WO2018121194A1 - 外引物可降解的巢式pcr - Google Patents

外引物可降解的巢式pcr Download PDF

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WO2018121194A1
WO2018121194A1 PCT/CN2017/114762 CN2017114762W WO2018121194A1 WO 2018121194 A1 WO2018121194 A1 WO 2018121194A1 CN 2017114762 W CN2017114762 W CN 2017114762W WO 2018121194 A1 WO2018121194 A1 WO 2018121194A1
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primer
pcr
amplification
external
reaction solution
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PCT/CN2017/114762
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French (fr)
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何玉贵
江洪
曲越
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北京泰格瑞分子检验有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of nucleic acid amplification PCR technology for molecular biology and molecular examination, and particularly relates to the field of single-tube nested PCR which is modified by external primer pre-amplification to improve sensitivity and modify primer enzymatic hydrolysis to limit its non-specificity.
  • the amplification process is generally denatured by the target DNA: the double strand is dissociated into a single strand by heating at 94 ° C; the primer is annealed (refolded): the temperature is lowered to about 54 ° C to allow the excess primer to bind to the complementary sequence of the single strand of the template DNA; primer extension : The temperature is raised to about 72 °C, the DNA single-stranded template-primer conjugate is under the action of heat-resistant DNA polymerase, dNTP is used as the reaction material, the target sequence is the template, and the principle of base reverse pairing and semi-reserved replication is followed.
  • Super-sensitivity exponential amplification or geometrical series amplification is the core essence or advantage of PCR.
  • the target molecule is amplified by 2 n series, and the conventional 30 thermal cycles are 2 30 approximately equal to 10 9 magnification.
  • the amplification amplification is like atomic fission.
  • the chain reaction is unimaginable, and it is also unmatched by the general linear amplification detection method.
  • the sensitivity is far greater than the amplification factor of the common detection method, and nearly 40 cycles can reach the fek fg/ml or single digit molecular level detection. This is also the root cause of PCR becoming the most widely used core technology and even how many of them are the most descriptive.
  • exponential amplification also leads to high specificity and stubborn non-specificity of PCR.
  • PCR specificity is directly derived from the specificity of the primer sequence.
  • One-point primer sequence non-specific mismatch amplification or even complete paired amplification is only linear amplification. Naturally, it is far behind the exponential amplification or geometrical series amplification. Any decrease in primer specificity or mismatch with the target-dot point causes the amplification rate to fall, but not far from the specific index or geometric progression.
  • the PCR amplification assay has sufficiently high specificity.
  • the non-specific causes of various conventional test methods exist in PCR technology, but after all, they cannot keep up with exponential amplification.
  • primers with a number of bases that are continuously reverse-complementary at the 3' end are primer-dimer PD amplification for about a few to a few cycles in a background PCR amplification reaction without template; a pair of conventional designs
  • the primers showed significant PD non-specific amplification in the 30 cycles of the background PCR reaction, and covered low-concentration samples of thousands of target molecules located in the 30-38 cycle, which is the main PCR for only 30 cycles. the reason.
  • Pre-amplification requires removal of at least 20-50 fold dilutions or purification of the target product by gel electrophoresis to remove the effect of the outer primers to eliminate the increased non-specificity of the system, which not only increases the operational complexity but also results in post-PCR processing. Cross contamination risk.
  • fluorescent dye SYBR Green I real-time fluorescent PCR and various fluorescent nucleic acid probe PCR (BioTechniques 1997, 22: 130-138) and nested fluorescent PCR (Malaria Journal 2014, 13: 393).
  • Dye real-time fluorescent PCR was detected by SYBR Green I binding product DNA double-stranded groove to increase the emission fluorescence by 1000-fold, but non-specific amplification products also combined with emission fluorescence, and non-specific amplification after 30 cycles also covered low concentration.
  • Amplification reduces the detection sensitivity; on the other hand, a certain degree of non-specific polymerization, extension amplification is also generated between the excess primer and the probe DNA strand, a PCR background system without a target template, a pair of primers and any A fluorescently labeled TaqMan probe produced a non-specific amplification curve of about Ct 33-35, and as the same PCR amplification was repeated repeatedly, the peak fluorescence absorption peak of the amplification plateau became higher and higher, and the Ct value gradually moved forward. Produce a certain sample to detect false positives. At present, the limited sensitivity and certain non-specificity of real-time fluorescent PCR still hinder the detection of very low concentration samples or single molecule detection.
  • the quantitative test method has long been the concept of dilution and quantitation, that is, the dilution method is used to dilute the target molecule by sample dilution until the target molecule is reacted, and then the dilution ratio is diluted; further, the digitization based on sample limiting dilution and Poisson distribution counting appears. Quantitative PCR concept.
  • the sample is diluted and dispersed to a large number of micro-separating units so that theoretically each detecting unit contains 0- a target molecule, which quantifies the target molecule by detecting the unit reaction signal 0-1 count and limited dilution factor, in order to facilitate the automated operation of the computer 0 or 1 mode, but only if the detection method itself is for 1 molecule or 0 molecules are reliably distinguished.
  • traditional chemical reactions, enzyme immunoassays, etc. are nanogram-level low-sensitivity detection methods in which the signal intensity is linearly proportional to the content of the molecule to be measured and the signals are simply added.
  • the linear traditional low-sensitivity method can not distinguish one target molecule. It is different from the 0 molecule, so it cannot be applied to the digital quantitative detection method of the limiting dilution method.
  • Sykes et al. (Biotechniques 13: p444) initially attempted quantitative nested PCR based on sample limiting dilution and Poisson distribution counting, and first proposed the concept of digital quantification, but due to the amplification of multiple primer aggregates in such PCR systems.
  • the serious non-specificity of the cross-contamination of aerosol gel outside the system offsets the amplification of secondary amplification.
  • the absolute Ct value of the absolute hot start background of the PCR after thermal denaturation is only pushed back 1-3 cycles.
  • the temperature is lower than 40 °C, the Taq activity is very low.
  • the low temperature start is not the non-specific formation of PD.
  • the test uses various non-specific measures such as changing PCR components, adding various chemical reagents, etc., which are substantially parallel to specific and non-specific amplification, and significantly inhibit PCR non-specificity and interfere with target-specific amplification to varying degrees. Efficiency, and vice versa, does not affect target amplification efficiency and is difficult to inhibit PCR non-specificity.
  • the non-specificity of the index caused by the primer is also derived from its base sequence, and a pair of completely identical primers are absolutely non-specific.
  • Hands technology Homo-Tag assisted non-dimer system, Nucleic Acids Res. Vol. 25, No16: p3235-3241 uses the same homologous primer tag to compete for free primers by binding the two ends of the single strand of the primer dimer, which not only significantly inhibits PD is non-specific and also does not selectively reduce target-specific amplification efficiency.
  • the single-stranded binding protein Single Strand Binding-protein (SSB), gene 32 protein and full primer binding to the antisense base Oligo can significantly reduce the optimized primer non-specificity, but also seriously affect the target-specific amplification efficiency and amplification curve. Linear relationship.
  • the middle part of the same primer pair and the middle antisense base Oligo were combined to eliminate endogenous PD non-specific amplification interference in 40 cycles of real-time fluorescent PCR; endogenous non-specific
  • the heterogeneous PD aerosol glue continues to be a template for subsequent PCR cross-contamination.
  • dU is used instead of dT substrate to form dU-containing product, and dU-degrading UDG enzyme degrades dU product aerosol glue.
  • a third-generation absolute quantitative PCR method for counting using a microfluidic or microdroplet method in the field of analytical chemistry, dispersing a large number of diluted nucleic acid solutions into microreactor units or droplets, and a nucleic acid template for each reaction unit The number is less than or equal to one.
  • the amplified product is hybridized with the added fluorescent probe, and the reaction unit of one nucleic acid molecule template is amplified to give a fluorescent signal, and the reaction unit without the template is not amplified without a fluorescent signal.
  • the nucleic acid concentration of the original solution can be deduced, and the absolute quantification of the starting DNA template can be achieved by counting and Poisson distribution statistics.
  • PCR and real-time fluorescent PCR are still not enough sensitivity for single-molecule detection, and pre-amplification amplification is needed to further improve system sensitivity; however, PCR is limited to non-specific coverage of low-concentration samples, hypersensitive PCR or even single molecules. Effective detection depends on both PCR sensitization and non-specific removal.
  • the "external primer degradable nested PCR" of the present invention adopts a UDG or RNase degradation strategy after pre-amplification of the outer RNA-containing base or dU base primer without increasing the non-specificity of subsequent primer polymerization in the inner side amplification,
  • the outer primers were pre-expanded for 15 cycles of finite pre-amplification to sensitize the internal primers for real-time PCR; the external primers were pre-expanded for 20 cycles and the amplification was too large and the internal primers were quantified by fluorescent PCR.
  • nested double-amplification PCR digital quantification method can be used as an important clinical sample, nucleic acid reference and scientific research
  • the manual/manual d-PCR method of the sample provides pre-study tools and development ideas for the development of a faster and more efficient super-cell microcapsule/microdroplet microfluidic device.
  • the "external primer degradable nested PCR" of the present invention uses a dU or RNA base external primer to pre-amplify the UDG or RNase degradation strategy without increasing the subsequent inner amplification of the primers to polymerize non-specific, lateral primer pre-
  • the finite pre-amplification of 10-15 cycles can be used to sensitize the primers for real-time PCR; the external primers are pre-expanded for 15-20 cycles and the amplification is too large, and the internal primers are quantitatively quantified by fluorescent PCR.
  • nested double-amplification PCR digital quantification method can be used as an important clinical sample, nucleic acid reference and scientific research
  • the manual/manual d-PCR method of the sample provides pre-study tools and development ideas for the development of a faster and more efficient super-cell microcapsule/microdroplet microfluidic device.
  • the outer primer degradable nested PCR is characterized in that the following steps are performed in the same reaction tube/well:
  • External primer amplification an external primer PCR reaction solution is added to the reaction tube/well, and the PCR reaction solution is sealed by adding mineral oil to complete a predetermined amplification procedure; wherein at least the PCR reaction solution used in the external primer is used.
  • One end of the outer primer contains one or more dU bases and/or RNA base modifications in the middle, and the outer primer PCR reaction solution uses Taq, K Taq or HK Taq, Mg 2+ and some or all of the dNTPs substrate.
  • the initial concentration is greater than the concentration required for the external primer PCR system to complete the predetermined PCR amplification procedure; the excess is used for the next primer amplification in the reaction tube/well;
  • (2) amplification of the inner primer adding the inner primer PCR reaction solution under the mineral oil surface layer of the reaction tube/well for completing the above external primer amplification, so that the outer primer PCR reaction solution is diluted by 2-4 times, and added
  • the internal primer PCR reaction solution contains UDG and/or RNase, and the final concentration of Mg 2+ and part or all of the dNTPs substrate is much lower than the concentration required for the internal primer PCR system to complete the predetermined PCR amplification procedure; Prior to PCR amplification of the primers, the primers were digested at 50 ° C for 2-10 minutes to degrade the outer primers.
  • the inner primer used in the inner primer PCR reaction solution is a middle homologous primer.
  • the primer used in the PCR reaction solution of the external primer contains at least one 1-3 dU base and/or RNA base modification at the distal end to the middle of the primer.
  • only one side of the outer primer in the outer primer PCR reaction solution contains the dU base and/or RNA base modification; the other side inner primer and its ipsilateral outer primer share the same sequence primer.
  • outer primer degradable nested PCR according to any one of the above, wherein the outer primer has a dU base or an RNA base modification at the 1-2 base position of the 3' end, and is provided in the primer portion.
  • the second and third base modifications are provided.
