Kit for detecting genotypes of human mitochondrial ND3 gene at 10191rst locus based on HRM method and method of kit

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 high sensitivity and specificity and low cost

Inactive Publication Date: 2020-09-18
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 ApaI, and DNA sequencing is not performed 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 genotypes of human mitochondrial ND3 gene at 10191rst locus based on HRM method and method of kit
  • Kit for detecting genotypes of human mitochondrial ND3 gene at 10191rst locus based on HRM method and method of kit
  • Kit for detecting genotypes of human mitochondrial ND3 gene at 10191rst locus based on HRM method and method of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: A kit and method for detecting the genotype of the mitochondrial 8993 site

[0031] 1. Embryo biopsy procedure

[0032] Biopsy fluid and operation dish preparation

[0033] Biopsy fluid: polar body (fertilization medium), cleavage stage embryo (cleavage medium), blastocyst (blastocyst medium)

[0034] Biopsy operating dish: Falcon351006 dish, with the patient's name marked on the bottom of the dish. Make three 10ul biopsy drops and one 10ul PVP drop, seal the oil, and place in a 37°C, 5% CO2, 5% O2, 90% N2 incubator for standby.

[0035] 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.

[0036] Embryo biopsy procedure

[0037] Turn on the switches of the inverted mirror and the operating ...

Embodiment 2

[0065] Example 2: A kit and method for detecting the genotype of the mitochondrial 8993 site

[0066] A kit for detecting the genotype of the 10191 site of the human mitochondrial ND3 gene based on the HRM method, including PCR primers for amplifying the 10191 site of the mitochondrial ND3 gene;

[0067] The PCR primers include: upstream primer F: 5'-AACTCAACGGCTACATAG-3';

[0068] Downstream primer R: 5'-TTTATGGAGAAAGGGACG-3'.

[0069] The preferred technical scheme is: also includes 2×PCR reaction MIX, 1×EvaGreen fluorescent dye, MgCl 2 , positive control substance and sterile water; 2×PCR reaction MIX including Taq DNA polymerase and dNTPs, Taq DNA polymerase and dNTPs were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., Taq DNA polymerase content 5U / μl, dNTPs Content 2.5mM; 1×EvaGreen fluorescent dye concentration is 5μmol / L; MgCl 2 The concentration is 2.5mM; positive control substances include mitochondrial ND3 gene 10191 site TT type plasmid, mitoc...

Embodiment approach

[0078] Preferred embodiment: the fluorescent PCR amplification conditions: the first stage 95°C / 5min, the second stage 95°C / 10sec, 53-60°C / 20sec, 72°C / 30sec, wherein the annealing temperature starts from the first cycle Each cycle increases by 0.5°C, reaches 60°C and maintains until the end of the 40th cycle, the third stage is 95°C / 1min, 40°C / 1min, 65°C / 1sec.

[0079] Preferred embodiment: 2×PCR reaction MIX, 1×EvaGreen fluorescent dye, MgCl 2 , positive control substance and sterile water; 2×PCR reaction MIX including Taq DNA polymerase and dNTPs, Taq DNA polymerase and dNTPs were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., Taq DNA polymerase content 5U / μl, dNTPs Content 2.5mM; 1×EvaGreen fluorescent dye concentration is 5μmol / L; MgCl 2 The concentration is 2.5mM; positive control substances include mitochondrial ND3 gene 10191 site TT-type plasmid, mitochondrial ND3 gene 10191 site CC-type plasmid.

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Abstract

The present invention discloses a kit for detecting genotypes of human mitochondrial ND3 gene at a 10191rst locus based on a HRM method. The kit comprises PCR primers for amplifying the mitochondrialND3 gene at the 10191rst locus; and the PCR primers comprise an upstream primer F: 5'-AACTCAACGGCTACATAG-3'; and a downstream primer R: 5'-TTTATGGAGAAAGGGACG-3'. The HRM technology is not limited by loci of bases or types of the bases, does not need to synthesize expensive probes, only needs to dissolve a reaction product in one step after a reaction is completed, then uses an instrument to analyze a dissolution curve and completes genotyping and scanning of mutation sites.

Description

technical field [0001] The invention relates to a kit and a method for detecting the genotype of the 10191 site of human mitochondrial ND3 gene based on the HRM method, and belongs 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 synd...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6858
CPCC12Q1/6883C12Q1/6858C12Q2600/156
Inventor 纪冬梅邹薇薇曹云霞盛旋李云飞宗凯刘雅静邓晓红陈逸青章志国魏兆莲王喆周平余晓峰
Owner THE FIRST AFFILIATED HOSPITAL OF ANHUI MEDICAL UNIV
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