CN102352350A - Same sequence primer transpositional nucleic acid amplification technology - Google Patents

Abstract

The invention provides a same sequence primer transpositional nucleic acid amplification technology, which comprises the steps of: (1) linking the artificial sequence to the upstream of the target specific primer 5', and forming a chimeric primer; (2) taking the chimeric primer as an amplification target template to produce a secondary template with the two ends having artificial sequences; and (3) taking the artificial sequences with the same sequence of 65%-75% as the transpositional primer amplification secondary template. The technology is applicable to clinic molecular diagnosis, basically can utilize the primer design to prevent the false positive result from generating, and is particularly applicable to communicable disease nucleic acid detection and accurate diagnosis. The technology is particularly suitable to multiple PRC amplification technology, is nearly designed by aiming at the multiple PRC amplification technology, and the redundant and extraordinarily complicated primers of the multiple PRC amplification technology can only adopt the primer transpositional design to eliminate the nonspecific amplification of the primer polymer.

Description

A kind of with preface primer metathetical nucleic acid amplification technologies

Technical field:

The invention belongs to the nucleic acid amplification technologies field of molecular biology and molecular diagnosis field, be specifically related to a kind of nucleic acid amplification technologies that adopts a pair of intermediate sequence major part same primers as for polymerization non-specific amplification between the minimizing primer.

Background technology:

Nucleic acid amplification technologies originated from 1971, the nucleic acid amplification in vitro imagination that Korana proposes: " through the DNA sex change, the primer hybridization with suitable extends primer with archaeal dna polymerase, and constantly repeats this process and just can clone the tRNA gene ".Also be development, sophisticated together along with the progress of modern molecular biology.Nineteen eighty-three, the Kary Mullis of U.S. PE-Cetus company human genetic research chamber has produced the inspiration of nucleic acid amplification when the research method for nucleic acid sequencing: in test tube, simulate reproduction process in the natural DNA body.Its ultimate principle: a kind of appropriate condition---template DNA is provided; Oligonucleotide primer, archaeal dna polymerase, suitable buffer system; The temperature and time of DNA sex change, renaturation and extension, the section of DNA molecule of known two terminal sequences of amplification with just can becoming geometricprogression.Through a series of explorations, development and heat-resisting polymerase, the invention of thermal cycler, application, Cetus company applied for first PCR patent of invention (US Patent 4,683,202) in 1987.

Three primitive reaction steps of--annealing--extension constitute by sex change in nucleic acid amplification PCR reaction: the 1. sex change of template DNA: the template DNA of intending amplification is after being heated to 94 ℃ of left and right sides certain hours; Template DNA double-stranded DNA double-stranded or that form through pcr amplification is dissociated; Make it to become strand; So that it combines with primer, for the lower whorl reaction is prepared; 2. the annealing (renaturation) of template DNA and primer: template DNA is after heat denatured becomes strand, and temperature is reduced to about 54 ℃, and primer combines with the complementary sequence pairing of template DNA strand; 3. the extension of primer: be warming up to about 72 ℃ dna profiling--primer binding substances is a reaction raw materials with dNTP under the effect of hot resistant DNA polymerase; Target sequence is a template; Honor is followed reverse pairing of base and semiconservative replication principle, and by 5 '-3 ' direction synthetic new and semiconservative replication chain template DNA chain reverse complemental, constantly three processes are extended in recirculation sex change--annealing--; Just can obtain more " semiconservative replication chain ", and this new chain can become next round-robin template again.General PCR carries out 30 circulations and amplifies more than millions of times with regard to goal gene expanded being the index amplification, and sharp increase adds with regard to the utmost point to surpass 30 circulation primer dimer non-specific amplifications.React the final available Y=of DNA cloning amount (1+X) ⁿCalculate.Copy number behind the Y representation DNA fragment amplification, X represent to put down (Y) all each amplification efficiency, and n represents cycle index.The theoretical value of average amplification efficiency is 100%, but av eff does not reach theoretical value in real reaction.Initial reaction stage PCR product increases gradually; It is logarithmic phase that the increase of the certain circulation of entering back target sequence dna fragmentation is exponential form; Along with the accumulation of amplified production and the consumption of PCR composition; The dna fragmentation that is increased no longer is index to be increased, and gets into linear growth phase or stationary phase, reaches " retention effects " plateau.

From hot spring, extract the discovery of other heat-resisting polymerases such as Taq hot resistant DNA polymerase and pfu, Vent, Tth in the aquatic thermophilic bacteria of an isolating strain (thermus aquaticus) uses along with 1985-1988 Saiki etc.; Round pcr is ripe, practical gradually; And because of its highly sensitive, easy and simple to handlely propagate the whole world fast, thereby be called " PCR explode year " in 1989.Round pcr has become the most important key foundation technology of life science, and therefore Kary Mullis also wins Nobel chemistry Prize in 1993.

In afterwards 20 years, reach tens of kinds of PCR and newly improve, novel method continues to bring out, and is invented; Comprise reverse transcription PCR (RT-PCR), original position PCR, ligase chain reaction (Ligase chain reaction, LCR); Mark PCR (Labeled primers, LP-PCR), inverse PCR (reverse PCR, two primers outside unknown nucleotide sequence increases); Asymmetric PCR (asymmetric PCR), touchdown PCR (touchdown PCR), recombinant PCR (recombinant PCR), nest-type PRC (nest PCR); Multiplex PCR (multiplex PCR), immunity-PCR (immuno-PCR), mRNA difference PCR; (Stranddisplacement amplification SDA), relies on amplification (the Nucleic acid sequence-basedamplification of nucleotide sequence in chain replacement amplification; NASBA), transcribe dependence amplification system (Transcript-based amplification system, TAS); Q β replicative enzyme (Q-beta replicase) catalysis RNA amplification, and rolling circle amplification (Rolling circle amplification, RCA); The isothermal duplication of ring mediation (Loop mediated isothermal amplification, LAMP) etc., the development of various especially in recent years real-time fluorescence PCRs (Real-time PCR) technology has realized the leap from the qualitative test to the accurate quantification; Like optical dye SYBRGreen I real-time fluorescence PCR and various fluorescent probe Taqman (Hydrolysisprobe), FRET Hybriditionprobes, and Molecular Beacons PCR; See summary (July 2005 for Maisa L.Wong and Juan F.Medrano, BioTechniques 39:75-85) for details.Nucleic acid amplification technologies has not only greatly promoted gene clone technology and has also improved nucleic acid detecting sensitivity, efficient, and PCR uses and has been extended to biological various fields.Round pcr has not been the monotechnics method, but comprises the new subject of series of theories, methodology and application.Summarize in detail in PCR books (Huang Liuyu etc. " PCR state-of-the-art technology principle, method and application " Chemical Industry Press 2005); PCR is widely used in life sciences such as molecular cloning, order-checking, recombination, protein engineering; Reach numerous detection Application Areass such as medical treatment, agricultural, herding, environmental protection, food, become the most crucial basic technology of 21st century biology.The patent that the whole world is relevant with PCR so far reached thousands of more than.

To sum up; PCR is detected at conventional end eventually can only qualitative analysis; Insufficient sensitivity; Be lower than that 1000 copy number samples have been surveyed not otherwise the primer dimer non-specific amplification causes false positive results, and a difficult problem such as false positive due to the amplified production steam fog glue recontaminate, conventional PCR adds the product gel electrophoretic detection and is difficult to be suitable for applying detection such as Clinical Laboratory.Higuchi in 1992 etc. have proposed to adopt dynamic PCR method and closed fluoroscopic examination mode that goal gene quantity is analyzed first, for a difficult problem that solves conventional P CR has proposed new approaches.The real-time fluorescence PCR quantitative analysis is directly related and quantitative with initial target gene amount through real-time detection amplified production amount (product mark fluorescent intensity).The fluorescent signal that the amplified production amount increases reaches the cycle number Ct value (Cycle threshold) of logarithmic phase and there is the negative correlation linear relationship in the logarithm of the initial copy number of target template.Be that starting template dilution reaches the required amplification cycles of same logarithmic phase fluorescence intensity for one times and will increase a cycle number (Ct).The signal that amplified production increases both can show through product D NA binding fluorescent dyes, like the real-time fluorescence PCR (US Patent6,569,627) based on optical dye SYBR Green I; Can adopt the fluorescent probe of band quenching group to detect again.Promptly can be activated by the fluorescent probe of cancellation, develop fluorescence labeling probe and real-time fluorescence quantitative PCR technology (Livak KJ, the et al. of Taq enzymic hydrolysis like nineteen ninety-five U.S. PE company through degraded; 1995; Genome Res:4:357-362), applied for hydrolysis probes (trade(brand)name: TaqMan) PCR patent of invention (US Patent 6,485 in 1997; 903); And Epoch company increases the MGB probe (US Patent 7,205,105) of joint efficiency on hydrolysis fluorescent probe basis.Also can be activated by the fluorescent probe of cancellation through secondary structural change; Molecular beacon Molecular beacon (Tyagi S; Et al; 1996, Nat Biotechnol 14:303-308) a kind of like this hybridization probe of loop-stem structure has just been applied for Molecular beacon patent of invention (US Patent 05925517) in 1999.Other double cross (FRET) probe, scorpion shape (Scorpion) probe, Sunrise-Primer, use range such as Lux Pimers are confined to certain application, the not obvious hydrolysis probes TaqMan method that is superior to.The real-time fluorescence PCR method sensitivity of optical dye SYBR Green I can reach several copies, and quantitatively accurately, but the false positive of primer dimer amplification combined with fluorescent has limited its clinical application; And hydrolysis probes TaqMan real-time fluorescence PCR has increased by one specific probe hybridization, and non-specific primer dimer amplification does not produce fluorescence, is widely used in Clinical Laboratory, but exists sensitivity to hang down an one magnitude yet, and quantitative linearity concerns out of true.

