CN106834441B

CN106834441B - Digital quantitative PCR method for same-sequence double amplification

Info

Publication number: CN106834441B
Application number: CN201611243465.5A
Authority: CN
Inventors: 江洪; 曲越; 曲超琪
Original assignee: Macao Emperor Digital Gene Co Ltd
Current assignee: Macao Emperor Digital Gene Co Ltd
Priority date: 2016-12-28
Filing date: 2016-12-28
Publication date: 2020-02-28
Anticipated expiration: 2036-12-28
Also published as: CN106834441A

Abstract

The invention discloses a digital quantitative PCR method of homosequence double amplification, which is a quantitative PCR method of a multiple-ratio dilution sample and is characterized in that 3' end is firstly used for shortening 3-6b basic groupsThe short target primer of (2) is pre-amplified for 10-15 cycles and pre-amplified for 10²‑10⁴And diluting the pre-amplification solution by 10-20 times by using the long target primer PCR reaction solution with the same sequence in the middle part in the same tube, then performing fluorescence PCR for 30-35 cycles, performing non-heat-cover amplification under the sealing of mineral oil, wherein the tail ends of the pre-amplification short primers are 5-8b in the same sequence and are homologous with the long target primer with the same sequence in the middle part, so that the primers are not easy to polymerize, and the diluted short primers have no non-specificity in the 35 cycles, so as to ensure that the molecules of more than or equal to 1 are effectively detected and the molecules of 0 are not subjected to amplification reaction.

Description

Digital quantitative PCR method for same-sequence double amplification

Technical Field

The invention belongs to the technical field of nucleic acid amplification PCR (polymerase chain reaction) of molecular biology and molecular inspection, and particularly relates to the field of digital PCR (polymerase chain reaction) for improving sensitivity by pre-amplifying a same primer with a slightly short tail end and limiting non-specificity of a part of same-sequence primer so as to dilute and quantify.

Background

The quantitative detection method comprises a standard curve quantitative method, an internal standard quantitative method and a dilution quantitative concept method. The dilution quantification is a quantitative digitization method for diluting target molecules by a sample multiple ratio until no target molecule reaction occurs and then measuring the dilution ratio, or digital quantification detection or digital diagnosis is a method for diluting and dispersing the sample to a large number of micro separation units under the condition that a positive single molecule or more than one molecule is ensured to have a detection signal and is determined as 1, and a negative absolute no reaction signal and is determined as 0, so that each detection unit theoretically contains 0-1 target molecules, and the target molecules are quantified by counting the detection unit reaction signals by 0-1 and the limiting dilution ratio, thereby facilitating the automatic operation of a computer 0 or 1 mode, but the premise is that the detection method can reliably distinguish 1 molecule or 0 molecule. However, the conventional chemical reaction and enzyme immune reaction are low-sensitivity detection methods in which the detection signal intensity is linearly proportional to the content of the molecule to be detected and the signals are simply added, for example, a typical reaction formula of a target molecule A and a reagent molecule B are added to form a product C, the amount of the target molecule A can be measured by adding the amount of the reagent B consumed or the amount of the molecule C generated, but the sensitivity, i.e., the minimum detection amount, is usually greater than the nanogram level (ng/ml), for example, 1 nanogram of water molecules is about 3 × 10¹³The number of molecules can be seen that the target molecules which are lower than the sensitivity range of the detection method can not be detected and are easy to have false negative reaction, and the traditional low-sensitivity method can not distinguish the difference between 1 target molecule and 0 molecule, so that the method can not be suitable for limited rare target moleculesThe digital quantitative detection method of the release method.

PCR technology for exponential amplification of nucleic acid produced in the last 80 th century, and 2 thereofⁿ(n is the number of amplification cycles) the amplification factor can detect target molecules as low as tens or even several copies, however, the sensitivity of 30 cycles of amplification is still not enough to detect the difference between 1 target molecule and 0 molecule, and over 30 cycles of amplification brings serious nonspecific and false positive; and such first generation final/end point PCR has non-uniform final yield due to too large variation of the same amount of sample after amplification and super amplification, which makes it difficult to monitor the amount of product for quantitative detection. The subsequent two rounds of amplification nested PCR application (Porter-Jordan K., et al, 1990, J.MedVirol30 (2): 85-91) with over 40 total cycles of amplification of extremely high sensitivity for single target molecule detection provides possibility, pre-amplification of outer primer dimer product can not become two rounds of amplification template, but different sequence of outer primer greatly increased system primer polymerization between nonspecific, need pre-amplification of post-gel electrophoresis purification to eliminate outer primer nonspecific; in 1992, Sykes et al (Sykes P.J., et al, 1992, BioTechniques 13: 444-. Subsequently, Higuchi develops a new way, and the real-time fluorescence PCR method of simultaneous amplification and "closed-tube" fluorescence detection reduces cross contamination of post-PCR treatment, while the relative quantification of the number of initial templates is achieved by exponential phase determination of the amplification curve, i.e. the number of amplification cycles in the exponential phase is inversely related to the number of initial templates, but the real-time fluorescence PCR using a fluorescent dye still produces non-specific amplification of primer dimers at 30 amplification cycles, thereby interfering with the quantification of low-concentration target molecules. In 1997, real-time fluorescent PCR (Wittwer C., et al, 1997, BioTechniques 22: 130-138) with a series of probe methods that do not hybridize with non-specific amplification significantly reduced non-specific reactions, especially with hydrolysis probe Taqman method, developed gradually and widely used in clinical tests, and has been recognized as a second generation q-PCR quantitative PCR technology. But require quantitative referenceCalibration of the standard curve or internal standard with reference and limited sensitivity is still insufficient to distinguish between 1 target molecule and 0 molecule and still has some limitations for digital quantification applications.

Another non-specific reason for limiting digital PCR is numerous, and most studies focus on hot start of PCR to overcome possible non-specific amplification due to low temperature primer binding, and measures taken include wax-coated Mg²⁺The ionic heat release and polymerase Taq modification inhibition comprise KlenaTaq with N-terminal deletion, an anti-Taq enzyme antibody, Taq enzyme inhibition oligonucleotide Aptamar, a pentane tetroxide heat-activated primer and other heat-starting methods. However, the amplification of one cycle is doubled/once at most by cold start, which is far from the nonspecific exponential amplification. And the Ct value of the absolute hot start background of the PCR added with complete components manually after thermal denaturation is only deducted by 1-3 cycles, the activity of Taq enzyme is very low when the temperature is lower than 40 ℃, and the low-temperature start is not a key reason for nonspecific formation of PD.

