluating the skills of a maker preparing a reagent composition, or a device capable of appropriately evaluating in-plane uniformity of a test device, inter-test-device performance, and inter-test-facility performance.
Drawings
Fig. 1 is a perspective view illustrating an example of the device of the present disclosure.
Fig. 2 is a side view illustrating an example of the apparatus of the present disclosure.
Fig. 3 is a plan view illustrating an example of the apparatus of the present disclosure.
Fig. 4 is a plan view illustrating another example of the apparatus of the present disclosure.
Fig. 5 is a plan view illustrating another example of the apparatus of the present disclosure.
Fig. 6 is a plan view illustrating another example of the apparatus of the present disclosure.
Fig. 7 is a plan view illustrating another example of the apparatus of the present disclosure.
Fig. 8 is a diagram illustrating an example of a calibration curve generated using the apparatus of fig. 7.
Fig. 9 is a plan view illustrating another example of the apparatus of the present disclosure.
Fig. 10 is a plan view illustrating another example of the apparatus of the present disclosure.
Fig. 11 is a plan view illustrating another example of the apparatus of the present disclosure.
Fig. 12 is a plan view illustrating another example of the apparatus of the present disclosure.
Fig. 13 is a plan view illustrating another example of the apparatus of the present disclosure.
FIG. 14 is a graph plotting examples of the relationship between the frequency and fluorescence intensity of cells in which DNA replication has occurred.
Fig. 15A is an example diagram illustrating an example of a solenoid valve type discharge head.
Fig. 15B is an example diagram illustrating an example of a piezoelectric type discharge head.
Fig. 15C is an explanatory view illustrating a modified example of the piezoelectric type discharge head illustrated in fig. 15B.
Fig. 16A is an explanatory diagram plotting examples of voltages applied to the piezoelectric element.
Fig. 16B is an exemplary diagram plotted against another example of the voltage applied to the piezoelectric element.
Fig. 17A is an example diagram illustrating an example of a droplet state.
Fig. 17B is an example diagram illustrating an example of a droplet state.
Fig. 17C is an example diagram illustrating an example of a droplet state.
Fig. 18 is a schematic diagram illustrating an example of a dispensing device configured to sequentially land droplets into an orifice.
Fig. 19 is an example diagram illustrating an example of a droplet forming apparatus.
Fig. 20 is a diagram illustrating a hardware block of a control unit of the droplet forming apparatus of fig. 19.
Fig. 21 is a diagram illustrating a functional block of a control unit of the droplet forming apparatus of fig. 19.
Fig. 22 is a flowchart illustrating an example of the operation of the droplet forming apparatus.
Fig. 23 is an explanatory view illustrating a modified example of a liquid droplet forming apparatus.
Fig. 24 is an explanatory diagram illustrating another modified example of the liquid droplet forming apparatus.
Fig. 25A is a diagram illustrating a case where two fluorescent particles are contained in a flying droplet.
Fig. 25B is a diagram illustrating a case where two fluorescent particles are contained in a flying droplet.
Fig. 26 is a graph plotting an example of the relationship between the luminance Li when the particles do not overlap each other and the actually measured luminance Le.
Fig. 27 is an explanatory view illustrating another modified example of the liquid droplet forming apparatus.
Fig. 28 is an explanatory view illustrating another example of a droplet forming apparatus.
Fig. 29 is an exemplary view illustrating an example of a method for counting cells that have passed through a micro flow path.
Fig. 30 is an exemplary view illustrating an example of a method for capturing an image of a vicinity portion of a nozzle portion of a discharge head.
FIG. 31 is a graph plotting the relationship between the probability P (>2) and the average cell number in FIG. 31.
Fig. 32 is a block diagram illustrating an example of a hardware configuration of a maker skill evaluation device.
Fig. 33 is a diagram illustrating an example of a functional configuration of a maker skill evaluation device.
Fig. 34 is a flowchart illustrating an example of a process performed according to the maker skill assessment program.
Fig. 35 is a block diagram illustrating an example of a hardware configuration of the test apparatus performance evaluation apparatus.
Fig. 36 is a diagram illustrating an example of a functional configuration of a test apparatus performance evaluation apparatus.
Fig. 37 is a flowchart illustrating an example of a process performed according to the test apparatus performance evaluation program.
FIG. 38 is a view illustrating an example of a temperature control method of real-time PCR.
FIG. 39 is a diagram illustrating an example of a plate on which real-time PCR has been performed.
Fig. 40 is a diagram illustrating an example of the result of fig. 39.
Fig. 41 is a diagram illustrating an example of the result of fig. 39.
FIG. 42 is a graph plotting the results of FIG. 39.
FIG. 43 is a graph plotting a relationship between the copy number having a variation according to the Poisson distribution and the coefficient of variation CV.
Detailed Description
(device)
The device of the invention comprises a plurality of wells, and two or more groups into which the wells are divided, the two or more groups of wells differing in composition. The device further includes other components as desired.
Preferably, the device of the present disclosure comprises a plurality of wells, a reagent composition comprising a specific copy number of amplifiable reagents disposed in each well; and two or more groups into which the wells are divided, the two or more groups having amplifiable reagents arranged at the same specific copy number but differing in composition of reagent compositions other than the specific copy number.
The apparatus of the present disclosure is based on the following findings. Existing dilution methods may potentially result in the inability to fill some parts with (multiple) copies, or may result in the inability to arrange copies as designed when the absolute number to be arranged is only one copy. Therefore, the performance of the measurement system cannot be evaluated with high accuracy.
The apparatus of the present disclosure is also based on the following findings. With existing standard nucleic acid kits, the problem of possible variations of sample nucleic acids without regard to the low copy number range cannot be overcome.
The apparatus of the present disclosure is also based on the following findings. The existing methods for evaluating a measurement system evaluate the measurement system based only on temperature as an indirect factor, and cannot appropriately evaluate the influence of manual operation on the measurement system.
The device of the present disclosure, which includes two or more sets of wells, the specific copy numbers of the amplifiable reagents contained in the reagent compositions between the sets being the same, but the reagent compositions other than the specific copy numbers being different in composition, can be used to appropriately assess the skill of the manufacturer that prepared the reagent compositions. Note that the copy number of amplifiable reagents may be correlated with the number of molecules.
Preferably, the device of the present disclosure comprises a plurality of wells, each well having a specific copy number of amplifiable reagents disposed therein; and two or more groups into which the wells are divided, the two or more groups differing in the specific copy number of the amplifiable reagent.
The apparatus of the present disclosure is based on the following findings. Existing dilution methods may result in parts that cannot be filled with (multiple) copies, or may result in copies that cannot be laid out as designed when the absolute number to be laid out is only one copy. Thus, existing dilution methods cannot be used for the evaluation and quantitative evaluation of in-plane properties of test devices.
The apparatus of the present disclosure is also based on the following findings. Existing methods utilizing manipulation are techniques related to methods for preparing sample solutions. The performance evaluation of the test device is not described or suggested. Furthermore, these techniques have problems in terms of throughput.
The device of the present disclosure, which includes two or more groups into which a plurality of wells of amplifiable reagents are divided, each arranged at a specific copy number, such that the specific copy numbers of the amplifiable reagents differ between the groups, makes it possible to appropriately evaluate the in-plane uniformity of a test device, the inter-test-device performance, and the inter-test-facility performance.
When the same base sequence is not introduced in plural number into one molecule, "the number of molecules" may be used synonymously with "copy number".
< well >
For example, the shape, number, volume, material, and color of the holes are not particularly limited and may be appropriately selected depending on the intended purpose.
The shape of the well is not particularly limited, and may be appropriately selected depending on the intended purpose, as long as a reagent composition containing a specific copy number of the amplifiable reagent can be disposed in the well. Examples of the shape of the hole include: concave, such as flat bottom, round bottom, U-bottom, and V-bottom; and a portion on the substrate.
The number of pores is preferably 2 or more, more preferably 5 or more, and still more preferably 50 or more.
A multi-well plate having a well number of 2 or more is suitable.
Examples of multi-well plates include 24-well, 48-well, 96-well, 384-well, or 1,536-well plates.
The pore volume is not particularly limited and may be appropriately selected depending on the intended purpose, and is preferably 10 microliters or more but 1,000 microliters or less in consideration of the amount of the sample used in a general nucleic acid testing device.
The material of the pores is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of the material of the pores include polystyrene, polypropylene, polyethylene, fluorine resin, acrylic resin, polycarbonate, polyurethane, polyvinyl chloride, and polyethylene terephthalate.
Examples of the color of the hole include a transparent color, a translucent color, a coloring, and a full shading color.
The wettability of the pores is not particularly limited and may be appropriately selected depending on the intended purpose. The wettability of the pores is preferably hydrophobic. When the wettability of the well is hydrophobic, adsorption of the amplifiable reagent to the inner wall of the well can be reduced. Further, when the wettability of the well is hydrophobic, the amplifiable reagent, the primer and the amplifying agent in the well may move in a solution state.
The method of imparting hydrophobicity to the inner wall of the pores is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of the method include a method of forming a fluororesin coating film, fluorine plasma treatment, and embossing treatment. Specifically, by applying the hydrophobicity-imparting treatment that imparts a contact angle of 100 degrees or more, a decrease in amplifiable reagents due to liquid overflow can be suppressed, and an increase in the degree of uncertainty (or coefficient of variation) can be suppressed.
< substrate >
The device is preferably a flat plate-like device obtained by providing holes in a base material, but may be a connection-type hole tube such as an 8-piece tube.
For example, the material, shape, size, and structure of the base material are not particularly limited and may be appropriately selected depending on the intended purpose.
The material of the base material is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of the material of the substrate include semiconductors, ceramics, metals, glass, quartz glass, and plastics. Of these materials, plastic is preferred.
Examples of the plastic include polystyrene, polypropylene, polyethylene, fluorine resin, acrylic resin, polycarbonate, polyurethane, polyvinyl chloride, and polyethylene terephthalate.
The shape of the substrate is not particularly limited and may be appropriately selected depending on the intended purpose. For example, plate-like and flat plate shapes are preferable.
The structure of the substrate is not particularly limited and may be appropriately selected depending on the intended purpose, and may be, for example, a single-layer structure or a multi-layer structure.
< identification means >
Preferably, the device comprises an identification unit enabling identification of information about the specific copy number of the amplifiable reagent.
The identification unit is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of the identification unit include a memory, an IC chip, a barcode, a QR code (registered trademark), a radio frequency identifier (hereinafter also referred to as "RFID"), a color code, and a print body.
The arrangement position of the identification units and the number of the identification units are not particularly limited and may be appropriately selected depending on the intended purpose.
Examples of the information stored in the identification unit include not only information on the specific copy number of the amplifiable reagent, but also the analysis result (e.g., activity value and emission intensity), the number of the amplifiable reagent (e.g., cell number), whether the cell is live or dead, the copy number of the specific base sequence, which well of the plurality of wells is filled with the amplifiable reagent, the kind of the amplifiable reagent, the measurement date and time, and the name of the person in charge for measurement.
The information stored in the identification unit may be read using various reading units. For example, when the identification unit is a barcode, a barcode reader is used as the reading unit.
The method for writing information in the identification unit is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of such methods include manual entry, methods of directly writing data by a droplet forming device configured to count the number of amplifiable reagents during dispensing of the amplifiable reagents into the wells, transmission of data stored in a server, and transmission of data stored in a cloud system.
< other Member >
The other members are not particularly limited and may be appropriately selected depending on the intended purpose. Examples of other members include a sealing member.
Sealing means
Preferably, the apparatus includes a sealing member to prevent foreign materials from being mixed into the hole and the filling material from flowing out.
Preferably the sealing member is configured to be able to seal at least one aperture and is separable at the perforation to be able to individually seal or open each aperture respectively.
The shape of the sealing member is preferably a cap shape matching the inner diameter of the hole or a film shape covering the opening of the hole.
Examples of the material of the sealing member include polyolefin resin, polyester resin, polystyrene resin, and polyamide resin.
Preferably, the sealing member has a film shape capable of sealing all the holes at once. It is also preferred that the sealing member is configured to have different adhesive strengths to the hole that needs to be reopened and the hole that does not need to be reopened so that the user can reduce misuse.
The device of the present disclosure includes a plurality of wells, and reagent compositions disposed in the plurality of wells and each comprising a specific copy number of an amplifiable reagent.
Copy number refers to the number of target or specific base sequences in the amplifiable reagents contained in a well.
The target base sequence refers to a base sequence including a defined base sequence in at least the primer and probe regions. Specifically, a base sequence having a defined total length is also referred to as a specific base sequence.
The specific copy number refers to the above-mentioned copy number in which the number of the target base sequence is specified with a certain level of accuracy or more.
This means that a specific copy number is considered to be the number of specific sequences actually contained in a well. That is, the specific copy number in the present disclosure is more accurate or more reliable as a quantity than a predetermined copy number (calculated estimated value) obtained according to the existing serial dilution method, and is a controlled value that does not depend on the poisson distribution even if the value is specifically in a low copy number range of 1,000 or less. When it is said that the specific copy number is a controlled value, it is preferable that the coefficient of variation CV, which represents an uncertainty, approximately satisfies CV <1/√ x, or CV ≦ 20% with respect to the average copy number x. Thus, the use of a device comprising a well containing a specific base sequence at a specific copy number allows qualitative or quantitative tests to be performed on a sample containing a target base sequence more accurately than ever.
When the number of the target base sequence and the number of the nucleic acid molecules comprising the sequence coincide with each other, "copy number" and "molecule number" may be associated with each other.
Specifically, for example, in the case of norovirus, when the number of viruses is 1, the number of nucleic acid molecules is 1, and the specific copy number is 1. In the case of GI stage yeast, when the number of yeast cells is 1, the number of nucleic acid molecules (the number of identical chromosomes) is 1, and the specific copy number is 1. In the case of human cells at G0/GI stage, when the number of human cells is 1, the number of nucleic acid molecules (the number of identical chromosomes) is 2, and the copy number is 2.
