CN104450918B - The method in detection FGF13 Exon 2 mutational site and test kit thereof - Google Patents
The method in detection FGF13 Exon 2 mutational site and test kit thereof Download PDFInfo
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Abstract
The invention discloses a kind of method detecting FGF13 Exon 2 mutational site and test kit thereof, the method comprising the steps of: 1) extracts sample DNA, as DNA profiling；2) utilize the ARMS primer as shown in SEQ ID NO.1 3 and the probe as shown in SEQ ID NO.11, DNA profiling is carried out fluorescent PCR amplification；3) fluorescence intensity of the reaction system in the amplification of detection fluorescent PCR, and cycle-index Ct value required when reaching, according to fluorescence intensity, the threshold value set judges the sudden change in FGF13 Exon 2 mutational site.This test kit includes: the ARMS primer shown in SEQ ID NO.1 3 and a probe as shown in SEQ ID NO.11.The shortcomings such as the present invention can overcome existing sequencing technologies small throughput, time-consuming length, power of test limited, and there is quick, flux height, sensitivity advantages of higher.
Description
Technical field
The present invention relates to a kind of method detecting gene mutation and test kit thereof, particularly relate to a kind of detection FGF13 and (become fiber
Growth factor-21 3) method in Exon 2 (EXON 2) mutational site and test kit thereof.
Background technology
FGF13 is FGF11 subfamily member, this kind of FGF molecule for want of signal peptide and cannot secrete, only at intracellular
Wave function.Research finds, the chain dysnoesia of FGF13 with X-is relevant, and on the mankind and mice, FGF13 gene mapping is at X
Chromosome q26 section, the hereditary variation in this region may cause Borjeson-Forssman-Lehmann (BFLS) syndrome.Little
The neurodevelopment research of Mus finds, FGF13 is microtubule stabilization albumen, can be polymerized and stablize micro-pipe.FGF13 regulates brain skin
Layer and Development of Hippocampal Neurons and migration, FGF13 disappearance can hinder neuron to convert to bipolarity from pluripolarity, also result in aixs cylinder
Or the excessive branch of leading projection, thus slow down the migration of neuron, cause cerebral cortex and hippocampal formation abnormal, cortex thalamus
The axiramificate of nerve tract increases.The ability of learning and memory of FGF13 knock-out mice is by obvious damage.Clinical case research finds,
FGF13 gene mutation is closely related with children's intelligence obstacle.These find that prompting FGF13 is a kind of important regulation and control neurodevelopment
With the key molecule affecting dysnoesia syndrome.
One point mutation of research display FGF13 gene 5 ' UTR causes FGF13 protein expression amount in 293T cell line
Decline, and corresponding mRNA level in-site does not change.This site is positioned in FGF13 gene 2 exon, genome
Physical location is chrX:138286301, sports a C > G point mutation.Large sample amount Mass screening finds, in 302 example intelligence
In dysplasia infant, suddenly change in this site with the presence of 3 male's infants.The father of 3 infants does not the most carry this sudden change
Site, 3 mothers are then the heterozygote carrying mutational site, and this illustrates the heritability in this site.It addition, right
In 1245 samples according to group, only 2 women are to carry the heterozygote in this site, and its frequency is far smaller than dysnoesia group.
These data prove that this mutational site plays an important role for intelligent development obstacle.
Amplification refractory mutation system (amplification refractory mutation system, ARMS) is on the basis of PCR
Grow up identifies mutational site or the technology of polymorphism.ARMS is successfully used for Polymorphism Analysis, including somatic cell
Sudden change and germinal mutation.The advantage of this technology is can successfully to tell the sudden change of low content under a large amount of wild DNA backgrounds.
ARMS system is based on 3 ' end mispairing principles, i.e. when amplified reaction, if 3 ' end base pairs form mispairing, in nothing 3 '-5 '
In the PCR system of 5 prime excision enzyme activity, chain extension reaction can be because of 3 '-5 ' obstacle of phosphodiester bond formation and be obstructed, therefore, only
When template strand is specific allele, just can produce special amplified production.For avoiding primer to exist with during target DNA mispairing
Mispairing extends, and 2-3 position can be held in the 3 ' of primer to introduce base mismatch, make to form multiple mispairing between same template thus stop mistake
Join extension.
