primer 5' end reverse complement fluorescent PCR

Abstract

The application discloses a primer 5' end reverse complementary fluorescent PCR, belongs to the field of molecular biology-nucleic acid amplification technology, and is characterized in that a pair of primers are added with a sequence reverse complementary fluorescent PCR amplification at the 5' end, which brings about end complementarity between amplification products, and the 3' end of a target product can also be used as a template and a primer to increase the amplification efficiency. 9‑12 On the basis of the conventional PCR, the sensitivity is increased by 50 times, or 5 cycles of amplification can be saved under the condition of the same sensitivity; on the other hand, the 5' end complementarity of the primer cannot be extended, but can inhibit the polymerization of the original primer to the 3' end, and 5 cycles of non-specific amplification can be avoided. The primer with the same sequence in the middle and the primer with the A adenine at the last 1-2 bases of the 3' end + UDG enzyme dU instead of dT of the amplification product, and mineral oil sealing can prevent the generation of primer dimers PD in the PCR reaction cycle and eliminate the cross contamination of product aerosol glue.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of fluorescent PCR technology in molecular biology and nucleic acid detection, and specifically to a single-tube one-step real-time fluorescent PCR for increasing sensitivity and limiting primer non-specificity by adding complementary sequences to the 5' end of primers to exceed exponential amplification. BACKGROUND

[0002] PCR is a nucleic acid amplification technology in vitro (in test tube) that simulates natural gene replication based on the DNA double helix model and semi-conservative replication principle established by Watson in 1953. In 1971, Khorana published a paper on nucleic acid amplification in vitro (J. Molec. Biol., 56:341). However, it was not until 1985 that Mullis of Cetus Company in the United States thought of the essence of PCR - exponential amplification or geometric series amplification, and realized its great power, that a pair of specific primers were combined to replicate the target molecule gene, simulating the natural DNA semi-conservative replication PCR technology. After a series of developments and the invention and application of heat-resistant polymerase and thermal cycler, Cetus Company applied for the first PCR invention patent (US Patent 4,683,202) in 1987.

[0003] Amplification utilizes efficient dissociation by thermal cycling. Generally, the target DNA is denatured into single strands at 94°C, annealed and recombined at about 54°C to pair the excess primers with the complementary sequences of the template DNA single strand, and the primer extension is carried out at about 72°C, where the DNA single strand template-primer combination is extended by the action of heat-resistant DNA polymerase, with dNTP as the reaction raw material, the target sequence as the template, and the primer end extended in the 5'→3' direction to synthesize a new complementary strand to the template DNA chain, with half new chain and half mother chain semi-conservative replication. The three processes of denaturation, annealing, and extension are repeated continuously, and the new chain can become the template for the next cycle, and the target molecule produces more new chains in a 2 n geometric progression.

[0004] Various PCR methods, constantly emerging, improvements, inventions, including reverse transcription RT-PCR, in situ PCR, ligase chain reaction (LCR), labeled primers-PCR, reverse PCR, asymmetric PCR, touchdown PCR, recombinant PCR, multiplex PCR, immuno-PCR, mRNA differential PCR, strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-dependent amplification system (TAS), Q-beta replicase catalyzed RNA amplification, rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), etc. Because the conventional end PCR is not uniform in yield due to the large change after the same amount of sample is super amplified by PCR, it is difficult to quantitatively detect, and traditional end / terminal PCR can only distinguish specific and non-specific amplification bands by gel electrophoresis after PCR; Real-time PCR technology in 1997 realizes the leap from qualitative determination to accurate quantification by dynamically detecting the number of cycles in the logarithmic phase of amplification, which is inversely proportional to the initial amount of target (inverse logarithmic relationship); according to the light-emitting principle, there are two categories of real-time fluorescent PCR: dye method real-time fluorescent PCR with SYBR Green I and other dyes that enhance the emission fluorescence by 1000 times after binding to the small groove of double-stranded product DNA, and various fluorescently labeled hybridization probe real-time PCR (Bio Techniques 1997, 22: 130-138).

[0005] Exponential amplification or geometric amplification of super sensitivity is the most core nature or advantage of PCR, and the target molecule is amplified by 2 n times per cycle, and the conventional 30 thermal cycle reactions are 30 about equal to 10 9The amplification factor of the PCR is far greater than that of the atomic fission chain reaction, and the sensitivity of the PCR is far greater than that of the general linear amplification method. The real-time fluorescent PCR can reach fg / ml or even close to single molecule detection after 40 cycles. The sensitivity of these PCR methods is still insufficient, and then the nested PCR with two-step amplification of outer and inner primers (J Med Virol, 30-2: 85) appeared. The outer primer is pre-amplified for 15-20 cycles to improve the sensitivity, and then the inner primer is continuously amplified for 25-30 cycles to exceed the high sensitivity of 40 cycles of exponential amplification, which even provides the possibility of detecting a single target molecule. However, the outer primer increases the complexity of the system and the non-specificity of the two-round PCR. The residue of the outer primer promotes the non-specific amplification of the inner primer, which increases the non-specificity of the general conventional primer from the background Ct value of 30 cycles to the background non-specificity before the Ct value of 25 cycles. The increased non-specificity of the two-round PCR offsets the amplification effect of the first round or the outer primer pre-amplification.

[0006] Therefore, the excessive 40 cycles and excessive primers also bring about the stubborn non-specificity of the PCR. The non-specificity of the PCR is also derived from the primer sequence, and any decrease in the specificity of the primer or any mismatch with the target will cause the amplification rate to fall. Even the complete matching of one end of the amplification is only linear amplification, which is far behind the specific exponential amplification or geometric amplification. On the contrary, as long as the target is exponentially amplified or geometrically amplified, the PCR amplification detection has high enough specificity. The non-specificity of various conventional detection methods is also present in the PCR technology, but it ultimately cannot catch up with the exponential amplification. Only one pair of non-specific binding primers can be exponentially amplified, and the dimer primer (PD) amplification and PD product re-amplification are the essence of PCR non-specificity and the fundamental reason why it is difficult to overcome. The 3' end reverse complementarity of a pair of primers is the main cause of exponential non-specificity. Generally, a few bases of the 3' end continuous reverse complementarity of the primer will appear primer dimer PD amplification from several to ten cycles in the background PCR amplification reaction without template. A pair of conventionally designed primers will appear significant PD non-specific amplification at 30 cycles of the background PCR reaction, which will cover the detection of low-concentration samples of several thousand target molecules at 30-38 cycles. This is also the main internal reason why the conventional terminal PCR is only performed for 30 cycles of reaction.

[0007] The current real-time PCR still has limited sensitivity and some degree of primer non-specificity, which hinders the detection of very low concentration samples or single molecule detection. A pair of highly homologous primers has no primer non-specificity, and the elimination of primer exponential non-specificity can only be traced back to its base sequence. Hands technology (Homo-Tag assisted non-dimer system, Nucleic Acids Res. Vol. 25, No 16: p3235-3241) uses completely homologous primers Tag to compete with free primers by binding to the single-stranded ends of primer dimers, which not only significantly inhibits PD non-specificity, but also reduces the efficiency of target-specific amplification without selection. Single-stranded binding protein Single Strand Binding-protein (SSB), gene 32 protein and oligo can significantly reduce the optimized primer non-specificity, which also seriously affects the target-specific amplification efficiency and the linearity of the amplification curve. Chinese patents (CN 201010105371.8 and PCT / CN2013 / 088054) use partially homologous primers to partially inhibit PD amplification, and placing "homology" in the middle of the primer 3' end can maximize the selective inhibition of PD amplification without affecting the target amplification efficiency; PCR adds an antisense base-containing oligo that pairs with the middle homologous site of the primer, which only retains the binding function and has no template and primer effect, which can further amplify this effect selectively. On the basis of the conventional primer design principle without 3' end reverse complement, the middle part of the homologous primer pair and the hybridization of the middle antisense base oligo can eliminate the endogenous PD non-specific amplification interference in the real-time fluorescence PCR 40-cycle reaction. Because UDG can effectively degrade the target amplification product, but the enzymatic degradation of the non-a end primer dimer produces primer "fragments" with complementary bases, which are easily re-polymerized and ineffective; the PD product using a end primer can use dU instead of dT substrate to form a dU-containing dimer PD at the junction, and UDG enzyme can effectively degrade the a end primer of the exogenous dU-containing primer dimer PD product aerosol glue (Chinese patent CN 201810001086.8).