In addition, in the case of a GI stage yeast into which the target base sequence was introduced at two positions, when the number of yeast cells was 1, the number of nucleic acid molecules (the number of identical chromosomes) was 1, and the copy number was 2.
In the present disclosure, a particular copy number of an amplifiable reagent may also be referred to as an absolute number of amplifiable reagents.
When there are multiple wells containing amplifiable reagents, it is at least desirable to have the same specific copy number of amplifiable reagents in the wells.
When referring to the same specific copy number of amplifiable reagents contained in a well, it is meant that the amount of amplifiable reagents is within a tolerance, wherein the variation occurs when the amplifiable reagents are filled in the device. Whether or not the variation in the amount of the amplifiable reagent is within the allowable range can be judged based on the following negative information.
The information on the specific copy number of the amplifiable reagent is not particularly limited, and may be appropriately selected depending on the intended purpose, as long as the information is information on the amplifiable reagent in the device. Examples of the information include information on the negative, information on the support described below, and information on the amplifiable reagent.
"failure to qualify" is defined in ISO/IEC Guide 99:2007[ International Voltage building of Metrology-bases and general definitions and related metrics (VIM) ] as a "parameter characterizing the variation or dispersion of values accompanying a measurement that reasonably correlates with the measurement".
Here, the "numerical value reasonably related to the measured quantity" refers to a true value candidate of the measured quantity. That is, the uncertainty refers to information about a variation in measurement results due to operations and devices involved in the preparation of the measurement target. The greater the uncertainty, the greater the variation in the expected measurement.
For example, the uncertainty may be a standard deviation obtained from the measurement results, or a half value of the reliability level, which is expressed as a numerical range in which a true value is contained with a predetermined probability or higher.
The non-certainty can be calculated, for example, according to a method based on Guide to the Expression of Ucertainity in Measurement (GUM: ISO/IEC Guide 98-3) and Japan
By converting the non-certainty to a standard non-certainty, all non-certainty due to factors such as operation and measurement can be expressed as the same level of reliability. The standard does not necessarily indicate the variation of the mean value of the measured values.
In an example method for calculating the denial, for example, a factor that may cause the denial is extracted, and the denial (standard deviation) due to the corresponding factor is calculated. Then, the synthesis is unsuccessfully calculated due to the corresponding factor according to a square sum method to calculate the synthesis criterion unsuccessfully. In the calculation of the synthesis criterion, the sum of squares method is used. Therefore, among the factors causing the non-certainty, the factors causing the non-certainty small enough can be ignored.
In the device of the present disclosure, the coefficient of variation of the amplifiable reagent filled in the well may be used as information about the uncertainty.
The coefficient of variation refers to a relative value of variation in the number of cells (or the number of amplifiable reagents) filled in each recess, wherein the variation occurs when the cells are filled in the recesses. That is, the coefficient of variation refers to the filling accuracy in terms of the number of cells (or amplifiable reagents) filled in the recess. The coefficient of variation is a value obtained by dividing the standard deviation σ by the average value x. Here, the coefficient of variation CV is a value obtained by dividing the standard deviation σ by the average copy number (average filling copy number) x. In this case, a relational expression represented by the following
[ mathematical formula 1]
In general, the cells (or amplifiable reagents) have a random distribution-Poisson distribution-in the dispersion. Therefore, in a random distribution state by the continuous dilution method, i.e., poisson distribution, it can be considered that the standard deviation σ satisfies the relational expression with the average copy number x shown in the
[ mathematical formula 2]
[ mathematical formula 3]
TABLE 1
As can be understood from the results of table 1 and fig. 43, when filling, for example, 100 copy numbers of amplifiable reagents into wells according to the serial dilution method, the final copy number of the amplifiable reagents to be filled in the reaction solution has a Coefficient of Variation (CV) of at least 10%, even when other accuracies are ignored.
With respect to the copy number of the amplifiable agent, it is preferred that the coefficient of variation CV and the average specific copy number x of the amplifiable agent satisfy the relationship: CV <1/√ x, and more preferably CV <1/2 √ x.
With respect to the information on the non-certainty, it is preferable that the device includes a plurality of wells each containing an amplifiable reagent therein, and the information on the non-certainty includes information on the non-certainty of the device as a whole based on a specific copy number of the amplifiable reagent contained in each well.
There are some conceivable causes of the failure. For example, in a preparation process in which an intended amplifiable reagent is introduced into cells and the cells are dispensed while counting the number of cells, examples of conceivable factors include the number of amplifiable reagents in the cells (e.g., the cell cycle of the cells), the unit configured to arrange the cells in the device (including any operational results of the inkjet device or portions of the device, such as the operational timing of the device and the number of cells contained in the droplets when the cell suspension is formed into a droplet form), the frequency with which the cells are arranged in place in the device (e.g., the number of cells arranged in the wells), and contamination due to destruction of the cells in the cell suspension and mixing of the amplifiable reagents in the cell suspension (hereinafter also described as contamination mixing).
Examples of the information on the amount of the amplifiable reagent as the information on the amplifiable reagent include information on the unsuitability of the amount of the amplifiable reagent contained in the device.
In addition to a specific copy number of amplifiable reagents, the reagent composition also comprises components required for amplification of an amplifiable reagent (e.g., a nucleic acid), and for example, comprises primers and an amplification agent.
The primer is a synthetic oligonucleotide having a complementary base sequence including 18 or more but 30 or less bases, and is specific to a template DNA of a Polymerase Chain Reaction (PCR). A pair of primers, i.e., a forward primer and a reverse primer, is provided at two positions in such a manner as to sandwich a region to be amplified.
Examples of the amplification agent for, for example, Polymerase Chain Reaction (PCR) include an enzyme such as DNA polymerase, a substrate such as four bases (dGTP, dCTP, dATP and dTTP), Mg2+(2mM magnesium chloride) and a buffer to maintain an optimal pH (pH of 7.5 to 9.5).
The state of the amplifiable agent, the primer and the amplifying agent in the well is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the states of the amplifiable reagents, primers and amplifiable agents may be in solution or in a solid state. The state of the amplifiable agent, the primer and the amplification agent is particularly preferably a solution state in terms of ease of use. In solution, the user can directly use the amplifiable reagents, primers and amplifiable reagents for testing. In terms of transportation, the state of the amplifiable agent, the primer and the amplifying agent is particularly preferably a solid state, and more preferably a dry state. In the solid dry state, the reaction speed of the amplifiable reagent decomposed by, for example, a decomposing enzyme can be reduced, and the storage stability of the amplifiable reagent, the primer and the amplification agent can be improved.
It is preferable that appropriate amounts of amplifiable reagents, primers and amplification agents are filled in the device in a solid dry state so that the amplifiable reagents, primers and amplification agents in the form of reaction solutions can be directly used by dissolving the amplifiable reagents, primers and amplification agents in a buffer or water immediately before using the device.
The drying method is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of the drying method include freeze drying, heat drying, hot air drying, vacuum drying, steam drying, suction drying, infrared drying, drum drying, and spin drying.
The device comprises two or more sets of wells, the specific copy number of the amplifiable reagent between the sets being the same, but the composition of the reagent composition being different except for the specific copy number. For example, when the substrate of the device is a plate comprising a plurality of wells, the sets of "zones" are formed one by one on the plate. Two or more regions of the reagent composition differing in the specific copy number of the amplifiable reagent may be adjacent to each other or may be separated from each other.
For example, a difference in composition other than a particular copy number of an amplifiable agent means that any other primer or combination of amplifiable agents may be provided that may or may not include nucleic acid that serves as an amplifiable agent. For example, a first set of devices includes (1) compositions comprising nucleic acids, primers, and amplification agents, a second set of devices includes (2) compositions comprising nucleic acids and primers, and a third set of devices includes (3) compositions comprising only nucleic acids. In preparing the reagent composition, the manufacturer adds at least either one of the primer and the amplification agent to the compositions of (2) and (3) by a manual operation, and uses the reagent composition for the PCR reaction. Thus, the device enables, for example, the skill of a manufacturer who prepares the reagent composition to be assessed.
When nucleic acids are used as amplifiable reagents, examples of the skill of the manufacturer include pipetting, the amount of amplifiable to be prepared, and adding reagents to the well plate.
In preparation, the manufacturer may know the type, amount and concentration of nucleic acids, primers (or primer sets including multiple primers), and amplification agents to be included in each set of the device used. This allows the manufacturer to also prepare reagent compositions in a manner suitable for the kind, amount and concentration in each group and use the reagent compositions for PCR reactions. This enables the skill of the maker to be evaluated more appropriately.
Further, the following mode may be adopted: the device comprises a plurality of wells and reagent compositions disposed in the plurality of wells, and each reagent composition comprises at least a composition selected from the group consisting of an amplifiable reagent, a primer, and an amplifiable agent, wherein the reagent compositions comprise two or more groups that differ in composition.
This configuration allows the skill of the manufacturer preparing any composition to be evaluated.
Furthermore, it is preferred that the device comprises sets that differ from each other in the specific copy number of the amplifiable reagent. That is, it is preferred that there be more than two specific copy numbers of amplifiable reagents between one well and any other well(s). Examples of combinations of two or more specific copy numbers include combinations of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, combinations of 1, 3, 5, 7, and 9, and combinations of 2, 4, 6, 8, and 10.
In the device of the present disclosure, it is preferred that the specific copy number of the amplifiable reagent in one well is 10N1And the specific copy number of the amplifiable reagent in the other well is 10N2(wherein N1 and N2 are consecutive integers). Examples of such combinations of specific copy numbers include 1, 10100 and 1,000, and combinations of 100, 1,000, 10,000, 100,000 and 1,000,000. Thus, calibration curves for a wide range from low to high copy numbers can be easily generated by the apparatus of the present disclosure.
The devices of the present disclosure include two or more sets of wells that differ in the specific copy number of the amplifiable reagents. For example, when the substrate of the device is a plate comprising a plurality of wells, the "zones" of each set are formed one by one on the plate. In a "region" formed by two or more groups of amplifiable reagents that differ in specific copy number, the wells may be adjacent to each other or may be separated from each other.
Thus, using the device of the present disclosure, when it is found that a test device performing real-time PCR (a performance assessment) comprises one well (failing well) that is not suitable as compared to another well that exists at a different location and shares the same specific copy number, it can be determined whether to recalibrate the test device by real-time PCR or to exclude the failing well from actual sample application. In addition, by periodically measuring a test device using the device of the present disclosure, information on temporal changes in Ct values of in-plane positions of the test device can be obtained, and in-plane characteristics of the test device can be evaluated based on the information. Furthermore, the use of devices arranged with the same specific copy number enables a comparison between the test device subjected to measurement and another test device.
Preferably, at least one of the sets of wells is a set in which the specific copy number of the amplifiable reagent is close to the detection limit.
The limit of detection (LOD) represents the minimum copy number of amplifiable agent detectable by the method of detecting an amplifiable agent (e.g., a nucleic acid). The detection limit is not particularly limited, may be appropriately selected according to the measurement method, and may be, for example, the average ± 3 σ.
In the present specification, when there are sample groups different in specific copy number and each sample set includes 21 samples having the same specific copy number, and any one sample group results in undetectable (corresponding to 2 σ judged at a probability of 95%) from one of the 21 samples, the minimum copy number in the specific copy number of such sample group can be used as a detection limit.
Approaching the detection limit means that the copy number is within ± 1 of the detection copy number limit.
It is assumed that the device of the present disclosure includes at least a set in which a specific copy number of the amplifiable reagent approaches the detection limit. For example, assume that the device of the present disclosure includes sets of specific copy numbers of amplifiable reagents of "1", "2", "3", "4", and "5", respectively. In this case, when the test device for performance evaluation using the device of the present disclosure is proved to be capable of amplifying an amplifiable reagent in a group having a specific copy number of "3" or more but is not capable of amplifying an amplifiable reagent in a group having a specific copy number of "2" or less, it can be shown that the lower limit value of the detection limit indicating the specific copy number of the amplifiable reagent in the test device is "3". Further, when another similar test device for performance evaluation using the device of the present disclosure was demonstrated to be able to amplify an amplifiable reagent in a group having a specific copy number of "4" or more but not in a group having a specific copy number of "3" or less, it could be shown that the lower limit value of the detection limit indicating the specific copy number of the amplifiable reagent in the detection device was "4". Thus, the minimum specific copy number of amplifiable reagents detectable by the test device may be determined.
Preferably, at least one of the groups of amplifiable reagents arranged at a specific copy number is a group in which the specific copy number of amplifiable reagents is greater than the copy number of the quantitation limit.
The limit of quantitation (LOQ) represents the minimum copy number of an amplifiable reagent that can be quantitated by a method that can quantitate an amplifiable reagent (e.g., a nucleic acid), where "quantifiable" indicates that the quantitation result can be sufficiently reliable. The limit of quantification is not particularly limited and may be appropriately selected according to the measurement method.
In the present specification, a value representing the deviation of the copy number from the linearity of a calibration curve generated using samples formed from a plurality of molecular species (e.g., a plurality of nucleic acid samples having different specific molecular numbers (having different copy numbers)) can be used as a limit of quantification. Alternatively, the uncertainty of the calibration curve may be represented by a CV value. In a graph in which CV values are plotted by representing the copy number on the horizontal axis and the Ct value on the vertical axis, for example, a value (copy number) with a CV value lower than 5% or 10% may be used as a limit of quantification.
In quantitative evaluation, the number of molecules (copy number or concentration) corresponding to the Ct value, rather than the Ct value itself, can be obtained from the calibration curve and PCR efficiency. Therefore, the limit of quantitation can be set based on the CV value converted to the number of molecules (copy number or concentration).
When the device of the present disclosure includes at least a group in which the specific copy number of an amplifiable reagent (e.g., a nucleic acid) is a copy number greater than the limit of quantitation, for example, the minimum specific copy number of an amplifiable reagent that can ensure quantitative detection by a test device can be determined, as if one test device can be found that can ensure quantitative detection when the specific copy number of an amplifiable reagent is 10 or more and another test device can be found that can ensure quantitative detection when the specific copy number of a nucleic acid is 20 or more.