Be different from conventional PCR and detect end reaction-plateau amplified production eventually, generally PCR 25 to 30 the circulation products amounts that only increase are just enough; And real-time fluorescence PCR need detect the Ct40 cycle number that is low to moderate 10 copy target molecules; Label probe PCR such as TaqMan need increase at least 40 and circulate, based on the real-time fluorescence PCR of optical dye SYBR Green I in order to bring into play more highly sensitive 45 circulations of need increasing usually.Therefore the real-time fluorescence PCR technology exists more serious primer dimer non-specific amplification problem; There is the homology more than 25% in a pair of primer of extremely excessive only four kinds of base permutation and combination; Also has 25% complementarity; Primer extends below the formation primer dimer to 3 ' terminal a small amount of complementary hybridization back in the polysaccharase effect, in thermal cycling subsequently, is that template is by free a large amount of non-specific amplifications of primer and combination dye with the dimer.Do not add template based in the dyestuff SYBR Green I real-time fluorescence PCR experiment; Most a pair of primers produce dimeric background cycle threshold (Ct value) generally near 30 circulations; Some primer is to background Ct value even less than 25 circulations; Be positioned within target molecule detection by quantitative scope Ct value 15-37 circulation or " the gold detection window ", seriously disturb the quantitative and weak positive of lower concentration (copy number) target molecule to misread.Also there is the primer dimer problem in probe for real-time fluorescence PCR such as the commonly overlooked TaqMan of being in fact, just loses the primer dimer emitting fluorescence, but can competitive PCR system composition, reduces amplification efficiency and causes sensitivity to reduce, and the weak positive is prone to omission; Quantitatively inaccurate, linear relationship and repeatability are not good yet.

Real-time fluorescence PCR " closed tube analysis " in most cases and not exclusively airtight; A difficult problem that also has amplified production steam fog glue recontaminate; Remove the screw-cap extracapillary, most of 0.2ml PCR test tubes or 96 orifice plates are when 95 ℃ of sex change of thermal cycling; Just have some aerosol glue excessive (squeezing) under the HTHP and go out Guan Gai, an aerosol glue particle just contains 10 ⁵Individual molecule copy; The high density that not only the contains aerosol glue positive target molecule that increased; More be primer dimer amplification or the polymeric amplification of primer-probe that produces between excessive a pair of primer, and each detection reaction pipe/Kong Douhui produce the non-specific index amplification of primer dimer.Along with repeating same PCR, the pollutent of leakage can obtain index amplification times without number, gather, and real-time fluorescence PCR subsequently is not since 0 circulation but since PCR end loop number last time, pollutent is more and more.Amplified production steam fog glue recontaminate generally adopts dUTP to replace the dTTP product to combine uridylic-DNA-glycosyl enzyme (UDG/UNG again; US Patent 6; 090,553) selectivity pollution degradation product, UDG/UNG is PCR thermally denature inactivation subsequently; But it is limited in real work, a large amount of primer dimers to be contained dUTP amplified production Degradation, can not eliminate or postpone SYBR Green I real-time fluorescence PCR background primer Ct value.

Summary of the invention

Make nucleic acid amplification technologies such as SYBR Green I real-time fluorescence PCR be unfavorable for the limitation that clinical detection is analyzed in order to overcome PCR false positive results that primer dimer causes, the present invention " a kind of with preface primer metathetical nucleic acid amplification technologies " does not form dimer or a pair of primer PCR background Ct value highly similar more than 70% in the later experimental result of 50-60 cycle number according to single primer self.Employing is replaced as a pair of about 70% primer with preface with the conventional primer in one or both sides, carries out the nucleic acid PCR amplification again, neither disturbs target molecule amplification, and its background is a straight line in PCR45 circulation again basically, does not have the non-specific amplification value to increase.Auxiliary again modify to handle with UNG with a MO sealing end primer slowly-releasing warm start and PCR toughener DMF down make the nucleic acid applying detection in PCR reacts, not have primer dimer to accumulate; The multiple sealing of PCR does not have aerosol glue to produce or only do not have the aerosol glue of amplification; Just in case possible product is revealed also can be further by the effective enzymolysis of UNG; Multiple assurance does not produce the false positive nonspecific reaction, and it is absolute reliable that nucleic acid amplification is detected.

Provided by the invention a kind of with preface primer metathetical nucleic acid amplification technologies, its step comprises: (1) connects artificial sequence at the target special primer 5 ' upper reaches, form chimeric primers; (2) produce the secondary template of two ends band artificial sequence with said chimeric primers amplified target template; (3) with 65%-75% with the artificial sequence of preface as displacement primer amplification secondary template.

Further, it is one-sided target special primer metathetical nucleic acid amplification technologies, its displacement design of primers formula: with X _nRepresent one section base of the direct target special primer of a side, middle X _TConfirming as should be with the base of preface, then Y _nRepresent opposite side target special primer sequence base, with X _n, X _T, Y _nRepresent any A/G/C/T, A is an adenine base;

Direct target distinguished sequence primer X:5 '-X ₁X ₂X ₅X _TX _Tx _AX _AX _n-3 ',

Chimeric primers Y _s: 5 '-y _ny _N-1Y _N-5X _TX+ _T(A) AX _n+ AAY ₁Y ₂Y _N-1Y _n-3 ', small letter y represents the Y complementary base,

Displacement primer S _y: 5 '-y _ny _N-1Y _N-5X _TX _T(A) AX _n-3 ', X _AVariation is A.

The displacement primer: chimeric primers is all by 2-20: 1 prepares in advance.

Further, it is a bilateral target special primer metathetical nucleic acid amplification method, and its multiple target primer lower concentration and excessive amplimer simplification strategy are particularly suitable for multiplex PCR and detect.Its displacement design of primers formula: with X _1-nRepresent the direct target special primer of side sequence base, then Y _1-nRepresent opposite side target special primer sequence base, S _1-nBe a pair of 70% function sequence base with preface, A is an adenine base.

One side chimeric primers X _s: 5 '-S ₁S ₆S _N-1S _n+ AAX ₁X ₂X _N-1X _n-3 ',

One side changes primer S _T: 5 '-S ₁S ₆S _N-1S _n-3 ',

Opposite side chimeric primers Y _s:

5 '-y _ny _N-1Y _N-5S ₆S _N-AS _n+ AAY ₁Y ₂Y _N-1Y _n-3 ', small letter y represents the Y complementary base,

Opposite side displacement primer S _Y: 5 '-y _ny _N-1Y _N-5S ₆S _N-AS _n-3 ', S _N-1Variation is A.

The displacement primer: chimeric primers is all by 5-50: 1 prepares in advance.

Further, said amplification reaction system adds PCR toughener 1%-2% N DMF.

Further, the chimeric tieing of said chimeric primers place introduces deoxyadenine dA base as much as possible, so that the dUTP base that chimeric primers polymer non-specific amplification product contains is many as far as possible, density is more concentrated.

Further, said chimeric primers two ends head and the tail are designed with the 4-6base base complementrity in advance, and said head and the tail complementary chimeric primers forms head and the tail at ambient temperature from the loop-stem structure of hybridizing similar panhandle of bonded or molecular beacon appearance.

Further, said box composition comprises: nucleic acid extracting reagent, dNTPs, archaeal dna polymerase and damping fluid thereof, reversed transcriptive enzyme and damping fluid thereof, upstream primer F/ downstream primer R, standard control thing, purified water dH ₂O and MO.

Further, one of which end primer adopts the slowly-releasing warm start, and the warm start of said primer slowly-releasing is that primer is adsorbed in the primer sustained release dosage in advance.

Further, said primer sustained release dosage is the oligomeric base microballoon of nanometer to micron particles material, weak negative ion fiber element or the synthetic of positive electric charge a little less than 20% dextran solution, natural viscose glue, weak negative ion exchange resin, the surface band.

Further, nanometer to the micron particles material of positive electric charge was a chitosan a little less than be with on said surface.

The non-specific high background of nucleic acid amplification round pcr is mainly increased repeatedly, is accumulated and cause by primer dimer (Primer Dimer).Reagent, the measure of all inhibition primer dimer PD non-specific amplifications that adopt at present lack special specific aim to the effect between primer-primer and the target-primer, do not have the restriction that at all solves the PD non-specific amplification.The present invention " a kind of with preface primer metathetical nucleic acid amplification technologies " solves special problem from the primer self of key.According to arbitrarily always more or less there is primer dimer in primer in SYBR Green I real-time fluorescence PCR detects; And the above complementation of short of continuous three bases each other of any single primer; The experiment prerequisite that just always has no dimer to form; Provide a kind of 5 ' in addition artificial sequence target special primer that passes through to come preparatory amplified target template, adopt the 70% a pair of artificial aligning primer PCR with preface again behind the secondary template of the in addition artificial sequence of generation, its primer is right; Approximate single primer, thus significantly reduce the design amplification method that dimer forms.

The present invention " a kind of " with preface primer metathetical nucleic acid amplification technologies; Its essential characteristic is the same preface primer Replacement Strategy of nucleic acid amplification technologies; Earlier add the artificial sequence of the preceding paragraph at the target special primer 5 ' upper reaches; Produce the secondary template of the artificial sequence of two ends band with this preparatory amplified target template, re-use 70% with the artificial sequence of preface as PCR primer amplification secondary template.Its key feature also is to adopt a pair of height to replace the amplification of target special primer with the artificial primer of preface, partly with not producing the primer dimer non-specific amplification in the circulation of preface primer PCR.Be applicable to that the various round pcrs that primer combines, extends comprise isothermal amplification technique.

" a kind of " with preface primer metathetical nucleic acid amplification technologies; One end primer slowly-releasing warm start of nucleic acid amplification technologies; Described primer slowly-releasing is that primer is adsorbed in a certain solid matter surface in advance or is included in a certain certain material, can reversible adsorption attract the material or the material of thing to be called the primer sustained release dosage, does not get into the PCR reaction solution under the slowly-releasing primer normal temperature of application of sample in advance amplification can not be carried out; Primer is released into PCR reaction solution startup PCR operation fully after the thermally denature, and otherwise disturbs PCR.Described primer sustained release dosage generally comprises than great and liquid that viscosity is strong like 20% VISOSE (Dextran) liquid; Natural viscose glue etc.; Nanometer to micron particles material such as chitosan (Chitosan) with positive electric charge a little less than the band of surface; A little less than negative ion exchange resins/fibers plain, the oligomeric base microballoon of synthetic.