[0008] There is also a dilution quantitative method, i.e. a dilution quantitative method that calculates the dilution ratio by diluting the target molecules in the sample by a certain ratio until there are no target molecules for reaction, and then calculating the dilution ratio. Subsequently, the concept of digital quantitative PCR based on sample limited dilution and Poisson distribution counting appeared. That is, under the condition that a positive single molecule or more than one molecule has a detection signal and is defined as 1, and a negative absolute non-reaction signal is defined as 0, the sample is diluted and dispersed into a large number of micro-separation units so that each detection unit theoretically contains 0-1 target molecules. Through the detection of 0-1 counting and limited dilution ratio, the target molecules are quantified, which facilitates the automatic operation of the computer 0 or 1 mode, but the prerequisite is that the detection method itself can reliably distinguish between 1 molecule or 0 molecules. However, traditional chemical reactions, enzyme immunoassay, etc. are all nanogram-level low-sensitivity detection methods with linear proportion between detection signal intensity and content of the target molecules and simple signal addition. The linear traditional low-sensitivity method cannot distinguish the difference between 1 target molecule and 0 molecules, so it cannot be applied to the digital quantitative detection method of limited dilution method. In 1992, Sykes et al. (Biotechniques 13: p444) initially tried to quantitatively nest PCR based on sample limited dilution and Poisson distribution counting, and first proposed digital quantitative fluorescent PCR, but due to the serious non-specificity of multi-primer polymerization amplification in the PCR system and cross-contamination of aerosol glue outside the system, the amplification effect of the secondary amplification was offset.

[0009] Further, Vogelstein & Kinzler reported the quantitative PCR of oncogenic mutant gene ras in microliter 96-well plates in 1999 and first proposed the concept of digital PCR (PNAS, USA 96:9236). d-PCR is a third-generation absolute quantitative PCR method based on single-molecule PCR counting. Microfluidic or micro-droplet methods in analytical chemistry are used to disperse a large amount of diluted nucleic acid solution into micro-reactor units or micro-droplets, and the number of nucleic acid templates in each reaction unit is less than or equal to one. After PCR thermal cycling, the amplified product is hybridized with the added fluorescent probe. The reaction unit with one nucleic acid molecule template has amplification and gives a fluorescent signal, and the reaction unit without template has no amplification and no fluorescent signal. According to the dilution ratio and the volume of the reaction unit, the concentration of the original solution can be calculated, and through counting and Poisson distribution statistics, the absolute quantification of the starting DNA template can be realized. In recent years, with the development of microfluidic devices with million-level (i.e. nanoliter) and even billion-level reaction unit (picoliter) volumes (Anal. Chem. 2011, 83:8604), d-PCR has begun to be applied.

[0010] Therefore, the sensitivity of conventional PCR and real-time fluorescent PCR for single molecule detection is still insufficient, and super-sensitivity amplification is needed to further improve the system sensitivity. However, PCR is limited by the covering of non-specific detection of low-concentration samples, and super-sensitivity PCR even single molecule effective detection depends on PCR sensitivity and non-specific removal. The present application "fluorescent PCR with 5' end reverse complementary primer" uses 5' end sequence complementary primer PCR amplification, which brings end complementary between amplification products, and the 3' end of the target product can also be used as a template and primer to increase the amplification efficiency, with >2 n exponential amplification strategy to increase sensitivity. On the other hand, the 5' end complementarity of the primer does not extend, but inhibits the non-specific polymerization between the 3' ends of the original primer; and combined with UDG and mineral oil sealing to completely eliminate primer dimer PD aerosol glue non-specific amplification. The "fluorescent PCR with 5' end reverse complementary primer" quantitative method can be used as a d-PCR value method for nucleic acid standard reference, important scientific research samples and ultra-low copy clinical samples, and also provides technical methods and sharp tools for faster and more efficient super single unit microcapsule / microdroplet d-PCR microfluidic device.

[0011] REFERENCES

[0012] (1)Porter-Jordan k;et al.,1990,J.Med Virol 30(2):85-91.

[0013] (2)Sykes,P.J.;et al.,1992,BioTechniques 13:444-449.

[0014] (3)Wittwer,C.;et al.,1997,BioTechniques 22:130-138.

[0015] (4)Brownie J.,et al,1997,Nucleic Acids Res.Vol.25,No16:3235-3241.

[0016] (5)Vogelstein,B.;Kinzler,K.W.1999,PNAS,USA 96:9236-9241.

[0017] (6)Hindson B.J.,et al.,2011,Anal.Chem.83:8604-8610.

[0018] (7)Tran M.T.,2014,Malaria Journal 13:393。 SUMMARY

[0019] The application "fluorescent PCR with 5' end reverse complementary primers" is characterized by adding a sequence reverse complementary to the 5' end of a pair of primers for PCR amplification, which brings about the complementarity between the ends of the amplified products, and the 3' ends of the target products can also serve as templates and primers to increase the amplification efficiency, with the amplification sensitivity increased by more than 2 n orders of magnitude, or the number of cycles can be reduced under the same sensitivity. On the other hand, the complementarity of the 5' end of the primers does not extend, but instead inhibits the non-specific polymerization between the 3' ends of the original primers. In combination with the primers ending with the 1st or 2nd reverse complementary base A at the 3' end and the UDG enzyme to digest dU instead of dT in the amplified products, and the mineral oil to block the products, especially the primer dimer PD aerosol glue pollution.

[0020] has the following technical approaches:

[0021] (1) A pair of primers with a short reverse complementary artificial sequence of 5-10b added to the 5' end of each primer, according to the phenomenon that the 3' ends of the amplified product chains are complementary to the primers and serve as templates for the next cycle of reactions, the 3' ends of the amplified product chains of a pair of 5' complementary primers are also complementary to each other accordingly. The 3' ends of the amplified products not only serve as templates for the binding primers in the subsequent cycles, but also can serve as templates and primers to bind and extend, which is more than the exponential amplification of a pair of primers, and the amplification efficiency is more than 2 n orders of magnitude or geometric progression, achieving enhanced PCR amplification sensitivity.

[0022] (2) Using the sequence of the 5' end of the primer as "artificial complementary sequence", a 5-8b

[0023] reverse complementary sequence corresponding to the 5' end sequence of one primer is added to the front of the 5' end of the other primer in the 5'-3' direction, and vice versa, a 5' reverse complementary sequence of two segments is formed to increase the sensitivity, and the original primer and the added sequence share a segment of the 5' sequence of the original primer. A short 4b sequence restriction enzyme site can be inserted between the original primer sequence and the added sequence to make the lengths of the amplified products consistent after enzyme digestion, facilitating electrophoresis recovery and helping to degrade PD product pollution.

[0024] According to the "fluorescent PCR with 5' end reverse complementary primers", the upper and lower original primers have a 6-8b same sequence in the middle and a primer ending with the 1st or 2nd reverse complementary base A at the 3' end, which is used to further reduce the degree of primer dimerization and to push back the background amplification Ct value of primer dimerization; an antisense base of 5-9b in the middle is added to the middle of the primer to interfere with the Oligo oligonucleotide enhancer, which selectively inhibits the background Ct value of primer dimer amplification in the PCR cycle reaction.

[0025] The "5' end reverse complementary fluorescent PCR" is characterized in that the tail base A of the 6-8b same sequence A ending primer in the original primer is changed to an RNA base immediately before or after the tail base A, so that the A base ending and the RNA base jointly digest the primer dimer PD product cross contamination.

[0026] The "5' end reverse complementary fluorescent PCR" is characterized in that the primer design is based on the modification and supplement of some background reduction, 5' end reverse complementary primer design principles on the basis of conventional primer selection:

[0027] 1) Select a pair of target-specific (conservative) gene span 100-300bp both ends sequence 17-22 nucleotide base length as upstream and downstream primers, and the 3' end of the primer cannot have continuous reverse complement;

[0028] 2) Select 6-8b "inverted repeat" within 300bp of the target gene as the middle same sequence primer, which is placed in the middle part of the primer 2-6 bases away from the 3' end;

[0029] 3) 5' end reverse complement - adopt 5-7b opposite original primer 5' reverse sequence as artificial sequence added to the front of the opposite primer, and a short 4b sequence restriction enzyme site is inserted between the artificial added sequence and the original primer sequence;

[0030] 4) The 3' last base of a pair of primers is selected as A / AC ending, so that the dU corresponding to the amplification product is located in the middle of the primer dimer connection; do not choose G ending with strong hydrogen bond "mismatch" more, also do not choose T ending with weak hydrogen bond / poor specificity, and more cannot adopt double GG / TT ending.

