阅读说明：本技术 反应处理装置 (Reaction processing apparatus ) 是由 福泽隆 川口磨 于 2018-06-18 设计创作，主要内容包括：反应处理装置(100)包括：流路(12),其供样品(50)移动；反应处理容器(10),其包括被设置在流路(12)的两端的一对第一空气连通口(24)及第二空气连通口(26)；温度控制系统(102),其在流路(12)中的第一空气连通口(24)与第二空气连通口(26)之间提供中温区域(38)、以及高温区域(36)；以及送液系统(120),其为了使样品(50)在流路(12)内移动及停止而进行空气的喷出及吸引。反应处理容器(10)的一对空气连通口中距高温区域(36)较远的第一空气连通口(24)经由管(130)与送液系统(120)连通。反应处理容器(10)的一对空气连通口中较接近高温区域(36)的第二空气连通口(26)被开放在大气压中。(A reaction processing device (100) is provided with: a flow path (12) through which a sample (50) moves; a reaction processing container (10) having a pair of first and second air communication ports (24, 26) provided at both ends of a flow path (12); a temperature control system (102) that provides a medium-temperature region (38) and a high-temperature region (36) between a first air communication port (24) and a second air communication port (26) in a flow path (12); and a liquid feeding system (120) which performs air ejection and suction for moving and stopping the sample (50) in the flow path (12). A first air communication port (24) of the pair of air communication ports of the reaction processing container (10), which is farther from the high-temperature region (36), is communicated with the liquid feeding system (120) via a pipe (130). A second air communication port (26) of the pair of air communication ports of the reaction processing container (10) which is closer to the high temperature region (36) is opened to the atmospheric pressure.)

1. A reaction processing apparatus, comprising:

a reaction processing container including a flow path through which a sample moves, and a pair of air communication ports provided at both ends of the flow path,

a temperature control member that provides a first temperature region maintained at a first temperature, a second temperature region maintained at a second temperature higher than the first temperature, and

a liquid feeding system for ejecting and sucking air to move and stop the sample in the flow path;

a liquid supply system for supplying a liquid to the reaction processing container, the liquid supply system being connected to the one of the pair of air communication ports located farther from the second temperature range;

the air communication port of the pair of air communication ports of the reaction processing container, which is closer to the second temperature range, is opened to atmospheric pressure.

2. The reaction processing apparatus according to claim 1,

the liquid feeding system includes:

a chamber having an inner space with a certain volume, and a first air port and a second air port communicating the inner space with the outside,

a first pump configured to eject air from the first air port to an internal space of the chamber, an

A second pump configured to discharge air from the second air port into an internal space of the chamber;

a first air port of the chamber is communicated with an air communication port, which is farther from the second temperature range, of the pair of air communication ports of the reaction processing container through the pipe;

the second air port of the chamber is opened to the atmospheric pressure;

the first pump and the second pump are controlled to alternately perform an air discharge operation.

3. The reaction processing apparatus according to claim 1 or 2,

a filter is provided at least at one end of the flow path farther from the second temperature range, among both ends of the flow path of the reaction processing container.

Technical Field

The present invention relates to a Reaction processing apparatus used for Polymerase Chain Reaction (PCR).

Background

Genetic testing is widely used in various medical fields for testing, identification of crops and pathogenic microorganisms, safety evaluation of foods, and testing of pathogenic viruses and various infectious diseases. In order to detect a minute amount of DNA with high sensitivity, a method of analyzing a substance obtained by amplifying a part of DNA is known. Among them, the method using PCR is a remarkable technique for selectively amplifying a certain portion of a very small amount of DNA extracted from a living body or the like.

PCR is a reaction as follows: a predetermined thermal cycle is applied to a sample in which a biological sample containing DNA and a PCR reagent composed of primers, an enzyme, or the like are mixed, and denaturation, annealing, and extension reactions are repeatedly performed, thereby selectively amplifying a specific portion of DNA.

In PCR, a reaction is generally performed by placing a predetermined amount of a target sample in a reaction processing container such as a PCR tube or a microplate (microwell) having a plurality of wells, but in recent years, a method of performing a reaction using a reaction processing container (also referred to as a chip) having a fine flow path formed on a substrate has been put into practical use (for example, patent document 1).

[ Prior art documents ]

[ non-patent document ]

Patent document 1 Japanese patent laid-open publication No. 2009-232700

Disclosure of Invention

[ problems to be solved by the invention ]

In addition, since PCR can synthesize millions of copies of DNA and is very sensitive to contamination, contamination of products of a reaction performed before the reaction becomes a problem when the reaction is started from an extremely small amount of sample DNA. The phenomenon that the product of the reaction carried out before affects the next reaction is called "carry-over". It is very important to prevent or reduce contamination. This is because, for example, the previous reaction product containing the target sequence is mixed with only 1 copy in the reaction system, and there is a possibility that the interpretation of the result will be incorrect.

The present invention has been made in view of such circumstances, and an object thereof is to provide a reaction processing apparatus capable of preventing or at least suppressing contamination during PCR.

[ means for solving the problems ]

In order to solve the above problems, a reaction processing apparatus according to an aspect of the present invention includes: a reaction processing container including a flow path through which a sample moves, and a pair of air communication ports provided at both ends of the flow path; a temperature control member that provides a first temperature region maintained at a first temperature and a second temperature region maintained at a second temperature higher than the first temperature between the pair of air communication ports in the flow path; and a liquid feeding system for ejecting and sucking air to move and stop the sample in the flow path. Of the pair of air communication ports of the reaction processing container, the air communication port located farther from the second temperature range is communicated with the liquid feeding system via a pipe, and of the pair of air communication ports of the reaction processing container, the air communication port located closer to the second temperature range is opened to the atmospheric pressure.