Other researches for inhibiting the non-specificity of the PCR have few reports, and various measures for controlling the non-specificity, such as changing PCR components, adding various chemical reagents and the like, are tried, so that the specific amplification and the non-specific amplification are basically parallel, the non-specificity of the PCR is obviously inhibited, the target specific amplification efficiency is also interfered to different degrees, and the non-specificity of the PCR is difficult to inhibit without influencing the target amplification efficiency. The non-specificity of the index caused by the primer is also caused by the base sequence of the primer, and a pair of completely identical primers has no non-specificity of the primer at all. The Hands technology (Homo-Tag assisted non-dimer system, Brown ie J., et al, 1997, Nucleic acids Ser. No. Vol.25, No 16: 3235-3241) adopts a completely homologous primer Tag to significantly inhibit PD non-specificity and not to selectively reduce the target-specific amplification efficiency by self-binding of the two ends of the primer dimer single strand to the competitive free primer. The Single-stranded Binding protein Single Strand Binding-protein (SSB), the gene 32 protein and the full primer Binding antisense base Oligo can obviously reduce and optimize the non-specificity of the primer, but seriously affect the target specific amplification efficiency and the linear relation of the amplification curve. Chinese patents (CN 201010105371.8 and PCT/CN2013/088054) adopt partial same-sequence primers to partially inhibit PD amplification according to the fact that a pair of completely same-sequence primers has no non-specificity phenomenon, and the ' same sequence ' is arranged at the middle part of the primers and is close to the 3 ' end, so that PD amplification can be selectively inhibited to the maximum extent without influencing the target amplification efficiency; the addition of Oligo containing an antisense base, which binds to the middle part of the primer at the position corresponding to the central part of the primer, in PCR enables further specific amplification of the effect, since it retains only the binding function and does not have the effect of the template and the primer. Based on the design principle of a non-terminal reverse complementary conventional primer, a middle part of homologous primer pairs and an Oligo combined with a middle antisense base are selected to have no interference of PD non-specific amplification in 40 cycles of real-time fluorescent PCR.

1999, Vogelstein&Kinzler reports oncogenic mutant gene ras quantitative PCR and a digital PCR concept (Vogelstein B.; Kinzler K.W.1999, PNAS, USA 96: 9236-. After PCR thermocycling reaction, the amplified product hybridizes with the added fluorescent probe, the reaction unit with one nucleic acid molecular template has amplification and gives a fluorescent signal, and the reaction unit without template has no amplification and no fluorescent signal. The nucleic acid concentration of the original solution can be calculated according to the dilution ratio and the volume of the reaction unit, and absolute quantification of the initial DNA template can be realized through counting and Poisson distribution statistics. The key to realize digital PCR is that the fluorescent probe specifically hybridizes to ensure that no (0) template reaction unit has no fluorescent signal; secondly, the reaction units of the dPCR apparatus are sufficiently large to ensure that the detection unit does not exceed one template, so the reaction units are further miniaturized to the maturity of microfluidic million-level reaction units, namely nanoliter volume reaction apparatuses, and recently micro-dripping million-level reaction units, namely picoliter volume apparatuses (hindson b.j., et al., 2011, anal. chem.83: 8604-. The concentration is less than 10% lower due to the very large span of the target molecule content in the sample⁰1copy/ml, high concentration greater than 10¹⁰copies/ml, concentrated specimens often require millions of reaction cells, i.e., pico-liter volume microfluidic or microdroplet devices, to be able to dispense each reaction cellThe number of nucleic acid templates (2) is less than or equal to one, which results in limited application due to the extreme pursuit of reaction units. Even for some over-concentrated specimens, pre-dilution of 1 to several orders of magnitude is necessary before million reaction unit d-PCR can be used.

The conventional PCR and the real-time fluorescence PCR still have slightly insufficient sensitivity to single-molecule detection, and pre-amplification is needed to further improve the system sensitivity; however, PCR is limited to the detection of low concentrations of non-specific sample, and efficient detection of single molecules ultimately depends on the avoidance or elimination of non-specificity.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a digital quantitative PCR method for same-sequence double amplification.

In order to achieve the purpose, the invention adopts the following technical scheme:

according to a first aspect of the present invention, there is provided a digital quantitative PCR method for double amplification in the same sequence, which performs double amplification PCR for a dilution sample with a multiple ratio, comprising the steps of: the method comprises the steps of pre-amplifying by using a short target primer with 3' end shortened by 3-6b base in a small-volume PCR reaction solution for 10-15 cycles to increase sensitivity, then diluting pre-amplification solution by using middle homosequence target primer PCR solution with non-shortened end in the same tube for 10-20 times of large-volume fluorescent PCR30-35 cycles, performing non-hot-cover amplification under a mineral oil closed condition, ensuring that the molecule is not less than 1 to be effectively detected and the template is not 0 to be subjected to non-specific reaction by using homosequence double-amplification PCR with total reaction more than 40 cycles, and calculating target molecule quantification according to the times and volumes of sample diluted to zero target molecule.

In one embodiment of the invention, the sample is diluted by multiple times and subjected to two-round PCR double amplification, and the first round is to pre-amplify the 14-18base short primer with shortened 3' end of the target primer by 2-5 mul PCR solution for 12-14 thermal cycles to pre-amplify the sample by 1-2 x 10²-10⁴Adding 17-24base long target fluorescent PCR reaction solution with the same sequence as the middle part 5-8b to dilute the pre-amplification reaction solution by 10-20 times, performing 20-100 mul second round fluorescent PCR for 30-32 thermal cycles, performing non-thermal cover amplification under the condition of mineral oil sealing, and ensuring that single target molecules and more than single target molecules are detected to be '1'; background '0' has no non-specific reaction; according to the sampleThe digital PCR method for dilution quantitative detection of sample target content is calculated by the times and volumes of dilution to zero target molecules.

In another embodiment of the present invention, a set of simple digital quantitative PCR samples are diluted in gradient, the samples are diluted in gradient in multiple ratios in EP tube, and the samples are taken x 10⁰～×10^-5Beginning 10X (times) dilution for 5 times and then 2X (equal ratio) dilution for 5 gradients, ensuring that the turning point of a target molecule tube is diluted until no target molecule tube falls in the gradient range, adding 0.5-1 mu L of 5X pre-amplification short primer PCR reaction solution, 2-5 mu L diluted sample and 30-50 mu L mineral oil into a PCR tube, sealing, and carrying out 10-5 thermal cycle first-round PCR non-calotte amplification of annealing 14-17base short primers at 45-50 ℃; 18 to 45/95 μ L of 1 Xlong primer PCR reaction solution was added to the same tube in the first round, inserted under the layer of mineral oil with the sample application tip and added, and subjected to 30 to 35 thermal cycles of annealing at 54 to 60 ℃ for 17 to 24base long primers, and thermal cap-free amplification in the second round of PCR.

In another embodiment of the invention, after 10 times of serial dilution of 9 or more steps, the dilution gradient of each step is equally distributed to a limited number of reaction holes/reaction units, two-round double-amplification PCR statistical quantification of a 96-pore plate and a 384-pore plate is carried out, 9 groups of gradient repeated PCR of 10 times of dilution of the sample in advance are adopted, the theoretical number of target molecules of one group of 10 PCR is between 0 and 1, the effect equivalent to that of a ten-thousand or even million-level reaction unit microfluidic or micro-droplet device can be obtained, and 100 times of dilution or division of two levels is added on the basis; for example, the second round of 1 XPCR reaction solution can be added with 1 XPYRBR Green I fluorescent dye for carrying out the fluorescent PCR detection in the second round, or after the second round of PCR reaction, each hole is added with 2-5 μ L of EB (0.5 μ g/mL) below the mineral oil layer, and the mixture is placed in a 96-hole plate for color counting under an ultraviolet lamp or detection by a fluorescent photometer.

