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Primer and method for detecting CYP2C9*2 gene polymorphism

A gene polymorphism and amplification primer technology, which is applied in the field of primers for detecting CYP2C9*2 gene polymorphism, can solve the problems of high cost of gene chip design, lack of specificity, high detection cost, etc., and achieve good accuracy , good specificity and high sensitivity

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

AI Technical Summary

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 prior art lacks a primer and method thereof with good specificity, high sensitivity, and detection of CYP2C9 gene polymorphism

Method used

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  • Primer and method for detecting CYP2C9*2 gene polymorphism
  • Primer and method for detecting CYP2C9*2 gene polymorphism
  • Primer and method for detecting CYP2C9*2 gene polymorphism

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Example 1 Primers

[0021] The inventor designed a large number of primers for the gene polymorphism site of CYP2C9*2, and selected primers with good specificity through optimization and comparison of primer reaction conditions. 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 CYP2C9*2, the upstream primer is 5'-CAGCAATGGAAAGAAATGGAAGG-3'(SEQ ID NO.1), the downstream primer is 5'-CAGTAAGGTCAGTGATATGGAGTAG-3'(SEQ ID NO.2), and the SNaPshot PCR primer is 5'-TTTTTTTTTTGGAAGAGGAGCATTGAGGAC- 3' (SEQ ID NO. 3). 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 in chr10:96701959-96702133 , the length is 175bp, the result is as follows figure 1 As shown, the amplified fragments of all primers covered the corresponding detection sites CYP2C9 *2, 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 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 image 3 shown. From image 3 It can be seen that the relative position of each product peak and the bases incorporated in the sequencing...

Embodiment 3

[0033] Example 3 Specificity of the method for detecting CYP2C9*2 gene polymorphism

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

[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 17 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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PUM

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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 [0001] The invention belongs to the technical field of biological detection, in particular to a primer and a method for detecting CYP2C9*2 gene polymorphism. 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 had 33.7% and 78.1% less w...

Claims

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Application Information

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