And ARMS-qPCR system adds specific fluorogenic probe in ARMS system, specificity and the spirit of testing result can be improved
Sensitivity.Thus, based on ARMS-qPCR system, the method detecting FGF13 Exon 2 mutational site has
There are research further and the value of application, in order to can simply and quickly detect FGF13 Exon 2 mutational site.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of method detecting FGF13 Exon 2 mutational site and examination thereof
Agent box.The method is fluorescence PCR detecting method based on ARMS-qPCR system, its can overcome existing sequencing technologies small throughput,
The shortcomings such as time-consuming length, power of test are limited, and there is quick, flux height, sensitivity advantages of higher.
For solving above-mentioned technical problem, the method in the detection FGF13 Exon 2 mutational site of the present invention, including following
Step:
1) sample DNA is extracted, as DNA profiling；
2) utilize one group of ARMS primer as shown in SEQ ID NO.1-3 and a probe as shown in SEQ ID NO.11 (special
The opposite sex dicyclo probe), to step 1) DNA profiling carry out fluorescent PCR amplification (i.e. carry out fluorescent PCR amplification sudden change base
Because of sequence)；
3) fluorescence intensity of the reaction system in the amplification of detection fluorescent PCR, and the threshold value time institute set is reached according to fluorescence intensity
The cycle-index Ct value needed judges the sudden change in FGF13 Exon 2 mutational site.
Described step 1) in, sample includes: blood or saliva.
Described step 2) in, 5 ' ends and the 3 ' ends of described specificity dicyclo probe are connected to fluorophor and quenching group；
Wherein, the 5 ' fluorophors held include: FAM, HEX, CY5 or ROX, preferably FAM, HEX or ROX；3 ' hold
Quenching group comprises the steps that BHQ (such as BHQ 1 etc.)；
The reaction system that cumulative volume is 20 μ l of described fluorescent PCR amplification is as follows:
Wherein, the 1 × PCR buffer in the amplification of described fluorescent PCR, MgCl2, each dNTP and Taq enzyme and water can be all from
In LIFE TECH company.
The reaction condition of described fluorescent PCR amplification is as follows:
95 DEG C of denaturations 10 minutes；15 circulations, 94 DEG C of degeneration 30 seconds, 60 DEG C of annealing extend 1 minute；35 circulations, 94 DEG C
Degeneration 30 seconds, 60 DEG C of annealing extend 1 minute；And the fluorescence of annealing stage detection FAM and HEX in rear 35 circulations
Signal, or the fluorescence signal of detection FAM and ROX.
Described step 2) in, also include: utilize the Quality Control gene primer as shown in SEQ ID NO.13-14 and SEQ ID NO.15
Shown Quality Control gene probe is to step 1) DNA profiling carry out fluorescent PCR amplification.
Described step 3) in, it is judged that the standard of the sudden change in FGF13 Exon 2 mutational site is:
The fluorescence signal of FAM Yu HEX of the reaction system in detection fluorescent PCR amplification, or FAM's with ROX is glimmering
Optical signal, with HEX or ROX signal reach set threshold value (12 < Ct < 20) time, show loading amount of DNA in allowed band,
FAM signal results is credible；Cycle-index Ct value required when reaching the threshold value of setting using FAM judges as sudden change yin and yang attribute
Standard, Ct value > 20: negative (unmutated)；Ct value < 20: positive (sudden change).
Table 1 sequence
It addition, the present invention also provides for a kind of detection kit for said method, comprising: shown in SEQ ID NO.1-3
ARMS primer and a probe as shown in SEQ ID NO.11.
Described test kit may also include that shown in the Quality Control gene primer as shown in SEQ ID NO.13-14 and SEQ ID NO.15
Quality Control gene probe.
Further, described test kit may also include that the reagent required for PCR reaction, such as conventional PCR buffer, MgCl2、
DNTPs and Taq enzyme.
In the present invention, according to human fibroblast's growth factor FGF-2 13 Exon 2 mutational site wild type gene sequence
Row and corresponding mutant gene sequence, design one group of ARMS primer and a specificity dicyclo probe, and prepare fluorescent PCR expansion
Increase the reaction system of mutational site gene order, and expand gene sequence to be measured with above-mentioned primer and specificity dicyclo probe simultaneously
Row, utilize the hybridization of dicyclo probe and amplified production, the fluorescence intensity of detection reaction system, thus finally carry out FGF13 gene
The detection in exon 2 mutational site.
