CN103233064B

CN103233064B - Primer probe system, method and kit for detecting epidermal growth factor receptor exon 19 and 21 mutations

Info

Publication number: CN103233064B
Application number: CN201310092075.2A
Authority: CN
Inventors: 陈晓琦; 樊青霞; 赵亚敏; 郑玉玲; 王峰
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-03-21
Filing date: 2013-03-21
Publication date: 2014-09-24
Anticipated expiration: 2033-03-21
Also published as: CN103233064A

Abstract

The present invention relates to a primer probe system, a method and a kit for detecting epidermal growth factor receptor exon 19 and 21 mutations. According to the method, an asymmetric PCR technology, a nucleic acid isothermal amplification technology and a fluorescence detection technology are organically integrated, wherein the asymmetric PCR amplification technology and special primers are adopted to obtain a lot of single-stranded DNA so as to overcome disadvantages of low efficiency linear amplification of 3WJ (an isothermal digestion nucleic acid detection technology), and specificity and simplicity of the 3WJ technology are adopted, such that a reaction temperature can be controlled at about 30 DEG C, enzyme activity reduction can not be generated, expensive and precise temperature control equipment is not required during a detection process, and discrimination can be achieved only through the simple fluorescence detection instrument with the special probe design. According to the present invention, advantages of rapidness, simpleness, specificity, sensitivity, and the like are provided.

Description

Detect primer probe system, method and the test kit of ErbB1 9,21 exons mutations

Technical field

The present invention relates to technical field of bioengineering, be specifically related to a kind of primer probe system, method and test kit that detects ErbB1 9,21 exons mutations.

Background technology

Lung cancer (Lung cancer) especially non-small cell carcinoma (Non-small cell lung cancer) is one of the most pernicious tumour of Present Global.Although traditional chemotherapy improves constantly, the prognosis of patients with terminal is poor.Along with the raising of the level of molecular engineering, taking EGFR(EGF-R ELISA) outstanding day by day in NSCLC treatment as targeted drug-TYR kinase inhibitor (TKI) for the treatment of target spot, wherein Gefitinib and erlotinib treatment NSCLC have been widely used in NSCLC treatment.Along with the accumulation of clinical data, in the treatment of use NSCLC, to TKI, treatment responds some people.Analyze discovery by these TKI being treated to patient's EGFR gene effectively, nearly all have EGFR transgenation.A large amount of research shows that patients with lung cancer EGFR transgenation mainly occurs on 18 ~ 21 exons, and wherein more than 90% 5 bases of 19 exons 1 that sport lack and 21 exon L858R sudden changes.These sudden changes are relevant to the curative effect of TKI, and possible reason is the protein structure that these sudden changes have changed EGFR ATP-land, make this region be easy to be combined with TKI, thereby improved the drug effect of TKI treatment.Therefore before using clinically TKI treatment, detect patient EGFR transgenation, very necessary.Due in China, EGFR transgenation mainly concentrates on 19 exons and 21 exons.Therefore detect fast and accurately EGFR sudden change and instruct clinical application, the process that improves the personalized medication of China is extremely important.

Come out from round pcr, this art is widely used in molecular diagnosis.The sudden change detection technique of PCR-based technology has obtained development: PCR-RFLP rapidly, allele-sepecific PCR, TaqMan probe, ARMS, PNA-LNA clamp fluorescent PCR, Cycleave probe PCR, Nanofluidic Digital PCR Arrays, MassArray and DNA chip etc.PCR-RFLP is that early stage experimental development detects SNP typing method, it depends on selectional restriction restriction endonuclease dexterously, and to cut out short fragment size for identifying mutational site polymorphic, but this method needs the product after electrophoretic separation enzymic digestion, and this is by the automatization of the flux of serious limited reactions and operation.ARMS technology and allele-sepecific PCR are roughly the same, utilize the circumscribed activity of archaeal dna polymerase disappearance 3 '～5 ', the primer of 3 ' end mispairing is lower than the extension speed of normal primer, in the time that mispairing number reaches certain rigorous degree, 3 ' end cannot extend, if PCR has band, corresponding sudden change is described.ARMS technique sensitive and special higher, has been successfully applied to clinical detection SNP.

