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Primer and method for simultaneously detecting polymorphism of CYP2C*2 and CYP2C*3 genes

A gene polymorphism and genotyping technology, applied in biochemical equipment and methods, microbial measurement/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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Problems solved by technology

[0004] 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 polymorphism of CYP2C*2 and CYP2C*3 genes
  • Primer and method for simultaneously detecting polymorphism of CYP2C*2 and CYP2C*3 genes
  • Primer and method for simultaneously detecting polymorphism of CYP2C*2 and CYP2C*3 genes

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Embodiment 1

[0020] Example 1 Primers

[0021] The inventor designed a large number of primers for the gene polymorphism sites of CYP2C9*2 and CYP2C9*3. Through optimization and comparison of the primer reaction conditions, the primers were screened out with good specificity, no cross-reaction and very close PCR reaction conditions. primers. 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. All primer sequences provided by the present invention are compared through UCSC database, and there is no known SNP site.

[0022]

Embodiment 2

[0023] The specificity of embodiment two primers

[0024] The primers provided by the present invention were blasted in UCSC, and the results were as follows: 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: CYP2C9 *2 and CYP2C9 *3, No other homologous genes.

[0025] The PCR amplification primers in Table 1 were used to amplify and Sanger sequence the detection samples respectively. The sequencing results showed that the amplified fragments of each primer were the same as CYP2C9 *2 and CYP2C9 *3 The gene reference sequence matches, the result is as follows figure 2 shown. Using the SNaPshot PCR primers in Table 1, the SNaPshot method is used for detection, and the results are as follows im...

Embodiment 3

[0033] Example three detection CYP2C9 *2 and CYP2C9 *3 Specificity of the method for genetic polymorphism

[0034] The specificity of this assay is defined as the negative coincidence rate. The CYP2C9*2 430 C / C genotype accounted for the vast majority, and the CYP2C9*3 1075A / A genotype accounted for the vast majority. Therefore, in this test, the detection of CYP2C9*2 430 C / C genotype is defined as a negative result, and the detection of CYP2C9*3 1075 A / A genotype is defined as a negative result.

[0035] 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 11 samples without mutation (negative), which was consistent with the results shown by the Sanger sequencing method, as shown in Table 4. The specificity of this detection method is 100%.

[0036]

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Abstract

The invention provides a primer for simultaneously detecting polymorphism of CYP2C*2 and CYP2C*3 genes and belongs to the technical field of biological detection. The primer comprises a PCR amplification primer and a SNaPshot PCR primer. By the adoption of the primer, specific detection of polymorphism of the CYP2C*2 and CYP2C*3 genes can be achieved, cross reaction does not occur, and accuracy is good.

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 CYP2C9*2 and CYP2C9*3 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 patien...

Claims

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Application Information

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