Analysis method and system for analyzing nucleic acid amplification reaction
阅读说明：本技术 用于分析核酸扩增反应的分析方法和系统 (Analysis method and system for analyzing nucleic acid amplification reaction ) 是由 R.诺贝尔 于 2015-08-26 设计创作，主要内容包括：本发明涉及用于分析核酸扩增反应的分析方法和系统。本发明涉及一种以在较宽范围的分析物浓度里改善通过实时核酸扩增反应RNR(诸如聚合酶链式反应PCR)量化的线性(精确性和/或准确性)和/或限制存在干扰性物质的效应为目的的分析方法,其通过以与增长曲线与包含至少两个不同阈值水平的在RNR循环范围里的组合阈值函数(CTF)的交叉对应的循环数目测定RNR的量化循环数目(Cq)进行,所述量化循环数目(Cq)指示增长曲线的定量和/或定性分析结果,所述增长曲线指示RNR循环范围里对于RNR的每个扩增反应循环,分析物的荧光发射强度。(The present invention relates to an analysis method and system for analyzing a nucleic acid amplification reaction. The invention relates to an analytical method with the aim of improving the linearity (accuracy and/or precision) of the quantification of RNRs by real-time nucleic acid amplification reactions (such as polymerase chain reaction PCR) and/or limiting the effect of the presence of interfering substances in a wide range of analyte concentrations by determining the quantification cycle number (Cq) of RNRs, which indicates the result of the quantitative and/or qualitative analysis of the growth curve, by the cycle number corresponding to the intersection of the growth curve with a Combined Threshold Function (CTF) in the RNR cycle range comprising at least two different threshold levels, which indicates the intensity of the fluorescence emission of the analyte for each amplification reaction cycle of RNRs in the RNR cycle range.)
1. An assay method for real-time nucleic acid amplification reaction RNR, the method comprising:
-obtaining the fluorescence emission intensity of the analyte for each amplification reaction cycle of the RNR;
-creating a growth curve (200), the growth curve (200) being indicative of the fluorescence emission intensity over a range of RNR cycles;
-calculating a Combined Threshold Function (CTF) over the RNR cycles, the Combined Threshold Function (CTF) comprising at least two different threshold levels, wherein the Combined Threshold Function (CTF) depends on the number of cycles of the RNR, the Combined Threshold Function (CTF) transitioning from an initial threshold level (y1) to a final threshold level (y2), the initial threshold level (y1) being different from the final threshold level (y2), the initial threshold level (y1) being B + r · I, wherein B is a baseline function, the baseline being an initial part of a growth curve showing no signal increase, r being a positive percentage parameter, and I being truncated extrapolation to cycle number 0, the final threshold level (y2) being y (x) B + p · G ═ B + p · S (S-B), wherein B is a baseline function, S is a saturation line of a growth curve, g is the increase from baseline to saturation line, and p is a parameter between 0% and 100%, wherein the initial threshold level (y1) is higher than the final threshold level (y2), wherein the combined threshold function is selected from the group of three functions comprising:
i. with a threshold level from the initial (y)1) To a final threshold level (y)2) Linear transformation ofCTF according to
Wherein x is1Is the number of early reaction cycles, x, equal to 0or 12Is a number of late reaction cycles, y, of more than 301Is the initial threshold level, y2Is the final threshold level;
having a threshold level (y) from the initial threshold level1) To a final threshold level (y)2) According to the CTF of the polynomial transition of
Where p is a fixed polynomial order, where p is 2 for a quadratic polynomial function;
have a threshold level (y) from the initial1) To a final threshold level (y)2) Of an exponential transition of CTF according to
And
-determining a quantified number of cycles (Cq) of the RNR, which indicates a quantitative and/or qualitative analysis result of a growth curve (200) as a number of cycles corresponding to an intersection of the growth curve (200) with the Combined Threshold Function (CTF).
2. The assay according to claim 1, wherein:
-the initial threshold level (y1) comprises at least one method appropriate for the early reaction cycle of the RNR; and/or
-the final threshold level (y2) comprises at least one method appropriate for the late reaction cycle of the RNR.
3. The assay according to claim 1 or 2, wherein:
-said initial threshold level (y1) having a starting point (T)1) The starting point (T)1) An end point (T) having a y-seat height above the final threshold level (y2)2) Resulting in a reduced Combined Threshold Function (CTF).
4. An assay method according to any one of claims 1-3, wherein said combination comprises at least one threshold level appropriate for the early response cycle of said RNR and at least one threshold level appropriate for the late response cycle of said RNR.
5. The assay according to any one of claims 1-4, wherein the real-time nucleic acid amplification reaction RNR is real-time polymerase chain reaction PCR.