In the device of the present disclosure, preferably at least one of the well groups is a negative control group in which the specific copy number of the amplifiable reagent is 0.
In the case where at least one of the well groups in the device of the present disclosure is set as a negative control group in which the specific copy number of the amplifiable reagent is 0, any detection of the amplifiable reagent from the negative control group indicates that the detection system (reagent or device) is abnormal. In the case where the negative control group is provided in the apparatus, the user can immediately recognize the problem when it occurs, and can stop measuring and checking the source of the problem.
In the device of the present disclosure, preferably at least one of the well groups is a positive control group in which the specific copy number of the amplifiable reagent is 100 or more.
In the case where at least one of the well groups in the device of the present disclosure is set as a positive control group in which the specific copy number of the amplifiable reagent is 100 or more, any failure to detect (the case of) the amplifiable reagent from the positive control group indicates that the detection system (reagent or device) is abnormal. In the case where the positive control group is set in the apparatus, the user can immediately recognize the problem when it occurs, and can stop measuring and checking the source of the problem.
In the device of the present disclosure, it is further preferred that at least one of the sets of wells is a set having the smallest number of copies (a set having the smallest number of copies) except for the negative control group, and the set having the smallest number of copies is located at least in a well at the periphery of the near (appoximately) device.
The nearly peripheral holes refer to the outermost peripheral holes and the inward rows of holes from the outermost periphery among the two-dimensionally arranged holes on the device. Examples of outermost rows of holes and rows of holes inward from the outermost periphery include the first row, or the higher ordinal row but at row 47, or the lower ordinal row.
Unlike the holes located near the center of the device, the holes located at the outermost periphery of the device are free of other holes on the outside and define a boundary between the device and the exterior of the device. Therefore, (1) the outermost peripheral well physically has uneven heat conduction on the device, and (2) the outermost peripheral well is susceptible to the temperature fluctuation factor of the PCR device because these wells are provided on the periphery of the temperature control member, which is a part constituting the PCR device. Therefore, by setting at least one of the well groups to the group with the smallest copy number (the group with the smallest copy number) in the device of the present disclosure except for the negative control group and by setting the group with the smallest copy number to the well at least approximately at the periphery of the device, it is possible to detect a malfunction of the PCR device with higher sensitivity.
Preferably, the amplifiable reagent is a nucleic acid. Preferably, the nucleic acid is introduced into the nucleus of a cell.
Nucleic acid-
Nucleic acids refer to polymeric organic compounds: wherein the nitrogenous base, the sugar and the phosphate derived from purine or pyrimidine are regularly bonded to each other. Examples of nucleic acids also include nucleic acid fragments or nucleic acid analogs or analogs of nucleic acid fragments.
The nucleic acid is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of nucleic acids include DNA, RNA and cDNA.
The nucleic acid or nucleic acid fragment may be a natural product obtained from a living organism, or a processed product of a natural product, or a product produced by a genetic recombination technique, or a chemically synthesized artificially synthesized nucleic acid molecule. One of these nucleic acids may be used alone, or two or more of these nucleic acids may be used in combination. With the artificially synthesized nucleic acid molecule, impurities can be suppressed and the molecular weight can be set to a low level. This makes it possible to improve the initial reaction efficiency.
An artificially synthesized nucleic acid refers to an artificially synthesized nucleic acid that is produced to have the same components (base, deoxyribose, and phosphate) as a naturally occurring DNA or RNA. Examples of the artificially synthesized nucleic acid include not only a nucleic acid having a base sequence encoding a protein but also a nucleic acid having an arbitrary base sequence.
Examples of analogues of nucleic acids or nucleic acid fragments include nucleic acids or nucleic acid fragments bonded to non-nucleic acid components, nucleic acids or nucleic acid fragments labeled with a labeling agent such as a fluorescent dye or an isotope (e.g., a primer or probe labeled with a fluorescent dye or a radioisotope), and artificial nucleic acids, i.e., nucleic acids or nucleic acid fragments in which the chemical structure of some of the component nucleotides is changed (e.g., PNA, BNA, and LNA).
The form of the nucleic acid is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of nucleic acid forms include double-stranded nucleic acids, single-stranded nucleic acids, and partially double-stranded or single-stranded nucleic acids. Circular or linear plasmids may also be used.
The nucleic acid may be modified or mutated.
Preferably, the nucleic acid has a specific base sequence. The term "specifically" means "specifically designated".
The specific base sequence is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of the specific base sequence include a base sequence used for infectious disease testing, a non-natural base sequence which does not exist in nature, a base sequence derived from an animal cell, a base sequence derived from a plant cell, a base sequence derived from a fungal cell, a base sequence derived from a bacterium, and a base sequence derived from a virus. One of these base sequences may be used alone, or two or more of these base sequences may be used in combination.
When a non-natural base sequence is used, the specific base sequence preferably has a GC content of 30% or more but 70% or less, and preferably has a constant GC content (for example, see SEQ ID NO. 1).
The base length of the specific base sequence is not particularly limited and may be appropriately selected depending on the intended purpose, and may be, for example, a base length of 20 base pairs (or mers) or more but 10,000 base pairs (or mers) or less.
When a base sequence for infectious disease test is used, the base sequence is not particularly limited, and may be appropriately selected depending on the intended purpose, as long as the base sequence includes a specific base sequence of a target infectious disease. Preferably, the base sequence includes a base sequence specified in an official analysis method or an official announcement method (for example, see SEQ ID NO.2 and 3).
The nucleic acid may be a nucleic acid derived from the cell used, or a nucleic acid introduced by a transgene. When a nucleic acid introduced by a transgene and a plasmid are used as the nucleic acid, it is preferable to confirm that one copy of the nucleic acid is introduced per cell. The method for confirming the introduction of 1 copy of the nucleic acid is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of the method include a sequencer, a PCR method, and a Southern blotting method.
One or two or more nucleic acids having a specific base sequence may be introduced through a transgene. Also in the case where only one kind of nucleic acid is introduced by transgene, base sequences of the same kind may be introduced in tandem according to the intended purpose.
The method of transgene is not particularly limited, and may be appropriately selected depending on the intended purpose, as long as the method can introduce a desired copy number of a specific nucleic acid at a desired position. Examples of such methods include homologous recombination, CRISPR/Cas9, CRISPR/Cpf1, TALEN, zinc finger nucleases, Flip-in, and Jump-in (Jump-in). In the case of yeast fungi, homologous recombination is preferred among these methods in terms of high efficiency and easy control.
A carrier-
It is preferable to treat the amplifiable reagent in a state of being carried on a carrier. When the amplifiable agent is a nucleic acid, the preferred form is that the nucleic acid is carried (or more preferably encapsulated) by a carrier (carrier particle) having a particle shape.
The carrier is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of carriers include cells, resins, liposomes, and microcapsules.
-cell-
By cell is meant a structural functional unit that comprises an amplifiable agent (e.g., a nucleic acid) and forms an organism.
The cells are not particularly limited and may be appropriately selected depending on the intended purpose. All kinds of cells can be used, whether the cells are eukaryotic cells, prokaryotic cells, multicellular biological cells, and unicellular biological cells. One of these kinds of cells may be used alone, or two or more of these kinds of cells may be used in combination.
The eukaryotic cell is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of eukaryotic cells include animal cells, insect cells, plant cells, fungi, algae, and protozoa. One of these kinds of eukaryotic cells, or two or more of these kinds of eukaryotic cells may be used in combination. Among these eukaryotic cells, animal cells and fungi are preferable.
The adherent cells may be primary cells directly taken from a tissue or organ, or may be cells obtained by passaging primary cells directly taken from a tissue or organ several times. The adherent cells may be appropriately selected depending on the intended purpose. Examples of adherent cells include differentiated cells and undifferentiated cells.
The differentiated cells are not particularly limited and may be appropriately selected depending on the intended purpose. Examples of differentiated cells include: hepatocytes, which are parenchymal cells of the liver; an astrocyte cell; kupffer cells; endothelial cells, such as vascular endothelial cells, sinus endothelial cells, and corneal endothelial cells; a fibroblast cell; osteoblasts; osteoclasts; periodontal ligament-derived cells; epidermal cells, such as epidermal keratinocytes; epithelial cells, such as tracheal epithelial cells, intestinal epithelial cells, cervical epithelial cells, and corneal epithelial cells; a mammary gland cell; a pericyte; muscle cells, such as smooth muscle cells and cardiac muscle cells; a renal cell; pancreatic islet cells; nerve cells, such as peripheral nerve cells and optic nerve cells; chondrocytes; and bone cells.
The undifferentiated cell is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of undifferentiated cells include: pluripotent stem cells such as embryonic stem cells belonging to undifferentiated cells, and mesenchymal stem cells having pluripotency; unipotent stem cells, such as vascular endothelial progenitor cells with unipotent properties; and iPS cells.
The fungus is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of fungi include mold and yeast fungi. One of these kinds of fungi may be used alone, or two or more of these kinds of fungi may be used in combination. Among these species of fungi, yeast fungi are preferred because the cell cycle is adjustable and haploids can be used.
The cell cycle refers to a cell proliferation process in which a cell undergoes cell division, and a cell (daughter cell) resulting from the cell division becomes a cell (mother cell) that undergoes another cell division to generate a new daughter cell.
The yeast fungus is not particularly limited and may be appropriately selected depending on the intended purpose. For example, yeast fungi that are synchronously cultured to be synchronized at G0/G1 and fixed at G1 are preferable.
Further, as yeast fungi, for example, Bar 1-deficient yeasts having enhanced sensitivity to a pheromone (sex hormone) which controls the G1 phase cell cycle are preferable. When the yeast fungus is a Bar1 deficient yeast, the abundance ratio of the yeast fungus with uncontrolled cell cycle can be reduced. This makes it possible, for example, to prevent an increase in the number of specific nucleic acids in the cells contained in the well.
The prokaryotic cell is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of prokaryotic cells include eubacteria and archaea. One of these kinds of prokaryotic cells may be used alone, or two or more of these kinds of prokaryotic cells may be used in combination.
As the cell, a dead cell is preferable. The use of dead cells prevents cell division after fractionation.
As the cell, a cell that can emit light when receiving light is preferable. With cells that emit light when subjected to light, the cells can be dropped into the wells while having a high degree of precise control over the number of cells.
Receiving light means receiving light.
Optical sensors refer to passive sensors configured to collect with a lens any light in the visible to near infrared, short wavelength infrared and thermal infrared range of wavelengths longer than visible light rays visible to the human eye to obtain a target cell shape, for example in the form of image data.
Cells that emit light upon exposure to light- -
The cell that can emit light when receiving light is not particularly limited, and may be appropriately selected depending on the intended purpose, as long as the cell can emit light when receiving light. Examples of the cells include cells stained with a fluorescent dye, cells expressing a fluorescent protein, and cells labeled with a fluorescent-labeled antibody.
The cell site stained with a fluorescent dye, expressing a fluorescent protein, or labeled with a fluorescent-labeled antibody is not particularly limited. Examples of such cellular sites include whole cells, nuclei, and membranes.
-fluorescent dyes- -
Examples of fluorescent dyes include fluorescein, azo dyes, rhodamine, coumarin, pyrenes, cyanine (cyanines). One of these fluorescent dyes may be used alone, or two or more of these fluorescent dyes may be used in combination. Among these fluorescent dyes, fluorescein, azo dyes, and rhodamine are preferable, and eosin, evan blue, trypan blue, rhodamine 6G, rhodamine B, and rhodamine 123 are more preferable.
As the fluorescent dye, commercially available products can be used. Examples of commercially available products include the product name: EOSIN Y (available from Wako Pure Chemical Industries, Ltd.); the product name is as follows: EVANS BLUE (available from Wako pure chemical Industries, ltd.), product name: TRYPAN BLUE (available from Wako Pure chemical industries, ltd.); the product name is as follows: RHODAMINE 6G (available from Wako Pure Chemical Industries, ltd.); the product name is as follows: RHODAMINE B (available from Wako Pure Chemical Industries, ltd.); and product name: RHODAMINE 123 (available from Wako Pure Chemical Industries, ltd.).
- -fluorescent protein-
Examples of fluorescent proteins include Sirius, EBFP, ECFP, mTurquoise, TagCFP, AmCyan, mTFP1, Midorisis Cyan, CFP, TurboGFP, AcGFP, TagGFP, Azami-Green, ZsGreen, EmGFP, EGFP, GFP2, HyPer, TagYFP, EYFP, Venus, YFP, PhiYFP-m, TurboYFP, ZsYellow, mBanana, Kusabiarange, mOrange, TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, mStrery, TurboFP602, mRFP1, Red2, KirKirKillecKirmPer, KirmPer, KirmPy, KirmPol, KirmPp, Kirke-Msry, Kirke-Msrep, Kirbep, Kirke. These fluorescent proteins may be used alone, or two or more of these fluorescent proteins may be used in combination.
- -fluorescent-labeled antibody- -
The fluorescent-labeled antibody is not particularly limited, and may be appropriately selected depending on the intended purpose, as long as the fluorescent-labeled antibody is fluorescently labeled. Examples of fluorescently labeled antibodies include CD4-FITC and CD 8-PE. One of these fluorescently labeled antibodies may be used alone, or two or more of these fluorescently labeled antibodies may be used in combination.
In the free state, the volume average particle diameter of the cells is preferably 30 micrometers or less, more preferably 10 micrometers or less, and particularly preferably 7 micrometers or less. When the volume average particle diameter of the cells is 30 μm or less, the cells can be suitably used for an ink-jet method or a droplet discharge unit such as a cell sorter.
The volume average particle diameter of the cells can be measured by, for example, the following measurement method.
10 microliters of the resulting dyed yeast dispersion was extracted and poured onto a plastic slide formed of PMMA. The volume average particle size of the CELLs can then be measured using an AUTOMATED CELL COUNTER (product name: COUNTESS AUTOMATED CELL COUNTER, available from Invitrogen). The cell number can be obtained by a similar measurement method.
