CN102352411A - Multiplex polymerase chain reaction (PCR) amplification method and reagent kit - Google Patents
Multiplex polymerase chain reaction (PCR) amplification method and reagent kit Download PDFInfo
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Abstract
The invention discloses a multiplex polymerase chain reaction (PCR) amplification method and a reagent kit for multiplex PCR amplification. The multiplex PCR amplification method comprises the step of designing public primers and multiplex primers with the public primers. The public primer design needs to follow two rules that: (1) under any optimized amplification conditions, no specific products are produced during the amplification of the public primers and templates to be measured; and (2) when the specific products are obtained through the amplification of the multiplex primers with the public primers, the public primers have the capability of amplifying the specific products, and then, a multiplex PCR system is configured for amplification. Because of the introduction of the public primers, the sequence specificity primer pair concentration in the reaction system is reduced by the level of more than four pairs, the possibility of the multiplex primers in the low concentration to generate the mutual action in the same reaction system is greatly reduced, the problem of the technical bottle neck in the multiplex PCR of mutual interference of different primer pairs can be solved, and the precondition can also be created for adding new primer pairs and increasing the detection gene number.
Description
Technical field
The invention belongs to the gene test field, the test kit that is specifically related to a kind of multiplex PCR amplification method and is used for the multiplex PCR amplification.
Background technology
Multiplex PCR (multiplex PCR); Claim multi-primers PCR or composite PCR again, it is in same PCR reaction system, to add primer more than two pairs, amplifies the PCR reaction of a plurality of nucleic acid fragments simultaneously; Its reaction principle, reaction reagent is identical with general PCR with operating process.
In the multi-PRC reaction, generally comprise following reagent: primer is to, dNTP, MgCl more than two pairs
2, PCR damping fluid, template DNA, Taq archaeal dna polymerase and other adjuvants (DMSO, glycerine, BSA); Usually in order to obtain reasonable amplification; Can be to the usage quantity of reagent, the reaction conditions of pcr amplification; For example elongating temperature, extension time, annealing time, annealing temperature, PCR cycle number etc. are optimized.
In the prior art, selection of primers is deferred to simple rule usually: primer length is 18-24bp or longer, and GC content is 35%-60%, 55 ℃-58 ℃ or higher of annealing temperatures.Longer primer (like the primer of DMD gene, 28-30bp) makes to be reflected at and carries out under the higher annealing temperature and produce less non-specific product.Use software Primers to calculate fusing point and detect the interaction between possible primer.Use the BLAST instrument in the NCBI sequence library, primer to be compared, to detect possible tumor-necrosis factor glycoproteins to many.
As one of important deriving technology of PCR, multiplex PCR has many advantages: (1) high efficiency, reduce operating process, and can detect a plurality of genes or fragment simultaneously; (2) economical and convenient property can save time and reagent cost greatly.This technology has been widely used in fields such as biotechnology, quarantine and medical science detection at present.Yet because the multiplex PCR principle of work is in same PCR reaction system, to add manyly to increase the simultaneously PCR reaction of a plurality of dna fragmentations of primer, therefore many in the same reaction system are prone to interact to primer, like formation hairpin structure, dimeric structure etc.Usually, multi-primers concentration is 5-12pmol in the multi-PRC reaction system, and primer is to many more with the primer amount, and the interaction between the primer is obvious more, thereby influences pcr amplification efficient.