The liquid feeding system may include: a chamber having an inner space with a certain volume, and a first air port and a second air port which communicate the inner space with the outside; a first pump configured to eject air from the first air port to an internal space of the chamber; and a second pump configured to eject air from the second air port to the internal space of the chamber. The first air port of the chamber is communicated with an air communication port, which is located farther from the second temperature range, of the pair of air communication ports of the reaction processing container through the pipe, and the second air port of the chamber is opened to the atmospheric pressure.

The reaction processing vessel may be provided with a filter at least at one end of the flow path which is farther from the second temperature range, among both ends of the flow path.

Effects of the invention

According to the present invention, a reaction processing apparatus capable of preventing or at least suppressing contamination in PCR can be provided.

Drawings

Fig. 1 (a), 1 (b) and 1 (c) are diagrams for explaining a reaction processing container that can be used in the reaction processing apparatus according to the embodiment of the present invention.

Fig. 2 is a plan view of a substrate included in the reaction processing container.

Fig. 3 is a cross-sectional view of the reaction processing vessel, illustrating the relationship between each part and the main surface of the substrate.

FIG. 4 is a view schematically showing the state in which a sample is introduced into a reaction processing vessel.

FIG. 5 is a schematic view for explaining a reaction treatment apparatus according to an embodiment of the present invention.

Fig. 6 is a schematic diagram for explaining the configuration of the liquid feeding system.

FIG. 7 is a view showing a state in which a heat cycle is applied to a sample in a flow path of a reaction processing vessel.

FIG. 8 is a diagram showing the results of PCR amplification in the reaction processing apparatus according to the present embodiment.

FIG. 9 is a diagram showing the results of PCR amplification in the reaction processing apparatus according to the present embodiment.

FIG. 10 is a table showing Ct values corresponding to 6 samples having different initial sample concentrations.

FIG. 11 is a graph showing a standard curve in which the relationship between the initial concentration and the Ct value shown in FIG. 10 is plotted.

FIG. 12 is a diagram for explaining a reaction treatment apparatus of a comparative example.

FIG. 13 is a diagram showing the results of PCR amplification performed by the reaction processor of the comparative example.

FIG. 14 is a diagram for explaining another embodiment of the reaction treatment apparatus.

Detailed Description

The reaction treatment apparatus according to the embodiment of the present invention will be described below. The same or equivalent components, members, and processes shown in the respective drawings are denoted by the same reference numerals, and overlapping descriptions are appropriately omitted. The embodiments are not intended to limit the invention, and are merely examples, and not all the features and combinations described in the embodiments are essential contents of the invention.

Fig. 1 (a), 1 (b) and 1 (c) are views for explaining a reaction processing container 10 that can be used in the reaction processing apparatus according to the embodiment of the present invention. Fig. 1 (a) is a plan view of the reaction processing container 10, fig. 1 (b) is a front view of the reaction processing container 10, and fig. 1 (c) is a bottom view of the reaction processing container 10. Fig. 2 is a plan view of the substrate 14 included in the reaction processing container 10.

Fig. 3 shows a schematic cross-sectional view for explaining the reaction processing container 10. The reaction processing vessel 10 is composed of: a resinous substrate 14 having a groove-like flow path 12 formed on a lower surface 14a thereof; a flow path sealing film 16 which is adhered to the lower surface 14a of the substrate 14 to seal the flow path 12; a first sealing film 18 which is also attached to the lower surface 14a of the substrate 14 to seal the first air communication port 24 and the second air communication port 26; and a second sealing film 20 attached to the upper surface 14b of the substrate 14 for sealing the first sample introduction port 45 and the second sample introduction port 46. Fig. 3 is a diagram illustrating how they are arranged with respect to the substrate 14.

The substrate 14 is preferably made of a material that is stable against temperature changes and is not easily corroded by the sample solution used. Further, the substrate 14 is preferably formed of a material having good moldability, good transparency and barrier properties, and low autofluorescence. As such a material, in addition to inorganic materials such as glass and silicon (Si), resins such as acrylic, polypropylene, and silicone are included, and among them, cycloolefin polymer resin (COP) is preferable. Examples of the dimensions of the substrate 14 include a long side 76mm, a short side 26mm, and a thickness 3 mm.

A groove-like flow path 12 is formed in the lower surface 14a of the substrate 14. In the reaction processing vessel 10, most of the flow channel 12 is formed in a groove shape exposed to the lower surface 14a of the substrate 14. This is because the molding can be easily performed by injection molding using a mold or the like. In order to seal the groove and use it as a flow path, a flow path sealing film 16 is attached to the lower surface 14a of the substrate 14. The cross-sectional shape of the groove is not particularly limited, and may be rectangular or "U" shaped (circular). In order to improve the mold release property during molding, the mold may have a shape tapered in the depth direction from the lower surface 14a, and may have a mesa shape, for example. An example of the dimensions of the flow channel 12 is 0.7mm in width and 0.7mm in depth at maximum.

The flow path sealing film 16 may have adhesiveness on one main surface, or may have a functional layer that exhibits adhesiveness or adhesiveness by pressing, energy irradiation such as ultraviolet rays, heating, or the like formed on one main surface, and the flow path sealing film 16 has the following functions: can be easily brought into close contact with the lower surface 14a of the substrate 14 to be integrated therewith. The flow path sealing film 16 is preferably made of a material that also contains an adhesive and has low self-fluorescence. In this regard, a transparent film made of a resin such as cycloolefin polymer, polyester, polypropylene, polyethylene, or acrylic is preferable, but is not limited thereto. The flow path sealing film 16 may be made of a plate-like glass or resin. In this case, since the rigidity can be expected, it contributes to preventing the reaction processing container 10 from being bent or deformed.

A first air communication port 24 communicating with one end 12a of the flow path 12 is formed in the substrate 14. Similarly, a second air communication port 26 communicating with the other end 12b of the flow path 12 is formed. The pair of first and second air communication ports 24 and 26 are formed so as to be exposed to the lower surface 14a of the substrate 14. That is, the surfaces of the pair of first and second air communication ports 24 and 26 exposed are the same as the surfaces on which the flow paths 12 are formed.