Beneficial effects of the present invention is as follows:
1) flux is high: can disposably analyze 48 samples；
2) highly sensitive: 20ng genomic DNA can complete precisely to analyze；
3) specificity is good, and precision is high, and detection success rate is 100%；
4) pollute less: detection process is stopped pipe detection, reduces the probability polluted；
5) simple to operate quickly, whole detection process is the shortest；
6) polymorphic type pattern detection: the DNA sample as originated the complex samples such as blood sample, saliva sample all can detect；
7) safety: whole system does not comprise poisonous and harmful substance, avoids electrophoresis detection, to experimenter and environment non-hazardous.
Accompanying drawing explanation
The present invention is further detailed explanation with detailed description of the invention below in conjunction with the accompanying drawings:
Fig. 1 is the standard curve under the conditions of different plasmid template copy number；
Fig. 2 be plasmid template be 1000 copies and 100 copy repeated testing results, wherein, 1 is plasmid template 1000
Copy, 2 copy for plasmid template 100；
Fig. 3 is homozygous mutation sample amplification curve diagram；
Fig. 4 is wild sample amplification curve diagram；
Fig. 5 is heterozygous sample amplification curve diagram.
Detailed description of the invention
Below in conjunction with specific embodiment, such scheme is described further.Should be understood that these embodiments are merely to illustrate the present invention
And do not limit the scope of the invention.
Mutant plasmid that the present invention builds with genetic engineering and wild plasmid, as template, build mankind FGF13EXON2 and suddenly change position
Point ARMS real-time PCR detection system, with FAM for detection object, by the optimum organization of special ARMS primer with
And fluorescent probe detection system optimizes, thus realizing quick and precisely, simply, flux detects.
The method in detection FGF13 EXON2 mutational site, comprises the following steps:
1. for mutational site design synthesis ARMS primer combination and probe
According to the sequence in FGF13 EXON2 mutational site, design the combination of multipair ARMS primer and probe for mutational site,
Pass through optimum experimental, it is achieved highly sensitive and specific detection.Mutational site information refers to the base of the band horizontal line in table 2.
For mutational site sequence, application primer 5.0 primer-design software designs the combination of multipair ARMS primer and probe, draws
Thing and probe sequence are as shown in table 2.Wherein, primer Actin forward and Actin reverse, as Quality Control gene primer, visits
Pin Actin Probe is as Quality Control gene probe, to carry out Quality Control.
Table 2
2, sample to be tested preparation and extraction
Business-like DNA extraction kit is used to extract sample DNA, concrete operations reference reagent box description.
3, fluorescent PCR amplification, sets up amplification reaction system
The DNA sample (i.e. DNA profiling) that will obtain in step 2, through UV spectrophotometer measuring and read its content.
DNA profiling is diluted to 10ng/ μ L.
PCR amplification (cumulative volume 20 μ L) is carried out according to following amplification system:
Wherein, the 1 × PCR buffer in the amplification of described fluorescent PCR, MgCl2, each dNTP and Taq enzyme and water can be all from
In LIFE TECH company.
Real-time PCR reactions condition is: 95 DEG C of denaturations 10 minutes；94 DEG C of degeneration of 15 circulations 30 seconds, 60 DEG C of annealing extensions 1
Minute；94 DEG C of degeneration of 35 circulations 30 seconds, 60 DEG C of annealing extend 1 minute；Annealing stage detection FAM in rear 35 circulations
With the fluorescence signal of HEX, or detection FAM and ROX fluorescence signal.
4, detection fluorescence signal, the standard that period Ct value required during to reach to set threshold value judges as result:
Wherein, as a example by the fluorescence signal of FAM and HEX, specific as follows:
FAM and the HEX fluorescence intensity of detection reaction system, when reaching to set threshold value (12 < Ct < 20) with HEX signal, table
Bright DNA applied sample amount is in allowed band, and FAM signal is credible.Needed for reaching to set threshold value using FAM, Ct value is as judging mark
Accurate:
Amplification sudden change (mutation) site Ct value < 20, and with HEX signal Ct value absolute difference be less than 4, expansion
Increase the Ct value in wild type (wild) site > 20 and difference with amplification mutation site Ct value be more than 7, for homozygous mutant；
Amplification wild site Ct value < 20, and with HEX signal Ct value absolute difference be less than 4, amplification mutation
The Ct value in site > 20 and difference with amplification mutation site Ct value be more than 7, for wild type；
The Ct value in amplification mutation and wild site is respectively less than 20, both ct value difference values less than 7, and both Ct values all with
The Ct value absolute difference of HEX signal is less than 4, then be judged to heterozygous.