Described " a kind of " with preface primer metathetical nucleic acid amplification technologies, a side target special primer metathetical nucleic acid amplification technologies, it is difficult for producing false positive diagnoses, is fit to clinical communicable disease and detects.Its displacement design of primers formula:

With X _nRepresent one section base of the direct target special primer of a side, middle X _TConfirming as should be with the base of preface, then Y _nRepresent opposite side target special primer sequence base, with X _n, X _T, Y _nRepresent any A/G/C/T, A is an adenine base.

Direct target distinguished sequence primer X:5 '-X ₁X ₂X ₅X _TX _Tx _AX _AX _n-3 ',

Chimeric primers Y _s: 5 '-y _ny _N-1Y _N-5X _TX _T(A) AX _n+ AAY ₁Y ₂Y _N-1Y _n-3 ', small letter y represents the Y complementary base,

Displacement primer S _y: 5 '-y _ny _N-1Y _N-5X _TX _T(A) AX _n-3 ', X _AVariation is A.

Described " a kind of " with preface primer metathetical nucleic acid amplification technologies, bilateral target special primer metathetical nucleic acid amplification technologies, its multiple target primer lower concentration and excessive amplimer simplification strategy are particularly suitable for multiplex PCR and detect.Its displacement design of primers formula: with X _1-nRepresent the direct target special primer of side sequence base, then Y _1-nRepresent opposite side target special primer sequence base, S _1-nBe a pair of 70% function sequence base with preface, A is an adenine base.

One side chimeric primers X _s: 5 '-S ₁S ₆S _N-1S _n+ AAX ₁X ₂X _N-1X _n-3 ',

One side changes primer S _T: 5 '-S ₁S ₆S _N-1S _n-3 ',

Opposite side chimeric primers Y _s:

5 '-y _ny _N-1Y _N-5S ₆S _N-AS _n+ AAY ₁Y ₂Y _N-1Y _n-3 ', small letter y represents the Y complementary base,

Opposite side displacement primer S _Y: 5 '-y _ny _N-1Y _N-5S ₆S _N-AS _n-3 ', S _N-1Variation is A.

Described " a kind of with preface primer metathetical nucleic acid amplification technologies " a pair of displacement primer is 65%-70% with the preface rate, postpones with in advance with this low Ct value with preface rate height adjustable specific amplified Ct value, to accept or reject balance.

Described " a kind of " with preface primer metathetical nucleic acid amplification technologies; Amplification reaction system adds PCR toughener 1%-2% N (DMF); Preferred final concentration 1%-1.2%DMF; Add how many adjustable specific amplified Ct values of toughener DMF and postpone with in advance, to accept or reject balance with this low Ct value.

Described " a kind of " with preface primer metathetical nucleic acid amplification technologies; Ankylose becomes the chimeric tieing of chimeric primers place to introduce deoxyadenine dA base as much as possible; So that make chimeric primers polymer non-specific amplification product contain dUTP base many as far as possible, that density is more concentrated, improve the uridylic-more effective enzymolysis of DNA-glycosyl enzyme (UDG/UNG) that adds in advance.

Described " a kind of " with preface primer metathetical nucleic acid amplification technologies; Ankylose becomes chimeric primers two ends head and the tail to be designed with the 4-6base base complementrity in advance; Head and the tail complementary chimeric primers can form head and the tail at ambient temperature from the loop-stem structure of hybridizing similar panhandle of bonded or molecular beacon (Molecular beacon) appearance; Not with other primer or irrelevant nucleic acid nonspecific reaction, the PCR thermally denature starts specific hybrid, amplification.

Described " a kind of " with preface primer metathetical nucleic acid amplification technologies, its technology is used for gene detecting kit, and the box composition comprises: nucleic acid extracting reagent; DNTPs; Archaeal dna polymerase and damping fluids thereof such as Taq, reversed transcriptive enzyme and damping fluid thereof, upstream primer F/ downstream primer R; The standard control thing, purified water dH ₂O, MO.

The present invention " a kind of with preface primer metathetical nucleic acid amplification technologies ", its ultimate principle, operation and application all are same as general conventional nucleic acid amplification round pcr, just select different with preface primer metathetical use-pattern.Though carry out the nucleic acid PCR amplification after also having the experiment employing that target distinguished sequence primer is replaced as the primer that is different from target sequence, do not see the method for replacing amplification for the big portion that reduces the primer dimer non-specific amplification with the preface primer at present as yet.Content of the present invention mainly is to write, test and use with optical dye SYBRGreen I real-time fluorescence PCR technology; It is simple, directly perceived that reason is based on dyestuff SYBR Green I real-time fluorescence PCR know-why; Quantitative data is analyzed easily, is not that expression technology of the present invention only limits to this technology of SYBR Green I real-time fluorescence PCR.The present invention " a kind of with preface primer metathetical nucleic acid amplification technologies " is applicable to the various nucleic acid amplification technologies of primer hybridization, extension and solves the root problem of its primer dimer non-specific amplification.

The nucleic acid amplification system generally is made up of template, primer, substrate and polysaccharase and corresponding damping fluid; Wherein primer is not only determining the specificity of detection; And amplified production directly comes from primer extension; The target nucleic acid amplification is that being converted to molecule number is 5 * 6.02 * 10 because due to the excessive tens billion of times primer effect, a pair of amplimer often uses 5 μ M/L or 5pM/ μ l concentration (reaction final concentration 0.1 μ M/L or 0.1pM/ μ l) for millions of times ¹⁷/ L or 5 * 6.02 * 10 ¹¹/ μ l, its number is in excess in template concentrations far away, even final amplified production molecule number also exceeds more than a lot of times.Every necessary 3-5 μ M/L of a pair of primer or 3-5pM/ μ l be specific amplification target masterplate effectively, and it is that the generation dimer is extended in the phase mutual cross that extremely excessive a pair of primer 3 ' end like this has complementation, more in a large number by non-specific amplification.Primer dimer form generally be by its 3 ' terminal a plurality of complementary bases oppositely match, primer extension forms dimer each other; Primer between a plurality of continuous reverse complemental bases can evade through conventional primer design method; But there be 1-2 complementary sequence inevitable; DNA chain ribose has 5 ' 3 ' directivity and base does not have directivity, primer to 3 ' terminal minority base complementrity can by a primer bending turn to the terminal outer a plurality of discontinuous complementary bases in back 3 ' make a concerted effort combine, extend and produce dimer, in thermal cycling subsequently, be that template is by free a large amount of non-specific amplifications of primer and combination dye with the dimer; Do not add template based on the control experiment of dyestuff SYBR Green I real-time fluorescence PCR background in; The primer dimer non-specific amplification generally begins to get into increased logarithmic phase 30 left and right sides thermal cyclings, and non-specific amplification begins seriously, for general PCR; 30 thermal cycling amplified production amounts have enough been used; PCR can be through with mostly, if do not have the pollution of dimer amplified production and pollute quilt amplification repeatedly, primer dimer is not too big with interior PCR influence for 30 thermal cyclings of great majority.Most a pair of primers produce dimeric background cycle threshold (Ct value) generally near 30-32 circulation; 30-40 thermal cycling of real-time fluorescence PCR still is positioned at 1000-10 copy target molecule sensing range or gold detection window phase, and therefore serious interference lower concentration target molecule accurate quantification and weak positive sample are accurately qualitative.It is heat-resisting polymerase part katalysis when being lower than primer intermolecular hybrid Tm value temperature in theory that primer forms dimer, but the background primer Ct value of thermally denature heat start PCR also only postpones 1-3 cycle number, how Ct33 circulate about.Therefore warm start only can destroy a pair of primer 3 ' terminal indivedual 1-2 bases hybridization, and the most of origin cause of formation of primer dimer also should be that terminal minority complementary base relends the generation dimer of making a concerted effort to combine and extend that helps a plurality of discontinuous forward parallel complementary bases between 3 ' terminal outer primer when coming from 54 ℃ of thermal cycling primer annealing temperature.

Usually also having what ignored by everybody is that probe for real-time fluorescence PCR such as TaqMan also exists the primer dimer problem, just loses the primer dimer emitting fluorescence, but can exhaust PCR system composition, reduces amplification efficiency.Also have complementary hybridization between probe such as more serious is TaqMan and the excessive primer, extend; Do not adding template; The Ct value that only adds the SYBR GreenI PCR in real time of a pair of primer and TaqMan probe is advanced to about 25 circulations; Explain that primer adds probe PCR and can form " dimer, the tripolymer of band probe sequence "; " two, tripolymer " of non-specific amplification competition subsequently combines a large amount of TaqMan probes, causes weak positive low copy template to be difficult for bonding probes and to cause sensitivity to reduce and weak positive omission jointly.

The primer of nucleic acid amplification system, substrate and polysaccharase and corresponding damping fluid buffer and Mg ²⁺The ion composition all is long-term experiment optimum result, allows the scope of variation very narrow, and the effect that simple concentration changes primer dimer non-specific amplification and target-specific amplification basically all is parallel effects.As use the conventional primer that reduces a half strength can significantly reduce dimeric formation amount, but the low specific target template amplification quantity of also simultaneously parallel utmost point sharp fall can not be carried out normal PCR.Change polysaccharase and corresponding damping fluid buffer and Mg ²⁺, K ⁺The ion effect basically also is an equifinality.But for the conventional real-time fluorescence PCR of the short template of amplification 100-200bp especially TaqMan probe method, use reduction by one semi custom concentration dNTP substrate can improve the part amplification efficiency.Nucleic acid amplification technologies usually adopts multiple PCR toughener; Like trimethyl-glycine (Betaine); DMSO 99.8MIN. (DMSO); Increase amplification efficiencies such as first/ethanamide, parallel 1 the Ct value of precontract that pushes away are mainly still effectively promoted the amplification of unwinding of template secondary structure, but the two property reagent destruction of Betaine MO sealing process.We find that final concentration 1%-2% N (DMF) effect is better, 1-3 Ct value before can be parallel pushing away and do not disturb the sealing of MO, but do not have specificity yet.Seek to solve the key measure of primer dimer non-specific amplification and must match base complementrity hybridization and go to explore from primer with self special rule that the preface base is repelled each other.