TaqMan probe is a kind of oligonucleotide probe, be connected with fluorescence report group, and 5 ' end is connected with fluorescent quenching group at 3 ' end of probe.When probe is complete, the fluorescent signal of reporter group transmitting is quenched group and absorbs, in the time of pcr amplification, 5 ' 5 prime excision enzyme activity of Taq enzyme can cut away in connection with the quenching group of the probe to target dna strand, the fluorescence report group of probe is separated with fluorescent quenching group, thereby fluorescence monitoring system can receive fluorescent signal.By using different fluorescence report groups, in same PCR, can detect different enzymes simultaneously and cut digestion allele-specific probe.This 5 ' excision enzyme experiment is own through being successfully used for distinguishing the allelotrope that only has a base difference.

Cycleave probe PCR, the similar Taqman probe of know-why, just probe contains a rNTP base in SNP complementation, when existing sudden change to be, rNTP and sudden change pairing, RNaseH identifies and cuts rNTP, the fluorescence report group of probe is separated with fluorescent quenching group, and fluorescence produces.

PNA-LNA-clamp technology, adopts NestPCR technology and PNA-LNA-clamp probe, the sealing wildness SNP of PNA-LNA-clamp probe specificity, thus stop wild nature template amplification to reach the object of amplification sudden change.Although this technology is very sensitive, experience twice PCR is easily polluted, and probe design is also restricted.

Although above-mentioned SNP detection technique susceptibility and specificity are very high, these Technology Needs accurate expensive temperature control and fluorescence detection device.Therefore the SNP detection method of, developing a kind of economical and practical type has very important meaning.The detection technique of SNP sudden change at present, gold standard is " DNA sequencing " still, but DNA sequencing susceptibility is lower, the more difficult resolution of nucleic acid mutation of <20%, and the cycle is long.

Nucleic acid constant-temperature amplification technology is cut detection technique (3WJ, three way junction) invader technology as constant temperature enzyme and is employed gradually sudden change detection.3WJ is the propositions such as Amicarelli G, its know-why is: with the core component of two probe PA and PB composition 3WJ, PB is combined with template as grappling probe, and PA is as differentiating probe identification SNP, 3 ' end is with BHQ cancellation group, and restriction enzyme site 5 ' end upstream is with the FAM of fluorescence group; Between PA and PB, contain 6 ~ 8 complementary bases, wherein comprise a restriction enzyme cleavage site (5 '-GTAC-3 '), when SNP to be detected exists, PA and PB and target sequence form stable " Y " type structure, cancellation group-BHQ identify and cut to restriction restriction endonuclease as CviQ1, fluorescence generation.

Although be that JP and 3WJ technology can be good at differentiating SNP polymorphism separately, its linear amplification efficiency, susceptibility does not reach the ability that detects clinical sample DNA far away, the more important thing is that it can only detect single stranded DNA.

Summary of the invention

The technical problem to be solved in the present invention has been to provide the primer probe system of a kind of high specificity, sensitive detection ErbB1 9,21 exons mutations, and asymmetric PCR and nucleic acid constant-temperature amplification, detection technique of fluorescence are carried out in conjunction with having designed a kind of quick, simple, special, sensitive, stable, method of detecting cheaply ErbB1 9,21 exons mutations, and design the test kit of simple, practical detection ErbB1 9,21 exons mutations according to the method.

For solving the problems of the technologies described above, the technical solution used in the present invention is:

Design a kind of primer probe system that detects ErbB1 9,21 exons mutations, comprise the asymmetric PCR primer that following amplification EGFR 19 exon E746～A750del sudden change and EGFR 21 exon L858R suddenly change, and the 3WJ probe matching with its primer is as follows:

Wherein, in the 11st the flag F AM(fluorescence group of probe A), at 3 ' end mark DABCY; At the 7th the flag F AM of probe A ＇, at 3 ' end mark DABCY.

A method that detects ErbB1 9,21 exons mutations, comprises the steps:

(1) carry out according to a conventional method testing sample processing;

(2) pcr amplification: upstream primer A and downstream primer B with above-mentioned detection 19 DEL form following asymmetric PCR system amplification EGFR19 exon E746～A750del:

5 × buffer, 10 microlitres

The MgCl that 25 millis rub ₂2.5 microlitre

DNTP 1 microlitre that 10 millis rub

10 micro-upstream primer A 3 microlitres that rub

10 micro-downstream primer B 0.2 microlitres that rub

Taq DNA Polymerase 0.3 microlitre of 500 units

DdH ₂o 33 microlitres

Amount to 50 microlitres

Pcr amplification condition is as follows:

With the upstream and downstream primer in the upstream primer A ＇ of above-mentioned detection L 858R, the above-mentioned asymmetric PCR system of replacement that downstream primer B ＇ is corresponding, the asymmetric PCR system forming is by the same pcr amplification condition 21 exon L858R that increase;

(3) 3WJ detects: upwards walk respectively in two kinds of products of gained and directly add the corresponding probe system concentrated solution of 10ul, the 3WJ probe system that forms respectively 60 microlitres contains:

10 × Buffer, 6 microlitres

100 × BSA, 0.6 microlitre

10 micro-probe A rubbing or A ＇ 1.2 microlitres

10 micro-probe B rubbing or B ＇ 1.2 microlitres

DdH ₂o 51 microlitres

3WJ condition is: incubate for 37 DEG C and bathe 20min, 95 DEG C of 5min termination reactions detect fluorescence;

(4) result judgement: produce if any fluorescence, exist corresponding exons mutation.

Detect a test kit for ErbB1 9,21 exons mutations, at least comprise following reagent:

(1) form the needed 2mM Mg of asymmetric PCR system of above-mentioned amplification EGFR19 exon E746～A750del ²⁺, 200nM dNtP, 300nM upstream primer, 30nM downstream primer, Taq enzyme, and form the needed 1 × BSA of its corresponding 3WJ probe system, restriction restriction endonuclease, probe A and probe B;

(2) form the needed 2mM Mg of asymmetric PCR system of above-mentioned amplification 21 exon L858R ²⁺, 200nM dNtP, 300nM upstream primer, 30nM downstream primer, Taq enzyme, and form the needed 1 × BSA of its corresponding 3WJ probe system, restriction restriction endonuclease, probe A ＇ and probe B ＇.

The present invention has actively useful effect:

With traditional detection method of gene mutation comparison, the inventive method has quicker, simple, special, sensitive, stable advantage.

Asymmetric PCR and nucleic acid constant-temperature amplification, detection technique of fluorescence have been carried out organic integration by the inventive method: on the one hand apply asymmetric PCR amplification technique and special primer obtains a large amount of single stranded DNAs, made up 3WJ(constant temperature enzyme and cut nucleic acid detection technique) the deficiency of inefficient linear amplification; On the other hand applied 3WJ technology specificity and simplicity, make temperature of reaction can be controlled in 30 ^oabout C, and do not worry in testing process, not needing the activity decreased of enzyme expensive and accurate temperature controlling instruments, special probe design to make to need only simple fluoroscopic examination instrument and just can differentiate.

The design feature of technical solution of the present invention makes this technology be not limited to a kind of restriction enzyme, and up to the present the restriction enzyme of the marketization has exceeded hundred kinds.

Technical solution of the present invention has two important innovations, and (1) has separated nucleic acid hybridization surveyed area and signal produces region; (2) can detect the nucleic acid samples in all kinds, various sources.

Brief description of the drawings

Fig. 1 is the asymmetric PCR primer of amplification EGFR19 exon E746～A750del sudden change and the configuration picture of probe.

Fig. 2 is the asymmetric PCR primer of amplification EGFR21 exon L858R sudden change and the configuration picture of probe.

Fig. 3 is susceptibility and the specificity collection of illustrative plates that joint probe (JP) detects EGFR sudden change.In figure, A and B are the collection of illustrative plates that detects EGFR L858R; C and D are the collection of illustrative plates that detects EGFR 19DEL; A and C are fluorescence intensity figure in detection of dynamic JP reaction system; When B and D reach plateau for reaction, the fluorescence intensity figure that mini microplate reader detects.

Fig. 4 is the amplification of APCR-JP method and the collection of illustrative plates that detects EGFR sudden change.In figure, A, B are the electrophorogram that corresponds respectively to the APCR primer of L858R and 19DEL, and the primer concentration in 1～8 road is followed successively by 1:1,5:1, and 10:1,20:1,50:1,100:1,200:1,400:1,9 roads are PCR negative control; As can be seen from the figure when primer concentration is respectively 5:1,10:1, when 20:1 and 50:1, is easy to detect ssDNA; C, D are fluoroscopic examination intensity collection of illustrative plates, in the time that primer concentration is 10:1, and fluorescence intensity maximum, L858R(C), 19DEL (D).

Fig. 5 direct sequencing (A) and APCR-JP system of the present invention (B) detect the contrast collection of illustrative plates of L858R cell content.As can be seen from the figure, APCR-JP system can be found 5% mutant cell, and direct sequencing can only detect more than 20% mutant cell.

