CN115141878A

CN115141878A - Method and device for detecting relative quantity of nucleic acid sample, storage medium and electronic equipment

Info

Publication number: CN115141878A
Application number: CN202110346328.9A
Authority: CN
Inventors: 唐向荣
Original assignee: Shanghai Kangpai Nn Medical Technology Co ltd; Jiangsu Purisian Biotechnology Co ltd
Current assignee: Shanghai Kangpai Nn Medical Technology Co ltd; Jiangsu Purisian Biotechnology Co ltd
Priority date: 2021-03-31
Filing date: 2021-03-31
Publication date: 2022-10-04

Abstract

The present disclosure provides a relative quantitative detection method and device for nucleic acid samples, and relates to the technical field of biological detection. Respectively carrying out dual-channel cyclic amplification on dual-target plasmid standard products with different gradients and nucleic acid samples to be detected by using a probe method, wherein the standard products comprise reference genes and target genes in a ratio of 1:1; respectively obtaining normalized fluorescence values of each cycle of amplification of the standard substance with different gradients and the nucleic acid sample to be detected; determining the amplification efficiency ratio of the two fluorescence channels based on the fluorescence growth curves of the standard substances with different gradients; determining the ratio of the luminescence coefficients of the two fluorescence channels according to the newly added fluorescence ratio of the standard substance in the first amplification round with stable amplification efficiency and the amplification efficiency ratio of the standard substance in the two fluorescence channels; and determining the relative proportion of the target gene and the reference gene in the nucleic acid sample to be detected according to the newly increased fluorescence ratio and the amplification efficiency ratio of the nucleic acid sample to be detected in the second amplification round with stable amplification efficiency in combination with the luminescence coefficient ratio, so as to more accurately perform relative quantitative detection on the nucleic acid sample.

Description

Method and device for relative quantitative detection of nucleic acid sample, storage medium and electronic equipment

Technical Field

The present disclosure relates to the field of biological detection technologies, and in particular, to a method and an apparatus for relatively quantitatively detecting a nucleic acid sample, a storage medium, and an electronic device.

Background

The fluorescence quantitative qPCR technology (RTFQ PCR) is a revolutionary breakthrough in nucleic acid detection and quantitative analysis, and is introduced by the American Applied Biosystems company in 1996. The qPCR has the advantages of high detection speed, wide linear range, high flux and no pollution, and from birth to the present, the qPCR is one of the most widely applied tools in biological and medical research as the most sensitive molecular biology detection technology.

The physical basis of qPCR is that the fluorescence intensity of a fluorescent substance is directly proportional to the concentration of the substance at lower concentrations; when the number of cycles reaches a threshold, the DNA of different initial template amounts N0 all grows exponentially to the same amount with fixed efficiency. On the one hand, the detection sensitivity of the qPCR is greatly improved, but the core problem of quantitative detection of the qPCR is that the amplification efficiency needs to be accurately detected, because the deviation of the amplification efficiency value is exponentially and cumulatively amplified to influence the detection result of the qPCR. However, according to percentage calculation, the current trace loading STD is more than 5%, and the fluctuation of the corresponding efficiency detection can reach +/-4% (if the theoretical value is 1, the corresponding fluctuation is +/-8%). Literature 1 (The Digital MIQE Guidelines: minimum Information for Publication of Quantitative Digital PCR Experiments) dPCR Guidelines indicate that in The current qPCR system, absolute quantification refers to The ratio of The target gene in The measured sample relative to a standard curve derived from a series of gradient standards (as does relative quantification) that were previously quantified using independent methods. Thus, the number of target genes in the measurement sample can be estimated by the proportional relationship between the standard substance and the measurement sample without detecting the amplification efficiency. The existing qPCR standard curve method is explicitly indicated in the document 1dPCR guide, with eight replicates for CNV detection, with a resolution limit of 25%. Since the amplification efficiency cannot be accurately detected, the qPCR result cannot be repeated in different scenarios in case there are factors that affect the amplification efficiency.

There are many reports of studies attempting to obtain the amplification efficiency of qPCR by fluorescence intensity variation. XIAOYU RAO et al, in document 2 (A New Method for Quantitative reactive time Polymer Chain Reaction Data analysis. JOURNAL OF COMPUTATIONAL BIOLOGY), adopt E _n ＝(F _n -F _n-1 ) /(F _n-1 -F _n-2 ) Performing efficiency detection, and selecting four rounds of F before and after CT value _n And (4) calculating, wherein efficiency changes of the four rounds among different samples are nonlinear and asynchronous, and the obtained result cannot represent actual amplification efficiency and the proportion thereof. Rutledge et al, in document 3 (A kinetic-based molecular model for the polymerase chain reaction and the application to high-capacity absolute-time PCR), assume that the amplification efficiency decreases linearly after reaching a Threshold (Threshold), thus reversing Emax, this method assumes that the conditions are not physically based, nor is the quantification advantageous compared to the standard gradient dilution method. Recently Yulia Panina in document 4 (Pairwise efficacy: a new chemical amplification to qPCR data analysis of the precision of the amplification curve) adopted a greater amount of standard dilution to reduce the STD of the amplification efficiency of the traditional method, not only the sample addition is tedious, but also the sample cannot be processed in such a way, and actually only the precision of the amplification efficiency of the standard can be improved. In the above documents, SYBRGreen, a non-specific fluorescent dye, is used for fluorescence detection, and the linear relationship between fluorescence increase and DNA concentration increase is not strict enough, which is the biggest bottleneck of the detection methods.

Document 5 Imperial Philips corporation (CN.102395977B) adopts E _n ＝(F _n -F _b )/(F _n-1 -F _b ) Calculating amplification efficiency, calculating efficiency values in several cycles around CT value, comparing with which efficiency value to obtain gradient dilution sample data with small dispersion, and making the methodThe quantitative relation between fluorescence increase and DNA copy is not accurately analyzed, qPCR data analysis is tried to be carried out completely by a pure mathematical algorithm, the fluctuation of a detection result is extremely large, and a linear result cannot be obtained by the method even if a gradient standard substance is detected.

The core factors affecting the resolution and accuracy of qPCR detection are hardware, algorithm. The sample adding error is large, and the precision of the equipment is insufficient; the amplification efficiency algorithm is not accurate, and the difference between the amplification efficiencies of the sample and the standard cannot be detected. Fundamental changes are needed to improve the detection limit on the basis of the existing qPCR equipment, and a brand-new data analysis method is needed to measure and calculate the amplification efficiency and optimize errors.

Reference documents

1.Jim F.Huggett et al.2013The Digital MIQE Guidelines:Minimum Information for Publication of Quantitative Digital PCR Experiments dPCR.Clinical Chemistry 59:6

892–902(2013)

2.XIAYU RAO et al.2013A New Method for Quantitative RealTime Polymerase Chain Reaction Data Analysis.JOURNAL OF COMPUTATIONAL BIOLOGY

Volume 20,Number 9,2013

3.Rutledge et al.2008A kinetic-based sigmoidal model for the polymerase chain reaction and its application to high-capacity absolute quantitative real-time PCR.BMC Biotechnology 2008,Vol.8(1),pp.47

4.Yulia Panina et al.2019Pairwise efficiency:a new mathematical approach to qPCR data analysis increases the precision of the calibration curve assay.BMC Bioinformatics(2019)20:29

CN.102395977B Royal Philips electronics, inc

It is to be noted that the information disclosed in the above background section is only for enhancement of understanding of the background of the present disclosure, and thus may include information that does not constitute prior art known to those of ordinary skill in the art.

Disclosure of Invention

The present disclosure aims to provide a method and apparatus for quantitatively detecting a nucleic acid sample, a method and apparatus for detecting a cyclic amplification efficiency, a storage medium, and an electronic device, which overcome, at least to some extent, the problem of inaccurate quantitative detection of a nucleic acid sample in the related art.

Additional features and advantages of the disclosure will be set forth in the detailed description which follows, or in part will be obvious from the description, or may be learned by practice of the disclosure.