The present invention " a kind of " with preface primer metathetical nucleic acid amplification technologies; It is not have any a pair of conventional primer of continuous complementary each other according to one; In the real-time SYBR Gree of the nucleic acid that does not add positive template I fluorescent PCR amplification system; Its background that only forms by primer dimer amplification Ct value general most in the 30-32 cycle number left and right sides (see figure 1), be positioned at low copy number template 10-1000copy/ secondary response sensing range just, disturb the specific amplification detection; The background Ct value of the double amount of single primer self is the straight line that does not have amplification in 100 circulations mostly generally; Perhaps a pair of height homology is promptly greater than after often being positioned at the 60-70 circulation with the background Ct value of preface primer more than 70%, outside minimum copy number sensing range.Therefore one to open one's minds be exactly to select try one's best homology and a pair of primer PCR of minimum complementary as far as possible, generally can postpone background Ct value several cycles, make it to drop on after the detection window.But the homology of a pair of natural primer can not be higher than 50%; Still have background Ct value 33-37 not wait; And along with repeating carrying out repeatedly of same detection, the crossed contamination of micro-aerosol glue also can be accumulated and background Ct value can move forward gradually, produces the false positive reaction (see figure 2).So adopt the as far as possible approximate single primer of a pair of artificial aligning primer of 70% homologous; Under the not obvious preceding topic that influences target-specific amplification Ct value; In the PCR reaction cycle, eliminate the amplification of primer dimer, stop continuously, accumulate during same amplification repeatedly the source of aerosol glue stain.

The present invention " a kind of " with preface primer metathetical nucleic acid amplification technologies; It is that the PCR specificity is by one section target distinguished sequence decision of primer 3 ' end according to two; Primer 5 ' is held the characteristic that can add the irrelevant sequence of the preceding paragraph, and the target distinguished sequence one or both sides primer of selecting 5 ' end front is added the irrelevant artificial sequence formation chimeric primers of the preceding paragraph does not influence the target molecule specific amplification.As only a side adds artificial sequence and then chooses 70% of the special Ct value sequence of opposite side target and partly add 30% variation as adding the synthetic side chimeric primers of artificial sequence; All add artificial sequence like both sides and then choose a specific function or rare sequence such as T7 promotor, a wherein row culture 30% variation, synthetic both sides chimeric primers as adding artificial sequence.At first add artificial PERMUTATION OF SEQUENCES template, again to replace nucleic acid amplifications such as template SYBR Green I real-time fluorescence quantitative PCR with the artificial sequence of adding of preface as the displacement primer more than 70% with the preparatory amplified target sequence generation of the chimeric primers of the conventional concentration of 1/5-1/20 band.Target distinguished sequence primer can not be replaced as fully and disturb amplification with the preface primer in order to avoid displacement template two ends self are hybridized; Some in addition be replaced as 80% and also obviously disturb carrying out and the accurate quantification of normal PCR with a pair of primer of preface, replace how many ratios and need between special Ct value and background Ct value, accept or reject balance with preface.A pair of 70% partly generally can postpone 1-2 circulation of specific C t value with the nucleic acid PCR amplification of preface displacement primer and the conventional concentration chimeric primers of 1/5-1/20; Can partly be remedied by N PCR tougheners such as (DMF); But what bring is not have primer dimer amplification, background PCR to react in the whole thermal cycling to be a straight line, to have solved the puzzlement of visiting false positive results.

The possible reason of nucleic acid amplification false positive results also comprises: non-specific hybridization, the amplification of (1) excessive primer nothing to do with DNA masterplate; At first design of primers is except that considering target gene; Big section continuous base sequence of all nucleic acid DNAs possibility cross hybridizations all must be got rid of outside the primer option in the sample; Even exist a small amount of hybridization of individual regions also only to cause linear amplification, non-specific amplification out-focus like this; Possibly produce the exponential amplification of a pair of primer hybridization hardly.Secondly, because of the target template concentrations is very low, the irrelevant at random sequence DNA of excessive primer and lower concentration carries out also loseing in 100 circulations has amplification Ct value, and this false positive probability is extremely low.(2) amplified production aerosol glue recontaminate, its major ingredient is a primer dimer, also comprises positive amplified production fragment.Prevent that key that aerosol glue overflows diffusion from being to adopt screw-cap PCR pipe, add airtight measure such as MO sealing; Even but PCR reaction solution surface spill when adding one deck MO also because of application of sample/MO or the liquid level tube wall on residual PCR mixed solution effective pcr amplification still; And have product aerosol glue to overflow, cause same repetition PCR crossed contamination next time.A measure that prevents this pollution is to adopt dUTP to replace the substrate (dNTP) of dTTP simultaneously; DUTP does not influence normal pcr amplification; But the UDG enzyme liberating that the glue stain of PCR product aerosol is all added by amplification system because of the strand/double-stranded DNA that contains dUTP in advance; The UDG enzyme can be from containing six dna fragmentation excision dUTP more than the base of dUTP in theory, and for more effective greater than the positive target molecule fragment of 100bp, and actual PCR is extremely limited for primer dimer effect degraded in detecting; Possible primer dimer is too short, contains dUTP density and measures big inadequately.

The present invention " a kind of " with preface primer metathetical nucleic acid amplification technologies, its content key is that a side primer and bilateral primer metathetical are replaced the PCR strategy, reached a side and bilateral displacement primer design, with preface primer metathetical nucleic acid amplification experimental implementation with the preface primer; The UNG enzymolysis auxiliary with the end primer slowly-releasing warm start under the MO sealing, that DMF toughener and chimeric primers variation are modified.And the nucleic acid amplification reaction condition, combine the measure of various PCR complex optimum again.

One side is replaced the design of primers scheme with preface:

For generally single (/ substance) detection by quantitative PCR and conventional RT-PCR reaction; Answer first-selected this kind relatively than easy scheme; Choose one section the most special sequence fragment of target; From sense strand sequence screening go out the to try one's best two hop counts base district of continuous reverse complemental; The upper reaches just are primer sequence with the sense strand; Its downstream change into and form local several a pair of height homology primers with the preface base continuously that have behind the antisense strand primer sequence, consider simultaneously primer between continuous complementary base is arranged as far as possible less.With this to primer sequence as the basis, a side A is directly as target distinguished sequence primer; Opposite side B target special primer then is replaced as and the artificial aligning primer of A primer sequence 70% with preface; Just replace a side and will synthesize two primers; Article one, synthetic B side target special primer and add in its 5 ' end front A side 70% with the artificial sequence of preface as the special longer chimeric primers of target, use increase in advance secondary (displacement) template of 1-5 the artificial sequence of circulation generation one end band of conventional concentration (the 5 μ M) chimeric primers of 1/5-1/20; Article one, synthetic and A side 70% are carried out conventional pcr amplification with the artificial aligning primer of preface with A side target distinguished sequence primer and are replaced template.3 ' holds the selection of target distinguished sequence longer slightly than opposite side target special primer in the chimeric primers, so that the annealing temperature that increases in advance can carry out preparatory amplification and displacement amplification more than high 6-8 ℃ step by step in same test tube.The double-stranded DNA template select that side change all can, mainly, notice that some pathogeny template possibly be DNA normal chain or minus strand by the test decision; The RNA template then preferably selects its antisense strand as chimeric primers, so that rt, chimeric primers rt cDNA chain is after a circulation 30-60 minute, conventional PCR.

What need with the position of preface ratio and variation that the special Ct value of root a tree name is not obvious to be postponed and background Ct value is accepted or rejected balance between about 45 for primer displacement.After the direct target distinguished sequence of one side primer is confirmed; 9-13 base is constant as " same preface " in the middle of general; Become one or two adenine base A and its 5 ' end become five reverse complemental bases of chimeric primers 3 ' least significant end together along several first to the 5th base artificial sequence from its 3 ' terminal three bit bases second from the bottom; Artificial sequence after the variation is added in opposite side target distinguished sequence primer 5 ' end front forms head and the tail complementary chimeric primers, add two or three adenine base A between the chimeric joint area.Why all make a variation for VITAMIN B4 A base is to contain the higher dUTP of density for chimeric primers non-specific amplification dimer product aerosol glue, so that more effectively degraded by the UDG enzyme.Head and the tail complementary chimeric primers can form the loop-stem structure that head and the tail are hybridized similar panhandle of bonded or molecular beacon (Molecular beacon) appearance at ambient temperature; Not with other primer or irrelevant nucleic acid nonspecific reaction, the PCR thermally denature starts specific hybrid, amplification.The synthetic displacement of about 30% the artificial sequence of same preface of making a variation primer, chimeric primers increase in advance generate secondary (displacement) template of band with the artificial sequence of preface after, replace template with direct target distinguished sequence primer pcr amplification.If middle long and disturb specific amplification Ct value, then can replace template two ends self and hybridize probability to reduce amplification be VITAMIN B4 A with the base aritifical variant in preface base middle with the preface base.

One side is replaced the primer formula with preface: with X _nRepresent one section base of the direct target special primer of a side, middle X _TConfirming as should be with the base of preface, then Y _nRepresent opposite side target special primer sequence base, with X _n, X _T, Y _nRepresent any A/G/C/T, A is an adenine base.

Direct target distinguished sequence primer X:5 '-X ₁X ₂X ₅X _TX _TX _AX _n-3 ',

Chimeric primers Y _s: 5 '-y _ny _N-1Y _N-5X _TX _TAX _n+ AAY ₁Y ₂Y _N-1Y _n-3 ', small letter y represents the Y complementary base,

Displacement primer S _Y: 5 '-y _ny _N-1Y _N-5X _TX _TAX _n-3 ', X _AVariation is A.