In another embodiment of the present invention, after 10 times of serial dilution of 9 steps is performed on the sample to be tested, the dilution gradient of each step is reduced to reduce the 10 times of volume and equally distribute to 10000 reaction unit polydimethylsiloxane microporous chips, and an in situ PCR instrument is used for two-round nested PCR digital quantification.

According to another aspect of the inventionThe invention provides a gene detection kit for the digital quantitative PCR method of the same-sequence double amplification, which comprises the following components: nucleic acid extraction reagent, dNTPs and dUTP, UDG enzyme, Taq, HKTAQ enzyme and buffer solution thereof, pre-amplification short primer F/R, long target primer F/R, dye EB and SYBR Green I, and purified water dH₂O, mineral oil.

According to the fact that the completely homologous primers completely have no primer dimer non-specificity phenomenon, the partially homologous primer pairs can partially reduce the amplification degree of the primer dimer PD; similarly, a slightly shorter preamplification primer with the target primer can reduce the degree of non-specific amplification of polymerization between primers due to homology or "identity" with the target primer. The inner side or the second round of PCR of the nested or two-round amplification PCR increases the system complexity due to the residual first round or outer side primer, thereby increasing the nonspecific property of the two-round PCR, and the nonspecific amplification of the background Ct value of the general conventional primer for 30 cycles is promoted to the background nonspecific amplification before the Ct value is less than 25 cycles; the increased non-specificity of nested or two rounds of PCR negates the amplification of the first round or outer primer preamplification. The ' same sequence ' of partial same sequence primer is arranged in the middle and 3 ' end of the primer pair to reduce the amplification of primer dimer PD to the maximum extent, the background Ct value of a pair of middle 6-8b same sequence target primer is usually pushed to be more than 36 cycles later or close to the Ct value for 40 cycles; and then, the tail segment ' same sequence ' short primer pre-amplification strategy which is the same as the target primer and only shortens 3-6b from the 3 ' end is combined, so that the diluted pre-amplified short primer can eliminate the polymerization non-specific amplification among the primers to the maximum extent due to the ' same sequence ', the absolute non-polymerization non-specific amplification among the primers in 30-35 thermal cycle reactions in the second round of double-amplification PCR is ensured, and the non-specificity of a non-target template ' 0 ' reaction is improved compared with the nested double-amplification PCR without gel electrophoresis.

One pair of primers has high enough specificity, the PCR specificity is directly derived from the specificity of the primers, the primer at one end is not specifically mismatched at all, even the completely paired amplification is only linear amplification, and naturally, the exponential amplification or geometric series amplification cannot be achieved. Any point of primer specificity is reduced or does not match with the target one, so that the amplification rate falls down and is far from catching up with specific exponential or geometric series amplification; conversely, exponential amplification also gives a high degree of specificity to PCR, provided that it is sufficiently specific to amplify exponentially or geometrically. On the basis of high enough specificity, a pair of nested PCR primers is added without help, and a plurality of pairs of primers with different sequences increase the non-specificity of polymerization between the primers. In cases where the 3 'end shortened preamplified short primer non-specific extension products, including short primer dimers, are not suitable templates for long target primers (most importantly 3' end unpaired), two rounds of double amplification with the same sequence primer are both sufficiently specific and minimally non-specific between primers.

Therefore, in order to overcome the nonspecific barrier of the background reaction of the existing digital PCR '0' and the limitation of the number of reaction units of the microfluidic or micro-droplet device. The invention relates to a digital quantitative PCR method of homosequence double amplification, which carries out two rounds of PCR double amplification on a series of samples diluted by times, but the conventional nested outer primers are replaced by short primers with the 3' tail ends of target primers shortened, and the first round of pre-amplification is carried out for 10-15 cycles to pre-amplify 10²-10⁴Adding a part of target primer fluorescent PCR reaction solution with the same sequence to dilute the pre-amplification reaction solution by 10 times or 20 times to perform 30-35 cycles of second round of fluorescent PCR, and ensuring that more than one single target molecule can be detected to be 1; the shortened target primer is not easy to polymerize and amplify due to the same sequence as the long target primer, so that the polymerization non-specific amplification among the primers is absolutely not promoted after the shortened target primer is diluted by 10-20 times, and meanwhile, in order that the fluorescence PCR reaction of the target primer does not have non-specific background amplification in 35 cycles, the target primer adopts a middle same sequence strategy, namely a short primer end same sequence strategy, so that the background of 0 is ensured to have no non-specific reaction; the absolute content of the target molecules in the sample can be calculated according to the times and volumes of the target molecules diluted by the sample to zero. In the two-round amplification PCR, the first round and the final round of PCR have nonuniform yield due to overlarge change of samples with the same amount after PCR amplification, so that the real-time fluorescence PCR quantification of the second round of PCR is difficult. The hypersensitive two-round double-amplification PCR is more suitable for a digital quantitative PCR method of gradient dilution; however, two rounds of operation are not conducive to the use of a superunit microencapsulated/microdroplet digital PCR device, and therefore, manual double-dilution quantitative PCR is preferred.

Firstly, a group of simple digital quantitative PCR of sample gradient dilution is carried out, the sample is subjected to multiple ratio gradient dilution in an EP tube, and the target is ensured to be keptThe turning point of the molecular tube is in the gradient range from the diluted molecular tube to the non-target molecular tube. Therefore, the samples with lower concentration were judged to be diluted 10 × (times) for 5 gradients and then 2 × (equal ratios) for 5 times; and for most biological samples, the sample is multiplied by 10^-3The assay was performed by starting a 10 × (fold) dilution 5 times and then 2 × (equal ratio) dilutions 5 gradients or one gradient apart. The traditional two-round PCR operation mode is improved, a short 14-18base first round pre-amplification primer and a long 17-24base second round target primer are designed, the annealing temperature of the target primers is 5-10 ℃ higher than that of the pre-amplification primers, two rounds of PCR reaction are carried out in a PCR tube, 0.5-1 mu L of short primer 5 XPCR reaction solution is firstly added into each tube of the first round PCR, the bottom tip of the tube is added with 2-5 mu L of diluted sample solution, 30-50 mu L of mineral oil layer is gently added along the tube wall to seal, and 10-15 thermal cycle amplifications of annealing at 45-50 ℃ are carried out; in the second round of PCR, 18. mu.L to 45/95. mu.L of 1 Xlong primer PCR reaction solution was added to the same well of the previous round, and carefully added by inserting the tip of a sample gun under the layer of mineral oil, and 30 to 35 thermal cycles of annealing at 54 ℃ to 60 ℃ were carried out. The slightly low temperature annealing amplification of the first round of PCR short primers will increase some single primers and non-target template linear amplification, but will not focus to generate exponential non-specific amplification sufficient for fluorescence detection; subsequent high temperature annealing amplification of the longer target primers in the second round of PCR will further exclude these small amounts of non-target amplified DNA, and the specificity is still better than that of the normal PCR. The most fundamental advantage of this improved dual amplification PCR protocol is the elimination of primer dimer non-specific amplification, and conventional primer pairs often must be greater than 4. mu.M concentration and more than 30 thermal cycles of amplification to produce detectable primer dimer amplification. The short primer can not generate detectable primer dimer in 10-15 thermal cycles of the first round, the short primer is diluted by 10-20 times after entering the second round of PCR, the concentration is only less than 0.5 mu M, the Tm value of the short primer is lower than the annealing temperature of the second round of PCR by more than 10 ℃, and the amplification of the primer dimer can not be detected from the beginning to the end of the two rounds of PCR by the short primer under the limit of the triple action of the diluted primer concentration, the low Tm value and the homology of the short primer and the long target primer; while the middle homologous target primer pair at 5. mu.M concentration was only involved in 30-35 thermal cycles of the second round of PCR and was generally not able to produce detectable primer dimers. And the cross contamination of aerosol outside the PCR system is isolated by matching with closed mineral oil.