[0031] The "5' end reverse complementary fluorescent PCR" is characterized in that the primer concentration is 3-6μM as 50× times or the final concentration is 0.1μM, and the optimal primer concentration is adjusted according to the minimum concentration detection requirement of different samples and the system background value.

[0032] The "5' end reverse complementary fluorescent PCR" is characterized in that the PCR reaction solution is added with betaine (Betain) and polyanion polyphosphate (PPA) universal PCR enhancer, which enhances the target amplification efficiency and inhibits the non-specific amplification of primer dimer PD in two directions.

[0033] The "5' end reverse complementary fluorescent PCR" is characterized in that the combination of mineral oil sealed PCR reaction is used to adopt a component such as a primer containing 10% sucrose to release slowly and combine with heat start PCR to reduce the production of aerosol glue or produce invalid expansion aerosol glue.

[0034] The "primer 5' end reverse complementary fluorescent PCR" is characterized in that: in order to prevent the micro-pollution of target molecule aerosol glue to reagents, 0.2%-2% v / v E. coli UDG enzyme is added to the PCR reagent components / each component, and 0.1%-1.0% (v / v) target sequence restriction endonuclease mixed enzyme liquid is added at a dilute concentration which does not affect target amplification, and the cross contamination of target template molecules is digested by enzyme at room temperature RT for 0.5-2 hours before mixing the PCR components, or at 37°C for twenty minutes to forty minutes.

[0035] The "primer 5' end reverse complementary fluorescent PCR" is characterized in that: the PCR water and buffer use cheap chemical method to replace enzyme method to digest pollution, and fresh purified water is digested by 0.1‰-0.2‰ (v / v) i.e. one ten-thousandth, two dilution 10% sodium hypochlorite solution at room temperature for 1-2 days, and then boiled to remove sodium hypochlorite after opening the cover; 10×Taq buffer is added with 0.05‰-0.1‰ (v / v) exonuclease ExoIII and stored in cold storage.

[0036] The "primer 5' end reverse complementary fluorescent PCR" is characterized in that: a closed independent liquid preparation, sample adding and amplification room are used for physical space isolation, the PCR reagent components / each component are mixed in the independent liquid preparation room, and sample DNA purification and sample adding are carried out in the separated sample adding room, a filter tip is used for sample adding and thrown into 5% sodium hypochlorite waste liquid after use, and the laboratory uses pure 1-2% sodium hypochlorite for digestion and 70% alcohol for cleaning before and after use, and ultraviolet lamp and ozone are used for disinfection before and after the experiment.

[0037] The "primer 5' end reverse complementary fluorescent PCR" is characterized in that: the PCR method operation steps are as follows:

[0038] 1) 2 μL of the buffer release primer containing sucrose / + dye is added to the bottom of each PCR reaction tube;

[0039] 2) the remaining PCR reaction reagents are mixed, and 18 / 13 μL of the reaction mixture is sucked to the middle of the wall of the PCR reaction tube;

[0040] 3) 30 μL of mineral oil is added along the upper part of the wall of the PCR tube, and centrifuged momentarily, which can be placed at 37°C for at most 20-40 minutes;

[0041] 4) 5 / 10 μL of DNA solution extracted from the sample, quantitative standard, negative control or positive control is added to the surface under the mineral oil layer, and a filter tip is used for sample adding, and a new tip is used for each tube;

[0042] 5) avoid mixing to avoid destroying the buffer release layer, tightly cover the PCR reaction tube cap, high-speed short-time centrifugation, and as soon as possible or immediately perform PCR reaction, heat denaturation mixing buffer release layer to start PCR, and amplify 30-40 thermal cycles.

[0043] The "primer 5' end reverse complementary fluorescent PCR" is characterized in that: the chain replacement thermostable polymerase is applied to sample ratio dilution unit digital PCR, amplification detection dispersed unit nucleic acid amplification generates a fluorescent signal, and no nucleic acid amplification has no fluorescent signal, according to the dilution ratio and the reaction unit volume, the nucleic acid concentration of the original solution is calculated, and the absolute quantification of the starting DNA template is realized through counting and Poisson distribution statistics.

[0044] According to the "primer 5' end reverse complementary fluorescent PCR", the method is applied to a nucleic acid detection kit, and the kit components include: sample nucleic acid extraction reagent, dNTPs and dUTP, Taq enzyme and its buffer, chimeric 5' complementary primer F / R, dye SYBR Green I, PCR enhancer; eUDG enzyme, restriction endonuclease and ExoIII are pre-added to each PCR component / component, chemical treatment water dH2O, mineral oil.

[0045] The exponential amplification or geometric amplification of the polymerase chain reaction PCR brings super-sensitivity in detection, but the sensitivity is still not enough for detecting several or even single molecules, and it is difficult to perform digital detection dPCR; at the same time, the exponential or geometric amplification also brings the primer dimer non-specific amplification with a background of 30 cycles, which leads to the recontamination of the PCR product aerosol glue as a secondary template, and even under the condition of mineral oil sealed PCR reaction, the residual trace reaction exponential amplification on the oil layer is also a difficult-to-overcome leakage pollution source; some concepts and methods in the linear detection field are completely not applicable to the exponential amplification detection field.

[0046] The "primer 5' end reverse complementary fluorescent PCR" of the application, one of the conventional PCR primer pairs (such as the upstream primer) is combined with the target product template after extension and synthesis of a new replication chain (a meaningful chain), and a segment at the 3' end of the new chain has a complementary sequence relationship with the other primer (such as the downstream primer); the corresponding downstream primer extends and synthesizes a new chain (an anti-meaning chain) whose 3' end is complementary to the sequence of the upstream primer; the 3' ends of the pair of new replication chains are the primer binding templates for the next cycle reaction. According to this phenomenon, if a pair of primers are added with a reverse complementary artificial sequence slightly shorter than the length of the primer at the 5' end (the sequence added at the 5' end generally does not affect PCR amplification), the corresponding 3' ends of the pair of new replication chains are complementary to each other; the 3' ends of the amplification products not only serve as the templates for the subsequent cycle binding primers, but also can serve as templates for each other, primers for binding and extension; compared with the simple exponential amplification of a pair of primers, the mutual amplification between the products is more, and the amplification efficiency is far more than >2 nExponential or geometric series, to achieve enhanced PCR amplification sensitivity. Primer 5 'end complementary binding will not extend instead of inhibiting the original primer 3 'end between the polymerization of non-specific, but the inhibition of primer dimer PD is not as strong as the middle of the primer sequence.

[0047] Although the primer pair 5 'end plus artificial complementary sequence sensitivity PCR amplification efficiency, but the length of the about 40 base primer will also increase the dimer amplification non-specific. In order to reduce the length of the primer 5 'complementary strategy is to use the primer 5 'own sequence as the "artificial complementary sequence", set the corresponding 5-10b reverse complementary sequence of one primer 5 'end sequence to the other primer 5 'end in front of the 5 '-3' direction; otherwise, the reverse complementary sequence of the other primer 5 'end is added to the corresponding primer 5 'front. In this way, the 5 'end "artificial complementary sequence" includes the reverse sequence of the opposite primer 5 'end + the original primer 5 'own sequence, and the primer and the "artificial sequence" share a segment of the primer 5 'own sequence. Thus, the 5 'end complementary primer can be reduced to about 30 bases, and a short 4b restriction enzyme sequence can be inserted between the original primer sequence and the artificially added sequence, so that the length of the amplified product is consistent after enzyme digestion, facilitating electrophoresis recovery, or pre-setting the restriction enzyme digestion + UDG enzyme digestion measure to prevent cross contamination of the amplified product.