A first filter 28 is provided between the first air communication port 24 and the one end 12a of the flow path 12 in the substrate 14. A second filter 30 is provided between the second air communication port 26 and the other end 12b of the flow path 12 in the substrate 14. The pair of first and second filters 28 and 30 provided at both ends of the flow channel 12 is excellent in low impurity characteristics, and can prevent contamination by allowing only air to pass therethrough, so that the amplification and detection of the target DNA are not hindered by PCR, or the quality of the target DNA is not deteriorated. As the filter material, for example, a material obtained by subjecting polyethylene to a water repellent treatment can be used, but as long as the above function is provided, it can be selected from known materials. The first filter 28 and the second filter 30 are sized to be accommodated in the filter installation space formed in the substrate 14 without a gap, and may have a diameter of 4mm and a thickness of 2mm, for example.

Further, the flow path 12 becomes 2 systems immediately before the second filter. Some of the evaporated sample may be deposited in the flow path as droplets, and particularly, the deposited droplets may interfere with the liquid feeding of the sample in the vicinity of the second filter 30 which is intended to maintain a relatively high temperature, and 2 systems are formed to compensate for this.

The flow path 12 includes a reaction region between the pair of first air communication ports 24 and the second air communication port 26, in which a plurality of levels of temperature can be controlled by a reaction processing device described later. By continuously moving the sample back and forth in the reaction region where the temperatures of the plurality of levels are maintained, thermal circulation can be provided to the sample.

When a reaction processing apparatus described later is mounted with the reaction processing container 10, the reaction region of the flow path 12 includes a reaction region (hereinafter referred to as "high temperature region 36") maintained at a relatively high temperature (about 95 ℃) and a reaction region (hereinafter referred to as "intermediate temperature region 38") maintained at a lower temperature (about 62 ℃) than the high temperature region 36. The high temperature region 36 is located on the right side of the flow path 12 in the drawing, and has one end communicating with the second air communication port 26 via the second filter 30 and the connection flow path 261 between the second filter 30 and the second air communication port 26, and the other end communicating with the medium temperature region 38 via the connection flow path 40. The intermediate temperature region 38 is located at the center of the flow path 12 on the paper surface, and has one end communicating with the high temperature region 36 via a connection flow path 40 and the other end communicating with the low temperature region 34 described later via a connection region 41.

The high temperature region 36 and the medium temperature region 38 each include a continuously-folded serpentine flow path formed by combining a curved portion and a straight portion. In the case of the serpentine flow path, there are advantages as follows: the limited effective area of the heater and the like constituting the temperature control means described later can be effectively used, and the fluctuation of the temperature in the reaction region can be easily reduced, and the physical size of the reaction processing vessel can be reduced, which contributes to the miniaturization of the reaction processing apparatus. On the other hand, the connection flow path 40 may be a straight flow path.

The flow path 12 also includes a region maintained at a lower temperature than the medium temperature region 38 (hereinafter referred to as "low temperature region 34"). In the low temperature region, the temperature may be controlled to be maintained at a temperature lower than that in the medium temperature region, or may not be particularly controlled. The low temperature region 34 is located on the left side of the flow path 12 on the paper surface, and has one end communicating with the first air communication port 24 via the first filter 28 and the connection flow path 241 between the first air communication port 24 and the first filter 28, and the other end communicating with the medium temperature region 38 via the connection region 41.

The low temperature region 34 is used for so-called dispensing for extracting a predetermined amount of sample. The low temperature region 34 includes: a dispensing flow path 42 for specifying a predetermined amount of a sample; 2 branch channels (a first branch channel 43 and a second branch channel 44) which branch from the dispensing channel 42; a first specimen inlet 45 disposed at an end of the first branch channel 43; and a second sample introduction port 46 disposed at an end of the second branch channel 44. The first sample introduction port 45 communicates with the dispensing flow path 42 via the first branch flow path 43. The second sample introduction port 46 communicates with the dispensing channel 42 via the second branch channel 44. The dispensing flow path 42 is formed as a serpentine flow path so that a predetermined amount of sample is dispensed in a minimum area. The first sample introduction port 45 and the second sample introduction port 46 are formed so as to be exposed to the upper surface 14b of the substrate 14. That is, the surfaces of the first sample introduction port 45 and the second sample introduction port 46 which are exposed are the surfaces opposite to the surfaces on which the flow paths 12 are formed. When the branching point of the first branch channel 43 from the dispensing channel 42 is the first branching point 431 and the branching point of the second branch channel 44 from the dispensing channel 42 is the second branching point 441, the volume of the sample supplied to the PCR is determined approximately by the volume in the dispensing channel 42 between the first branching point 431 and the second branching point 441.

As described above, the first filter 28 and the second filter 30 are exposed on the lower surface 14a of the substrate 14. Since the flow path 12 is formed on the lower surface 14a of the substrate 14, the flow path sealing film 16 for sealing the flow path 12 may have an outer shape such that the first filter 28 and the second filter 30 are simultaneously sealed as shown in fig. 1 (c). The corners may also be rounded so that they are difficult to peel. In this embodiment, a polypropylene film 9795 manufactured by 3M corporation is used as the flow path sealing film 16.

Further, although the first air communication port 24 and the second air communication port 26 are also exposed to the lower surface 14a of the substrate 14, the first sealing film 18 which is a separate body from the flow path sealing film 16 is used to seal them, as shown in fig. 1 (c).

Further, in order to seal the first sample introduction port 45, the second sample introduction port 46, and the connection flow paths 241 and 261, as shown in fig. 1 (a), a second sealing film 20 is attached to the upper surface 14b of the substrate 14. In a state where the first sealing film 18 and the second sealing film 20 are bonded in addition to the flow path sealing film 16, the entire flow path becomes a closed space.