Secondly, carrying out statistical quantification on a double-amplification digital PCR 96 pore plate, and firstly carrying out 10-time continuous dilution of 9 steps or adding several steps on a sample (I) to be detected with unknown concentration: taking 9 plastic EP tubes of 1.5mL, adding a certain microliter of purified water into each tube, adding 1/10 sample stock solution to be measured into the water of the 1 st tube, and gently mixing by using a suction head to obtain stock solution multiplied by 10^-1Twice, 1/10 volumes of 10 times diluted liquid were taken from the 1 st tube, added to the 2 nd tube water, and lightly mixed to original liquid × 10^-2Double, changing one suction head per tube, stock solution x 10^-3... the original solution is multiplied by 10^-9And (II) adding the first round of 5 × PCR reaction solution and mineral oil: taking a sharp-bottomed 96-well PCR reaction plate (8X 12 wells), adding 0.5/1. mu.L of 5X PCR reaction solution containing short primers into each well, and (III) diluting the sample by doubling the ratio: adding multiple gradient diluent below the mineral oil layer to each group of holes of the PCR plate corresponding to each EP tube diluent sample to generate a first round of PCR mixed solution, A_1-10Adding 2-5 mul/hole stock solution x 10^-9Multiple gradient dilution in mineral oil at the bottom of the well, B_1-10Adding 2-5 mul/hole stock solution x 10^-8Duplicate gradient dilution, C..... H, etc., 2-5. mu.L/well stock solution X10 added to the remaining A-E11-12 wells^-1Adding two-hole positive control, two-hole negative control and two-hole system background control to the double-gradient diluent, and adding 30-50 microliter of mineral oil to each hole of a 96-hole PCR plate for 10-15 first-round PCR pre-amplification of denaturation at 94 ℃ for 20 seconds, annealing at 45-50 ℃ for 60 seconds and extension at 72 ℃ for 30 seconds; (V) adding 18. mu.L-45/95. mu.L/well of 1 XPCR reaction solution containing long target primers to a 96-well plate, inserting the reaction solution under a mineral oil layer with a sample application tip, and carefully adding the reaction solution, and performing a second round of PCR amplification of 30-35 thermal cycles of denaturation at 94 ℃ for 20 seconds, annealing at 54 ℃ -60 ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds. Two rounds of amplification, adding up to more than 40 cycles, provide sensitivity to the detection of a single molecule; the detectable non-specific amplification interference of polymers among primers can not be generated in 30-35 cycles of long primer amplification after the first cycle of short amplification dilution is less than 15 cycles; then, 9 sets of repeated PCR samples diluted by 10 times are used, and the theoretical number of target molecules of 10 PCR sets is 0-1. Improved double amplification PCR, such as adding 1 XSSYBR Green I fluorescent dye to the second round of 1 XPCR reactionPerforming fluorescent PCR detection in the second round; or adding 2-5 μ L EB (0.5 μ g/mL) into each well after the second round of PCR reaction, placing the mixture under the mineral oil layer, and placing the mixture in a 96-well plate to perform color counting under an ultraviolet lamp or detecting the mixture by a fluorescence photometer; or after PCR reaction, red and green molecular beacon probes with different wavelengths aiming at wild and mutant genes are added into each hole, so that mutation quantification can be realized under a high wild background. And (4) interpretation of results: a set of 10 PCRs with 7 or more than 7 amplification fluorescence reactions is defined as 1 target molecule/well sample, a set of 10 PCRs with 3 and less than 3 amplification fluorescence reactions is defined as 0 target molecule/well L sample, a set of 10 PCRs with 4-6 amplification fluorescence reactions is defined as 0.5 target molecule/well sample, and the absolute number of initial target template molecules, plus or minus 5-10 molecules, is calculated by multiplying the sample dilution times by the 1 target molecule set or 0.5 target molecule set.

If further more accurate quantitative detection is required, based on the above d-PCR, the sample is diluted to about 0.5 target molecules/well at a time, an equal amount of 2 × first round PCR reaction solution is added and dispersed to the whole 96-well plate, two rounds of modified double amplification d-PCR are performed, the result data is counted in Poisson distribution, and the absolute number of the initial target template molecules is +/-1-2 molecules.

Pre-testing of the impact of pre-amplification on the background of fluorescence target PCR:

the pre-amplification mainly influences the background non-specific amplification of the second round PCR target primer through the concentration of the pre-amplification primer, the number of pre-amplification cycles, the PD background of the pre-amplification primer dimer and whether the pre-amplification primer dimer and the second round PCR target primer are in same sequence interference. If the pre-amplification primers are removed by gel electrophoresis of the pre-amplification products in nested and double-amplification PCR, or the pre-amplification primers and the products are diluted by more than 20 times, the background of the second round PCR target primers is not influenced by the pre-amplification, and the background is the system background. However, if the pre-amplification reaction is not diluted or removed enough, the number of pre-amplification cycles is too large, so that the number of pre-amplification non-specific products is too large, the non-specific amplification of the second round PCR target primer is increased, and the background of the nested or double-amplification PCR system is increased.

The condition of diluting the fixed pre-amplification product by 10-20 times, and the background of 30 cycles of Ct value of the second round PCR primer determines that the second round PCR can only carry out 30-35 cycles; the test of the proper pre-amplification reaction cycle number, and the short primer pre-amplification cycle number without influencing the background of the second round PCR target primer is the key for determining the nonspecific detection of the digital PCR. Therefore, a pair of optimally designed middle homosequence primer pairs is selected, the target-free template pre-amplification with shortened target primer 3' end is tested for different cycles of 0, 10, 15 and 20, and the background Ct value is measured by SYBR Green I real-time fluorescence PCR in a second round after the dilution by 10 times. The following PCR procedure and primers for hepatitis B virus X gene (one) were used, and the results are shown in the following table:

and (4) conclusion: the influence of pre-amplification less than or equal to 15 thermal cycles on the system background is small, and the fluorescence PCR amplification is shown in figure 1.

