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Primer combination and applications thereof

A technique of primer composition and reaction, applied in the field of biology, to achieve the effect of convenient and quick method and low cost

Inactive Publication Date: 2017-06-20
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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

Method used

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  • Primer combination and applications thereof
  • Primer combination and applications thereof
  • Primer combination and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Apply the method of the present invention to the peripheral blood of 5 cases of hypertrophic cardiomyopathy probands (except collecting the demographic data of HCM patients, also collecting its electrocardiogram and echocardiogram) clinically confirmed in Department of Cardiovascular Medicine, First People's Hospital of Yunnan Province. Real-time fluorescent PCR mutation screening was performed on the blood genome to find possible pathogenic variants associated with the pathogenesis of HCM.

[0078] A method for detecting hotspot mutations of hypertrophic cardiomyopathy-related pathogenic genes, comprising the following steps:

[0079] (1) Genome extraction: 1 ml of peripheral venous blood was extracted from 5 probands with clinically confirmed hypertrophic cardiomyopathy, anticoagulated with EDTA, and the whole genome was extracted using a commercial Miniprep Kit (Axygen, USA). After agarose gel electrophoresis, the concentration and OD value were determined, OD 260 / 2...

Embodiment 2

[0094] Apply the method of the present invention to the peripheral blood of 5 cases of hypertrophic cardiomyopathy probands (except collecting the demographic data of HCM patients, also collecting its electrocardiogram and echocardiogram) clinically confirmed in Department of Cardiovascular Medicine, First People's Hospital of Yunnan Province. Real-time fluorescent PCR mutation screening was performed on the blood genome to find possible pathogenic variants associated with the pathogenesis of HCM.

[0095] A method for detecting hotspot mutations of hypertrophic cardiomyopathy-related pathogenic genes, comprising the following steps:

[0096] (1) Genome extraction: 1 ml of peripheral venous blood was extracted from 5 probands with clinically confirmed hypertrophic cardiomyopathy, anticoagulated with EDTA, and the whole genome was extracted using a commercial Miniprep Kit (Axygen, USA). After agarose gel electrophoresis, the concentration and OD value were determined, OD 260 / 2...

Embodiment 3

[0109] After condition optimization, the repeatability verification of the primer-probe sequence described in step (4) was carried out 3 times of repeated experiments (inter-batch and intra-batch repetition). Observe the Ct value of the template, and calculate its coefficient of variation, coefficient of variation (p)=standard deviation (SD) / mean (X), test the sensitivity and repeatability of the detection method.

[0110] The results are shown in Table 1-2:

[0111] Table 1

[0112]

[0113]

[0114] Table 2

[0115]

[0116] It can be seen from Table 1-2 that the inter-assay and intra-assay repetition coefficients of variation are both less than 2%, and have good inter-assay and intra-assay repeatability.

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Abstract

The invention relates to the technical field of biology, in particular relates to medical molecular diagnosis and biotechnology, and specifically relates to a primer group and applications of the primer group. A primer combination comprises at least one group of five groups of real-time fluorescent PCR primers capable of detecting five hotspot mutation sites including MYH7-c.1987C>T, TNNI3-c.370G>C, MYH7-c2155C>T, TNNI3-c.433C>G and PRKAG2-c.298G>A, and Taqman-MGB probe sequences. The primer combination can detect a plurality of case samples at one time, also can realize the combined detection of five mutation sites on each sample, and has the features of being simple and convenient, rapid, accurate and economical, and thus a novel method is established for the clinical early molecular diagnosis and prevention of hypertrophic cardiomyopathy.

Description

technical field [0001] The present invention relates to the field of biotechnology, to medical molecular diagnosis and biotechnology, in particular to a primer set and its application, in particular to a primer composition for detecting hotspot mutations of pathogenic genes related to hypertrophic cardiomyopathy and its application . Background technique [0002] Hypertrophic cardiomyopathy (HCM) is the most common hereditary heart disease, mainly manifested as left ventricular or biventricular asymmetric hypertrophy, and less patients show left ventricular outflow tract obstruction. The main pathological features are diffuse hypertrophy, deformity, nuclear enlargement, deep staining and myocardial fiber disorder of cardiomyocytes. The clinical manifestations of HCM range from no symptoms to dyspnea, syncope, chest pain and even sudden cardiac death and fatal arrhythmia. [0003] HCM is the first inherited heart disease whose etiology has been elucidated from a genetic per...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/156C12Q2531/113C12Q2561/101Y02A50/30
Inventor 夏雪山赵跃冯悦
Owner KUNMING UNIV OF SCI & TECH
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