Bilateral is replaced design of primers with preface:

Be fit to multiplex PCR and isothermal transcription RNA amplification technique, a series of in recent years isothermals (/ constant temperature) gene amplification technology has also had significant progress.Unlike the PCR reaction, need the working cycle of tens temperature variation of experience.The whole process of isothermal amplification need not to carry out under the special amplification instrument all in single temperature, makes them simplify greatly the requirement of required instrument, and significantly shorten detection time, thereby is suitable for on-the-spot fast inspection or bedside detection.Wherein the transcribe rna amplification technique is high because of RNA and dna double molality plate amplification sensitivity again, becomes clinical application and detects first-selected.The amplification method of transcriptive intermediate (being the transcribe rna amplification technique) comprises TMA and NASBA, TMA (US Patent:5,766; 849) be the acting in conjunction of a kind of AMV/M-MLV of utilization reversed transcriptive enzyme and 2 kinds of enzymes of T7 RNA polymerase; The reactive system of cloning RNA or DNA under isothermal condition, the principle that increases basically is: target sequence under the reversed transcriptive enzyme effect, with the chimeric primer of T7 promotor serve as that rt is carried out in guiding; After the RNA enzyme H activity of reversed transcriptive enzyme is degraded the RNA on the heterozygosis chain; The double-stranded DNA of synthetic terminal band T7 promotor, and under the effect of T7 RNA polymerase, a dna molecular is transcribed out 700 target RNA sequences; Part RNA can be used as template again and carries out next one circulation, and entire reaction is an autocatalysis process.The principle of NASBA [US Patent:7,074,558] is similar with TMA, but only primer 5 ' end adds the T7 promoter sequence, so DNA only a chain transcribe; Also different on nucleic acid extraction and amplified production and signal detecting method.Yet; Isothermal (/ constant temperature) gene amplification technology temperature of reaction generally is starkly lower than thermal cycling PCR reaction; Make a pair of excessive, long chimeric primers intermolecular hybrid combine probability to be significantly higher than the warm start situation of conventional P CR reaction; The non-specific index amplification of the primer dimer that increases is further efficiently amplified by the dual amplification of the transcriptive intermediate of high sensitivity, very easily produces false positive.

The present invention " a kind of with preface primer metathetical nucleic acid amplification technologies " integrates this two kinds of transcribe rna amplification techniques; Choose one section the most special sequence fragment of target; Therefrom filter out two a hop counts base district of the continuous reverse complemental of trying one's best; The upper reaches just are primer sequence with the sense strand, and its downstream change into and form local several a pair of height homology primers with the preface base continuously that have behind the antisense strand primer sequence, consider simultaneously primer between continuous complementary base is arranged as far as possible less.With this to primer sequence as the basis, antisense strand primer 5 ' end is added that complete T7 promoter sequence is chimeric is reverse transcriptase primer; Opposite side sense strand primer 5 ' end adds the T7 promoter sequence of 30% variation; 9-13 base is constant as " same preface " in the middle of the T7 sequence; Become one or two adenine base A and its 5 ' end become five reverse complemental bases of sense strand primer 3 ' least significant end together along several first to the 5th base artificial sequence from its 3 ' terminal three bit bases second from the bottom; T7 sequence after the variation is added in sense strand primer 5 ' end forms head and the tail complementary chimeric primers, add two or three adenine base A between the chimeric joint area.Use conventional concentration (the 5 μ M) chimeric primers of a pair of 1/5-1/20; Wherein chimeric reverse transcriptase primer reverses the cDNA of synthetic 5 ' end band T7 promotor; And under the effect of T7 RNA polymerase, a DNA transcribes out hundreds of times of a large amount of target RNA sequences, and part RNA can be used as template again and carries out next one circulation; Rt becomes the cDNA of band T7 promotor and is synthesized secondary template DNA by the amplification of sense strand chimeric primers, and carries out pcr amplification secondary (displacement) template by a pair of 70% with preface T7 displacement primer.So carry out dual DNA and the RNA template amplification efficiently amplifies.

Multiplex PCR is with a plurality of target genes and overlaps the round pcr that target special primer PCR system is placed on while augmentation detection in the reaction tubes more; Not only there is amplification problem nonparallel, asymmetric competition in existing " multiplex PCR " method; The system that is difficult to carry out " face " of the parallel amplification of more target gene detects, and the most basic still extremely excessive manyly the target special primer is mixed in a PCR reactive system produces serious primer-oligomerization body non-specific amplification problem.Too much assorted excessively in order to solve multiple PCR primer; With many target special primer 5 ' end is all added with a pair of 70% a plurality of chimeric primers of function sequence synthesizing series with preface; It is to contain various many cover target special primer sequences that all chimeric primers 3 ' are held half; 5 ' holds half all to contain with a pair of 70% function sequence with preface, synthetic a pair of 70% with the artificial function sequence of preface as the multiple displacement primer.Adopt and many conventional concentration (the 5 μ M) chimeric primers of 1/10-1/50 is increased or the multiple target gene of rt in advance; Produce two ends all with a pair of 70% multiple displacement target template, replace primer PCR amplification multiple displacement target template with preface with a pair of conventional concentration 70% with the artificial sequence of preface.Thus, many target special primer has been reduced excessive concentrations, last has reduced too much kind to the high density amplimer, significantly reduces primer-oligomerization body non-specific amplification.Select multiple target template size in advance, amplified production just can carry out multiplex PCR according to the product clip size and detect.Adopt SYBR Green I real-time fluorescence PCR and combine the melting curve analysis, different Tm values are arranged, can carry out the heavy multiplex PCR of 3-4 and detect according to different sizes of product fragment and structure; Adopt multiplex PCR to add product agarose gel electrophoresis detection method and can carry out the heavy multiplex PCR detection of 4-5; Adopting multi-fluorescence mark TaqMan real-time fluorescence PCR can carry out the heavy multiplex PCR of 5-6 at present detects; Adopting multiplex PCR to combine the solid phase probe hybridization can carry out the heavy multiplex PCR of 50-100 detects; Adopt multiplex PCR to combine to reach 140 heavy multiple detections on the liquid detection chip Luminex of the coding microball technicism.But multiplex PCR generally only can carry out 50 heavy interior multiple reactions simultaneously.Isothermal transcription RNA amplification technique also can adopt the present invention to carry out detecting with preface primer displacement multiplex amplification.

Bilateral is replaced the primer formula with preface: with X _1-nRepresent the direct target special primer of side sequence base, then Y _1-nRepresent opposite side target special primer sequence base, S _1-nBe a pair of 70% function sequence base with preface, A is an adenine base.

One side chimeric primers X _s: 5 '-S ₁S ₆S _N-1S _n+ AAX ₁X ₂X _N-1X _n-3 ',

One side changes primer S _T: 5 '-S ₁S ₆S _N-1S _n-3 ',

Opposite side chimeric primers Y _s:

5 '-y _ny _N-1Y _N-5S ₆S _N-AS _n+ AAY ₁Y ₂Y _N-1Y _n-3 ', small letter y represents the Y complementary base,

Opposite side displacement primer S _Y: 5 '-y _ny _N-1Y _N-5S ₆S _N-AS _n-3 ', S _N-1Variation is A.

The present invention also can be applicable to reporter gene metathetical multiplex amplification with preface primer displacement amplification technique and detects.Up to now, it all is the one section direct PCR greater than 100bp DNA between the known array of two ends of directly increasing that all round pcrs and various are improved one's methods, and promptly detects the A gene A gene entire segment that just increases, and specificity is decided by the two ends primer sequence.For applying detection such as the many Clinical Laboratorys that need not take full-length gene order, food safety checks, the long intermediate sequence except that primer sequence partly is not play specific action.And other general nucleic acid detection method such as nucleic acid probe hybridization detection technique adopt the short nucleic acid Oligo of marks such as isotropic substance, fluorescence and the hybridization of target gene distinguished sequence all only to need one section 20 sequence to 30base length to get final product; But the sensitivity of hybridization probe signal plus is well below the exponential amplification of PCR.It is only to select a bit of target gene distinguished sequence (40-60bp is long) that the present invention adopts reporter gene metathetical PCR approach; Be replaced as band segment target sequence through target distinguished sequence hybridization with target homologous 100-300bp report sequence (planting system's conserved sequence farthest) not; Amplification report sequence is indicated the indirect PCR of target gene indirectly again.Be similar to preface primer bilateral displacement PCR; The left and right sides two portions that a bit of target gene distinguished sequence are divided into similar upstream primer and downstream primer; A left side is a upstream probe with the sense strand partly; Right half is downstream probe with the antisense strand, with this to probe sequence as the basis, a side probe 5 ' end is added that complete function or short rare sequence are as chimeric probe/primer; Opposite side probe 5 ' end front adds that function or the rare sequence of irrelevant sequence of a segment length and 30% variation are as chimeric report sequence; Chimeric report sequence is that an end contains the irrelevant double-stranded DNA of one section 60-250bp target-specific that target specific probe and the other end contain the function or the rare sequence of 30% variation, is added target specific probe and function sequence and is increased and make by the irrelevant dna primer of the amplification outside.Use short chimeric probe of the conventional concentration of a pair of 1/5-1/20 (5 μ M) and chimeric report length dna amplification or rt acquisition secondary total length report template in advance, conventional again pcr amplification secondary report template comes indication target gene indirectly.Linked enzyme/thermostable ligase is connected breach between chimeric probe and the chimeric reporter dna behind chimeric report length dna and the specific target gene recombination of a pair of short chimeric probe and 5 ' end phosphorylation; Form/be replaced as secondary complete report template, conventional again pcr amplification secondary report template comes indication target gene indirectly.No matter the reporter dna of the artificial designed size of the corresponding a plurality of differences of all replaceable one-tenth of multiple target gene size carries out conventional multiplex PCR and combines product agarose gel electrophoresis detection method to do the multiple detection of different big or small reporter dnas; Or combine solid phase reporter probe (bigger gradually report sequence) hybridization to do multiple reporter probe hybridization detection.Insufficient is that this method can not be suitable for real-time fluorescence PCRs such as optical dye SYBR Green I.But to the old sample legal medical expert verification of traces of degraded, the food ingredients of dark ripe processing or the check of pollutent trace, and high-throughput multis such as multiplex amplification detection nonparallel, asymmetric competition is especially effectively unique.