The invention relates to a digital quantitative PCR method of homosequence double amplification, which comprises the following operation steps:

(1) the method comprises the following steps of improving double-amplification d-PCR target primer selection and short target primer design and verification:

the key to the success of the double amplification PCR is the quality of the primer selection, especially the primer of the double amplification PCR pre-amplification needs more than 40 thermal cycles in two rounds to generate the non-specific amplification of the primer dimer PD, thereby covering the background PCR reaction without the template. Primer pairs that are more than 70% homologous according to the Hands (Nucleic Acids Res., 1997, 25-16: 3215) technique do not produce primer dimers; we await a tabulation test by placing the native homologous 6-8base sequence in the middle of 3-6b from the 3' end of the primer, so that one pair of partially homologous primers will generally generate PD amplification after 6-10 thermal cycles to reduce the probability of PD generation in the whole PCR system, and then pre-amplification primer 10-fold dilution and the Tm value 10 degrees lower than that of the second round of PCR will generate PD amplification. The pre-amplification primer selection is designed on the principle of short primer with shortened 3-6b at the 3' end of the preferred middle 6-8b homologous target primer restriction sequence.

Is the design of the pre-amplification short and long target primers chosen to result in PD amplification or under what conditions do they result in PD amplification? The final choice of which must be determined by preliminary experiments. The PD amplification is verified by adopting a template-free blank system PCR without a sample, 5 double-amplification PCR reactions are matched, the first round of PCR is respectively amplified for 0-10-15-20 thermal cycles by using a common PCR instrument 1-5 tube, the second round of PCR is completely amplified for 40 cycles by adopting SYBR Green I real-time fluorescence PCR, the optimal cycle condition of the first round of PCR of a test tube without PD or with the latest PD amplification is selected, and meanwhile, the optimal cycle condition of the second round of PCR which is performed for a plurality of cycles without PD amplification can be exactly known through the real-time fluorescence PCR.

The first round of template-free pre-amplification primer general PCR: 5 times of pre-amplified short primer PCR mixed solution with the volume of 5 mu L is prepared according to the following formula,

taking 1-5 PCR tubes with the volume of 0.2mL, sequentially adding 5 mu L of PCR mixed solution, respectively adding 30 mu L of mineral oil into each tube, performing 94 ℃ denaturation for 2 minutes, performing 20 thermal cycles of first round PCR non-calotte amplification with 94 ℃ denaturation for 20 seconds, 45-48 ℃ annealing for 60 seconds and 72 ℃ extension for 30 seconds, and manually taking out the No. 1-5 PCR tubes after 0-10-15-20 thermal cycles of 72 ℃ extension are completed.

Second round SYBR Green I real-time fluorescent PCR: 5 times of long target primer PCR mixed solution with the volume of 50 mu L is prepared according to the following formula,

sucking 45 mu L/tube of fluorescent PCR reaction solution containing long target primer, inserting sample-adding suction head under No. 1-5 tube mineral oil layer, carefully adding, performing 94 ℃ denaturation for 2 min, 40 thermal cycles of 94 ℃ denaturation for 20 sec, 54-60 ℃ annealing for 30 sec, and 74 ℃ extension for 30 sec, and performing second round of real-time fluorescent PCR amplification without thermal cap.

Once the first optimal cycle number of the short primer pair is verified, the verification is not required to be performed again and/or each time the d-PCR detection is applied, and the determined first and second optimal cycle numbers are fixedly used.

(2) Sample DNA/RNA extraction and purification:

extracting animal DNA by a microspheric method: using 0.2-1M guanidinium isothiocyanate and 0.2-1 g% SDS for lysis, nucleic acid was bound to polystyrene microspheres (0.1-0.4mg/mL) containing 0.1M 4-hydroxyethylpiperazine ethanesulfonic acid (pH6.5) under 0.1-0.3M sodium salt for silanization of surface hydroxyl groups (Melzak et al, 1996), washed with 0.1-0.3M sodium chloride buffer less than pH6.0, and eluted with TE buffer greater than pH 8.5.

DNA extraction by plant CTAB method: the plant sample is smashed in a sterilization mortar by adding liquid nitrogen, then 500 mu L of cetyltrimethylammonium bromide CTAB is added for grinding, then the plant sample is transferred into a 1.5mL plastic EP tube for 1 hour in water bath at 65 ℃, chloroform-isoamyl alcohol (24+1) with the same volume is added for extraction once, the micro-magnetic spheres are combined or 2 times of alcohol salt solution is added for precipitation, and 40 mu L of TE is added for elution or DNA dissolution.

And (3) rapidly extracting RNA: adding 0.1ml of lysis solution (/ Trizol)1ml (0.5ml of 4M GTC solution +0.5ml of water-saturated phenol), vortexing, adding 0.1ml of chloroform, mixing, centrifuging at high speed for 10 min, precipitating the lysis supernatant with equal amount of isopropanol, washing with 70% cold ethanol, adding 50. mu.l of DEPC-treated dH₂O dissolves the RNA. Or cracking the supernatant and adding a micro-magnetic ball to crosslink and solidify one end of the primer for purification. (GTC solution: 4M guanidinium isothiocyanate +0.1mM DTT +0.2 g% SDS dissolved at 65 ℃ C.)

(3) Sample nucleic acid 10-fold dilution gradient first round PCR: the samples to be tested were diluted 10-fold in 9 steps according to the following table,

pipe number	1	2	3	4	5	6	7	8	9
										Concentration of	Original x 10^-1	×10^-2	×10^-3	×10^-4	×10^-5	×10^-6	×10^-7	×10^-8	×10^-9

Taking 9 plastic EP tubes of 1.5mL, adding 45 mu L of purified water into each tube, adding 5 mu L of sample stock solution to be detected into the water of the 1 st tube, and gently mixing the sample stock solution with a suction head to obtain stock solution multiplied by 10^-15 μ L of 10-fold diluted solution from the 1 st tube was added to the 2 nd tube water and mixed lightly to give stock solution × 10^-2Double, changing one suction head per tube, stock solution x 10^-3... the original solution is multiplied by 10^-9。

The 5 × PCR reaction solution was prepared according to the following formulation:

taking a 96-well PCR reaction plate (8 × 12 wells) with a sharp bottom, and firstly, 1 μ L/well of 5 × PCR reaction solution A_1-10Adding 5 mul/well stock solution x 10^-9Diluting gradient liquid by 10 times at the bottom of mineral oil lower tube, B_1-10Adding 5 mul/well stock solution x 10^-810-fold dilution gradient, c..... H, etc., 5 μ L/well stock solution × 10 for the remaining a-E11-12 wells^-1Diluting the gradient solution by 10 times, and adding two-well positive control, two-well negative control and two-well system background dH₂And (4) performing O control. Then 30. mu.L of mineral oil was added, and after denaturation at 95 ℃ for 2 minutes, 10 to 15 cycles of first PCR apyro-amplification of denaturation at 94 ℃ for 20 seconds, annealing at 48 ℃ for 60 seconds, and extension at 72 ℃ for 30 seconds were carried out.

(4) Second round inner primer PCR after first round product dilution: preparing a second round of 1 XPCR reaction solution according to the following formula,

adding 50 mu L/hole of 1 XPCR reaction solution containing inner primers into a 96-well plate, taking 50 mu L of 1 XPCR reaction solution from a sample adding suction head, cleaning the outer surface of the suction head as clean as possible, inserting the suction head under a mineral oil layer, adding the suction head in a hole, and replacing the suction head with the suction head in another hole; after denaturation at 94 ℃ for 2 min, a second round of PCR apyro-PCR amplification with 30-35 thermal cycles of denaturation at 94 ℃ for 20 sec, annealing at 54-60 ℃ for 30 sec, and extension at 74 ℃ for 30 sec was performed.

