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Primer and method for simultaneously detecting VKORC1 and CYP2C9 gene polymorphisms

A gene polymorphism and genotype technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of high cost of gene chip design, high detection cost, lack of specificity, etc. , to achieve good accuracy, high sensitivity and good specificity

Inactive Publication Date: 2015-10-21
GUANGZHOU KINGMED DIAGNOSTICS CENT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Chinese patent application 201310533548.8 discloses a specific primer pair and probe for CYP2C9 and VKORC1 gene chip detection, but the design cost of the gene chip is high and the detection cost is expensive
The existing technology lacks a CYP2C9 and VKORC1 gene polymorphism detection primer and its method with good specificity, high sensitivity, and simultaneous detection of multiple sites

Method used

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  • Primer and method for simultaneously detecting VKORC1 and CYP2C9 gene polymorphisms
  • Primer and method for simultaneously detecting VKORC1 and CYP2C9 gene polymorphisms
  • Primer and method for simultaneously detecting VKORC1 and CYP2C9 gene polymorphisms

Examples

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Effect test

Embodiment 1

[0022] Example 1 Primers

[0023] The inventor designed a large number of primers for the gene polymorphism sites of VKORC1 and CYP2C9, and through optimization and comparison of primer reaction conditions, primers with good specificity, no cross-reaction and very close PCR reaction conditions were screened out. The primers provided by the present invention are shown in Table 1, including PCR amplification primers and SNaPshot PCR primers, and the PCR amplification primers and SNaPshot PCR primers are corresponding. For VKROC1-1639G>A, the upstream primer is 5'-GCCAGCAGGAGAGGGAAATA-3'(SEQ ID NO.1), the downstream primer is 5'-AGTTTGGACTACAGGTGCCT-3'(SEQ ID NO.2), and the SNaPshot PCR primer is 5'- ATAGGCGTGAGCCACCGCACC-3' (SEQ ID NO. 7). All primer sequences provided by the present invention are compared through UCSC database, and there is no known SNP site.

[0024]

Embodiment 2

[0025] The specificity of embodiment two primers

[0026] The primers provided by the present invention were blasted in UCSC, and the results were as follows: VKORC1 The -1639G>A amplified fragment is located at chr16: 31107523-31107812, with a length of 290bp; CYP2C9 *2 The amplified fragment is located between chr10:96701959-96702133, with a length of 175bp; CYP2C9 *3 The amplified fragment is located at chr10:96740948-96741101, with a length of 154bp; the result is as follows figure 1 As shown, the amplified fragments of all primers covered the corresponding detection sites: VKORC1 -1639G>A, CYP2C9 *2 and CYP2C9 *3, No other homologous genes.

[0027] The PCR amplification primers in Table 1 were used to amplify and Sanger sequence the test samples respectively. The sequencing results showed that the fragments amplified by each primer were consistent with the reference sequences of the VKORC1 and CYP2C9 genes. The results were as follows: figure 2 shown. Us...

Embodiment 3

[0035] Example 3 Specificity of the method for detecting VKORC1 and CYP2C9 gene polymorphisms

[0036] The specificity of this assay is defined as the negative coincidence rate. VKORC1 -1639 A / A genotype accounted for the vast majority (82.1%), VKORC1-1639 A / G genotype accounted for 17.9%, CYP2C9*2 430 C / C genotype accounted for the vast majority, and CYP2C9*3 1075A / A gene type accounts for the vast majority. Therefore, this test defines the detection of VKORC1 -1639 A / A genotype as a negative result, the detection of CYP2C9*2 430 C / C genotype as a negative result, and the detection of CYP2C9*3 1075 A / A genotype Defined as a negative result.

[0037] The SNaPshot method provided by the present invention was used to detect 18 samples, and at the same time, the Sanger sequencing method was used for verification. The SNaPshot sequencing method detected a total of 9 samples without mutation (negative), which was consistent with the results shown by the Sanger sequencing metho...

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Abstract

The invention belongs to the technical field of biological detection, and provides a primer for simultaneously detecting VKORC1 and CYP2C9 gene polymorphisms. The primer comprises a PCR amplification primer and a SNaPshot PCR primer. The primer can achieve specific detection on VKORC1 and CYP2C9 gene polymorphisms, causes no cross reaction and is good in accuracy.

Description

technical field [0001] The invention belongs to the technical field of biological detection, in particular to a primer and a method for simultaneously detecting VKORC1 and CYP2C9 gene polymorphisms. Background technique [0002] There are currently 30 CYP2C9 alleles discovered, among which CYP2C9*l (wild type), CYP2C9*2 (c.430C>T, Argl44Cys, rs1799853), CYP2C9*3 (c.1075A>C, Ile359Leu, rs1057910 ) is the most common. [0003] The gene frequency of CYP2C9*2 and CYP2C9*3 varies greatly among different races and different ethnic groups, and the allele frequency in Chinese population is much lower than that in Caucasians. Studies have shown that the CYP2C9 gene mutation status is closely related to the clinical drug sensitivity and toxicity of warfarin drugs. Patients with variant alleles have significantly lower warfarin metabolic enzyme activity than wild-type patients, and their hemorrhage rate The risk increased by 2-3 times, and CYP2C9*1 / *3 and CYP2C9*3 / *3 patients h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q1/6858C12Q2600/106C12Q2600/156C12Q2600/16
Inventor 赵薇薇胡昌明燕启江郭周萍
Owner GUANGZHOU KINGMED DIAGNOSTICS CENT
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