Primer pair for detecting CYP2C9 genetic typing through pyrosequencing method and kit

A pyrosequencing method and a genotyping technology, which are applied in the field of primer pairs and kits for detecting CYP2C9 genotyping by pyrosequencing, and can solve the problems of low sensitivity of CYP2C9 genotyping, long detection period, complicated operation, etc. problems, to achieve the effect of operating high-throughput sample detection, short reaction time, and simple sample processing

Inactive Publication Date: 2016-03-02
CHANGSHA 3G BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] 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 provides a met

Method used

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  • Primer pair for detecting CYP2C9 genetic typing through pyrosequencing method and kit
  • Primer pair for detecting CYP2C9 genetic typing through pyrosequencing method and kit
  • Primer pair for detecting CYP2C9 genetic typing through pyrosequencing method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1: the preparation of kit

[0062] 1. Design and synthesis of primers and probes

[0063] For the polymorphic sites CYP2C9*2 and CYP2C9*3 alleles of the human CYP2C9 gene, specific mutation sites were selected, and primers were designed using PyroMarkAssayDesign2.0 software; the amplification primers and sequencing primers were first purified by PAGE, and then Purified by HPLC, wherein the 5' ends of SEQ ID NO.1 and SEQ ID NO.5 were labeled with biotin.

[0064] Table 1. Mutation site and type:

[0065] Mutation

[0066] The amplified sequence is shown in Table 2:

[0067] Table 2. Specific amplification primers and primer sequences

[0068]

[0069] 2. Selection of reference substance

[0070] A synthetic oligonucleotide chain TAYGGTTTGCAcontrololigo was used as the quality control substance; DNase / RNase-Free water was used as the blank control substance.

[0071] 3. Composition of PCR reaction solution

[0072] Table 3. Composition of PCR...

Embodiment 2

[0078] Embodiment 2: the use of kit

[0079] 1. Sample testing

[0080] Dissolve the dry powder of the primers (the validity period of the primers is 1 month after dissolution). Prepare the system according to the number of templates: take the PCR reaction solution, add dissolved primers, uracil DNA glycosylase, and TaqDNA polymerase, 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.

[0081] The main components of CYP2C9*2 and CYP2C9*3 systems are as follows:

[0082] Table 5. Main components of CYP2C9*2 system

[0083]

[0084] Table 6. Main components of CYP2C9*3 system

[0085]

[0086]

[0087] The system reaction procedure is as follows:

[0088] Table 7. PCR reaction program

[0089]

[0090] After the amplification is completed, check the PCR results on agarose gel to proceed to the next step.

[00...

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Abstract

The invention relates to a primer pair for detecting CYP2C9 genetic typing through a pyrosequencing method and a kit, and belongs to the technical field of in vitro nucleic acid detection. The primer pair comprises a CYP2C9*2 forward amplification primer, a CYP2C9*2 reverse amplification primer, a CYP2C9*2 sequencing primer, a CYP2C9*3 forward amplification primer, a CYP2C9*3 reverse amplification primer and a CYP2C9*3 sequencing primer. Biotin labeling is conducted at the 5' end of the CYP2C9*2 forward amplification primer and the 5' end of the CYP2C9*3 reverse amplification primer. The kit comprises the amplification primers, a PCR reaction solution 1, a PCR reaction solution 2, the sequencing primers, uracil DNA glycosylase and Taq polymerase. The kit has the advantages of being accurate in detection result, high in specificity, short in detection period, easy to operate and capable of effectively meeting the clinical examination requirement.

Description

technical field [0001] The invention relates to the technical field of in vitro nucleic acid detection, in particular to a pair of primers and a kit for detecting CYP2C9 genotyping by pyrosequencing. Background technique [0002] Cytochrome P4502C9 (CytochromeP4502C9, CYP2C9) accounts for 20% of the total amount of cytochrome P450 in liver microsomal, 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. [0003] The CYP2C9 gene has a high degree of genetic polymorphism, and its most common gene mutation sites are CYP2C9*2 (rs1799853) and CYP209*3 (rs1057910), and the enzyme activities encoded by them are 30% and 80% lower than wild-type CYP2C9*1, respectively. %, affecting the metabolism of its substrate drugs in the body, resulting in individual differences in drug efficacy and toxicity, resulting in a lower dose of warfarin for individuals with CYP2C9 gene mutations. Ind...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 滕祥云黄少亚
Owner CHANGSHA 3G BIOTECH
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