(5) d-PCR final product fluorescence color development and counting quantification:

in the second round of PCR reaction, 4 mu L of ethidium bromide EB (0.5 mu g/mL) is added into each hole after the reaction of dye SYBR Green I, and the mixture is placed in a 96-hole plate to be subjected to color counting under an ultraviolet lamp with the wavelength of 254nm or to be detected by a fluorescence photometer; or after PCR reaction, red and green molecular beacon probes with different wavelengths for wild and mutant genes are added into each hole to carry out mutation quantitative detection under the high wild background. And (4) interpretation of results: a set of 10 PCRs having 7 or more than 7 amplification fluorescent reactions defined as 1 target molecule/2.0. mu.L sample, a set of 10 PCRs having 3 and less than 3 amplification fluorescent reactions defined as 0 target molecule/2.0. mu.L sample, and a set of 10 PCRs having 4 to 6 amplification fluorescent reactions defined as 0.5 target molecule/2.0. mu.L sample, the absolute number of initial target template molecules can be calculated by multiplying the sample dilution factor by 1 target molecule set or 0.5 target molecule set, with a plus or minus error of 5 to 10 molecules. The critical set of PCR products for which the 0-1 results were not clearly judged were further examined by 1.5% Agarose gel electrophoresis.

The invention has the beneficial effects that:

the digital quantitative PCR method for the same-sequence double amplification of the invention adopts the 3' end shortened 3-6base short primer pre-amplification to improve the sensitivity and the fluorescence PCR of the target primer with the same part and the same sequence to inhibit the non-specificity thereof so as to carry out the limited dilution quantitative PCR, and the pre-amplified short primer does not promote the polymerization non-specific amplification of the target primer due to the same sequence with the target primer. Meanwhile, in order to overcome the nonspecific limitation of d-PCR and the limit of a reaction unit of a microfluidic or micro-droplet device, the operation improvement comprises that a single-hole or single-tube two-step primer is diluted and the annealing temperature is increased so as to ensure that the first-step pre-amplification primer fails during the second-step amplification and does not interfere with the polymerization nonspecific amplification of the primer, and the primer dimer PD is completely prevented from nonspecific amplification by the double-amplification d-PCR of two rounds of dilution and mineral oil sealing; the operation comprises that the sample is firstly diluted by 9 or more orders of magnitude, then the diluted specimen of each order of magnitude is distributed to a limited number of reaction holes/reaction units, and the micro-dripping of million-level reaction units and the effect of the micro-fluidic chip device can be achieved by a 96-pore plate, a 384-pore plate or a polydimethylsiloxane micro-pore chip in total; and adding a two-stage multiple dilution or division on the basis of the obtained product. The two-wheel double-amplification PCR digital quantitative method can be used as the manual/manual d-PCR of important temporary inspection samples, nucleic acid reference products and scientific research samples, and provides early research tools and development ideas for the development of faster and efficient super-unit micro-encapsulated/micro-encapsulated micro-fluidic devices.

Drawings

FIG. 1: the background of the diluted 10-fold fluorescence PCR is Ct values of 38, 34.5, 32 and 22 cycles after 0, 10, 15 and 20 cycles of template-free pre-amplification, and the background of the double-amplification PCR system is not obviously influenced or increased only when the pre-amplification is not more than 15 cycles.

Detailed Description

The following examples further illustrate the contents of this patent but should not be construed as limiting the patent. Modifications or substitutions to methods, conditions, steps and applications of the invention may be made without departing from the spirit and substance of the invention.

Example 1

Hepatitis B Virus (HBV) dilution method digital quantitative double amplification dPCR:

viral Hepatitis B (Hepatitis B for short) is a worldwide infectious disease caused by Hepatitis B Virus (HBV), and the world health organization WHO reports that about 20 million people worldwide have Hepatitis B virus infection. The hepatitis B infection rate of people in China is very high (nearly 10%), and the liver cancer with the first listed tumor mainly caused by hepatitis B virus is the first, thus greatly harming the health of people. The existing hepatitis B detection methods mainly comprise an enzyme immunoassay method, an radioimmunoassay method, a chemiluminescence method, an immunofluorescence method, a nucleic acid amplification (PCR) fluorescence quantitative method and the like. The traditional enzyme immunoassay method is wide in application but insufficient in sensitivity; the real-time fluorescent PCR quantitative and digital quantitative PCR method can accurately determine the virus load of hepatitis B patients, and has irreplaceable important functions on the judgment of virus replication level of infected patients, disease infectivity and antiviral drug curative effect monitoring. This example is a dilution method of Hepatitis B Virus (HBV) and a digital quantitative double-amplification PCR, wherein the X region (1545-₁₉Vector plasmid pHBx (MW 2.2X 10)⁶) As positive control sequence of hepatitis B virus.

AB 493827X region (1588-

CACTTCGCTT CACCTCTGCA……ACTAGGAGGC TGTAGGCATAAATTG

Selecting a section as much as possible "Same sequence"a long target primer with a base placed in the middle of the primer pair, and a pre-amplified short primer placed at the 3' end; the primer dimer PD amplification can be reduced to the greatest extent in turn, and according to the design principle, the sequences of the pre-amplified short primer pair and the long target primer pair are as follows: (underlining)Sequence ofIn the same order in the middle part)

Pre-amplification short primer pair DhbF: 5'-ctt cgc ttc acc tc-3'

DhbR：5’-tta tgc cta cag cct c-3’

Long target primer pair HBxF: 5' -ctt cgc ttcacc tct gca-3’

HBxR：5’-tta tgc cta caa cct cct a-3’

Because the same sequence cctc in the middle part of the primer pair is too short, the homologous cctc 5' side g of the long target primer HBxR is mutated into an a base, the primer binding annealing temperature is reduced a little, but the homologous sequence ca and the increased actc homologous sequence obviously inhibit the background non-specific amplification of the long target primer pair PD.

(1) Extracting DNA of a blood sample:

one-step boiling purification method comprises adding 50-100 μ l serum into equal amount of boiling buffer (mixing microbeads thoroughly before use, sucking with big mouth sucker), mixing lightly, placing in boiling water bath for 10 min, cooling at 4 deg.C for 10 min, centrifuging at high speed for 10 min, and collecting supernatant 5 μ l.

Two-step PEG precipitation method, using PEG to precipitate HBV from weak positive specimen, taking 500. mu.l serum, adding 500. mu.l 2 XPEG liquid (16% w/v PEG &0.7M NaCl), Vortex mixing, high speed centrifuging for 10 minutes, discarding 950. mu.l supernatant, concentrating precipitate, keeping 50. mu.l, adding 50. mu.l boiling buffer solution, and taking 5-10. mu.l gradient dilution sample adding by the same boiling method. However, the recovery rate of PEG precipitation is 50% -60% on average, and the loss must be calculated by quantitative detection.

Micromagnetic bead binding method, commercially available extraction cartridge.

2 × boiling buffer: 0.02N NaOH, 0.02% SDS (w/v), 25mM KoAc, 10mM (NH)₄)₂SO₄0.5% G25(v/v), 0.5M Betaine, 0.5% Glycerol (v/v) and 0.05% Gelatin (w/v).