6. A system for real-time nucleic acid amplification reaction RNR for analysis of an analyte, the system comprising:
-a detection system (130) configured to obtain a fluorescence emission intensity of an analyte for each amplification reaction cycle of the RNR;
-a processing unit (145) configured to:
creating a growth curve (200), the growth curve (200) indicating the fluorescence emission intensity over a reaction cycle range of the RNR;
providing a Combined Threshold Function (CTF) in the RNR cycle range, the Combined Threshold Function (CTF) comprising at least two different threshold levels, wherein the Combined Threshold Function (CTF) depends on the number of cycles of the RNR, the Combined Threshold Function (CTF) transitioning from an initial threshold level (y1) to a final threshold level (y2), the initial threshold level (y1) being different from the final threshold level (y2), the initial threshold level (y1) being B + r-I, wherein B is a baseline function, the baseline being an initial portion of a growth curve showing no signal increase, r being a positive percentage parameter, and I being truncated extrapolated to a cycle number 0, the final threshold level (y2) being y (x) B + p G B + p (S-B), wherein B is a baseline function, S is a saturation line of a growth curve, g is the increase from baseline to saturation line, and p is a parameter between 0% and 100%, wherein the initial threshold level (y1) is higher than the final threshold level (y2), wherein the combined threshold function is selected from the group of three functions comprising:
i. with a threshold level from the initial (y)1) To a final threshold level (y)2) According to the CTF of linear transition of
Wherein x is1Is the number of early reaction cycles, x, equal to 0or 12Is a number of late reaction cycles, y, of more than 301Is the initial threshold level, y2Is the final threshold level;
having a threshold level (y) from the initial threshold level1) To a final threshold level (y)2) According to the CTF of the polynomial transition of
Where p is a fixed polynomial order, where p is 2 for a quadratic polynomial function;
have a threshold level (y) from the initial1) To a final threshold level (y)2) Of an exponential transition of CTF according to
And
-determining a quantified number of cycles (Cq) of the RNR, which indicates the result of a quantitative and/or qualitative analysis of a growth curve (200) as the number of cycles corresponding to the intersection of the growth curve (200) with the Combined Threshold Function (CTF).
7. The system according to claim 6, wherein the Combined Threshold Function (CTF) is provided by the processing unit by reading data and/or instructions from an electronic memory of the system describing the Combined Threshold Function (CTF) and/or by calculating the Combined Threshold Function (CTF).
8. The system according to claim 6 or 7, wherein the real-time nucleic acid amplification reaction RNR is real-time polymerase chain reaction PCR.
9. An assay method for real-time nucleic acid amplification reaction RNR, the method comprising:
-obtaining the fluorescence emission intensity of the analyte for each amplification reaction cycle of the RNR;
-creating a growth curve (200), the growth curve (200) being indicative of the fluorescence emission intensity over a range of RNR cycles;
-calculating a Combined Threshold Function (CTF) over the RNR cycles, the Combined Threshold Function (CTF) comprising at least two different threshold levels, wherein the Combined Threshold Function (CTF) depends on the number of cycles of the RNR, the Combined Threshold Function (CTF) transitioning from an initial threshold level (y1) to a final threshold level (y2), the initial threshold level (y1) being different from the final threshold level (y2), the initial threshold level (y1) being B + r · I, wherein B is a baseline function, the baseline being an initial part of a growth curve showing no signal increase, r being a positive percentage parameter, and I being truncated extrapolation to cycle number 0, the final threshold level (y2) being y (x) B + p · G ═ B + p · S (S-B), wherein B is a baseline function, S is a saturation line of a growth curve, g is the increase from baseline to saturation line, and p is a parameter between 0% and 100%, wherein the initial threshold level (y1) is lower than the final threshold level (y2), wherein the combined threshold function is selected from the group of three functions comprising:
i. with a threshold level from the initial (y)1) To a final threshold level (y)2) According to the CTF of linear transition of
Wherein x is1Is the number of early reaction cycles, x, equal to 0or 12Is a number of late reaction cycles, y, of more than 301Is the initial threshold level, y2Is the final threshold level;
having a threshold level (y) from the initial threshold level1) To a final threshold level (y)2) According to the CTF of the polynomial transition of
Where p is a fixed polynomial order, where p is 2 for a quadratic polynomial function;
have a threshold level (y) from the initial1) To a final threshold level (y)2) Of an exponential transition of CTF according to
And
-determining a quantified number of cycles (Cq) of the RNR, which indicates a quantitative and/or qualitative analysis result of a growth curve (200) as a number of cycles corresponding to an intersection of the growth curve (200) with the Combined Threshold Function (CTF).
