CN104988145A - Primer and method for detecting CYP2C9*2 gene polymorphism - Google Patents
Primer and method for detecting CYP2C9*2 gene polymorphism Download PDFInfo
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- CN104988145A CN104988145A CN201510384305.1A CN201510384305A CN104988145A CN 104988145 A CN104988145 A CN 104988145A CN 201510384305 A CN201510384305 A CN 201510384305A CN 104988145 A CN104988145 A CN 104988145A
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Abstract
The invention provides a primer for detecting CYP2C9*2 gene polymorphism. The primer for detecting CYP2C9*2 gene polymorphism comprises a PCR amplification primer and a SNaPshot PCR primer. The primer for detecting CYP2C9*2 gene polymorphism belongs to the technical field of biological detection. The primer for detecting CYP2C9*2 gene polymorphism can achieve the specific detection on the CYP2C9*2 gene polymorphism, and the accuracy is good.
Description
Technical field
The invention belongs to technical field of biological, particularly relate to a kind of primer and the method that detect CYP2C9*2 gene pleiomorphism.
Background technology
The CYP2C9 allelotrope found at present has 30 kinds, wherein with CYP2C9*l (wild-type), CYP2C9*2 (c.430C>T, Argl44Cys, rs1799853), CYP2C9*3 (c.1075A>C, Ile359Leu, rs1057910) the most common.
CYP2C9*2 and CYP2C9*3 gene frequency differs greatly between different ethnic group and different nationalities, and the gene frequency in Chinese population is well below white people.Research shows, CYP2C9 transgenation state and warfarin class clinical drug Drug Sensitivity and Toxicity Relationships close, carry the allelic patient of variability, the activity of its warfarin metabolic enzyme is starkly lower than wild-type, and its hemorrhage danger increases 2-3 doubly, CYP2C9*1/*3 with CYP2C9*3/*3 patient is compared with wild-type patient, and warfarin medication will reduce by 33.7% and 78.1% respectively.
Chinese patent application 201310533548.8 disclose a kind of Auele Specific Primer for CYP2C9 and VKORC1 genechip detection to and probe, but the design cost of gene chip is high, and testing cost is expensive.Prior art lacks primer and the method thereof that a species specificity is good, highly sensitive, can realize CYP2C9 genetic polymorphism detection.
Summary of the invention
For solving problems of the prior art, the invention provides a kind of primer and the method that detect CYP2C9*2 gene pleiomorphism, there is the advantages such as specificity is good, highly sensitive, accuracy is good.The site that the present invention detects comprises CYP2C9*2 (430C>T, Argl44Cys, rs1799853).
The invention provides a kind of primer detecting CYP2C9*2 gene pleiomorphism, comprise pcr amplification primer and SNaPshot PCR primer; Described pcr amplification primer comprises: for the upstream primer 5 '-CAGCAATGGAAAGAAATGG of CYP2C9*2
AAGG-3 ' (SEQ ID NO.1) and downstream primer 5 '-CAGTAAGGTCAGTGATATGGAGTAG-3 ' (SEQ ID NO.2); Described SNaPshot PCR primer comprises: for SNaPshot the PCR primer 5 '-TTTTTTTTTGGAAGAGGAGCATTGAGGAC-3 ' (SEQ ID NO.3) of CYP2C9*2.
Adopt technique scheme, the primer of detection CYP2C9*2 gene pleiomorphism provided by the invention, can realize the specific detection of CYP2C9*2 gene pleiomorphism, accuracy is good.
Correspondingly, the present invention also provides a kind of method detecting CYP2C9*2 gene pleiomorphism, comprises the steps: A) extract DNA sample; B) adopt the pcr amplification primer described in claim 1 to carry out pcr amplification, purifying is carried out to amplified production; C) adopt the SNaPshot PCR primer described in claim 1 to carry out SNaPshot pcr amplification, purifying is carried out to amplified production; D) capillary electrophoresis technique detects, and analyzes detected result, determines SNP site and genotype thereof.
Preferably, described steps A) in DNA sample be the DNA sample of EDTA anticoagulation cirumferential blood.
