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Mitochondrial T3866C detection kit of Leber disease, and application thereof

A T3866C, detection kit technology, applied in the direction of microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of strict PCR amplification conditions, increase of false negatives, etc., and achieve intuitive result interpretation, low cost, and detection process. easy effect

Active Publication Date: 2015-01-28
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method can detect positive sites in a short period of time, the PCR amplification conditions are strict, and the probability of false negatives is greatly increased

Method used

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  • Mitochondrial T3866C detection kit of Leber disease, and application thereof
  • Mitochondrial T3866C detection kit of Leber disease, and application thereof
  • Mitochondrial T3866C detection kit of Leber disease, and application thereof

Examples

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Effect test

Embodiment 1

[0038] Example 1 A kit for detecting mitochondrial DNA T3866C mutation

[0039] see figure 1 , the kit provided by the present invention consists of DNA extraction mixture 1, PCR mixture 2 for amplifying T3866C, outer primer 3 designed for T3866C, inner primer 4 designed for T3866C, restriction endonuclease 5, positive control 6 , negative control 7 and box body 8, DNA extraction mixture 1, PCR mixture 2 for amplifying T3866C, outer primer 3 designed for T3866C, inner primer 4 designed for T3866C, restriction endonuclease 5, positive control 6. The negative control 7 is placed in the box body 8 respectively. Among them, the DNA extraction mixture 1 is mainly composed of cell lysate and solution I (the main component is proteinase K); the PCR mixture 2 for amplifying T3866C is mainly composed of dNTP (deoxymononucleotide), 10×PCR buffer, MgCl 2 , triple distilled water and Taq Enzyme (DNA polymerase) composition, outer primer 3 designed for T3866C has outer forward primer ...

Embodiment 2

[0041] Example 2 Detection of a Family with Primary Leber's Disease Carrying Mitochondrial Gene T3866C Mutation

[0042] 1. Test samples

[0043] A pedigree with primary Leber's disease carrying the T3866C mutation was selected. See his pedigree Figure 4 . This family presents a typical maternal inheritance, and the only clinical symptom is elevated blood pressure in all patients, but the degree of elevated blood pressure of each affected member in the family varies. The total number of members in this family is 58, including 27 members of the maternal line, and 8 persons suffering from Leber's disease.

[0044] 2. Extraction of Genomic DNA

[0045]Samples from 10 subjects were obtained respectively (including the collection of capillaries of III-1, II-2, III-3, III-7 and IV-1, a drop of venous blood filter paper; IV-2 and 3 were hair follicles ), use clean scissors to cut the blood filter paper into pieces of paper about 1cm2 in size, put them into 1.5ml EP tube (Eppen...

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Abstract

The invention provides a mitochondrial T3866C detection kit of Leber disease, and an application thereof. The detection kit is composed of a DNA extraction mixed solution, a PCR mixed solution used for amplifying T3866C, outer primers and inner primers designed aiming at T3866C, a restriction endonuclease Bgl II, a positive control, and a negative control. According to the invention, genomic DNA is rapidly extracted from small amounts of samples such as blood samples, hair with follicle, oral mucosa smears, or saliva. Therefore, a problem of DNA extraction in primary Leber disease patient blood of traditional methods is solved, pain of patient to be detected is reduced, a problem of cross-region sample transferring is solved, enzyme digestion specificity is improved, false negative is avoided, and detection result stability is improved. The kit provided by the invention is rapid, simple, accurate, and economical. With the kit, primary-Leber-disease-related mtDNAT3866C mutation large-scale screening and preventive inspection can be implemented.

Description

technical field [0001] The invention belongs to the field of biological technology, relates to a kit for detecting the mitochondrial DNA T3866C mutation related to primary Leber's disease, and also relates to a detection method of mitochondrial gene mutation related to primary Leber's disease, in particular to Detection of mitochondria ND1 The method for the T3866C mutation, and the application of the above method or the above kit in detecting the mitochondrial DNA T3866C mutation associated with primary Leber's disease. Background technique [0002] Leber's Hereditary Optic Neuropathy (Leber's Hereditary Optic Neuropathy, LHON, referred to as Leber's disease) is a type of optic neuropathy, mainly involving the retina and the anterior part of the sclera cribriform plate macular bundle fibers, leading to maternal genetic disease of optic nerve degeneration, Seriously affect people's normal production and life. According to the latest statistics from the Ministry of Health (...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 蒋萍萍管敏鑫冀延春梁敏张娟娟徐静
Owner ZHEJIANG UNIV
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