10. A system for real-time nucleic acid amplification reaction RNR for analysis of an analyte, the system comprising:
-a detection system (130) configured to obtain a fluorescence emission intensity of an analyte for each amplification reaction cycle of the RNR;
-a processing unit (145) configured to:
creating a growth curve (200), the growth curve (200) indicating the fluorescence emission intensity over a reaction cycle range of the RNR;
providing a Combined Threshold Function (CTF) in the RNR cycle range, the Combined Threshold Function (CTF) comprising at least two different threshold levels, wherein the Combined Threshold Function (CTF) depends on the number of cycles of the RNR, the Combined Threshold Function (CTF) transitioning from an initial threshold level (y1) to a final threshold level (y2), the initial threshold level (y1) being different from the final threshold level (y2), the initial threshold level (y1) being B + r-I, wherein B is a baseline function, the baseline being an initial portion of a growth curve showing no signal increase, r being a positive percentage parameter, and I being truncated extrapolated to a cycle number 0, the final threshold level (y2) being y (x) B + p G B + p (S-B), wherein B is a baseline function, S is a saturation line of a growth curve, g is the increase from baseline to saturation line, and p is a parameter between 0% and 100%, wherein the initial threshold level (y1) is lower than the final threshold level (y2), wherein the combined threshold function is selected from the group of three functions comprising:
i. with a threshold level from the initial (y)1) To a final threshold level (y)2) According to the CTF of linear transition of
Wherein x is1Is the number of early reaction cycles, x, equal to 0or 12Is a number of late reaction cycles, y, of more than 301Is the initial threshold level, y2Is the final threshold level;
having a threshold level (y) from the initial threshold level1) To a final threshold level (y)2) According to the CTF of the polynomial transition of
Where p is a fixed polynomial order, where p is 2 for a quadratic polynomial function;
have a threshold level (y) from the initial1) To a final threshold level (y)2) Of an exponential transition of CTF according to
And
-determining a quantified number of cycles (Cq) of the RNR, which indicates the result of a quantitative and/or qualitative analysis of a growth curve (200) as the number of cycles corresponding to the intersection of the growth curve (200) with the Combined Threshold Function (CTF).
Technical Field
The present invention relates to an analytical method for a real-time nucleic acid amplification reaction RNR (such as polymerase chain reaction PCR) and a system for a nucleic acid amplification reaction for analyzing an analyte (such as for detecting the presence of and/or measuring the amount of an analyte in a sample by a nucleic acid amplification reaction).
Background
In vitro nucleic acid amplification techniques include a number of approaches based on different approaches. One approach involves methods to multiply DNA or RNA sequences so that they can be more easily detected for various procedures or tests. For example, in vitro amplification of nucleic acids can be accomplished using one of the following methods: polymerase chain reaction PCR, Ligase Chain Reaction (LCR), or isothermal Transcription Mediated Amplification (TMA). In all of the above mentioned approaches, the nucleic acid is subjected to repeated amplification cycles.
One technique that is often used for a variety of medical, biological, or industrial applications is the PCR technique. This technique relies on thermal cycling, including cycles of repeated heating and cooling for enzymatic replication of nucleic acids to amplify specific regions of a nucleic acid strand. As PCR progresses, the resulting DNA itself serves as a template for replication, thereby initiating a chain reaction in which the DNA template is exponentially amplified, doubling (at least theoretically) with each reaction cycle.
Nucleic acid amplification techniques include many variations. One of these variants is called real-time nucleic acid RNR and is based on amplifying the nucleic acid in a sample and simultaneously detecting its presence and, in addition, in order to quantify its concentration in the sample.
There are several known methods for detecting products in quantitative nucleic acid amplification. According to one of these methods, to indirectly measure the amount of nucleic acid during the RNR, the fluorescence emission intensity of the analyte is obtained, and a growth curve (growth curve) is created that indicates the fluorescence emission intensity over the range of the RNR cycle. The quantification cycle cq (quantification cycle cq) in each reaction was determined using the increase in the growth curve signal above the initial fixed baseline.
The determination of the quantification cycle Cq based on the growth curve using various CT methods is the subject of several invention applications:
EP 2719770 a1 discloses a method for detecting the presence of an analyte in a sample and/or measuring the amount of an analyte (quality) by PCR and a corresponding analyzer. In particular, a growth curve signal normalization is disclosed that takes into account the maximum growth value of the signal.
US7788039B2 discloses a method for determining the amount of a target nucleic acid in a sample. The initial amount of the target is calculated from a calibration equation using the initial amount of the standard, the target, and the standard growth curve value, wherein the calibration equation is a non-linear equation.
EP1798652B1 discloses a mathematical algorithm for creating a growth signal from measurement data.
EP1798542a1 shows a method for determining the presence of nucleic acids in a sample, wherein a numerical value is selected based on an increasing signal and the presence of nucleic acids is determined by comparing the selected numerical value with a calibrated numerical value.
“Analyzing real-time PCR data by the comparative CTmethod ", Thomas D Schmittgen et al, Nature Protocols 3, -1101-TSummary of the methods.
Nucleic acid amplification systems are commercially available from various manufacturers, based on various nucleic acid amplification methods and used in medical applications, such as the detection of infectious diseases. Therefore, the reliability and accuracy of nucleic acid amplification methods are of the greatest interest.
Accordingly, embodiments of the present invention are directed to providing improved analytical methods using nucleic acid amplification and corresponding systems that achieve improved robustness (robustness), accuracy, precision, linearity, and qualitative discrimination (discrimination) of nucleic acid amplification assays.
Summary of The Invention
According to an embodiment of the present invention, an analytical method for real-time polymerase chain reaction is provided for detecting the presence and/or measuring the amount of an analyte in a sample, as claimed in claim 1. Furthermore, according to claim 13, a system for a nucleic acid amplification reaction for analyzing an analyte is provided. Further embodiments of the invention are given in the dependent claims.
Embodiments of the present invention relate to analytical methods for real-time nucleic acid amplification of RNR using a Combined Threshold Function (CTF) within the cycle range of RNR (RNR cycle range), the CTF comprising at least two different threshold levels (threshold levels). The CTF is obtained by calculation based on at least two different threshold levels.