For example; Application number is 201010153921.3; Publication number is that the Chinese invention patent Shen Qing Publication specification sheets of 101824489A discloses a kind of triple PCR test kit that can differentiate endogenous and exogenous avian leukosis virus simultaneously; Its composition comprises: archaeal dna polymerase, dNTP, primer, PCR damping fluid, distilled water, and described primer is made up of 3 pairs of primers; Particularly, said triple PCR reaction system in the PCR reaction system of 25 μ L, contains rTaq archaeal dna polymerase 2.0u; DNTP (2.5mmol/L) 2 μ L; PCR damping fluid 2.5 μ L; Each 12pmol of primer P-F and P-R, primer A-R and each 5pmol of A-F, primer E-R and each 2.5pmol of E-F; Template 100ng.Response procedures is: 95 ℃ of 10min; 94 ℃ of 1min, 50 ℃ of 1min, 72 ℃ of 40s, 30 circulations; 72 ℃ of 10min.(1mol probably is 6.02 * 10
23Individual copy, 1pmol is approximately 6.02 * 10
11Individual copy)
In addition, leukaemic's genetics feature abnormalities is complicated and changeable, comprises chromosomal transposition, disappearance, sudden change, inversion and amplification etc.The multiple RT-PCR technology can effectively remedy the deficiency of chromosome karyotype analysis once detecting multiple common leukemia fusion gene simultaneously in the experiment, has improved the recall rate of invisible translocation chromosome.At present; Clinical leukemia molecule diagnostic method commonly used mainly is the reverse transcription multi-PCR detection method of representative with Poul Jorgensen; These class methods can detect clinical common 29 kinds of fusion genes; Generally reaction system is divided into eight pipes; Every pipe detects dissimilar fusion gene and product clip size and has notable difference, judges the fusion gene type according to amplified production reaction tubes position, place and clip size.Owing to many in same reaction system primer is prone to interact; As form hairpin structure, dimeric structure etc.; Thereby influence amplification efficiency; This is to be divided into the major cause that eight pipes detect; But this has also caused such detection method complicated, loaded down with trivial details simultaneously; Operator's relevant speciality is had relatively high expectations, be difficult to promote.
Summary of the invention
The test kit that goal of the invention of the present invention provides a kind of multiplex PCR amplification method and is used for the multiplex PCR amplification; Reduce the concentration of multi-primers in the multi-PRC reaction system; Interaction between primer is reduced, do not create conditions for more primers influence amplification efficiency in same reaction system, working simultaneously.
For reaching the foregoing invention purpose, the technical scheme that the present invention adopts is: a kind of multiplex PCR amplification method may further comprise the steps:
(A), be template with the cDNA of goal gene or goal gene, the design consensus primer is right; CDNA with goal gene or goal gene is a template, and according to the fragment design multi-primers of required amplification, said multi-primers comprises that Auele Specific Primer is right more than two pairs; 3 ' the end that connects the sense primer of consensus primer centering at each 5 ' end to the sense primer of Auele Specific Primer centering; 3 ' the end that connects the antisense primer of consensus primer centering at each 5 ' end to the antisense primer of Auele Specific Primer centering; The Auele Specific Primer that obtains having consensus primer is right, and all have the right Auele Specific Primer of consensus primer has consensus primer to formation multi-primers;
Said consensus primer design should be followed two principle: under the amplification condition of (1) any optimization, do not have the specificity product when consensus primer and template to be measured increase; (2) when the multi-primers amplification that has consensus primer obtained the specificity product, consensus primer was to having the ability of the said specificity product of amplification;
(B), configuration multi-PRC reaction system; Said multi-PRC reaction system comprises: dNTP, MgCl2, PCR damping fluid, template DNA, Taq archaeal dna polymerase; Said multi-PRC reaction system also comprises: consensus primer to, have a multi-primers of consensus primer; Wherein, The right concentration of consensus primer is 0.25～0.6 μ mol/L, and each bar has the concentration 5 * 10 of the Auele Specific Primer of consensus primer
-8～5 * 10
-10μ mol/L;
(C), according to prior art, multi-PRC reaction is optimized, carry out pcr amplification according to the condition after optimizing.
Further in the technical scheme, above-mentioned amplified production is identified.
The present invention requires to protect a kind of multiple PCR reagent kit that detects leukemia fusion gene simultaneously, and said multiple PCR reagent kit comprises: conventional multiplex PCR assembly, to the multi-primers of common leukemia fusion gene; Comprise that also consensus primer is right, said consensus primer is to comprising following nucleotide sequence: SEQ ID NO.1: the sense primer 5 ' of consensus primer centering-GCATGTATAGAACATAAGGTGTCTC-3 '; SEQ ID NO.2: the antisense primer 5 ' of consensus primer centering-GAGACACCTTATGTTCTATACATGC-3 '.