  • the outer primer degradable nested PCR according to any one of the above, wherein the initial concentration of dA, dG, and dC in the PCR reaction solution of the external primer is twice that of dT, and the initial concentration of Mg 2+ is 7 mM.
  • the added internal primer PCR reaction solution contains no Mg 2+ and conventional substrates dA, dG, dC, only dUTP, the concentration and volume of the dUTP and the dT added to the external primer PCR reaction solution. quite.
  • the UDG enzyme contained in the PCR reaction solution of the internal primer has an initial concentration of 0.0125-0.0625 U/ ⁇ l in the mixed reaction solution, that is, the original concentration of 5 U/ ⁇ l is diluted 80-400 times;
  • the initial concentration of the RNaseA enzyme contained in the mixed reaction solution is 0.4-40 ug/ml, that is, the original concentration is 4 mg/ml diluted 10 2 -10 4 times, or the initial concentration of the contained RNase I in the mixed total reaction solution is 0.031U/ ⁇ l.
  • said outer primer is degradable in nested PCR
  • External primer amplification first add 10 ⁇ L of the double-stranded PCR reaction solution to the bottom of the tube, add 10 ⁇ L of the diluted sample solution, and then add 50 ⁇ L of mineral oil layer along the tube wall to seal for 15-20 cycles.
  • Ordinary PCR amplification first add 10 ⁇ L of the double-stranded PCR reaction solution to the bottom of the tube, add 10 ⁇ L of the diluted sample solution, and then add 50 ⁇ L of mineral oil layer along the tube wall to seal for 15-20 cycles.
  • Ordinary PCR amplification Ordinary PCR amplification
  • the sample to be tested is diluted into a stock solution ⁇ 10 -1 , ⁇ 10 -2 , ⁇ 10 -3 , ⁇ 10 -4 , ⁇ 10 -5 , ⁇ 10 Dilutions of 9 concentration steps of -6 , ⁇ 10 -7 , ⁇ 10 -8 and ⁇ 10 -9 , taking ⁇ 10 -2 , ⁇ 10 -3 , ⁇ 10 -4 , ⁇ 10 -5 , ⁇ 10 - 6 , ⁇ 10 -7 , ⁇ 10 -8 , ⁇ 10 -9 of the test sample dilution and the positive control and the sterile water are separately dispensed as templates to the in situ PCR instrument for the external primer amplification and the internal primer Amplification.
  • the inner primer is amplified for 30-35 cycles, 0.5 ⁇ g/mL of EB2-5 ⁇ L is added to each layer of the mineral oil layer, and the reaction tube is colored under UV light or detected by a fluorometer.
  • the external primer degradable nested PCR is used to detect hepatitis B virus, and the inner primer pair is:
  • HBcF 5'-aat gcc cct atc tta tca a–3',
  • HBcR 5'–gat tga gat ctt atg cga c–3', where the 13th a is designed for c artificial variation.
  • the outer primer pair is: WhBcF: 5'–gtg gat tcg cac Ucc Tc–3',
  • the reverse primer is HBcR or dU modified HBcR.
  • a method for detecting a food transgene by detecting a promoter CaMV 35S characterized in that, in any of the above methods, the internal primer is:
  • CaMV F 5'-gaa ggt ggc tcc tac aa-3'
  • CaMV R 5'-tcc acg atg ctc ctc gt-3';
  • the external primers are:
  • WCaMV F 5'-tca ctt tat tgt gaa gaU tag U-3',
  • WCaMV R 5'-cca ctt gct Utg aag acG t-3'.
  • a kit for performing the nested PCR method of any of the above-described external primer degradable or the method for detecting the transgenic food of the food characterized in that the outer primer and the inner primer are contained.
  • the kit further comprises a pre-matched external primer PCR reaction solution for amplification of the external primer and a pre-matched internal primer PCR for amplification of the internal primer The reaction solution.
  • the invention relates to "external primer degradable nested PCR", which is characterized in that the sample is pre-amplified by a PCR of an external primer containing one or more dU bases and/or RNA bases at the end of the primer to the middle of the mineral oil.
  • the single-tube two-wheeled nested PCR containing the UDG and RNase-containing primers containing the UDG and RNase primers was added by PCR, and the gel was electrophoresed by external primer hydrolysis or a large amount of 20-50 ⁇ dilution.
  • the inner primer PCR solution does not need to contain Mg2+, and the primer PCR reaction solution is added on the mineral oil level.
  • the residual liquid was inactivated without Mg 2+ , and the substrate dU was used instead of dT in the primer solution to add the UDG enzymatic hydrolysis system to prevent external contamination of the product aerosol.
  • the central primer strategy in the middle of the sequence was used to further reduce the non-specificity of endogenous PD.
  • the root primer "degradable nested PCR” is characterized in that the dU/RNA base is placed at the 3' end/end of the outer primer and 1-3 in the middle, and the outer primer is preferably 1-3 modified bases.
  • Primer 3' is the last 1-2 base position, one modified base, the second and third modified bases are scattered in the primer, and the outer primer pair has at least one 3' end containing dU/RNA or upstream and downstream external primer 3'
  • the end of the primer contains 1 dU base;
  • the outer primer PCR solution contains an excess of dNTPs, such as 2 ⁇ dNTP (dA, dG, dC 0.25 mM, of which dT0.125 mM) and Mg 2+ (3.5 mM) and conventional Taq / Klen Taq/HK Taq pre-amplified under mineral oil tight conditions.
  • Excessively large primer fluorescence PCR can only be quantified by dilution digital method; containing E.coli UDG or RNaseA/I enzyme, only 5mM dUTP (final 0.125mM) and no Mg 2+ 10 ⁇ buffer and Taq, middle-order internal primer
  • the PCR solution is added to the surface of the mineral oil of the PCR tube. The tip of the tip should not contact the pre-expansion reaction solution.
  • the diluted pre-expansion solution is started by internal PCR hot mixing.
  • the second round of real-time fluorescent PCR reaction is 30-35 cycles, the second round. 50 before PCR 2-40 min digestion dU / RNA primer.
  • Root ⁇ "external primer degradable nested PCR", characterized by semi-nested PCR only one or one end of the set of different internal and external primers, the other end of the same sequence of primers; only one end of the external primer dU / RNA base modified in minerals Pre-amplification under oil-tight conditions to improve the sensitivity of the system; external primers and partial homologous reactions were prepared by pre-adding 2 ⁇ times Mg 2+ pre-amplification PCR and fluorescent primers containing UDG/RNase and no Mg 2+ internal primers. Primer strategies limit the non-specificity of fluorescent PCR within its primers.
  • the root primer "degradable nested PCR” is characterized by a special case of semi/nested PCR.
  • the distance between the inner and outer primers is also close.
  • the unilateral internal and external primers are close to or even several bases overlap, RNA/dU base.
  • the external sequence of the base modification serves as the set external primer, and the unmodified internal sequence is the set internal primer; the other side of the conventional internal and external primers, the two-round amplification of the nested PCR sensitization and the dU-UDG plus mineral oil closure thoroughly Eliminate the non-specificity of internal and external primer polymerization.
  • the “external primer degradable nested PCR” it is characterized by a set of gradient dilution simple digital quantitative PCR, and the sample is subjected to a ratio dilution in the EP tube, and the sampling sample is 1 ⁇ 10 0 to ⁇ 10 -9 and starts to be 10 ⁇ .
  • the sample to be tested is subjected to 10-fold serial dilution of 9 steps, and the dilution gradient of each step is distributed to 96-10000 reaction units by a reduction of 10 times volume.
  • a dimethylsiloxane microplate was used to perform two-way nested PCR digital quantification using an in situ PCR machine.
  • the method is characterized in that the method is used for a gene detection kit, and the kit components include: nucleic acid extraction reagent, dNTPs and dUTP, UDG, RNase I/A enzyme, Taq, HKTaq enzyme and buffer thereof Liquid, pre-expanded external primer F+R, internal primer F+R, dye EB and SYBR Green I, purified water dH 2 O, mineral oil.
  • kit components include: nucleic acid extraction reagent, dNTPs and dUTP, UDG, RNase I/A enzyme, Taq, HKTaq enzyme and buffer thereof Liquid, pre-expanded external primer F+R, internal primer F+R, dye EB and SYBR Green I, purified water dH 2 O, mineral oil.
  • Two-well amplification of nested PCR can detect the extremely high sensitivity of a single molecule, but the residual concentration of the external primer and the number of first-round cycles and product accumulation are the main reasons for the non-specific increase of subsequent primer primers; gel electrophoresis purification target product To remove the residual external primer and its product PD, or the first round of PCR product diluted 20-50 times or more to dilute the external primer and its PD is a common method to exclude the first round of PCR non-specific interference, but increased the risk of contamination after pre-expansion operation.
  • the UDG or RNaseA/I enzyme to efficiently digest the dU or RNA base in the nucleic acid, introduce the optimized primers containing the dU or RNA base primers and then add them to the UDG or RNaseA/I.
  • the oligonucleotide fragment below 8 bases does not increase PCR non-specificity at all, and the pre-expanded product is used as an in-house PCR template without degradation due to the use of dNTP (without dU), achieving a single tube reaction without pre-amplification purification or large dilution.
  • Two-step amplification of nested PCR in order to prevent leakage of contamination during the addition of the internal reaction solution, the internal expansion PCR component is used to double-add the external expansion reaction solution in advance, and the residual PCR solution is added to the residual liquid on the mineral oil layer.
  • Invalid amplification coupled with the in-house expansion of dU and UDG systems to prevent exogenous contamination of the product aerosol; the use of medial primers in the middle of the sequence further reduces endogenous PD non-specificity.
  • the "external primer degradable nested PCR" of the present invention is a single-tube two-step amplification PCR, and the external primer containing dU or RNA base (after the RNA base is placed after dC/dT) is pre-amplified by external PCR and then added by adding The internal PCR solution of UDG or RNaseA/I enzyme degrades the outer primer without affecting the subsequent inner amplification.
  • the primers are non-specific, and the modified base is placed at the 3' end/end of the outer primer and 1-3 in the middle, preferentially placed.
  • the 3' end is used to maximize the enzymatic hydrolysis of the external primer PD, and the second is placed in the middle to minimize the enzymatic fragment; the external primer is pre-expanded and the internal primer reaction solution is diluted 2-4 ⁇ times and UDGR and NaseA/I.
  • Internal amplification after enzymatic hydrolysis In order to prevent leakage of contamination during the addition of the internal reaction solution, the substrate of the internal primer PCR solution and the Mg 2+ were added to the pre-expanded PCR solution 2 ⁇ times to make the PCR solution lack Mg 2+ and the residual liquid on the oil layer was not amplified.
  • the buffer containing the UDG or RNaseA/I enzyme, only the substrate dUTP and no Mg 2+ and the Taq, the middle primer PCR solution are added to the surface of the mineral oil of the PCR tube (the tip of the tip should not contact the pre-expansion reaction solution)
  • the diluted pre-expanded solution was started by internal PCR hot mixing, and real-time fluorescent PCR was carried out for 30-35 cycles under mineral oil sealing. Two-round amplification nested PCR with a total reaction of more than 40 cycles ensures that there is no non-specific reaction between ⁇ 1" molecule and no template "0", and the target molecule can be extrapolated according to the dilution and dilution of the sample to zero target molecules. content.
  • the modified primers and conventional oligonucleotides containing dU or RNA bases in this patent are synthesized by Bioengineering Engineering (Shanghai) Co., Ltd., and the enzyme RNaseA is also purchased from Bioengineering Biotechnology (Shanghai) Co., Ltd.; enzymes E.Coli UDG and RNaseI It was purchased from NEB (Beijing) Co., Ltd.; the fluorescent PCR instrument was SLAN-96P (Shanghai Hongshi Medical Technology Co., Ltd.).