According to one aspect of the present disclosure, there is provided a method for relative quantitative detection of a nucleic acid sample, comprising:

respectively carrying out dual-channel cyclic amplification on dual-target plasmid standard products with different gradients and nucleic acid samples to be detected by using a probe method, wherein the standard products comprise reference genes and target genes in a ratio of 1:1, the nucleic acid sample to be detected comprises a reference gene and a target gene;

respectively obtaining normalized fluorescence values of each cycle of amplification of the standard substance with different gradients and the nucleic acid sample to be detected;

determining the amplification efficiency ratio of a target gene channel and a reference gene channel based on the fluorescence growth curves of the standard substances with different gradients; wherein the fluorescence growth curve is obtained from the normalized fluorescence values;

determining the ratio of the luminescence coefficients of the target gene channel and the reference gene channel according to the newly added fluorescence ratio of the standard in the first amplification round with stable amplification efficiency and the amplification efficiency ratio of the standard in the target gene channel and the reference gene channel;

and determining the relative proportion of the target gene and the reference gene in the nucleic acid sample to be detected according to the newly increased fluorescence ratio, the amplification efficiency ratio and the luminous coefficient ratio of the nucleic acid sample to be detected in the second amplification round with stable amplification efficiency.

In one embodiment, the probe method is a hydrolysis-specific probe method.

In one embodiment, determining the amplification efficiency ratio of the reference gene channel and the gene channel of interest based on the fluorescence increase curves of the standards of different gradients comprises:

obtaining fluorescence growth curves of different gradients of the standard product according to the normalized fluorescence value;

are respectively provided withDetermining the translation distance delta X of the coincidence of the fluorescence increase curves of the standard substance between the respective gradients in the target gene channel and the reference gene channel _T And Δ X _R ；

According to the formula Δ X _T /ΔX _R ＝lgE _R /lgE _T According to the dilution factor N corresponding to the gradient, according to

Determining the amplification efficiency ratio E of the target Gene channel to the reference Gene channel _T /E _R 。

In one embodiment, determining the amplification efficiency ratio of the gene channel of interest and the reference gene channel based on the fluorescence increase curves of the standards of different gradients comprises:

obtaining the fluorescence growth curve of the standard substance with different gradients according to the normalized fluorescence value;

performing second-order derivation on the fluorescence increasing curve, and judging the turn number Xmax corresponding to the turning point with the maximum change of the fluorescence increasing efficiency according to the second-order derivation;

setting a threshold value within the range of Xmax, and obtaining CT values of the standard substance with different gradients in a target gene channel and a reference gene channel;

respectively calculating the Delta CT value Delta CT between different gradients in the same channel of the target gene channel and the reference gene channel _T ，ΔCT _R ；

According to Delta CT _T /ΔCT _R And the formula Δ CT _T /ΔCT _R ＝lgE _R /lgE _T According to the corresponding dilution factor N

In one embodiment, determining the ratio of the luminescence coefficients of the target gene channel and the reference gene channel according to the newly added fluorescence ratio of the standard in the first amplification round with stable amplification efficiency and the amplification efficiency ratio of the standard in the target gene channel and the reference gene channel comprises:

x in target and reference Gene channels of the Standard _max Maximum round of calculation within the range the newly increased fluorescence ratio F of the target gene channel and the reference gene channel _XT /F _XR Wherein Xmax is the inflection point-corresponding round where the fluorescence increase efficiency changes most;

according to formula F _XT /F _XR ＝(N _T /N _R )*(F _ST /F _SR )*(E _T /E _R ) ^X+1 In standard N _T ＝N _R And the ratio of the amplification efficiencies of the target gene channel and the reference gene channel, determining the ratio of the luminescence coefficients of the target gene channel and the reference gene channel _ST /F _SR 。

and after second-order derivation is carried out on the original fluorescence data, calculating the ratio of the luminescence coefficients of the target gene channel and the reference gene channel within the range of Xmax, wherein the Xmax is the turn corresponding to the inflection point with the maximum change of the fluorescence growth efficiency.

In one embodiment, obtaining normalized fluorescence values for each cycle of amplification for different gradients of the standard and test nucleic acid samples comprises:

for each gradient of standard or test nucleic acid sample:

obtaining the fluorescence value f of the nucleic acid sample in each cycle of amplification ₁ ，f ₂ ，f ₃ …f _x Wherein f is _x As the fluorescence value f detected in the x-th round of amplification _x ＝L*C ₀ *f _b +L*C ₀ *f _s *k*x+L*f _s *N ₀ *(E ^x -1) wherein L is the excitation light intensity, f _b Is the background fluorescence luminescence coefficient, C, of the probe molecule ₀ K is the efficiency of hydrolysis of free probe in each amplification run, f is the initial number of probe molecules _s Is the luminous coefficient of a single fluorophore and x is the reactionRun of time, N ₀ Is the initial copy number of the nucleic acid and E is the qPCR amplification efficiency;

using f _x Mean value of the linear phase F _B And after normalization and fluorescence background subtraction and free probe hydrolysis are carried out on the slope K, obtaining a normalized new fluorescence value:

F _X ＝f _x -f _x-1 ＝N ₀ *(E ^x -E ^x-1 )*F _S -K

wherein, F _S ＝f _s /(C ₀ *f _b )，F _B For relative fluorescence background, K is the relative hydrolysis efficiency of the free probe.

According to still another aspect of the present disclosure, there is provided a nucleic acid sample relative quantitative detection apparatus comprising:

the circulating amplification equipment is used for respectively carrying out double-channel circulating amplification on double-target plasmid standard products with different gradients and a nucleic acid sample to be detected by using a probe method, wherein the standard products comprise reference genes and target genes in a ratio of 1:1, a nucleic acid sample to be detected comprises a reference gene and a target gene;

the fluorescence value acquisition equipment is used for respectively acquiring normalized fluorescence values of each cycle of amplification of the standard substance with different gradients and the nucleic acid sample to be detected;

the amplification efficiency ratio acquisition module is used for determining the amplification efficiency ratio of the target gene channel and the reference gene channel based on the fluorescence growth curves of the standard products with different gradients; wherein the fluorescence growth curve is obtained from normalized fluorescence values;

the luminescence coefficient ratio acquisition module is used for determining the luminescence coefficient ratio of the target gene channel and the reference gene channel according to the newly increased fluorescence ratio of the standard in the first amplification round with stable amplification efficiency and the amplification efficiency ratio of the standard in the target gene channel and the reference gene channel;

and the relative proportion obtaining module is used for determining the relative proportion of the target gene and the reference gene in the nucleic acid sample to be detected according to the newly increased fluorescence ratio, the amplification efficiency ratio and the luminous coefficient ratio of the nucleic acid sample to be detected in the second amplification round with stable amplification efficiency.

According to still another aspect of the present disclosure, there is provided an electronic device including: a processor; and a memory for storing executable instructions for the processor; wherein the processor is configured to execute the method for the relative quantitative detection of nucleic acid samples described above via execution of the executable instructions.

According to yet another aspect of the present disclosure, there is provided a computer-readable storage medium having stored thereon a computer program which, when executed by a processor, implements the above-described method for relative quantitative detection of nucleic acid samples.

According to the nucleic acid sample quantitative detection method device, the storage medium and the electronic equipment provided by the embodiment of the disclosure, by setting the proportion of the reference gene and the target gene in the standard substance, arranging the gradient of the standard substance and the nucleic acid sample to be detected consistently, and adopting the same double-channel cyclic amplification, the relative proportion of the target gene and the reference gene in the nucleic acid sample to be detected can be compared accurately by comparing the nucleic acid sample to be detected with the standard substance.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present disclosure and together with the description, serve to explain the principles of the disclosure. It is to be understood that the drawings in the following description are merely exemplary of the disclosure, and that other drawings may be derived from those drawings by one of ordinary skill in the art without the exercise of inventive faculty.