The concentration of the cells in the cell suspension is not particularly limited, may be appropriately selected depending on the intended purpose, and is preferably 5 × 1045 × 10 above cell/mL8cell/mL or less, more preferably 5 × 104cells/mL above but 5 × 107cell/mL or less, when the number of cells is 5 × 104cells/mL above but 5 × 108When the cell/mL ratio is less than or equal to the predetermined cell/mL ratio, the cells can be contained in the discharged droplet without fail. The CELL number can be measured with an automatic CELL COUNTER (product name: COUNTESS AUTOMATED CELL COUNTER, available from Invitrogen) in the same manner as the volume average particle diameter is measured.
The number of cells of the cell containing the nucleic acid is not particularly limited, and may be appropriately selected depending on the intended purpose, so long as the number of cells is plural.
-resins-
The material, shape, size, and structure of the resin are not particularly limited, and may be appropriately selected depending on the intended purpose, as long as the resin can carry an amplifiable agent (e.g., a nucleic acid).
-liposomes-
Liposomes are lipid vesicles formed from a lipid bilayer comprising lipid molecules. In particular, liposomes refer to lipid-containing closed vesicles that include a space separated from the external environment by a lipid bilayer created based on the polarity of hydrophobic and hydrophilic groups of lipid molecules.
Liposomes are closed vesicles formed of lipid bilayers using lipids, and include an aqueous phase (inner aqueous phase) in the space of the closed vesicles. The internal aqueous phase comprises, for example, water. Liposomes can be unilamellar (unilamellar or unilamellar with a single bilayer) or multilamellar (multilamellar with an onion-like structure comprising multiple bilayers, with the individual layers separated by aqueous layers).
As liposomes, liposomes that can encapsulate amplifiable agents (e.g., nucleic acids) are preferred. The encapsulation form is not particularly limited. By "encapsulated" is meant the form in which the nucleic acid is contained in the internal aqueous and liposomal layers. Examples of such forms include forms in which nucleic acid is encapsulated in an enclosed space formed by a layer, forms in which nucleic acid is encapsulated in a layer itself, and combinations of these forms.
The size (average particle diameter) of the liposome is not particularly limited as long as the liposome can encapsulate an amplifiable agent (e.g., a nucleic acid). The liposomes preferably have a spherical form or a form close to spherical form.
The components (layer components) constituting the lipid bilayer of the liposome are selected from lipids. As the lipid, any lipid soluble in a mixed solvent of a water-soluble organic solvent and an ester-based organic solvent can be used. Specific examples of the lipid include phospholipids, lipids other than phospholipids, cholesterol, and derivatives of these lipids. These components may be formed from one component or from a plurality of components.
Microcapsules-
The microcapsule refers to a minute particle having a wall material and a hollow structure, and may encapsulate an amplifiable agent (e.g., nucleic acid) in the hollow structure.
The microcapsule is not particularly limited, and, for example, the wall material and the microcapsule size may be appropriately selected depending on the intended purpose.
Examples of the wall material of the microcapsule include polyurethane resin, polyurea-polyurethane resin, urea-formaldehyde resin, melamine-formaldehyde resin, polyamide, polyester, polysulfone amide, polycarbonate, polysulfonate, epoxy resin, acrylate, methacrylate, vinyl acetate, and gelatin. One of these wall materials may be used alone, or two or more of these wall materials may be used in combination.
The size of the microcapsule is not particularly limited and may be appropriately selected depending on the intended purpose, as long as the microcapsule can encapsulate an amplifiable reagent (e.g., a nucleic acid).
The method for producing the microcapsules is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of the method include an in-situ method, an interfacial polymerization method, and a coagulation method.
Fig. 1 is a perspective view illustrating an example of the device of the present disclosure. Fig. 2 is a side view of the device of fig. 1. In the
Fig. 3 is a plan view illustrating an example of the apparatus of the present disclosure. In the apparatus of FIG. 3, 10 copies of a specific copy number of nucleic acid and primers were located on almost the entire surface of a 96-well plate. At the center, negative controls (NTCs) of 0 copies were located at two locations, Positive Controls (PCs) with 1,000 copies of nucleic acid and primers and amplification agents were located at two locations.
Real-time PCR was performed using the apparatus of fig. 3. The measurement results of Ct (Threshold Cycle) values are shown in FIG. 4. The average Ct value was 37.1. The standard deviation of the Ct value was 1.00. The CV value [ (standard deviation/average Ct value) × 100] was 2.70. In fig. 4, "UD" indicates that no Ct value is detected.
For example, in real-time PCR, first, a master mix (available from Thermo Fisher Scientific Inc., TAQMAN Universal PCR master mix) (1. mu.l), forward and reverse primers (0.5 nmol each) for amplifying a specific base sequence, and a probe (0.4nmol) are added to each sample in the apparatus. Amplification and detection can then be performed using a real-time PCR device (available from ThermoFisher Scientific inc.,
The Ct value represents the cycle number at the time when the fluorescence signal of the reaction crosses the threshold line. Since the Ct value linearly decreases to the logarithm of the target initial amount, the initial copy number of DNA can be calculated based on the Ct value.
The threshold line represents the signal level at which a statistically significant increase from the calculated baseline signal was observed, and means the threshold for real-time PCR reactions.
Therefore, the in-plane characteristics of the device can be evaluated based on, for example, the average Ct value, the standard deviation of the Ct value, the CV value [ (standard deviation/average Ct value) 100], and [ (Ct value (maximum) -Ct value (minimum))/2 · average Ct value ] × 100.
In the device of fig. 5, 3 copies of a specific copy number of nucleic acid, primers, and amplification agent are located on almost the entire surface of a 96-well plate. At the center of the plate, negative controls (NTCs) of 0 copies were located at four positions.
Real-time PCR was performed using the apparatus of fig. 5 to measure Ct values and calculate average Ct values. A pore with a particular copy number (3 copies) is "omicron" when its Ct value is within 10% of the average Ct value. When the Ct value of a well with a specific copy number (3 copies) is greater than 10% of the average Ct value, the well is shown to be "x". The results are shown in FIG. 6. In fig. 6, "UD" indicates that no Ct value is detected.
As a result, the Ct values at four wells on the periphery of the 96-well plate were greater than 10% of the average Ct value. Indicating that these four wells in the periphery are not suitable for low copy numbers. Therefore, it can be judged whether calibration is again performed by real-time PCR or whether the four peripheral wells are not used for actual samples.
For example, by performing measurement for a certain period of time using the apparatus described with reference to fig. 3 to 6, the temporal change in the Ct value can be obtained. Therefore, as with the in-plane characteristics of the test device, when a value greater than 10% of the average Ct value is obtained as the Ct value of the aperture, calibration of the test device may be performed or measures may be taken without using the test site. Further, since the specific copy number of the arranged copies is an absolute value, using a device having copies arranged at the same specific copy number enables performance comparison between test devices.
In the 96-well plate shown in fig. 7, specific copy numbers of nucleic acids, primers and amplification agents of 2 copies, 3 copies, 5 copies, 10 copies, 20 copies, 50 copies and 100 copies were arranged, and negative control (NTC) of 0 copies and Positive Control (PC) of 500 copies of nucleic acids and primers were arranged at the center.
Real-time PCR was performed using the apparatus of fig. 7 to measure Ct values. A calibration curve plotting Ct values on the vertical axis and copy number on the horizontal axis is shown in fig. 8. Coefficient of correlation (R)2) Used as a performance index for the test device. In this case, the correlation coefficient is 0.99. The correlation coefficient is a value indicating the degree of coincidence of data with the calibration curve, and reflects the linearity of the calibration curve. The minimum number of copies located at the outermost periphery (in this example, 2 copies) can serve as an index of in-plane uniformity.
In the device of fig. 9, nucleic acids, primers, and amplification agents of specific copy numbers of 2 copies, 10 copies, and 100 copies were located at the center of a 96-well plate, and 0-copy negative control (NTC) and 500-copy nucleic acid Positive Control (PC) and primers were located at the center. The minimum copy number (in this example, 2 copies) located on the outermost periphery and the approximate periphery including a horizontal row and two vertical rows inward from the outermost periphery can serve as an indicator of in-plane uniformity.
In the apparatus of fig. 10, nucleic acids, primers, and amplification agents of specific copy numbers of 2 copies, 10 copies, and 100 copies were located in 20 wells of each specific copy number in a 96-well plate, the minimum copy number (in this example, 2 copies) was located in the outermost periphery, and the well of specific copy number of 100 copies also served as a Positive Control (PC). The minimum number of copies located at the outermost periphery (in this example, 2 copies) can serve as an index of in-plane uniformity.
In the apparatus of FIG. 11, nucleic acids and primers of specific copy numbers of 3 copies, 5 copies, 10 copies and 100 copies are located in a 96-well plate, and the minimum copy number (in this case, 2 copies) is located at the outermost periphery and in a cross shape. A 0 copy negative control (NTC) and a 500 copy nucleic acid Positive Control (PC), primers and amplification reagents were arranged.
In the apparatus of fig. 12, nucleic acids and primers of specific copy numbers of 2 copies, 5 copies, 10 copies, and 100 copies are located in a 96-well plate, and the minimum copy number (in this example, 2 copies) is located at the outermost periphery. The 0 copy negative control (NTC) and the 500 copy nucleic acid Positive Control (PC) were centered with primers.
In the apparatus of FIG. 13, nucleic acids, primers and amplification agents at specific copy numbers of 2 copies, 10 copies and 100 copies are located in the center of a 384-well plate, and the minimum copy number (in this case, 2 copies) is located in the rest of the plate. The minimum number of copies (in this example, 2 copies) located on the outermost periphery and the near periphery including 5 horizontal rows and 8 vertical rows inward from the outermost periphery can serve as an indicator of in-plane uniformity.
By performing the measurement for a certain period of time using the apparatus described with reference to fig. 7 and 9 to 13, the temporal change in the Ct value can be obtained. Therefore, like the in-plane characteristic of the test apparatus, when a value deviating from the quality control value is obtained, calibration of the test apparatus may be performed or a measure not using the measurement position may be taken. Further, since the copy number of the arranged copies is an absolute value, performance comparison between test apparatuses can be achieved using apparatuses having copies arranged at the same copy number.
< method for producing apparatus >
The following will describe a method of preparing using a device including specific nucleic acid cells.
The device preparation method of the present disclosure comprises: a cell suspension preparation step of preparing a cell suspension comprising a plurality of cells containing a specific nucleic acid and a solvent; a droplet landing step of discharging the cell suspension in the form of droplets so that the droplets land successively in the wells of the plate; a cell count step of counting the number of cells contained in the droplet with a sensor after the droplet is discharged and before the droplet is dropped into the hole; and a nucleic acid extraction step of extracting nucleic acid from the cells in the wells, preferably including a step of calculating the degree of certainty of the estimated number of nucleic acid in the cell suspension preparation step, the droplet landing step, and the cell number counting step, an output step, and a recording step, and further including other steps as necessary.
< method for producing cell suspension >)
The cell suspension preparation step is a step of preparing a cell suspension comprising a plurality of cells containing a specific nucleic acid and a solvent.
Solvent refers to the liquid used to disperse the cells.
Suspension of a cell suspension refers to a state in which cells are present in a solvent in a dispersed manner.
The preparation refers to the preparation operation.
Cell suspensions
The cell suspension comprises a plurality of cells comprising a specific nucleic acid and a solvent, preferably comprising an additive, and further comprising other components as required.
The plurality of cells comprising a particular nucleic acid are as described above.
-solvent- -
The solvent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of the solvent include water, a culture solution, a separation solution, a diluent, a buffer, an organic matter dissolving solution, an organic solvent, a polymer gel solution, a colloidal dispersion solution, an electrolytic aqueous solution, an inorganic salt aqueous solution, a metal aqueous solution, and a mixture of these liquids. One of these solvents may be used alone, or two or more of these solvents may be used in combination. Of these solvents, water and a buffer are preferable, and water, Phosphate Buffered Saline (PBS), and Tris-EDTA buffer (TE) are more preferable.
Additives- -
The additive is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of additives include surfactants, nucleic acids, and resins. One of these additives may be used alone, or two or more of these additives may be used in combination.
The interfacial active agent can prevent cells from aggregating with each other and improve continuous loading stability.
The surfactant is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of the surfactant include ionic surfactants and nonionic surfactants. One of these surfactants may be used alone, or two or more of these surfactants may be used in combination. Among these surfactants, nonionic surfactants are preferable because proteins are not modified or deactivated by the nonionic surfactants, although depending on the added amount of the nonionic surfactants.
Examples of ionic surfactants include sodium fatty acid, potassium fatty acid, sodium alpha-sulfo fatty acid ester, sodium linear alkyl benzene sulfonate, sodium alkyl sulfate, sodium alkyl ether sulfate, and sodium alpha-olefin sulfonate. One of these ionic surfactants may be used alone, or two or more of these ionic surfactants may be used in combination. Among these ionic surfactants, sodium fatty acid is preferable, and Sodium Dodecyl Sulfate (SDS) is more preferable.
Examples of nonionic surfactants include alkyl glycosides, alkyl polyoxyethylene ethers (e.g., BRIJ series), octylphenol ethoxylates (e.g., TRITON X series, IGEPAL CA series, NONIDET P series, and NIKKOL OP series), polysorbates (e.g., TWEEN series, such as TWEEN 20), sorbitan fatty acid esters, polyoxyethylene fatty acid esters, alkyl maltosides, sucrose fatty acid esters, glycoside fatty acid esters, glycerin fatty acid esters, propylene glycol fatty acid esters, and fatty acid monoglycerides. One of these nonionic surfactants may be used alone, or two or more of these nonionic surfactants may be used in combination. Among these nonionic surfactants, polysorbate is preferred.
The content of the surfactant is not particularly limited and may be appropriately selected depending on the intended purpose, and is preferably 0.001 mass% or more but 30 mass% or less with respect to the total amount of the cell suspension. When the content of the surfactant is 0.001% by mass or more, the effect of adding the surfactant can be obtained. When the content of the surfactant is 30% by mass or less, aggregation of cells can be suppressed, so that the number of nucleic acid molecules in the cell suspension can be strictly controlled.
