CN106222252B - The multiple PCR primer and detection method of three kinds of integron related elements are detected simultaneously - Google Patents
The multiple PCR primer and detection method of three kinds of integron related elements are detected simultaneously Download PDFInfo
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Abstract
The present invention relates to bacterial drug resistance research fields, specifically a kind of multiple PCR primer and multi-PCR detection method for three kinds of integron related elements in detection bacterium simultaneously, the multiple PCR primer detects 1 class integron (Int1) while designing and screen including the use of primer-design software Primerplex 2.61, 4 class Super integrons (SXT) and the combination of the multiple PCR primer in the 1 common area of type insetion sequence (ISCR1), multiplex PCR detection architecture is established using the primer of design, the multi-PCR detection method for three kinds of integron related elements is established by the optimization to primer volume and annealing temperature, compared with prior art, the present invention has the advantages that can three kinds of integron phases in detection bacterium simultaneously using the multiple PCR method established of the present invention Element is closed, has many advantages, such as easy to operate, quick, high specificity.
Description
Technical field
The present invention relates to bacterial drug resistance research fields, specifically, being that one kind is used for while detecting 1 class integron
(Int1), the multiple PCR primer and detection method of 4 class Super integrons (SXT) and the 1 common area of type insetion sequence (ISCR1).
Background technique
Antibacterials cure the use in clinical and food animal-breeding in people, have saved the life of hundreds of millions of people, have also pushed away
The development of terrestrial and aquatic animal aquaculture is moved；At the same time, but also bacterial drug resistance problem is on the rise.Integron is
Mediate one of the generation of Gram-negative bacteria antibiotic resistance and fast-spreading important mechanisms.It is a kind of natural of bacterium
Clone and expression system, can under the catalysis of integrase mediated drug resistance box gene capture, integration and excision, cause drug resistant gene
It is propagated rapidly in flora.Currently, the relevant integron of drug resistance reported at present includes five classes, wherein 1 class integron is facing
It is most commonly seen in bed antibody-resistant bacterium.The drug resistant gene box type up to more than 100 that 1 class integron carries, the antimicrobial being related to
Object includes: beta-lactam, aminoglycoside, sulfamido, amphenicols, aminoglycoside, rifomycins etc..Some multiple
Usually there are Duan Changwei 2154bp, the DNA of 513 amino acid of codified in the downstream of miscellaneous 1 class integron 3 ' end conserved region
Sequence, therefore referred to as Orf513 are named as the 1 common area of type insetion sequence (Insertion sequence again later
common region,ISCR1).ISCR1 is a kind of to shift moving for its adjacent drug resistant gene by " rolling ring swivel base " mechanism
Genetic elements；A large amount of evidence shows that the Spreading and diffusion of ISCR1 and a variety of Drug-resistant genes are closely related, as chlorine is mould
Plain class, trimethoprim class, quinolones, aminoglycoside resistant gene and A class and C class beta-lactam enzyme gene etc..IV type is whole
Zygote, that is, Super integron SXT is found in comma bacillus for the first time, it is considered to be the principal element for instructing host gene to evolve, it can
Carry drug resistant gene, a box genes up to a hundred such as virulence gene, bacterium can be made to adapt to the variation of environment rapidly, be bacteria live and into
The important genetic elements changed.Currently, above-mentioned three kinds of integron related elements are a large amount of in the vibrios in a variety of sources at home and abroad
Report, the type for being related to drug resistant gene includes: amphenicols drug resistant gene (cat, floR), beta-lactam drug resistant gene
(blaPER, blaVIM-1, blaOXA-10), aminoglycoside resistant (aadA1, aacA3, aadB, strA/B), Tetracyclines
Drug resistant gene (tetA), quinolones drug resistant gene (qnrVC), rifomycins drug resistant gene (arr), trimethoprim drug resistance base
Because of (dfrA1, dfr16), Sulfonamides-resistant genes (sul1, sul2) etc..Carrying out sea-farming source vibrios drug resistance in this laboratory
During Journal of Sex Research, from Jiangsu, Shandong, Liaoning, Fujian and Hainan etc. area separation Vibrio in detect Int1,
The prevalence of SXT, ISCR1, but the presence that other types integrate subcomponent (such as 2 and 3 class integron) is not detected.However, arriving mesh
Before until there are no a kind of methods that can detect simultaneously these three elements of Int1, SXT, ISCR1.Therefore it is same to need to establish a kind of energy
When detect the multiple PCR method of 1 class integron Int1,4 class Super integron SXT, the common area ISCR1 of 1 type insetion sequence, with reality
The simplicity of existing above-mentioned three kinds of elements and quick detection.