In the reaction processing apparatus 100 of the present embodiment, a pressurizing pump (described later) as a liquid feeding means is connected only to the first air communication port 24, and the second air communication port 26 is opened to the atmospheric pressure. The connection of the pressure pump to the first air communication port 24 and the opening of the second air communication port 26 are performed as follows: the first sealing film 18 is peeled off at the corresponding portions to expose the first air communication port 24 and the second air communication port 26. The first sealing film 18 may be provided with a label 181 having no adhesiveness so as to be easily grasped with fingers and easily peeled. Further, the communication ports may be opened by punching holes with a needle or the like at the positions corresponding to the first air communication port 24 and the second air communication port 26 of the first sealing film 18. In this case, the first sealing film 18 is preferably made of a material and thickness that facilitate the piercing of the needle. Conversely, the second air communication port 26 may be connected to a pressure pump, and the first air communication port 24 may be opened to the atmospheric pressure.

The sample passing through the first sample introduction port 45 and the second sample introduction port 46 is introduced into the reaction cuvette 10 so that only the corresponding portions of the first sample introduction port 45 and the second sample introduction port 46 are temporarily peeled off from the substrate 14 within a range where the connection flow paths 241 and 261 of the second sealing film 20 are not exposed, and after a predetermined amount of sample is introduced, the second sealing film 20 is adhered back to the upper surface 14b of the substrate 14. Therefore, it is preferable that the second sealing film 20 has adhesion such that it is durable against multiple cycles of adhesion and peeling. In addition, the second sealing film 20 may be a film to which a new film is attached after the sample is introduced, and in this case, the importance of the characteristics relating to the repeated attachment/detachment can be relaxed.

The first sealing film 18 and the second sealing film 20 may have an adhesive layer formed on one main surface thereof or a functional layer that exhibits adhesiveness or adhesiveness by pressing, as in the case of the flow path sealing film 16. In this regard, a transparent film made of a resin such as cycloolefin polymer, polyester, polypropylene, polyethylene, or propylene is preferable, but is not limited thereto. Further, as described above, it is preferable that the adhesion and other properties are not deteriorated to such an extent that the use is affected even by the multiple sticking/peeling, but in the case of a method in which a new film is stuck after a sample or the like is introduced for peeling, the importance of the properties relating to the sticking/peeling can be relaxed. In the present embodiment, a polypropylene film 9793 manufactured by 3M corporation or the like is used as the first sealing film 18 and the second sealing film 20.

Next, a method of using the reaction processing vessel 10 configured as described above will be described. First, a sample to be amplified by thermal cycling is prepared. Examples of the sample include a sample obtained by adding a fluorescent probe, a thermostable enzyme, and 4 types of deoxynucleoside triphosphates (dATP, dCTP, dGTP, and dTTP) as PCR reagents to a mixture containing one or two or more types of DNA. Further, a primer which causes a specific reaction is mixed with the DNA to be subjected to the reaction treatment. A commercially available reagent set for real-time PCR can also be used.

Next, in the second sealing film 20, only the corresponding portions of the first sample inlet 45 and the second sample inlet 46 are peeled from the substrate 14 within a range where the connecting flow paths 241 and 261 are not exposed, and the first sample inlet 45 and the second sample inlet 46 are exposed and opened.

The second sealing film 20 may be provided with a non-adhesive label 201 so as to be easily handled by fingers and easily peeled off. The operator grasps the label 201 and peels the second sealing film 20, for example, to a broken line (a) shown in fig. 1 (a).

Subsequently, the sample is introduced into the sample introduction port by a pipette (ピペッター), a burette, a syringe, or the like. Fig. 4 schematically shows a state where the sample 50 is introduced into the reaction processing vessel 10. In fig. 4, the film and the like are not shown in order to explain the relationship between the introduced sample 50 and the reaction processing container 10. The sample 50 is introduced into the dispensing flow path 42 from either the first sample introduction port 45 or the second sample introduction port 46. The method of introduction is not limited to this, and an appropriate amount of the sample 50 may be directly introduced with a pipette or a burette, for example. In the sample introduced from any one of the sample introduction ports, the sample remaining in excess of the volume of the branched flow path accumulates in the other sample introduction port. Therefore, the sample introduction port portion may be formed to have a fixed space for the purpose of utilizing it as a kind of container. As described later, the sample 50 filled in the dispensing flow path 42 between the first branch point 431 and the second branch point 441 is supplied to PCR by being pressurized from the first air communication port 24. In this way, the low temperature region 34 of the reaction processing vessel 10 functions as a dispensing function for extracting a predetermined amount of sample.

Next, the second sealing film 20 is attached back to the substrate 14, and the first sample introduction port 45 and the second sample introduction port 46 are sealed. The label 201 may also be cut off in advance, as it is no longer necessary after sealing. Instead of sticking the peeled-off second sealing film 20, a new second sealing film 20 may be stuck. Thus, the introduction of the sample 50 into the reaction processing container 10 is completed.

The dispensing function in the reaction processing vessel does not hinder the introduction of a sample by precisely dispensing the sample with a pipette. In this case, since the sample is dispensed in advance, the tip of the sample introduced from any one of the sample introduction ports may not reach the branch point on the other sample introduction port side.

Fig. 5 is a schematic diagram illustrating the reaction processing apparatus 100 according to the embodiment of the present invention. In the above-described embodiment of the reaction processing container 10, the flow path 12, the first air communication port 24, and the second air communication port 26 are present on the front surface 14a of the reaction processing container 10, and therefore, in actuality, the flow path 12 and the connection portion from the liquid feeding system 120 are disposed on the same side of the substrate 14, and the following points are noted here: for ease of understanding of the drawings, the case where the connection portion between the flow path 12 and the liquid delivery system 120 is described as being disposed on a different side of the substrate 14 and the case where each temperature region exists between a pair of air communication ports is conceptually and easily understood.