[0048] Principle diagram Figure 1 is to use cartoon to further illustrate the 5 'end complementary primer PCR product end complementary relationship, with various lines, representing the 5 'end complementary primer and its amplification: long solid line represents the target gene DNA, arrow short solid line represents the upstream and downstream primer sequence, arrow represents the 3 'end, the arrow direction is the DNA extension, synthesis direction; attached Figure 1 b the lower part is the repeated two sets of primer amplification and replication product: virtual long line represents the newly synthesized replication chain or product template, AGCTCA base letter with 5 represents the "artificial complementary sequence" added in front of the 5 'end of the original primer (short arrow represents), forming the 5 'end complementary chimeric primer; 5 indicates the 5 'end, the oblique base letter indicates the 5-10b reverse complementary base sequence. The chimeric primer itself will become the 5 'end / terminal part of the newly synthesized replication chain, and the last base of the 3 'end of the newly synthesized replication sequence corresponding to the "artificial complementary sequence" added after the chimeric primer is also the reverse complement of the replication / copy. The end / terminal of the newly synthesized replication chain / product chain can be used as a template for each other and as a primer for each other for thermal cycle amplification, without free primer or independent primer for additional amplification.

[0049] Primer index non-specificity is derived from the reverse complement of the 3' end of a pair of primers, which extends to the non-specificity of the 3' end of the over-concentration. Although the reverse complement of the 3' consecutive bases can be avoided by primer design, the 3' most base on one side is not limited by space and is prone to mismatching and binding. There is always unavoidable mismatching and binding of the last two bases between any primers, which brings about 30Ct value of background non-specificity. A pair of central 6-8b homologous primers in Chinese patent (PCT / CN2013 / 088054) can destroy the mismatching of the last two bases of the 3' end and the 3' external base binding force, partially reduce / push back the primer dimer 7-10Ct value, and further add the antisense base 5-9b central interference to selectively inhibit the endogenous primer dimer non-specific amplification in the PCR reaction. Generally, inverted repeats with a spacing of less than 300b are searched from the target sequence as the central 6-8b homologous primers to design the primer pair. The 5' end complementary primer 5' polymerization combined with the non-extended 5' end can inhibit the non-specificity between the 3' ends of the original primer pair, not only advancing the minimum sensitivity of single molecule detection to 30 cycles, but also pushing back the endogenous primer dimer PD background non-specificity to 40-45 cycles of reaction.

[0050] Fluorescent PCR non-specificity not only comes from endogenous PD production, but also from exogenous PD product aerosol glue recontamination, which interferes with each other. The source of endogenous PD non-specificity cannot be eliminated, and it is difficult to eliminate product aerosol glue. On the contrary, exogenous aerosol glue cannot be controlled, and endogenous PD non-specificity cannot be studied. Because the dU product of ordinary primer dimer PD is still a primer binding template, or the dU product of non-A ending primer dimer PD is more likely to polymerize with complementary bases, and the enzyme reaction cannot be 100% effective under the condition of extremely excessive product aerosol glue. Incomplete enzyme reaction will cause 10 9-12 Exponential amplification has limited effect; therefore, the 3' A ending of the primer corresponds to dU, which is located at the connection of the dimer primer. The PD product with dU removed in the middle lacks bases that can be paired and templates that can be copied, so that the exogenous aerosol glue PD loses the template effect. Under the condition of mineral oil sealed PCR, the use of one component such as one primer containing 10% sucrose layered slow release-thermal start can reduce the production of aerosol glue or invalid amplification aerosol glue, which is also a prerequisite for effective enzyme degradation and pollution. The mineral oil layer does not affect the light path transmission or fluorescence intensity.

[0051] Molecular detection of aerosol glue pollution is a difficult to eradicate controversial issue, aerosol glue molecules in addition to small molecules PD also include purified target molecules, especially quantitative standard plasmid is also commonly filtered, easy cross contamination of PCR reagents; placed in purified water is also easy to melt aerosol glue molecules; and long-term contamination with freezer storage. To prevent such aerosol glue cross contamination, production of PCR reagents must be sterile nucleic acid-free environment, fresh purified water reagent components / components pre-join 0.2% -2% v / v E. coli UDG enzyme; (add does not affect the target amplification of dilute concentration of target sequence restriction endonuclease 0.1% -1% v / v 1-2 or more mixed solution), using PCR reagent dispensing enzyme digestion of nucleic acid molecules cross contamination. A large number of PCR water can also add 0.1‰ about one ten-thousandth volume of sodium hypochlorite stock solution (10%) digestion for a long time, autoclaving / boiling to remove sodium hypochlorite; preparation of 10x Taq buffer buffer plus 0.05‰ -0.1‰ volume of exonuclease ExoIII. Application detection must be liquid, sample addition, amplification of separate space, PCR liquid mixing to sample addition before the pre-chamber temperature 30-60 minutes using pre-digestion of molecular pollution; sample addition is PCR thermal denaturation inactivation of pre-set enzyme.

[0052] The "primer 5' end reverse complementary fluorescent PCR" primer design principle of the application: on the basis of the current primer selection principle, (1) generally select target specific (conservative) sequence 17-22 nucleotide base length, the length of the upstream and downstream primer difference can not be greater than 3 base bases, both T m(2) G+C content should be in 40%-60%, four bases distribution / pairing should be uniform, avoid the appearance of four or more than four bases same repeat, and sequence simple repeat secondary structure; (3) a pair of primers cannot have 3 base or 3 base or more continuous reverse complement, especially the reverse complement of the 3' end of the primer; (4) the 3' end base of the primer, especially the last and the second last base should be correctly paired with the target, and the 3' last base of each primer should be G / C as far as possible, but cannot be NNGC or NNCG end (so-called GC / CG clamp), and also cannot be T end with poor specificity. There are certain cognitive differences in the existing primer selection principles, and some new background reduction and directional complement rules need to be modified and supplemented on the basis of the continuous reverse complement of the 3' end of the conventional primer: (1) middle sequence - select 6-8b "inverted repeat" in the middle of the 3' end of a pair of primers; (2) 5' end reverse complement - use 5-8b opposite original primer 5' reverse sequence as artificial sequence added in front of the opposite primer, and insert a short 4b sequence restriction enzyme site between the artificial added sequence and the original primer sequence; (3) the last or second last base of the 3' end of a pair of primers is selected as A end to make the corresponding dU of the product located in the middle of the primer connection of the dimer, and the uracil-DNA glycosylase UDG enzyme hydrolysis primer end A corresponding to the middle dU of the PD product lacks replication template base and cannot be PD amplified; (4) the 3' last base of each primer is preferably selected as A / AC end, neither G end with strong hydrogen bond "mismatch" nor T end with weak hydrogen bond and poor specificity, and more cannot use repeated double GG / TT end.

[0053] The "fluorescent PCR of 5' end reverse complement of primer" of the application uses PCR primers or conventional oligonucleotides synthesized by Shengong Biotechnology (Shanghai) Co., Ltd., heat-resistant Taq polymerase, and substrate dNTP including dUTP, which are also purchased from Shengong Biotechnology (Shanghai) Co., Ltd.; Enzyme E. Coli UDG and restriction endonuclease are purchased from NEB (Beijing) Company; fluorescent dye SYBR Green I is purchased from Invitrogen Company; and fluorescent PCR instrument SLAN-96P (Shanghai Hongshi Medical Technology Co., Ltd.) is used.

[0054] The following formulation is the standard procedure for preparing SYBR Green I fluorescent PCR reaction solution. Each single reaction (per kit) specifies the concentration and amount of each PCR component to be added. The sustained-release primers contain 10% sucrose by weight. The formulation is for 10 reactions per × 10x reaction to facilitate calculation of one set of test reaction solutions. The corresponding 50x or 100x volume PCR components per single reaction (per kit) are kit components. The formulation includes dNTP (dU instead of dT), Taq, 10x buffer, and 25x PCR enhancer, which together equal a 5x concentrated reaction mixture. For this dye-based fluorescent PCR, a series of standard DNA gradient dilutions and 5 / 10 μl volumes of purified DNA from the target molecule sample are added to a standard 25 μl system for fluorescent PCR. Alternatively, the target template can be omitted to test the primer dimer PD background and its impact on background amplification in a template-free PCR system. The dye-based SYBR Green I fluorescent PCR is the most powerful tool for observing various factors that affect fluorescent PCR. Its principle and operation are the most direct and simple, and its linear relationship and coefficient of variation (CV) are the best. However, this invention does not mean that it is limited to SYBR Green I real-time fluorescent PCR.

