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Primer combination for detecting VCL gene mutation and application thereof

A primer combination and primer set technology are applied in the field of medical molecular biology to achieve the effects of low cost and convenient and quick method.

Pending Publication Date: 2020-03-06
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the MGB probe has not been used for the detection of DCM at present.

Method used

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  • Primer combination for detecting VCL gene mutation and application thereof
  • Primer combination for detecting VCL gene mutation and application thereof
  • Primer combination for detecting VCL gene mutation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Application of whole exome sequencing technology to 12 probands with dilated cardiomyopathy who were clinically diagnosed in the Department of Cardiovascular Medicine, First People's Hospital of Yunnan Province (in addition to collecting the demographic data of DCM patients, other data were also collected. Electrocardiogram and echocardiogram) were used to screen the whole exon of the human genome for mutations to find possible pathogenic variants associated with the pathogenesis of hereditary DCM.

[0063] A. Genome extraction: For probands with clinically confirmed dilated cardiomyopathy, 1mL of peripheral venous blood was drawn, anticoagulated with EDTA, and the whole genome was extracted with a commercial Miniprep Kit (Axygen, USA), and agarose After gel electrophoresis, the concentration and OD value are measured, and the OD260 / 280 is between 1.8-2.0.

[0064] B. Use enzyme digestion or physical methods to disrupt the genome and recover the target DNA fr...

Embodiment 2

[0072] Example 2: Detection VCL Real-time quantitative PCR method for c.439G>A mutation in gene

[0073] (1) Using the genome in Step A of Example 1 as a template, perform PCR amplification to construct wild-type, homozygous mutation and heterozygous mutation positive quality controls;

[0074] Wherein, the nucleic acid sequence of the primers amplified by PCR is as follows, and the amplified length is 256bp:

[0075] SEQ ID NO:1 5'-TAGAATGAAAATATGTTGAATGCTGCACATC-3'

[0076] SEQ ID NO:2 5'-GGCTCCAACATGGACAATCCTTA-3'

[0077] The reaction system is: template DNA 1µL, primer (3.2 pmol / uL) 1µL, PCR enzyme master mix 10µL, ddH 2 O 7 µL.

[0078] The reaction conditions for PCR amplification are as follows:

[0079]

[0080] After the PCR amplification reaction was completed, agarose gel electrophoresis was performed, and the electrophoresis results were as follows: Figure 4 As shown, the size of the target fragment is consistent with the expected size and purified; afte...

Embodiment 3

[0098] Example 3: 16 patients with dilated cardiomyopathy from known (Sanger sequencing) genotypes were tested using the primer set of the present invention VCL , c. 439G>A mutation site for mutation detection

[0099] The genotypes of the 16 patients with dilated cardiomyopathy used in this example were PCR amplified with reference to the reaction system and conditions in Example 2 by using the primer sets of SEQ ID NO: 1 and SEQ ID NO: 2, 2% agarose After gel electrophoresis to identify fragments of the desired size, it was determined by Sanger sequencing.

[0100] Using the genomes of the whole blood samples of the above 16 patients as a template, the primer set of the present invention was used for real-time fluorescent quantitative PCR detection. For the PCR amplification conditions and system, see step (2) of Example 2, and the results are shown in Figure 15 ; Figure 15 The genotyping scatter plot for detection of variant sites showed that there were 10 cases of GG g...

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Abstract

The invention discloses a primer combination for detecting VCL gene mutation. The primer combination comprises a primer group and a probe group for detecting c.439G>A mutation in a VCL gene. Accordingto the invention, new mutation of a pathogenic gene of dilated cardiomyopathy is obtained by using a whole exome sequencing technology, and new mutation of VCL-c.439G>A is simply, conveniently, quickly and accurately detected by a fluorescent quantitation PCR technology. The primer combination can be used to test a plurality of case samples at one time, and a new method is established for clinical early molecular diagnosis and prevention of dilated cardiomyopathy.

Description

technical field [0001] The invention belongs to the field of medical molecular biology, relates to medical molecular diagnosis and biotechnology, in particular to a method for detecting VCL A primer composition for gene mutation, and the application of the primer combination in assisting clinical diagnosis, prevention and treatment of dilated cardiomyopathy (DCM). Background technique [0002] Dilated cardiomyopathy (DCM) is a common genetic heart disease, which is the main cause of heart failure and heart transplantation. The pathological feature of dilated cardiomyopathy is the enlargement of left ventricle or double ventricle, which leads to cardiac systolic dysfunction. Its greatest feature is clinical genetic heterogeneity, which is mainly caused by gene mutations. The first pathogenic variant was discovered in 1990. According to reports from Olmstead County, USA from 1975 to 1984, the incidence of dilated cardiomyopathy is about 1 / 2700, more relevant evidence shows th...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6883C12Q1/6858C12Q2600/156C12Q2531/113C12Q2561/101
Inventor 夏雪山陈一波冯悦贾圆圆刘丽赵跃
Owner KUNMING UNIV OF SCI & TECH
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