In the technique scheme, said conventional multiplex PCR assembly comprises: cDNA template, archaeal dna polymerase, dNTP, MgCl
2, PCR damping fluid, distilled water.
In the technique scheme, said multi-primers to common leukemia fusion gene is right to the Auele Specific Primer of E2A/PBX1, TEL/AML1, SIL/TAL1, TLS/ERG, the conventional leukemia fusion gene of HOX11 respectively.In the optimized technical scheme, said multi-primers to common leukemia fusion gene is specifically shown in following nucleotide sequence:
。
In the optimized technical scheme, when using the multiple PCR reagent kit detection leukemia fusion gene of above-mentioned detection leukemia fusion gene, the consumption of consensus primer is 5-12pmol in the PCR reaction system of per 20 μ L.
In the optimized technical scheme, when using the multiple PCR reagent kit of above-mentioned detection leukemia fusion gene to detect leukemia fusion gene, to have the amount of the Auele Specific Primer of consensus primer be 1 * 10 to each bar in the PCR reaction system of per 20 μ L
-6～1 * 10
-8Pmol
Because the technique scheme utilization, the present invention compared with prior art has advantage:
1. the present invention is because the introducing of consensus primer; Make the interior sequence specific primers of reaction system to 4 above logarithm ranks of density loss; Interactional probability appears in the multi-primers of lower concentration like this in same reaction system will reduce greatly; Not only can solve different primers to the technical bottleneck in this multiplex PCR of phase mutual interference, also for add new primer to, increase and detect gene dosage and create precondition.
Since multi-primers to about 6 the above logarithm ranks of density loss; Different primers reduce phase mutual interference probability greatly; The present invention can merge into a plurality of independent reaction pipes in the reaction tubes and expand; Thereby significantly reduce amount of reagent; Can reduce the amount of reagent more than 40% as two reaction tubess are merged into a reaction tubes, have good economic benefit.
Description of drawings
Fig. 1 is a consensus primer design cycle among the embodiment;
Fig. 2 is the principle of work synoptic diagram of consensus primer among the embodiment;
Fig. 3 is the electrophorogram of pcr amplification product among the embodiment.
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is further described:
Embodiment one:
By the multiplex PCR utilisation technology system of consensus primer mediation, with multi-primers amount in the multi-PRC reaction system of 20 μ L from 10
12Copy is reduced to 10
4Below the copy, significantly reduce in the same reaction system many to the interaction between primer; Simultaneously, owing to many interaction between primer is reduced greatly, this reaction system can become the reaction system of " open ", makes that the new detection gene of increase becomes possibility in same reaction system.
Present embodiment carries feasibility and the efficient that E2A/PBX1 fusion gene patient sample checking multi-primers combines the principle of work of consensus primer with clinical definite.Consensus primer is filtered out by phage genome, does not have the specificity product with people's gene group pcr amplification.Consensus primer and have the multi-primers sequence of consensus primer and the fusion gene of respective detection is seen table 1.
Table 1 consensus primer and have the multi-primers sequence of consensus primer
In the technique scheme; In order to further specify the consensus primer designing principle; Be that template increases with human genome or its cDNA be example; Screening consensus primer sequence from phage genome: in ncbi database, search the phage genome sequence; With its high conservative; And nucleotide sequence is that the distinctive zone of phage is a template, designs the primer sequence of one section 22-28bp size; Through the homology of BLAST software this sequence of comparison in human genome, and carry out PCR through different human genomes with its cDNA and verify, the primer that filters out efficiency optimization is to right as preferred consensus primer; Mentality of designing is referring to accompanying drawing 1.