[0055] The standard SYBR Green I fluorescent PCR reaction solution is formulated according to the table below:

[0056]

[0057] ※10×Taq buffer: 0.6M Tris-Cl (pH8.3), 100mM KCl, 50mM (NH4)2SO4.

[0058] Mix the sustained-release primer (0.5 volume of 5–6 μM primer + 0.5 volume of 20% sucrose) with 1.0 volume of 25 × SYBR Green I. Add the sample in the following order:

[0059] (1) Add 2 μL of slow-release primer + dye to the bottom of each PCR reaction tube (no need to change the pipette tip);

[0060] (2) Add 18 / 13 μL of the remaining reaction mixture to the middle of the PCR reaction tube wall (be careful, no need to change the pipette tip);

[0061] (3) Carefully add 30 μL of mineral oil along the upper part of the PCR reaction tube wall, centrifuge briefly, and place at RT / 37℃ for up to 60 minutes;

[0062] (4) Finally, add 5 / 10 μL of DNA solution / quantitative standard / negative control / positive control of the extracted sample below the surface of the mineral oil (use a filter tip, and change the tip for each tube. After use, discard the tip into the 5% sodium hypochlorite waste liquid container and seal it).

[0063] (5) Do not mix well to avoid destroying the slow-release layer, cover the PCR reaction tube cap, and immediately perform the PCR reaction. Heat denaturation and mixing of the slow-release layer to start PCR, and amplify 30-40 thermal cycles.

[0064] Instrument standard PCR program settings: first denature at 94°C for 3-5 minutes, then run 30-40 thermal cycles of 94°C denaturation for 20 seconds, 53-55°C annealing for 20-30 seconds, 72°C extension for 30 seconds, and read the fluorescence value of the oil-sealed PCR non-thermal cover amplification. For special test purposes such as testing optimized primer PCR background, the number of thermal cycles can be increased. The instrument baseline is set at the first 0-9 cycles to avoid the detection window moving forward by 5 cycles after sensitization. The threshold is set at 10 times the standard deviation of the baseline fluorescence value, and the cycle number when the fluorescence reaches the threshold is the Ct value. Samples with a Ct value less than 30 cycles are positive, and samples with a Ct value greater than 32 cycles, including 32, are negative; To confirm samples with a Ct value of 30-32 gray area, a melting curve reaction can be set after the fluorescence PCR reaction.

[0065] The sensitization PCR conditions and boundaries of the 5' artificial complementary sequence primer and the common primer 5' sequence were tested using the standard fluorescence PCR program.

[0066] I. Sensitization effect of 5' artificial complementary sequence primer PCR:

[0067] "Artificial sequence" tries to use rare gene sequences that are not easily encountered in the test environment. This time we use a segment of the transgenic promoter CaMV sequence; The target molecule primer selects the inverted and repeated conservative sequence of the hepatitis B virus HBV core antigen, which has been repeatedly verified; The 5' artificial complementary sequence primer and the conventional HBV primer designed by chimeric design are as follows:

[0068] 5CmHBcF:

[0069] 5CmHBcRa:

[0070] (Black bold letters represent the CaMV promoter sequence, thin line letters represent the HBV sequence, and the underlined part is the same sequence),

[0071] HBVcF: 5'-atg ccc ct a tct tat caa c-3'

[0072] HBVcRa: 5'-gat tga g at ctt at g cga c-3'

[0073] First, compare the two groups of primers with the same amount of template DNA amplification sensitivity, because the conventional primer 5 μM (50 times) for single molecule detection concentration slightly insufficient and more than 6 μM primer PD non-specific too serious, the test primers according to the above formula using 5 μM concentration, loading standard (II about 1 ng / ml) plasmid 5 μL, 40 thermal cycles real-time fluorescence PCR. Results see attached Figure 2 , 5' artificial complementary sequence primer Ct value 14.5 than conventional HBV primer Ct value 20.0 ahead of nearly 5.6 cycles Ct value, generally 6.6 cycles Ct value amplification 10 2 = 100 times, 5' complementary PCR sensitivity about 5.6 cycles Ct value = 50 times amplification; note that in the original amplification 10 9-12 times again multiplied by 50 times. Or the same sensitivity can reduce the reaction 5-5.5 cycles.

[0074] Again, 5' artificial complementary sequence primer standard 10 times dilution gradient:

[0075] According to the following simple table to prepare 5 / PCR reaction mixture, XN represents a single time multiplied by all the PCR reaction tube number or total number of tests. This test the following primer R as a slow-release primer, its plus equal to 20% sucrose and then add double the amount of dye. In turn, each tube plus 2 μL slow-release primer, 18 μL PCR reaction solution, 30 μL mineral oil, finally add the standard II-V samples 5 μL DNA under the oil layer, after instantaneous high speed centrifugation immediately PCR.

[0076] Reagent name Primer F dNTP Taq enzyme 10 x Taq buffer PCR enhancer [dH2O] Total volume Aliquot volume (μL) 0.5×N 0.5×N 1×N 2.5×N 1×N 12.5×N 18×N

[0077] Amplification gradient curve see attached Figure 3 , standard II, III, IV, V sample Ct value was 15, 18.4, 22.3, 25.5 cycles, different concentrations of 5' artificial sequence complementary PCR sensitivity about 5 cycles Ct value ≈ 50 times amplification, every 10 times dilution still interval 3.3 cycles Ct value, the gradient interval does not change.

[0078] II, 5' sequence of the common primer 5' complementary primer fluorescence PCR range:

[0079] This scheme is designed to add 5-7b reverse sequence to the 5' end of the upstream primer in front of a ccgg restriction enzyme hpaII site; similarly, add 5-7b reverse sequence to the 5' end of the upstream primer in front of a ccgg restriction enzyme hpaII site; form a pair of about 30 b long chimeric primers. With this 5' complementary primer fluorescence PCR test application boundary.

[0080] The chimeric primer with 5' sequence of common primer designed in this project adopts a conserved sequence in S region of Hepatitis B virus (HBV) and a conserved sequence in C region of HBV; a sequence in S region (nt: 155-835) of HBV is cloned into pUC 19 The vector plasmid pHBs is used as positive control of HBV, and a sequence in C region (1901-2497) of HBV is cloned into pUC 19 The vector plasmid pHBc (MW 2.1 x 10 6 ) is used as positive control sequence of HBV.

[0081] The sequence in S region (nt: 410-660) of HBV is used as target-specific primer amplification sequence:

[0082] 5' HBVsF:

[0083] 5' HBVsR:

[0084] The sequence in C region (nt: 2307-2433) of HBV is used as middle homologous target primer sequence:

[0085] 5' HBVcF:

[0086] 5' HBVcRa:

[0087] (black bold letters represent hpaII restriction enzyme site, thin line letters represent HBV sequence, and underlined part is homologous),

[0088] Comparison of sensitization effect of chimeric primer with various concentrations with that of conventional primer without 5' complementarity:

[0089] The test variable, i.e. various concentrations of primer, is added to 5 μL sample solution for PCR as Test subject The slow-release primer is also added with water to 5 μL. The PCR reaction mixture solution without primer x N (x 20 times) with reduced water amount is prepared to 15 μL x 20, and half of it (10 times) is used for 150 μL reaction solution, to which 1 μL pHBs and 0.5 μL pHBc standard IV (10 pg / ml, standard VI / time, Ct 34.5) are added. The x 10 times reaction solution without standard is used as background test of various concentrations of 5' complementary primer. The standard and background Ct values of 5' complementary primer with various concentrations of 3, 3.5, 4, 4.5, 5 and 6 μM are determined by fluorescence PCR, and the standard and background Ct values of conventional primer with various concentrations of 5, 6, 7, 8, 9 and 10 μM are determined by fluorescence PCR. The results of Ct values of chimeric primer group of 5' HBVsF / 5' HBVsR are shown in Table 3 and Figure 1, and the results of Ct values of conventional primer group are shown in Table 4 and Figure 2. Figure 4, reducing the amount of primer to 3 μM does not affect the efficiency of the amplification.