  • the test method uses fluorescent dye SYBR Green I real-time fluorescent PCR because its principle and operation are the most direct and simple, and the linear relationship and coefficient of variation CV are the best. It does not mean that the "external primer degradable nested PCR" of the present invention is limited to SYBR Green I. Real-time fluorescent PCR.
  • Enzyme UDG or RNaseA dilution gradient to modify the external primers a series of UDG or RNaseA gradient dilutions were added to the dU or RNA-containing primers (using the first example) direct fluorescent PCR test, according to the following 10 recipes to prepare PCR The solution, in which the template was added to the PCR solution 10 times at a time, with 5 ⁇ l of enzyme UDG or RNaseA gradient as the test target:
  • Enzyme RNaseA (original concentration 4mg/ml) dilution gradient corresponding to the amplification Ct value is shown in Table 2 below. As a result, RNaseA is diluted to 10-4 times to almost completely inhibit the amplification of RNA-containing primers, and 10 to 5 times still inhibits RNA primer amplification. increase.
  • the external primers were pre-expanded for 5, 10, 15, and 20 cycles, and the real-time fluorescent PCR of the primers was compared with the direct real-time fluorescent PCR Ct value comparison test to determine the required pre-amplification magnification.
  • the first round contains dU or RNA external primers.
  • General PCR Prepare 2 ⁇ pre-expanded external primer PCR reaction solution 10 ⁇ l/per reaction according to the following formula, add the same amount of sample target Template 10 ⁇ l per reaction.
  • the final concentration is 3.5 mM of Mg 2+ ; 50 ⁇ l of mineral oil is added to each tube and sealed without a hot lid to pre-amplify 5-20 different cycles.
  • the second round of primer SYBR Green I real-time fluorescent PCR 1 ⁇ target inner PCR reaction mixture was prepared according to the following formula, but the nested second round PCR primers were doubled, and the single-sided nested PCR was only doubled on one side; After the amount was added, it was a 1 ⁇ PCR reaction.
  • the inner PCR reaction solution (corresponding to the inner primer 2 ⁇ on the degradation side of the external primer and sharing the inner primer 1 ⁇ ), the tip of the sample tip should not contact the pre-expansion reaction solution, one tube is replaced with a tip, and the diluted pre-expansion solution is subjected to intra-PCR.
  • the second round of real-time fluorescent PCR which was annealed for 30 seconds and extended at 74 ° C for 30 seconds, was not amplified by hot lid.
  • comparing the Ct value of the original ⁇ 10 -3 pre-expanded 10-20 cycles is similar to the Ct value of the original PCR original ⁇ 10 -3 - the original ⁇ 10 -1 , and the pre-amplification is estimated to be 10, 15, and 20 cycles in turn.
  • Approximately 10 1 , 10 2 , and 10 3 magnifications are enlarged, and some templates are enlarged by 1-2 times.
  • the pre-amplification of 15 cycles of finite pre-amplification is suitable for sensitization of 30-35 cycles of primer-quantitative PCR; the external primer pre-expansion 20 cycles of pre-amplification variable is too large and the inner primer fluorescence PCR is preferably 30 cycles of digitization.
  • the contrast PCR of the sample target template was not added, and the same primers were pre-expanded for 0, 5, 10, 15, and 20 cycles, and the same internal primers were used for PCR to perform the non-cutting group background Ct value.
  • UDG and RNase I digestion groups are all straight-line non-amplified Ct values; see Figure 4 PCR background amplification curve, nested PCR for external primer degradation is not non-specific.
  • the second round of PCR reaction can change the dye SYBR Green I reaction, add 4 ⁇ L of ethidium bromide EB (0.5 ⁇ g/mL) to the mineral oil layer per well, and set the PCR tube or 96-well plate to develop color count under UV light of 254 nm wavelength. Detected by fluorophotometer; or by adding red and green molecular beacon probes for different wavelengths of wild and mutant genes in each tube or well after PCR reaction, quantitative detection of mutations in high wild background can be performed. The critical set of PCR products that were unclear for the 0-1 results were further tested by 1.5% Agarose gel electrophoresis.
  • Digital PCR (digital PCR), a new method for nucleic acid detection and quantitative analysis, can be used as an alternative to traditional real-time quantitative PCR to achieve absolute quantification and detection of rare alleles.
  • Digital PCR works by dividing a DNA or cDNA sample into a number of separate, parallel PCR reactions.
  • the initial constant concentration of Mg 2+ and some or all of the dNTPs substrate exceeds/is much lower than the concentration required for the PCR system to perform the predetermined PCR amplification those skilled in the art can determine the PCR system prepared by the skilled artisan based on their technical knowledge and The dNTPs substrate and Mg 2+ concentration required for the upcoming PCR amplification.
  • One skilled in the art can readily determine how many dNTPs are added to a particular PCR amplification system to form more than the desired concentration and below the desired concentration, where the concentration is zero, which is a special case well below the desired concentration.
  • the concentration of four kinds of dNTPs is 20 ⁇ mol/L, which can substantially satisfy the synthesis of 2.6 ⁇ g DNA or a 400 bp sequence of 10 pmol/L.
  • 2 ⁇ (or 2 times) external primer PCR reaction solution refers to the pre-dosed PCR reaction system, each component is added at twice the normal concentration, and it needs to be diluted 2 times before use to start the PCR reaction, usually Dilution is carried out by adding other ingredients such as a template, sterile water or the like.
  • 1 ⁇ (or 1 ⁇ ) internal primer PCR reaction solution refers to the pre-dosed PCR reaction system, each component is added at a normal concentration, without dilution, the PCR reaction can be directly initiated.
  • Single-sided nested PCR One primer sequence is shared by the lateral side of the outer primer pair and the corresponding side of the inner primer.
  • Central homologous primers 6-8 identical bases in the upper and lower primers.
  • the "initial concentration" of the various components of the PCR reaction solution means that the PCR reaction is started in the completed PCR system before the start of the PCR reaction.
  • the concentration of each component will gradually decrease or change during the PCR reaction and will not reflect the initial addition amount.
  • pUC19 vector plasmid purchased from NEB (Beijing) Company;
  • E.coli UDG Uracil-DNA-glycosylase, the original concentration of the reagent is: 5U/ ⁇ l, purchased from NEB (Beijing) Co., Ltd.;
  • RNase I ribonuclease I, purchased from NEB (Beijing) Co., Ltd., reagent stock solution, 5000 U/ml
  • RNaseA ribonuclease A, endonuclease, purchased from Bioengineering Biotechnology (Shanghai) Co., Ltd., with a reagent stock solution concentration of 4 mg/ml.
  • modified primers and conventional oligonucleotides containing dU or RNA bases of this patent are synthesized by Bioengineering Engineering (Shanghai) Co., Ltd.
  • the fluorescent PCR instrument uses SLAN-96P (Shanghai Hongshi Medical Technology Co., Ltd.).
  • test method uses fluorescent dye SYBR Green I real-time fluorescent PCR because its principle and operation are the most direct and simple, and the linear relationship and coefficient of variation CV are the best, but other types of real-time fluorescent PCR techniques can also be used in the present invention.
  • Figure 3 Fluorescence amplification curve of the number of pre-expansion cycles required for nested PCR: original template ⁇ 10 -3 pre-expanded 10-20 cycles of fluorescence amplification Ct values 25.71, 23.43, 18.59 and direct fluorescent PCR original ⁇ 10 - 3 —The original C10 values of ⁇ 10 -1 are approximated by 26.19, 22.80, and 19.04. The pre-amplification of 10, 15, and 20 cycles is approximately 10 1 , 10 2 , and 10 3 times in magnification, and the template is more magnified twice.
  • Hepatitis B HBV sensitization semi-nested fluorescent PCR gradient multi-dilution two-order amplification curve and Ct value are equivalent to direct fluorescence PCR two orders of magnitude amplification curve and Ct value, pre-amplification can be proportionally sensitized Fluorescent PCR.
  • Hepatitis B virus is a worldwide class III infectious disease infected by Hepatitis B virus (HBV). According to the World Health Organization (WHO), about 2 billion people worldwide carry hepatitis B virus. The prevalence of hepatitis B infection in our population is very high (nearly 10%), and liver cancer caused mainly by hepatitis virus has ranked first in the tumor, which greatly harms the health of the people.
  • the detection methods of hepatitis B mainly include 5 items/7 items of enzyme immunoassay, chemiluminescence method, immunofluorescence method, nucleic acid amplification (PCR) fluorescence quantification method and the like. Traditional enzyme immunoassay is widely used, but its sensitivity is insufficient. Real-time fluorescent PCR and digital quantitative PCR can accurately determine the viral load of hepatitis B patients, determine the level of viral replication in infected patients, and transmit infectious and antiviral drugs. Monitoring has an important role that cannot be replaced.
  • the hepatitis B virus (HBV) external primer degradable semi-nested PCR and the Hepatitis B virus (HBV) C region (1905-2498) sequence was cloned into the pUC 19 vector plasmid pHBc (MW 2.1 ⁇ 10 6 ) as a positive control sequence for hepatitis B virus.
  • the test does not affect amplification efficiency variation where a base to increase the "same sequence" number, 7b can be selected with the middle order Sex-reduced primer dimer PD non-specific nearly 10 Ct values, the sequence of the target primer pair and the outer primer pair are as follows: (the underline sequence is the middle order)
  • HBcR 5'–gat tga g at ctt at g cga c–3' (formerly c-person variation a)
  • the R primer can share HBcR or dU to modify HBcR.
  • Simple boiling lysis method take 50 ⁇ l-100 ⁇ l serum and add the same amount of boiling lysate (mix the microbeads thoroughly before use, cut the big mouth to absorb), mix gently, put in boiling water bath for 10 minutes, and then cool at 4 °C for a short time. After centrifugation for 10 minutes, 5 ⁇ l of the supernatant was taken. Or a large amount of the lysed supernatant can be further purified by the micromagnetic ball reagent.
  • Virus PEG precipitation method weakly positive specimens were precipitated with HBV by PEG. 500 ⁇ l of serum was added with 500 ⁇ l of 2 ⁇ PEG solution (16% w/v PEG & 0.7 M NaCl), mixed with Vortex, centrifuged at high speed for 10 minutes, and the supernatant was discarded 950 ⁇ l. After leaving 50 ⁇ l of the precipitate, add 50 ⁇ l of boiling lysate, and the same as above, boil, and take 5-10 ⁇ l of gradient dilution to add. This PEG precipitation recovery was 50%.
  • Hepatitis B virus HBV core antigen C gene cloning plasmid pHBc (0.1 ⁇ g/ml) was used as a template for 10 ⁇ (double) shaving dilution: 0.1 ⁇ g/ml ⁇ 10 -1 , ⁇ 10 -2 , ⁇ 10 -3 , ⁇ 10 -4 , ⁇ 10 -5 , ⁇ 10 -6 , ⁇ 10 -7 ,
  • the comparison gradient was diluted by 3 orders of magnitude to 0.1 ⁇ g/ml ⁇ 10 -4 -10 -7 pre-expansion (real-time fluorescent PCR (inner primer) after 15 cycles of external primer:
  • the first round of degradable external primer PCR reaction solution was prepared according to the nested PCR standard formula.
  • the external primers were pre-amplified for 15 cycles with mineral oil sealed.
  • the 1 ⁇ internal primer PCR reaction mixture was prepared according to the following formula, and the single-sided nested PCR only doubled the concentration of one primer (the double-sided PCR of the second round PCR primers must be doubled):
  • the pre-expanded tube was added with 20 ⁇ l of the inner PCR reaction solution containing UDG enzyme under the surface of the mineral oil (the tip of the sample was not exposed to the pre-expansion reaction solution), and the real-time fluorescent PCR of the Acer SLAN-96P fluorescence PCR instrument was carried out for 35 cycles.
