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Primer combination for detecting TNNI3K gene mutation and application thereof

A technology of primer combination and primer set, which is applied in the field of medical molecular biology, can solve problems such as the detection of dilated cardiomyopathy, and achieve the effect of convenient, quick and low cost

Pending Publication Date: 2020-02-28
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, it has been reported that fluorescent quantitative PPCR technology can be used to detect hotspot gene mutation sites in hypertrophic cardiomyopathy, but there is no report on the detection of dilated cardiomyopathy.

Method used

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  • Primer combination for detecting TNNI3K gene mutation and application thereof
  • Primer combination for detecting TNNI3K gene mutation and application thereof
  • Primer combination for detecting TNNI3K gene mutation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] 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.

[0061] 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.

[0062] B. Use enzyme digestion or physical methods to disrupt the genome and recover the target DNA fr...

Embodiment 2

[0070] Embodiment 2: Detection TNNI3K Fluorescent quantitative PCR method for c.1910C>T mutation in gene

[0071] (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;

[0072] Wherein, the nucleic acid sequence of the primers amplified by PCR is as follows, and the amplified length is 464bp:

[0073] SEQ ID NO:5: 5'-GAGGAACTGAACAGCCACA-3'

[0074] SEQ ID NO:6: 5'-CTTACCTCCCTCTTTTGCCA-3';

[0075] The reaction system is: template DNA 1µL, primer (3.2 pmol / uL) 1µL, PCR enzyme master mix 10µL, ddH 2 O 7 µL.

[0076] The reaction conditions for PCR amplification are as follows:

[0077]

[0078] 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; after t...

Embodiment 3

[0096] Example 3: 16 patients with dilated cardiomyopathy with known (Sanger sequencing) genotypes were tested using the primer set of the present invention TNNI3K , c.1910C>T mutation site for mutation detection

[0097] The genotypes of the 16 patients with dilated cardiomyopathy used in this example were amplified by PCR with reference to the reaction system and conditions in Example 2 by using the primer sets of SEQ ID NO: 5 and SEQ ID NO: 6, 2% agarose After gel electrophoresis to identify fragments of the desired size, it was determined by Sanger sequencing.

[0098] 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 10 cases had CC genot...

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Abstract

The invention discloses a primer combination for detecting TNNI3K gene mutation. The primer combination is characterized by comprising a primer group and a probe set which are used for detecting c.1910C>T mutation in TNNI3K gene. The primer combination has the advantages that the all-exon sequencing technology is used to acquire dilated cardiomyopathy pathogenic gene new mutation, and the fluorescent quantitative PCR technology is used to simply, fast and accurately detect the TNNI3K-c.1910C>T new mutation; multiple case samples can be detected in one step, and a new method is built for the 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 TNNI3K 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] Hereditary cardiomyopathy is a cardiovascular disorder caused by genetic factors and environmental factors. It can be divided into dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM) and restrictive cardiomyopathy from the mentality and function of the ventricles. Cardiomyopathy (RCM) and arrhythmogenic right ventricular cardiomyopathy (ARVC). Hypertrophic cardiomyopathy and dilated cardiomyopathy are the main types of inherited cardiomyopathy. Hypertrophic cardiomyopathy is the leading cause of sudden death in adolescents and young athletes. Its molecular genetics is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6883C12Q1/6858C12Q2600/156C12Q2531/113C12Q2561/101
Inventor 冯悦肖慧夏雪山贾圆圆刘丽赵跃
Owner KUNMING UNIV OF SCI & TECH
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