[0090] Table 3:

[0091] The Ct values of the middle homologous 5' HBVcF / 5' HBVcRa chimeric primer group are shown in Table 4 below and the accompanying Figure 5 , only 2.5 μM delays 1 cycle, and 3-6 μM does not affect the efficiency of the amplification; the middle homologous primer greatly reduces non-specific effects.

[0092] Table 4:

[0093] Compared with the non-5' complementary conventional primer, because the conventional primer 5 μM (50x) is slightly insufficient for detection of low concentration samples, the primer concentration gradient is compared with more than 5 μM, and the Ct values of the HBVcF / HBVcRa chimeric primer group are shown in Table 5 below and the accompanying Figure 6 , the conventional primer increases the target amplification efficiency with increasing concentration, but also increases the primer background non-specificity. The 5' reverse complementary primer gradually increases the amplification product end to make up for the primer consumption, and the 5' complementary primer for fluorescence PCR requires less primer than conventional PCR, only 3-5 μM primer is enough for single molecule detection, and is not affected by primer non-specificity within 35-40 cycles.

[0094] Table 5:

[0095] The gradually increasing end of the amplification product makes up for the primer consumption, and the 5' complementary primer for fluorescence PCR requires less primer than conventional PCR, only 3-5 μM primer is enough for single molecule detection, and is not affected by primer non-specificity within 35-40 cycles.

[0096] Three, digital quantitative PCR by dilution method:

[0097] Before the application of digital PCR in microfluidic or microdroplet systems, manual dilution quantitative PCR is performed first. Taq polymerase + strand displacement thermostable polymerase (S. D. Polymerase, Bioron, Cat No: 108702) is applied to the sample dilution unit digital PCR, and dNTP is used (because dUTP inhibits S. D. Polymerase to some extent), and the sensitivity is further improved by one or more levels. The dispersed unit for amplification and detection has a fluorescent signal generated by nucleic acid amplification, and no fluorescent signal without nucleic acid amplification. According to the dilution ratio and the volume of the reaction unit, the nucleic acid concentration of the original solution is calculated, and through counting and Poisson distribution statistics, the absolute quantification of the initial DNA template is realized.

[0098] Firstly, the sample is parallelly diluted in two groups of gradient. The sample is diluted in EP tube with ratio gradient. The turning point of the tube with target molecule is diluted to the tube without target molecule, which is in the gradient range. Therefore, the sample with lower concentration is diluted 10 times (ratio) for several times (5 times) and then diluted 2 times (equivalent ratio) for 5 times. For most biological samples, the sample is diluted 10 times (ratio) from the sample first and then diluted 2 times (equivalent ratio) for 5 times. -2 -×10 -3 The 10 times (ratio) dilution detection is started, and then the range of 2 times (equivalent ratio) dilution detection is determined. The digital fluorescence PCR is sequentially added with 2 μL buffer release primer at the bottom of the tube, 13 μL PCR reaction solution along the tube wall, 30 μL mineral oil, and finally 10 μL of 10 times (ratio) dilution gradient and 2 times (equivalent ratio) dilution gradient under the oil layer. After the transient high-speed centrifugation, the PCR is performed. BRIEF DESCRIPTION OF DRAWINGS

[0099] Figure 1 The cartoon of 5' complementary primer PCR is shown. The long solid line represents the target gene DNA. The short arrow solid line represents the upstream and downstream primer sequence, and the arrow represents the 3' end. The arrow direction is the DNA extension and synthesis direction. The long dashed line represents the newly synthesized replication chain or product. The oblique base letter represents the "artificial complementary sequence" added at the 5' front end of the original primer. The letter indicates the reverse complementary base. The 5' complementary (letter) corresponding to the 3' end of the product chain can be used as a template and primer for thermal cycle amplification.

[0100] Figure 2 The same standard sample is amplified, and the Ct value of the 5' reverse complementary sequence primer C is 14.5, which is 5.6 cycles earlier than the conventional HBV primer Ct value 20.0. The 5.6 cycle Ct value of the 5' complementary PCR is converted to about 50 times of the sensitivity.

[0101] Figure 3 The standard sample gradient 5' complementary PCR amplification curve is shown. The Ct values of sample I, sample III, sample IV, and sample V are 15, 18.4, 22.3, and 25.5 cycles, respectively. The interval of 3.3 cycles Ct value is still maintained after dilution of 10 times. The gradient interval is the same as that of the conventional PCR, and the ratio and the repeatability are not changed.

[0102] Figure 4 The 5' HBVsF / 5' HBVsR chimeric primer group Ct value result is shown. The amount of primer is reduced to 3 μM, and the target Ct value is 26.5. The sensitivity amplification efficiency is not affected.

[0103] Figure 5. Fluorescent PCR test 5' complementary primer 5' HBVcF / 5' HBVcRa various 2.5, 3, 3.5, 4, 4.5, 5, 6 μM, etc. concentration of standard and background Ct value; compared with conventional primer sample of various 5, 6, 7, 8, 9, 10 μM, etc. concentration test its pHBc standard and background Ct value. Middle sequence 5' HBVcF / 5' HBVcRa chimeric primer group Ct value results, only 2.5 μM delay 1 cycle, and 3-6 μM do not affect the efficiency of the amplification; middle sequence inhibition of non-specific effect.

[0104] Figure 6 . Fluorescent PCR test conventional primer various 5, 6, 7, 8, 9, 10 μM, etc. concentration of standard and background Ct value, the results of conventional primer with increasing concentration and improve the efficiency of target amplification, but also parallel increase primer background nonspecific.

[0105] Figure 7 . Primer 5' HBVcF / 5' HBVcRa gradient I-VI corresponding Ct value 10.3, 13.8, 17.2, 20.5, 23.6, 26.6, 29.5, while the conventional primer HBVcF / HBVcRa I Ct value is 15.6, 5' end reverse complementary primer about 5 Ct value.

[0106] Figure 8 . Conventional primer real-time fluorescent PCR standard I-VII amplification curve. Specific embodiments:

[0107] I. Hepatitis B virus (HBV) sensitized real-time fluorescent PCR:

[0108] Hepatitis B (HBV) is a global Class III infectious disease caused by the hepatitis B virus (HBV). According to the World Health Organization (WHO), approximately 2 billion people worldwide carry the HBV virus. The infection rate of HBV in my country is very high (nearly 10%), and liver cancer, primarily caused by the hepatitis B virus, has become the leading cause of cancer death, posing a significant threat to public health. Currently, the main detection methods for HBV include enzyme immunoassay (5 / 7 items), chemiluminescence immunoassay, immunofluorescence assay, and quantitative PCR. Traditional enzyme immunoassay is widely used, but its sensitivity is insufficient. Real-time quantitative PCR and digital quantitative PCR can accurately determine the viral load in HBV patients, playing an irreplaceable role in assessing viral replication levels, disease infectivity, and monitoring the efficacy of antiviral drugs. For nucleic acid amplification, a conserved sequence from the S region and a conserved sequence from the C region of HBV are typically selected. We cloned a sequence from the HBV C region (nt: 1901-2497) into pUC. 19 Vector plasmid pHBc (MW 2.1×10⁻⁶) 6 () as a positive control sequence for hepatitis B virus.

[0109] HBV core antigen C region 5' reverse complementary primer:

[0110] Select an inverted repeat / "primer" from the conserved region of HBV C segment. Same sequence "The bases are placed in the middle of the primer pair as the original primer sequence. Then, the reverse 5' sequence of the upstream primer, gcat t, is added to the front of the 5' end of the downstream primer, separated by a ccgg. The reverse 5' sequence of the downstream primer, c tca atc ​​t, is added to the front of the 5' end of the upstream primer, separated by a ccgg, to form a chimeric primer pair:"

[0111] Because the number of five homologous bases in the middle sequence is insufficient, a single base is mutated to increase the "target amplification efficiency" without affecting the target amplification efficiency. Same sequence "The 7b homolog in the middle can selectively reduce the primer dimer's PD nonspecificity by up to 10 Ct values. The reverse complementary primer pair sequence at the 5' end of the target template is as follows: (underlined)" Sequence (in the same order as the middle section)

[0112] 5'HBVcF:

[0113] 5'HBVcRa:

[0114] (Bold letters represent the restriction enzyme hpaII site, thin lines represent the HBV sequence, and underlined parts indicate the same sequence.)