  • Transgenic technology breaks through the limitations of natural resources, greatly improves the agricultural benefits and the output and quality of agricultural products. It also brings about the biosafety of genetically modified foods, and is increasingly receiving widespread attention from governments and the public in the world. Followinged by food The demand for quantitative detection of transgenic components and absolute quantitative detection of dPCR has also increased rapidly. Genetically modified crops have grown from major crops such as cotton, soybeans and corn to dozens of GM crop varieties; most of the genetically modified varieties are herbicide-resistant, insect-resistant, or herbicide-resistant and insect-resistant, and have also expanded to change. A series of new transgenic varieties such as linolenic acid, high lysine, delayed softening and antiviral.
  • the following partial sequence of the transgenic CaMV 35S promoter was selected (the CaMV 35S promoter sequence was numbered V100141.1 in Genbank, and the selected position was 7155-7334).
  • Positive control The above selected sequence was used as a template and cloned into the pUC 19 vector as a plasmid pCaMV control.
  • CaMV F 5'-gaa ggt g gc tcc t ac aa-3'
  • the solid plants were ground in liquid nitrogen, 0.2-0.3 g plus 1 ml of 2% cetyl ammonium bromide (CTAB) in a 65 ° C water bath for 20-60 minutes, and an equal volume of chloroform (1/24 isoamyl alcohol) was mixed. After centrifugation, the supernatant was extracted and 2 volumes of absolute ethanol were precipitated at -20 ° C. After centrifugation, the supernatant was discarded, and the DNA was dissolved in 50 ⁇ L of TE buffer.
  • CTAB cetyl ammonium bromide
  • Soybean oil sample DNA extraction column purification :
  • DNA extraction method Take the Golden Dragon Fish brand edible genetically modified soybean oil 10mL plus 10mL of n-hexane in a beaker for magnetic stirring for 2hr, add 20mL of PBS and continue to stir for 3hr, transferred to 50mL plastic Tube centrifugation 12000g ⁇ 20mins, carefully remove the lower aqueous phase, transfer a new 50mL tube and add an equal volume of isopropanol and mix, set -20 ° C for 20mins and then centrifuge 12000g ⁇ 20mins, discard the supernatant, precipitate plus dH 2 O 100 ⁇ L Dissolve, add 500 ⁇ L of binding buffer to the spin column, wash the column twice with the wash solution, and add 50 ⁇ L of TE to elute the DNA after the column is dried.
  • the purified sample DNA is subjected to a dilution quantitative nested PCR reaction.
  • the purified sample DNA is ⁇ 10 -1 , ⁇ 10 -2 , ⁇ 10 -3 , ⁇ 10 -4 , ⁇ 10 -5 , ⁇ 10 -6 , ⁇ 10 -7 , ⁇ 10 -8 , ⁇ 10 -9
  • Sequence 9 times dilution for 9 steps Take 9 1.5mL plastic EP tubes, add 18 ⁇ L purified water to each tube, take 2 ⁇ L sample solution to be added to the first tube water, and mix gently with the tip, this is the original solution ⁇ 10 -1 times, take 2 ⁇ L of 10 times diluted solution from the first tube into the second tube water, and lightly mix it into the original solution ⁇ 10 -2 times, change one nozzle per tube, the original solution ⁇ 10 -3 ... and so on...
  • Stock solution ⁇ 10 -9 Take 9 1.5mL plastic EP tubes, add 18 ⁇ L purified water to each tube, take 2 ⁇ L sample solution to be added to the first tube water, and mix gently with the tip, this is the original solution ⁇ 10 -1 times,
  • the PCR product tube was read by a fluorescence detector (Xi'an Tianlong Company) to read the relative fluorescence absorption value.
  • the original solution ⁇ 10 -2 to ⁇ 10 -4 tube fluorescence value > 3000 is determined as: 1 target molecule / 10 ⁇ L sample; the original liquid ⁇ 10 -5 to ⁇ 10 -9 tube fluorescence value ⁇ 50 is negative, that is, 0 target molecules /10 ⁇ L sample.

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Abstract

本申请提供了一种酶解代替纯化的单管两轮PCR，其特征是引物末端至中部含1个或以上dU及RNA碱基的外引物PCR于矿物油密闭下预扩放大后，其RNA/dU外引物被加入UDG及RNA酶的内引物荧光PCR酶解，以及预设外引物PCR液中过量底物和Mg2+使内PCR液不用含Mg2+，加内PCR液时矿物油层面上残留液无Mg2+而无效扩增，加上内扩UDG系统防止产物气雾胶外源污染；并采用中部同序的内引物策略进一步减少内源PD非特异性，确保≥"1"分子有效检测和"0"无扩增反应的数字dPCR方法。

Description

外引物可降解的巢式PCR 技术领域：
本发明属于分子生物学及分子检验的核酸扩增PCR技术领域，具体涉及修饰外引物预扩增来提高灵敏度及修饰引物酶解而限制其非特异性的单管巢式PCR领域。
背景技术：
多聚酶链反应PCR背景可追溯至其历史发展源头，1953年Watson建立DNA双螺旋模型及半保留复制法则为PCR扩增奠定了其分子基础，甚至1971年Khorana就已发表了一核酸体外扩增(J.Molec.Biol.,56:341)相关论文；但直到1985年美国Cetus公司Mullis想到了PCR本质-指数扩增或几何级数式放大的巨大威力,才发明了一对特异引物结合、复制靶分子基因,体外(于试管内)模拟天然基因半保留复制的PCR技术，再经过一系列发展和耐热聚合酶、热循环仪的发明、应用，Cetus公司于1987年申请了首个PCR发明专利(US Patent 4,683,202)。扩增过程一般经靶DNA变性：加温94℃使双链解离成为单链；引物退火(复性)：降温至54℃左右使过量引物与模板DNA单链的互补序列配对结合；引物延伸：升温至72℃左右，DNA单链模板—引物结合物在耐热DNA聚合酶的作用下，以dNTP为反应原料，靶序列为模板，遵循碱基反向配对与半保留复制原理，引物末端按5'-3'方向延伸、合成一条新的与模板DNA链反向互补的半保留复制链,不断重复循环变性--退火--延伸三过程，这种新链又可成为下次循环的模板，靶分子以2n级数产生更多的“半保留复制”新链。
超级灵敏度的指数放大或几何级数放大是PCR最核心的本质或优点，靶分子以2n级数放大,常规30个热循环反应230约等于109放大倍数,扩增放大倍数似原子裂变链式反应难以想象,也是一般线性放大检测方法无可比拟的,灵敏度远远大于普通检测方法的放大倍数,近40个循环反应可达飞克fg/ml或个位数分子水平检测。这也是PCR成为应用最广的最核心技术甚至多少个最形容也绝不过分的根本原因。同样，指数放大亦带来PCR高度特异性和顽固的非特异性，PCR特异性直接来源于引物序列的特异性,一端引物序列一点非特异错配扩增甚至完全配对扩增也仅是线性放大,自然远远赶不上指数扩增或几何级数放大，引物特异性任何一点降低或与靶一丁点不匹配导致扩增速率落下了,而远远赶不上特异的指数或几何级数放大；反过来说,只要是靶指数扩增或几何级数放大，PCR扩增检测就具有足够高特异性。非特异性同理，各种常规检验方法遇见的非特异性原因虽也存在于PCR技术,但终究赶不上指数扩增放大，仅一条引物非特异结合线性扩增远远赶不上指数非特异性,只有一对引物均非特异结合、 延伸的扩增才是唯一指数非特异性原因,引物间互为模板、互为引物的二聚体PrimerDimer(PD)指数扩增是PCR非特异性本质,也是其难以克服的根本原因。一般3'末端数个碱基连续反向互补的引物对于不加模板的本底PCR扩增反应中约几个—十几个循环处即出现引物二聚体PD扩增；一对常规设计的引物于本底PCR反应30个循环开始出现显著的PD非特异性扩增，并遮盖位于30-38循环的数千以下靶分子的低浓度样本检测，这也是普通PCR只进行30个循环反应的主要原因。
