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Rapid detection kit for gene polymorphism of CYP2C9 and/or VKORC1

A VKORC1G-1639A and kit technology, applied in the field of molecular biology, can solve the problems of increasing cross-contamination, nucleic acid loss, etc., and achieve the effect of avoiding the use of harmful reagents, reducing the sample size, and enhancing the activity of DNA polymerase

Active Publication Date: 2018-03-27
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] One of the objectives of the present invention is to provide a kit that can be used for direct detection of CYP2C9 and / or VKORC1 gene polymorphisms in blood, aiming at solving the problem of performing nucleic acid analysis on blood in advance when performing fluorescent quantitative PCR amplification on blood in the prior art. Extraction and purification treatment, resulting in a large loss of nucleic acid, and increased cross-contamination and the use of toxic reagents, in addition, it also has the advantages of high amplification efficiency, high sensitivity, and good specificity

Method used

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  • Rapid detection kit for gene polymorphism of CYP2C9 and/or VKORC1
  • Rapid detection kit for gene polymorphism of CYP2C9 and/or VKORC1
  • Rapid detection kit for gene polymorphism of CYP2C9 and/or VKORC1

Examples

Experimental program
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Embodiment 1

[0038] The embodiment of the present invention provides a kit for direct detection of CYP2C9 and / or VKORC1 gene polymorphism in blood, comprising the following components:

[0039] Hot start taq DNA polymerase, blood direct PCR buffer (10X), primer pair, probe pair, dNTPs, UNG enzyme.

[0040] The primer pair is a CYP2C9C430T primer, a CYP2C9A1075C primer and a VKORC1G-1639A primer, and the primers are shown in Table 3:

[0041] Table 3 gene primer sequence

[0042]

[0043] The probe pair is CYP2C9C430T probe, CYP2C9A1075C probe and VKORC1G-1639A probe, and the specific probe sequences are shown in Table 4: the 5' of the probe is marked with VIC or FAM, and the 3' of the probe is marked with MGB.

[0044] Table 4 gene probe sequence

[0045]

[0046]

[0047] The blood direct PCR buffer (10X) is: 100mM Tris-HCL, 500mM KCl, 20mM MgCl 2 , 50% glycerin, 25% DMSO, 0.5% Tween-20, pH8.3.

[0048] The method for using the kit comprises the following steps:

[0049] (1) p...

Embodiment 2

[0067] Embodiment 2 kit of the present invention is for the sensitivity of standard substance

[0068] In this embodiment, the CYP2C9 gene C430T site wild type (C / C) is taken as an example, and different concentrations of standard substances are used, respectively 10 7 copies / ul, 10 6 copies / ul, 10 5 copies / ul, 10 4 copies / ul, 10 3 copies / ul for fluorescent quantitative PCR amplification experiments.

[0069] The kit used in this example is the same as the probe and primer for detecting the C430T site of the CYP2C9 gene in Example 1, and other components are also the same.

[0070] The detection method adopted in this embodiment is the same as that in Embodiment 1.

[0071] see results Image 6 As shown, it can be seen from the CYP2C9 gene C430T site wild-type (C / C) standard product amplification curve that different concentrations of standard products (respectively 10 7 copies / ul, 10 6 copies / ul, 10 5 copies / ul, 10 4 copies / ul, 10 3 copies / ul) is a parallel standar...

Embodiment 3

[0072] Example 3 Selection of CYP2C9 and / or VKORC1 gene polymorphism detection specific primer sequences

[0073] Taking the two polymorphic sites of CYP2C9 gene C430T and A1075C as an example, respectively design specific primer sequences, and use the forward or reverse complementary sequence of the target sequence where the mutation site is located as a template to target wild C430T and A1075C respectively. Type and mutant design specific probe sequences, including preferred specific primers and probe sequences in Example 1 of the present invention and 2 alternative specific primers and probe sequences, as shown in Tables 3 and 4. The detection method is as described in Example 1.

[0074] Table 3 Primer Sequence

[0075]

[0076]

[0077] Table 4 specific probe sequence

[0078]

[0079]

[0080] In the research and development stage of the present invention, a series of primers and probes are first screened, and then a set of primers and probes with the stron...

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Abstract

The invention discloses a kit for detecting gene polymorphism of CYP2C9 and / or VKORC1. The kit comprises the following components: hot start taq DNA polymerase, a blood direct PCR buffer (10X), a primer pair, a probe pair, and a UNG enzyme. The kit is simple, high efficient, and safe: the product can be used for directly carrying out fluorescence PCR amplification of blood samples without nucleicacid extraction, purifying and other steps, risks of cross contamination of templates are reduced, and usage of phenol and other harmful reagents are avoided; the kit has high sensitivity: blood samples which are low to 2[mu]l can be accurately detected, and required sample amount is low; the specificity is good: specific primers and parting probes are designed aiming at CYP2C9C430T, CYP2C9A1075Cand VKORC1G-1639A, and corresponding polymorphism can be identified by specific amplification.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a rapid detection kit for CYP2C9 and / or VKORC1 gene polymorphism. Background technique [0002] Warfarin is a new generation of anticoagulant, which can inhibit the synthesis of coagulation factors in liver cells by competitively antagonizing the effect of vitamin K, and reduce the platelet aggregation reaction induced by thrombin, so it has anticoagulant and antiplatelet aggregation functions. [0003] As the first oral anticoagulant, warfarin has been widely used clinically for anticoagulant treatment of various diseases, but the pharmacological effects of warfarin are easily affected by many factors, with large individual differences, narrow therapeutic window, and half effective dose The INR level of the median lethal dose is only about 1 times different, and even a small dose change may cause adverse reactions such as bleeding, so dose ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2531/113C12Q2521/531C12Q2527/125
Inventor 彭璨璨许嘉森吴诗扬刘苏燕刘志明
Owner SUREXAM BIO TECH
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