The reaction processing apparatus 100 of the present embodiment includes: a container installation unit (not shown) in which the reaction processing container 10 is installed; a temperature control system 102; and a CPU 105. As shown in fig. 5, the temperature control system 102 is configured to: the high temperature region 36 in the flow path 12 of the reaction vessel 10 can be maintained and controlled at about 95 ℃, the medium temperature region 38 can be maintained and controlled at about 62 ℃, and the low temperature region 34 can be maintained and controlled at about 30 ℃ to 40 ℃ with high accuracy for the reaction vessel 10 provided in the vessel installation part.

The temperature control system 102 adjusts the temperature of each of the thermal cycling zones, and specifically includes: a high-temperature heater 104 for heating the high-temperature region 36 of the flow path 12; a medium-temperature heater 106 for heating the medium-temperature region 38 of the flow path 12; a low-temperature heater 112 for heating the low-temperature region 34 of the flow path 12; temperature sensors (not shown) such as thermocouples for measuring actual temperatures of the respective temperature zones; a high-temperature heater driver 108 for controlling the temperature of the high-temperature heater 104; a medium-temperature heater driver 110 that controls the temperature of the medium-temperature heater 106; and a low-temperature heater driver 114 that controls the temperature of the low-temperature heater 112. Actual temperature information measured with the temperature sensor is sent to the CPU 105. The CPU105 controls each heater driver based on the actual temperature information of each temperature region so that the temperature of each heater becomes a predetermined temperature. Each heater may be, for example, a resistance heating element, a peltier element, or the like. Alternatively, the temperature control system 102 may further include other components for improving the temperature controllability of the various temperature zones.

The reaction processing apparatus 100 of the present embodiment further includes a liquid feeding system 120, and the liquid feeding system 120 performs ejection and suction of air so as to move and stop the sample 50 in the flow path 12 of the reaction processing container 10.

Fig. 6 is a schematic diagram for explaining the configuration of the liquid feeding system 120. Fluid delivery system 120 includes a chamber 125; a first pump 122; a second pump 124; a first pump driver 126 for driving the first pump 122; a second pump driver 128 for driving the second pump 124; and a tube 130. The first pump driver 126 and the second pump driver 128 are controlled by the CPU 105.

The chamber 125 has: an inner space 125a having a fixed volume; and a first air port 125b and a second air port 125c which communicate the internal space 125a with the outside. The first pump 122 and the second pump 124 are disposed in the internal space 125a of the chamber 125. The first pump 122 and the second pump 124 may be, for example, a micro booster pump formed of a diaphragm pump. As the first pump 122 and the second pump 124, for example, a micro booster pump (model MZB1001T02) manufactured by mitsubishi corporation, inc. The first pump 122 is configured to: the air ejected from the ejection port 122a is ejected from the first air port 125b of the chamber 125. The second pump 124 is configured to: the air ejected from the ejection port 124a is ejected from the second air port 125c of the chamber 125.

The first air port 125b of the chamber 125 communicates with the first air communication port 24, which is one of the pair of air communication ports of the reaction processing container 10 that is located farther from the high temperature region 36, via the pipe 130. On the other hand, the second air port 125c of the chamber 125 is opened to the atmospheric pressure. In such a connected state, the CPU105 controls the first pump 122 and the second pump 124 to alternately perform the air discharge operation through the first pump driver 126 and the second pump driver 128. When the discharge operation of the first pump 122 is performed and the second pump 124 is stopped, the pressure in the pipe 130 becomes positive, and air is discharged from the pipe 130 to the first air communication port 24 of the reaction processing container 10. On the other hand, when the first pump 122 is stopped and the second pump 124 is operated to discharge air, the air is discharged from the second air port 125c of the chamber 125, so that the internal space 125a of the chamber 125 becomes a negative pressure, and therefore the inside of the tube 130 also becomes a negative pressure, and the air is sucked into the tube 130 from the first air communication port 24 of the reaction processing container 10. By the ejection or suction of air by the tube 130, the sample 50 can be moved in the flow path in a reciprocating manner, and repeatedly exposed to each temperature region of the flow path 12 of the reaction processing container 10, and as a result, a thermal cycle can be provided to the sample 50. More specifically, the target DNA in the sample 50 is selectively amplified by repeating the steps of denaturing in the high temperature region 36 and annealing and extension in the medium temperature region 38. In other words, the high temperature region 36 may be considered a denaturation temperature region, and the medium temperature region 38 may be considered an annealing/extension temperature region. Further, the time of residence in each temperature region can be appropriately set by changing the time at which the sample 50 stops at a predetermined position of each temperature region.

The reaction processing apparatus 100 of the present embodiment further includes a fluorescence detector 140. As described above, in the sample 50, a predetermined fluorescent probe is added. Since the intensity of the fluorescence signal emitted from the sample 50 increases as the DNA amplification progresses, the intensity value of the fluorescence signal can be used as an index for determining the progress of PCR or the termination of the reaction.

As the fluorescence detector 140, an optical fiber type fluorescence detector FLE-510 manufactured by nippon nit co can be used, which is a very compact optical system capable of rapid measurement and detecting fluorescence in both bright and dark places. The optical fiber type fluorescence detector can be tuned in advance so that the wavelength characteristics of the excitation light/fluorescence are suitable for the characteristics of fluorescence emitted from the sample 50, can provide an optimum optical system and detection system for samples having various characteristics, and is suitable for detecting fluorescence from a sample existing in a small or thin region such as a flow path because the diameter of light rays by the optical fiber type fluorescence detector is small, and has excellent response speed.

The optical fiber type fluorescence detector 140 includes: an optical head 142; a fluorescence detector driver 144; and an optical fiber 146 connecting the optical head 142 and the fluorescence detector driver 144. The fluorescence detector driver 144 includes a light source for excitation light (an LED or a light source adjusted to emit laser light or other specific wavelength), a fiber-optic wavelength division multiplexer, a photoelectric conversion element (a photodetector such as a PD, APD, or photomultiplier) (none of which is shown), and the like, and the fluorescence detector driver 144 is configured by a driver or the like for controlling these. The optical head 142 is configured by an optical system such as a lens, and functions to irradiate the excitation light to the sample in a directional manner and to collect fluorescence emitted from the sample. The collected fluorescence is separated from the excitation light by a fiber type wavelength division multiplexer inside the fluorescence detector driver 144 through the optical fiber 146 and converted into an electric signal by a photoelectric conversion element.