FIG. 1 is a flow chart of a method for relative quantitative detection of a nucleic acid sample according to an embodiment of the present disclosure;

FIG. 2 is a flow chart showing the determination of the amplification efficiency ratio of a gene channel of interest and a reference gene channel in one embodiment of the present disclosure;

FIG. 3 shows a flow chart for determining the amplification efficiency ratio of a gene channel of interest and a reference gene channel in another embodiment of the present disclosure;

FIG. 4 is a flow chart showing the determination of the ratio of the luminescence coefficients of a target gene channel and a reference gene channel in one embodiment of the present disclosure;

FIG. 5 is a schematic diagram showing a structure of a relative quantitative detection apparatus for a nucleic acid sample according to an embodiment of the present disclosure; and

fig. 6 shows a block diagram of an electronic device in an embodiment of the present disclosure.

Detailed Description

Example embodiments will now be described more fully with reference to the accompanying drawings. Example embodiments may, however, be embodied in many different forms and should not be construed as limited to the examples set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of example embodiments to those skilled in the art. The described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

Furthermore, the drawings are merely schematic illustrations of the present disclosure and are not necessarily drawn to scale. The same reference numerals in the drawings denote the same or similar parts, and thus their repetitive description will be omitted. Some of the block diagrams shown in the figures are functional entities and do not necessarily correspond to physically or logically separate entities. These functional entities may be implemented in the form of software, or in one or more hardware modules or integrated circuits, or in different networks and/or processor devices and/or microcontroller devices.

The inventors of the present invention found that:

1) Through analysis of the change of fluorescence intensity in the qPCR process, the fluorescence in the qPCR process is found to come from three parts, the first is background fluorescence, and the excitation light intensity L is multiplied by the number of probe molecules C ₀ And background fluorescence luminance coefficient f _b Obtaining; second, hydrolysis of free probe releases fluorescence, which is multiplied by the excitation light intensity by the number of probe molecules C ₀ Fluorescence dye monomolecular luminescence coefficient f _S The hydrolysis ratio k of the free probe in each amplification reaction and the reaction result obtained in each amplification reaction are reflected; thirdly, the fluorescence released and accumulated by the displacement hydrolysis of the probe in each round of replication reaction is multiplied by the excitation light intensitySingle-molecule luminous coefficient of light dye, initial copy number of nucleic acid template and amplification multiple E ^x Thus, the compound is obtained. Thus, equations were obtained that accurately describe the change in fluorescence growth in qPCR: f. of _x ＝L*C ₀ *f _b +L*C ₀ *f _s *k*x+L*f _s *N ₀ *(E ^x -1). For each replicate, the mean value F of the fx linear stage (typically 6. Ltoreq. N. Ltoreq.24) is used _B After normalization with slope K, fluorescence background is subtracted, and hydrolysis of free probe is carried out, and fluorescence value F is added _X ＝f _x -f _x-1 ＝N ₀ *(E ^x -E ^x-1 )*F _S -K(F _X For the corresponding cumulative newly-increased fluorescence of x rounds, F _S ＝f _s /(C ₀ *f _b )，F _B Relative fluorescence background value, K is relative hydrolysis efficiency of free probe), that is, the relative newly increased fluorescence generated by DNA replication is equal to the initial copy number and amplification multiple E ^X Multiplication by the luminescence coefficient under the same amplification conditions, F _S Is a constant value.

2) The amplification efficiency is reduced from cycle to cycle as the qPCR fluorescence increases and shows exponential rise, so that the maximum cycle for maintaining stable amplification efficiency must be determined in data analysis of qPCR, and the qPCR fluorescence increase curve can be accurately segmented and interpreted. The invention is used for f of different rounds of each multiple hole _x Performing a second derivative according to f _n The second derivative of (a) determines the round corresponding to the inflection point where the fluorescence increase efficiency changes most, i.e., the maximum round x where the DNA replication efficiency remains stable _max Only at x _max The previous run fluorescence growth equation holds.

3) At x _max Within the range, newly increased fluorescence F _X ’＝N ₀ *F _S *(E ^x ) This equation is the theoretical basis for quantitative detection of qPCR at present. However, in practical application, E needs to be obtained by performing gradient dilution on a sample to draw a standard curve, and the dilution factor depends on the accuracy of sample addition, which results in that the amplification efficiency obtained by the current detection method has a fluctuation range of ± 5%; in addition, there is no method for accurately detecting the luminescence coefficient of a single fluorescent probe molecule, so that accurate absolute quantification cannot be performed. The invention adopts double-channel fluorescence detection and utilizes the double-target plasmid standard substance, can accurately detect the amplification efficiency ratio of the reference gene R and the target gene T and the luminescence coefficient ratio of an amplification system thereof while accurately correcting the sample adding error. Therefore, the relative proportion of the target gene and the reference gene can be accurately detected by combining the ratio of the luminescence coefficient and the amplification efficiency ratio of the two fluorescence channels of the sample in the double-channel fluorescence detection of the sample to be detected, and the precision and the accuracy can be improved to the level of 1% compared with the theoretical limit (distinguishing the difference of 25%) of the traditional detection method.

Hereinafter, each step of the method for detecting a nucleic acid sample in the present exemplary embodiment will be described in more detail with reference to the drawings and examples.

FIG. 1 shows a flow chart of a method for relative quantitative detection of a nucleic acid sample in an embodiment of the disclosure.

As shown in fig. 1, S102, performing dual-channel circular amplification on the dual-target plasmid standard product and the nucleic acid sample to be detected with different gradients by using a probe method, wherein the standard product comprises a reference gene and a target gene at a ratio of 1:1, the nucleic acid sample to be detected comprises a reference gene and a target gene, and the ratio of the reference gene to the target gene in the nucleic acid sample to be detected is detected. The probe method may be a hydrolysis-specific probe method, such as the TaqMan probe method. In one embodiment, the standard and the nucleic acid sample to be tested have two gradients, one corresponding to no dilution of the standard and one corresponding to 1-fold, 2-fold, 5-fold, 10-fold, 12-fold, 15-fold or 20-fold dilution of the standard. In one embodiment, there are 3 gradients for the standard and the test nucleic acid sample, corresponding to 1-fold, 3-fold, and 10-fold dilutions, respectively. In one embodiment, the number of gradients in the standard and the nucleic acid sample to be tested is the same, e.g., there are 5 gradients, but the dilution factor for the gradient of the standard is not the same as the dilution factor for the nucleic acid sample to be tested. In one embodiment, the number of gradients in the standard and the number of gradients in the test nucleic acid sample are different; for example, the standard has 3 gradients, while the nucleic acid sample to be tested may have 2 gradients, or 5 gradients, and the dilution may be the same or different from that of the standard.

S104, obtaining the normalized fluorescence values of the standard substance with different gradients and the nucleic acid sample to be detected amplified in each cycle respectively. For the standard and the test nucleic acid samples, each gradient corresponds to a dilution factor.

S106, determining the amplification efficiency ratio of the target gene channel and the reference gene channel based on the fluorescence growth curves of the standard substances with different gradients; wherein the fluorescence growth curve is obtained from normalized fluorescence values. Various embodiments for determining the amplification efficiency ratio will be described later.

S108, determining the ratio of the luminescence coefficients of the target gene channel and the reference gene channel according to the newly added fluorescence ratio of the standard substance in the target gene channel and the reference gene channel of the first amplification round with stable amplification efficiency and the amplification efficiency ratio of the standard substance in the target gene channel and the reference gene channel.

And S110, determining the relative proportion of the target gene and the reference gene in the nucleic acid sample to be detected according to the newly added fluorescence ratio of the target gene channel and the reference gene channel of the nucleic acid sample to be detected in the second amplification round with stable amplification efficiency, the amplification efficiency ratio and the luminous coefficient ratio. In one embodiment, xmax is the turn corresponding to the inflection point where the fluorescence increase efficiency of the nucleic acid sample to be detected changes most, and the second amplification turn with stable amplification efficiency is the maximum turn X within the range of Xmax, so that a more accurate result can be obtained. It will be appreciated by those skilled in the art that the second amplification round in which the amplification efficiency is stable may also employ other rounds within the range of Xmax.