It addition, for the judging such as the criterion of above-mentioned FAM and HEX of fluorescence signal of FAM and ROX.
Embodiment 1
(site to be measured is C, treats location for one to use the plasmid template containing FGF13 Exon 2 mutational site
It is G at Dian), utilize primer and probe in table 2, carry out qPCR respectively with the optimal ARMS primer of optimized choice.
Wherein, for the wild plasmid FGF13 wild vector (site to be measured is C) related in following table, prominent
Modification plasmid FGF13 mutation vector (site to be measured is G) all can be according to conventional plasmid construction method and utilize
PCR cloning process prepares.
1) plasmid processes and extracting:
The extraction of plasmid uses the plasmid extraction kit of TIANGEN (HighPure Plasmid Kit, DP116) to carry out, tool
Body operating procedure refers to product description.Carried DNA is dissolved in Tris-HCl (10mmol/L, pH8.0), ultraviolet spectrometry
Photometer detection sample quality also measures concentration.Then, by Sample Dilution to 10ng/ μ L.Take 1 μ L and carry out PCR reaction.
2) PCR amplification (qPCR system, cumulative volume 20 μ L) is carried out according to following amplification system
Wherein, the 1 × PCR buffer in the amplification of described fluorescent PCR, MgCl2, each dNTP and Taq enzyme and water can be all from
In LIFE TECH company.
3) PCR reaction condition is 95 DEG C of denaturations 10 minutes, and 94 DEG C of degeneration of 15 circulations 30 seconds, 60 DEG C of annealing extend 1 point
Clock；94 DEG C of degeneration of 35 circulations 30 seconds, 60 DEG C of annealing extend 1 minute；Annealing stage detection FAM in rear 35 circulations
Fluorescence signal and HEX fluorescence signal.
4) detection fluorescence signal, the standard (with reference to above-mentioned) judged as result according to Ct value.
5) optimize ARMS primer, primer in table is divided into three groups.
Group 1:FGF13 Wild C, FGF13 Mutation G, FGF13 RV；
Group 2:FGF13 Wild-2 C, FGF13 Mutation-2 G, FGF13 RV；
Group 3:FGF13 Wild-1 C, FGF13 Mutation-1 G, FGF13 RV.
Downstream primer is FGF13 RV, and probe is FGF13 Probe, and in group, forward primer is with downstream primer pairing detection phase
Answer mutational site.In other qPCR systems, composition immobilizes, result as shown in Table 3-5, primer sets 1 (FGF13 Wild C,
FGF13 Mutation G, FGF13 RV) result is optimal.
Table 3 primer sets 1 is with the FGF13probe probe fluorescent PCR testing result with the plasmid template containing mutational site as template
Table 4 primer sets 2 is with the FGF13probe probe fluorescent PCR testing result with the plasmid template containing mutational site as template
Table 5 primer sets 3 is with the FGF13probe probe fluorescent PCR testing result with the plasmid template containing mutational site as template
6) sensitive analysis: plasmid template being started dilution from 10000 copies, until being diluted to 5 copies, then distinguishing
Detecting it, result shows that the fluorescence PCR method of the present invention is highly sensitive, the corresponding plasmid sample of primer detection, 5 copies
(as shown in Figure 1) can be detected.
7) repeated experiment: each reaction is separately added into positive control template plasmid DNA 1000 and copies, 100 copies, repeat
Carrying out fluorescent PCR amplification for 10 times, 10 Ct value differences are less than 0.5 circulation (as shown in Figure 2).
Testing result shows, the detection system of the present invention select optimum ARMS primer to and probe (i.e. SEQ ID NO.1-3
Probe shown in shown primer and SEQ ID NO.11), mutational site can be accurately identified, the sensitivity of detection can reach 5 and copies
Shellfish.
Embodiment 2
Use the present invention to detect clinical sample, obtain the peripheral blood sample of person to be measured, extract DNA, utilize in the present invention special
It is detected by ARMS primer and fluorescent probe PCR system, and carries out sequence verification simultaneously.