Each component of reaction system: the polysaccharase premixed liquid (comprises Mg
2+, dNTPs, reaction buffer and archaeal dna polymerase) 10 μ l; The multi-primers that has consensus primer, each bar have the Auele Specific Primer 1 * 10 of consensus primer
4Copy); Sense primer 5～the 12pmol (10 of consensus primer centering
12The copy order of magnitude); Antisense primer 5～the 12pmol (10 of consensus primer centering
12The copy order of magnitude); CDNA template 1 μ l; DdH
2O 7 μ l; Cumulative volume 20 μ l.Wherein, the multi-primers concentration that has a consensus primer only is 10
4Copy.Reaction conditions is: 95 ℃ of 2min, 94 ℃ of 10s, 56 ℃ of 10s, 72 ℃ of 10s, 72 ℃ of 2min; 35 circulations.
The cardinal principle of above-mentioned multiplex PCR is: add the consensus primer sequence at original multi-primers two ends, consensus primer is 1: 10 with the multi-primers concentration ratio that has consensus primer in the reaction system
-8, because it is extremely low to have the multi-primers aequum of consensus primer, its amplified production copy number only need satisfy the PCR reaction and get final product (10
2-10
5Copy), the multi-primers concentration that therefore is connected with consensus primer can be reduced to 10
4Copy interactional probability occurs and also reduces greatly between the primer, this does not disturb the creation precondition for many to primer mutually in same reaction system.Amplification is early stage, and consensus primer can't not worked because of there being complementary sequence, and the multi-primers amplified production that has a consensus primer all has the complementary sequence of consensus primer.Along with the carrying out of reaction, the multi-primers that has consensus primer exhausts because of concentration is very low very soon, and its amplified production promptly can be used as the template of consensus primer, utilizes the required special product of consensus primer amplification; Above-mentioned principle is referring to accompanying drawing 2.
Above-mentioned pcr amplification product is identified; The gained result is referring to Fig. 3; The 2nd swimming lane is for only adding the negative control of consensus primer among Fig. 3; The 3rd swimming lane is for adding 3-10 multi-primers in the table 4 (do not comprise and detect E2A/PBX1 fusion gene primer) and consensus primer, and the 4th swimming lane is for adding 1-10 multi-primers and consensus primer in the table 4.The result shows that the 2nd and the 3rd swimming lane is not seen product, and the amplified production of swimming lane 4 conforms to the expection size, and turns out to be the E2A/PBX1 fusion gene through order-checking.
In sum, in this reaction system, the multi-primers concentration of 20 μ L success by usually used 10
12Copy is reduced to 10
4Copy proves the feasibility of consensus primer in multiplex PCR and the research of leukemia fusion gene Application of Diagnosis.
SEQUENCE?LISTING
< 110>University Of Suzhou
< 120>a kind of multiplex PCR amplification method and test kit
<160> 12
<170> PatentIn?version?3.5
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Claims (6)
1. a multiplex PCR amplification method is characterized in that, may further comprise the steps:
(A), be template with the cDNA of goal gene or goal gene, the design consensus primer is right; CDNA with goal gene or goal gene is a template, and according to the fragment design multi-primers of required amplification, said multi-primers comprises that Auele Specific Primer is right more than two pairs; The 3' end that connects the sense primer of consensus primer centering at each 5' end to the sense primer of Auele Specific Primer centering; The 3' end that connects the antisense primer of consensus primer centering at each 5' end to the antisense primer of Auele Specific Primer centering; The Auele Specific Primer that obtains having consensus primer is right, and all have the right Auele Specific Primer of consensus primer has consensus primer to formation multi-primers;
Said consensus primer design should be followed two principle: under the amplification condition of (1) any optimization, do not have the specificity product when consensus primer and template to be measured increase; (2) when the multi-primers amplification that has consensus primer obtained the specificity product, consensus primer was to having the ability of the said specificity product of amplification;
(B), configuration multi-PRC reaction system; Said multi-PRC reaction system comprises: dNTP, MgCl2, PCR damping fluid, template DNA, Taq archaeal dna polymerase; Said multi-PRC reaction system also comprises: consensus primer to, have a multi-primers of consensus primer; Wherein, The right concentration of consensus primer is 0.25～0.6 μ mol/L, and each bar has the concentration 5 * 10 of the Auele Specific Primer of consensus primer
-8～5 * 10
-10μ mol/L;
(C), according to prior art, multi-PRC reaction is optimized, carry out pcr amplification according to the condition after optimizing.