The amplification efficiency ratio E of the two fluorescence channels _TS /E _RS Luminescence coefficient F _ST /F _SR Substituting the ratio into a newly added fluorescence ratio equation of the maximum round x with stable amplification efficiency of two channels of the nucleic acid sample to be detected: f _XTS /F _XRS ＝(N _TS /N _RS )*(F _ST /F _SR )*(E _TS /E _RS ) ^X+1 The relative proportion N of the target gene and the reference gene in the nucleic acid sample to be detected can be calculated _TS /N _RS 。

In the above embodiment, by setting the ratio of the reference gene to the target gene in the standard, setting the gradient of the standard and the gradient of the nucleic acid sample to be detected to be consistent, and performing the same two-channel cyclic amplification, the relative ratio of the target gene to the reference gene in the nucleic acid sample to be detected can be accurately compared by comparing the nucleic acid sample to be detected with the standard.

FIG. 2 is a flow chart showing the determination of the amplification efficiency ratio of a gene channel of interest and a reference gene channel in one embodiment of the present disclosure.

As shown in fig. 2, a fluorescence increase curve of the standard with different gradients is obtained according to the normalized fluorescence value of the standard S202. And (5) drawing fluorescence growth curves of the standard substance and the sample with different gradients according to fn of each hole.

S204, respectively determining the translation distance delta X of the standard substance coincident with the fluorescence increase curves between the respective gradients in the reference gene channel and the target gene channel _R And Δ X _T . Respectively calculating the translation distance for the standard product to coincide with the fluorescence growth curve between the gradients in different fluorescence channels, and setting the translation distance of the fluorescence growth curve between different gradients in the reference gene channel as DeltaX _R The translation distance of the fluorescence growth curve between different gradients in the target gene channel is delta X _T 。

S206, according to the formula delta X _T /ΔX _R ＝lgE _R /lgE _T According to the dilution factor N corresponding to the gradient, according to

Determining the amplification efficiency ratio E of the target Gene channel to the reference Gene channel _T /E _R . The amplification efficiencies of the reference gene channel and the target gene channel are respectively E _R And E _T When the amplification efficiency of both channels is constant at the same dilution factor,. DELTA.X _T /ΔX _R ＝lgE _R /lgE _T I.e. from Δ X _T /ΔX _R According to the corresponding dilution factor, calculate E _T /E _R This is the ratio of the amplification efficiencies of the two fluorescence channels.

In the above-described embodiment, the amplification efficiency ratio of the target gene channel and the reference gene channel can be accurately determined by using the measured values of the standard substance in the reference gene channel and the target gene channel.

FIG. 3 shows a flow chart for determining the amplification efficiency ratio of a gene channel of interest and a reference gene channel in another embodiment of the present disclosure.

As shown in fig. 3, S302, performing a second derivative on the fluorescence increase curve, and determining the turn Xmax corresponding to the inflection point with the maximum change in fluorescence increase efficiency according to the second derivative;

s304, setting a threshold value within the range of Xmax, and acquiring CT values CT of the standard substance with different gradients in the reference gene channel and the target gene channel _R ，CT _T ；

S306, respectively calculating the Delta CT values between different gradients in the same channel of the reference gene channel and the target gene channel _R ，ΔCT _T ；

S308, according to the Delta CT _T /ΔCT _R And the formula Δ CT _T /ΔCT _R ＝lgE _R /lgE _T According to the corresponding dilution factor N

In the above embodiment, the CT values of the standard substance in the reference gene channel and the target gene channel are obtained by obtaining the CT values of the standard substance with different gradients _R ，CT _T And the delta CT value between different gradients of the standard substance can accurately determine the amplification efficiency ratio of the reference gene channel and the target gene channel.

In the case of two gradients, the difference between the two gradient CT values; with 3 or more gradients, by applying a respective Δ CT _R Or Delta CT _T And a more accurate difference value can be obtained by averaging.

FIG. 4 is a flowchart illustrating the determination of the ratio of the luminescence coefficients of the target gene channel and the reference gene channel according to an embodiment of the present disclosure. In this example, the ratio of the luminescence coefficients of the target gene channel and the reference gene channel was determined based on the newly increased fluorescence ratio of the standard in the first amplification round in which the amplification efficiency was stable and the amplification efficiency ratio of the standard in the target gene channel and the reference gene channel.

As shown in FIG. 4, S402, X in the target gene channel and the reference gene channel of the standard _max Calculating the newly increased fluorescence ratio F of the target gene channel and the reference gene channel in the maximum turn X in the range _XT /F _XR (deducting fluorescence background and free probe hydrolysis fluorescence), wherein Xmax is the turn corresponding to the inflection point with the maximum change of the fluorescence increase efficiency, and the first amplification turn with stable amplification efficiency is the maximum turn X;

according to formula F _XT /F _XR ＝(N _T /N _R )*(F _ST /F _SR )*(E _T /E _R ) ^X+1 In standard N _T ＝N _R And the ratio of the amplification efficiencies of the target gene channel and the reference gene channel, determining the ratio of the luminescence coefficients of the target gene channel and the reference gene channel _ST /F _SR . The ratio of the luminescence coefficients of the two fluorescence channels under the same amplification conditions was constant.

In the above embodiment, the ratio of the luminescence coefficients of the target gene channel and the reference gene channel is determined by using the maximal round newly-increased fluorescence ratio value with stable amplification efficiency, so that a relatively good experimental effect can be obtained. It will be appreciated by those skilled in the art that other rounds within the Xmax range may be employed for the first round in which the amplification efficiency is stable.

for each gradient of standard or test nucleic acid sample:

obtaining the fluorescence value f of the nucleic acid sample during each cycle of amplification ₁ ，f ₂ ，f ₃ …f _x Wherein f is _x The fluorescence value detected in the x round circulation amplification is the fluorescence value; f. of _x ＝L*C ₀ *f _b +L*C ₀ *f _s *k*x+L*f _s *N ₀ *(E ^x -1), wherein L is the excitation light intensity, f _b Is background fluorescence of probe moleculesCoefficient, C ₀ K is the efficiency of hydrolysis of free probe in each amplification run, f is the initial number of probe molecules _s Is the luminous coefficient of a single fluorophore, x is the reaction run, N ₀ Is the initial copy number of the nucleic acid and E is the qPCR amplification efficiency;

using f _x Mean value F of the linear phases (in general 6. Ltoreq. N.ltoreq.24) _B And after normalization and fluorescence background subtraction and free probe hydrolysis are carried out on the slope K, obtaining a normalized new fluorescence value:

F _X ＝f _x -f _x-1 ＝N ₀ *(E ^x -E ^x-1 )*F _S -K

wherein, F _S ＝f _s /(C ₀ *f _b ) And K is the hydrolysis efficiency of the free probe after normalization.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended that the experiments below be all experiments performed but are merely experiments that may be performed. The experimental method in which the specific conditions are not specified in the experimental examples is usually carried out under the conventional conditions or under the conditions recommended by the manufacturers.

Example 1: calculation of plasmid standard product double-channel amplification efficiency ratio

1. Preparation of standards

After preparing the target gene and reference gene fragments connected in equal proportion (1) by using a PCR method, inserting the target fragment into a T vector, picking a single clone, sequencing, and amplifying the prepared verified double-target standard plasmid 1 (SEQ ID NO: 1) in Escherichia coli.

The target gene plasmid (SEQ ID NO: 2) and the reference gene plasmid (SEQ ID NO: 3) were constructed, respectively. The target gene plasmid and the plasmid reference gene are mixed according to the ratio of 1:1, mixing to obtain a nucleic acid sample 1; plasmid target genes and plasmid reference genes were expressed as 2:1 proportion to obtain a plasmid nucleic acid sample 2.

2. Respectively diluting the plasmid standard substance 1 and the nucleic acid samples 1 and 2 by 100 times, and circularly amplifying the templates before and after dilution

In the experiment, a double fluorescent quantitative PCR experiment and result analysis are carried out by using a SLAN-96S fluorescent quantitative PCR instrument of Shanghai Hongshi medical science and technology Limited company.

3. The reaction system mixed solution (25 uL/tube) is prepared according to the table 1 to carry out the fluorescent quantitative PCR reaction, the PCR reaction conditions are set according to the table 2, and HEX is selected as the target gene channel, and FAM is selected as the reference gene channel. The plasmid standard 1 and the nucleic acid samples 1 and 2 were subjected to qPCR reaction in two gradients, 8 replicates each.