(2) A set of 10 gradient HBV miniprep dpcrs:

diluting 10 μ l of diluted purified sample with 90 μ l of water sequentially with 10 μ l of diluent by 10 × (times) for 5 gradients, and diluting 100 μ l of diluent with 100 μ l of water by 2 × (equal ratio) for 5 times; and for most biological samples, the sample is multiplied by 10^-3Start 10 × (fold) dilution 5 times, and 2 × (equal ratio) dilutions 5 gradients; mu.l of each of the two-fold gradient solutions was pre-amplified. This example uses a 70-fold dilution of plasmid pHBx miniprep DNA (70. mu.g/ml) from 1. mu.g/ml.times.10^-51-10 times of dilution tube gradient; and one positive HBV serum DNA X10^-3Tube 10-20 fold gradient.

The short target primer pre-amplification PCR reaction, 1 mul of short primer 5 XPCR reaction solution (1: 1 volume 10mM dNTP: 5 MuM short primer F: R and: 2 volume H.K.Taq and 10 Xbuffer mixed in equal amount and diluted by one time) is added into each tube of the first round of pre-amplification PCR, 30 mul of mineral oil is ensured to be sealed, a tube of suction head is inserted into the lower part of a mineral oil layer, 5 mul of gradient solution with each time ratio is added, and the suction head of a sample adding gun is gently inserted into the lower part of the mineral oil layer to be carefully added. No-heat-cap amplification using a common PCR instrument: denaturation at 94 ℃ for 4 min, followed by 13 cycles of 94 ℃ for 20 sec, 48 ℃ for 60 sec, 72 ℃ for 30 sec; the annealing temperature of the reaction is slightly higher than the set Tm value of the short primer by 2-5 ℃ but the reaction time is slightly longer.

The second round of long target primer fluorescence PCR is to prepare 20 times of long target primer fluorescence PCR mixed solution with the volume of 50 mu L according to the following SYBR Green I fluorescence PCR formula, add 50 mu L of 1X long primer PCR reaction solution into the same PCR tube in the previous round, insert the reaction solution into the mineral oil layer by using a suction head of one tube, and add the reaction solution to 30 thermal cycle non-thermal-cover amplifications at 54 ℃.

Denaturation at 94 ℃ for 4 min, followed by 31 cycles of 94 ℃ for 20 sec, 54 ℃ for 30 sec, 74 ℃ for 30 sec; fluorescence PCR end reaction fluorescence values relative fluorescence absorbance values were read using a fluorescence detector from the company Siansilong. The result shows that relative to the fluorescence absorption value, the number of plasmid tubes 1-7 is more than 3000, the number of plasmid tubes 8 is 2000, the number of plasmid tubes 9-10 is less than 50, and the calculation shows that the number of plasmid tubes is 1 mu g/ml multiplied by 10^-5The plasmid was about 10⁹Copying; positive HBV serum tube 11-12 is greater than 3000, tube 13-20 is less than 50, and estimated HBV is about 10²⁺³And (6) copying.

Example 2

(II) food transgenic promoter dPCR quantitative detection:

the transgenic technology breaks through the limitation of natural resources, greatly improves the agricultural benefit, the yield and the quality of agricultural products, brings the biological safety problem of transgenic food, and is increasingly concerned by governments and social public of all countries in the world. Along with this, the demand for quantitative detection of food transgenic components and absolute quantitative detection of dPCR has also rapidly increased. Transgenic crops have evolved from major crops such as soybean, corn, cotton, etc. to tens of transgenic crop species; the transgenic types are mostly various herbicide resistant types, insect resistant types or composite type of herbicide resistant and insect resistant types, and have also been expanded to a series of novel transgenic types with changed linolenic acid content, high lysine type, delayed maturity, softness and virus resistance and the like. A certain transgenic target molecule is selected as a transgenic qPCR or dPCR quantitative detection, which is not easy to be widely used for detection, however, mosaic virus CaMV 35S promoter is adopted for expression and regulation of most transgenic types, and the common promoter sequence can be preferentially used as a primary screening tool for detection of the transgenic qPCR or dPCR. Transgenic soybean oil is used as an application example to perform dPCR detection on a CaMV 35S promoter.

(1) Designing and verifying a short target primer and a long target primer of a CaMV 35S promoter sequence:

primer pairs that follow the Hands technique that more than 70% of the homologous primer pairs do not generate primer dimer rules and primer pairs that are 3-5b from the middle natural homologous 6-8base sequence from the 3' end of the primer generally follow a PD strategy of about 6-10 cycles. The selection of inverted repeat from the CaMV 35S promoter sequence, i.e. a pair of 6base homologous sequences as long target primers, is as follows:

CaMV F：5’-g aag gtggct cctaca a-3’

CaMV R：5’-tc cac gatgct cctcgt-3’

and 3' end shortened 3-5b short target primer pair:

DCaMV F：5’-gg aag gtg gct cct-3’

DCaMV R：5’-ttc cac gat gct cct-3’

(2) DNA extraction and column purification of soybean oil samples:

the DNA extraction method according to (Chinese agricultural science 2007, 40 (5): 1069): adding 10mL of edible transgenic soybean oil of Gonglongyu brand into 10mL of n-hexane, magnetically stirring for 2hr, adding 20mL of PBS, stirring for 3hr, transferring into 50mL plastic tube, centrifuging for 12000g × 20min, carefully taking out lower water phase, transferring into a new 50mL tube, adding equal volume of isopropanol, mixing, and standing at-20 deg.C^℃X 20mins recentrifugation12000g × 20mins, discarding the supernatant, precipitating and adding dH₂O100. mu.L was dissolved, 500. mu.L of the binding buffer was added to the column, the column was washed twice with the washing solution, and after flash drying of the column, 50. mu.L of TE was added to elute the DNA.

taking a 96-well PCR reaction plate (8 × 12 wells) with a sharp bottom, and adding 1 μ l/well of 5 × PCR reaction solution A to each well_1-10Add 5. mu.l/well stock solution X10^-9Diluting gradient liquid by 10 times at the bottom of mineral oil lower tube, B_1-10Add 5. mu.l/well stock solution X10^-810-fold dilution gradient, C..... H, etc., 5. mu.l/well stock solution × 10 for the remaining A-E11-12 wells^-1Diluting the gradient solution by 10 times, and adding two-well positive control, two-well negative control and two-well system background dH₂And (4) performing O control. After further addition of 30. mu.l of mineral oil and denaturation at 95 ℃ for 2 minutes, 13 cycles of first PCR apyro amplification with denaturation at 94 ℃ for 20 seconds, annealing at 48 ℃ for 60 seconds and extension at 72 ℃ for 30 seconds were carried out.

after 50. mu.l/well of the 1 XPCR reaction solution containing the inner primer was added to a 96-well plate, the plate was inserted under a mineral oil layer with a sample-adding tip, and denaturation was carried out at 94 ℃ for 2 minutes, and then second PCR amplification was carried out by 33 cycles of denaturation at 94 ℃ for 20 seconds, annealing at 54 ℃ for 30 seconds, and extension at 74 ℃ for 30 seconds without a heat cap.