Step is as follows:
1) sample process and DNA extraction:
Business-like DNA extraction kit is used to extract sample DNA, concrete operations reference reagent box description.
Sample DNA is diluted to 10ng/ μ L.
2) PCR amplification (cumulative volume 20 μ L) is carried out according to following amplification system
Wherein, the primer in above-mentioned PCR amplification is the primer as shown in SEQ ID NO.1-3 and SEQ ID NO.13-14 institute
The Quality Control gene primer shown, probe is the probe as shown in SEQ ID NO.11 and the Quality Control gene as shown in SEQ ID NO.15
Probe.
It addition, PCR amplification in 1 × PCR buffer, MgCl2, each dNTP and Taq enzyme and water can be both from LIFE TECH
Company.
3) PCR reaction condition is 95 DEG C of denaturations 10 minutes, and 94 DEG C of degeneration of 15 circulations 30 seconds, 60 DEG C of annealing extend 1 point
Clock；94 DEG C of degeneration of 35 circulations 30 seconds, 60 DEG C of annealing extend 1 minute；Annealing stage detection FAM in rear 35 circulations
With HEX fluorescence signal.
4) detection fluorescence signal, the standard judged as result according to Ct value
The fluorescence intensity of FAM and HEX of detection reaction system, when reaching to set threshold value (12 < Ct < 20) with HEX signal,
Show DNA applied sample amount in allowed band, FAM signal is credible.Needed for reaching to set threshold value using FAM, Ct value is as result
Criterion:
The Ct value in amplification mutation site < 20, and it is less than 4 with the absolute difference of the Ct value of HEX signal, and expand wild
The Ct value in site > 20 and difference with amplification mutation site Ct value be more than 7, for homozygous mutant, amplification figure refers to Fig. 3；
Amplification wild site Ct value < 20, and with HEX signal Ct value absolute difference be less than 4, amplification mutation
The Ct value in site > 20 and difference with amplification mutation site Ct value be more than 7, for wild type, amplification figure refers to Fig. 4；
The Ct value in amplification mutation and wild site is respectively less than 20, and both Ct value absolute difference are less than 7, and both Ct
Value is all less than 4 with the Ct value absolute difference of HEX signal, then be judged to heterozygous, and amplification figure refers to Fig. 5.
5) testing result shows that testing result of the present invention is consistent with DNA sequencing result: have 8 example heterozygosis in 1225 example samples, 4
Example homozygous mutation (as shown in table 6).
Table 6
Embodiment 3
Use the present invention to detect saliva sample, after saliva sample extracting DNA, utilize in the present invention special ARMS primer and glimmering
It is detected by light probe PCR system, and carries out sequence verification simultaneously.
Step is as follows:
1) sample process and DNA extraction:
Business-like DNA extraction kit is used to extract sample DNA, concrete operations reference reagent box description.
Sample Dilution is to 10ng/ μ L.
2) PCR amplification (cumulative volume 20 μ L) is carried out according to following amplification system
Wherein, the primer in above-mentioned PCR amplification is the primer as shown in SEQ ID NO.1-3 and SEQ ID NO.13-14 institute
The Quality Control gene primer shown, probe is the probe as shown in SEQ ID NO.11 and the Quality Control gene as shown in SEQ ID NO.15
Probe.
It addition, PCR amplification in 1 × PCR buffer, MgCl2, each dNTP, Taq enzyme and water can be both from LIFE TECH
Company.
3) PCR reaction condition is 95 DEG C of denaturations 10 minutes, and 94 DEG C of degeneration of 15 circulations 30 seconds, 60 DEG C of annealing extend 1 point
Clock；94 DEG C of degeneration of 35 circulations 30 seconds, 60 DEG C of annealing extend 1 minute；Annealing stage detection FAM in rear 35 circulations
With HEX fluorescence signal.