Preferably, state step B) in pcr amplification reaction condition comprise: Step 1:98 DEG C of for 3min; Step 2:98 DEG C for 10 s; Step 3:58 DEG C of for 30s; Step 4:72 DEG C of for 1min; Step 5:Go to step 2,29 times; Step 6:72 DEG C of for 5min; Step 7:25 DEG C forever.
Preferably, described step C) in SNaPshot pcr amplification reaction condition comprise: Step 1:96 DEG C of for 10s; Step 2:55 DEG C for 5 s; Step 3:60 DEG C of for 30s; Step 4:Go to step 1,25 times; Step 5:4 DEG C forever.
Preferably, described step D) in, adopt GENEMAPPERID V4.1 software to analyze detected result.
In addition, the present invention also provides the purposes of the primer detecting CYP2C9*2 gene pleiomorphism in preparation detection CYP2C9*2 gene pleiomorphism reagent.
Compared with prior art, the invention has the beneficial effects as follows: the invention provides a kind of primer detecting CYP2C9*2 gene pleiomorphism, the specificity of this primer is good, and accuracy is good, improves detection efficiency.In addition, present invention also offers a kind of SNaPshot method detecting CYP2C9*2 gene pleiomorphism, by using specific pcr amplification primer and SNaPshot PCR primer, there is the advantages such as specificity is good, highly sensitive, accuracy is good, guidance can be provided for the clinical application of warfarin.
Accompanying drawing explanation
The amplified fragments of Fig. 1 CYP2C9*2 gene primer provided by the invention.
The part sequencer map of Fig. 2 CYP2C9*2 gene primer amplified production.
The CYP2C9*2 genetic polymorphism detection result figure of Fig. 3 sample.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further details.
embodiment one primer
Contriver, for the gene polymorphism sites of CYP2C9*2, devises a large amount of primer, by the optimization of primer reaction conditions with compare, has filtered out the primer that specificity is good.Primer provided by the invention is as shown in table 1, comprises pcr amplification primer and SNaPshot PCR primer, and pcr amplification primer and SNaPshot PCR primer are corresponding.For CYP2C9*2, upstream primer is 5 '-CAGCAATGGAAAGAAATGGAAGG-3 ' (SEQ ID NO.1), downstream primer is 5 '-CAGTAAGGTCAGTGATATGGAGTAG-3 ' (SEQ ID NO.2), and SNaPshot PCR primer is 5 '-TTTTTTTTTGGAAGAGGAGCATTGAGGAC-3 ' (SEQ ID NO.3).All primer sequences provided by the invention all by the comparison of UCSC database, without known SNP site.
the specificity of embodiment two primer
Primer provided by the invention is carried out Blasting in UCSC, and result is as follows:
cYP2C9* 2 amplified fragments are positioned at
chr10:96701959-96702133between, length is 175bp, and as shown in Figure 1, the amplified fragments of all primers all covers corresponding detection site to result
cYP2C9* 2, without other homologous gene.
In use table 1 pcr amplification primer respectively to detection sample increase and Sanger order-checking, sequencing result show, each primer amplification fragment with
cYP2C9* 2 gene reference sequence coincide, result as shown in Figure 2.Use SNaPshot PCR primer in table 1, SNaPshot method detects, and result as shown in Figure 3.As can be seen from Figure 3, the base that the relative position at each product peak and sequencing reaction mix conforms to expection, and without other Interference Peaks.
the detection of embodiment two CYP2C9*2 gene pleiomorphism
Extract the DNA sample of EDTA anticoagulation cirumferential blood, extracting method with reference to TIANamp Blood DNA Kit(purchased from Tiagen, article No. DP318) specification sheets, DNA sample is diluted to 100ng/ μ L, for subsequent use.
Pcr amplification adopts Q5
?warm start surpasses fidelity 2X Master Mix(purchased from NEB company, article No. M0494L), reaction system is as shown in table 2, and the concentration of pcr amplification primer is 5pmol/uL.Pcr amplification reaction condition comprises: Step 1:98 DEG C of for 3min; Step 2:98 DEG C for 10 s; Step 3:58 DEG C of for 30s; Step 4:72 DEG C of for 1min; Step 5:Go to step 2,29 times; Step 6:72 DEG C of for 5min; Step 7:25 DEG C forever.After pcr amplification terminates, get 2.0 μ L ExoSAP-IT(purchased from Affymetrix, article No. 78205) and 3.0 μ L ddH
2o, mixing, adds pcr amplification product 2.0 μ L, mixes micro-centrifugal; Endonuclease reaction is carried out, program: 37 DEG C, 15 min in PCR instrument; 80 DEG C, 15min; 4 DEG C, insulation.