relative fluorescence absorbance values were read on a fluorescence detector from the company Siansilong after the second PCR reaction in 96-well plates. Counting and interpretation: a set of 10 PCRs with 7 or more than 7 amplification fluorescent reactions is defined as 1 target molecule/5 μ L sample, a set of 10 PCRs with 3 and less than 3 amplification fluorescent reactions is defined as 0 target molecule/5 μ L sample, and a set of 10 PCRs with 4-6 amplification fluorescent reactions is defined as 0.5 target molecule/5 μ L sample, and the absolute number of initial target template molecules, plus or minus 5-10 molecules, can be deduced by multiplying the sample dilution factor by 1 target molecule set or 0.5 target molecule set. Results negative wells and fluorescence values from groups A-E<50，F_1-10Group has 2 holes > 3000, G_1-10Group 7 wells are more than 3000, and both group H and positive wells are more than 3000; estimate G_1-10The group contained 1 target promoter molecule/5. mu.L, soybean oil 10mL contained 10X 1000/5X 10³2 × 10 times⁶Promoter transgenic molecules, or containing 2X 10⁵Transgenic molecules/mL soybean oil.

Sequence listing

Thymus thick plant Thymus officinalis code Co Ltd

<120> digital quantitative PCR method of same-sequence double amplification

<160>9

<210>1

<211>45

<212>DNA

<213> partial sequence of Hepatitis B Virus (HBV)

<400>1

cacttcgctt cacctctgca--- ---actaggaggc tgtaggcata aattg 45

<210>2

<211>14

<212>DNA

<213> Artificial sequence

<400>2

ctt cgc ttc acc tc 14

<210>3

<211>16

<212>DNA

<213> Artificial sequence

<400>3

tta tgc cta cag cct c 16

<210>4

<211>18

<212>DNA

<213> Artificial sequence

<400>4

ctt cgc ttc acc tct gca 18

<210>5

<211>19

<212>DNA

<213> Artificial sequence

<400>5

tta tgc cta caa cct cct a 19

<210>6

<211>17

<212>DNA

<213> Artificial sequence

<400>6

gaa ggt ggc tcc tac aa 17

<210>7

<211>17

<212>DNA

<213> Artificial sequence

<400>7

tcc acg atg ctc ctc gt 17

<210>8

<211>14

<212>DNA

<213> Artificial sequence

<400>8

gga agg tgg ctc ct 14

<210>9

<211>15

<212>DNA

<213> Artificial sequence

<400>9

ttc cac gat gct cct 15

Claims

1. A digital quantitative PCR method of homosequence double amplification is characterized in that the method is the double amplification quantitative PCR of a multiple dilution sample, and comprises the following steps: the method comprises the steps of pre-amplifying for 10-15 cycles in a small-volume PCR reaction solution by using a short target primer with a 3' end shortened by 3-6b base, then diluting the pre-amplified solution for 10-20 times of a large-volume fluorescent PCR for 30-35 cycles in the same tube by using a middle homosequence target primer PCR solution with a non-shortened end, amplifying without a hot cover under a mineral oil closed condition, ensuring effective detection of molecules more than or equal to 1 and no non-specific reaction of a template 0 by using a homosequence double-amplification PCR with a total reaction of more than 40 cycles, and calculating target molecule quantification according to the dilution multiple and the volume of a sample, wherein the method is non-disease diagnosis, and the short target primer is the terminal homosequence short target primer.

2. The digital quantitative PCR method of the same-sequence double amplification according to claim 1, wherein the sample is diluted by multiple times for two PCR double amplification, and the first round is pre-amplified by using 2-5 μ L of 14-18base short primers with shortened 3' end of the target primer for 12-14 thermal cycles to pre-amplify the PCR solution with 1-2 x 10²-10⁴Adding 17-24base long target fluorescent PCR reaction solution with the same sequence as the middle part 5-8b to dilute the pre-amplification reaction solution by 10-20 times, performing 20-100 mu L of second round fluorescent PCR for 30-32 thermal cycles, performing non-thermal cover amplification under the condition of mineral oil sealing, and ensuring that single target molecules and more than single target molecules are detected to be '1'; background '0' has no non-specific reaction; and (3) calculating the dilution quantitative detection digital PCR method of the sample target content according to the times and the volumes of the sample diluted to zero target molecules.

3. The method of claim 1, wherein the method is a set of sample dilution gradientsEasy digital quantitative PCR, sample is diluted in EP tube in multiple ratio gradient, sample is taken x 10⁰～×10^-5Beginning 10X (times) dilution for 5 times and then 2X (equal ratio) dilution for 5 gradients, ensuring that the turning point of a target molecule tube is diluted until no target molecule tube falls in the gradient range, adding 0.5-1 mu L of 5X pre-amplification short primer PCR reaction solution, 2-5 mu L of diluted sample and 30-50 mu L of mineral oil into a PCR tube, sealing, and carrying out 12-14 thermal cycle first round PCR non-calotte amplification of short primer annealing at 45-50 ℃; 18. mu.L to 45/95. mu.L of 1 Xlong primer PCR reaction solution was added to the same tube in the first round, inserted under the layer of mineral oil with the sample-adding tip, and subjected to 30-35 thermal cycles of annealing the long target primers at 54 ℃ to 60 ℃ for the second round PCR amplification without heat cap.

4. The digital quantitative PCR method of the same-sequence double-amplification according to claim 1, wherein the dilution gradient of each step is equally distributed to a limited number of reaction wells/reaction units after 10-fold serial dilution of 9 or more steps is performed on a sample to be tested, two-round double-amplification PCR statistical quantification is performed on a 96-well plate and a 384-well plate, 9 sets of gradient repeated PCR diluted by 10 times in advance of the sample are adopted, the theoretical number of target molecules of 10 PCR sets is between 0 and 1, the efficacy equivalent to that of a ten-thousand or even million-level reaction unit microfluidic or micro-titration device can be obtained, and 100-fold dilution or two-level dilution or cut-off is added on the basis; for example, the second round of 1 XPCR reaction solution can be added with 1 XPYRBR Green I fluorescent dye for carrying out the fluorescent PCR detection in the second round, or after the second round of PCR reaction, each hole is added with 2-5 μ L of EB (0.5 μ g/mL) below the mineral oil layer, and the mixture is placed in a 96-hole plate for color counting under an ultraviolet lamp or detection by a fluorescent photometer.

5. The digital quantitative PCR method of the same-sequence double-amplification as claimed in claim 1, wherein the sample to be tested is subjected to 10-fold serial dilution of 9 steps, then the dilution gradient of each step is reduced by 10-fold volume and is equally distributed to 10000 polydimethylsiloxane microporous chips as reaction units, and an in-situ PCR instrument is used for two-round nested PCR digital quantification.

6. For use in claims 1-5The gene detection kit for the digital quantitative PCR method of the double-amplification in the same sequence is characterized by comprising the following components: nucleic acid extraction reagent, dNTPs and dUTP, UDG enzyme, Taq, HKTAQ enzyme and buffer solution thereof, pre-amplification short primer F/R, long target primer F/R, dye EB and SYBR Green I, and purified water dH₂O, mineral oil.