Summary of the invention
The purpose of the present invention is to provide one kind can detect detection 1 class integron (Int1), 4 class Super integrons simultaneously
(SXT) and the multiple PCR primer and detection method in the 1 common area of type insetion sequence (ISCR1), with it is easy, easily detect it is above-mentioned
Popularity of three kinds of elements in bacterium.
For achieving the above object, the first aspect of the present invention provides a kind of whole for three kinds in detection bacterium simultaneously
The design method of the multiple PCR primer of zygote related elements, comprising: utilize multiple PCR primer design software Primerplex
2.61 (PREMIER Biosoft) design while detection 1 class integron integrase gene int1,4 class Super integron integrases
Gene intSXTIt is combined with the multiple PCR primer of the common area's transposase gene ISCR1 of 1 type insetion sequence, is formed by primer combination
The primer base number for being included is 18-20, and the melting temperature Tm value of primer is 49.6 DEG C -50.7 DEG C, and the GC% of primer is
40%-55%.
The second aspect of the present invention, provide it is a kind of using above-mentioned design method screen be used for and meanwhile detect detection 1
The multiple PCR primer of class integron (Int1), 4 class Super integrons (SXT) and the 1 common area of type insetion sequence (ISCR1), including
1 class integron integrase gene int1,4 class Super integron integrase gene intSXTWith the common area's transposase of 1 type insetion sequence
The primer of gene ISCR1.
Wherein, the upstream and downstream primer of 1 class integron integrase gene int1 is respectively as follows:
P1:5 '-CGAACCGAACAGGCTTAT-3 ' (SEQ ID NO.1)
P2:5 '-GGATACTTCCGCTCAAGG-3 ' (SEQ ID NO.2)
4 class Super integron integrase gene intSXTUpstream and downstream primer be respectively as follows:
P3:5 '-CTCTACCTACAGCAGGAACG-3 ' (SEQ ID NO.3)
P4:5 '-TCTCAGTGATTTTTGACTCG-3 ' (SEQ ID NO.4)
The upstream and downstream primer of the common area's transposase gene ISCR1 of 1 type insetion sequence is respectively as follows:
P5:5 '-AGACGATACGCTGACTCA-3 ' (SEQ ID NO.5)
P6:5 '-AGAATCTGCTCAATGACCTT-3 ' (SEQ ID NO.6).
Preferably, the corresponding pcr amplified fragment size of the 1 class integron integrase gene primer int1 is 569bp, institute
State 4 class Super integron integrase gene intSXTThe corresponding pcr amplified fragment size of primer is 430bp, and 1 type is inserted into sequence
Arranging the corresponding pcr amplified fragment size of common area's transposase gene ISCR1 primer is 651bp.
The third aspect of the present invention provides a kind of above-mentioned multiple PCR primer of application three kinds of integrons in detection bacterium simultaneously
The multi-PCR detection method of related elements, the present invention detect 1 class integron integrase gene int1,4 by multiplex PCR simultaneously
Class Super integron integrase gene intSXTWith the optimum response item of the common area's transposase gene ISCR1 method of 1 type insetion sequence
The measurement of part carries out the optimization of multiplex PCR optimum annealing temperature, primer concentration, template concentrations etc. using 3 pairs of primers, then root
The measurement that multiplex PCR optimum cycle number is carried out according to experimental result, obtained preferably three kinds of integrons in detection bacterium simultaneously
The multi-PCR detection method of related elements includes the following steps:
A) the initial gross separation of bacterium: extracting genomic DNA from the bacterium of culture, is used as pcr template；
B) multiplexed PCR amplification, comprising: a, multiplexed PCR amplification reaction system: taking 0.5 μ L of said gene group DNA profiling,
Premix Ex TaqTMEach 0.3 μ L of 12.5 μ L, primer P1, P2 (10pmol/L), each 0.6 μ L of primer P3, P4 (10pmol/L),
Each 0.6 μ L of primer P5, P6 (10pmol/L), ultrapure water are settled to 25 μ L.B, amplification reaction condition: 95 DEG C of initial denaturation 5min, 95
DEG C denaturation 30s, 50 DEG C of annealing 30s, 72 DEG C of extensions 35s, circulation 35 times, 72 DEG C of terminals extension 10min；C, by the amplification of step b
Product carries out electrophoresis detection and interpretation of result.