The nucleic acid is not particularly limited, and may be appropriately selected depending on the intended purpose, as long as the nucleic acid does not affect the detection of the detection target nucleic acid. Examples of nucleic acids include ColE1 DNA. With such a nucleic acid, it is possible to prevent a nucleic acid having a specific base sequence from attaching to the wall surface of the well.
The resin is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of the resin include polyethylene imide.
Other materials- -
The other materials are not particularly limited and may be appropriately selected depending on the intended purpose. Examples of other materials include crosslinking agents, pH adjusting agents, preservatives, antioxidants, osmotic pressure adjusting agents, wetting agents, and dispersing agents.
< method for dispersing cells >
The cell dispersion method is not particularly limited, and may be appropriately selected depending on the intended purpose. Examples of the method include a medium method such as a bead mill, an ultrasonic method such as an ultrasonic homogenizer, and a method using a pressure difference such as a French press. One of these methods may be used alone, or two or more of these methods may be used in combination. Among these methods, the ultrasonic method is more preferable because the ultrasonic method has low damage to cells. With the media approach, high crushing forces may damage cell membranes or cell walls, and the media may mix as contaminants.
< method of cell selection >
The cell screening method is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of the method include screening by wet sorting, cell sorter and filter. One of these methods may be used alone, or two or more of these methods may be used in combination. Among these methods, screening by a cell sorter and a filter is preferable because the method has low damage to cells.
The number of nucleic acids having a base sequence of interest among the number of cells contained in the cell suspension is estimated, preferably by measuring the cell cycle of the cells.
Measuring the cell cycle means quantifying the number of cells due to cell division.
Estimating the number of nucleic acids means obtaining the copy number of nucleic acids based on the number of cells.
The number of cells to be counted is not necessarily the number of cells, but may be the number of target base sequences. In general, it is considered that it is reliable that the number of target base sequences is equal to the number of cells because the cells selected as the counted cells are cells each containing one target base sequence (═ 1 target base sequence/cell), or because one target base sequence is introduced into each cell by gene recombination. However, nucleic acid replication occurs in cells such that the cells undergo cell division in a particular cycle. The cell cycle varies depending on the cell type. By extracting a predetermined amount of the solution from the cell suspension and measuring the cycles of a plurality of cells, it is possible to calculate the degree of certainty of the expected value and the estimated value of the number of target base sequences contained in one cell. This can be achieved, for example, by observing the nuclear stained cells with a flow cytometer.
The degree of certainty refers to the probability of occurrence of a particular event predicted in advance when there is a possibility that some event will occur.
Calculation means that a desired value is obtained by a calculation operation.
FIG. 14 is a graph plotting an example of the relationship between the frequency and the fluorescence intensity of cells in which replication of a target base sequence has occurred. As shown in fig. 14, two peaks appear on the histogram based on the presence or absence of the target base sequence copy. Thus, the percentage of cells in which replication of the target base sequence has occurred can be calculated. Based on the calculation result, the average number of target base sequences contained in one cell can be calculated. The estimated number of the target base sequence can be calculated by multiplying the number of counted cells by the obtained average number of the target base sequences.
The manipulation to control the cell cycle is preferably performed prior to the preparation of the cell suspension. By uniformly preparing the cells in a state before replication or in a state after replication, the number of target base sequences can be calculated more accurately based on the number of cells.
It is preferable to calculate the degree of certainty (probability) of the estimated specific copy number. By calculating the degree of certainty (probability), the degree of certainty can be expressed and output as a variance or standard deviation based on these values. When the effects of multiple factors are to be superimposed, the square root of the sum of the squares of the usual standard deviations can be used. For example, the positive response rate (correct answer) of the number of discharged cells, the number of DNAs in the cells, and the landing ratio of the discharged cells in the well can be used as the factors. A high impact factor may be selected for the calculation.
< droplet landing step >)
The droplet landing step is a step of discharging the cell suspension in the form of droplets so that the droplets land in the wells of the plate one after another.
A droplet refers to a liquid aggregate formed by surface tension.
Discharging means causing the cell suspension to fly in the form of droplets.
"sequential" means "in order".
Landing refers to the arrival of a droplet at an orifice.
As the discharge unit, a unit configured to discharge the cell suspension in the form of droplets (hereinafter also referred to as "discharge head") may be suitably used.
Examples of a method of discharging the cell suspension in the form of droplets include an on-demand method and a continuous method based on an ink-jet method. In these processes, in the case of a continuous process, there is a tendency that: the dead volume of the cell suspension used is high due to, for example, empty discharge until the discharge state becomes stable, adjustment of the amount of droplets, and continuous formation of droplets even during transfer between wells. In the present disclosure, in terms of cell number regulation, it is preferable to suppress the influence caused by dead volume. Therefore, of the two methods, the on-demand method is more preferable.
Examples of the on-demand method include various known methods such as a pressure application method of applying pressure to a liquid to discharge the liquid, a thermal method of discharging the liquid by causing film boiling by heating, and an electrostatic method of attracting droplets by electrostatic attraction to form the droplets. Among these methods, the pressure-applying method is preferable for the following reasons.
In the electrostatic method, it is necessary to arrange electrodes in a manner facing a discharge unit configured to hold a cell suspension and form droplets. In the device manufacturing method, a flat plate for receiving droplets is arranged at a facing position. Therefore, it is preferable not to provide an electrode to increase the latitude of the flat panel configuration.
In thermal methods, there is a risk that local heating concentrations may affect cells as biological material, as well as a risk of fouling of the heater section. The thermal influence depends on the use of the included components or the plate. Thus, there is no need to totally exclude the thermal method. However, the pressure-applying method is preferable because the heater portion of the pressure-applying method is less likely to be fouled than the thermal method.
Examples of the pressure applying method include a method of applying pressure to a liquid using a piezoelectric element, and a method of applying pressure using a valve such as a solenoid valve. Fig. 15A to 15C illustrate configuration examples of a droplet generating apparatus that can be used to discharge droplets of a cell suspension.
Fig. 15A is an example diagram illustrating an example of a solenoid valve type discharge head. The solenoid valve type discharge head includes a motor 13a, a solenoid valve 112, a liquid chamber 11a, a cell suspension 300a, and a nozzle 111 a.
As the solenoid valve type discharge head, for example, a dispenser available from Tech Elan LLC may be suitably used.
Fig. 15B is an example diagram illustrating an example of a piezoelectric type discharge head. The piezoelectric type discharge head includes a piezoelectric element 13b, a liquid chamber 11b, a cell suspension 300b, and a nozzle 111 b.
As the piezoelectric type discharge head, for example, a single cell printer available from Cytena GmbH can be suitably used.
Any of these discharge heads may be used. However, the pressure application method by the solenoid valve cannot repeatedly form droplets at high speed. Therefore, in order to increase the board production throughput, the piezoelectric method is preferably used. The piezoelectric type discharge head using the common piezoelectric element 13b may cause cell concentration unevenness due to sedimentation, or may have nozzle clogging.
Therefore, a more preferable configuration is the configuration shown in fig. 15C. Fig. 15C is an example diagram of a modified example of the piezoelectric type discharge head using the piezoelectric element shown in fig. 15B. The discharge head of fig. 15C includes a piezoelectric element 13C, a liquid chamber 11C, a cell suspension 300C, and a nozzle 111C.
In the discharge head of fig. 15C, when a voltage is applied to the piezoelectric element 13C from a control device not illustrated, a compressive stress in the horizontal direction of the drawing sheet is applied. This may deform the film in the up-down direction of the drawing sheet.
Examples of any other method than the on-demand method include a continuous method for continuously forming droplets. When a droplet is pushed out from a nozzle by pressurization, a continuous method applies regular fluctuation with a piezoelectric element or a heater, so that minute droplets can be continuously formed. Further, the continuous method can select whether to drop the flying liquid droplets into the hole or to recover the liquid droplets in the recovery unit by controlling the discharge direction of the liquid droplets with the applied voltage. This method is used in cell sorters or flow cytometers. For example, a device available from Sony Corporation under the name CELL SORTER SH800Z may be used.
FIG. 16A is a schematic view showingExample graphs plotting examples of voltages applied to piezoelectric elements. Fig. 16B is an example diagram illustrating another example of plotting voltages applied to the piezoelectric element. Fig. 16A plots the drive voltage for forming a droplet. According to high or low level (V) of voltageA、VBAnd VC) Droplets may be formed. Fig. 16B plots the voltage used to stir the cell suspension without discharging droplets.
During the period when the liquid droplet is not discharged, the input of a plurality of pulses not high enough to discharge the liquid droplet enables the cell suspension in the liquid chamber to be stirred, so that the occurrence of concentration distribution caused by cell sedimentation can be suppressed.
A droplet forming operation of the discharge head that can be used in the present disclosure will be described below.
The discharge head can discharge a droplet by applying a pulse voltage to upper and lower electrodes formed on the piezoelectric element. Fig. 17A to 17C are example diagrams illustrating a state of a droplet corresponding to a time.
In fig. 17A, first, after a voltage is applied to the
Next, as shown in fig. 17B, the liquid is continuously pushed out through the nozzle portion for a while until the pressure is relaxed upward, so that the liquid droplets are grown.
Finally, as shown in fig. 17C, when the membrane 12C returns to the initial state, the liquid pressure near the interface between the cell suspension and the membrane 12C is reduced, thereby forming a droplet 310'.
In the device manufacturing method, a flat plate formed with holes is fixed on a movable platform, and droplets are sequentially landed in recesses by a combination of driving the platform and forming the droplets by a discharge head. A method of moving a tablet in conjunction with a mobile platform is described herein. However, naturally, the discharge head may be moved.
The flat plate is not particularly limited, and a plate commonly used in the field of biology and formed with holes may be used.
The number of holes in the flat plate is not particularly limited and may be appropriately selected depending on the intended purpose. The number of holes may be singular or plural.
Fig. 18 is a schematic diagram illustrating an example of a
As shown in fig. 18, a
In the
The control device 900 may be configured to include, for example, a CPU, a ROM, a RAM, and a main memory. In this case, various functions of the control apparatus 900 may be realized by a program recorded in, for example, a ROM, read out into a main memory, and executed by a CPU. However, part or all of the control apparatus 900 may be implemented by only hardware. Alternatively, the control device 900 may be configured with, for example, a plurality of devices physically.
When the cell suspension is caused to fall into the well, it is preferable to cause the droplets to be discharged into the well to land in such a manner that a plurality of levels are obtained.
Multiple levels refer to multiple references that serve as criteria.
As the plurality of levels, it is preferable that a plurality of cells including a specific nucleic acid in the well have a predetermined concentration gradient. With a concentration gradient, nucleic acids can advantageously be used as reagents for a calibration curve. The plurality of levels may be controlled using values counted by the sensor.
As the plate, for example, 1-well microtube, 8-tube, 96-well plate and 384-well plate are preferably used. When the number of wells is plural, the same number of cells may be distributed to the wells of these plates, or a different level of cell number may be distributed to the wells. There may be one well that does not contain cells. Specifically, in order to prepare a plate for evaluating a real-time PCR device or a digital PCR device configured to quantitatively evaluate the amount of nucleic acid, it is preferable to allocate a plurality of levels of nucleic acid numbers. For example, it is conceivable to prepare a plate in which cells (or nucleic acids) are distributed at 7 levels, i.e., about 1 cell, 2 cells, 4 cells, 8 cells, 16 cells, 32 cells, and 64 cells. With such a plate, quantitative, linear and evaluation lower limits of, for example, a real-time PCR device or a digital PCR device can be checked.
< cell count step >)
The cell count step is a step of counting the number of cells contained in the droplet by the sensor after the droplet is discharged and before the droplet falls into the hole.
A sensor refers to a device configured to convert mechanical, electromagnetic, thermal, acoustic, or chemical properties of natural phenomena or artifacts or spatial information/temporal information indicated by these properties into signals (as different media easily processed by a human or a machine) by using some scientific principles.
Counting refers to counting the number.
The cell number counting step is not particularly limited and may be appropriately selected according to the intended purpose, as long as the cell number counting step counts the number of cells contained in the droplet using the sensor after the droplet is discharged and before the droplet lands in the hole. The cell number counting step may include an operation of observing the cells before the discharge and an operation of counting the cells after the landing.
In order to count the number of cells contained in the droplet after the droplet is discharged and before the droplet falls into the well, it is preferable to observe the cells in the droplet at a time when the droplet is just in a position above the opening of the well and it is expected that the droplet will enter the well on the plate without fail.
Examples of the method for observing cells in the droplet include an optical detection method and an electrical or magnetic detection method.
Optical detection method
Referring to fig. 19, fig. 23, and fig. 24, the optical detection method will be described below.
Fig. 19 is an example diagram illustrating an example of a
In fig. 19, a liquid obtained by dispersing cells in a predetermined solution after fluorescent staining of the cells with a specific pigment is used as a cell suspension. The cells are counted by irradiating the liquid droplets formed by the discharge head with light having a specific wavelength and emitted from a light source, and detecting fluorescence emitted by the cells with a light receiving element. Here, in addition to a method of staining cells with a fluorescent dye, autofluorescence emitted by molecules initially contained in the cells may be used. Alternatively, a gene for producing a fluorescent protein (for example, GFP (green fluorescent protein)) may be introduced into a cell in advance so that the cell can emit fluorescence.
Light irradiation means applying light.
The
The
The
The driving
By supplying a drive signal from the
When a piezoelectric element is used as the driving
The light source 30 is configured to irradiate the flying droplet 310 with light L. The flying state is a state from when the droplet 310 is discharged from the
Preferably, the beam diameter of the light L is about 10 to 100 times the diameter of the droplet 310. This is to ensure that the droplet 310 is irradiated with the light L from the light source 30 without fail even when the position of the droplet 310 fluctuates.
However, it is not preferable if the beam diameter of the light L is much larger than 100 times the diameter of the droplet 310. This is because the energy density of the light irradiating the liquid droplet 310 is reduced, thereby reducing the light amount of the fluorescence Lf emitted under the light L serving as the excitation light, so that the light receiving element 60 detects the fluorescence Lf.