TABLE 1 fluorescent quantitative PCR reaction System in example 1

4. The fluorescent quantitative PCR reaction was performed, and the PCR reaction conditions were set as shown in Table 2.

TABLE 2 fluorescent quantitative PCR reaction conditions

5. Obtaining the fluorescent quantitative PCR amplification result, and sequentially obtaining the fluorescent value f of each group of the two gradients of the plasmid standard substance and 8 multiple wells ₁ ，f ₂ ，f ₃ …f _n Wherein f is _n The fluorescence value detected in the nth cycle of amplification is shown. For example, the fluorescence values of each duplicate well of plasmid standard 1 in each group of the HEX fluorescence channels are shown in table 3.

Table 3 plasmid standard 1 in example 1 f in each cycle of HEX fluorescence channel _n Value of

6. Different gradients of f according to plasmid Standard 1 _n Drawing fluorescence growth curves with different gradients, respectively calculating the translation distance for enabling the fluorescence growth curves of the standard product to coincide among the respective gradients in different fluorescence channels, and setting the translation distance of the fluorescence growth curve among the different gradients in a reference gene channel (FAM channel) as DeltaX _R The translation distance of the fluorescence increase curve between different gradients in a target gene channel (HEX channel) is delta X _T Obtaining Δ X _R And Δ X _T . The amplification efficiencies of the reference gene and the target gene channel are respectively E _R And E _T When the amplification efficiency of both channels is constant at the same dilution factor (100 times), Δ X _T /ΔX _R ＝lgE _R /lgE _T I.e. by Δ X _T /ΔX _R The ratio of (A) is determined by the corresponding dilution factor (100 times) _T /E _R The ratio of (a) to (b), which is the amplification efficiency ratio of two fluorescence channels of plasmid standard 1, is shown in table 4.

TABLE 4. DELTA.X of plasmid Standard 1 obtained in example 1 _R And Δ X _T And amplification efficiency ratio E _T /E _R

Example 2: calculation of the ratio of the luminescence coefficients of the fluorescence channel of the target gene and the fluorescence channel of the reference gene

1. Plasmid Standard 1 two fluorescence channels X obtained in example 1 _max RangeInner 18 maximal rounds with newly added fluorescence F _18T Subtracting the 17 th round fluorescence value from the 18 th round fluorescence value and the hydrolysis fluorescence of the free probe in the current round (calculating the slope of the fluorescence in the linear interval of the fluorescence increase curve through linear regression, namely the hydrolysis fluorescence value in each round), and calculating the newly added fluorescence ratio F of the two fluorescence channels _18T /F _18R From F _18T /F _18R ＝(N _T /N _R )*(F _ST /F _SR )*(E _T /E _R ) ¹⁹ In standard N _T ＝N _R From example 1, the amplification efficiency ratio E of plasmid Standard 1 is known _T /E _R The ratio of the luminescence coefficients F of the two fluorescence channels of the plasmid standard 1 can be calculated _ST /F _SR I.e. F _ST /F _SR ＝(F _18T /F _18R )/[(N _T /N _R )*(E _T /E _R ) ¹⁹ ]As shown in table 5.

TABLE 5 luminescence coefficient ratio of target gene to reference gene in plasmid Standard 1

Example 3: calculation of copy number ratio of target Gene to reference Gene in plasmid nucleic acid samples 1, 2

1. It is known that the ratio of the luminescence coefficients of the two fluorescence channels is constant under the same amplification conditions, and the ratio of the copy numbers of the target gene and the reference gene in the PCR amplification experiments of the plasmid nucleic acid samples 1 and 2 in example 1 can be determined from the ratio of the luminescence coefficients of the target gene and the reference gene obtained in example 2.

2. Obtaining the fluorescent quantitative PCR amplification result in example 1, and sequentially obtaining the f of the multiple wells under different gradients of the plasmid nucleic acid samples 1 and 2 _n Wherein f is _n The fluorescence value detected in the nth cycle of amplification is shown.

3、F according to different gradients of plasmid nucleic acid samples 1, 2 _n Drawing fluorescence growth curves of different gradients, respectively calculating translation distances for enabling the fluorescence growth curves of the plasmid nucleic acid samples among the gradients in different fluorescence channels to coincide, and setting the translation distance of the fluorescence growth curve among the different gradients in a reference gene channel (FAM channel) as DeltaX _R The translation distance of the fluorescence increase curve between different gradients in a target gene channel (HEX channel) is delta X _T Obtaining Δ X _R And Δ X _T . The amplification efficiencies of the reference gene and the target gene channel are respectively E _R And E _T When the amplification efficiency of both channels is constant at the same dilution factor (100 times), Δ X _T /ΔX _R ＝lgE _R /lgE _T I.e. by Δ X _T /ΔX _R The ratio of (A) is determined by the corresponding dilution factor (100 times) _T /E _R The ratio of (A) to (B) is the amplification efficiency ratio of the two fluorescence channels of the plasmid nucleic acid samples 1 and 2, as shown in Table 6.

TABLE 6. DELTA.X of plasmid nucleic acid samples 1, 2 obtained in example 1 _R And Δ X _T And amplification efficiency ratio

4. Plasmid nucleic acid sample 1 two fluorescence channels X _max Maximum 18 rounds in range, newly increasing fluorescence F _18T Subtracting the 17 th round fluorescence value and the current round free probe hydrolysis fluorescence from the measured 18 th round fluorescence value, and calculating the newly added fluorescence ratio F of the two fluorescence channels _18T /F _18R (ii) a Plasmid nucleic acid sample 2 two fluorescence channels X _max Maximum run in range 19 new fluorescence F _19T Subtracting the 18 th round fluorescence value from the 19 th round fluorescence value and the current round free probe hydrolysis fluorescence, and calculating the newly added fluorescence ratio F of the two fluorescence channels _19T /F _19R . The ratio of the luminescence coefficients of the two fluorescence channels under the amplification conditions is known from example 2, by equation F _XTS /F _XRS ＝(N _TS /N _RS )*(F _ST /F _SR )*(E _TS /E _RS ) ^X+1 Can obtain N _TS /N _RS ＝(F _XTS /F _XRS )/[(F _ST /F _SR )*(E _TS /E _RS ) ^X+1 ]I.e., the copy number ratio of the target gene to the reference gene in the plasmid nucleic acid samples 1 and 2, are shown in tables 7 and 8.

TABLE 7 plasmid nucleic acid sample 1 copy number ratio of target gene to reference gene

TABLE 8 plasmid nucleic acid sample 2 copy number ratio of target gene to reference gene

5. The ratio of the target gene to the reference gene in the mixed plasmid nucleic acid sample 2 is known to be 2 times that in the plasmid nucleic acid sample 1 (in this experiment, a precision balance of ten-thousandth is used for accurate weighing and uniform mixing, that is, the ratio is 2 ± 0.00001), and the actual ratio is calculated to be 2.004543 according to the experimental results, and the deviation is only 0.227139%, as shown in table 9.

TABLE 9 calculated ratios and deviations of the experimental results for plasmid nucleic acid samples 1, 2

N of plasmid nucleic acid sample 1 _TS /N _RS	0.745563
		N of plasmid nucleic acid sample 2 _TS /N _RS	1.494514
(N of plasmid nucleic acid sample 2 _TS /N _RS ) V (plasmid nucleic acid sample 1N _TS /N _RS )	2.004543
		Experimental calculated bias for plasmid nucleic acid samples	0.227139％

Example 4 calculation of amplification efficiency ratio and luminescence coefficient ratio of plasmid Standard 1 and amplification efficiency ratio of plasmid nucleic acid samples 1 and 2 and copy number ratio of target Gene to reference Gene by Δ CT value

1. The amplification efficiency ratio and the luminescence coefficient of plasmid standard 1 were calculated using the Δ CT values, and are shown in table 10.