The invention has the advantages that:
Multiplex PCR detection architecture is established using the primer of design, is established by the optimization to primer volume and annealing temperature
For the multi-PCR detection method of three kinds of integron related elements, compared with prior art, established using the present invention multiple
PCR method can three kinds of integron related elements in detection bacterium simultaneously, have many advantages, such as easy to operate, quick, high specificity.
Detailed description of the invention
Fig. 1 multiplex PCR detects the electrophoresis result of three kinds of integron related elements in different bacterium.
Specific embodiment
It elaborates below with reference to embodiment to specific embodiment provided by the invention.
Embodiment 1
1) it designs while detecting using multiple PCR primer design software Primerplex 2.61 (PREMIER Biosoft)
1 class integron integrase gene int1,4 class Super integron integrase gene intSXTWith the common area's transposase of 1 type insetion sequence
The multiple PCR primer of gene ISCR1 combines；
2) optimal primer combination is screened from the result that multiple PCR primer design software Primerplex 2.61 is provided；
3) provide it is a kind of using above-mentioned multiple PCR primer simultaneously in detection bacterium three kinds of integron related elements it is optimal
Multi-PCR detection method.
The step 1) is directed to primer with reference to sending out on three kinds of corresponding GenBank of integron related elements
The gene order of table, the i.e. corresponding sequence of 1 class integron integrase gene int1 are that KU130396.1,4 class Super integrons are whole
Synthase gene intSXTCorresponding sequence is GQ495075.1, the corresponding sequence of the common area's transposase gene ISCR1 of 1 type insetion sequence
Column are KT725789.1, can detect 1 class integron simultaneously using the design of multiple PCR primer design software Primerplex 2.61
Integrase gene int1,4 class Super integron integrase gene intSXTWith the common area's transposase gene ISCR1 of 1 type insetion sequence
Specific multiple PCR primer combination.
Remarks: in text of the present invention, P1 and P2 is according to the upstreams of 1 class integron integrase gene int1 sequence design
Primer P1 and downstream primer P2, P3 and P4 are according to 4 class Super integron integrase gene intSXTThe upstream primer of sequence design
P3 and downstream primer P4, P5 and P6 are the upstream primer according to the common area's transposase gene ISCR1 sequence design of 1 type insetion sequence
P5 and downstream primer P6.
After the multiple PCR primer of design synthesis is carried out best pairing screening experiment by the step 2), by reacting item
Part, comparative test and verification test obtain the optimal multiple PCR primer combination of high specificity of the present invention.Wherein, 1 class
The corresponding pcr amplified fragment size of integron integrase gene primer is 569bp, the 4 class Super integron integrase bases
Because of intSXTThe corresponding pcr amplified fragment size of primer is 430bp, the common area's transposase gene of 1 type insetion sequence
The corresponding pcr amplified fragment size of ISCR1 primer is 651bp.Specific primer sequence is as follows:
The upstream and downstream primer of 1 class integron integrase gene int1:
P1:5 '-CGAACCGAACAGGCTTAT-3 ' (SEQ ID NO.1)
P2:5 '-GGATACTTCCGCTCAAGG-3 ' (SEQ ID NO.2)
4 class Super integron integrase gene intSXTUpstream and downstream primer:
P3:5 '-CTCTACCTACAGCAGGAACG-3 ' (SEQ ID NO.3)
P4:5 '-TCTCAGTGATTTTTGACTCG-3 ' (SEQ ID NO.4)
The upstream and downstream primer of the common area's transposase gene ISCR1 of 1 type insetion sequence:
P5:5 '-AGACGATACGCTGACTCA-3 ' (SEQ ID NO.5)
P6:5 '-AGAATCTGCTCAATGACCTT-3 ' (SEQ ID NO.6).