The light L emitted by the light source 30 is preferably pulsed light. Preferably, for example, solid-state lasers, semiconductor lasers and dye lasers are used. When the light L is pulsed light, the pulse width is preferably 10 microseconds or less, more preferably 1 microsecond or less. The energy per unit pulse is preferably above about 0.1 microjoules, more preferably above 1 microjoule, although it depends to a large extent on the optical system, e.g. whether or not there is a spot light.
The light receiving element 60 is configured to receive fluorescence Lf emitted by the fluorescent-stained cell 350 after absorbing the light L as the excitation light when the fluorescent-stained cell 350 is contained in the flying droplet 310. Since the fluorescence Lf is emitted from the fluorescent-stained cells 350 in all directions, the light-receiving element 60 can be disposed at any position where the fluorescence Lf can be received. Here, in order to improve the contrast, it is preferable to arrange the light receiving element 60 at a position where direct incidence of the light L emitted from the light source 30 to the light receiving element 60 does not occur.
The light receiving element 60 is not particularly limited and may be appropriately selected according to the intended purpose, as long as the light receiving element 60 is an element capable of receiving the fluorescence Lf emitted from the fluorescent-stained cells 350. Such an optical sensor is preferred: configured to receive fluorescence from cells in the droplet when the droplet is illuminated with light having a particular wavelength. Examples of the light receiving element 60 include one-dimensional elements such as a photodiode and a photosensor. When high sensitivity measurements are required, it is preferable to use a photomultiplier tube and an Avalanche photodiode. As the light receiving element 60, two-dimensional elements such as a CCD (charge coupled device), a CMOS (complementary metal oxide semiconductor), and a gate CCD (gate CCD) can be used.
The fluorescence Lf emitted by the fluorescently stained cells 350 is weaker than the light L emitted by the light source 30. Therefore, a filter configured to narrow the wavelength range of the light L may be mounted on the front stage (light receiving surface side) of the light receiving element 60. This enables the light receiving element 60 to obtain an extremely high contrast image of the fluorescent-stained cell 350. As the optical filter, for example, a notch filter configured to reduce a specific wavelength range including the wavelength of the light L may be used.
As described above, the light L emitted by the light source 30 is preferably pulsed light. The light L emitted by the light source 30 may be continuously oscillating light. In this case, it is preferable to control the light receiving element 60 to be able to receive light at the timing when the flying liquid droplet 310 is irradiated with the continuously oscillating light, so that the light receiving element 60 receives the fluorescence Lf.
The control unit 70 has a function of controlling the driving
Fig. 20 is a diagram illustrating a hardware block of a control unit of the droplet forming apparatus of fig. 19. Fig. 21 is a diagram illustrating functional blocks of a control unit of the droplet forming apparatus of fig. 19. Fig. 22 is a flowchart illustrating an example of the operation of the droplet forming apparatus.
As shown in fig. 20, the control unit 70 includes a CPU 71, a ROM72, a RAM 73, an I/F74, and a bus 75. The CPU 71, ROM72, RAM 73, and I/F74 are coupled to each other via a bus 75.
The CPU 71 is configured to control various functions of the control unit 70. The ROM72 serving as a storage unit is configured to store programs to be executed by the CPU 71 to control various functions of the control unit 70 and various information. The RAM 73 serving as a storage unit is configured to be used as a work area of the CPU 71, for example. The RAM 73 is also configured to be able to store predetermined information for a temporary period of time. I/F74 is an interface configured to couple droplet-forming
As shown in fig. 21, the control unit 70 includes, as functional blocks, a discharge control unit 701, a light source control unit 702, and a cell number counting unit (cell number sensing unit) 703.
The number of cells (number of particles) counted by the
In step S11, the discharge control unit 701 of the control unit 70 outputs a discharge instruction to the
Next, in step S12, in synchronization with the discharge of the liquid droplets 310 (in synchronization with the drive signal supplied from the
Here, the light emission of the light source 30 is synchronized not with the
For example, the velocity v of the liquid droplet 310 to be discharged when the drive signal is supplied to the
Next, in step S13, cell number counting section 703 of control section 70 counts the number of fluorescent-stained cells 350 contained in droplet 310 based on the information from light-receiving element 60 (the case where the number is zero is also included). The information from the light receiving element 60 indicates the brightness (light amount) and area value of the fluorescent-stained cells 350.
The cell number counting unit 703 can count the number of the fluorescent-stained cells 350 by, for example, comparing the amount of light received by the light receiving element 60 with a predetermined threshold value. In this case, a one-dimensional element may be used or a two-dimensional element may be used as the light receiving element 60.
When a two-dimensional element is used as the light receiving element 60, the cell number counting unit 703 may utilize a method of performing image processing to calculate the brightness or area of the fluorescent-stained cells 350 based on the two-dimensional image obtained from the light receiving element 60. In this case, the cell number counting unit 703 can count the number of the fluorescent-stained cells 350 by: the brightness or area value of the fluorescent-stained cell 350 is calculated through image processing, and the calculated brightness or area value is compared with a predetermined threshold value.
The fluorescently stained cells 350 can be cells or stained cells. Stained cells refer to cells stained with a fluorescent dye or cells that can express a fluorescent protein.
The fluorescent dye that stains the cells is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of fluorescent pigments include fluorescein, rhodamine, coumarin, pyrenes, cyanine, and azo pigments. One of these fluorescent dyes may be used alone, or two or more of these fluorescent dyes may be used in combination. Among these fluorescent pigments, eosin, evans blue, trypan blue, rhodamine 6G, rhodamine B and rhodamine 123 are more preferable.
Examples of fluorescent proteins include Sirius, EBFP, ECFP, mTurquoise, TagCFP, AmCyan, mTFP1, Midorisis Cyan, CFP, TurboGFP, AcGFP, TagGFP, Azami-Green, ZsGreen, EmGFP, EGFP, GFP2, HyPer, TagYFP, EYFP, Venus, YFP, PhiYFP-m, TurboYFP, ZsYellow, mBanana, Kusabiarange, mOrange, TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, mStrery, TurboFP602, mRFP1, Red2, KirKirKillecKirmPer, KirmPer, KirmPy, KirmPol, KirmPp, Kirke-Msry, Kirke-Msrep, Kirbep, Kirke. One of these fluorescent proteins may be used alone, or two or more of these fluorescent proteins may be used in combination.
In this way, in the
That is, the droplet-forming
Fig. 23 is an example diagram illustrating a modified example of
In the
For example, in the layout of fig. 19, when the nozzle 111 and the landing target are close to each other, there is a risk of interference between the landing target and the optical system (specifically, the light receiving element 60) of the
That is, by changing the layout of the light receiving elements 60 as shown in fig. 23, it is possible to reduce the distance (gap) between the landing target on which the liquid droplets 310 land and the nozzle 111, and suppress landing at an erroneous position. Thereby, the dispensing accuracy can be improved.
Fig. 24 is an example diagram illustrating another modified example of
Fluorescent Lf1And Lf2Represents the portion of fluorescence emitted in all directions from the fluorescently stained cells 350. The light receiving elements 60 and 61 may be disposed at any position where fluorescence emitted in different directions by the fluorescent-stained cells 350 can be received. Three or more light receiving elements may be disposed at positions where fluorescence emitted in different directions by the fluorescent-stained cells 350 can be received. The light receiving elements may have the same specification or different specifications.
In the case of one light receiving element, when a plurality of fluorescent-stained cells 350 are contained in the flying droplet 310, there is a risk that the cell number counting unit 703 may erroneously count the number of fluorescent-stained cells 350 contained in the droplet 310 (a risk that a counting error may occur) because the fluorescent-stained cells 350 may overlap each other.
Fig. 25A and 25B are diagrams illustrating a case where two fluorescence-stained cells are contained in a droplet in flight. For example, as shown in FIG. 25A, there may be fluorescently stained cells 3501And 3502Overlap with each other, or as shown in FIG. 25B, there may be fluorescently stained cells 3501And 3502And do not overlap each other. By providing two or more light receiving elements, the influence of overlapping of the fluorescent-stained cells can be reduced.
As described above, the cell number counting unit 703 can count the number of fluorescent particles by: calculating a brightness or area value of the fluorescent particles through image processing, and comparing the calculated brightness or area value with a predetermined threshold value.
When two or more light-receiving elements are mounted, occurrence of a counting error can be suppressed by employing data indicating the maximum value among luminance values or area values obtained from these light-receiving elements. This will be described in more detail with reference to fig. 26.
Fig. 26 is a graph plotting an example of the relationship between the luminance Li when the particles do not overlap each other and the actually measured luminance Le. As shown in fig. 26, Le is equal to Li when the particles in the droplet do not overlap each other. For example, in the case where the brightness of one cell is assumed to be Lu, when the number of cells per droplet is 1, Le is equal to Lu, and when the number of cells per droplet is n (n: natural number), Le is equal to nLu.
However, in practice, when n is 2 or more, since the particles may overlap with each other, the actually measured luminance is Lu ≦ Le ≦ nLu (halftone dot-like grid portion in FIG. 26). Therefore, in the case where the number of cells per droplet is n, the threshold value can be set to, for example, (nLu-Lu/2). ltoreq.threshold (nLu + Lu/2). When a plurality of light receiving elements are mounted, occurrence of a counting error can be suppressed by employing the maximum value among data obtained from these light receiving elements. Area values may be used instead of brightness.
When a plurality of light receiving elements are mounted, the number of cells can be judged according to an algorithm for estimating the number of cells based on a plurality of shape data to be obtained.
It is understood that the droplet forming device 401B can further reduce the frequency of occurrence of erroneous counting of the number of the fluorescent-stained cells 350 by a plurality of light receiving elements configured to receive fluorescent light emitted in different directions by the fluorescent-stained cells 350.
Fig. 27 is an example diagram illustrating another modified example of
The droplet discharge unit 10C includes a liquid chamber 11C, a film 12C, and a driving element 13C. The liquid chamber 11C has an atmosphere exposure portion 115 at the top, the atmosphere exposure portion 115 being configured to expose the inside of the liquid chamber 11C to the atmosphere, and bubbles mixed in the cell suspension 300 can be evacuated through the atmosphere exposure portion 115.
The membrane 12C is a membrane-like member fixed to the lower end of the liquid chamber 11C. A nozzle 121 as a through hole is formed at substantially the center of the membrane 12C, and the vibration of the membrane 12C causes the cell suspension 300 held in the liquid chamber 11C to be discharged through the nozzle 121 in the form of droplets 310. Since the droplets 310 are formed by the inertia of the vibration of the membrane 12C, the cell suspension 300 can be discharged even when the cell suspension 300 has a high surface tension (high viscosity). The planar shape of the membrane 12C may be, for example, a circle, but may also be, for example, an ellipse or a quadrangle.
The material of the film 12C is not particularly limited. However, if the material of the membrane 12C is very flexible, the membrane 12C is likely to vibrate, and it is not easy to be able to stop the vibration immediately when discharge is not required. Therefore, a material having a certain hardness is preferable. As the material of the membrane 12C, for example, a metal material, a ceramic material, and a polymer material having a certain hardness can be used.
Specifically, when cells are used as the fluorescent-stained cells 350, the material of the membrane is preferably a material having low adhesion to cells or proteins. Overall, the adhesiveness of the cells is weighed out depending on the contact angle of the material with respect to water. When a material has high hydrophilicity or high hydrophobicity, the material has low adhesion to cells. As the material having high hydrophilicity, various metal materials and ceramics (metal oxide) can be used. As the material having high hydrophobicity, for example, a fluororesin may be used.
Other examples of such materials include stainless steel, nickel, and aluminum, as well as silica, alumina, and zirconia. In addition, it is conceivable to reduce cell adhesion by coating the surface of the material. For example, the surface of the material may be coated with a metal or metal oxide material as described above, or with a synthetic phospholipid polymer (e.g., LIPIURE, available from NOF Corporation) that mimics a cell membrane.
The nozzle 121 is preferably formed to have a substantially perfect circular through hole at substantially the center of the film 12C. In this case, the diameter of the nozzle 121 is not particularly limited, but is preferably twice or more the size of the fluorescent-stained cells 350 to prevent the nozzle 121 from being clogged with the fluorescent-stained cells 350. When the fluorescent-stained cells 350 are, for example, animal cells, particularly human cells, the diameter of the nozzle 121 is preferably 10 micrometers or more, more preferably 100 micrometers or more, depending on the cells used, because the human cells generally have a size of about 5 micrometers to 50 micrometers.
On the other hand, when the liquid droplet is extremely large, it is difficult to achieve the purpose of forming a fine liquid droplet. Therefore, the diameter of the nozzle 121 is preferably 200 μm or less. That is, in the droplet discharge unit 10C, the diameter of the nozzle 121 is generally in the range of 10 μm to 200 μm.
The driving element 13C is formed on the lower surface of the film 12C. The shape of the drive element 13C may be designed to match the shape of the membrane 12C. For example, when the planar shape of the film 12C is a circular shape, it is preferable to form the driving element 13C having a ring-shaped (annular) planar shape around the nozzle 121. The driving method for driving the driving element 13C may be the same as the driving method for driving the driving
The
For example, both the discharge waveform and the agitation waveform may be rectangular waves, and the drive voltage for the agitation waveform may be set lower than the drive voltage for the discharge waveform. This makes it possible to form the droplet 310 without applying the agitation waveform. That is, the vibration state (vibration degree) of the film 12C can be controlled according to the driving voltage.
In the droplet discharge unit 10C, the driving element 13C is formed on the lower surface of the film 12C. Therefore, when the
Here, the fluorescent-stained cells 350 are moved upward from the lower position to generate convection in the liquid chamber 11C, thereby stirring the cell suspension 300 containing the fluorescent-stained cells 350. The flow from the lower portion to the upper portion in the liquid chamber 11C causes the settled aggregated fluorescently stained cells 350 to be uniformly dispersed in the liquid chamber 11C.