The calculation can be performed before or after obtaining the intensity of the fluorescence emission and creating the growth curve. For example, the calculation is performed once, and the resulting CTF is stored in an electronic memory (electronic memory) so that it is read out when it is needed to determine the number of quantization cycles Cq of the RNR. Alternatively, the CTF is calculated after the intensity of the fluorescence emission is obtained and/or after a growth curve is created, at the moment the number of quantification cycles is determined.
Embodiments of the present invention are particularly advantageous because combining at least two different threshold levels into a CTF enables an improvement in RNR accuracy over a wider range of analyte concentrations and/or limits the effect of the presence of interfering substances.
Furthermore, embodiments of the present invention are particularly advantageous because false negatives of weak analyte concentrations and the occurrence of false Cq values of high analyte concentrations are prevented, thus increasing the reliable working range of RNR assays.
According to an embodiment of the invention, the at least one CT threshold level is a constant value over the range of RNR cycles. The constant value can be given by a relative intercept increment (RII method), a partial growth threshold (PGT method), a multiple of the standard deviation of the growth curve above the baseline (multiple of the stationary deviation of the growth curve), a manually set threshold (manual set threshold) or a constant function (constant function).
According to an embodiment of the invention, the CTF is obtained from at least two different threshold levels by combining the threshold levels in the RNR cycle range, such as by superposition (superposition), linear or non-linear combination. Alternatively or additionally, the CTFs may be obtained by dividing the range of RNR cycles into RNR cycle inter-cycle regions cycle inter) and calculating separate CTFs per inter-region (calculating separate a separate CTF per inter-using at least two different threshold levels per inter-region), wherein the at least two different threshold levels may vary between the inter-regions.
According to an embodiment of the invention, the CTF is a combination (such as a linear combination) of two threshold levels over the entire RNR cycle. One of the threshold levels delivers a major contribution to the CTF in early RNR reaction cycles (such as for numbers of cycles below 10,15or 20), while One of the threshold levels delivers a major contribution to the CTF (One of the threshold levels a dominant contribution to the CTF at early RNR reaction cycles to the CTF at early reaction cycles of the RNR, such as for numbers of cycles greater than 30,40or 50, sub-as cycle number below 10,15or 20, One of the threshold levels a dominant contribution to the CTF at last reaction cycles of the RNR, sub-as cycle number below 10,15or 20, 40or 50). According to an embodiment of the present invention, the CTF transitions from an initial threshold level to a final threshold level over the RNR cycle. In other words, the contribution of the initial threshold level to CTF may be dominant for the RNR early response cycles (for cycle numbers 0or 1), and the contribution of the final threshold level to CTF may be substantially lower than the contribution of the initial threshold level (such as 0) for the RNR early response cycles (such as for cycle numbers 0or 1). Similarly, the final threshold level of the RNR late-reaction cycle may contribute substantially more to CTF than the initial threshold level. The transition of the CTF from the initial threshold level to the final threshold level over the RNR cycle may be a one-step or multi-step transition (step or multi-step transition), a linear transition (linear transition), a polynomial transition (polynomial transition), an exponential transition (exponential transition), or a combination thereof.
According to an embodiment of the invention, the initial threshold level itself comprises a first combination a of at least two different threshold levels. Alternatively/additionally, the final threshold level itself comprises a second combination B of at least two different threshold levels, wherein the threshold level combined into the initial threshold level may be the same or a different threshold level than the threshold level combined into the final threshold level.
According to an embodiment of the invention, one of the initial and final threshold levels is not a combination of threshold levels but a single threshold level.
According to an embodiment of the invention, the initial threshold level comprises at least one threshold level suitable for accurately determining the number of quantification cycles of a strong growth curve, i.e. a growth curve obtained for high analyte concentrations and/or low concentrations of interfering substances.
In another aspect, the final threshold level comprises at least one threshold level suitable for accurately determining the number of quantification cycles for late growth (i.e., low analyte concentration and/or high concentration of interfering species). Due to the transition of CTF from the initial threshold level to the final threshold level over the RNR cycles, an accurate determination of the number of quantification cycles is achieved for both early and late signal increases.
According to an embodiment of the invention, the initial threshold level has a starting point with a y-coordinate lower than the end point of the final threshold level, resulting in an increasing combined threshold function with a positive slope or the initial threshold level has a starting point with a y-coordinate higher than the end point of the final threshold level, resulting in a combined threshold function with a decreasing combined threshold function, i.e. a combined threshold function with a negative slope over the range of the RNR cycle (the initial threshold level having a starting point with a y-coordinate above the end point of the final threshold level), resulting in a combined threshold function with a negative slope (the initial threshold level having a negative slope with an end point of the initial threshold level having a positive slope or the initial threshold level having a positive slope with a positive or negative threshold level having a positive or negative threshold value of the initial threshold level having a positive or negative threshold level with a positive or negative threshold level of the initial threshold level having a positive or negative threshold level of the initial threshold level a dividing combined threshold function, i.e. in a combined threshold function as a negative slope over the RNR cycle range).