In the reaction processing apparatus 100 of the present embodiment, the optical head 142 is configured to: fluorescence from the sample 50 in the flow path connecting the high temperature region 36 and the medium temperature region 38 can be detected. Since the reaction progresses due to the sample 50 repeatedly reciprocating in the channel and the predetermined DNA contained in the sample 50 is amplified, the progress of the amplification of the DNA can be known in real time by monitoring the change in the amount of the detected fluorescence. In the reaction processing apparatus 100 of the present embodiment, as will be described later, the output value from the fluorescence detector 140 is used to control the movement of the sample 50. The fluorescence detector is not limited to the optical fiber type fluorescence detector as long as it can function to detect fluorescence from a sample.

Referring to fig. 5, the operation of providing a thermal cycle to the sample 50 by the reaction processing apparatus 100 will be described. The reaction processing container 10 filled with the sample 50 is set in the reaction processing apparatus 100, the first air communication port 24 of the reaction processing container 10 is connected to the first air port 125b of the chamber 125 via the tube 130, and the second air communication port 26 of the reaction processing container 10 is opened to the atmospheric pressure. Then, by operating only the first pump 122, the inside of the tube 130 becomes a positive pressure, and the sample 5 in the low temperature region 34 is moved to the medium temperature region 38 or the high temperature region 36. When the sample 50 is moved from the high temperature region 36 to the medium temperature region 38, only the second pump 124 is operated. As a result, a negative pressure is generated in the tube 130, and the sample 50 moves from the high temperature region 36 to the medium temperature region 38. On the other hand, when the sample 50 is moved from the medium temperature region 38 to the high temperature region 36, only the first pump 122 is operated. Thus, the inside of the tube 130 becomes a positive pressure, and the sample 50 moves from the medium temperature region 38 to the high temperature region 36. Then, by alternately operating the first pump 122 and the second pump 124, the sample 50 is continuously reciprocated between the high temperature region 36 and the medium temperature region 38, and a thermal cycle can be provided to the sample 50.

Fig. 7 is a diagram showing a case where heat circulation is provided to the sample in the flow path of the reaction processing vessel 10. In the reaction processing vessel 10 shown in fig. 7, the sample 50 filled in the dispensing channel 42 between the first branch point 431 and the second branch point 441 of the low temperature region 34 moves between the high temperature region 36 and the medium temperature region 38.

As described above, in the reaction processing apparatus 100 of the present embodiment, the liquid feeding system 120 is connected to only the first air communication port 24 located farther from the high temperature region 36 via the pipe 130, and the second air communication port 26 located closer to the high temperature region 36 is opened to the atmospheric pressure. In the contamination in the PCR, it is considered that a part of the amplified DNA fragments float or adhere to the air in the tube 130 in the form of suspended particles or the like and are mixed into the sample in the reaction processing container 10. It is considered that more suspended particles of DNA fragments are generated than when the liquid in which the DNA fragments are mixed is vaporized. As for the vaporization phenomenon, it is needless to say that the higher the vapor pressure of the sample (solution) is, the more likely the vaporization phenomenon is, the more likely the air communication port on the high temperature region 36 side (i.e., the second air communication port 26 close to the high temperature region 36) is opened, and the liquid feeding system 120 is made to communicate only with the air communication port on the medium temperature region 38 side (i.e., the first air communication port 24 farther from the high temperature region 36) where the vaporization phenomenon is relatively less likely to occur, whereby the DNA fragments are less likely to be taken into the tube 130 as suspended particles, and contamination can be prevented or at least suppressed.

In order to confirm the effect of the reaction processing apparatus 100 configured as described above, an experiment was performed in which a specific strain was amplified by PCR using the reaction processing apparatus 100 of the present embodiment. The samples for PCR were adjusted as follows. Here, reference is made to the contents described in "Rapid Escherichia coli assay by genetic screening method" (http:// www.city.osaka.lg.jp/suido/cmsfiles/contents/0000245/245422/6-250227. pdf) published in the homepage of the Water course office of Osaka City.

(i) For the forward primer, 5'-GTG TGA TAT CTA CCC GCT TCG C-3' (SEQ ID NO: 1) was used; for the reverse primer, 5'-AGA ACG GTT TGT GGT TAA TCA GGA-3' (SEQ ID NO: 2) was used, and 5 '-FAM-TCGGCA TCC GGT CAG TGG CAG T-MGB-3' (SEQ ID NO: 3) was selected as a TaqMan (registered trademark) probe. The concentration of each primer (final concentration) was 900nM (nM: nanomole/liter), 900nM, and 250nM, respectively.

(ii) (ii) the template DNA of the region amplified in (i) above was sent to a synthetic DNA manufacturer at 1X 10 ⁶The concentration of Copies/. mu.L was determined. Next, a standard specimen (1X 10) composed of a 10-fold dilution series was prepared ¹～1×10 ⁶Copies/μL)。

(iii) Further, SpeedSTAR manufactured by Takara-bio K.K. (registered trade name) at 0.1U/. mu.L was added to the mixture in accordance with the instructionsTrademark) HS DNA Polymerase as a DNA Polymerase was mixed with X10 Fast buffer I attached thereto and dNTP Mix and added to the reagent. In addition, [ U/. mu.L ]]The "U" in (1) is a unit of the amount of enzyme activity, and 1 μmol (1 × 10) can be converted within 1 minute under optimum conditions ^－6mol) of substrate is defined as 1U, and the amount of enzyme can be roughly expressed.

