Kid and method for detecting coagulation factor V gene prothrombin and application of kit

A technology of gene polymorphism and blood coagulation factor, which is applied in the field of R506Q polymorphism site genotype kit and blood coagulation factor V gene polymorphism detection kit, can solve the problem that APTT cannot be extended, and achieve clinical guidance Medication, reduction of medical costs, high efficiency effect

Inactive Publication Date: 2009-12-02
BEIJING HUAANFO BIOMEDICAL RES CENT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Arg506→Gln leads to prolongation of APC-inactivated FV, a result that makes FV resistant to exogenous APC and prevents APTT from prolonging

Method used

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  • Kid and method for detecting coagulation factor V gene prothrombin and application of kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The present invention will be further described in detail below by taking the R506Q positive control plasmid at position 506 of the blood coagulation factor V gene as an example.

[0055] 1. Primers and templates

[0056] Gene fragment amplification uses the following primers:

[0057] The forward primer is: 5'-TGCCCAGTGCTTAACAAGACCA-3';

[0058] The reverse primer is: 5'-CTTGAAGGAAATGCCCCATTA-3'.

[0059] The template uses human genomic DNA (No. AI011611, concentration 15ng / μl)

[0060] 2. Gene fragment amplification

[0061] To configure the PCR amplification mixture:

[0062] Template (1515ng / μl): 3μl;

[0063] 10×PCRbuffer(-MgCl 2 ): 1 μl;

[0064] 50mM MgCl 2 : 0.3 μl;

[0065] 10mM dNTPs: 0.2μl;

[0066] 20 μM primer mix: 0.2 μl;

[0067] 5U / μl Taq enzyme: 0.05μl;

[0068] Sterile double distilled water: 5.25 μl;

[0069] Total: 10 μl.

[0070] The PCR amplification conditions are:

[0071] Pre-denaturation at 94°C for 5 minutes; denaturation at 94°...

Embodiment 2

[0102]The present invention will be further described in detail below by taking the prothrombin gene polymorphism genotype detection process of individuals with familial thrombosis tendency as an example.

[0103] We used this kit to detect the prothrombin genotypes of 10 individuals with familial thrombosis tendency. The procedure is as follows:

[0104] 1. Sample processing:

[0105] Since the sample is genomic DNA, use a UV spectrophotometer to detect the DNA concentration of the sample, and dilute the DNA concentration to 30-100ng / ml

[0106] 2. Configure the configuration of the specific PCR amplification mixture of coagulation factor V gene

[0107] Configure gene amplification mixture: PCR reaction mixture: 2.5ul;

[0108] Specific primer + probe: 1ul;

[0109] H2O: 6.5ul;

[0110] Total: 10ul.

[0111] 3. Polymerase chain reaction (PCR) amplification of specific coagulation factor V gene fragments:

[01...

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Abstract

The invention relates to specific primer and probe adopting a PCR method to detect coagulation factor V R506Q gene prothrombin, application of the specific primer and probe in preparing a reagent which detects coagulation factor V R506Q gene polymorphism through PCR amplified nucleic acid samples, the kit which detects coagulation factor V R506Q gene polymorphism and the application of the kit. The invention belongs to the pharmaceutical field.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a detection kit, method and application of coagulation factor V gene polymorphism. More specifically, the present invention relates to a kit, method and application for analyzing the genotype of the R506Q polymorphic site of the gene encoding blood coagulation factor V by polymerase chain reaction method. Background technique [0002] Congenital thrombotic disease, also known as hereditary thrombosis tendency, is a type of disease caused by a specific disorder in a certain link of the coagulation, anticoagulation and fibrinolytic system. The main clinical manifestation is recurrent venous thrombosis, most of which are congenital. Hereditary, often presents in adolescence. According to statistics in some parts of Western Europe, the incidence rate is above 20 / 100,000, surpassing congenital bleeding disorders. [0003] The hypercoagulable state and thrombosis secondary to some diseases...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 张彦明王燕于多徐希平
Owner BEIJING HUAANFO BIOMEDICAL RES CENT
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