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Primer pairs and kit for detecting warfarin medication related gene polymorphism

A gene polymorphism and kit technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. and other problems, to achieve the effect of high-throughput sample detection, simple sample processing, and high sensitivity

Inactive Publication Date: 2018-07-13
CHANGSHA 3G BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] In order to solve the technical problems of low accuracy of detection results, complicated operation procedures and low sensitivity in the process of detecting VKORC1 and CYP2C9 gene polymorphisms related to warfarin medication in the prior art, the present invention provides a A primer pair and kit for detecting warfarin-related VKORC1-1639, CYP2C9*2 and CYP2C9*3 gene polymorphisms with simple procedures, high sensitivity, strong specificity, fast sequencing speed, and effectively meeting the requirements of clinical testing; ARMS combined with QPCR technology to achieve

Method used

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  • Primer pairs and kit for detecting warfarin medication related gene polymorphism
  • Primer pairs and kit for detecting warfarin medication related gene polymorphism
  • Primer pairs and kit for detecting warfarin medication related gene polymorphism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1: the preparation of kit (30 tests / box)

[0068] 1. Design and synthesis of primers and probes

[0069] For human VKORC1 gene mutant 1639 and CYP2C9 gene mutants *2 and *3, specific mutation sites were selected, and the selected primers and probes were designed at the VKORC1 mutation site and nearby conserved regions to avoid unwanted occurrence in the primer-binding region. SNP (retrieve the SNP of the target gene sequence through the online NCBI website), and perform Primer Blast 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 the binding region; the amplification primers and fluorescent probes are first purified by PAGE, and then purified by HPLC. The target probes are SEQ ID NO: 2, SEQ ID The 5' ends of NO:6 and SEQ ID NO:10 are labeled with a fluorescent reporter group (FAM), the 5' end of the internal reference probe SEQ ID NO:15 ...

Embodiment 2

[0111] Embodiment 2: the use of kit

[0112] 1. Sample testing

[0113] Prepare the system according to the number of templates: take the PCR reaction solution, add the corresponding mixed solution, blank control substance, and add sample DNA, blank control substance or positive control substance as templates to form a PCR reaction system. Perform PCR amplification according to the PCR reaction procedure.

[0114] The main components of the PCR system are as follows:

[0115] Table 13. Main components of PCR system

[0116]

[0117]

[0118] The system reaction procedure is as follows:

[0119] Table 14. PCR reaction program

[0120]

[0121] 2. ABI7500 fluorescent quantitative PCR

[0122] Press the power button on the right to start ABI 7500. After starting up, the "power" indicator on the left end of the machine is always on. Open the door of the chamber, put the prepared reagents into the chamber, and remember where you put it.

[0123] 1) Double-click the...

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Abstract

The invention relates to primer pairs and a kit for detecting warfarin medication related gene polymorphism, and belongs to the technical field of in vitro nucleic acid detection, wherein the primer pair comprises amplification primers respectively for alleles such as VKORC1-1639, CYP2C9*2 and CYP2C9*3, and GAPDH internal reference gene, and the kit comprises PCR reaction solutions containing theamplification primers. According to the present invention, the kit has high sensitivity and is suitable for the detection of low-content mutation samples, wherein the sensitivity can be up to 1 / 10000,and the minimum detection limit is only 1-2 copies; compared to the sequencing method, the method of the present invention has the following advantages that the detection result can be real-timely observed, the product does not require gel electrophoresis detection, and the completely closed tube operation is performed so as to effectively reduce the risk of PCR product contamination; and the kitfurther has the fast detection speed, and is 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 pair and a kit for detecting warfarin drug-related gene polymorphisms. Background technique [0002] Warfarin is a dicoumarin derivative and is currently one of the most widely used oral anticoagulant drugs in clinical practice for the prevention and treatment of deep vein thrombosis, pulmonary embolism, heart valve replacement and thrombosis caused by atrial fibrillation form. The therapeutic window of warfarin is narrow, and a very small dose may cause adverse reactions, and the same effect can be achieved in different individuals, and the difference between high and low doses can be more than 10 times. [0003] Vitamin K epoxide reductase complex 1 (VKORC1) is a key enzyme in the vitamin K cycle. Warfarin blocks vitamin K from participating in the catalytic reaction of carboxylase in the form of cofactor due to the inhibition of this enzyme, a...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/686C12Q1/6883C12Q2600/106C12Q2600/156C12Q2535/137C12Q2563/107C12Q2545/114
Inventor 滕祥云尹贞毛平道黄少亚
Owner CHANGSHA 3G BIOTECH
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