According to an embodiment of the present invention, the combined threshold function is calculated by a linear, polynomial, exponential, logarithmic or other transition between two y-coordinates using the y-coordinate of the starting point and the y-coordinate of the ending point. For example, the two y-coordinates are stored in and read from an electronic memory, whereby the combination threshold function is calculated by means of linear interpolation (interpolation) between the two y-coordinates.
According to an embodiment of the invention, the CTF is a constant (constant) within the RNR cycle. For example, one constant is provided for each threshold level, and the combined threshold function is calculated as a linear combination of two constants (such as an average or weighted average of the two constants). For example, one of the threshold levels may be suitable for accurately determining the number of quantification cycles of early signal increase, while the other of the two threshold levels may be suitable for accurately determining the number of quantification cycles of late signal increase, and thus for CT determination of RNR late reaction cycles.
According to another aspect of the present invention, there is provided a system for a nucleic acid amplification reaction for analyzing an analyte, the system comprising: a detection system configured to obtain a fluorescence emission intensity of the analyte for each amplification reaction cycle of the RNR, and a processing unit configured to: creating a growth curve indicative of the fluorescence emission intensity over a reaction cycle of the RNR, providing a combined threshold function CTF comprising at least two different threshold levels; and determining a quantified cycle number Cq of RNR, which indicates the quantitative and/or qualitative analysis result of the growth curve as the cycle number corresponding to the intersection of the growth curve with the combined threshold function (the system comprising: a detection system configured to access the interaction of the growth curve with the combinatorial threshold function of the RNR and a processing unit configured to access the interaction of the growth curve with the interaction of the propagation of the interaction of the cycle of the RNR and the processing unit, and the method of the quantitative and/or qualitative analysis result of the interaction of the RNR, the method of the growth curve, the method of the growth of the RNR, the method of the growth of the RNR, the method of the growth of the RNR, the growth of the growth function).
According to an embodiment of the invention, the processing unit provides a combined threshold function by reading data from an electronic memory of the system describing the combined threshold function and calculating the Combined Threshold Function (CTF) using this data and/or the growth curve. For example, data stored in the electronic memory indicates the y-coordinates of the start point of the initial threshold level and the end point of the final threshold level, and the combined threshold function calculation by the processor is performed by means of interpolation between the y-coordinates within the range of the RNR cycle.
Embodiments of the present invention may be particularly advantageous because the accuracy and precision of the analysis may be increased over a wide range of analyte concentrations and/or increased robustness against interferents (as the acquisition and precision of the analysis mass information over a wide range of analytes and/or the distribution of the interference).
Brief Description of Drawings
Embodiments of the invention will be described in more detail hereinafter, by way of example only, with reference to the accompanying drawings, in which:
FIG. 1 is a block diagram showing a schematic of an RNA analyzer for analyzing a sample, such as a biological sample,
FIG. 2 schematically shows the RNR growth curve for the analyte for each RNR cycle,
FIG.3A schematically shows two different RNR growth curves, two threshold levels and the resulting CTF,
FIG. 3B schematically shows two different RNR growth curves, two threshold levels and the resulting CTF,
figure 4 shows an alternative CTF to the growth curve of figures 3A and 3B,
FIG. 5 shows a window of CTFs (negatively sloped CTFs) with negative slopes displayed by the analyzer,
fig. 6 shows a window of CTFs with negative slopes displayed by the analyzer, which transition from RII as the initial threshold level to PGT as the final threshold level.
FIG. 7 shows a window of CTFs with positive slopes displayed by the analyzer,
figure 8 shows an example of a linear and quadratic term (quadratic) and exponential transition between the initial and final threshold levels,
figure 9 shows various growth curves for a dilution series,
FIG. 10 shows various Cs obtained using known threshold levels and inventive CTFsqThe comparison of the values is carried out,
FIG. 11 shows C as a function of concentration for a known threshold level and inventive CTFqVarious calibration curves of values (calibration curve),
figure 12 shows the growth curve of the interferent,
FIG. 13 shows thresholds of interferents, and
FIG. 14 shows C as a function of the growth curve of the interferentqThe value is obtained.
Detailed Description
Throughout the following detailed description of embodiments of the invention, identical or similar elements are designated with identical reference numerals.
The term "baseline" refers to the initial portion of the growth curve that does not show an increase in signal.
As understood herein, the term "clipping" or "clipping value" refers to a signal offset value that increases the curve at an early cycle number (i.e., 0 obtained by extrapolation).
The term "saturation line" refers to the portion of the growth curve in the plateau (plateau region) after the exponential growth phase (which is also referred to as the exponential amplification phase) and after the flattening off phase.
As used herein, the term "threshold level" encompasses a threshold level of a growth curve.
As understood herein, "combined threshold function" or "CTF" encompasses a mathematical function over a cyclic range consisting of at least two different threshold levels. According to one embodiment, the CTF may be a linear or logarithmic combination of two different threshold levels.
In one embodiment, the Y-value (i.e., threshold level) returned by the CTF for a given X-value (i.e., number of cycles) may transition from one to the other of two different threshold levels.
According to one embodiment, the CTF may transition from a first combination (e.g., linear or logarithmic) of at least two different threshold levels to a second combination (e.g., linear or logarithmic) of at least two different threshold levels.
