Primer composition, kit composed of primer composition and application

The technology of a primer composition and a kit is applied in the biological field to achieve the effect of low cost and convenient and quick method

Active Publication Date: 2017-08-18
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 composition, kit composed of primer composition and application
  • Primer composition, kit composed of primer composition and application
  • Primer composition, kit composed of primer composition and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Apply the method of the present invention with reference to the 2011 American Heart Association hypertrophic cardiomyopathy diagnostic guidelines, 1 case of familial hypertrophic cardiomyopathy proband (the patient signed an informed consent, And through the approval of the Hospital Medical Ethics Committee) the peripheral blood genome was screened for mutations by real-time fluorescent PCR to find possible pathogenic variants associated with the pathogenesis of HCM.

[0051] A detection method for hypertrophic cardiomyopathy-related pathogenic gene TAZ, c.622A>G mutation, comprising the following steps:

[0052] (1) Genome extraction: From a proband with clinically confirmed familial hypertrophic cardiomyopathy, 1 ml of peripheral venous blood was drawn, anticoagulated with EDTA, and a commercial small-volume genome extraction kit (Axygen, USA) was used. After extracting its whole genome, after agarose gel electrophoresis, the concentration and OD value were determined...

Embodiment 2

[0066] Apply the method of the present invention with reference to the 2011 American Heart Association hypertrophic cardiomyopathy diagnostic guidelines, 1 case of familial hypertrophic cardiomyopathy proband (the patient signed an informed consent, And through the approval of the Hospital Medical Ethics Committee) the peripheral blood genome was screened for mutations by real-time fluorescent PCR to find possible pathogenic variants associated with the pathogenesis of HCM.

[0067] A method for detecting hotspot mutations of hypertrophic cardiomyopathy-related pathogenic genes, comprising the following steps:

[0068] (1) Genome extraction: From a proband with clinically confirmed familial hypertrophic cardiomyopathy, 1 ml of peripheral venous blood was drawn, anticoagulated with EDTA, and a commercial small-volume genome extraction kit (Axygen, USA) was used. After extracting its whole genome, after agarose gel electrophoresis, the concentration and OD value were determined,...

Embodiment 3

[0084] 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.

[0085] The repeated results between batches and within batches are shown in Table 1-Table 2 respectively:

[0086] Table 1

[0087]

[0088] Table 2

[0089]

[0090]

[0091] It can be seen from Table 1-Table 2 that the inter-assay and intra-assay repetition coefficients of variation are all less than 2%, which has good inter-assay and intra-assay repeatability.

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Abstract

The invention belongs to the technical field of biology, relates to medical molecular diagnosis and biotechnology and relates to a primer composition, a kit composed of the primer composition and application. The primer composition comprises a real-time fluorescence PCR (Polymerase Chain Reaction) primer for detecting a new mutation site c.622 greater than G of a TAZ gene and a Taqman-MGB (Minor Groove Binder) probe sequence. The primer composition can be used for detecting a plurality of case samples and has the characteristics of simplicity, rapidness, accuracy and economical efficiency and the like; a new method is established for clinical early-stage molecular diagnosis and prevention of hypertrophic cardiomyopathy.

Description

technical field [0001] The invention belongs to the field of biotechnology, relates to medical molecular diagnosis and biotechnology, relates to a primer composition and a kit and application thereof, in particular to a primer and a probe for detecting the new mutation c.622A>G of TAZ gene. 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 genetic heart disease whose etiology has been elucidated from a genetic perspective, and is one of the leading causes o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/156C12Q2561/101C12Q2563/107C12Q2545/113
Inventor 夏雪山赵跃冯悦
Owner KUNMING UNIV OF SCI & TECH
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