TABLE 10 Delta CT, amplification efficiency ratio, and luminescence coefficient of plasmid standards

	Plasmid Standard 1
		ΔCT _T	6.85771
ΔCT _R	6.81579
		Amplification efficiency ratio E _T /E _R	0.995879
Luminous coefficient ratio F _ST /F _SR	0.545701

2. The amplification efficiency ratio of the plasmid nucleic acid samples 1 and 2 and the copy number ratio of the target gene to the reference gene are calculated by using the Delta CT value. It is known that the ratio of the target gene to the reference gene in the mixed plasmid nucleic acid sample 2 is 2 times that in the plasmid nucleic acid sample 1, and the actual ratio is 2.004553 calculated by the experimental results and the deviation is 0.227667%, as shown in table 11, the deviation of the calculated result of the plasmid nucleic acid sample is slightly larger than the calculated result using the Δ X value.

TABLE 11 Δ CT, amplification efficiency ratio, calculated ratio of experimental results and deviation of plasmid nucleic acid samples 1, 2

If a traditional standard curve method is used, only a standard substance gradient curve is drawn, and quantitative calculation of all samples is uniformly carried out according to standard substance efficiency, the ratio of the sample 1 to the sample 2 is 1.878, the quantitative deviation is 6.1%, the quantitative deviations of the two methods are 0.227139% and 0.227667%, respectively, and the quantitative precision of the method is far higher than that of the traditional standard curve method.

FIG. 5 is a schematic structural diagram of a quantitative nucleic acid sample detection device according to an embodiment of the present disclosure. As shown in fig. 6, the apparatus includes:

and the circulating amplification equipment 61 is used for respectively carrying out double-channel circulating amplification on the double-target plasmid standard substance and the nucleic acid sample to be detected with different gradients by using a probe method, wherein the standard substance comprises a reference gene and a target gene in a ratio of 1:1, a nucleic acid sample to be detected comprises a reference gene and a target gene;

a fluorescence value obtaining device 62 for obtaining normalized fluorescence values of each cycle of amplification of the standard substance and the nucleic acid sample to be detected with different gradients;

an amplification efficiency ratio obtaining module 63, configured to determine an amplification efficiency ratio between the target gene channel and the reference gene channel based on the fluorescence growth curves of the standards with different gradients; wherein the fluorescence growth curve is obtained from normalized fluorescence values;

a luminescence coefficient ratio obtaining module 64, configured to determine a luminescence coefficient ratio between the target gene channel and the reference gene channel according to the new amplification fluorescence ratio of the standard in the maximum round in which the amplification efficiency is stable and the amplification efficiency ratio of the standard in the target gene channel and the reference gene channel;

and a relative proportion obtaining module 65, configured to determine the relative proportion of the target gene and the reference gene in the nucleic acid sample to be detected according to the newly-increased fluorescence ratio value, the amplification efficiency ratio, and the light-emitting coefficient ratio of the nucleic acid sample to be detected in the maximum round in which the amplification efficiency is stable.

An electronic device 800 according to this embodiment of the invention is described below with reference to fig. 6. The electronic device 800 shown in fig. 6 is only an example and should not bring any limitations to the function and scope of use of the embodiments of the present invention.

As shown in fig. 6, the electronic device 800 is in the form of a general purpose computing device. The components of the electronic device 800 may include, but are not limited to: the at least one processing unit 810, the at least one memory unit 820, and a bus 830 that couples the various system components including the memory unit 820 and the processing unit 810.

Where the memory unit stores program code, the program code may be executed by the processing unit 810 to cause the processing unit 810 to perform steps according to various exemplary embodiments of the present invention as described in the above-mentioned "exemplary methods" section of this specification. For example, processing unit 810 may perform the steps as shown in fig. 1.

The memory unit 820 may include readable media in the form of volatile memory units, such as a random access memory unit (RAM) 8201 and/or a cache memory unit 8202, and may further include a read only memory unit (ROM) 8203.

Storage unit 820 may also include a program/utility module 8204 having a set (at least one) of program modules 8205, such program modules 8205 including, but not limited to: an operating system, one or more application programs, other program modules, and program data, each of which, or some combination thereof, may comprise an implementation of a network environment.

Bus 830 may be any of several types of bus structures including a memory unit bus or memory unit controller, a peripheral bus, an accelerated graphics port, a processing unit, or a local bus using any of a variety of bus architectures.

The electronic device 800 may also communicate with one or more external devices 700 (e.g., keyboard, pointing device, bluetooth device, etc.), with one or more devices that enable a user to interact with the electronic device 600, and/or with any device (e.g., router, modem, etc.) that enables the electronic device 800 to communicate with one or more other computing devices. Such communication may occur via an input/output (I/O) interface 650. Also, the electronic device 800 may communicate with one or more networks (e.g., a Local Area Network (LAN), a Wide Area Network (WAN), and/or a public network, such as the internet) via the network adapter 860. As shown, the network adapter 860 communicates with the other modules of the electronic device 800 via the bus 830. It should be appreciated that although not shown in the figures, other hardware and/or software modules may be used in conjunction with the electronic device 600, including but not limited to: microcode, device drivers, redundant processing units, external disk drive arrays, RAID systems, tape drives, and data backup storage systems, among others.

Through the above description of the embodiments, those skilled in the art will readily understand that the exemplary embodiments described herein may be implemented by software, and may also be implemented by software in combination with necessary hardware. Therefore, the technical solution according to the embodiments of the present disclosure may be embodied in the form of a software product, which may be stored in a non-volatile storage medium (which may be a CD-ROM, a usb disk, a removable hard disk, etc.) or on a network, and includes several instructions to enable a computing device (which may be a personal computer, a server, a terminal device, or a network device, etc.) to execute the method according to the embodiments of the present disclosure.

In an exemplary embodiment of the present disclosure, there is also provided a computer-readable storage medium having stored thereon a program product capable of implementing the above-described method of the present specification. In some possible embodiments, aspects of the invention may also be implemented in the form of a program product comprising program code means for causing a terminal device to carry out the steps according to various exemplary embodiments of the invention described in the above-mentioned "exemplary methods" section of the present description, when the program product is run on the terminal device.

The program product for implementing the above method according to the embodiment of the present invention may employ a portable compact disc read only memory (CD-ROM) and include program codes, and may be run on a terminal device, such as a personal computer. However, the program product of the present invention is not limited in this respect, and in this document, a readable storage medium may be any tangible medium that can contain, or store a program for use by or in connection with an instruction execution system, apparatus, or device.

The program product may employ any combination of one or more readable media. The readable medium may be a readable signal medium or a readable storage medium. A readable storage medium may be, for example, but not limited to, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, or device, or any combination of the foregoing. More specific examples (a non-exhaustive list) of the readable storage medium include: an electrical connection having one or more wires, a portable disk, a hard disk, a Random Access Memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or flash memory), an optical fiber, a portable compact disc read-only memory (CD-ROM), an optical storage device, a magnetic storage device, or any suitable combination of the foregoing.

A computer readable signal medium may include a propagated data signal with readable program code embodied therein, for example, in baseband or as part of a carrier wave. Such a propagated data signal may take many forms, including, but not limited to, electro-magnetic, optical, or any suitable combination thereof. A readable signal medium may also be any readable medium that is not a readable storage medium and that can communicate, propagate, or transport a program for use by or in connection with an instruction execution system, apparatus, or device.

Program code embodied on a readable medium may be transmitted using any appropriate medium, including but not limited to wireless, wireline, optical fiber cable, RF, etc., or any suitable combination of the foregoing.

Program code for carrying out operations for aspects of the present invention may be written in any combination of one or more programming languages, including an object oriented programming language such as Java, C + + or the like and conventional procedural programming languages, such as the "C" programming language or similar programming languages. The program code may execute entirely on the user's computing device, partly on the user's device, as a stand-alone software package, partly on the user's computing device and partly on a remote computing device, or entirely on the remote computing device or server. In the case of a remote computing device, the remote computing device may be connected to the user computing device through any kind of network, including a Local Area Network (LAN) or a Wide Area Network (WAN), or may be connected to an external computing device (e.g., through the internet using an internet service provider).