Adopt SNaPshot
multiplex Kit(purchased from ABI company, article No. 4323151) increase, reaction system is as shown in table 3, and the concentration of SNaPshot PCR primer is 5pmol/uL.SNaPshot PCR reaction conditions comprises: Step 1:96 DEG C of for 10s; Step 2:55 DEG C for 5 s; Step 3:60 DEG C of for 30s; Step 4:Go to step 1,25 times; Step 5:4 DEG C forever.
In SNaPshot PCR primer, add 1.0 μ L SAP enzymes, react according to following program: 37 DEG C, 60min; 75 DEG C, 15min; 4 DEG C, insulation.After completion of the reaction, capillary electrophoresis technique detects, and adopt GENEMAPPERID V4.1 software to analyze to detected result, determine SNP site and genotype thereof, result as shown in Figure 3.
embodiment three detects the specificity of the method for CYP2C9*2 gene pleiomorphism
The specificity of this detection method is defined as negative match-rate.CYP2C9*2 430 C/C genotype accounts for the overwhelming majority, and CYP2C9*2 430 C/T genotype accounts for very small portion, and therefore, genotypic the detecting of CYP2C9*2 430 C/C is defined as negative findings by this detection.
Adopt SNaPshot method provided by the invention to detect 18 routine samples, adopt Sanger sequencing to verify simultaneously.SNaPshot sequencing detects sample totally 17 examples without sudden change (feminine gender), and the result shown with Sanger sequencing conforms to, as shown in table 4.The specificity of this detection method is 100%.
embodiment four detects the sensitivity of the method for CYP2C9*2 gene pleiomorphism
The sensitivity definition of this detection method is positive coincidence rate.Adopt SNaPshot method provided by the invention to detect 18 routine samples, adopt Sanger sequencing to verify simultaneously.SNaPshot sequencing detects sample totally 1 example of sudden change (positive), conforms to Sanger sequencing result, as shown in table 5.This detection method highly sensitive.
embodiment five detects the accuracy of the method for CYP2C9*2 gene pleiomorphism
The accuracy of this detection method is defined as the consistence of different methods detected result.18 routine samples are through SNaPshot method and Sanger sequencing, and different methods detected result is consistent, as shown in table 6.The accuracy of this detection method is 100%.
embodiment six detects the precision of the method for CYP2C9*2 gene pleiomorphism
The precision of this detection method is defined as carries out to sample the ability that duplicate detection obtains same result.This detection has been carried out between personnel, contrast experiment between the different hole of different time, different instrument, same sample, and result is as shown in table 7, and all results all show unanimously, and this detection precision is 100%.
Therefore, primer provided by the present invention and detection method can be used as one independently, widely used authentication method, solve CYP2C9*2 and carry out accurate Classification Identification problem, play the feature that PCR-SNaPshot PCR is accurate to described gene locus genotypic results, high-throughput operates, very large directive function can be played to the personalized medicine of warfarin, reduce the untoward reaction of warfarin.
Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, some simple deduction or replace can also be made, all should be considered as belonging to protection scope of the present invention.
SEQUENCE LISTING
<110> Guangzhou Kingmed Center for Clinical Laboratory Co., Ltd., Guangzhou medical university
<120> mono-kind detects primer and the method for CYP2C9*2 gene pleiomorphism
<130>
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 23
<212> DNA
<213> artificial sequence
<400> CYP2C9*2_F
cagcaatgga aagaaatgga agg 23
<210> 2
<211> 25
<212> DNA
<213> artificial sequence
<400> CYP2C9*2_R
cagtaaggtc agtgatatgg agtag 25
<210> 3
<211> 29
<212> DNA
<213> artificial sequence
<400> CYP2C9*2
tttttttttg gaagaggagc attgaggac 29
Claims (7)
1. detect a primer for CYP2C9*2 gene pleiomorphism, it is characterized in that: comprise pcr amplification primer and SNaPshot PCR primer; Described pcr amplification primer comprises: for upstream primer 5 '-CAGCAATGGAAAGAAATGGAAGG-3 ' and the downstream primer 5 '-CAGTAAGGTCAGTGATATGGAGTAG-3 ' of CYP2C9*2; Described SNaPshot PCR primer comprises: for SNaPshot the PCR primer 5 '-TTTTTTTTTGGAAGAGGAGCATTGAGGAC-3 ' of CYP2C9*2.