然而飞克fg/ml及个位数靶分子水平检测需要扩增至38个循环,为了解决30个循环PCR方法灵敏度仍不足的局限，随后开始出现外侧与内侧引物两步扩增的巢式PCR(J Med Virol,30-2:85)，其采用外侧引物预扩增15-30个循环来提高灵敏度，再加以内侧引物继续扩增25-30个循环以超过40个循环指数扩增的极高灵敏度甚至为单个靶分子检出提供了可能；且随着巢式PCR新反应成份的补充，也推迟了从指数形式或对数期进入所谓“停滞效应”平台期的时间，保持了2n级数的几何扩增效率。然而巢式PCR外侧引物增加了系统复杂性进而增加了两轮PCR非特异性，外引物的残留促进内侧引物非特异扩增，将一般常规引物本底Ct值30个循环的非特异性提升至Ct值﹤25个循环以前的本底非特异性；巢式或两轮PCR增加的非特异性抵消了第一轮或外侧引物预扩增的放大作用。预扩增需要取出至少稀释20-50倍以上或通过凝胶电泳纯化靶产物以除去外侧引物的影响才能消除系统增加的非特异性，不但增加操作复杂性而且PCR后处理亦带来产物气雾胶交叉污染风险。
随后其它许多PCR新方法，新改进也不断涌现,发明，包括反转录RT-PCR,原位PCR,连接酶链反应(LCR)，标记PCR(Labeled primers-PCR)，反向PCR(reverse PCR)，不对称PCR(asymmetric PCR)，降落PCR(touchdown PCR)，重组PCR(recombinant PCR)，多重PCR(multiplex PCR)，免疫-PCR(immuno-PCR)，mRNA差异PCR，链替换扩增(SDA)，依赖核酸序列的扩增(NASBA)，转录依赖的扩增系统(TAS)，Qβ复制酶(Q-beta replicase)催化RNA扩增,滚环扩增(RCA),环介导的等温扩增(LAMP)等。由于常规终末PCR因同样量的样本经PCR超级放大以后变化太大而产量不均一，致使难以定量检测，传统的终末/终点PCR只能PCR后凝胶电泳来区分特异与非特异扩增条带；1997年采用动态实时检测扩增对数期的循环数与靶初始量成反比(反对数关系)的实时荧光PCR(Real-time PCR)技术实现了从定性测定到精确定量的飞跃，根据发光原理不同分为荧光染料SYBR Green Ⅰ实时荧光PCR和各种荧光核酸探针PCR两大类(BioTechniques 1997,22:130-138)及巢式荧光PCR(Malaria Journal 2014,13:393)。染料法实时荧光PCR通过SYBR Green Ⅰ结合产物DNA双链小沟使发射荧光增强1000倍而检测，但非特异性扩增产物也结合发射荧光，同样30个循环后非特异扩增也遮盖了低浓度样本检测；探针法TaqMan等实时荧光PCR的靶特异探针不针对引物序列而绕 开了PD非特异性产物,但引物二聚体PD扩增于探针法荧光PCR不可见并不代表其不存在、不起作用，较高的Ct 30PD扩增竞争性抑制同引物的低浓度靶扩增而降低其检测灵敏度；另一方面,过量引物与探针DNA链之间也产生一定程度的非特异聚合、延伸的扩增，不加靶模板的PCR本底系统,一对引物与任意一个荧光标记TaqMan探针均产生约Ct 33-35的非特异性扩增曲线，且随着反复重复同一PCR扩增,其扩增平台期荧光吸收峰值越来越高,Ct值也逐渐前移，产生一定的样本检测假阳性。目前实时荧光PCR有限灵敏度和一定非特异性仍妨碍了极低浓度样本检测或单分子检出。
定量检验方法很早就出现过稀释定量法概念，即通过样本倍比稀释靶分子直至无靶分子反应再算稀释倍数的倍比稀释定量方法；进一步出现了基于样本有限稀释和泊松分布计数的数字化定量PCR理念。即确保阳性单个分子或单个以上分子均有检测信号而定为1、阴性绝对无反应信号而定为0的条件下，样本稀释、分散至大量的微型分隔单元使理论上每检测单元含0-1个靶分子，通过检测单元反应信号0-1计数和有限稀释倍数而对靶分子进行定量的方法,以便于计算机0或1模式的自动化操作,但前提必须是检测方法本身对1个分子或0分子可靠区分。然而传统的化学反应、酶免疫反应等均是检测信号强度与待测分子含量成线性比例且信号简单相加的纳克级低灵敏度检测方法，线性传统低灵敏度方法远远区分不了1个靶分子和0分子的差别，所以无法适用于有限稀释法的数字化定量检测方法。1992年,Sykes等人(Biotechniques13:p444)初步尝试了基于样本有限稀释和泊松分布计数的定量巢式PCR,并首次提出了数字化定量理念，但由于这类PCR系统内多引物聚合体扩增和体系外气雾胶交叉污染的严重非特异性而抵消了二次扩增放大作用。
限制数字PCR其它非特异性原因众多,大多研究焦点集中于PCR热启动来克服可能的引物低温结合所致非特异扩增，措施包括蜡包Mg2+离子热释放、聚合酶Taq修饰抑制包括端N缺失的KlenTaq,抗Taq酶抗体和Taq酶抑制寡核苷酸Aptamar,以及四氧化戊烷热激活引物等热启动方法。但低温启动最多也仅扩增一个循环放大一倍/一次,远赶不上非特异指数放大。且PCR在热变性后才手动加完全组份的绝对热启动本底Ct值也仅推后1-3个循环数，低于40℃度时Taq酶活性很低,低温启动不是PD非特异性形成的关键原因。试验使用各种控制非特异的措施如变化PCR组份、添加各种化学试剂等,其对特异与非特异扩增基本是平行的,显著抑制PCR非特异性也不同程度地干扰靶特异性扩增效率,反之不影响靶扩增效率又难以抑制PCR非特异性。引物引起的指数非特异性还是源于其碱基序列,一对完全同序引物绝没有引物非特异性。Hands技术(Homo-Tag assisted non-dimer system,Nucleic Acids Res.Vol.25,No16:p3235-3241)采用完全同序引物Tag通过引物二聚体单链两端自身结合竞争游离引物,不仅显著抑制PD非特异性,也都没有选择地减低靶特异性扩增效率。单链结合蛋白Single Strand  Binding-protein(SSB)、基因32蛋白和全引物结合反义碱基Oligo能显著降低优化的引物非特异性,但也严重地影响靶特异性扩增效率及扩增曲线的线性关系。
中国专利(CN 201010105371.8和PCT/CN2013/088054)根据一对完全同序引物绝没有非特异性现象，采用部分同序引物部分抑制PD扩增,将“同序”置于引物中部偏3'端能最大化地选择性抑制PD扩增而不影响靶扩增效率；PCR添加与引物中部同序部位配对结合的含反义碱基Oligo,因其仅保留结合功能而没有模板和引物作用,能进一步特异选择性地放大这种作用。在无末端反向互补常规引物设计原则基础上,选择中部部分同序引物对和结合中部反义碱基Oligo于实时荧光PCR 40个循环反应内消除内源PD非特异性扩增干扰；内源非特异性PD气雾胶持续成为后续PCR交叉污染模板，一般采用dU代替dT底物来形成含dU产物，水解dU的UDG酶降解dU产物气雾胶。
深一步Vogelstein & Kinzler于1999年报导微升级96孔板的致癌突变基因ras定量PCR及首创数字(digital)PCR概念(PNAS,USA 96:9236),d-PCR是一种基于单分子PCR方法来进行计数的第三代绝对定量PCR方法,采用分析化学领域的微流控或微滴化方法,将大量稀释后的核酸溶液分散至微反应器单元或微滴中,每个反应单元的核酸模板数少于或等于一个。经过PCR热循环反应之后,扩增产物与加入的荧光探针杂交,有一个核酸分子模板的反应单元有扩增而给出荧光信号,没有模板的反应单元没有扩增而无荧光信号。根据稀释比例和反应单元体积,就可以推算出原始溶液的核酸浓度,通过计数及泊松分布统计，就可以实现起始DNA模板的绝对定量。近年来缩微至微流控万级(即纳升)乃至百万级反应单元(皮升级)体积装置(Anal.Chem.2011,83:8604)的成熟而d-PCR开始走向应用。
所以，常规PCR及实时荧光PCR对单分子检测灵敏度仍稍显不够，需要预扩增放大来进一步提高系统灵敏度；但PCR又限制于非特异性对低浓度样本检测的遮盖，超敏PCR甚至单分子有效检测取决于PCR增敏和非特异性去除两个方面。本发明“外引物可降解的巢式PCR”采用末端含RNA碱基或dU碱基外引物于外侧预扩增后UDG或RNA酶降解策略而不增加随后的内侧扩增内引物聚合非特异性，外侧引物预扩近15个循环的有限预放大可增敏内引物荧光定量PCR；外引物预扩20个循环放大变化过大而内引物荧光PCR数字化法定量。两轮双扩增的巢式PCR以及UDG和矿物油封闭来彻底杜绝引物二聚体PD非特异性扩增；巢式双扩增PCR数字定量方法可作为重要的临检样本、核酸参考品和科研样本的手动/手工d-PCR方法,同时为更快捷、高效超级单元微囊/微滴化的微流控装置的研制提供前期研究工具和发展思路。
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发明内容
本发明“外引物可降解的巢式PCR”采用含dU或RNA碱基外引物于外侧预扩增后UDG或RNA酶降解策略而不增加随后的内侧扩增内引物聚合非特异性，外侧引物预扩10-15个循环的有限预放大可增敏内引物荧光定量PCR；外引物预扩15-20个循环放大变化过大而内引物荧光PCR数字化法定量。两轮双扩增的巢式PCR以及UDG和矿物油封闭来彻底杜绝引物二聚体PD非特异性扩增；巢式双扩增PCR数字定量方法可作为重要的临检样本、核酸参考品和科研样本的手动/手工d-PCR方法,同时为更快捷、高效超级单元微囊/微滴化的微流控装置的研制提供前期研究工具和发展思路。
本发明提供的技术方案概括如下：
外引物可降解的巢式PCR，其特征在于：在同一个反应管/孔中完成以下步骤：
(1)外引物扩增：在反应管/孔中加入外引物PCR反应液，并加入矿物油对PCR反应液进行密封，完成预定扩增程序；其中所述外引物PCR反应液中采用的至少一侧外引物的末端至中部含有1个以上dU碱基和/或RNA碱基修饰，所述外引物PCR反应液中使用Taq、K Taq或H.K Taq,Mg2+和部分或全部dNTPs底物的初始终浓度超出外引物PCR体系完成预定的PCR扩增程序所需的浓度；超出量用于该反应管/孔中下一步进行的内引物扩增；
(2)所述内引物扩增：于完成上述外引物扩增的反应管/孔的矿物油表层下加入内引物PCR反应液，使外引物PCR反应液被稀释2-4倍体积，加入的所述内引物PCR反应液中含有UDG和/或RNA酶，Mg2+和部分或全部dNTPs底物的终浓度远低于内引物PCR体系完成预定的 PCR扩增程序所需的浓度；启动内引物PCR扩增前，先在50℃下消化2-10分钟以降解所述外引物。
优选地，所述内引物PCR反应液中采用的内引物为中部同序引物。
优选地，其中所述外引物PCR反应液中采用的引物至少一侧引物末端至中部含有1-3个dU碱基和/或RNA碱基修饰。
优选地，所述外引物PCR反应液中仅单侧外引物含有所述dU碱基和/或RNA碱基修饰；其另一侧内引物及其同侧外引物共用同一序列引物。
上述任一所述的外引物可降解的巢式PCR，其特征在于，所述外引物3'末端倒数1-2碱基位置具有1个dU碱基或RNA碱基修饰，在引物中部设置有第2、3个碱基修饰。
上述任一所述的外引物可降解的巢式PCR，其特征在于：所述外引物PCR反应液中dA、dG、dC的初始终浓度是dT的2倍，Mg2+的初始终浓度为7mM。
优选地，加入的所述内引物PCR反应液中，不含Mg2+和常规底物dA、dG、dC，仅含dUTP，所述dUTP的浓度和体积与外引物PCR反应液中加入的dT相当。
优选地，所述内引物PCR反应液中含有的UDG酶，在混合而成的总反应液中的初始终浓度为0.0125-0.0625U/μl，即原浓度5U/μl稀释80倍至400倍；