2. multiple PCR reagent kit that detects leukemia fusion gene, said multiple PCR reagent kit comprises: conventional multiplex PCR assembly, to the multi-primers of common leukemia fusion gene; It is characterized in that said multiple PCR reagent kit comprises that also consensus primer is right, said consensus primer is to comprising following nucleotide sequence:
SEQ ID NO.1: the sense primer 5 ' of consensus primer centering-GCATGTATAGAACATAAGG TGTCTC-3 '; SEQ ID NO.2: the antisense primer 5 ' of consensus primer centering-GAGACACCTTA TGTTCTATACATGC-3 '; Said multi-primers comprises that Auele Specific Primer is right more than two pairs; The 3' end that connects the sense primer of consensus primer centering at each 5' end to the sense primer of Auele Specific Primer centering; The 3' end that connects the antisense primer of consensus primer centering at each 5' end to the antisense primer of Auele Specific Primer centering; The Auele Specific Primer that obtains having consensus primer is right, and all have the right Auele Specific Primer of consensus primer has consensus primer to formation multi-primers.
3. according to the said a kind of multiple PCR reagent kit that detects leukemia fusion gene of claim 2, it is characterized in that said conventional multiplex PCR assembly comprises: cDNA template, archaeal dna polymerase, dNTP, MgCl
2, PCR damping fluid, distilled water.
4. according to the said a kind of multiple PCR reagent kit that detects leukemia fusion gene of claim 2; It is characterized in that said multi-primers to common leukemia fusion gene is right to the Auele Specific Primer of E2A/PBX1, TEL/AML1, SIL/TAL1, TLS/ERG, the conventional leukemia fusion gene of HOX11 respectively.
5. according to the said a kind of multiple PCR reagent kit that detects leukemia fusion gene of claim 2, it is characterized in that the amount of consensus primer is 5～12pmol in the PCR reaction system of per 20 μ L.
6. according to the said a kind of multiple PCR reagent kit that detects leukemia fusion gene of claim 5, it is characterized in that to have the amount of the Auele Specific Primer of consensus primer be 1 * 10 to each bar in the PCR reaction system of per 20 μ L
-6～1 * 10
-8Pmol.
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| CN103937891B (en) * | 2014-04-16 | 2015-11-04 | 苏州大学 | A kind of multiple PCR reagent kit detecting leukemia fusion gene |
| CN103937891A (en) * | 2014-04-16 | 2014-07-23 | 苏州大学 | Multiplex PCR (polymerase chain reaction) kit for detecting leukemia fusion genes |
| CN104178566A (en) * | 2014-07-18 | 2014-12-03 | 中国水产科学研究院珠江水产研究所 | Multiplex fluorescence PCR (polymerase chain reaction) universal adapter for microsatellite detection, and detection method and application thereof |
| CN104178566B (en) * | 2014-07-18 | 2016-01-20 | 中国水产科学研究院珠江水产研究所 | A kind ofly can be used for multiple fluorescence PCR universal joint that micro-satellite detects and detection method and application |
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| CN108456726A (en) * | 2018-04-19 | 2018-08-28 | 深圳会众生物技术有限公司 | Spinal muscular atrophy genetic test probe, primer and kit |
| CN112481383A (en) * | 2020-12-22 | 2021-03-12 | 合肥艾迪康医学检验实验室有限公司 | ERG gene mRNA expression detection kit and detection method thereof |
| CN112795695A (en) * | 2020-12-31 | 2021-05-14 | 湖南中医药大学 | Fluorescent PCR detection kit and detection method for detecting new coronavirus |
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