Sequence listing

<110> Shanghai kang Panien medical science and technology Co., ltd

<120> nucleic acid sample relative quantitative detection method, device, storage medium and electronic apparatus

<130> LZ2100560CN01

<160> 9

<170> SIPOSequenceListing 1.0

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<213> Artificial sequence

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aatgggcgga ggagagtagt ctgaattggg ttatgaggtc ccctgcgggg tacctcacct 60

cagccattga actcacttcg ctggccgtga gtctgttcca agctccggca aaggaggcat 120

ccgccgggcc cctccccgaa gggcggggtc cacggcatct cctgcccagt ctgacctcgc 180

gcggagcccc gttctctggg aactcacctc cccgaagctc agggagagcc ctgttagggc 240

cgcctctggc cctagtctca gaccttccca agggacatgg gagtggagtg acaggacgca 300

ctcagctcgt ggccccactg atgagcttcc ctccgcccta tgggaaaaag tcatccttcc 360

cccattctcg cctctctcac ctcctcagac ccgtccaaga agcagggacc atggctggag 420

gaagaagaag aagcctatgg atggatggac ttcggccgcc gcagtgctga ggatgagaac 480

taacaatcct agaaccaagc ttcagagcct agccacctcc caccccactc cagccctgtc 540

ccctgaaaaa ctgatcaaaa ataaactagt ttccagtgga tcaatggact gtgtcagtgt 600

tgtagggcag aggaggggga ctcatctggg ggtgaagttg tggcagggag aagagctgag 660

tgcctcttag gggcagggac cctggctgat tcttcttggg tcccccggcc aaaccttacg 720

atgggatccc agcccgggag atccctgacc tgctggaaaa gggggagcgg ctgccccagc 780

cccccatctg caccattgat gtctacatga tcatggtcaa atgtgcgtgg ctgagctgtg 840

ctggctgcct ggaggagggt gggaggtcct gggtggagga gcccacaagg ggcatgaaag 900

gggaccagga tgtatgtaga cccaggagcc ctagtatgtt aggagcctca aaaccttctt 960

gtatcccttt tacagtcaaa gtccaaagcc actcttgagg aacactcttg tacaaaatta 1020

agctgggcac agtggctcat gcctgtaatc ccagtacttt tggaggctga ggtgggagga 1080

tcccttgaag ccaggagttc aagaccagcc tgggcaacat agtgagatcc tatctctaca 1140

aaaaataaaa aaattatctg ggtgtggtgg tgtgtgccag tagtcccagc tactcaggag 1200

aggctgaggc aggaagatca cttgagccta gtttaaggtt gcagtaagct atgattgcac 1260

cactgaaatc cagcctgggt gacagagcga aacctcatct caaaaaaata aaaaagcaaa 1320

caaaaagaaa aaaaaaatta aaagggaaac tagaagagat gccaaaggtt ctggctgaag 1380

accccagagt ctggtgctac ttctctacca cctgagggct ttgggctgtc ccttgggact 1440

gtctagacca gactggaggg ggagtgggag gggagaggca gcaagcacac agggcctggg 1500

actagcatgc tgacctccct cctgccccag gttggatgat tgactctgaa tgtcggccaa 1560

gattccggga gttggtgtct gaattctccc gcatggccag ggacccccag cgctttgtgg 1620

tcatccaggt actgggcctc tgtgccccat ccctgcctgt ggctaagagc accctcctgc 1680

agagggtggg aaggagagat gagtccagta tgccaggccc ctcacggaag gctgcatgct 1740

gggctgggga ggggccacca tcctgcctct ccttcctcca cagaatgagg acttgggccc 1800

agccagtccc ttggacagca ccttctaccg ctcactgctg gaggacgatg acatggggga 1860

cctggtggat gctgaggagt atctggtacc ccagcagggc ttcttctgtc cagaccctgc 1920

cccgggcgct gggggcatgg tccaccacag gcaccgcagc tcatctacca gggtcagtgc 1980

cctcggtcac actgtgtggc tgtctgctta cctcccccaa ccccggtgga ctagggtccc 2040

tttctctgat gttccctcaa ctgtcacctc tcaaggaaac cccattatcc ctacaaaaaa 2100

ttcttactgc cttccaaccc ctgtgacccc attctctcca cggtgactgt gtcatacccc 2160

aaaggtgacc tctgtttttc tcctgtgacc ctgtcacctt ccatggagtc cccatcccag 2220

atccgtgagt gacccccatc atgactttct ttcttgtccc cagagtggcg gtggggacct 2280

gacactaggg ctggagccct ctgaagagga ggcccccagg tctccactgg caccctccga 2340

aggggctggc tccgatgtat ttgatggtga cctgggaatg ggggcagcca aggggctgca 2400

aagcctcccc acacatgacc ccagccctct acagcggtac agtgaggacc ccacagtacc 2460

cctgccctct gagactgatg gctacgttgc ccccctgacc tgcagccccc agcctggtat 2520

ggagtccagt ctaagcagag agactgatgg gcaggggagg tgggaccttc agcccagggt 2580

ccactgtggg ggcagaggga gtggcagaga caccggggtt ccttccccta atgggtcacc 2640

ttctcttgac ctttcagaat atgtgaacca gccagatgtt cggccccagc ccccttcgcc 2700

ccgagagggc cctctgcctg ctgcccgacc tgctggtgcc actctggaaa ggcccaagac 2760

tctctcccca gggaagaatg gggtcgtcaa agacgt 2796

<210> 2

<211> 2090

<212> DNA

<213> Artificial sequence

<400> 2

ggccaaacct tacgatggga tcccagcccg ggagatccct gacctgctgg aaaaggggga 60

gcggctgccc cagcccccca tctgcaccat tgatgtctac atgatcatgg tcaaatgtgc 120

gtggctgagc tgtgctggct gcctggagga gggtgggagg tcctgggtgg aggagcccac 180

aaggggcatg aaaggggacc aggatgtatg tagacccagg agccctagta tgttaggagc 240

ctcaaaacct tcttgtatcc cttttacagt caaagtccaa agccactctt gaggaacact 300

cttgtacaaa attaagctgg gcacagtggc tcatgcctgt aatcccagta cttttggagg 360

ctgaggtggg aggatccctt gaagccagga gttcaagacc agcctgggca acatagtgag 420

atcctatctc tacaaaaaat aaaaaaatta tctgggtgtg gtggtgtgtg ccagtagtcc 480

cagctactca ggagaggctg aggcaggaag atcacttgag cctagtttaa ggttgcagta 540

agctatgatt gcaccactga aatccagcct gggtgacaga gcgaaacctc atctcaaaaa 600

aataaaaaag caaacaaaaa gaaaaaaaaa attaaaaggg aaactagaag agatgccaaa 660

ggttctggct gaagacccca gagtctggtg ctacttctct accacctgag ggctttgggc 720

tgtcccttgg gactgtctag accagactgg agggggagtg ggaggggaga ggcagcaagc 780

acacagggcc tgggactagc atgctgacct ccctcctgcc ccaggttgga tgattgactc 840

tgaatgtcgg ccaagattcc gggagttggt gtctgaattc tcccgcatgg ccagggaccc 900

ccagcgcttt gtggtcatcc aggtactggg cctctgtgcc ccatccctgc ctgtggctaa 960

gagcaccctc ctgcagaggg tgggaaggag agatgagtcc agtatgccag gcccctcacg 1020

gaaggctgca tgctgggctg gggaggggcc accatcctgc ctctccttcc tccacagaat 1080

gaggacttgg gcccagccag tcccttggac agcaccttct accgctcact gctggaggac 1140

gatgacatgg gggacctggt ggatgctgag gagtatctgg taccccagca gggcttcttc 1200

tgtccagacc ctgccccggg cgctgggggc atggtccacc acaggcaccg cagctcatct 1260

accagggtca gtgccctcgg tcacactgtg tggctgtctg cttacctccc ccaaccccgg 1320

tggactaggg tccctttctc tgatgttccc tcaactgtca cctctcaagg aaaccccatt 1380

atccctacaa aaaattctta ctgccttcca acccctgtga ccccattctc tccacggtga 1440

ctgtgtcata ccccaaaggt gacctctgtt tttctcctgt gaccctgtca ccttccatgg 1500

agtccccatc ccagatccgt gagtgacccc catcatgact ttctttcttg tccccagagt 1560

ggcggtgggg acctgacact agggctggag ccctctgaag aggaggcccc caggtctcca 1620

ctggcaccct ccgaaggggc tggctccgat gtatttgatg gtgacctggg aatgggggca 1680

gccaaggggc tgcaaagcct ccccacacat gaccccagcc ctctacagcg gtacagtgag 1740

gaccccacag tacccctgcc ctctgagact gatggctacg ttgcccccct gacctgcagc 1800

ccccagcctg gtatggagtc cagtctaagc agagagactg atgggcaggg gaggtgggac 1860

cttcagccca gggtccactg tgggggcaga gggagtggca gagacaccgg ggttccttcc 1920

cctaatgggt caccttctct tgacctttca gaatatgtga accagccaga tgttcggccc 1980

cagccccctt cgccccgaga gggccctctg cctgctgccc gacctgctgg tgccactctg 2040

gaaaggccca agactctctc cccagggaag aatggggtcg tcaaagacgt 2090

<210> 3

<211> 351

<212> DNA

<213> Artificial sequence

<400> 3

aatgggcgga ggagagtagt ctgaattggg ttatgaggtc ccctgcgggg tacctcacct 60

cagccattga actcacttcg ctggccgtga gtctgttcca agctccggca aaggaggcat 120

ccgccgggcc cctccccgaa gggcggggtc cacggcatct cctgcccagt ctgacctcgc 180

gcggagcccc gttctctggg aactcacctc cccgaagctc agggagagcc ctgttagggc 240

cgcctctggc cctagtctca gaccttccca agggacatgg gagtggagtg acaggacgca 300

ctcagctcgt ggccccactg atgagcttcc ctccgcccta tgggaaaaag t 351

<210> 4

<211> 22

<212> DNA

<213> Artificial sequence

<400> 4

ctaagcagag agactgatgg gc 22

<210> 5

<211> 21

<212> DNA

<213> Artificial sequence

<400> 5

aggtgaccca ttaggggaag g 21

<210> 6

<211> 22

<212> DNA

<213> Artificial sequence

<400> 6

agcccagggt ccactgtggg gg 22

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence

<400> 7

cgttctctgg gaactcacct 20

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence

<400> 8

tcctgtcact ccactcccat 20

<210> 9

<211> 23

<212> DNA

<213> Artificial sequence

<400> 9

agccctgtta gggccgcctc tgg 23