The present invention is with preface primer metathetical nucleic acid amplification experimental implementation, and is the same basically with general conventional PCR, and sample nucleic acid extraction purifying adopts the ordinary method preparation, does not have particular requirement.Sample DNA extracts the preparation method and comprises boiling method, the PEG precipitator method, alcohol precipitation, alkaline lysis, white enzyme K of dawn digestion method, phenol chloroform extraction method, centrifugal combination post method, and magnetic microballon combined techniques.The total RNA of sample extracts preparation general employing guanidinium isothiocyanate single stage method or the quick extracting of Trizol reagent, generally without purified mRNA, adopts PolyA Mierocrystalline cellulose or magnetic microballon solid phase method purifying in case of necessity.Reverse transcription (RT) gets final product stepwise reaction, also can with the PCR single step reaction.Rapid detection is selected one step RT-PCR single tube reaction; Single tube adds two kinds of different enzymes; The ThermoScript II (like AMV or M-MLV two mutants, SuperScript II/SuperScript ThermoScript II etc.) that promptly is used for RT; Not restriction of archaeal dna polymerase is used for PCR in real time and adopts heat-stable DNA polymerase (like Taq, Taq Plus etc.) during routine PCR.The heat-stable DNA polymerase activity is suppressed by its antibody in transcriptive process,reversed, gets into the high-temperature denatured ThermoScript II inactivation that makes of PCR process, also makes heat-stable DNA polymerase suppress the antibody inactivation simultaneously, and amplified reaction is carried out smoothly.Also have a kind of strategy to be to use and both have reverse transcriptase activity, have the Tth heat-resisting polymerase of dna polymerase activity again.

The present invention is with substrate and polysaccharase and the corresponding damping fluid buffer and the Mg of preface primer metathetical nucleic acid amplification PCR reaction system ²⁺Ionic concn is general with general common PCR reaction system.Substrate dNTP adopts dUTP to replace dTTP for preventing product aerosol glue crossed contamination (by the UNG enzymolysis), can reduce half application of sample to the short template amplification dNTP amount of 100bp.Auxiliary again with the side primer slowly-releasing warm start under the MO sealing; Direct target distinguished sequence primer (5 μ M) is added to the PCR pipe end (the corresponding 2-4 of adding times volume) as the slowly-releasing primer with 20% VISOSE (Dextran) dilution 2-4 after doubly at first; Primer is sunken to the PCR reaction solution that the pipe end adds after making and splashes the lip-deep liquid of sealing MO by VISOSE viscosity and high specific gravity and can not increase because of the disappearance composition, and sex change makes reaction solution heat start PCR behind mixing for 94 ℃.The slowly-releasing primer also can add final concentration 10%-20% nearly nano-scale chitosan (Chitosan) microballon (＜30 μ m or＞400 order particles; Be convenient to draw) with the micro sample-adding head; Chitosan bead surface positive charge normal temperature adsorbs primer and the complete heat of desorption startup of high temperature down; Microballon combines primer to spill residual particles centrifugal the sinking to the bottom fast to the liquid level tube wall, lifting slowly-releasing high reliability as because of application of sample/MO the time.The opposite side chimeric primers has warm start stern construction and displacement primer directly to add the PCR reaction mixture.Because regular-PCR damping fluid K ⁺Ion allows the scope of variation too narrow, and the present invention selects 10 * PCR buffer of an innovation for use: contain 0.6M Tris-Cl (pH8.2), 0.1M KCl, 50mM (NH ₄) ₂SO ₄, 20mM MgCl and 0.5% (v/v) Tween20 (or 1%Triton X-100) can bear the interference of impurity such as stronger ion and have higher amplification efficiency.The preparation PCR reaction solution that do not contain template separates on the tube wall of some distances above being added to the slowly-releasing primer of PCR test tube bottom then; Adding contrast or template samples DNA/RNA subsequently moves back on the tube wall of any again to the PCR test tube; Add the isopyknic MO of reaction solution (claiming Yellow Protopet 2A again) at last near the tube wall the PCR test tube mouth; Short fast centrifugal settling liquid, attention are made sure to keep in mind not mixing No Vortex! after adding all reagent In order to avoid destroy the primer slow release layer, cause the amplification of mixing reaction solution to be leaked.

Because the present invention has interference with preface primer displacement PCR to segment template specific amplification Ct value accuracy, makes the linear bad of quantitative criterion curve, accurate quantification has error.The PCR reaction solution adopts final concentration 1%-1.2% (v/v) DMF toughener (adding chimeric primers in advance) significantly to increase 1-5 cycle number of amplification Ct value, and the linear relationship of typical curve is obviously improved.But PCR toughener lack of specific such as DMF to the parallel promotion amplification with primer dimer background Ct value of the special Ct value of target template, must carefully be accepted or rejected balance between target-specific sensitivity and primer dimer background.Owing to preset a plurality of deoxyadenine dA of high-density successive base in the middle of the long chimeric primers of the present invention; Produce highdensity dU base in the middle of the synthetic primer dimer; The preventative UNG50 ℃ of pre-treatment possible aerosol glue stain of digestion in 2 minutes before the PCR reaction of PCR reaction solution (UNG of preparatory adding 1/10 in the Taq enzyme); Add the warm start of PCR primer slowly-releasing, add MO and blind nut or reverse cover PCR and manage the aerosol glue that multiple sealing does not have aerosol glue to produce or only do not have to increase, just in case possible product is revealed again further by the effective enzymolysis of UNG; Multiple assurance does not produce the false positive nonspecific reaction, and it is absolute reliable that nucleic acid amplification is detected.

The single reaction mixture of real-time fluorescence PCR:

During actually operating; At first preparation does not add 100 times * 19 μ l=1.9ml reaction mixtures of template; Both each composition added 100 times of single reaction volumes; Mixing, parallel packing 96 * 19 μ l are in the PCR of 96 orifice plates or 0.2ml reaction tubes, and every hole or every pipe add 4 μ l specimen dna/RNA or mock standard article more respectively.Each detection can be counted average Ct value by parallel 2-3 part * 25 μ l.

Plasmid mock standard curve: 1 μ g/ml * 10 ^-1, * 10 ^-2, * 10 ^-3, * 10 ^-4, * 10 ^-5, * 10 ^-6, * 10 ^-7Dilution.

Each reaction tubes is careful to add and the short fast centrifugal settling of the isopyknic MO of reaction solution, capping liquid, and (sky, Xi'an swells company's T L988, TL988-II type and MJ Inc.DNA Engine Option to last real-time fluorescence PCR appearance ^TM2).At first 94 ℃ of 50 ℃ of pre-reactions 2 minutes are 2 minutes, 45 circulations then: 94 ℃ 20 seconds, 54-57 ℃ 30 seconds, 72-74 ℃ 30 seconds.Read fluorescent value in 72-74 ℃.

The invention has the advantages that:

(1) is fit to clinical molecular diagnosis, can controls through design of primers basically and not produce false positive results, be specially adapted to the detection of nucleic acids of communicable disease and accurately diagnosis.

(2) especially be fit to the multiplex PCR amplification technique, the present invention almost makes to measure for the multiplex PCR amplification technique, and the too much assorted excessively primer of multiplex PCR amplification technique can only adopt primer displacement design of the present invention to eliminate primer-oligomerization body non-specific amplification.

Description of drawings:

Fig. 1. for not adding dna profiling, only contain the amplification background Ct of the fluorescent PCR system value of various primers.By the preferred HBV of general design of primers principle contain the part with preface primer background Ct value about 33-34, the background Ct value that a pair of primer adds probe moves forward to about 25 circulations.Generally after 45-50 circulates, and any most primer adds the double amount background of single primer self and is straight line, no Ct value with preface displacement primer background Ct value in the present invention.

Fig. 2. make 10 times of standard quantitative curves that dilute templates for the HBV primer real-time fluorescence PCR that adopts a pair of general primer principle of design to optimize, Far Left article one amplification curve is 0.1 μ g/ml template about 10 ⁸Copy, 10 times of dilutions thereupon, the last item amplification curve is the background contrast that does not have template, 30 circulations of the Ct of background contrast as a result value are with 10 ¹-10 ³Copy inseparablely, full weight stacks, and detects inaccurate and false positive.

Fig. 3. be known quantitative hepatitis B virus (HBV) cAg plasmid contrast pUC-HBcore1 μ g/ml about 10 ⁹Copy is made 10 times of standard quantitative curves that dilute templates, and Far Left article one amplification curve is 0.1 μ g/ml template about 10 ⁸Copy is done 10 times of dilutions thereupon, and the last item amplification curve is the background contrast that does not have template, and the Ct of background contrast as a result value has any to rise in the 43-45 circulation, and the Ct value does not almost increase in the PCR circulation.Standard quantitative curve Gradient distribution is especially hanged down copy gradient La Dekai near common fluorescent PCR, and 10 times of gradient dilutions of copy number can accurately be measured.Repeatedly repeated very good.

Fig. 4. be always pathogenic pearl of EV and the analysis of 71 hypotypes two-fold real-time fluorescence PCR, the totally one plasmid template single tube reaction of two cover PCR systems, the double amplification still obtains gradient preferably.

Fig. 5. for EV total with EV71 type two-fold real-time fluorescence PCR melting curve, the total Tm value of EV is that 87 ℃ and EV71Tm are 90.6 ℃, the two can obviously distinguish evaluation, and with primer dimer Tm value be that 78 ℃ of intervals are far away, can not misread.