Embodiment

Further set forth the present invention below in conjunction with specific embodiment.

The extraction of embodiment 1 clinical sample DNA

The present embodiment is to extract DNA from the paraffin-embedded tissue section of Patients with Non-small-cell Lung lung, and it is carried out quantitatively to the template detecting as PCR.Adopt the paraffin-embedded tissue of Takara company to extract test kit, specifically details are as follows:

(1) extract sample DNA

1. paraffin-embedded tissue is cut into 5 μ m(4～10 μ m) thick after, with sterilizing pincet, 1～3 piece of section is put into the 1.5 ml Microtube that TaKaRa DEXPAT Easy is housed, investing tissue at least needs 6mm × 6mm size.For preventing the pollution of DNA, by washing composition and H for slicing machine ₂o ₂sterilization, then clean with alcohol, cutting knife and pincet etc. also will operate equally, then uses UV to irradiate more than 10 minutes.

2. cover Microtube lid, 100 DEG C are heated 10 minutes, heat after approximately 5 minutes, and Microtube is turned upside down 2～3 times gently, fully mix.

Use Heat Block very convenient.

1.5 ml Microtube are awfully hot, note not scalding.

3. after heating, carry out immediately 17,000 turning, 4 DEG C centrifugal 10 minutes.

4. the Microtube after centrifugal transfers to immediately on ice and leaves standstill 5 minutes.

5. draw water layer with liquid-transfering gun, avoid being drawn onto Parafilm; While drawing water layer, can see the jelly above polymeric adsorbent, draw the water layer above jelly.

The template that the water layer of drawing can directly react as PCR.

While using the regular-PCR enzymes such as TaKaRa Ex Taq, DNA solution add-on is generally to add 5 μ l) in the 1/10(50 μ l PCR reaction system of PCR reaction cumulative volume.

Use Inhibitors is had and in add-on that the MightyAmp DNA Polymerase of very strong patience the carries out PCR when reaction DNA 4/10(50 μ l PCR reaction system for PCR reaction cumulative volume, adds 20 μ l) also can obtain good amplification.

(2) quantitative: the DNA of extraction is carried out quantitatively with ultraviolet spectrophotometer, and recording DNA concentration is 500ng/ul.

Embodiment 2 asymmetric PCR method amplification clinical sample DNA

Adopt the DNA sample extracting in primer provided by the present invention, probe amplification embodiment 1.Asymmetric PCR primer and probe (shown in SEQ ID NO:1～4) as shown in Figure 1 that the EGFR19 exon E746～A750del that wherein increases suddenlys change; Asymmetric PCR primer and probe (shown in SEQ ID NO:5～8) as shown in Figure 2 of the EGFR21 exon L858R that wherein increases sudden change.

With corresponding separately upstream primer and downstream primer, increase respectively EGFR19 exon E746～A750del and 21 exon L858R, asymmetric PCR amplification system is constructed as follows:

5X buffer 10 microlitres

MgCl ₂(25 millis rub) 2.5 microlitres

DNTP (10 millis rub) 1 microlitre

DNA template 2 microlitres

Upstream primer (10 micro-rubbing) 3 microlitres

Downstream primer (10 micro-rubbing) 0.2 microlitre

Polymerase(500 unit of Taq DNA) 0.3 microlitre

DdH ₂o 31 microlitres

Total amount 50 microlitres

Each upstream and downstream primer is pressed different ratios preparation, is respectively: 1:1, and 5:1,10:1,20:1,50:1,100:1,200:1 and 400:1, negative control group does not add DNA profiling.

Pcr amplification condition is in table 1:

Table 1 asymmetric PCR amplification condition

3WJ probe system: above-mentioned product directly adds 10ul probe system concentrated solution, the 3WJ probe system that forms 60ul contains:

Buffer(10X) 6 microlitres

BSA (100X) 0.6 microlitre

Probe A (10 micro-rubbing) 1.2 microlitres

Probe B (10 micro-rubbing) 1.2 microlitres

ddH ₂o 51 microlitres

Total amount 60 microlitres

3WJ condition is: incubate for 37 DEG C and bathe 20min, 95 DEG C of 5min termination reactions.