[0115] Where, the restriction enzyme hpaII sequence tcc gga homologous enhances the middle homologous of primer pair atc tta t Inhibition of PD effect. Virus DNA precipitation boiling method extraction and standard procedure sensitization fluorescence PCR:

[0116] (1) Serum specimen DNA extraction:

[0117] Simple boiling lysis method, take 50 μl-100 μl serum plus equal amount of boiling lysis solution (mix well before use, cut large mouth suction head to take), mix gently, place in boiling water bath for 10 minutes, after brief cooling at 4℃, high speed centrifugation for 10 minutes, take 5 μl supernatant for sampling. Or a large amount of lysis supernatant can be further purified by micro magnetic bead adsorption reagent.

[0118] Virus PEG precipitation method, weak positive specimen adopts PEG precipitation HBV, take 500 μl serum plus 500 μl 2×PEG solution (16% w / v PEG & 0.7M NaCl), vortex mix, high speed centrifugation for 10 minutes, discard 950 μl supernatant, concentrate the precipitate to 50 μl, then add 50 μl boiling lysis solution, the rest is the same as the boiling method, take 5-10 μl gradient dilution for sampling. The recovery rate of this PEG precipitation is 50%.

[0119] 2×Boiling lysis solution: 0.02N NaOH, 0.02% SDS (w / v), 25mM KoAc, 10mM (NH4)2SO4, 0.5% G25 (v / v), 0.5M Betaine, 0.5% Glycerol (v / v) and 0.05% Gelatin (w / v).

[0120] (2) Standard gradient and sample detection sensitization PCR reaction:

[0121] Prepare N / once single PCR reaction mixture according to the following brief table, ×N represents single time multiplied by all PCR reaction tube numbers or total detection numbers, the following downstream primer R is used as a slow-release primer, add equal amount of 20% sucrose, then add double amount of dye. Add 2 μL slow-release primer, 18 μL PCR reaction solution and 30 μL mineral oil to each tube in turn, and finally add 5 μL DNA of standard I-VI samples under the oil layer, high speed centrifugation immediately, and then PCR.

[0122] Reagent name Primer F dNTP Taq enzyme 10 x Taq buffer PCR enhancer [dH2O] Total volume Aliquot volume (μL) 0.5×N 0.5×N 1×N 2.5×N 1×N 12.5×N 18×N

[0123] Or add 5 μl sample DNA under the surface layer of mineral oil, high speed centrifugation immediately. Macrostone SLAN-96P fluorescence PCR instrument real-time fluorescence PCR, first denaturation at 95℃ for 3 minutes, then 35 thermal cycles of denaturation at 94℃ for 20 seconds, annealing at 54℃ for 20 seconds, extension at 72℃ for 30 seconds and reading of real-time fluorescence PCR without heat cover amplification.

[0124] HBV core antigen C gene cloning plasmid pHBc (0.1 μg / ml) as template for 10x (times) dilution series gradient I (0.1 μg / ml x 10 -1 -2 -3 -4 -5 -6 -7 The experimental results of 7 standard products 10-fold dilution curve concentration gradient corresponding to the gradient amplification curve are shown in Figure 7 Figure 7 The Ct values of 5' HBVcF / 5' HBVcRa gradient markers I-VII are 10.3, 13.8, 17.2, 20.5, 23.6, 26.6, and 29.5, while the Ct value of the conventional primer HBVcF / HBVcRa marker I is 15.6; the 5' end reverse complementary primer PCR is about 5 Ct values earlier. The amplification curves of the conventional primer real-time fluorescent PCR standard markers I-VII are shown in Figure 8, and the 5' end reverse complementary primer amplification is about 5 Ct values earlier. The 1000 cases of clinical samples from three first-class hospitals were detected, and the 5' end complementary primer was about 5 cycles or Ct values earlier than the conventional primer. Figure 8

[0125] II. Real-time fluorescent PCR quantitative detection of genetically modified food:

[0126] Genetic technology breaks through the limitations of natural resources, greatly improves agricultural efficiency and the yield and quality of agricultural products, but also brings the problem of biological safety of genetically modified food, which has attracted widespread attention from governments and the public around the world. Subsequently, the demand for quantitative detection of genetically modified ingredients in food and real-time fluorescent PCR has also rapidly increased. Genetically modified crops have developed from major crops such as soybeans, corn, and cotton to dozens of genetically modified crop types; most of the genetically modified types are various herbicide-resistant, insect-resistant, or herbicide- and insect-resistant composite types, and have also expanded to a series of new genetically modified types such as altered linolenic acid content, high lysine type, delayed softening, and virus resistance. It is not easy to choose a genetically modified target molecule as a universal method for real-time fluorescent PCR quantitative detection, but most genetically modified types use the cauliflower mosaic virus CaMV 35S promoter for expression and regulation, and the common promoter sequence can be used as a preliminary screening tool for real-time fluorescent PCR detection and quantitative detection of genetically modified food. This patent uses genetically modified soybeans / soybean oil as an application example to detect the CaMV 35S promoter by fluorescent PCR.

[0127] (1) Design of 5' end reverse complementary and 3' a tail primers for CaMV 35S promoter sequence:

[0128] ​​​​​​​The principle of more than 70% of the primer pairs will not produce primer dimer and the middle 3-5 base sequence of the 3' end of the primer will be pushed back about 6-10 cycles of PD strategy. The inverted repeat of the 6 base sequence of the 35S promoter sequence is selected as the target upstream and downstream F / R primer pair with a at the end:

[0129] 5'NcmF:

[0130] 5'NcmR:

[0131] (Black bold letters represent hpaII sites, thin line letters represent CaMV 35S sequences, and underlined sequences are 6b homologous),

[0132] Another primer pair including a CaMV 35S promoter sequence cloned into pUC 19 The vector plasmid pCaM (MW ~ 10 6 ) as a standard curve dilution gradient reference and positive control for transgenic CaMV.

[0133] 2. PCR component processing and preparation: After adding 1% volume of eUDG and 0.2% endonuclease mixture to each PCR component of primer 5' CamF / R, incubate at 37°C for 20-40 minutes, then prepare the 5' complementary primer PCR reaction solution according to the following table, and perform 35 cycles of standard program 53°C annealing. The sample Ct is controlled by the standard curve marker I (2.5 x 10 9 copies) - VI, with Ct values of 11, 14.5, 17.9, 21.2, 25.5, and 28.6.

[0134]

[0135] The standard 10x dilution curve value is stable and repeatable, and the sample transgenic preliminary detection content is calculated according to it.

[0136] If digital precise quantification detection is required, use SD Polymerase and dT instead of dU, and directly add sample dilution gradient solution to 20 μl reaction mixture / tube PCR, 30 μl mineral oil sealing, high-speed short-time centrifugation, and immediately dPCR.

[0137] (3) Plant CTAB method DNA extraction: plant samples were crushed in a sterile mortar with liquid nitrogen, then 500 μL of cetyltrimethylammonium bromide (CTAB) was added and ground, then transferred to a 1.5 mL plastic EP tube and incubated in a 65°C water bath for 1 hour, then extracted once with an equal volume of chloroform-isoamyl alcohol (24+1), precipitated with micro-magnetic balls or 2 times alcohol salt solution, eluted or dissolved with 40 μL of TE. Soybean oil DNA extraction method according to (Chinese Agricultural Sciences 2007, 40(5): 1069): take 10 mL of transgenic soybean oil and 10 mL of n-hexane in a beaker and stir magnetically for 2 hours, add 20 mL of PBS and continue stirring for 3 hours, transfer to a 50 mL plastic tube and centrifuge at 12000g for 20 minutes, carefully remove the lower aqueous phase, transfer to a new 50 mL tube, add an equal volume of isopropanol and mix well, place at -20°C for 20 minutes, then centrifuge at 12000g for 20 minutes, discard the supernatant, dissolve the precipitate with 100 μL of dH2O, add 500 μL of 5x DNA binding buffer to the DNA binding centrifugal column, wash the column twice with the washing solution, and elute the DNA from the column with 50 μL of TE. Purified plasmid pCaM (about ~ 0.4 μg / μl) was used as the stock solution and diluted 10 times in 8 steps according to Table 3 to make a standard curve gradient. Take 9 plastic PCR tubes, add 45 μL of purified water to each tube, take 5 μL of the original plasmid pCaM solution and add it to the first tube of water, and mix gently with a pipette. This is the original solution x 10 -1 Take 5 μL of the 10-fold dilution from the first tube and add it to the second tube of water, and mix gently. This is the original solution x 10 -2 Take a new pipette for each tube. The original solution x 10 -3 … and so on. The original solution x 10 -9 The tenth is the sample DNA tube.