含有的RNaseA酶在混合的总反应液中的初始浓度为0.4-40ug/ml，即原浓度4mg/ml稀释102-104倍，或含有的RNaseI在混合的总反应液中的初始浓度为0.031U/μl。
优选地，所述的外引物可降解的巢式PCR中，
(1)外引物扩增：先加入10μL 2倍浓度的所述外引物PCR反应液于管底，并加10μL稀释的样本液，再沿管壁加入50μL矿物油层密闭，进行15-20个循环的普通PCR外扩增；
(2)内引物扩增：于上一轮的反应管中加20μL的1倍浓度的所述内引物PCR反应液，其中可降解侧外引物同侧的内引物浓度是其它引物的2倍，进行30-35个热循环荧光PCR内扩增。
优选地，用于进行简易数字定量PCR，其中所述待测样本为原液被稀释为原液×10-1、×10-2、×10-3、×10-4、×10-5、×10-6、×10-7、×10-8、×10-9的9个浓度梯次的稀释液，取×10-2、 ×10-3、×10-4、×10-5、×10-6、×10-7、×10-8、×10-9的待测样本稀释液以及阳性对照以及无菌水作为模版分别分配至原位PCR仪进行所述外引物扩增和所述内引物扩增。
优选地，内引物扩增30-35个循环后，每孔加入0.5μg/mL的EB2-5μL于矿物油层下面,置反应管于紫外灯下显色计数或于荧光光度仪检测。
所述的外引物可降解的巢式PCR用于检测乙型肝炎病毒，内引物对为：
HBcF:5'—aat gcc cct atc tta tca a–3'，
HBcR:5'–gat tga gat ctt atg cga c–3'，其中第13位的a为c人工变异设计而来，
外引物对为：WhBcF:5'–gtg gat tcg cac Ucc Tc–3'，
反向引物为HBcR或dU修饰的HBcR。
一种通过检测启动子CaMV 35S进行食品转基因检测的方法，其特征在于：上述任一所述的方法，所述内引物为:
CaMV F：5'-gaa ggt ggc tcc tac aa-3'，
CaMV R：5'-tcc acg atg ctc ctc gt-3'；
外引物为：
WCaMV F：5'-tca ctt tat tgt gaa gaU tag U-3'，
WCaMV R：5'-cca ctt gct Utg aag acG t-3'。
一种用于进行上述任一所述外引物可降解的巢式PCR方法或所述的食品转基因检测的方法的试剂盒，其特征在于：包含所述外引物和、内引物。
优选地，所述的试剂盒，还包含用于所述外引物扩增的预配好的所述外引物PCR反应液和用于所述内引物扩增的预配好的所述内引物PCR反应液。
本发明“外引物可降解的巢式PCR”，其特征是样本经引物末端至中部含1个或以上dU碱基和/或RNA碱基的外引物PCR于矿物油密闭下预扩增后，其含dU和/RNA外引物PCR被加入的含UDG和RNA酶的内引物荧光PCR酶解的单管两轮巢式PCR，通过外引物水解来代替凝胶电泳纯化或大量20-50×稀释以消除外引物残留对内引物聚合非特异性影响，以及预设的外引物PCR液中过量底物dNTP和Mg2+使内引物PCR液不用含Mg2+，加内引物PCR反应液时矿物油层面上残留液无Mg2+而无效扩增，内引物PCR液中采用底物dU代替dT加 上UDG酶解系统防止产物气雾胶外源污染。并采用中部同序的内引物策略进一步减少内源PD非特异性。
根椐“外引物可降解的巢式PCR”，其特征是将dU/RNA碱基置于外引物3'末端/末端和中部1-3个,所述外引物1-3个修饰碱基首选引物3'最末1-2碱基位置1个修饰碱，第2、3个修饰碱基则散布引物中部，外引物对至少有一条3'最末端含dU/RNA或上下游外引物3'最末端均含1个dU碱基；外引物PCR液含过量的dNTP,例如2×dNTP(dA、dG、dC 0.25mM,其中dT0.125mM)和Mg2+(3.5mM)及常规Taq/Klen Taq/H.K Taq于矿物油密闭条件下预扩增，第一轮外引物预扩12-15个循环的有限预放大可作增敏的荧光定量PCR，外引物预扩18-20个循环放大变量过大而内引物荧光PCR只能稀释数字法定量；含E.coli UDG或RNaseA/I酶、仅5mM dUTP(终0.125mM)和无Mg2+10×buffer及Taq、中部同序的内引物PCR液等比加入PCR管矿物油表层下，吸头尖不要接触预扩反应液，稀释预扩液经内PCR热混匀启动，第二轮实时荧光PCR反应30-35个循环，第二轮PCR前50℃2-40分钟消化dU/RNA引物。
根椐“外引物可降解的巢式PCR”，其特征是半巢式PCR仅单侧或一端设置内外不同序列引物，另一端共用同一序列引物；仅一端外引物dU/RNA碱基修饰于矿物油密闭条件下预扩增来提高系统检测灵敏度；通过预加2×倍Mg2+预扩PCR和含UDG/RNA酶和无Mg2+的内引物荧光PCR酶解外引物和部分同序内引物策略限制其内引物荧光PCR非特异性。
根椐“外引物可降解的巢式PCR”，其特征作为半/巢式PCR一个特例，内外引物距离也可接近，极端例子单侧内外引物靠近乃至数个碱基重迭，RNA/dU碱基修饰的外序列作为设定的外引物，无修饰饰内序列为设定的内引物；另一侧常规内外引物，两轮扩增的巢式PCR增敏和dU-UDG加矿物油封闭来彻底杜绝内外引物聚合非特异性。
根据“外引物可降解的巢式PCR”，其特征是样本一组梯度稀释的简易数字定量PCR，样本于EP管进行倍比梯度稀释，取样本1×100～×10-9开始10×(即10倍)稀释5-10次或再2×(即等比)稀释5-10个梯度，确保有靶分子管稀至无靶分子管的转折点落在梯度范围内，外引物dU碱基修饰于矿物油密闭条件下预扩增15-20个热循环第一轮PCR无热盖扩增；于第一轮同一管中加含UDG和无Mg2+的内引物荧光PCR液,用加样吸头插入矿物油表层下面加入,进行30-35个热循环第二轮荧光PCR无热盖扩增。如第二轮PCR反应后每孔加入2-5μL的EB(0.5μg/mL)于矿物油层下面,置反应管于紫外灯下显色计数或于荧光光度仪检测。
根据“外引物可降解的巢式PCR”，其特征是待测样本进行9个梯次的10倍连续稀释后每个梯次的稀释梯度以缩小10倍体积等量分配至96-10000个反应单元聚二甲基硅氧烷微孔芯片，使用原位PCR仪进行两轮巢式PCR数字定量。
根据“外引物可降解的巢式PCR”，其特征在于其方法用于基因检测试剂盒,盒成份包括： 核酸提取试剂，dNTPs及dUTP，UDG、RNaseI/A酶，Taq，HKTaq酶及其缓冲液，预扩外引物F+R，内引物F+R，染料EB及SYBR Green I，纯化水dH2O，矿物油。
多聚酶链反应PCR指数扩增或几何级数放大带来检测超级灵敏度，但对数个甚至单个分子检出仍稍显灵敏度不够；同时指数或几何放大亦带来本底位于30个循环的引物二聚体非特异性扩增，导致PCR产物气雾胶作为二次模板再污染，即使矿物油密闭PCR反应情况下油层面上残留微量反应对指数放大PCR也是难以克服的交叉污染。巢式PCR两轮扩增放大能检出单个分子极高灵敏度，但外引物的残留浓度和首轮循环次数及产物累积依次是后续内引物PCR非特异性增加的主要原因；凝胶电泳纯化靶产物以除去残留外引物及其产物PD，或首轮PCR产物稀20-50倍以上以稀释外引物及其PD是排除首轮PCR非特异性干扰的常用方法，但增加了预扩后操作污染风险。根据UDG或RNaseA/I酶高效消化核酸中dU或RNA碱基的现象，引入含dU或RNA碱基外引物预扩增后被加入UDG或RNaseA/I的优化内引物PCR液消化、水解，试验8碱基以下寡核苷酸片段完全不增加PCR非特异性，预扩产物作为内扩PCR模板因使用dNTP(不含dU)而不降解，实现无需预扩后纯化或大量稀释的单管反应两步扩增巢式PCR；为防止加内反应液时泄露污染，采用内扩PCR成分预先以双倍量加入外扩反应液，加内PCR液时因缺完全成分而矿物油层面上残留液无效扩增，加上内扩dU及UDG系统防止产物气雾胶外源污染；采用中部同序的内侧引物进一步减少内源PD非特异性。
本发明“外引物可降解的巢式PCR”为单管两步扩增PCR，采用含dU或RNA碱基(RNA碱基置于dC/dT之后)外引物于外PCR预扩后通过加入含UDG或RNaseA/I酶的内PCR液使外引物降解而不影响随后的内侧扩增内引物聚合非特异性，修饰碱基置于外引物3'末端/末端和中部1-3个，优先置于3'末端以使外引物PD也能最大程度酶解，第二置于中部使酶解片段最小；外引物预扩后加内引物反应液小量稀释2-4×倍及UDGR及NaseA/I酶解后进行内扩增。为防止加内反应液时泄露污染，通过内引物PCR液的底物和Mg2+预先2×倍加入预扩PCR液使加内PCR液时缺Mg2+而油层面上残留液无效扩增，外引物PCR加2×倍dNTP(其中dT 1×)和2×Mg2+及Klen Taq(/HK Taq)于矿物油密闭条件下预扩增，外侧引物预扩12-15个循环的有限预放大可增敏内引物荧光定量PCR；外引物预扩18-20个循环放大变量过大而内引物荧光PCR最好数字化法定量；预扩超过20个循环则非特异PD急剧增加而无必要。含UDG或RNaseA/I酶、仅底物dUTP和无Mg2+的buffer及Taq、中部同序的内引物PCR液等比加入PCR管矿物油表层下(吸头尖不要接触预扩反应液)，稀释预扩液经内PCR热混匀启动，于矿物油密闭下实时荧光PCR 30-35个循环。总反应大于40个循环的两轮扩增巢式PCR确保≥“1”分子有效检出和无模板“0”无非特异性反应，根据样本稀释至零个靶分子的倍数、体积可推算靶分子含量。
在本专利“外引物可降解的巢式PCR”应用检测前，首先需要试验出UDG或RNaseA酶作用范围和增敏扩增或数字PCR定量所需的预扩循环数。
本专利含dU或RNA碱基的修饰引物和常规寡核苷酸由生工生物工程(上海)有限公司合成，酶RNaseA亦购自生工生物工程(上海)有限公司；酶E.Coli UDG及RNaseI购自NEB(北京)公司；荧光PCR仪器采用SLAN-96P(上海宏石医疗科技有限公司)。试验方法采用荧光染料SYBR Green Ⅰ实时荧光PCR是因为其原理及操作最直接简便而线性关系及变异系数CV最好，并不代表本发明“外引物可降解的巢式PCR”仅限于SYBR Green Ⅰ实时荧光PCR。
酶UDG或RNaseA稀释梯度作用修饰外引物的试验：将一系列UDG或RNaseA梯度稀释液分别加入含dU或RNA的引物(实例一中采用的)的直接荧光PCR试验，按以下10次配方制备PCR液，其中模板一次加入10次PCR液，以5μl酶UDG或RNaseA梯度液为试验目标：
Figure PCTCN2017114762-appb-000001
取UDG或RNaseA(依次按下表1和表2的梯度稀释液)5μl加入PCR管，再依次加入20μL/管的PCR反应液,再分别加入30μl/管的矿物油,进行50℃2分钟后94℃变性2分钟，40个热循环94℃变性20秒、54℃退火30秒、72℃延伸30秒的荧光PCR无热盖扩增，扩增曲线分别见附图1和附图2，UDG(原浓度5U/μl)稀释梯度对应扩增Ct值见下表1,结果UDG稀释至10-2倍完全抑制含dU引物扩增，至10-3倍仍小部分抑制dU引物扩增。
表1 UDG(原浓度5U/μl)稀释梯度对应扩增Ct值
Figure PCTCN2017114762-appb-000002
酶RNaseA(原浓度4mg/ml)稀释梯度对应扩增Ct值见下表2,结果RNaseA稀释至10-4倍几乎完全抑制含RNA引物扩增，至10-5倍仍大部分抑制RNA引物扩增。
表2 酶RNaseA(原浓度4mg/ml)稀释梯度对应扩增Ct值
Figure PCTCN2017114762-appb-000003
增敏预扩循环数或数字定量所需预扩循环数试验：
同样靶标样本，外引物预扩5、10、15、20个循环后内引物实时荧光PCR对比直接实时荧光PCR的Ct值比较试验，测定所需预扩放大倍数。按以下外引物可降解的巢式PCR配方配液，第一轮含dU或RNA外引物普通PCR:按以下配方制备2×的预扩外引物PCR反应液10μl/每反应,等量加样本靶模板10μl/每反应。
Figure PCTCN2017114762-appb-000004