As understood herein, the term "real-time nucleic acid amplification RNR cycle range" refers to a range of numbers of cycles performed to obtain an intensity value to create a growth curve. For example, the RNR cycle range may be a predetermined fixed number, such as 30,40, or 50. Setting the RNR cycle range is a compromise (tradeoff) between the throughput of the assay system used to analyze the nucleic acid amplification reaction (throughput) and the sensitivity of the assay system used to determine the number of quantification cycles of a weak signal.
According to an embodiment of the invention, the thresholding is performed with a relative truncation increasing RII. For example, RII is a parallel line (parallel) above the baseline.
According to an embodiment of the invention, the RII may have the form:
y(x)＝B+r·I (1)
b: base line function, usually linear
I: truncate the extrapolation to a number of cycles of 0
r: a positive percentage parameter, for example 50%.
According to an embodiment of the present invention, the thresholding may be performed with a partial growth threshold PGT that depends on the growth curve signal difference between the saturation line and the baseline, i.e. the growth from the baseline to the saturation line. The PGT may return (return) a constant Y-value over the entire range of the number of cycles.
According to an embodiment of the invention, the PGT may have the following form:
y(x)＝B+p·G＝B+p·(S-B) (2)
b: base line function
S: saturation line
G: increase from baseline to saturation line
p: parameters between 0% and 100%, for example 5%, are preferred.
According to an embodiment of the invention, the thresholding is given by the standard deviation of the growth curve above the baseline and may have the form:
y(x)＝B+f·D (3)
b: base line function
f: multiplication factor parameter (Multiplying factor parameter), e.g. 10
D: standard deviation of growth curve
According to an embodiment of the present invention, the thresholding method is given by a threshold value that is preset by the user or manually input.
According to an embodiment of the invention, the thresholding is a constant value over the range of the RNR cycle, such as:
y(x)＝C (4)
c: a predefined constant based on empirical data.
According to an embodiment of the present invention, the transition from one of the at least two different threshold levels (i.e. the initial threshold level) to the other of the at least two different threshold levels (i.e. the final threshold level) is by one or more steps, linear, polynomial and/or exponential transition, as shown in fig. 8.
E.g. with a threshold level y from the initial1To a final threshold level y2The linear transformed CTF of (a) may have the following form:
wherein:
x1: the number of early reaction cycles, such as 0or 1;
x2: number of late reaction cycles, such as 30,40or 50;
y1: an initial threshold level;
y2: the final threshold level.
According to an embodiment of the present invention, the CTF may have the following form for performing polynomial transformation:
wherein:
p: fixed polynomial order (Fixed polynomial) such as 2 (for quadratic polynomial function).
According to an embodiment of the present invention, the CTF may have the following form for performing the exponential transition:
FIG. 1 shows a schematic diagram of an RNR system 100 for amplifying and simultaneously quantifying analytes in real time. Using the RNR method, a signal is created and detected during the amplification process. Generally, the signal represents the amount of any analyte created during amplification and as such present in the sample. A sample is a liquid that may contain analytes and/or other products of an amplification reaction. The RNR system comprises a thermal cycler block 120, an excitation light source 110, a detector system 130 for collecting RNR signals in real time, and a data processing system 140, said data processing system 140 comprising a processing unit and a memory 150 for storing RNR signals and program instructions 160 for analysing the RNR signals, and a unit 170 for displaying the signals and outputting the result of the analysis.
The analyte is conjugated to a fluorescent dye and the sample is loaded into the thermal cycler block 120. Thermal cycler block 120 can be a conventional design sample block containing 96 wells and capable of holding up to 96 samples. The sample is illuminated with the excitation light source 110 and raw fluorescence data is measured for each number of RNR cycles by the RNR detector system 130. The RNR detector 130 is adapted to collect RNR fluorescent signals emitted by one or more fluorescent dyes. The measured data is collected in the data processing system memory unit 150 and may be displayed on the display unit 170 as an un-normalized (un-normalized) RNR growth curve, or alternatively as a normalized RNR growth curve.
FIG. 2 shows a schematic example of a growth curve 200 representing the RNR signal acquired for each RNR cycle. The graph includes a Cartesian (Cartesian) coordinate system, where the abscissa is designated with the x-axis and the ordinate is designated with the y-axis, and where x is the number of cycles of the RNR and y is the intensity of the fluorescent emission.
FIG. 2 also shows in dotted lines fluorescence intensity values 205, where each intensity value 205 has been obtained by performing a fluorescence emission intensity measurement on the ongoing amplification reaction. The intensity values 205are modeled using a mathematical growth curve model formula or another interpolation and/or interpolation (The intensity values 205are modeled using a mathematical growth curve model for use of The intensity or other of The interpolation and/or interpolation). The RNR signal is shown in fig. 2as an increase curve 200. Here, the truncated value 210 is an RNR signal offset value when the number of RNR cycles is 0.
Baseline 240 is the RNR signal during the initial cycle of the RNR reaction (typically measured between cycles 1 and 15), with no detectable increase in fluorescence due to the RNR reaction product. The baseline 240 is a baseline function B (see equations 1 to 3). The number of cycles of RNR signal beyond the baseline 240 of RNR response, i.e., the number of cycles for which there is a first detectable significant increase in fluorescence, here about c, is determined using a predefined threshold 220q22. The threshold 220 is given by CTF (see equations 5 to 7).
