CYP2C9 genotype detecting primer-probe set and kit

A primer-probe, genotyping technology, applied in recombinant DNA technology, microbial assay/inspection, biochemical equipment and methods, etc. problems, to achieve the effect of fast sequencing, short reaction time, and simple sample processing

Inactive Publication Date: 2017-01-25
CHANGSHA 3G BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In order to solve the technical problems of low sensitivity, long detection cycle, cumbersome operation and high cost in the detection of CYP2C9 genotyping by the above method, the present invention prov

Method used

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  • CYP2C9 genotype detecting primer-probe set and kit
  • CYP2C9 genotype detecting primer-probe set and kit
  • CYP2C9 genotype detecting primer-probe set and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1: the preparation of kit

[0064] 1. Design and synthesis of primers and probes

[0065] For the polymorphic sites CYP2C9*2 and CYP2C9*3 alleles of the human CYP2C9 gene, specific mutation sites were selected, and the selected primers and probes were designed at the CYP2C9 mutation site and the nearby conserved regions to avoid potential for primer binding. SNPs appear in the region (retrieve the SNP of the target gene sequence through the online NCBI website), and perform PrimerBlast through the online NCBI website to confirm the specific amplification of the primer pair. The probe is located in the region between a pair of primers, and avoids SNPs in its binding region; the amplification primers and fluorescent probes are first purified by PAGE, and then purified by HPLC, wherein the target probes SEQ ID NO.2 and SEQ ID NO .6 The 5' end is labeled with a fluorescent reporter group (FAM), the 5' end of the internal control probe SEQ ID NO.11 is labeled wit...

Embodiment 2

[0085] Embodiment 2: the use of kit

[0086] 1. Sample testing

[0087] Dissolve the dry powder of the primer probe (the validity period of the primer is 1 month after dissolution). Prepare the system according to the number of templates: take the PCR reaction solution, add dissolved primers and probes, aliquot the system, add sample DNA, blank control substance or positive control substance as templates, and form a PCR reaction system. Perform PCR amplification according to the PCR reaction procedure.

[0088] The main components of the CYP2C9*2 system are as follows:

[0089] Table 7. Composition of PCR reaction solution 1

[0090] raw material name Amount added (μL) 2x Mix Buffer (including ROX) 10 SEQ ID NO.3 0.75 SEQ ID NO.1 0.75 SEQ ID NO.2 0.5 SEQ ID NO.9 0.25 SEQ ID NO.10 0.25 SEQ ID NO.11 0.5 DNA template 1 Deionized water 11 total capacity 25

[0091] Table 8. Composition of PCR reaction s...

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Abstract

The invention relates to a CYP2C9 genotype detecting primer-probe set. The primer-probe set comprises primers and a probe of CYP2C9*2, primers and a probe of CYP2C9*3 and primers and a probe of internally-controlled GAPDH. The invention further relates to a CYP2C9 genotype detecting kit. The kit comprises a PCR reaction solution 1, a PCR reaction solution 2, a PCR reaction solution 3, a PCR reaction solution 4, a positive control substance and a blank control substance. The kit provided by the invention has high sensitivity up to one ten thousandth and the minimum detection limit of only 1-2 copies, and is especially applicable to detection of low-content mutant samples (such as serum or plasma); compared with a sequencing method, the invention has the advantages as follows: results obtained in the invention can be observed in real time, gel electrophoresis detection on a product is not required, the risks in contaminating the PCR product are effectively reduced through completely-closed operation, and the detection speed is high; therefore, the primer-probe set and the kit are suitable for high-throughput sample detection.

Description

technical field [0001] The invention relates to the technical field of in vitro nucleic acid detection, in particular to a primer probe set and a kit for detecting CYP2C9 genotyping. Background technique [0002] Cytochrome P450 2C9 (Cyt℃chrome P450 2C9, CYP2C9) accounts for 20% of the total amount of P450 in liver microsomes, and can catalyze the metabolism of many commonly used clinical drugs, and its importance in clinical drug metabolism has been paid more and more attention. CYP2C9 has a high degree of genetic polymorphism, and the more common gene mutants are CYP2C9*2 (rs1799853) and CYP209*3 (rs1057910), whose encoded enzyme activities are respectively 30% and 80% lower than wild-type CYP2C9*1, resulting in Individuals with CYP2C9 mutations require lower doses of warfarin. Individuals carrying these two alleles need a longer time to reach the steady-state concentration after taking warfarin, and have a higher risk of bleeding in the initial stage of use. Therefore, w...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2545/101
Inventor 滕祥云毛平道
Owner CHANGSHA 3G BIOTECH
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