(iv) To 24. mu.L of this reagent, 1. mu.L of the standard specimen prepared in (ii) at a different concentration was added to prepare a sample. Further, a sample to which a reagent for the standard specimen was not added was prepared as a negative control (ネガコン). The negative control is a control (sample) to be negative in an effect control experiment, and in this case, even when PCR is performed, a result that bacteria are not contained (negative) is obtained. On the contrary, the positive control (ポジコン) corresponds to the standard specimen, and the result of containing bacteria (positive) is obtained by performing PCR.

The sample prepared as described above was included at a concentration of 1X 10 ⁶Samples of the Copies/. mu.L specimens were used as positive control samples, and PCR amplification was performed alternately with the negative control samples. 15. mu.L of the sample was introduced into the reaction processing vessel 10 by pipette. Further, since the dispensing flow path 42 capable of dispensing is provided in the reaction processing container 10, erroneous determination of the result due to the difference in sample volume can be reduced even if the reaction processing container 10 is changed.

The sample subjected to the thermal cycle is repeatedly reciprocated between the high temperature region 36 and the medium temperature region 38 in the reaction processing vessel 10. The thermal cycle conditions were 1 cycle repeated, with the sample held for 10 seconds in the medium temperature region 38 (annealing/extension region) maintained at 62 ℃ and the sample held for 3 seconds in the high temperature region 36 (heat-denatured region) maintained at 95 ℃. The temperature of the low temperature region 34 is maintained at about 30 to 40 ℃.

FIG. 8 shows the result of PCR amplification performed by the reaction processing apparatus 100 according to the present embodiment. In fig. 8, the horizontal axis represents cycle number (C) and the vertical axis represents fluorescence signal intensity (arbitrary unit). The intensity of the fluorescence signal detected by the fluorescence detector 140 with respect to the number of cycles is measured by the reaction processing apparatus 100. When the specimen in the sample is amplified, the intensity of the fluorescence signal increases. FIG. 8 shows the results of alternating the PCR of the positive control sample and the PCR of the negative control sample 3 times each. As shown in fig. 8, it was found that the fluorescence signal intensity in the case of the positive control sample sharply increased from around 26 cycles, and the specimen in the positive control sample was amplified. On the other hand, it was found that the fluorescence signal intensity in the case of the negative control sample was almost constant (background level) even after 50 cycles, and the contamination (carry-over) was small to such an extent that it was not generated or was negligible.

FIG. 9 also shows the result of PCR amplification performed by the reaction processing apparatus 100 according to this embodiment. Here, the initial concentration of the catalyst is 1X 10 ¹～1×10 ⁶PCR was performed on samples of 6 specimens per mu L (10 times each) of Copies. As can be seen from fig. 9, as the initial sample concentration increases, the number of cycles in which the fluorescence signal intensity increases, that is, the number of cycles in which amplification of the sample starts decreases.

FIG. 10 shows the initial specimen concentration at 1X 10 ¹～1×10 ⁶Table of Ct values (threshold cycle number) for 6 specimens of Copies/. mu.l. Here, the Ct value was defined as 10% of the plateau of each amplification curve.

FIG. 11 shows a calibration curve plotting the initial concentration versus Ct value shown in FIG. 10. When the slope (S) of the calibration curve was found from FIG. 11, it was-3.55. In addition, when calculating PCR efficiency (10) ^(－1/S)When the expression was expressed in the case of-1), it was found that the PCR was performed satisfactorily at 91.2%.

Next, a description will be given of a comparative example. FIG. 12 is a diagram for explaining a reaction treatment apparatus 200 of a comparative example. The reaction processing apparatus 200 of this comparative example is different from the liquid feeding system 120 of the reaction processing apparatus 100 shown in FIG. 5 in the configuration of the liquid feeding system 220. Liquid feeding system 220 includes: a first pump 222; a second pump 224; a first pump driver 226 for driving the first pump 222; a second pump driver 228 for driving the second pump 224; a first tube 230; and a second tube 232.

In the liquid feeding system 220, the first pump 222 and the second pump 224 are not disposed in the chamber, but are disposed at atmospheric pressure. The first air communication port 24 of the reaction processing container 10 communicates with the discharge port of the first pump 222 via the first pipe 230, and the second air communication port 26 of the reaction processing container 10 communicates with the discharge port of the second pump 224 via the second pipe 232. Gaskets 234 and 236 or seals for ensuring airtightness may be disposed at the connection portion between the first air communication port 24 and the first pipe 230 and the connection portion between the second air communication port 26 and the second pipe 232, respectively.

In the liquid feeding system 220 configured and arranged as described above, when the first pump 222 and the second pump 224 are alternately operated to discharge, the sample 50 can be reciprocated in the flow path 12 of the reaction processing container 10.

FIG. 13 shows the result of PCR amplification performed by the reaction processor 200 of the comparative example. In fig. 13, the horizontal axis represents cycle number (C) and the vertical axis represents fluorescence signal intensity (arbitrary unit). For the samples, positive controls and negative controls obtained by adjustment in the same manner as in the experiment described in fig. 8 were prepared, and PCR was performed alternately 2 times each. From the amplification curve shown in FIG. 13, it was found that the same amplification results as those shown in FIG. 8 were obtained for the positive control, while the fluorescence signal intensity started to increase at 30 cycles or more for the negative control. The Ct values of the first negative control and the second negative control were 41.89 and 41.41, respectively. Regarding the level of the Ct value, the initial concentration of the specimen is approximately 1 × 10 in the calibration curve shown in FIG. 11 ²Copies/. mu.L corresponds, and in the reaction processing apparatus 200 of the comparative example, the initial concentration of the specimen is 1X 10 ²Below Copies/. mu.l, the following risks arise: it is unclear whether the assay is actually positive or detected as a result of contamination.

On the other hand, in the reaction processing apparatus 100 according to the embodiment of the present invention, since such contamination can be prevented, for example, even 1 × 10 ¹Initial concentrations on the Copies/μ L scale or below can also be quantified.