The maximum increase value 250 is the difference between the maximum intensity of the RNR signal at the plateau region 230 and the RNR signal at the baseline 240. The plateau phase 230 is the RNR signal during the final cycle of the RNR reaction.
The intensity of the RNR signal at the plateau region 230 is S, and the maximum increase value 250 is an increase G in the above equation 2.
Fig.3A shows a diagram with a diagram of a growth curve 200.1 and a further growth curve 200.2 shown in dashed lines. The graph includes a cartesian coordinate system with the abscissa indicated with the x-axis and the ordinate indicated with the y-axis similar to the graph shown in fig. 2, where x is the number of cycles of RNR and y is the intensity of the fluorescence emission.
The figure shows a first threshold level y1 and a second threshold level y 2.
The threshold value y1 can be input(entering) starting point T1(x1,y1) Defining; likewise, the threshold level y2 may be determined by inputting the end point T2(x2,y2) And (4) limiting.
In the embodiment considered herein, the starting point T of the threshold level y1 is decided1Is greater than the end point T2Y of (A) to (B)2Such that y1 is above another threshold level y2, as shown in fig. 3A.
The growth curve 200.1 originates from the sample concentration at the upper end (upper end) of the RNR dynamic range (dynamic range), whereas the sample concentration of the growth curve 200.2 is at the lower end (lower end) of the dynamic range. Thus, the exponential phase of the growth curve 200.1occurs in a much lower range of cycle numbers, as is the growth curve 200.2, as shown in FIG.3A (the explicit phase of the growth curve 200.1 occure in a multi-low cycle number range that is the case for the growth curve 200.2as irradiated in FIG. 3A).
C of the growth curves 200.1 and 200.2 is determined using only the threshold level y1qResults in the detection of c only for the growth curve 200.1qHowever, c of the growth curve 200.2 may not be detectedqThe value (or result in a very late detection) because the latter is too weak to reach the intensity y in its exponential phase and/or plateau region within the range of the RNR cycle1。
On the other hand, using only the threshold level y2 may result in c of the growth curve 200.2qDetection of value, but increasing c of curve 200.1qDetection of the value may be unreliable because y2Close to the baseline, so that the variation will lead to an inaccuracy C of the growth curve 200.1qAnd (6) detecting.
This situation is remedied by combining the threshold levels y1 and y2, which provide a combined threshold function ctf (x). In the example considered herein, the CTF conforms to equation 5 above, such that the CTF has an x-coordinate range x1To x2From y1To y2As shown in fig. 3A. This has the beneficial effect that both CTF and growth curves 200.1 and 200.2 intersect within their respective exponential growth phases, resulting inCorresponding number of quantization cycles cq1And cq2Reliable and accurate detection.
Thus, the combined threshold function ctf (x) is calculated by combining the threshold levels y1 and y 2. In the embodiments contemplated herein, this is one providing a direction from T along the x-axis1To T2Linear combinations of linear transitions of (a). In embodiments contemplated herein, the RNR cycle range may be set equal to the x-coordinate range x1To x2Because it is higher than the cycle x2The additional RNR cycles of (a) do not provide a significant contribution to the quantitative cycle number determination, but merely reduce system throughput.
The calculation of the CTF may be performed by the RNR system 100 (see fig. 1). For example, storing a point T in the memory 1501To T2The coordinates of (a). By executing program instructions 160, T is read from memory 1501To T2And a combined threshold function CTF is calculated, such as according to equation 5, 6 or 7. Alternatively, the combined threshold function CTF is stored in memory 150 by means of data describing the CTF (such as in tabular form).
In the example illustrated in fig.3A, the CTF transitions from an initial threshold level y1 to a final threshold level y 2. In the example considered here, the transition is linear and results in a CTF of negative slope, whereby the initial threshold level y1 provides the number of cycles x1Initial starting point T when equal to 01And the final threshold level y2 provides the number of cycles x2End point T of linear CTF2。
Fig. 3B shows alternative selections of levels y1 and y 2. In the example considered here, the use of only the threshold level y1 does not result in false negatives, as is the case with the fig.3A and 3B examples. However, the intersection of the growth curve 200.2 with the CTF threshold level y1 is only at the late cycle number Cq2I.e. at x-48 instead of x-40 (i.e. than C)q1At about nightOne cycle corresponding to a ratio between the first and second analyte concentrations of 106). This is disadvantageous because a relatively large number of cycles have to be performed for qualitative results, thereby reducingThe flux of the system was analyzed. Another drawback is that the intersection point between CT threshold level y1 and growth curve 200.2 may occur after the exponential growth phase of growth curve 200.2, in which the RNR method is in its flattening phase (i.e., the amount of nucleic acid is no longer doubled at each cycle), resulting in false CqThe value is obtained. This situation is improved by using the CTF derived from the combination of y1 and y2, since the intersection of the growth curve 200.2 and thus CqMove to x-40.