2. detect a method for CYP2C9*2 gene pleiomorphism, it is characterized in that: comprise the steps: A) extract DNA sample; B) adopt the pcr amplification primer described in claim 1 to carry out pcr amplification, purifying is carried out to amplified production; C) adopt the SNaPshot PCR primer described in claim 1 to carry out SNaPshot pcr amplification, purifying is carried out to amplified production; D) capillary electrophoresis technique detects, and analyzes detected result, determines SNP site and genotype thereof.
3. the method for detection CYP2C9*2 gene pleiomorphism according to claim 2, is characterized in that: described steps A) in DNA sample be the DNA sample of EDTA anticoagulation cirumferential blood.
4. the method for detection CYP2C9*2 gene pleiomorphism according to claim 2, is characterized in that: described step B) in pcr amplification reaction condition comprise: stage 1:98 DEG C 3min; Stage 2:98 DEG C 10s; Stage 3:58 DEG C 30s; Stage 4:72 DEG C 1min; Stage 5: turn back to stage 2,29 circulation; Stage 6:72 DEG C 5min; Stage 7:25 DEG C of insulation.
5. the method for detection CYP2C9*2 gene pleiomorphism according to claim 2, is characterized in that: described step C) in SNaPshot pcr amplification reaction condition comprise: stage 1:96 DEG C 10s; Stage 2:55 DEG C 5s; Stage 3:60 DEG C 30s; Stage 4: turn back to stage 1,25 circulation; Stage 5:4 DEG C of insulation.
6. the method for detection CYP2C9*2 gene pleiomorphism according to claim 2, is characterized in that: described step D) in, adopt GENEMAPPERID V4.1 software to analyze detected result.
7. the primer of detection CYP2C9*2 gene pleiomorphism according to claim 1 detects the purposes in CYP2C9*2 gene pleiomorphism reagent in preparation.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107287318A (en) * | 2017-07-11 | 2017-10-24 | 厦门美因生物科技有限公司 | A kind of SNP site detection solution system and kit |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101522911A (en) * | 2006-09-11 | 2009-09-02 | Inje大学校产学协力团 | Htsnps for determining a genotype of cytochrome P450 1a2, 2A6 and 2D6, PXR and UPD-glucuronosyltransferase 1A gene and multiplex genotyping methods using thereof |
| CN103233068A (en) * | 2013-04-19 | 2013-08-07 | 向华 | Primer system for detecting genetic polymorphic sites related to human cytochrome P450 and application of primer system |
| CN103820553A (en) * | 2014-02-27 | 2014-05-28 | 厦门大学附属中山医院 | Multiplex PCR combined pyrosequencing kit used for guiding individualized medication of warfarin and detection method thereof |
-
2015
- 2015-06-30 CN CN201510384305.1A patent/CN104988145A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101522911A (en) * | 2006-09-11 | 2009-09-02 | Inje大学校产学协力团 | Htsnps for determining a genotype of cytochrome P450 1a2, 2A6 and 2D6, PXR and UPD-glucuronosyltransferase 1A gene and multiplex genotyping methods using thereof |
| CN103233068A (en) * | 2013-04-19 | 2013-08-07 | 向华 | Primer system for detecting genetic polymorphic sites related to human cytochrome P450 and application of primer system |
| CN103820553A (en) * | 2014-02-27 | 2014-05-28 | 厦门大学附属中山医院 | Multiplex PCR combined pyrosequencing kit used for guiding individualized medication of warfarin and detection method thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107287318A (en) * | 2017-07-11 | 2017-10-24 | 厦门美因生物科技有限公司 | A kind of SNP site detection solution system and kit |
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