The step 3), multiplex PCR detect 1 class integron integrase gene int1, the integration of 4 class Super integrons simultaneously
Enzyme gene intSXTWith the measurement of the optimum reaction condition of the common area's transposase gene ISCR1 method of 1 type insetion sequence, comprising: first
First optimize multi-PRC reaction system, then optimizes primer concentration, template concentrations, the optimum annealing temperature of multi-PRC reaction system
Deng finally according to the measurement of experimental result progress multiplex PCR optimum cycle number.
It establishes multiplex PCR system: establishing the initial multiplex PCR system of 25 μ L: Premix ExTaqTM12.5 μ L, upstream and downstream are drawn
Each 0.5 μ L of object (10pM), 0.5 μ L of DNA profiling, ultrapure water are settled to 25 μ L；Wherein int1, intSXTDraw with the upstream and downstream of ISCR1
Object (10pM) volume is 0.5 μ L.
Initial p CR reaction system: grads PCR amplification program: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 50 DEG C of annealing
30s, 72 DEG C of extension 35s, 30 circulations；Last 72 DEG C re-extend 10min, 4 DEG C of preservations.Amplified production is solidifying through 1% agarose
After gel electrophoresis (150V, 30min), observed using gel imaging system as a result, being carried out according to result to multi-PRC reaction system excellent
Change.
The primer volume combination of optimization multiplex PCR system: in above-mentioned experimental basis, being fixed as 50 DEG C for annealing temperature,
Then to int1, intSXTThe volume combination of target gene primer (10pM) is optimized with ISCR1 tri-.In preset int1
(0.3 μ L, 0.4 μ L and 0.5 μ L), intSXT(0.4 μ L, 0.5 μ L and 0.6 μ L) and ISCR1 (0.4 μ L, 0.5 μ L and 0.6 μ L) volume
In range, comprehensive experiment of Three factors-levels is carried out, is combined with the optimal volume of each primer of determination: 0.3 μ L (int1), 0.6 μ
L(intSXT) and 0.6 μ L (ISCR1).
Optimize the annealing temperature of multiplex PCR system: in above-mentioned experimental basis, according to initial multi-PRC reaction system and
The optimal volume of each primer combines, and sets a series of annealing temperatures: 47.0 DEG C, 47.5 DEG C, 48.0 DEG C, 48.5 DEG C, 49.0 DEG C,
49.5 DEG C, 50.0 DEG C, 50.5 DEG C, 51.0 DEG C, 51.5 DEG C, 52.0 DEG C and 52.5 DEG C, to determine the best annealing of multi-PRC reaction
Temperature: 50 DEG C.
Optimize the cycle-index of multiplex PCR system: according to initial multi-PRC reaction system, the optimal volume group of each primer
It closes, optimal annealing temperature, sets a series of cycle-indexes: 28,29,30,31,32,33,34,35,36,37, it is multiple with determination
The optimum cycle number of PCR reaction: 35 times.
The step 3), multiplex PCR detect 1 class integron integrase gene int1, the integration of 4 class Super integrons simultaneously
Enzyme gene intSXTIt is 25 μ L with the reaction system of the common area's transposase gene ISCR1 method of 1 type insetion sequence, specific as follows:
Premix Ex TaqTM1 class integron each 0.3 μ L of integrase gene int1 upstream and downstream primer of 12.5 μ L, 10pmol/L,
The 4 class Super integron integrase gene int of 10pmol/LSXTThe 1 type insetion sequence of upstream and downstream primer each 0.6 μ L, 10pmol/L
Common area each 0.6 μ L of transposase gene ISCR1 upstream and downstream primer, 0.5 μ L of DNA profiling, ultrapure water are settled to 25 μ L, amplification condition
Are as follows: 95 DEG C of initial denaturations 5min, 95 DEG C of denaturation 30s, 50 DEG C of annealing 30s, 72 DEG C of extension 35s are recycled 35 times, and 72 DEG C of terminals extend
10min, by the above PCR product under 1 × TAE, 1.0% Ago-Gel, 150V voltage conditions electrophoresis 30min, then solidifying
It is observed in glue imaging system.