Wherein in each detection system, add corresponding detection primer and probe, the probe combinations of its amplimer and correspondence is in table 2, read plate instrument (Gemini EM, USA) and mini single passage luminoscope (OP-162, Opulen Technology with fluorescence imaging; China) detect fluorescence.

For the specificity of assessment joint probe method, corresponding artificial mutation template (MT) as shown in table 2 and wild-type sudden change template (WT) in above-mentioned reaction system, are added respectively, by this routine described method, it is detected, result as shown in Figure 3, this method energy specific detection is to artificial mutation template, similar (A and the C) of the fluorescence intensity of wild-type template and control group; Detection sensitivity, by ten footwork dilution sudden change templates, JP method can be found the sudden change template of 20nM level; A and B, detect EGFR L858R; C and D, detect EGFR 19DEL.A and C, fluorescence intensity in detection of dynamic JP reaction system; B and D, when reaction reaches plateau, the fluorescence intensity that mini microplate reader detects.

Table 2 is for detection of the primer probe combinations of 19 DEL and 21 L 858R sudden change

Analyze above-mentioned PCR product with 12% polyacrylamide and SYBR Gold dyeing and gel imaging system, result is as shown in A, B in Fig. 4 simultaneously; The amplification of APCR-JP method and detection EGFR sudden change, L858R (A) and 19DEL (B) APCR primer (as shown in table 2); Primer concentration (1～8 road) 1:1,5:1,10:1,20:1,50:1,100:1,200:1,400:1,9 roads are PCR negative controls; When primer concentration is 5:1,10:1, when 20:1 and 50:1, is easy to detect ssDNA; In the time that concentration is 10:1, fluorescence intensity maximum, L858R(C), 19DEL (D).

Embodiment 3 direct sequencing checking the inventive method

EGFR order-checking

(1) primer (using the 19DEL shown in table 2 and the sequencing primer separately of L858R)

(2) PCR system

Genomic dna (>50ng/ul): 3ul

Forward primer（10uM） 3ul

Reverse primer（10uM） 3ul

dNTP mix（10uM） 1ul

5× buffer 10ul

Mg ²⁺（25mM） 5ul

Gota Taq（5U/ul） 0.3ul

ddH ₂O 24.7ul

(3) PCR condition-Cold-PCR, as shown in table 3:

Table 3 PCR condition

(4) PCR product purification

Axygen PCR product purification test kit purifying.

(5) PCR sequencing reaction 10ul system:

Forward primer（3.2uM）1 ul

PCR purified product: 3ul

Bigdye3.1： 1ul

5X buffer： 1.5ul

ddH ₂O： 3.5ul

(6) sequencing reaction condition (in table 4):

Table 4 sequencing reaction condition

(7) order-checking: with reference to ABi3730XL order-checking flow process

(8) result: as shown in Figure 5, direct sequencing (A) and APCR-JP system (B) detect L858R cell content.APCR-JP system can be found 5% mutant cell, and direct sequencing can only detect more than 20% mutant cell.

The detection kit of embodiment 4 ErbB1 9,21 exons mutations

The know-why that content is recorded according to the present invention and concrete technical scheme, the detection kit of collecting following reagent and can be made into commercial ErbB1 9,21 exons mutations:

(1) form the needed 2mM Mg of asymmetric PCR system of aforementioned amplification EGFR19 exon E746～A750del ²⁺, 200nM dNtP, 300nM upstream primer, 30nM downstream primer, Taq enzyme, and form the needed 1 × BSA of its corresponding 3WJ probe system, restriction restriction endonuclease, probe A and probe B;

(2) form the needed 2mM Mg of asymmetric PCR system of aforementioned amplification 21 exon L858R ²⁺, 200nM dNtP, 300nM upstream primer, 30nM downstream primer, Taq enzyme, and form the needed 1 × BSA of its corresponding 3WJ probe system, restriction restriction endonuclease, probe A ＇ and probe B ＇;

(3) other the available routine biochemistry reagent needing in testing process.

Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.

