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Mitochondrial t12338c detection kit and application of leber disease

A technology of T12338C and detection kit, which is applied in the mitochondrial T12338C detection kit and application field of Leber disease, can solve the problems of increased false negatives, strict PCR amplification conditions, etc., and achieve low cost, intuitive result interpretation, and simple detection process Effect

Active Publication Date: 2015-10-07
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 t12338c detection kit and application of leber disease
  • Mitochondrial t12338c detection kit and application of leber disease
  • Mitochondrial t12338c detection kit and application of leber disease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 A detection kit for mitochondrial T12338C of Leber's disease

[0039] see figure 1 , the kit provided by the present invention consists of a DNA extraction mixture 1 placed in a box body 8, a PCR mixture 2 for amplifying T12338C, a pair of outer primers 3 designed for T12338C, a pair of inner primers 4 designed for T12338C, restriction endonuclease Psi Ⅰ5. It is composed of positive control 6 and negative control 7. Among them, DNA extraction mixture 1 is mainly composed of cell lysate and solution I (the main component is proteinase K); PCR mixture 2 reagents for amplifying T12338C include dNTP (deoxygenated mononucleotide), 10×PCR buffer, MgCl 2 , triple distilled water and Taq Enzyme (DNA polymerase), a pair of outer primers 3 designed for T12338C include outer forward primer F: TAT CAC TCT CCT ACT TAC AG (SEQ ID NO: 1), outer reverse primer R: AGA AGG ATA TAA TTC CTA CG (SEQ ID NO:2); A pair of inner primers 4 designed for T12338C include inner forw...

Embodiment 2

[0041] Example 2 Detection of a Family with Primary Leber's Disease Carrying Mitochondrial Gene T12338C Mutation

[0042] 1. Test samples

[0043] A pedigree with primary Leber's disease carrying the T12338C mutation was selected. See his pedigree Figure 4 . This family showed maternal inheritance, and all patients had visual impairment as the only clinical symptom, but the degree of visual impairment of the maternal members of the family varied. The total number of members in this family is 18, including 12 members of the maternal line, and 2 persons suffering from Leber's disease.

[0044] 2. Extraction of Genomic DNA

[0045]Samples from 4 subjects were obtained respectively (including the collection of capillaries of II-9, II-10 and a drop of venous blood filter paper from normal persons outside the family; III-5 was the collection of hair with hair follicles due to younger age), and the blood Cut the filter paper into pieces of about 1cm2 with clean scissors, put t...

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Abstract

The invention provides a mitochondrial T12338C 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 T12338C, a pair of outer primers and a pair of inner primers designed aiming at T12338C, a restriction endonuclease Psi I, 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 mtDNAT12338C mutation screening and preventive inspection can be implemented.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to mitochondrial DNA used for detecting Leber's disease ND5 The test kit for the T12338C mutation also relates to a method for detecting mitochondrial gene mutations associated with primary Leber's disease, in particular to the detection of mitochondria ND5 The method for the T12338C mutation, and the application of the above method or the above kit in detecting the mitochondrial DNA T12338C 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 maternal hereditary optic neuropathy, mainly involving the retina, the anterior part of the scleral cribriform plate macular bundle fibers, leading to maternal genetic disease of optic nerve degeneration , Seriously affecting people's normal production and life. According to the latest statistics ...

Claims

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Application Information

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