Swimming lane 1,2,3,4,5,6,7,8 and 16 in Fig. 1 shows different control (positive control, negative control and blank
Control) 1 class integron integrase gene int1,4 class Super integron integrase gene int in bacterial strain DNA profilingSXTIt is inserted with 1 type
Enter the multiplex PCR testing result of the common area's transposase gene ISCR1 of sequence, wherein M swimming lane is DL2000DNAMarker；
No. 1 swimming lane is the amplification of 1 class integron integrase gene int1 positive control strain, is about in molecular weight
Bright purpose amplified band is presented at 569bp, illustrates to contain 1 class integron in the positive control strain DNA sample of the present embodiment
Integrase gene int1；
No. 2 swimming lanes are 4 class Super integron integrase gene intSXTThe amplification of positive control strain, in molecular weight
Bright purpose amplified band is presented at about 430bp, illustrates super containing 4 classes in the positive control strain DNA sample of the present embodiment
Grade integron integrase gene intSXT；
No. 3 swimming lanes are the amplification of the common area's transposase gene ISCR1 positive control strain of 1 type insetion sequence, are being divided
Son amount is about that bright purpose amplified band is presented at 651bp, illustrates to contain 1 in the positive control strain DNA sample of the present embodiment
The common area's transposase gene ISCR1 of type insetion sequence；
No. 4 swimming lanes are 1 class integron integrase gene int1 and the common area's transposase gene ISCR1 sun of 1 type insetion sequence
Property control strain amplification, molecular weight be about 569bp and 651bp at bright purpose amplified band is presented, illustrate this reality
It applies and contains 1 class integron integrase gene int1 and the common area's swivel base of 1 type insetion sequence in the positive control strain DNA sample of example
Enzyme gene ISCR1；
No. 5 swimming lanes are 1 class integron integrase gene int1 and 4 class Super integron integrase gene intSXTIt is positive right
According to the amplification of bacterial strain, molecular weight be about 569bp and 430bp at bright purpose amplified band is presented, illustrate the present embodiment
Positive control strain DNA sample in contain 1 class integron integrase gene int1 and 4 class Super integron integrase genes
intSXT；
No. 6 swimming lanes are the common area's transposase gene ISCR1 of 1 type insetion sequence and 4 class Super integron integrase genes
intSXTThe amplification of positive control strain, molecular weight be about 651bp and 430bp at bright purpose amplified band is presented, say
It is super containing the common area's transposase gene ISCR1 of 1 type insetion sequence and 4 classes in the positive control strain DNA sample of bright the present embodiment
Grade integron integrase gene intSXT；
No. 7 swimming lanes are the common area's transposase gene ISCR1 of 1 type insetion sequence, 1 class integron integrase gene int1 and 4
Class Super integron integrase gene intSXTThe amplification of positive control strain, molecular weight be about 651bp, 569bp and
Bright purpose amplified band is presented at 430bp, illustrates to be inserted into sequence containing 1 type in the positive control strain DNA sample of the present embodiment
Arrange common area's transposase gene ISCR1,1 class integron integrase gene int1 and 4 class Super integron integrase genes
intSXT；
No. 8 swimming lanes are the amplification of negative control bacterial strain, do not occur bright purpose amplified band, illustrate the present embodiment
Negative control bacterial strain DNA sample in be free of the common area's transposase gene ISCR1 of 1 type insetion sequence, 1 class integron integrase base
Because of int1 and 4 class Super integron integrase gene intSXT；
No. 16 swimming lanes are the amplification of blank control sample, do not occur bright purpose amplified band, illustrate this implementation
Multiplex PCR system in example is not by the common area's transposase gene ISCR1 of 1 type insetion sequence, 1 class integron integrase gene
Int1 and 4 class Super integron integrase gene intSXTThe pollution of positive DNA.