That is, the driving
In this way, agitating the cell suspension 300 without the formation of the droplets 310 may prevent the fluorescently stained cells 350 from settling and aggregating on the membrane 12C, and may disperse the fluorescently stained cells 350 in the cell suspension 300 without unevenness. This can suppress clogging of the nozzle 121 and variation in the discharge number of the fluorescent-stained cells 350 in the droplet 310. This makes it possible to stably discharge the cell suspension 300 containing the fluorescent-stained cells 350 in the form of droplets 310 for a long time.
In the droplet-forming device 401C, bubbles may be mixed into the cell suspension 300 in the liquid chamber 11C. Also in this case, in the case where the atmosphere exposure section 115 is provided at the top of the liquid chamber 11C, the droplet-forming device 401C can evacuate the bubbles mixed in the cell suspension 300 to the outside air through the atmosphere exposure section 115. This enables the continuous stable formation of droplets 310 without the need to handle large volumes of liquid to evacuate air bubbles.
That is, when there is a mixed bubble at a position near the nozzle 121 or when there are a plurality of mixed bubbles on the film 12C, the discharge state is affected. Therefore, in order to stably form droplets for a long time, it is necessary to eliminate the mixed bubbles. Generally, the mixed bubbles present on the membrane 12C move upward autonomously or by the vibration of the membrane 12C. Since the liquid chamber 11C is provided with the atmosphere exposure portion 115, the mixed bubbles can be discharged through the atmosphere exposure portion 115. This makes it possible to prevent occurrence of empty discharge even when bubbles are mixed in the liquid chamber 11C, enabling continuous and stable formation of the liquid droplets 310.
At the time when no droplet is formed, the membrane 12C may be vibrated to such an extent that no droplet is formed, to actively move the bubble upward in the liquid chamber 11C.
Electrical or magnetic detection methods-
In the case of the electrical or magnetic detection method, as shown in fig. 28, a
< procedure for observing cells before discharging >
The operation of observing the cells before the discharge can be performed by, for example, a method of counting the cells 350' passing through the micro flow path 250 shown in fig. 29 or a method of capturing an image of a portion near the nozzle portion of the discharge head shown in fig. 30. The method of fig. 29 is a method used in a cell sorting device, and for example, CELLSORTER SH800Z available from Sony Corporation may be used. In fig. 29, a light source 260 emits laser light into the micro flow path 250, and a detector 255 detects scattered light or fluorescence passing through a condenser lens 265. This makes it possible to distinguish the presence or absence of cells or the kind of cells when forming droplets. Based on the number of cells that have passed through the microfluidic channel 250, the method can estimate the number of cells that have landed in the predetermined well.
As the discharge head 10' shown in fig. 30, a single cell printer available from Cytena GmbH can be used. In fig. 30, the number of cells that landed in a predetermined well can be estimated by: by capturing an image of a portion near the nozzle portion by the image capturing unit 255 'via the lens 265' before the discharge and estimating that the cells 350 ″ existing near the nozzle portion have been discharged based on the captured image, or by estimating the number of cells that are considered to have been discharged based on a difference between the images captured before and after the discharge. The method of fig. 30 is more preferable because the method enables on-demand droplet formation, whereas the method of fig. 29 of counting cells that have passed through a micro-flow path continuously generates droplets.
< procedure for counting cells after landing >
The operation of counting cells after landing can be performed by the following method: methods for detecting fluorescently stained cells using, for example, a fluorescent microscope to view wells in a plate. This method is described, for example, in Sangjun et al, PLoS One, volume 6(3), e 17455.
The method of observing cells before discharging droplets or after landing has the following problems. Depending on the kind of plate to be prepared, it is most preferable to observe the cells in the droplet being discharged. In the method of observing cells before ejection, the number of cells considered to have landed is counted based on the number of cells that have passed through the flow path and image observation before ejection (and after ejection). Therefore, it is not confirmed whether or not the cells have been actually discharged, and an unexpected error may occur. For example, there may be the following: since the nozzle portion is contaminated, the liquid droplets are not properly discharged, but adhere to the nozzle plate, so that the cells in the liquid droplets are not landed. Further, the following problems may occur: the cells are retained in a narrow area of the nozzle portion, or the discharge operation causes the cells to move beyond the assumption and move outside the observation range.
The method for detecting the cells after landing on the plate is also problematic. First, it is necessary to prepare a plate that can be observed with a microscope. As the plate that can be observed, a plate having a transparent flat bottom surface, particularly a plate having a bottom surface formed of glass, is generally used. However, there is a problem that such a special plate is not compatible with the use of a general hole. Further, when the number of cells is large, such as several tens of cells, there is a problem that the cells may overlap each other and thus cannot be counted correctly. Therefore, it is preferable to perform an operation of observing cells before discharge and an operation of counting cells after landing, in addition to counting the number of cells contained in the droplet by the sensor and the cell number counting unit after discharge of the droplet and before landing of the droplet in the hole.
As the light receiving element, a light receiving element including one or a small number of light receiving portions, such as a photodiode, an Avalanche photodiode, and a photomultiplier tube, may be used. In addition, a two-dimensional sensor including light receiving elements in a two-dimensional array, such as a CCD (charge coupled device), a CMOS (complementary metal oxide semiconductor), and a gate CCD, may be used.
When a light receiving element including one or a small number of light receiving portions is used, it is conceivable to judge the number of contained cells based on the fluorescence intensity using a calibration curve prepared in advance. Here, binary detection of the presence or absence of cells in flying droplets is commonly used. When the cell suspension is discharged in a state where the cell concentration is sufficiently low so that almost only 1 or 0 cells are contained in the droplet, sufficiently accurate counting can be obtained by binary detection. On the premise that cells are randomly distributed in a cell suspension, it is considered that the number of cells in a flying droplet conforms to a poisson distribution, and the probability P (>2) that two or more cells are contained in a droplet is represented by the following formula (1). FIG. 31 is a graph plotting the relationship between the probability P (>2) and the average cell number. Here, λ is a value that represents the average number of cells in the droplet and is obtained by multiplying the cell concentration in the cell suspension by the volume of the droplet discharged.
P(>2)＝1-(1+λ)×e-λ- - -formula (1)
< step of calculating the degree of certainty in the evaluation of nucleic acid in the cell suspension preparation step, droplet landing step, and cell number counting step > >)
The step of calculating the degree of certainty of the estimated number of nucleic acids in the cell suspension preparation step, the droplet landing step, and the cell number counting step is a step of calculating the degree of certainty in each of the cell suspension preparation step, the droplet landing step, and the cell number counting step.
The degree of certainty in the estimated number of nucleic acids can be calculated in the same manner as the degree of certainty in the step of preparing the cell suspension is calculated.
The timing of calculating the degree of certainty may be collectively performed in the next step of the cell number counting step, or may be performed at the end of each of the cell suspension preparation step, the droplet landing step, and the cell number counting step, so that the degree of certainty is added in the next step of the cell number counting step. In other words, the degree of certainty in these steps need only be calculated at any time before the summation is performed.
< output step >)
The outputting step is a step of counting the number of cells contained in the cell suspension dropped in the well by the output particle number counting unit based on a detection result measured by the sensor.
The count value is the number of cells contained in the well calculated by the particle number counting means based on the detection result measured by the sensor.
The output means that, upon receiving the input, the value counted by devices such as a motor, a communication device, and a calculator is transmitted in the form of electronic information to an external server serving as a count result storage unit, or the count value is printed as a printed matter.
In the outputting step, an observed value or an estimated value obtained by observing or estimating the number of cells or nucleic acids in each well of the plate during plate preparation is output to the external storage unit.
The outputting may be performed simultaneously with the cell count counting step, or may be performed after the cell count counting step.
< recording step >
The recording step is a step of recording the observed value or the estimated value output in the outputting step.
The recording step may be suitably performed by a recording unit.
The recording may be performed simultaneously with the outputting step, or may be performed after the outputting step.
Recording means not only supplying information to a recording medium but also storing information in a storage unit.
< nucleic acid extraction step >)
The nucleic acid extraction step is a step of extracting nucleic acid from cells in the well.
Extraction means to disrupt e.g. cell membranes and cell walls to sort out nucleic acids.
As a method for extracting nucleic acid from cells, a method of heat-treating cells at 90 ℃ to 100 ℃ is known. By heat treatment at 90 ℃ or lower, there is a possibility that DNA cannot be extracted. By heat treatment at 100 ℃ or higher, there is a possibility that DNA may be decomposed. Here, the heat treatment is preferably performed with the addition of a surfactant.
The surfactant is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of the surfactant include ionic surfactants and nonionic surfactants. One of these surfactants may be used alone, or two or more of these surfactants may be used in combination. Among these surfactants, nonionic surfactants are preferable because proteins are not modified and deactivated by nonionic surfactants although depending on the added amount of the nonionic surfactants.
Examples of ionic surfactants include sodium fatty acid, potassium fatty acid, sodium alpha-sulfo fatty acid ester, sodium linear alkyl benzene sulfonate, sodium alkyl sulfate, sodium alkyl ether sulfate, and sodium alpha-olefin sulfonate. One of these ionic surfactants may be used alone, or two or more of these ionic surfactants may be used in combination. Among these ionic surfactants, sodium fatty acid is preferable, and Sodium Dodecyl Sulfate (SDS) is more preferable.
Examples of nonionic surfactants include alkyl glycosides, alkyl polyoxyethylene ethers (e.g., BRIJ series), octylphenol ethoxylates (e.g., TRITON X series, IGEPAL CA series, NONIDET P series, and NIKKOL OP series), polysorbates (e.g., TWEEN series, such as TWEEN 20), sorbitan fatty acid esters, polyoxyethylene fatty acid esters, alkyl maltosides, sucrose fatty acid esters, glycoside fatty acid esters, glycerin fatty acid esters, propylene glycol fatty acid esters, and fatty acid monoglycerides. One of these nonionic surfactants may be used alone, or two or more of these nonionic surfactants may be used in combination. Among these nonionic surfactants, polysorbates are preferred.
The content of the surfactant is preferably 0.01 mass% or more but 5.00 mass% or less with respect to the total amount of the cell suspension in the well. When the content of the surfactant is 0.01% by mass or more, the surfactant can be effectively used for DNA extraction. When the content of the surfactant is 5.00 mass% or less, inhibition of amplification during PCR can be prevented. As a numerical range in which both effects can be obtained at the same time, a range of 0.01 mass% or more but 5.00 mass% or less is preferable.
The above method may not sufficiently extract DNA from cells having a cell wall. Examples of methods for this include osmotic shock procedures, freeze-thaw methods, enzymatic digestion methods, use of DNA extraction kits, sonication methods, French press methods, and homogenizer methods. Among these methods, the enzyme digestion method is preferable because the method can reduce the loss of the extracted DNA.
< other steps >
The other steps are not particularly limited and may be appropriately selected depending on the intended purpose. Examples of other steps include an enzyme inactivation step and a step of changing the composition except for the specific copy number of the amplifiable reagent.
An enzyme deactivation step
The enzyme inactivation step is a step of inactivating the enzyme.
Examples of the enzyme include dnase, rnase, and an enzyme for extracting nucleic acid in the nucleic acid extraction step.
The method of inactivating the enzyme is not particularly limited and may be appropriately selected depending on the intended purpose. Known methods can be suitably used.
< step of changing composition other than specific copy number of amplifiable reagent >
The step of altering the composition of the amplifiable agent other than a particular copy number means that any other primer or combination of amplification agents may be provided that may or may not include nucleic acids that serve as amplifiable agents. Specifically, one well includes (1) a composition comprising a nucleic acid, a primer, and an amplification agent, another well includes (2) a composition comprising a nucleic acid and a primer, and yet another well includes (3) a composition comprising only a nucleic acid. The producer of the reagent composition adds the primer and the amplification agent to the compositions of (2) and (3) by manual operation at the time of preparing the reagent composition, and uses the reagent composition for the PCR reaction.
The step of changing the composition of the amplifiable reagents other than the specific copy number may be performed by machine dispensing rather than manual operation. This allows for accurate dispensing of primers and amplification agents.
Thus, the device enables, for example, the skill assessment of the manufacturer who prepared the reagent composition.
The device of the present disclosure is widely used in, for example, biotechnology related industries, life science industries, and healthcare industries, and may be suitably used for, for example, instrument calibration or calibration curve generation and test device accuracy management.
In case the device is used for infectious diseases, the device is suitable for methods defined as official analytical methods or official publishing methods.
(method, device and program for evaluating skill of Producer)
The maker skill evaluation method of the present disclosure is a maker skill evaluation method for evaluating the skill of a maker who prepares a reagent composition, including a Ct value information obtaining step of: obtaining information about Ct values in an apparatus of the present disclosure using the apparatus of the present disclosure; and a skill evaluation step: evaluating the skill of the producer based on the information to obtain the Ct value; and further includes other steps as necessary.
The maker skill evaluation device of the present disclosure is a maker skill evaluation device configured to evaluate a skill of a maker that prepares a reagent composition, including a Ct value information obtaining unit configured to obtain information on a Ct value in the device of the present disclosure using the device of the present disclosure; and a skill evaluation unit configured to evaluate a skill of the maker based on the obtained information on the Ct value; and further includes other units as necessary.
The maker skill evaluation program of the present disclosure is a maker skill evaluation program for evaluating the skill of a maker who prepares a reagent composition, and causes a computer to execute a process including: obtaining information about the Ct value in the apparatus of the present disclosure using the apparatus of the present disclosure, and evaluating the skill of the producer based on the obtained information about the Ct value.
The control by the control unit such as the maker skill assessment apparatus of the present disclosure has the same meaning as performing the maker skill assessment method of the present disclosure. Therefore, the details of the maker skill assessment method will also be illustrated by the description of the maker skill assessment apparatus of the present disclosure. Further, the maker skill evaluation program of the present disclosure realizes the maker skill evaluation device of the present disclosure by using, for example, a computer as a hardware resource. Therefore, the details of the maker skill assessment program of the present disclosure will also be explained by the description of the maker skill assessment apparatus of the present disclosure.
< Ct value information obtaining step and Ct value information obtaining Unit >
The Ct value information obtaining step is a step of obtaining information on a Ct value in the apparatus of the present disclosure with the apparatus of the present disclosure, and is performed by a Ct value information obtaining unit.
