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Primer and kit for detecting CYP2C9 and VKORC1 gene polymorphism and PCR method of primer and kit

A gene polymorphism and kit technology, applied in biochemical equipment and methods, microbial measurement/testing, DNA/RNA fragments, etc., can solve the problem of inability to detect clinical specimens on a large scale at the same time, high price of testing instruments, and clinical popularization Low degree of problems, to avoid site mismatch, fast detection speed, good sensitivity

Active Publication Date: 2015-10-07
沈阳优吉诺生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0019] In view of this, the present invention provides a primer, kit and PCR method for detecting CYP2C9 and VKORC1 gene polymorphisms, to at least solve the complex interpretation of results, high price of detection instruments, difficult operation, and certain problems existing in previous kits. False negatives and false positives, high cost of testing, low clinical popularity, inability to test clinical specimens on a large scale at the same time, etc. One or more problems

Method used

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  • Primer and kit for detecting CYP2C9 and VKORC1 gene polymorphism and PCR method of primer and kit
  • Primer and kit for detecting CYP2C9 and VKORC1 gene polymorphism and PCR method of primer and kit
  • Primer and kit for detecting CYP2C9 and VKORC1 gene polymorphism and PCR method of primer and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0128] Example 1: Preparation of wild type and mutant positive plasmids for detection of CYP2C9 gene rs1057910 polymorphism

[0129] First, we call out the gene sequence before and after the rs1057910 polymorphic site of the CYP2C9 gene from the gene bank, and mark the polymorphic site with a double underline, at the appropriate position upstream and downstream of the rs1057910 site of the CYP2C9 gene (in bold and underlined marked), design a pair of cloning primers, the amplified fragment is 269bp, including the mutation site, the gene sequence is shown below, SEQ No.1:

[0130] catgattcatatacccctgaattgctacaacaaatgtgccatttttctccttttccatcagtttttacttgtgtcttatcagctaaagtcca ggaagagattgaacgtgtgattg gcagaaaccggagcccctgcatgcaagacaggagccacatgccctacacagatgctgtggtgcacgaggtccagagatac a ttgaccttctccccaccagcctgccccatgcagtgacctgtgacattaaattcagaaactatctcattcccaaggtaagtttgtttctcctacactgcaactccatgttttcgaagtccccaaattcatagtatcatttttaa acctctaccatcaccgggtgag agaagtgcataactcatatgtatggca...

Embodiment 2

[0146] Example 2: Design and specificity screening of CYP2C9 gene rs1057910 locus allele-specific primers (ASP)

[0147] For the CYP2C9 gene rs1057910, wild-type and a series of mutant-specific primers were designed as follows:

[0148] CYP2C9 gene rs1057910-WT-F: ggtgcacgaggtccagagattca (SEQ No.8)

[0149] CYP2C9 gene rs1057910-mut-F: ggtgcacgaggtccagagattcc (SEQ No.9)

[0150]CYP2C9 gene rs1057910-mut-F1: gtgcacgaggtccagagattcc (SEQ No. 10)

[0151] CYP2C9 gene rs1057910-mut-F2: tgcacgaggtccagagattcc (SEQ No. 11)

[0152] CYP2C9 gene rs1057910-mut-F3:ggtgcacgaggtccagagatagc (SEQ No.12)

[0153] CYP2C9 gene rs1057910-mut-F4: gtgcacgaggtccagagatagc (SEQ No. 13)

[0154] CYP2C9 gene rs1057910-mut-F5: tgcacgaggtccagagatagc (SEQ No. 14)

[0155] Simultaneously design and synthesize the first probe specific to CYP2C9 gene Taqman:

[0156] SEQ No. 15: FAM-ccaccagcctgccccatgcagtg-BHQ1.

[0157] Relevant primers and probes were synthesized at Sangon Bioengineering (Shanghai) C...

Embodiment 3

[0160] Example 3: Sensitivity screening of ASP at rs1057910 site of CYP2C9 gene

[0161] Then use mutant primers to pair with the first downstream primer SEQ No.16 respectively, and use mutant recombinant plasmids according to 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10, 0 for serial dilution, plus Taqman-specific probes, sensitivity verification was performed on a fluorescent quantitative PCR instrument. The mutation-specific primer can detect 100 copies of the mutant, so this primer is the best primer for detecting the rs1057910 site of the CYP2C9 gene screened according to our method, as shown in Table 3.

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Abstract

The invention discloses a primer and a kit for detecting CYP2C9 and VKORC1 gene polymorphism and a PCR method of the primer and the kit. A detection CYP2C9 gene comprises a wild type specificity upstream primer, a mutant type specificity upstream primer and a first sharing downstream primer; the wild type specificity upstream primer has a SEQ No.17 sequence, the mutant type specificity upstream primer has a SEQ No.14 sequence, and the first sharing downstream primer has a SEQ No.16 sequence; a detection VKORC1 gene comprises a wild type specificity upstream primer, a mutant type specificity upstream primer and a second sharing downstream primer; and the wild type specificity upstream primer has a SEQ No.38 sequence, the mutant type specificity upstream primer has a SEQ No.35 sequence, and the second sharing downstream primer has a SEQ No.37 sequence. The kit has the beneficial effects of being simple, rapid and accurate in detection, low in cost and the like.

Description

Technical field [0001] The invention involves the field of molecular biology gene testing. It specially provides a fast detection of CYP2C9 and VKORC1 gene polymorphism, kit and its PCR method for CYP2C9 gene RS1057910 and VKORC1 gene RS9923231 polymorphic sites. Background technique [0002] Huafalin is currently the most commonly used long -term oral anticoagulant drug in domestic and foreign. It is also the only vitamin K antagonist that is currently used in clinical practice.Artificial vascular transplantation and other postoperative anticoagulant therapy, as an important oral anticoagulant drug, has been used in clinical clinic for nearly 60 years.However, there are large differences in the effective treatment range of Huafalin and the dosage of different individuals. In addition to clinical factors such as the age, gender, weight, body surface area, nutritional state, liver and kidney function, diet, and drug use of patients with patientsIn addition to the influence, Hua Fa...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2531/113C12Q2535/125C12Q2561/101
Inventor 高劲松张英杰魏欣芳李星颐刘晓莹魏潇魏奇
Owner 沈阳优吉诺生物科技有限公司
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