As is clear from the amplification results shown in FIG. 8, in the reaction processing apparatus 100 of the present embodiment in which the liquid feeding system 120 is connected to only the first air communication port 24 of the reaction processing vessel 10, no contamination occurred in the negative control. On the other hand, as is clear from the amplification results shown in FIG. 13, in the reaction processing apparatus 200 of the comparative example in which the liquid feeding system 220 was communicated with the first air communication port 24 and the second air communication port 26 of the reaction processing container 10, contamination occurred in the negative control. Therefore, according to these experimental results, it is considered that the contamination in the PCR is caused by the contamination of the sample in the reaction processing container 10 due to the DNA fragments amplified in the positive control floating as aerosols in the air in the tube.

In the present embodiment, filters are provided at both ends of the flow path 12 of the reaction processing container 10. These filters are effective in preventing or suppressing contamination. In particular, when considering that it is difficult for DNA fragments to be taken into the tube 130 as aerosol particles, it is preferable to provide a filter (i.e., the first filter 28) at least at one end 12a of the flow path which is farther from the high-temperature region 36.

FIG. 14 is a diagram for explaining another embodiment of the reaction treatment apparatus. The reaction processing apparatus 400 shown in fig. 14 is an apparatus for thermally circulating a reaction processing vessel 510, and the reaction processing vessel 510 includes 3 stages of reaction regions, i.e., a high-temperature region 536, an intermediate-temperature region 538, and a low-temperature region 534. The reaction processing container 510 includes a first air communication port 524 and a second air communication port 526 at both ends of the flow path.

The reaction processing apparatus 400 includes 2 fluorescence detectors (a first fluorescence detector 251 and a second fluorescence detector 252). The first fluorescence detector 251 includes: a first optical head 151 for detecting fluorescence from the sample 50 passing through a region between the high temperature region 536 and the medium temperature region 538 of the channel of the reaction processing vessel 510; a first fluorescence detector driver 152; and a first optical fiber 153 connecting the first optical head 151 and the first fluorescence detector driver 152. The second fluorescence detector 252 includes: a second optical head 154 for detecting fluorescence from the sample 50 passing through a region between the medium temperature region 538 and the low temperature region 534 of the channel of the reaction processing container 510; a second fluorescence detector driver 155; and a second optical fiber 156 connecting the second optical head 154 and the second fluorescence detector driver 155.

Even in the reaction processing vessel 510 including 3 stages of reaction regions as shown in fig. 14, the liquid feeding system 120 is connected to the first air communication port 524, which is the air communication port located farther from the high temperature region 536, via the pipe 130, and the second air communication port 526, which is the air communication port located closer to the high temperature region 536, is opened to the atmospheric pressure, whereby the effect of preventing or suppressing contamination can be obtained.

In the reaction processing apparatus 400, the thermal cycle conditions are repeated for 1 cycle, for example, by holding the sample 50 in the high temperature region 536 maintained at 95 ℃ for 3 seconds, holding the sample 50 in the low temperature region 534 maintained at 55 ℃ for 10 seconds, and holding the sample 50 in the medium temperature region 538 maintained at 70 ℃ for 10 seconds. When PCR is performed in 3-step reaction regions, it is advantageous from the viewpoints of ease of adjustment of the sample 50, expansion of the selection range of additives, and the like. Further, the sample 50 may be held in the high temperature region 536 for about 2 minutes before starting the thermal cycle for activating the polymerase.

The present invention has been described above based on the embodiments. It should be understood by those skilled in the art that the present embodiment is merely an example, and various modifications can be made in the combination of the respective constituent elements and the respective processing procedures, and such modifications are also within the scope of the present invention.

[ description of reference numerals ]

10. 510 reaction processing container, 12 flow path, 14 substrate, 16 flow path sealing film, 18 first sealing film, 20 second sealing film, 24, 524 first air communication port, 26, 526 second air communication port, 28 first filter, 30 second filter, 34, 534 low temperature region, 36, 536 high temperature region, 38, 538 medium temperature region, 40, 41 connection region, 42 dispensing flow path, 43 first branch flow path, 44 second branch flow path, 45 first sample inlet, 46 second sample inlet, 50 sample, 100, 200, 400 reaction processing device, 102 temperature control system, 104 high temperature heater, 105CPU, 106 medium temperature heater, 108 high temperature heater driver, 110 medium temperature heater driver, 112 low temperature heater, 114 low temperature heater driver, 120, 220 liquid delivery system, 122, 222 first pump, 124, 224 second pump, 125 chamber, 125a internal space, and, 125b first air port, 125c second air port, 126, 226 first pump driver, 128, 228 second pump driver, 130, 230, 232 tube, 136, 234, 236 shim, 140 fluorescence detector, 142 optical head, 144 fluorescence detector driver, 146 optical fiber, 151 first optical head, 152 first fluorescence detector driver, 153 first optical fiber, 154 second optical head, 155 second fluorescence detector driver, 156 second optical fiber, 181, 201 label, 241, 261 connecting flow path, 251 first fluorescence detector, 252 second fluorescence detector, 431 first branch point, 441 second branch point.

[ Industrial availability ]

The present invention can be used for Polymerase Chain Reaction (PCR).

[ sequence Listing free text ]

Sequence number 1: forward PCR primer

Sequence number 2: reverse PCR primer

Sequence number 3: probe needle

[ sequence listing ] NSG-70055WO sequence listing txt

Sequence listing

<110> Nitri Kabushiki Kaisha (NIPPON SHEET GLASS COMPANY, LIMITED)

<120> REACTION processing apparatus (REACTION TREATMENT DEVICE)

<130>P0013463WO01

<150>JP 2017-123604

<151>2017-06-23

<160>3

<170>PatentIn version 3.5

<210>1

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Forward PCR Primer

<400>1

gtgtgatatc tacccgcttc gc 22

<210>2

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Reverse PCR Primer (Reverse PCR Primer)

<400>2

agaacggttt gtggttaatc agga 24

<210>3

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> TaqMan (R) Probe (TaqMan (R) Probe)

<400>3

tcggcatccg gtcagtggca gt 22