The Ct value represents the cycle number at the time when the fluorescence signal of the reaction crosses the threshold line. Since the Ct value linearly decreases to the logarithm of the initial amount of the target, the initial copy number of DNA can be calculated based on the Ct value.
The threshold line represents the signal level at which a statistically significant increase from the calculated baseline signal was observed, and means the threshold for real-time PCR reactions.
Baseline refers to the signal level in the initial cycle of PCR when there is little change in the fluorescence signal.
Examples of the information on the Ct value include a Ct value, an average Ct, a standard deviation, a CV value [ (standard deviation/average Ct) × 100] and [ (Ct (maximum) -Ct (minimum))/2 · average Ct ] × 100.
< skill evaluation step and skill evaluation Unit >
The skill evaluation step is a step of evaluating the skill of the preparation based on the information on the Ct value, and is performed by the skill evaluation unit.
Examples of the skill of the preparer include, with respect to the composition other than the nucleic acid in the device, the skill of the preparer for preparing the reagent composition by manual operation.
The skill of the manufacturer can be assessed based on the PCR reaction to prepare the reagent composition using the device of the present disclosure and comparison with Ct value information to be obtained.
To assess the skill of the manufacturer, the manufacturer may prepare the sample on the same device that provides the standard sample to which the sample is to be compared, or may prepare the sample on a different device. As a method of evaluating the skill of a maker by making the maker prepare a sample on the same device that provides a standard sample to which the sample is to be compared, for example, an automatic dispensing device or an authenticated technician prepares a reagent on a well, while the maker to be evaluated prepares a reagent on a different well of the same device. Then, Ct values obtained from PCR reactions in the respective wells were compared with each other. In this way, the skill of the manufacturer may be evaluated. As a method of evaluating the skill of a maker by making the maker prepare a sample on the same device that provides a standard sample to which the sample is to be compared, for example, an automatic dispensing device or an authenticated technician prepares a reagent on a first device, while the maker to be evaluated prepares a reagent on a second device. Then, Ct values obtained from the PCR reaction using the prepared first and second devices were compared with each other. In this way, the skills of the preparation to be evaluated can be evaluated.
< other Steps and other units >
The other steps and other units are not particularly limited and may be appropriately selected depending on the intended purpose. Examples of the other steps and other units include a display step and a display unit.
The procedure of the maker skill evaluation program of the present disclosure may be executed using a computer including a control unit constituting a maker skill evaluation device.
The hardware configuration and the functional configuration of the maker skill evaluation device will be described below.
< hardware configuration of Producer skill evaluation apparatus >
Fig. 32 is a block diagram illustrating an example of the hardware configuration of the maker
As shown in fig. 32, the maker
The CPU 101 is a processing device configured to execute various controls and operations. The CPU 101 realizes various functions by executing an OS (operating system) and programs stored in, for example, the main storage 102. That is, in the present example, the CPU 101 functions as the control unit 130 of the maker
The CPU 101 also generally controls the operation of the maker
The producer skill assessment program and various databases need not be stored, for example, in primary storage 102 or secondary storage 103. The maker skill evaluation program and various databases may be stored in, for example, any other information processing apparatus that is coupled to the maker
The main storage 102 is configured to store various programs and store, for example, data required for executing the various programs.
The main storage 102 includes a ROM (read only memory) and a RAM (random access memory) which are not shown.
The ROM stores, for example, various programs such as BIOS (basic input/output system).
The RAM is used as a work area to be developed when various programs stored in the ROM are executed by the CPU 101. The RAM is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of the RAM include DRAM (dynamic random access memory) and SRAM (static random access memory).
The secondary storage device 103 is not particularly limited and may be appropriately selected according to the intended purpose, as long as the secondary storage device 103 can store various information. Examples of secondary storage 103 include solid state drives and hard disk drives. As the secondary storage device 103, for example, portable storage devices such as a CD (compact disc) drive, a DVD (digital versatile disc) drive, and a BD (blu-ray (registered trademark) disc) drive can also be used.
As the output device 104, for example, a display and a speaker can be used. The display is not particularly limited, and an appropriate known display may be used. Examples of the display include a liquid crystal display and an organic EL display.
The input device 105 is not particularly limited, and an appropriate known input device may be used as long as the input device can receive various requests to the maker
The communication interface (communication I/F)106 is not particularly limited, and an appropriate known communication interface may be used. Examples of communication interfaces include wireless or wired communication devices.
The above-described hardware configuration can realize the processing function of the maker
< functional configuration of Producer skill evaluation apparatus >
Fig. 22 is a diagram illustrating an example of the functional configuration of the maker
As shown in fig. 22, the maker
The control unit 130 includes a Ct value information obtaining unit 131 and a skill evaluation unit 132. The control unit 130 is configured to control the maker
The storage unit 140 includes a Ct value information database 141 and a skill evaluation result database 142. Hereinafter, "database" may also be referred to as "DB".
The Ct-value-information obtaining unit 131 is configured to obtain information on a Ct value using data representing information on a Ct value stored in the Ct-value-information DB 141 of the storage unit 140. As described above, the Ct-value information DB 141 stores, for example, data representing Ct values previously obtained through experiments. Note that information on a Ct value associated with the apparatus may be stored in the Ct value information DB 141. The input to the DB may be performed via other information processing devices coupled with the maker
The skill evaluation unit 132 is configured to evaluate the skill of the preparation based on the information on the Ct value. Examples of specific methods for evaluating the skill of the manufacturer include a method of calculating a standard deviation from the obtained Ct values and evaluating the skill of the manufacturer based on the calculated standard deviation.
The result of evaluating the skill of the maker by the skill evaluation unit 132 is stored in the skill evaluation result DB 142 of the storage unit 140.
Next, a procedure of the maker skill evaluation program of the present disclosure will be described. Fig. 23 is a flowchart illustrating a procedure of a maker skill evaluation program in the control unit 130 of the maker
In step S110, the Ct-value-information obtaining unit 131 of the control unit 130 of the maker
In step S111, the skill evaluation unit 132 of the control unit 130 of the maker
In step S112, the control unit 130 of the maker
(test device Performance evaluation method, test device Performance evaluation device, and test device Performance evaluation program)
The test device performance evaluation method of the present disclosure is a test device performance evaluation method for evaluating performance of a test device configured to test a test target, including a Ct value information obtaining step of: obtaining information about the Ct value in the apparatus of the present disclosure using the apparatus of the present disclosure; and a performance evaluation step: evaluating the performance of the test device based on the information about the Ct value; and further includes other steps as necessary.
The test device performance evaluation device of the present disclosure is a test device performance evaluation device configured to evaluate the performance of a test device configured to test a test target, and includes a Ct value information obtaining unit configured to obtain information on a Ct value in the device of the present disclosure with the device of the present disclosure; and a performance evaluation unit configured to evaluate performance of the test device based on the information on the Ct value; and further includes other units as necessary.
The test apparatus performance evaluation program of the present disclosure is a test apparatus performance evaluation program for evaluating performance of a test apparatus configured to test a test target, and causes a computer to execute a process including: processing to obtain information on Ct values in the apparatus of the present disclosure using the apparatus of the present disclosure, and evaluating the performance of the test apparatus based on the information on Ct values.
The control performed by the control unit of the test apparatus performance evaluation apparatus of the present disclosure, for example, has the same meaning as the execution of the test apparatus performance evaluation method of the present disclosure. Therefore, the details of the test device performance evaluation method will also be explained by the description of the test device performance evaluation device of the present disclosure. Further, the test apparatus performance evaluation program of the present disclosure realizes the test apparatus performance evaluation apparatus of the present disclosure by using, for example, a computer as a hardware resource. Therefore, the details of the test device performance evaluation program of the present disclosure will also be explained by the description of the test device performance evaluation device of the present disclosure.
< Ct value information obtaining step and Ct value information obtaining Unit >
The Ct value information obtaining step is a step of obtaining information on a Ct value in the apparatus of the present disclosure with the apparatus of the present disclosure, and is performed by a Ct value information obtaining unit.
By performing real-time PCR using the apparatus of the present disclosure, Ct values can be obtained.
Examples of the information on the Ct value include an average Ct value, a standard deviation of the Ct value, a CV value [ (standard deviation/average Ct value) × 100] and [ (Ct value (maximum) -Ct value (minimum))/2 · average Ct value ] × 100.
Information about the Ct value may be obtained from each of two or more sets provided in the device and differing in the specific copy number of the amplifiable reagent.
< Performance evaluation step and Performance evaluation Unit >
The performance evaluation step is a step of evaluating the performance of the test apparatus based on the information on the Ct value, and is performed by the performance evaluation unit.
In qualitative assessment, real-time PCR was performed using the apparatus of the present disclosure to measure Ct values and calculate average Ct values. The in-plane properties can be evaluated by ranking the pores as "omicron" when the Ct value in the pores is within 10% of the mean Ct value, and as "x" when the Ct value in the pores is greater than 10% of the mean Ct value.
By measuring for a certain period of time using the apparatus of the present disclosure, the temporal variation of the Ct value can be obtained. Thus, as with the in-plane properties, when the Ct value of the well is greater than 10% of the average Ct value, calibration of the test device can be performed or measures taken without using the measurement site. Further, since the specific copy number of the arranged copies is an absolute value, using a device having copies arranged at the same specific copy number enables performance comparison between test devices.
In quantitative evaluation, the time variation of the Ct value can be obtained by measuring for a certain period of time using the apparatus of the present disclosure. Thus, as with the in-plane characteristic, when a value deviating from the quality control value is obtained, calibration of the device may be tested or measures taken without using the measurement position. Further, since the specific copy number of the arranged copies is an absolute value, using a device having copies arranged at the same copy number enables performance comparison between test devices.
In quantitative evaluation, the number of molecules (copy number or concentration) corresponding to the Ct value, rather than the Ct value itself, can be obtained from the calibration curve and PCR efficiency. Therefore, performance evaluation between test devices can be performed using (maximum-minimum)/2 · average × 100 calculated using a value such as the number of molecules (copy number or concentration) or a CV value converted into the number of molecules (copy number or concentration) and the number of molecules (converted into the copy number or concentration).
< other steps and other units >
The other steps and other units are not particularly limited and may be appropriately selected depending on the intended purpose. Examples of the other steps and other units include a display step and a display unit.
The procedure of the test apparatus performance evaluation program according to the present disclosure may be executed using a computer including a control unit constituting the test apparatus performance evaluation apparatus.
The hardware configuration and the functional configuration of the test apparatus performance evaluation apparatus will be described below.
< hardware configuration of test apparatus Performance evaluation apparatus >
Fig. 35 is a block diagram illustrating an example of the hardware configuration of the test apparatus
As shown in fig. 35, the test apparatus
The CPU 101 is a processing device configured to execute various controls and operations. The CPU 101 realizes various functions by executing an OS (operating system) and programs stored in the main storage 102, for example. That is, in the present example, the CPU 101 functions as the control unit 130 of the test apparatus
The CPU 101 also generally controls the operation of the test apparatus
The test device performance evaluation program and various databases need not be stored in, for example, the primary storage device 102 or the secondary storage device 103. The test device performance evaluation program and various databases may be stored, for example, in any other information processing device that is coupled to the test device
The main storage 102 is configured to store various programs and store, for example, data required for executing the various programs.
The main storage 102 includes a ROM (read only memory) and a RAM (random access memory) which are not shown.
The ROM stores, for example, various programs such as BIOS (basic input/output system).
The RAM is used as a work area to be developed when various programs stored in the ROM are executed by the CPU 101. The RAM is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of the RAM include DRAM (dynamic random access memory) and SRAM (static random access memory).
The secondary storage device 103 is not particularly limited and may be appropriately selected according to the intended purpose, as long as the secondary storage device 103 can store various information. Examples of secondary storage 103 include solid state drives and hard disk drives. As the secondary storage device 103, for example, portable storage devices such as a CD (compact disc) drive, a DVD (digital versatile disc) drive, and a BD (blu-ray (registered trademark) disc) drive can also be used.
As the output device 104, for example, a display and a speaker can be used. The display is not particularly limited, and an appropriate known display may be used. Examples of the display include a liquid crystal display and an organic EL display.
The input device 105 is not particularly limited, and an appropriate known input device may be used as long as the input device can receive various requests to the test device
The communication interface (communication I/F)106 is not particularly limited, and an appropriate known communication interface may be used. Examples of communication interfaces include wireless or wired communication devices.
The above-described hardware configuration can realize the processing function of the test apparatus
< functional configuration of test apparatus Performance evaluation apparatus >
Fig. 36 is a diagram illustrating an example of the functional configuration of the test apparatus
As shown in fig. 36, the test apparatus
The control unit 130 includes a Ct value information obtaining unit 131 and a performance evaluation unit 132. The control unit 130 is configured to control the test apparatus
The storage unit 140 includes a Ct value information database 141 and a performance evaluation result database 142. Hereinafter, "database" may also be referred to as "DB".
The Ct-value-information obtaining unit 131 is configured to obtain information on a Ct value using data representing information on a Ct value stored in the Ct-value-information DB 141 of the storage unit 140. As described above, the Ct-value information DB 141 stores, for example, data representing Ct values previously obtained through experiments. Note that information on a Ct value associated with the apparatus may be stored in the Ct value information DB 141. The input to the DB may be performed via other information processing apparatus coupled with the test apparatus
The performance evaluation unit 132 is configured to evaluate the performance of the test apparatus based on the information on the Ct value. Specific methods for evaluating the performance of the test device are described above.
The result of evaluating the performance of the test apparatus by the performance evaluation unit 132 is stored in the performance evaluation result DB 142 of the storage unit 140.
Next, a process procedure of the test apparatus performance evaluation program of the present disclosure will be described. Fig. 37 is a flowchart illustrating a procedure of a test apparatus performance evaluation procedure in the control unit 130 of the test apparatus
In step S110, the Ct-value-information obtaining unit 131 of the control unit 130 of the test-device-
In step S111, the performance evaluation unit 132 of the control unit 130 of the test apparatus
In step S112, the control unit 130 of the test apparatus