[0138] (4) Fluorescent PCR: components are digested and mixed as above, then 20 μl of the mixture is added to two groups of PCR tubes, followed by 30 μl of mineral oil, then 5 μl of the gradient or sample DNA, immediately PCR 95°C denaturation for 2 minutes, then 35 cycles of 94°C denaturation for 20 seconds, 53°C annealing for 30 seconds, and 72°C extension for 30 seconds, and read the real-time fluorescent PCR without a heat cover.

[0139] Transgene standard value:

[0140] Primer 5'NcmF / NcmR group 5' complementary primer PCR background is a straight line, no target-water control group 30 cycles later, straight line amplification without Ct value; low concentration target molecule gradient is uniform and highly reproducible. The DNA sample extracted and purified from soybean oil has a Ct value of about 17.8 cycles, and the standard curve gradient is about 5 x 10 7 copies / 10 mL sample, which is equivalent to about 2.5 x 10 5 / 5 μL, which is equivalent to about 2.5 x 105 Transgene molecules / mL soybean oil or 2.5 x 10 6 Transgene molecules / 10 mL soybean oil. SEQUENCE LISTING <110> JIANG HONG <120> Reverse complement fluorescent PCR of 5' end of primer <160> 10 <210> 1 <211> 35 <212> DNA <213> Artificial Sequence <400> 1 gga ttc cat tgc cca gat gcc cct atc tta tca ac 35 <210> 2 <211> 35 <212> DNA <213> Artificial Sequence <400> 2 ctg ggc aat gga atc cga ttg aga tct tat gcg ac 35 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <400> 3 atg ccc cta tct tat caa c 19 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <400> 4 gat tga gat ctt atg cga c 19 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <400> 5 ggc ttc cgg atc ctg ctg cta tgc ctc a 28 <210> 6 <211> 29 <212> DNA <213> Artificial Sequence <400>6 CAG GAT CCG GAA GCC CAC TCC CAT AGG AA 29 <210>7 <211>30 <212> DNA <213> Artificial Sequence <400>7 TCA ATC TCC GGA ATG CCC CTA TCT TAT CAA 30 <210>8 <211>30 <212> DNA <213> Artificial Sequence <400>8 GGC ATT CCG GAG ATT GAG ATC TTA TGC GAC 30 <210>9 <211>29 <212> DNA <213> Artificial Sequence <400>9 CGT AAG CCG GAT TGT GAA GAT AGT GGA AA 29 <210>10 <211>28 <212> DNA <213> Artificial Sequence <400>10 CAC AAT CCG GCT TAC TCT AGT GGA GAT A 28

Claims

1. A method of fluorescent PCR with 5' end reverse complementarity of primers, characterized in that: The method comprises the following steps: adding a sequence reverse complement at the 5' end of a pair of primers, and then performing fluorescent PCR amplification, so that the ends of the amplification products are complementary; the complement at the 5' end of the primers does not extend but inhibits the non-specific polymerization between the 3' ends of the original primers, and the last 1-2 bases of the 3' end of the primers are adenine and the amplification product is dU instead of dT, and the specific operation mode is as follows: (1) a pair of primers are added with a reverse complementary artificial sequence at the 5' end, and the length of the complementary artificial sequence is 5-10 b, and according to the phenomenon that the 3' end of the amplification product chain is complementary to the primer and is the template for the next cycle reaction, the 3' end of the amplification product chain of a pair of 5' complementary primers is also complementary to each other; Or (2) the sequence at the 5' end of the primer is used as an artificial complementary sequence, and a 5-8 b reverse complementary sequence corresponding to the sequence at the 5' end of one primer is added in front of the 5' end of the other primer in the 5'-3' direction, and vice versa, a 5' reverse complementary sequence is formed in front of the 5' end of the other primer, and the original primer and the added sequence share a sequence at the 5' end of the primer, and a short 4 b sequence restriction enzyme cutting site can be inserted between the sequence of the original primer and the added sequence, so that the length of the amplified product is consistent after enzyme cutting; The operation steps of the PCR method are as follows: (1) 2 μL of slow-release primers containing sucrose or slow-release primers containing sucrose and dyes are added to each PCR reaction tube at the bottom of the tube; (2) the remaining PCR reaction reagents are mixed, and 18 or 13 μL of the reaction mixture is taken and placed in the middle of the wall of the PCR reaction tube; (3) 30 μL of mineral oil sealing liquid is added along the upper part of the wall of the PCR reaction tube, high-speed centrifugation is performed, and the tube is placed at 37℃ for 20-40 minutes; (4) 5 or 10 μL of DNA solution extracted from the sample, quantitative standard, negative control or positive control is added to the surface under the mineral oil layer, and a filter core suction head is used for each tube; (5) avoid mixing to avoid damaging the slow-release layer, tightly cover the PCR reaction tube, high-speed centrifugation, and immediately perform PCR reaction, heat denaturation, mix the slow-release layer and start PCR amplification for 30-40 cycles; The tail base A of the primer with the same sequence A at the middle 6-8 b of the primer end is changed to RNA.

2. The method of fluorescent PCR with reverse complementarity of the 5' end of primers according to claim 1, characterized in that: The primer has the same sequence A at the middle 6-8 b of the primer end; An antisense base 5-9 b middle interference oligonucleotide enhancer complementary to the middle of the primer is added to selectively inhibit the amplification of the primer dimer background Ct value in the PCR reaction.

3. The method of fluorescent PCR with reverse complement of 5' end of primers according to claim 1, characterized in that, The concentration of the primer is 3-6 μM or the final concentration is 0.1 μM, and the optimal primer concentration needs to be adjusted according to the minimum concentration detection requirement of different samples and the system background value.

4. The method of fluorescent PCR with reverse complement of 5' end of primers according to claim 1, characterized in that: A PCR enhancer containing betaine and polyanion polyphosphate is added to the PCR reaction reagent.

5. The method of fluorescent PCR with reverse complement of 5' end of primers according to claim 1, characterized in that: Under the condition of combined mineral oil sealing PCR reaction, a primer layered slow-release combined with a hot start PCR is used.

6. The method of fluorescent PCR with reverse complement of 5' end of primers according to claim 1, characterized in that: PCR water and buffer use chemical method instead of enzyme digestion contamination, fresh purified water using add one or two volume ratio dilution of 10% sodium hypochlorite solution room temperature 1-2 days digestion of contaminated DNA, then open the cover and boil to remove sodium hypochlorite after adding 0.05‰-0.1‰(v / v) exonuclease ExoIII refrigeration for use.

7. The method of fluorescent PCR with reverse complement of 5' end of primers according to claim 1, characterized in that: The PCR method is operated in a closed independent liquid preparation room, a sample adding room and an amplification laboratory which are physically isolated from each other. The PCR reaction reagent components are mixed in the independent liquid preparation room, and sample DNA purification and sample adding are performed in the separated sample adding room. Filtered chip suction heads are used for sample adding, and are discarded into 5% sodium hypochlorite waste liquid after use. The laboratory is regularly disinfected with 1-2% sodium hypochlorite and cleaned with 70% alcohol before use. Ultraviolet lamps and ozone are used alternately for disinfection before and after the experiment.

8. The method of fluorescent PCR with reverse complementarity of the 5' end of primers according to claim 1, characterized in that: The chain replacement thermostable polymerase is used in sample dilution unit digital PCR, and dispersed units are detected by amplification of nucleic acid to generate fluorescent signals, and no nucleic acid amplification results in no fluorescent signals. According to the dilution ratio and the volume of the reaction unit, the nucleic acid concentration of the original solution is calculated, and the absolute quantification of the initial DNA template is realized through counting and Poisson distribution statistics.

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