Swimming lane 9,10,11,12,13,14 and 15 in Fig. 1 shows total to vibrios 1 type insetion sequence of progress is clinically separated
Same district transposase gene ISCR1,1 class integron integrase gene int1 and 4 class Super integron integrase gene intSXTIt is more
Weight PCR testing result illustrates the present embodiment wherein No. 9 swimming lanes are about that bright purpose amplified band is presented at 430bp in molecular weight
Be clinically separated in cholerae strain DNA sample containing 4 class Super integron integrase gene intSXT；
No. 10 swimming lanes are about that bright purpose amplified band is presented at 569bp in molecular weight, illustrate the clinic point of the present embodiment
Contain 1 class integron integrase gene int1 in arc of recess bacteria strain DNA sample；
No. 11 swimming lanes are about that bright purpose amplified band is presented at 651bp in molecular weight, illustrate the clinic point of the present embodiment
Contain the common area's transposase gene ISCR1 of 1 type insetion sequence in arc of recess bacteria strain DNA sample；
No. 12 swimming lanes molecular weight be about 569bp and 430bp at bright purpose amplified band is presented, illustrate the present embodiment
It is clinically separated in cholerae strain DNA sample and contains 1 class integron integrase gene int1 and 4 class Super integron integrase genes
intSXT；
No. 13 swimming lanes molecular weight be about 569bp and 651bp at bright purpose amplified band is presented, illustrate the present embodiment
It is clinically separated in cholerae strain DNA sample and contains 1 class integron integrase gene int1 and the common area's transposase of 1 type insetion sequence
Gene ISCR1；
No. 14 swimming lanes are about that bright purpose amplified band is presented at 569bp in molecular weight, illustrate the clinic point of the present embodiment
Contain 1 class integron integrase gene int1 in arc of recess bacteria strain DNA sample；
No. 15 swimming lanes are about that bright purpose amplified band is presented at 651bp in molecular weight, illustrate the clinic point of the present embodiment
Contain the common area's transposase gene ISCR1 of 1 type insetion sequence in arc of recess bacteria strain DNA sample.
The preferred embodiment of the present invention has been described in detail above, but the invention be not limited to it is described
Embodiment, those skilled in the art can also make various equivalent on the premise of not violating the inventive spirit of the present invention
Variation or replacement, these equivalent variation or replacement are all included in the scope defined by the claims of the present application.
Claims (4)
1. a kind of multiple PCR primer for three kinds of integron related elements in detection bacterium simultaneously, which is characterized in that including 1
Class integron integrase gene int1,4 class Super integron integrase gene intSXTWith the common area's transposase of 1 type insetion sequence
The primer of gene ISCR1；
Wherein, the upstream primer P1 and downstream primer P2 of the 1 class integron integrase gene int1 is respectively such as SEQ ID
Shown in NO.1 and SEQ ID NO.2；
The 4 class Super integron integrase gene intSXTUpstream primer P3 and downstream primer P4 respectively such as SEQ ID
Shown in NO.3 and SEQ ID NO.4；
The upstream primer P5 and downstream primer P6 of the common area's transposase gene ISCR1 of 1 type insetion sequence is respectively such as SEQID
Shown in NO.5 and SEQID NO.6.
2. the multiple PCR primer according to claim 1 for three kinds of integron related elements in detection bacterium simultaneously,
Be characterized in that: the corresponding pcr amplified fragment size of 1 class integron integrase gene int1 primer is 569bp, described
4 class Super integron integrase gene intSXTThe corresponding pcr amplified fragment size of primer is 430bp, and 1 type is inserted into sequence
Arranging the corresponding pcr amplified fragment size of common area's transposase gene ISCR1 primer is 651bp.
3. a kind of multiple PCR primer for three kinds of integron related elements in detection bacterium simultaneously as described in claim 1
Design method, which is characterized in that using primer-design software Primerplex2.61 design simultaneously detect 1 class integron integration
Enzyme gene int1,4 class Super integron integrase gene intSXTIt is more with the common area's transposase gene ISCR1 of 1 type insetion sequence
Weight PCR primer combination, being formed by primer and combining included primer base number is 18-20, the melting temperature Tm value of primer
It is 49.6 DEG C -50.7 DEG C, the GC% of primer is 40%-55%.
4. a kind of apply multiple PCR primer as described in claim 1 three kinds of integron related elements in detection bacterium simultaneously
Multi-PCR detection method, which comprises the steps of:
A) the initial gross separation of bacterium: extracting genomic DNA from the bacterium of culture, is used as pcr template；
B said gene group DNA profiling 0.5 μ L, Premix) multiplexed PCR amplification, comprising: a, multiplexed PCR amplification reaction system: are taken
Ex TaqTMPrimer P3, P4 each 0.6 the μ L, 10pmol/L of each 0.3 μ L of primer P1, P2 of 12.5 μ L, 10pmol/L, 10pmol/L
Each 0.6 μ L of primer P5, P6, ultrapure water is settled to 25 μ L；B, amplification reaction condition: 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation
30s, 50 DEG C of annealing 30s, 72 DEG C of extension 35s are recycled 35 times, and 72 DEG C of terminals extend 10min；C, by the amplified production of step b into
Row electrophoresis detection and interpretation of result.
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