Specific embodiment:

Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Under the situation that does not deviate from the present invention's spirit and essence, modification or replacement to the inventive method, condition, step and application are done all belong to scope of the present invention.

Instance one: the viruses of human hepatitis B detects with preface primer displacement fluorescent PCR:

Hepatitis B (abbreviation hepatitis B) is by hepatitis B virus (Hepatitis B virus, a kind of global communicable disease that HBV) causes.Hepatitis B infected rate is very high in population of China, has endangered the healthy of people greatly.At present the detection method of hepatitis B mainly contain enzyme immunoassay, put the method for exempting from, chemoluminescence method, immunofluorescence technique, nucleic acid amplification (PCR) fluorescent quantitation etc.Enzyme immunoassay is used wider, but the real-time fluorescence PCR analytical method can accurately be measured hepatitis B patient's virogene, and to the judgement of the infected's virus replication level, state of an illness infectivity and antiviral curative effect monitoring have irreplaceable vital role.

Hepatitis B virus (HBV) is a dsdna segment virus; Mainly contain three sections special conserved regions of kind type; Lay respectively at surface antigen HBsAg district, X district and core Core district, most of HBV real-time fluorescence PCR research is concentrated and is selected core Core district and surface antigen HBsAg district.There is positive and negative double-stranded DNA in HBV core Core district, and surface antigen HBsAg district only contains the minus-strand dna strand, and secondary structure also concentrates on this zone in a large number, influences pcr amplification efficient.Relatively more most of core Core district PCR primer sequences of delivering still exist to form the dimer complementary base sequences thereof between some tangible primers, even do not meet general design of primers generic principles, and the actual primer dimer of PCR on probation is mostly in 30 circulations place of Ct value.

The one section sequence in the selected hepatitis B virus of the present invention (HBV) core Core district (CDR:2306-2444) with preface primer metathetical nucleic acid amplification target distinguished sequence, shows that each 20 base Core district (nt:2306-2444) sequence of two ends are following as HBV:

AB540584 Core district (nt:2306-2444)

CAAATGCCCC?TATCTTATCA?AC------GCAGAAGATCTCAATCTC

HBV designs with preface primer displacement fluorescence PCR primer:

Root a tree name the present invention one side is with preface displacement design of primers scheme and contrast its formula,

Direct target distinguished sequence primer X:5 '-X ₁X ₂X ₅X _TX _TX _AX _n-3 ',

Chimeric primers Y _s: 5 '-y _ny _N-1Y _N-5X _TX _TAX _n+ AAY ₁Y ₂Y _N-1Y _n-3 ', small letter y represents the Y complementary base,

Displacement primer S _y: 5 '-y _ny _N-1Y _N-5X _TX _TAX _n-3 ', X _AVariation is A.

HBVc is following with preface displacement primer sequence:

HBVcF _s：5’- gtt?ga

aac?aaa?tgc?ccc

a tc?aac-3’

HBVcS _f：5’-gt?tga aac-3’

HBVcR：5’-gag

tgc-3’

Wherein F represents upstream primer sequence, F _sBe upper reaches chimeric primers just; R represents the direct target special primer in downstream, and runic is represented with the preface base, and underscore is that chimeric primers two ends are from hybridization sequences.

Another set of substitute chimeric primers and the displacement primer following: its Italic capitals for preface oversize artificially introduce one the variation base

2HBVcF _s：5’- gtt? aa?caa?atg?ccc?ct

at?caac-3’

2HBVcS _f：5’-gtt aa?c-3’

Known HBV positive sample serum boiling method supernatant with Clinical Laboratory is a template in addition, and pUC is cut and be cloned into to selected total length Core district (1900-2450) fragment both sides sequence anamorphic zone restriction enzyme site primer amplification template 450bp fragment with this fragment enzyme ₁₉Carrier is simulated positive control dna as hepatitis B.

(1) adopt a step boiling method: get 50 μ l serum and add 2 of 50 μ l * boil damping fluid (room temperature is deposited, with preceding abundant mixing microballon), light mixed, put boiling water bath 5 minutes, high speed centrifugation is 5 minutes after 4 ℃ of of short duration precoolings, gets supernatant application of sample 4 μ l.

Weak positive sample adopts PEG deposition HBV to concentrate, and get serum 500 μ l and add 500 μ l PEG liquid, the Vortex mixing, high speed centrifugation 10 minutes is abandoned supernatant, and deposition adds 25 μ l dH ₂Add 2 of 25 μ l * boil damping fluid, surplus the same boiling method, application of sample 4 μ l behind the O mixing.But PEG precipitate recovery rate average out to 60%, detection by quantitative must counting loss.

2 * boil damping fluid: 0.1M Tris, 2%NP40,0.01%SDS, 50mM KoAc and 8%Chelax-100 (400mesh).

(2) real-time fluorescence PCR reaction:

At first prepare the slowly-releasing primer; The chitosan powder that rinsing is good is dissolved in 2N hydrochloric acid HCl number hour to spending the night; Fully after the dissolving; Pour into the most fast in the sodium hydroxide NaOH beaker that contains big volume equivalent of just quick magnetic agitation, chitosan is condensed into several microns to 20 microns microparticle again, uses the centrifugal rinsing of neutral Tris-Glycine damping fluid repeatedly.The HBVcR primer is dissolved in purified water to 100 μ M stock solution earlier; Be diluted to 5 μ M application liquid with 20% glycan of crawling; Concentration 5 μ M primers and isopyknic 20%-40% (v/v) chitosan microparticle mixing generate 2.5 μ M, or mix and form rare four times of slowly-releasing HBVcR with isopyknic 20% glycan of crawling again.With before shaking! In order to avoid that the long storage time solids precipitation causes primer is inhomogeneous.Each PCR reaction tubes adds the rare four times of slowly-releasing HBVcR of 2 μ l to managing the end.

By following formula rate,

Preparation does not add TemplateN * or 100 * secondary response mixed solution; Mixing; Parallel packing 19 μ l are in 96 orifice plates of existing 2 μ l slowly-releasing primers or the PCR reaction tubes of 0.2ml, and every hole or every pipe add 4 μ l specimen dnas again, carefully add 20-25 μ l MO at last again in reaction surface sealing PCR reaction tubes.Each detection can be counted average Ct value, analytic statistics result by parallel 2-3 part * 25 μ l.

Last real-time fluorescence PCR appearance (grand company's T L988 type in sky, Xi'an and MJ Inc.DNA Engine Option ^TM2), press the working instructions operation.At first-66 ℃ of-94 ℃ of 50 ℃ of pre-reactions 2 minutes 2 minutes are 2 minutes, 45 circulations then: 94 ℃ 20 seconds, 56 ℃ 20 seconds, 74 ℃ 20 seconds.Read fluorescent value in 74 ℃.

(3) interpretation:

Adopt known quantitative hepatitis B virus (HBV) cAg plasmid contrast pUC-HBcore1 μ g/ml about 10 ⁹Copy is made 10 times of standard quantitative curves that dilute templates, and Far Left article one amplification curve is 0.1 μ g/ml template about 10 ⁸Copy, 10 times of dilutions thereupon, the last item amplification curve is the background contrast that does not have template, and the Ct of (see figure 3) background contrast as a result value has a bit in the 43-45 circulation, and the Ct value does not almost increase in the PCR circulation.Standard quantitative curve Gradient distribution is near common fluorescent PCR; Ct value spoke degree directly primer real-time fluorescence PCR is dispersed a little in middle gradient; Cycle number postpones 1 Ct value/each gradient, and linear relationship is still better generally, and 10 times of gradient dilutions of copy number can be accurately quantitative.

Inspection institute hepatitis B virus (HBV) nucleic acid quantification standard substance (lot number 0711) are positive with reference to article and quantitatively with reference to article L in the purchase ₁-L ₅The standard testing result basically identical, negative is baseline response with reference to article entirely.Clinical trial 400 routine positive samples 99% meet, and negative sample detects and do not occur false positive reaction so far as yet.Repeatedly repeated very good.

Instance two: HEV's pathogenic strain bifluorescence PCR detects:

In recent years hand foot mouth disease begins in China child popularly on a large scale, and case fatality rate is high.(EnteroVirus, EV) nucleic acid real-time fluorescence PCR detect and become control it infects popular key means its cause of disease enterovirus, and enterovirus EV is divided into serotype different more than 60 kinds at first ,-71 types that comprise Enterovirus 68.Based on the classification of its nucleotide sequence, people EV is divided into five types of A, B, C, D and PolioVirus again, and wherein main pathogenic strain Coxsackie A16 (CA16), EnteroVirus 71 types (EV71) are classified as HEV A type.Enterovirus EV genovariation is big, and only 5 ' UTR is conservative, and three sections all strain homology conserved regions are arranged, and the EV universal primer of delivering is all selected this conserved regions thereby the non-pathogenic strain of flase drop, can only differentiate as total EV and detect; The big VP of EV71 type diagnostic primers choosing variation ₁The serious again omission in district.

The enterovirus genome is compared repeatedly, finds in nt 434-448 sequence

Detect upstream primer for pathogenic strain CA16 and EV71 height homologous region can be used as pathogenic strain, choose second section all strain conserved regions nt 538-557 sequence Antisense strand 5 '-c acc caa Agt agt cGg ttc-3 ' is EV pathogenic strain rt and downstream primer, and amplification produces the 138bp fragment.

Conserved regions nt2757-2773 sequence gcc aac tgg

a ga of hypotype EV71 and antisense strand 5 '-ctc gca ggt g aa-3 ' of nt3018-3034 (ttcatgtcacctgc gag) are as EV71 hypospecificity amplimer, and amplification produces the 278bp fragment.There is a small amount of omission in hypotype EV71 Auele Specific Primer, but just can do the differential diagnosis of accurate EV71 hypotype with the coupling of total EV pathogenic strain special primer.