Alternatively, ctf (x) may conform to equation 6 or 7 above or it may be another monotonic or step-function.
According to an embodiment of the invention, the threshold level y1 conforms to equation 2, thus taking into account the signal level S of the saturation line in the plateau phase, while the threshold level y2 conforms to equation 1, which takes into account the truncated value I instead of S.
FIG. 4 shows combining thresholds y by means of linear combination1And y2Such as
CTF (x) ═ 0.5y1+0.5y2, which is a constant, and thus no CTF transition in this case, where threshold level y1 is appropriate for the RNR early reaction cycle (adequate) and threshold level y2 is appropriate for the RNR late reaction cycle, the combination of which results in an appropriate CTF over a wide range of cycles. For example, a suitable choice of y1 and y2 is to select y1 and y2 such that y1> y2, as illustrated in fig. 4.
The resulting CTF and the number of quantification (quantification) cycles C are shown in FIG. 4qSo that it is detected for the growth curves 200.1 and 200.2.
FIG. 5 shows a window as an output on display 170 (see FIG. 1) with a CTF having a similar function for detecting c as the embodiment of FIGS. 3A and 3Bq1And cq2Negative slope (negative slope).
Fig. 6 shows a further embodiment similar to the embodiment of fig.3A and 3B, wherein the initial threshold level y1 (which is the RII according to equation 1 above) is linearly transformed according to equation 5 above into the final threshold level y2 (which is the PGT according to equation 2 above).
Fig. 7 shows an embodiment in which the CTF has a positive slope (positive slope).
Fig. 8 shows the transition of the contributions of thresholds y1 and y2 to the CTF for linear, quadratic term (p ═ 2) and exponential transitions according to equations 5, 6 and 7, respectively.
Fig. 9 shows growth curves 200.1 to 200.7 for a dilution series, where the growth curve 200.1 is obtained for the highest analyte concentration and the continuous growth curves 200.2, 200.3, etc. are obtained for decreasing analyte concentrations, where the analyte concentration decreases by one order of magnitude from one growth curve to the next, illustrating the dynamic range of the analyzer (see RNR system 100 of fig. 1).
FIG. 10 shows c obtained from growth curves 200.1 to 200.7 for three casesq-the value:
i. curve 300 shows c obtained when using the RII alone according to equation 1 with r-50%qThe value is obtained.
Curve 302 shows c obtained when using PGT alone according to equation 2 with p 0.1qThe value is obtained.
Curve 304 is obtained when using a CTF that combines these RII and PGT (e.g. according to equations 5, 6 or 7) to produce approximately equidistant c for the dilution seriesqThe value is obtained.
As can be observed in FIG. 10, using a standard CT method with a fixed threshold height (e.g., RII 0.5) results in a relative C of the low concentration curve due to a less steep increase (less steep increase)qAnd (4) delaying. On the other hand, the early detection of C can be overcompensated using growth-dependent CT methods (e.g., PGT)qThe effect of the less significant increase in value is obtained.
Equidistant C obtained in case iiiqThe values reflect the constant relative concentration of the respective analyte (i.e. one order of magnitude for the continuous curve) corresponding to the resulting growth curves 200.1 to 200.7, due to the fact that the crossing point of the CTF with the respective growth curve is always within an exponential growth phase, regardless of the dilution degree of the analyte in an extremely wide concentration range. This in turn implies CqThe accuracy of the assay is substantially increased compared to the above cases i. The growth curve has the lowest amount of noise in the exponential growth phase and thus produces the most accurate results.
Fig. 11 shows the calibration curves of the data of fig. 10 when either the RII or the PGT are used alone and for the combined CTF curve, which provides an almost perfectly linear calibration curve, as is evident from fig. 11. If a single threshold method is used (such as RII or PGT alone), the calibration curve is compared to C in a logarithmic scaleqThe theoretical linear law relating values to the initial analyte concentration of the sample deviates significantly, as is evident from fig. 11.
FIG. 12 shows three growth curves 200.1, 200.2 and 200.3 for the same concentration of analyte but different concentrations of interfering substances affecting the RNR response.
FIG. 13 shows the resulting c obtained for the growth curves 200.1 to 200.3 of FIG. 12 if flat threshold method (RII) is used and when CTFs with negative slopes are used (such as according to equations 5, 6 or 7)qThe value is obtained.
As is evident from FIG. 13, if a single thresholding RII is used, C is obtained for the three growth curves 200.1, 200.2 and 200.3qThe values vary between 30 and 32, resulting in correspondingly large errors. In comparison, as also shown in fig. 13, C was obtained for these curves using a negative slope of CTFqValues yielded nearly identical C for all three curvesqThe accuracy is thus greatly improved even if interfering substances are present in the sample at various concentrations. This is also shown in FIG. 14, which shows c for the same sample concentration as a function of interferent concentrationqThe value is obtained.
List of reference numerals
100 RNR system
110 excitation source
120 thermal cycler unit
130 detection system
140 data processing system
145 processing unit
150 memory
160 program instruction (program instruction)
170 display
200 growth curve
205 intensity value
210 truncated value
220 threshold value
230 plateau region
240 base line
250 maximum growth curve
300 curve
Curve 302 (curve x)
Curve 304