※终浓度3.5mM of Mg2+ ；每管加50μl矿物油密闭后无热盖预扩增5-20不同循环数。
第二轮内引物SYBR Green I实时荧光PCR:按以下配方制备1×靶内侧PCR反应混合液,但巢式第二轮PCR引物须双倍，单侧巢式PCR仅一侧引物双倍；等量加入后为1×PCR反应。
Figure PCTCN2017114762-appb-000005
Figure PCTCN2017114762-appb-000006
※10×Taq buffer：0.6M Tris-Cl(pH8.3),100mM KCl,50mM(NH4)2SO4。
为防止加样时残留液扩增泄露，从管底依次加10μl修饰外引物及HK Taq预扩2×倍PCR反应液(终浓度dNTP2×但dT 1×)，等量10μl纯化样本，沿管壁加50μl矿物油密闭下普通PCR预扩10-15个循环；再小心吸头于矿物油表层下加入等比20μl含UDG酶及仅底物dUTP和无Mg2+的buffer及Taq的1×倍内侧PCR反应液(对应外引物降解侧的内引物2×而共用内引物1×)，加样吸头尖不要接触预扩反应液，一管换一吸头，稀释预扩液经内PCR热混匀启动，于矿物油密闭下实时荧光PCR50℃2分钟后再94℃预变性2分钟,进行30-35个热循环94℃变性20秒、54-60℃
退火30秒、74℃延伸30秒的第二轮实时荧光PCR无热盖扩增。
结果采用靶模板质粒(pHBc)稀释梯度：原、原×10-1、原×10-2、原×10-3、无pHBc直接实时荧光PCR对应的Ct值19.04、22.80、26.19、31.46、—(无)；
将同样量pHBc原×10-2、原×10-3巢式荧光PCR预扩5-20循环后所对应的Ct值见表3：，扩增曲线见附图3。
Figure PCTCN2017114762-appb-000007
所以，对比原×10-3预扩10-20个循环的Ct值与直接PCR原×10-3—原×10-1的Ct值类同，推算预扩增10、15、20个循环依次约放大101、102、103倍，较浓一些模板多放大1-2倍。预扩近15个循环的有限预放大适合增敏30-35个循环内引物荧光定量PCR；外引物预扩20 个循环预放大变量过大而内引物荧光PCR最好30个循环数字化法定量。
而同样PCR反应条件下，不加样本靶模板的对比PCR，同样外引物预扩0、5、10、15、20个循环后，相同的内引物荧光PCR进行非酶切组本底Ct值为35.0、32.0、27.5、21.5、18，随预扩循环数增加的非特异性反应遮盖靶特异性扩增；UDG和RNase I酶切组全部为直线无扩增Ct值；见说明书附图4巢式PCR本底扩增曲线，外引物降解的巢式PCR无非特异性。
在应用数字PCR于微流控或微滴化装置之前，先行手动倍比稀释定量PCR。首先样本一组梯度稀释的简易数字定量PCR，样本于EP管进行倍比梯度稀释，确保有靶分子管稀至无靶分子管的转折点落在梯度范围内。因此，判断浓度较稀的样本先10×(倍)稀释几(5)个梯度再2×(等比)稀释5次；而对于大多数生物样本先从样本×10-2-×10-3开始10×(倍)稀释检测再确定范围2×(等比)稀释检测。并改进传统的两轮PCR操作方式,设计第一轮预扩可降解的外引物和第二轮内侧引物,于PCR单管两轮PCR反应,第一轮PCR每管先加入10μL的外引物2×PCR反应液于管底尖，和加10μL稀释样本液,再轻轻沿管壁加入50μL矿物油层密闭,进行15-20个循环的普通PCR外扩增；第二轮PCR于上一轮同一孔中加20μL的1×内侧PCR(降解侧引物2×)反应液，加样枪吸头插入矿物油表层下小心加入，进行30-35个热循环荧光PCR内扩增。配合矿物油密闭隔绝PCR系统外气雾胶交叉污染和内引物PCR的UDG系统去除残留气雾胶污染。
d-PCR终产物荧光显色及计数定量：
第二轮PCR反应可改染料SYBR Green I反应后每孔加入4μL的溴化乙锭EB(0.5μg/mL)于矿物油层下面,置PCR管或96孔板于254nm波长紫外灯下显色计数或于荧光光度仪检测；或PCR反应后每管或孔加入针对野生与突变基因不同波长的红绿两种分子信标探针可进行高野生背景下突变定量检测。对于0-1结果判断不清楚的临界组PCR产物可进一步进行1.5％Agarose凝胶电泳检测。
本发明用到的术语或定义：
EB：是溴化乙锭(Ethidium bromide)
dPCR：数字PCR(digital PCR)，是一种核酸检测和定量分析的新方法，可以作为传统实时定量PCR的替代方法，以实现绝对定量及稀有等位基因的检测。数字PCR的工作原理在于将DNA或cDNA样品分割为许多单独、平行的PCR反应。
“Mg2+和部分或全部dNTPs底物的初始终浓度超出/远低于PCR体系完成预定的PCR扩增所需的浓度”：本领域技术人员根据其技术常识可以确定其配制的PCR体系及即将进行的PCR扩增中需要的dNTPs底物和Mg2+浓度。本领域技术人员可以很容易确定特定的PCR扩增体系中加入多少dNTPs能够形成超过所需浓度以及低于所需浓度，其中浓度为0，是远 低于所需浓度的特例。本发明中，例如在100μl的反应体系中,4种dNTP的浓度为20μmol/L,可基本满足合成2.6μg DNA或10pmol/L的400bp序列。例如，本发明中的特定实施例一和二中的第一轮外引物扩增中，dT的终浓度5/(0.5*20)＝1.25毫摩尔/L为“所需的浓度”，其它A,G,C的浓度为T的二倍即10/(0.5*20)＝2.5毫摩尔/L，为“超过所需浓度的浓度”。
2×(或2倍)外引物PCR反应液：指预配配好的PCR反应体系中，每种成分都按常规浓度的2倍加入，使用前需要稀释2倍后才可启动PCR反应，通常通过加入其它成分，例如模板、无菌水等进行稀释。
1×(或1倍)内引物PCR反应液：指预配配好的PCR反应体系中，每种成分都按常规浓度加入，不需要稀释，可直接启动PCR反应。
单侧巢式PCR:外侧引物对中一侧和内侧引物对应侧共用一条引物序列。
中部同序引物：上、下游引物中部有6-8个同序碱基。
PCR反应液的各种成分的“初始终浓度”(例如dUTP的初始终浓度、RNaseA酶的初始终浓度、UDG酶的初始终浓度)是指PCR反应启动之前在已经调配好的完整PCR体系中；各种成分的浓度在PCR反应过程中会逐渐减少或变化，无法反映初始加入量。
pUC19载体质粒：购于NEB(北京)公司；
E.coli UDG：尿嘧啶DNA糖基酶(Uracil-DNA-glycosylase)，试剂原浓度为：5U/μl，购于NEB(北京)公司；
RNase I：核糖核酸酶I，购自NEB(北京)公司，试剂原液，5000U/ml
RNaseA:核糖核酸酶A，内切核酸酶，购自生工生物工程(上海)有限公司,试剂原液浓度为4mg/ml。
本专利含dU或RNA碱基的修饰引物和常规寡核苷酸由生工生物工程(上海)有限公司合成。
荧光PCR仪器采用SLAN-96P(上海宏石医疗科技有限公司)。
试验方法采用荧光染料SYBR Green Ⅰ实时荧光PCR是因为其原理及操作最直接简便而线性关系及变异系数CV最好，但是本发明也可以采用其它类型实时荧光PCR技术。
附图说明:
图1.UDG作用范围：稀释至10-2倍无扩增即完全抑制含dU引物扩增；至10-3倍Ct18.9对比无UDG的Ct16，仍小部分抑制dU引物扩增。
图2.RNaseA作用范围：稀释至10-4倍Ct33.83几乎完全抑制含RNA引物扩增，至10-5倍Ct28.97对比无UDG的Ct16，仍大部分抑制RNA引物扩增。
图3.巢式PCR所需预扩循环数的荧光扩增曲线：原模板×10-3预扩10-20个循环的荧光扩增Ct值25.71、23.43、18.59与直接荧光PCR原×10-3—原×10-1的Ct值26.19、22.80、19.04近似，推算预扩增10、15、20个循环依次约放大101、102、103倍，较浓一些模板多放大一倍。
图4.巢式PCR本底扩增曲线对比：同样外引物预扩0、5、10、15、20个循环后，相同的内引物荧光PCR进行非酶切组本底Ct值为35.0、32.0、27.5、21.5、18，随预扩循环数增加的严重非特异性扩增；UDG/和RNase I酶切组全部为直线无扩增Ct值；外引物降解的巢式PCR无任何非特异性。
图5.乙肝HBV增敏半巢式荧光PCR梯度多稀释两个数量级的扩增曲线及Ct值与直接荧光PCR浓两个数量级的扩增曲线及Ct值相当，预扩可对应比例地增敏荧光PCR。
具体实施例：
以下实施例进一步说明本专利的内容，但不应理解为对本专利的限制。在不背离本专利精神和实质的情况下，对本专利方法、条件、步骤及应用所作的修改或替换，均属于本发明专利的范围。
实施例1 乙型肝炎病毒(HBV)外引物可降解的半巢式PCR
乙型病毒性肝炎(简称乙肝)由乙型肝炎病毒(Hepatitis B virus,HBV)感染的一种世界性III类传染性疾病，据世界卫生组织WHO报道全球约20亿人携带乙肝病毒。我国人群中乙肝感染率非常高(近10％)，主要由肝炎病毒引发的肝癌已位列肿瘤首位，极大的危害了人民的身体健康。目前乙肝的检测方法主要有酶免疫法5项/7项、化学发光法、免疫荧光法、核酸扩增(PCR)荧光定量法等。传统酶免疫法应用较广，但灵敏度不足；实时荧光PCR定量和数字定量PCR法可以精确测定乙型肝炎病人的病毒载量,对感染者病毒复制水平的判断,病情传染性及抗病毒药物疗效监测具有无法替代的重要作用。
本实施例乙肝病毒(HBV)外引物可降解的半巢式PCR，选取乙型肝炎病毒(Hepatitis Bvirus,HBV)C区(1905-2498)序列克隆至pUC19载体质粒pHBc(MW 2.1×106)作为乙肝病毒阳性对照序列。
AB540584 C区(2272-2434)
Figure PCTCN2017114762-appb-000008
在选取区域上选取一段倒置重复引物同序碱基置于靶引物对中部作为靶内侧引物，试验不影响扩增效率情况下变异一个碱基以增加“同序”数，中部7b同序能选择性减低引物二聚体PD非特异性近10个Ct值，靶内引物对和外引物对序列如下:(下划线序列为中部同序)
内引物对  HBcF:5'—aat gcc cct atc tta tca a–3'
          HBcR:5'–gat tga gat ctt atg cga c–3'(原c人为变异a)
外引物对  WhBcF:5'–gtg gat tcg cac dUcc Tc–3'
          R引物可共用HBcR或dU修饰HBcR。
(1)血标本DNA提取：
简易煮沸裂解法，取50μl-100μl血清加等量的煮沸裂解液(用前充分混匀微珠,剪大口吸头吸取)，轻混,置沸水浴10分钟,经4℃短暂冷确后高速离心10分钟，取上清5μl加样。或大量裂解上清可再经微磁球试剂进一步纯化。
病毒PEG沉淀法，弱阳性标本采用PEG沉淀HBV,取血清500μl加500μl 2×PEG液(16％w/v PEG & 0.7M NaCl)，Vortex混匀，高速离心10分钟，弃上清950μl，浓缩沉淀留50μl后加50μl煮沸裂解液，余同上煮沸法,取5-10μl梯度稀释加样。此PEG沉淀回收率为50％。2×煮沸裂解液:0.02N NaOH,0.02％SDS(w/v),25mM KoAc,10mM(NH4)2SO4,0.5M Betaine,0.5％Glycerol(v/v)和0.05％Gelatin(w/v)。
(2)乙肝病毒HBV增敏半巢式荧光PCR：
方法：
先从管底加10μl含dU外引物及HK Taq预扩2×倍PCR反应液(dNTP中dA、dG、dC的浓度是dT的两倍)，再加等量10μl纯化样本，沿管壁加50μl矿物油密闭下普通PCR预扩12-15个循环；
再小心用吸头于矿物油表层下加入20μl含UDG酶及仅底物dUTP和无Mg2+的buffer及Taq的1×倍内侧PCR反应液(可降解侧外引物侧的内引物的浓度是共用内引物的两倍)，加样吸头尖不要接触预扩反应液，实时荧光PCR 30-35个循环。
乙肝病毒HBV核心抗原C基因克隆质粒pHBc(0.1μg/ml)为模板作10×(倍)剃度稀释：0.1μg/ml×10-1、×10-2、×10-3、×10-4、×10-5、×10-6、×10-7,
