Kit for detecting genotype of mitochondrion locus 3243 and method

A mitochondrial and genotyping technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problem that the purpose of primer detection cannot be judged, and achieve the effect of simple operation and high sensitivity

Inactive Publication Date: 2020-12-22
THE FIRST AFFILIATED HOSPITAL OF ANHUI MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And the judgment of the result depends on the result of digestion with endonuclease Apa Ⅰ, and DNA sequencing is not carried out for final judgment. Therefore, it is impossible to judge whether the primers using this method achieve the purpose of detection

Method used

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  • Kit for detecting genotype of mitochondrion locus 3243 and method
  • Kit for detecting genotype of mitochondrion locus 3243 and method
  • Kit for detecting genotype of mitochondrion locus 3243 and method

Examples

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Effect test

Embodiment 1

[0036] Example 1: A kit and method for detecting the genotype of the mitochondrial 3243 site

[0037] 1. Embryo biopsy procedure

[0038] 2. Preparation of biopsy solution and operating dish

[0039] Biopsy fluid: polar body (fertilization medium), cleavage stage embryo (cleavage medium), blastocyst (blastocyst medium).

[0040] Biopsy operating dish: Falcon351006 dish, with the patient's name marked on the bottom of the dish. Make three 10 microliter biopsy drops and one 10 microliter PVP drop, seal in oil, and place at 37°C, 5% CO 2 , 5%O 2 , 90%N 2 The incubator is spare.

[0041] Embryo culture dish after biopsy: Nunclon108173 dish, use balanced culture medium (polar body-fertilization medium, cleavage embryo-cleavage fluid, blastocyst-blastocyst fluid) to make several (according to the number of living embryos) 35ul Culture the drops, seal the oil, and put them in the incubator for later use.

[0042] 3. Embryo Biopsy Operation Steps

[0043] Turn on the switches ...

Embodiment 2

[0074] Example 2: A kit and method for detecting the genotype of the mitochondrial 3243 site

[0075] A kit for detecting the genotype of the mitochondrial 3243 site, comprising PCR primers and Taqman-MGB probes for amplifying the 3243 site of the mitochondrial ATP6 gene;

[0076] The primers include: upstream primer F: 5'-CCACACCCACCCAAGAACAG-3';

[0077] Downstream primer R: 5′-GGGTATGTTGTTAAGAAGAGGAATTGA-3;

[0078] The probes include:

[0079] Taqman-MGB probe PA: 5′-FAM-TTTGTTAAGATGGCAGAGC-MGB-3′;

[0080] Taqman-MGB probe PG: 5'VIC-TTGTTAAGATGGCAGGGC-MGB-3'.

[0081] A preferred embodiment is: it also includes a PCR master mix containing PCR buffer, Taq DNA polymerase, MgCl 2 and dNTPs. PCR buffer, TaqDNA polymerase, dNTPs and MgCl 2 Purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., the PCR buffer is 10xPCR buffer, the content of TaqDNA polymerase is 5U / μl, the content of dNTPs is 2.5mM, MgCl 2 Content 1.5mM.

[0082] A preferred embodiment is: a...

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Abstract

The invention relates to a kit for detecting a genotype of a mitochondrion locus 3243. The kit comprises a PCR primer and a probe Taqman-MGB for amplifying a locus 3243 of a mitochondrion gene ATP6. Afluorescent quantitative PCR method is one of detecting means most extensively applied in the field of molecular diagnosis and is high in sensitivity and simple and convenient in operation, and the kit is applicable to large-scale clinical application. A furrow conjugate on a probe MGB can increase a Tm value of the probe, the probe MGB is designed to be shorter than an ordinary probe Taqman, andthe kit is free of background fluorescence, has the capacity for being capable of distinguishing base difference and is very applicable to SNP typing detection.

Description

technical field [0001] The invention relates to a kit and a detection method for detecting the genotype of the 3243 point of mitochondria, belonging to the technical field of gene detection. Background technique [0002] Leigh syndrome (LS) is the most common mitochondrial disorder in infancy and childhood, characterized by bilateral symmetrical necrotic lesions in the brainstem, basal ganglia, thalamus, and spinal cord. Inheritance of LS includes autosomal recessive inheritance, X-linked recessive inheritance, and maternal inheritance, because the mitochondrial oxidative phosphorylase composition is encoded by both the nuclear and mitochondrial genomes. According to foreign research data, mitochondrial genome mutations account for about 10% of the etiology of Leigh syndrome. Among them, point mutations are the most common, and very few are caused by insertions and large deletions. So far, 19 mitochondrial gene mutations related to Leigh syndrome have been reported. Among ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883
CPCC12Q1/6883C12Q2600/156
Inventor 纪冬梅曹云霞宗凯邹薇薇盛旋李云飞陈逸青刘雅静王喆邓晓红章志国魏兆莲周平余晓峰
Owner THE FIRST AFFILIATED HOSPITAL OF ANHUI MEDICAL UNIV
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