4) detection fluorescence signal, the standard judged as result according to Ct value
FAM and the HEX fluorescence intensity of detection reaction system, when reaching to set threshold value (12 < Ct < 20) with HEX signal,
Show DNA applied sample amount in allowed band, FAM signal is credible.Needed for reaching to set threshold value using FAM, Ct value is as result
Criterion:
The Ct value in amplification mutation site < 20, and it is less than 4 with the absolute difference of the Ct value of HEX signal, and expand wild
The Ct value in site > 20 and difference with amplification mutation site Ct value be more than 7, for homozygous mutant；
Amplification wild site Ct value < 20, and with HEX signal Ct value absolute difference be less than 4, amplification mutation
The Ct value in site > 20 and difference with amplification mutation site Ct value be more than 7, for wild type；
The Ct value in amplification mutation and wild site is respectively less than 20, and both Ct value absolute difference are less than 7, and both Ct
Value is all less than 4 with the Ct value absolute difference of HEX signal, then be judged to heterozygous.
5) testing result shows that testing result of the present invention is consistent with DNA sequencing result: have 2 example heterozygous mutants in 8 example samples, 1
Example homozygous mutation (as shown in table 7).
Table 7
6) experimental system of the present invention can be used for detection of complex sample, and such as saliva sample, sample is drawn materials convenient, and noinvasive sampling,
Patient's no pain.The detection time only needs 100 minutes.Therefore, the present invention is time saving and energy saving, and accuracy is high, can meet clinical noinvasive
Quickly detection.
Embodiment 4
According to the detection method in above-described embodiment, in order to easier and be used for quickly detecting testing sample (such as saliva or blood
Liquid etc.), reagent used in the detection method of the present invention is prepared as a kind of test kit.
The component of this test kit is that the ARMS primer as shown in SEQ ID NO.1-3 and one are as shown in SEQ ID NO.11
Probe.
It addition, as required, this test kit may also include that the Quality Control gene primer as shown in SEQ ID NO.13-14 and SEQ ID
Quality Control gene probe shown in NO.15.
Further, described test kit may also include that the reagent required for PCR reaction, such as conventional PCR buffer, MgCl2、
DNTPs and Taq enzyme.
Claims (8)
1. the method in the detection FGF13 Exon 2 mutational site of a non-diagnostic purpose, it is characterised in that include
Following steps:
1) sample DNA is extracted, as DNA profiling；
2) one group of ARMS primer as shown in SEQ ID NO.1-3 and a probe as shown in SEQ ID NO.11 are utilized,
To step 1) DNA profiling carry out fluorescent PCR amplification；
3) fluorescence intensity of the reaction system in the amplification of detection fluorescent PCR, and the threshold value time institute set is reached according to fluorescence intensity
The cycle-index Ct value needed judges the sudden change in FGF13 Exon 2 mutational site.
2. the method for claim 1, it is characterised in that:
Step 2) in, 5 ' ends and the 3 ' ends of probe are connected to fluorophor and quenching group；Wherein, fluorophor is selected from:
FAM, HEX, CY5 or ROX；Quenching group is selected from: BHQ.
3. method as claimed in claim 2, it is characterised in that: described fluorophor is FAM, HEX or ROX；Cancellation
Group is BHQ1.
4. the method for claim 1, it is characterised in that: described step 2) in, the cumulative volume of fluorescent PCR amplification is
The reaction system of 20 μ l is as follows:
Add water and complement to 20 μ l；
Step 2) in, the reaction condition of fluorescent PCR amplification is as follows:
95 DEG C of denaturations 10 minutes；15 circulations, 94 DEG C of degeneration 30 seconds, 60 DEG C of annealing extend 1 minute；35 circulations, 94 DEG C
Degeneration 30 seconds, 60 DEG C of annealing extend 1 minute；And the fluorescence of annealing stage detection FAM and HEX in rear 35 circulations
Signal, or the fluorescence signal of detection FAM and ROX.
5. the method for claim 1, it is characterised in that: described step 2) in, also include: utilize such as SEQ ID
Quality Control gene primer shown in NO.13-14 and the Quality Control gene probe shown in SEQ ID NO.15 are to step 1) DNA profiling
Carry out fluorescent PCR amplification.
6. the detection kit for the method as described in any one of Claims 1 to 5, it is characterised in that including: SEQ
ARMS primer shown in ID NO.1-3 and a probe as shown in SEQ ID NO.11.
7. test kit as claimed in claim 6, it is characterised in that: described test kit also includes: such as SEQ ID NO.13-14
Quality Control gene probe shown in shown Quality Control gene primer and SEQ ID NO.15.
8. test kit as claimed in claim 6, it is characterised in that: described test kit also includes: PCR buffer, MgCl2、
DNTPs and Taq enzyme.
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