梯度0.1μg/ml×10-1-10-4直接一步实时荧光PCR；
对比梯度多稀释3个数量级至0.1μg/ml×10-4-10-7预扩(外引物15个循环后实时荧光PCR(内引物)：
按巢式PCR标准配方制备第一轮可降解外引物PCR反应液，
Figure PCTCN2017114762-appb-000009
Figure PCTCN2017114762-appb-000010
于矿物油密闭下外引物普通PCR预扩增15个循环。
按以下配方制备1×内引物PCR反应混合液,单侧巢式PCR仅一侧引物浓度为双倍(双侧巢式的第二轮PCR上下游引物均须双倍，)：
Figure PCTCN2017114762-appb-000011
※10×Taq buffer：0.6M Tris-Cl(pH8.3),100mM KCl,50mM(NH4)2SO4。
预扩管于矿物油表层下加入20μl含UDG酶的内侧PCR反应液(加样吸头尖不要接触预扩反应液)，宏石SLAN-96P荧光PCR仪实时荧光PCR 35个循环。
结果见附图5：本发明的单侧巢式PCR对多稀释三个数量级模板(0.1μg/ml×10-4-10-7)的扩增曲线及Ct值与直接荧光PCR对浓三个数量级的模板(0.1μg/ml×10-1-10-4)的扩增曲线及Ct值相当，外引物预扩可对应比例地增敏荧光PCR。
实施例二 食品转基因启动子外引物可降解的巢式dPCR检测豆油转基因分子含量
转基因技术突破自然资源的限制，大幅度提高了农业效益和农产品产量、品质，同时亦带来了转基因食品生物安全问题,并日益受到世界各国政府和社会公众的广泛关注。随之,食品 转基因成份定量检测及dPCR绝对定量检测的需求也快速上升。转基因作物已从棉花、大豆、玉米等主要农作物发展至数十种转基因作物种类；转基因类型大多为各种抗除草剂型、抗虫型、或抗除草和抗虫复合型，且也已拓展至改变亚麻酸含量、高赖氨酸型、延迟熟软及抗病毒等一系列新型转基因种类，因此，选择某一特定转基因分子作为靶基因进行qPCR或dPCR定量检测已不太容易广泛检测使用，然而多数转基因型均采用花叶病毒CaMV 35S启动子表达、调控，采用共同的启动子序列可以优先作为转基因qPCR或dPCR检测的通用靶标。本专利以转基因豆油为应用实施例来进行CaMV 35S启动子dPCR检测。
(1)CaMV 35S启动子序列外引物和内引物设计：
选取转基因CaMV 35S启动子如下部分序列(CaMV 35S启动子序列在Genbank中的编号为V100141.1，选取位置为7155-7334)，
Figure PCTCN2017114762-appb-000012
阳性对照：以上述选取序列作为模板并克隆至pUC19载体为质粒pCaMV对照。
选择其倒置重复inverted repeat即引物对6base同序序列作为内引物如下:
CaMV F：5'-gaa ggt ggc tcc tac aa-3'
CaMV R：5'-tcc acg atg ctc ctc gt-3'
以及修饰外引物对：
WCaMV F：5'-tca ctt tat tgt gaa gaU tag U-3'
WCaMV R：5'-cca ctt gct Utg aag acg U-3'(下划线序列为同序)
(2)植物标本DNA提取：
CTAB简便提取法：
固体植物于液氮中磨碎，0.2-0.3g加1ml的2％十六烷基溴化铵(CTAB)65℃水浴20-60分钟，等体积的氯仿(1/24异戊醇)混旋、离心，抽提上清加2倍体积的无水乙醇-20℃沉淀，离心后弃上清，DNA溶于50μL的TE缓冲液。
豆油样本DNA抽提及柱纯化：
根据(中国农业科学2007,40(5):1069)提取DNA方法：取金龙鱼牌食用转基因大豆油10mL加10mL正己烷于烧杯中磁力搅拌2hr,加20mL的PBS继续搅拌3hr，转入50mL塑料管离心12000g×20mins，小心取出下层水相，转入一新50mL管加等体积的异丙醇并混匀，置-20℃20mins再离心12000g×20mins，弃上清，沉淀加dH2O 100μL溶解，加500μL结合缓冲液过离心柱，洗液洗柱两次，柱瞬离干燥后加50μL的TE洗脱DNA。纯化的样本DNA进 行稀释定量巢式PCR反应。
(3)按以下配方制备2×PCR反应液10次单个反应:
Figure PCTCN2017114762-appb-000013
纯化的样本DNA按原液×10-1、×10-2、×10-3、×10-4、×10-5、×10-6、×10-7、×10-8、×10-9顺序作9个梯次10倍稀释：取9个1.5mL塑料EP管，每管先加入18μL纯化水,取2μL待测样本原液加入第1管水中、并用吸头轻轻混匀、此为原液×10-1倍，从第1管中取2μL的10倍稀液加入第2管水中、并轻混为原液×10-2倍,每管换一吸头,原液×10-3…依次类推…原液×10-9。
取10个微型PCR反应管,每管均先加10μl的2×PCR反应液，再加10μl的稀释原液×10-2、原液×10-3、…依次类推…原液×10-9,最后加两管10μl阳性对照(质粒pCaMV)和10μl dH2O作为阴性本底对照；再加入50μl矿物油，进行95℃变性2分钟后,进行20个热循环94℃变性20秒、54℃退火30秒、72℃延伸30秒的第一轮普通PCR扩增。
(4)第一轮普通PCR反应后，按以下配方配制第二轮1×PCR反应液10次×20μl,
Figure PCTCN2017114762-appb-000014
取内侧引物1×PCR反应液20μl/管,用加样吸头插入矿物油表层下面小心加入,预先50℃2分钟后94℃变性2分钟,再进行30个热循环94℃变性20秒、53℃退火30秒、74℃延伸30 秒的第二轮荧光PCR无热盖扩增。
(5)d-PCR终产物荧光显色及计数定量：
第二轮PCR反应后，PCR产物管于荧光检测仪(西安天隆公司)来读取相对荧光吸收值。
计数判读标准:
原液×10-2至×10-4管荧光值﹥3000定为：1个靶分子/10μL样本；原液×10-5至×10-9管荧光值﹤50定为阴性，即0个靶分子/10μL样本。
可算出原液×10-4的反应管的10样本稀释液中至少含1个靶启动子分子；即10Ml被检豆油中含10×1000/10×104倍＝1.0×107拷贝CaMV 35S启动子的转基因分子，或约含1.0×106转基因分子/mL豆油。

Claims (15)

  1. 外引物可降解的巢式PCR，其特征在于：在同一个反应管/孔中完成以下步骤：
    (1)外引物扩增：在反应管/孔中加入外引物PCR反应液，并加入矿物油对PCR反应液进行密封，完成预定扩增程序；其中所述外引物PCR反应液中采用的至少一侧外引物的末端至中部含有1个以上dU碱基和/或RNA碱基修饰，所述外引物PCR反应液中使用Taq、K Taq或H.K Taq,Mg2+和部分或全部dNTPs底物的初始终浓度超出外引物PCR体系完成预定的PCR扩增程序所需的浓度；超出量用于该反应管/孔中下一步进行的内引物扩增；
    (2)所述内引物扩增：于完成上述外引物扩增的反应管/孔的矿物油表层下加入内引物PCR反应液，使外引物PCR反应液被稀释2-4倍体积，加入的所述内引物PCR反应液中含有UDG和/或RNA酶，Mg2+和部分或全部dNTPs底物的终浓度远低于内引物PCR体系完成预定的PCR扩增程序所需的浓度；启动内引物PCR扩增前，先在50℃下消化2-10分钟以降解所述外引物。
  2. 根据权利要求1所述的外引物可降解的巢式PCR，其特征在于：所述内引物PCR反应液中采用的内引物为中部同序引物。
  3. 根椐权利要求1或2所述的外引物可降解的巢式PCR，其特征在于，其中所述外引物PCR反应液中采用的引物至少一侧引物末端至中部含有1-3个dU碱基和/或RNA碱基修饰。
  4. 根椐权利要求1或2的外引物可降解的单侧半巢式PCR，其特征在于：所述外引物PCR反应液中仅单侧外引物含有所述dU碱基和/或RNA碱基修饰；其另一侧内引物及其同侧外引物共用同一序列引物。
  5. 根椐权利要求1-4任一所述的外引物可降解的巢式PCR，其特征在于，所述外引物3'末端倒数1-2碱基位置具有1个dU碱基或RNA碱基修饰，在引物中部设置有第2、3个碱基修饰。
  6. 根椐权利要求1-5任一所述的外引物可降解的巢式PCR，其特征在于：
    所述外引物PCR反应液中dA、dG、dC的初始终浓度是dT的2倍，Mg2+的初始终浓度为3.5mM。
  7. 根椐权利要求6所述的外引物可降解的巢式PCR，其特征在于：加入的所述内引物PCR反应液中，不含Mg2+和常规底物dA、dG、dC，仅含dUTP，所述dUTP的浓度和体积与外引物PCR反应液中加入的dT相当。
  8. 根椐权利要求1所述的外引物可降解的巢式PCR，其特征在于:所述内引物PCR反应液中含有的UDG酶，在混合而成的总反应液中的初始终浓度为0.0125-0.0625U/μl，即原浓度5U/μl稀释80倍至400倍；
    含有的RNaseA酶在混合的总反应液中的初始浓度为0.4-40ug/ml，即原浓度4mg/ml稀释102-104倍，或含有的RNaseI在混合的总反应液中的初始浓度为0.031U/μl。
  9. 根椐权利要求1的外引物可降解的巢式PCR，其特征在于：
    (1)外引物扩增：先加入10μL 2倍浓度的所述外引物PCR反应液于管底，并加10μL稀释的样本液，再沿管壁加入50μL矿物油层密闭，进行15-20个循环的普通PCR外扩增；
    (2)内引物扩增：于上一轮的反应管中加20μL的1倍浓度的所述内引物PCR反应液，其中可降解侧外引物同侧的内引物浓度是其它引物的2倍，进行30-35个热循环荧光PCR内扩增。
  10. 根椐权利要求9的外引物可降解的巢式PCR，其特征在于，用于进行简易数字定量PCR，
    其中所述待测样本为原液被稀释为原液×10-1、×10-2、×10-3、×10-4、×10-5、×10-6、×10-7、×10-8、×10-9的9个浓度梯次的稀释液，取×10-2、×10-3、×10-4、×10-5、×10-6、×10-7、×10-8、×10-9的待测样本稀释液以及阳性对照以及无菌水作为模版分别分配至原位PCR仪进行所述外引物扩增和所述内引物扩增。
  11. 根椐权利要求10的外引物可降解的巢式PCR，其特征在于，内引物扩增30-35个循环后，
    每孔加入0.5μg/mL的EB2-5μL于矿物油层下面,置反应管于紫外灯下显色计数或于荧光光度仪检测。
  12. 根据权利要求1-11任一所述的外引物可降解的巢式PCR，其特征在于，用于检测乙型肝炎病毒，内引物对为：
    HBcF:5'—aat gcc cct atc tta tca a–3'，
    HBcR:5'–gat tga gat ctt atg cga c–3'，其中第13位的a为c人工变异设计而来，
    外引物对为：
    WhBcF:5'–gtg gat tcg cac Ucc Tc–3'，
    反向引物为HBcR或dU修饰的HBcR。
  13. 一种通过检测启动子CaMV 35S进行食品转基因检测的方法，其特征在于采用权利要求1-11任一所述的方法，所述内引物为:
    CaMV F：5'-gaa ggt ggc tcc tac aa-3'，
    CaMV R：5'-tcc acg atg ctc ctc gt-3'；
    外引物为：
    WCaMV F：5'-tca ctt tat tgt gaa gaU tag U-3'，
    WCaMV R：5'-cca ctt gct Utg aag acG t-3'。
  14. 一种用于进行权利要求1-12任一所述外引物可降解的巢式PCR方法或权利要求13所述的食品转基因检测的方法的试剂盒，其特征在于：包含所述外引物和、内引物。
  15. 根据权利要求14所述的试剂盒，其特征在于，还包含用于所述外引物扩增的预配好的所述外引物PCR反应液和用于所述内引物扩增的预配好的所述内引物PCR反应液。
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