SEQUENCE LISTING

<110> Chen Xiao fine jade

<120> detects primer probe system, method and the test kit of ErbB1 9,21 exons mutations

<130> /

<160> 16

<170> PatentIn version 3.2

<210> 1

<211> 22

<212> DNA

<213> upstream primer A

<400> 1

atcccagaag gtgagaaagt ta 22

<210> 2

<211> 20

<212> DNA

<213> downstream primer B

<400> 2

catcgaggat ttccttgttg 20

<210> 3

<211> 17

<212> DNA

<213> probe A

<400> 3

cggacatctt tttgtac 17

<210> 4

<211> 40

<212> DNA

<213> probe B

<400> 4

agtaacggaa aaccgttact gstacatatt gatagcgacg 40

<210> 5

<211> 21

<212> DNA

<213> upstream primer A ＇

<400> 5

tgaaaacacc gcagcatgtc a 21

<210> 6

<211> 20

<212> DNA

<213> downstream primer B ＇

<400> 6

tctcttccgc acccagcagt 20

<210> 7

<211> 13

<212> DNA

<213> probe A ＇

<400> 7

cccgcctttg tac 13

<210> 8

<211> 40

<212> DNA

<213> probe B ＇

<400> 8

agtaacggaa aaccgttact gstacatatt gatagcgacg 40

<210> 9

<211> 46

<212> DNA

<213> 19 DEL wild-type templates

<400> 9

ttcccgtcgc tatcaaggaa ttaagagaag caacatctcc gaaagc 46

<210> 10

<211> 42

<212> DNA

<213> 19 DEL saltant type templates

<400> 10

cccgtcgcta tcaaaacatc tccgaaagcc aacaaggaaa tc 42

<210> 11

<211> 40

<212> DNA

<213> L 858R wild-type template

<400> 11

caagatcaca gattttgggc tggccaaact gctgggtgcg 40

<210> 12

<211> 40

<212> DNA

<213> L 858R saltant type template

<400> 12

caagatcaca gattttgggc gggccaaact gctgggtgcg 40

<210> 13

<211> 19

<212> DNA

<213> 19 DEL order-checking upstream primers

<400> 13

catgtggcac catctcaca 19

<210> 14

<211> 18

<212> DNA

<213> 19 DEL order-checking downstream primers

<400> 14

gacccccaca cagcaaag 18

<210> 15

<211> 22

<212> DNA

The <213> L 858R upstream primer that checks order

<400> 15

cctcacagca gggtcttctc tg 22

<210> 16

<211> 21

<212> DNA

The <213> L 858R downstream primer that checks order

<400> 16

tggctgacct aaagccacct c 21

Claims

1. one kind is detected the primer probe system of ErbB1 9,21 exons mutations, comprise the asymmetric PCR primer that following amplification EGFR 19 exon E746～A750del sudden change and EGFR 21 exon L858R suddenly change, and the 3WJ probe matching with its primer:

。

2. detect a test kit for ErbB1 9,21 exons mutations, at least comprise following reagent:

(1) form the needed 2mM Mg of asymmetric PCR system of following amplification EGFR19 exon E746～A750del ²⁺, 200nM dNtP, 300nM upstream primer, 30nM downstream primer, Taq enzyme, and form the needed 1 × BSA of its corresponding 3WJ probe system, restriction restriction endonuclease, probe A and probe B:

5 × buffer, 10 microlitres

The MgCl that 25 millis rub ₂2.5 microlitre

DNTP 1 microlitre that 10 millis rub

The 10 micro-A of upstream primer as claimed in claim 13 microlitres that rub

The 10 micro-B of downstream primer as claimed in claim 1 0.2 microlitres that rub

Taq DNA Polymerase 0.3 microlitre of 500 units

DdH ₂o 33 microlitres

Amount to 50 microlitres

(2) form the needed 2mM Mg of asymmetric PCR system of following amplification 21 exon L858R ²⁺, 200nM dNtP, 300nM upstream primer, 30nM downstream primer, Taq enzyme, and form the needed 1 × BSA of its corresponding 3WJ probe system, restriction restriction endonuclease, probe A ＇ and probe B ＇:

5 × buffer, 10 microlitres

The MgCl that 25 millis rub ₂2.5 microlitre

DNTP 1 microlitre that 10 millis rub

10 micro-rub the A of upstream primer as claimed in claim 1 ＇ 3 microlitres

The 10 micro-B of downstream primer as claimed in claim 1 ＇ 0.2 microlitres that rub

Taq DNA Polymerase 0.3 microlitre of 500 units

DdH ₂o 33 microlitres

Amount to 50 microlitres;

In 60 microlitres, described 3WJ probe system contains:

10 × Buffer, 6 microlitres

100 × BSA, 0.6 microlitre

10 micro-probe A rubbing or A ＇ 1.2 microlitres

10 micro-probe B rubbing or B ＇ 1.2 microlitres

DdH ₂o 51 microlitres.