Claims

1. A method for the relative quantitative detection of a nucleic acid sample, comprising:

respectively carrying out dual-channel cyclic amplification on dual-target plasmid standard products with different gradients and nucleic acid samples to be detected by using a probe method, wherein the standard products comprise reference genes and target genes in a ratio of 1:1, detecting the ratio of a reference gene contained in the nucleic acid sample to be detected to a target gene;

respectively obtaining the normalized fluorescence values of each cycle of amplification of the standard substance and the nucleic acid sample to be detected with different gradients;

and determining the relative proportion of the target gene and the reference gene in the nucleic acid sample to be detected according to the newly increased fluorescence ratio of the nucleic acid sample to be detected in the second amplification round with stable amplification efficiency, the amplification efficiency ratio and the luminous coefficient ratio.

2. The method for relatively quantitatively detecting a nucleic acid sample according to claim 1, wherein the probe method is a hydrolysis-specific probe method.

3. The method for the relative quantitative detection of nucleic acid samples according to claim 1, wherein the determining the amplification efficiency ratio of the target gene channel and the reference gene channel based on the fluorescence increase curves of the standards with different gradients comprises:

obtaining fluorescence growth curves of different gradients of the standard according to the normalized fluorescence value of the standard;

respectively determining the translation distance delta X of the coincidence of the fluorescence growth curves of the standard substance between the respective gradients in the target gene channel and the reference gene channel _T And Δ X _R ；

4. The method for relatively quantitatively detecting a nucleic acid sample according to claim 1, wherein the determining the amplification efficiency ratio of the target gene channel and the reference gene channel based on the fluorescence growth curves of the standards with different gradients comprises:

setting a threshold value within the range of Xmax, and acquiring CT values CT of the standard substance with different gradients in a target gene channel and a reference gene channel _T ，CT _R ；

5. The method for relatively quantitatively detecting nucleic acid samples according to claim 3 or 4, wherein the determining the ratio of the luminescence coefficients of the target gene channel and the reference gene channel based on the newly added fluorescence ratio of the standard in the first amplification round in which the amplification efficiency is stable and the amplification efficiency ratio of the standard in the target gene channel and the reference gene channel comprises:

x at the target gene channel and the reference gene channel of the standard _max Calculating the newly increased fluorescence ratio F of the target gene channel and the reference gene channel in the maximum round X in the range _XT /F _XR Wherein Xmax is a turn corresponding to the turn with the maximum change in fluorescence growth efficiency, and the first amplification turn with stable amplification efficiency is the maximum turn X;

according to formula F _XT /F _XR ＝(N _T /N _R )*(F _ST /F _SR )*(E _T /E _R ) ^X+1 N in the Standard substance _T ＝N _R And the ratio of the amplification efficiencies of the target gene channel and the reference gene channel, determining the ratio of the luminescence coefficients of the target gene channel and the reference gene channel _ST /F _SR 。

6. The method for the relative quantitative determination of nucleic acid samples according to claim 1, wherein the obtaining of the normalized fluorescence values for each cycle of amplification of the standard and the test nucleic acid samples at different gradients comprises:

for each gradient of the standard or the test nucleic acid sample:

obtaining the fluorescence value f of the nucleic acid sample in each cycle of amplification ₁ ，f ₂ ，f ₃ …f _x Wherein f is _x As the fluorescence value f detected in the x-th round of amplification _x ＝L*C ₀ *f _b +L*C ₀ *f _s *k*x+L*f _s *N ₀ *(E ^x -1) the new fluorescence value F associated with this wheel _X ＝L*C ₀ *f _s *k+L*f _s *N ₀ *(E ^x -E ^X-1 ) Wherein L is the intensity of the excitation light, f _b Is the background fluorescence luminescence coefficient, C, of the probe molecule ₀ K is the efficiency of hydrolysis of free probe in each amplification run, f is the initial number of probe molecules _s Is a singleThe luminescence coefficient of the fluorescent group, x is the reaction run, N ₀ Is the initial copy number of the nucleic acid and E is the qPCR amplification efficiency;

F _X ＝f _x -f _x-1 ＝N ₀ *(E ^x -E ^x-1 )*F _S -K

7. A relative quantitative detection apparatus for a nucleic acid sample, comprising:

the cyclic amplification equipment is used for respectively carrying out dual-channel cyclic amplification on dual-target plasmid standard products with different gradients and nucleic acid samples to be detected by using a probe method, wherein the standard products comprise reference genes and target genes in a ratio of 1:1, the nucleic acid sample to be detected comprises a reference gene and a target gene;

fluorescence value acquisition equipment for respectively acquiring normalized fluorescence values of each cycle of amplification of the standard substance and the nucleic acid sample to be detected with different gradients;

the amplification efficiency ratio acquisition module is used for determining the amplification efficiency ratio of a target gene channel and a reference gene channel based on the fluorescence growth curves of the standard substances with different gradients; wherein the fluorescence growth curve is obtained from the normalized fluorescence values;

a luminescence coefficient ratio obtaining module, configured to determine a luminescence coefficient ratio between a target gene channel and a reference gene channel according to a newly added fluorescence ratio of the standard in a first amplification round in which amplification efficiency is stable and an amplification efficiency ratio of the standard in the target gene channel and the reference gene channel;

and the relative proportion obtaining module is used for determining the relative proportion of the target gene and the reference gene in the nucleic acid sample to be detected according to the newly increased fluorescence ratio of the nucleic acid sample to be detected in the second amplification round with stable amplification efficiency, the amplification efficiency ratio and the luminous coefficient ratio.

8. The relative quantitative nucleic acid sample detection device according to claim 7, wherein the probe method is a hydrolysis-specific probe method.

9. A computer device comprising a memory and a processor, the memory storing a computer program, wherein the processor implements the method of any one of claims 1 to 6 when executing the computer program.

10. A computer-readable storage medium, on which a computer program is stored, which, when being executed by a processor, carries out the method of any one of claims 1 to 6.