EV designs with preface primer displacement fluorescence PCR primer:

Root a tree name bilateral of the present invention is with preface displacement design of primers scheme and contrast its formula,

One side chimeric primers X _s: 5 '-S ₁S ₆S _N-1S _n+ AAX ₁X ₂X _N-1X _n-3 ',

One side changes primer S _T: 5 '-S ₁S ₆S _N-1S _n-3 ',

Opposite side chimeric primers Y _s:

5 '-y _ny _N-1Y _N-5S ₆S _N-AS _n+ AAY ₁Y ₂Y _N-1Y _n-3 ', small letter y represents the Y complementary base,

Opposite side displacement primer S _Y: 5 '-y _ny _N-1Y _N-5S ₆S _N-AS _n-3 ', S _N-1Variation is A.

Always/and the EV bilateral that causes a disease is following with preface displacement primer sequence: add T7 sequence

before the special primer sequence

ZEVF _s：5’-gaggac GaA?Gag?cta?gtt?agt?agt?cc t?c-3’

ZEVS _f：5’-gaggac

GaA?G-3’

ZEVR _s：5’-

GGA?Gc?acc?caa?agt?agt?cgg?ttc-3’

ZEVS _r：5’- GGA?G-3’

EV71 hypotype bilateral is following with preface displacement primer sequence: add the T7 sequence before the special primer sequence

EV71F _s：5’-gaggac GaA?Gcc?aac?tgg a?ga-3’

EV71S _f：5’-gaggac GaA?G-3’

EV71R _s：5’-

GGA?Gctc?gca?ggt

g?aa-3’

EV71S _r：5’- GGA?G-3’

The displacement primer: chimeric primers all prepared by 10: 1 in advance.

(1) RNA extracts: first-selected bleb liquid, throat swab, (or blood, cerebrospinal fluid), 3 days optional anus swabs or ight soil leach liquor (or cell culture) 0.1ml fall ill; Ight soil leach liquor palpus natural sedimentation 10 minutes; Get supernatant 0.1ml (or 0.1g solid sample) and add RNA lysate 1ml (4M GTC liquid+0.5ml water-saturated phenol of 0.5ml) sex change cracking, strong vortex oscillation adds the vibration of 100 μ l chloroforms again; High speed centrifugation 10 minutes is got and is reset and added 3 * binding buffer liquid (6M sodium iodide N _aI) move to commercial silica gel purification post (detail operations is undertaken by Qiagen/Tiangen company specification sheets), with lavation buffer solution (the 2M N that contains 70%EtOH _aI liquid) wash post twice, add the dH that 50 μ l DEPC handle ₂O wash-out, centrifugal collection purified RNA.Or the 2M sodium-acetate (PH4.0) of resetting and adding equivalent Virahol and 1/10 volume in the cracking puts-20 ℃ of centrifugations again in 2 hours, and 75% cold washing with alcohol once adds the dH that 50 μ l DEPC handle ₂The O dissolving.

(GTC liquid: 65 ℃ of dissolved 4M guanidinium isothiocyanate+0.1mM DTT and 0.5%Sarkosyl.)

(2) RT bifluorescence PCR

Two steps of rt-real-time fluorescence PCR merge the single tube reaction:

It is airtight that the reaction solution surface carefully adds 20ul MO along tube wall again!

During actually operating, at first preparation does not add Template100 times * 15 μ l=1.5ml reaction mixtures, both each composition added 100 times of single reaction volumes, mixing, parallel packing 96 * 15 μ l are in the PCR of 96 orifice plates or 0.2ml reaction tubes, every hole or every pipe add 10 μ l specimen dna/RNA or mock standard article more respectively.Each detection can be counted average Ct value by parallel 2 parts * 25 μ l.

Typical curve: mock standard (1 μ g/ml) * 10 ^-1, * 10 ^-2, * 10 ^-3, * 10 ^-4, * 10 ^-5, * 10 ^-6, * 10 ^-7Dilution.

(sky, Xi'an swells company's T L988, TL988-II type and MJ Inc.DNA Engine Option to last real-time fluorescence PCR appearance ^TM2).At first-52 ℃ of 80 ℃ of RNA sex change 4 minutes are 30 minutes, 40 circulations then, 95 ℃ 20 seconds, 54-56 ℃ 20 seconds, 74 ℃ 20 seconds).Read fluorescent value in 74 ℃.

(3) experimental result (seeing Fig. 4 Fig. 5) is analyzed:

Typical curve and Quality Control: EV part 5 '-UTR sequence and EV71 sequence are inserted pUC ₁₉Enzyme is cut carrier, the clone generates pUC-EV ₃L simulation male plasmid quantitatively contrasts the long 2.94kb of plasmid, molecular weight MW=1.8 * 10 ⁶, calculate 1ng=3.3 * 10 ⁸The copy molecule.Plasmid pUC-EV ₃L carries (Miniprep) preparation DNA, photometry density OD for a short time ₂₆₀/ OD ₂₈₀, by 1 OD ₂₆₀Value=50 μ g/ml DNA calculate content, and are diluted to 1 μ g/ml as quantitative reference standards with TE liquid.

Accompanying drawing 4. is always pathogenic pearl of EV and the analysis of 71 hypotypes two-fold real-time fluorescence PCR, the totally one plasmid template single tube reaction of two cover PCR systems, and dual amplification still obtains gradient preferably.Fig. 5. for EV total with EV71 type two-fold real-time fluorescence PCR melting curve, the total Tm value of EV is that 87 ℃ and EV71Tm are 90.6 ℃, the two can obviously distinguish evaluation, and with primer dimer Tm value be that 78 ℃ of intervals are far away, can not misread.Mock standard sample HEV concentration is good with measurement Ct value linear relationship as a result, detects accurately and reliably.

Claims (10)

1. one kind with preface primer metathetical nucleic acid amplification technologies, it is characterized in that its step comprises: (1) connects artificial sequence at the target special primer 5 ' upper reaches, form chimeric primers; (2) produce the secondary template of two ends band artificial sequence with said chimeric primers amplified target template; (3) with 65%-75% with the artificial sequence of preface as displacement primer amplification secondary template.

2. it is characterized in that with preface primer metathetical nucleic acid amplification technologies according to claim 1 is described that it is one-sided target special primer metathetical nucleic acid amplification technologies, its displacement design of primers formula: with X _nRepresent one section base of the direct target special primer of a side, middle X _TConfirming as should be with the base of preface, then Y _nRepresent opposite side target special primer sequence base, with X _n, X _T, Y _nRepresent any A/G/C/T, A is an adenine base;

Direct target distinguished sequence primer X:5 '-X ₁X ₂X ₅X _TX _Tx _AX _AX _n-3 ',

Chimeric primers Y _s: 5 '-y _ny _N-1Y _N-5X _TX _T(A) AX _n+ AAY ₁Y ₂Y _N-1Y _n-3 ', small letter y represents the Y complementary base,

Displacement primer S _y: 5 '-y _ny _N-1Y _N-5X _TX _T(A) AX _n-3 ', X _AVariation is A.

The displacement primer: chimeric primers is all by 2-20: 1 prepares in advance.

3. described with preface primer metathetical nucleic acid amplification technologies according to claim 1; It is characterized in that; It is a bilateral target special primer metathetical nucleic acid amplification method, and its multiple target primer lower concentration and excessive amplimer simplification strategy are particularly suitable for multiplex PCR and detect.Its displacement design of primers formula: with X _1-nRepresent the direct target special primer of side sequence base, then Y _1-nRepresent opposite side target special primer sequence base, S _1-nBe a pair of 70% function sequence base with preface, A is an adenine base.

One side chimeric primers X _s: 5 '-S ₁S ₆S _N-1S _n+ AAX ₁X ₂X _N-1X _n-3 ',

One side changes primer S _T: 5 '-S ₁S ₆S _N-1S _n-3 ',

Opposite side chimeric primers Y _s:

5 '-y _ny _N-1Y _N-5S ₆S _N-AS _n+ AAY ₁Y ₂Y _N-1Y _n-3 ', small letter y represents the Y complementary base,

Opposite side displacement primer S _Y: 5 '-y _ny _N-1Y _N-5S ₆S _N-AS _n-3 ', S _N-1Variation is A.

The displacement primer: chimeric primers is all by 5-50: 1 prepares in advance.

4. it is characterized in that with preface primer metathetical nucleic acid amplification technologies according to claim 1 is described that said amplification reaction system adds PCR toughener 1%-2% N DMF.

5. described with preface primer metathetical nucleic acid amplification technologies according to claim 1; It is characterized in that; The chimeric tieing of said chimeric primers place introduces deoxyadenine dA base as much as possible, so that the dUTP base that chimeric primers polymer non-specific amplification product contains is many as far as possible, density is more concentrated.

6. described with preface primer metathetical nucleic acid amplification technologies according to claim 1; It is characterized in that; Said chimeric primers two ends head and the tail are designed with the 4-6base base complementrity in advance, and said head and the tail complementary chimeric primers forms head and the tail at ambient temperature from the loop-stem structure of hybridizing similar panhandle of bonded or molecular beacon appearance.

7. one kind with preface primer metathetical nucleic acid amplification gene detecting kit, it is characterized in that said box composition comprises: nucleic acid extracting reagent; DNTPs, archaeal dna polymerase and damping fluid thereof, reversed transcriptive enzyme and damping fluid thereof; Upstream primer F/ downstream primer R, standard control thing, purified water dH ₂O and MO.

8. a nucleic acid amplification technologies is characterized in that, one of which end primer adopts the slowly-releasing warm start, and the warm start of said primer slowly-releasing is that primer is adsorbed in the primer sustained release dosage in advance.

9. described with preface primer metathetical nucleic acid amplification technologies according to claim 8; It is characterized in that said primer sustained release dosage is the oligomeric base microballoon of nanometer to micron particles material, weak negative ion fiber element or the synthetic of positive electric charge a little less than 20% dextran solution, natural viscose glue, weak negative ion exchange resin, the surface band.

10. it is characterized in that with preface primer metathetical nucleic acid amplification technologies according to claim 9 is described that nanometer to the micron particles material of positive electric charge is a chitosan a little less than the band of said surface.

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