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Kit for simultaneously detecting mutations in mitochondria DNA A1555G and C1494T and using method thereof

A technology of mitochondria and kits, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as troubles, achieve the effects of relieving pain, ensuring specificity and stability, and avoiding false negative results

Inactive Publication Date: 2012-01-04
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This blood collection method has brought some pain and injury to many subjects, especially infants and young children, and there are certain risks and troubles when transferring samples across regions

Method used

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  • Kit for simultaneously detecting mutations in mitochondria DNA A1555G and C1494T and using method thereof
  • Kit for simultaneously detecting mutations in mitochondria DNA A1555G and C1494T and using method thereof
  • Kit for simultaneously detecting mutations in mitochondria DNA A1555G and C1494T and using method thereof

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Experimental program
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Effect test

Embodiment 1

[0040] 1. Primer Design

[0041] Use Primer 5.0 software and Oligo7.0 software to assist in the design of improved primers, according to the published Human Mitochondrial DNA Cambridge Reference Sequence (Human Mitochondrial DNA Revised Cambridge Reference Sequence, GenBank accession number: NC_012920.1) or SEQ ID NO: 7 in the sequence listing To design, the design scheme of the primers is as follows:

[0042] (1) For the mtDNA 1494 site, we designed two downstream primers N1 and M1 with the same length and 3′ ends corresponding to the wild-type G at the 1494 site and the mutant A at the 1494 site. A mismatch was introduced at the fourth base at the 3' end of the primer, and a phosphorothioate modification was introduced between the first and second bases at the 3' end to enhance specificity. The wild-type reverse primer N1 at position 1494 thus designed is mtDNA nt1494-nt1516, namely 5′-CCT TTG AAG TAT ACT TGA GAA G a G-3' (a is the phosphorothioate modification position), ...

Embodiment 2

[0073] 1. In vitro detection of maternally inherited drug-induced deafness mitochondrial DNA A1555G and C1494T mutation kit (100 parts) contains the same components as in Example 1.

[0074] 2. Detection object

[0075] 15 specimens of sporadic deafness patients without genetic association were selected, and the hair with follicles of these 15 subjects were obtained respectively.

[0076] 3. Detection method

[0077] Before extracting the genomic DNA of hair follicle cells, the hair needs to be pretreated accordingly: wash the hair (with hair follicles) once with 70% ethanol, and then rinse the hair twice with distilled water; put 2~ 4 hairs, the hair follicles are placed at the bottom of the EP tube, and the part of the hair higher than the test tube is cut off with clean scissors. Add 600 μl of cell lysate and 3 μl of solution I to a 1.5ml EP tube containing hair (with hair follicles), mix well, digest and lyse at a constant temperature of 55°C for 2-3 hours; / L potassium...

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Abstract

The invention provides a kit for simultaneously detecting mutations in mitochondria DNA A1555G and C1494T related to maternally inherited drug-induced deafness and a using method thereof. The kit comprises a reagent for extracting sample genomic DNA, a PCR amplification reactive reagent, a primer mixed liquor, a positive control template and a negative control template. The using method of the kit mainly comprises the following steps: using blood, hair with follicle, oral mucosa doctor blade, saliva, and the like, as a sample; adopting a proteinase K digestion pyrolysis method to extract genomic DNA; and then simultaneously detecting mutations in A1555G and C1494T by multiple allele specific PCR. The kit is used for detecting the mutations in mitochondria DNA A1555G and C1494T related to maternally inherited drug-induced deafness and is more rapid, economical and simpler than the single detection for mutation in A1555G or C1494T, and the kit has low requirements for equipment and environment and is conducive to promotion and application.

Description

technical field [0001] The invention relates to a kit for simultaneously detecting mitochondrial DNA A1555G and C1494T mutations related to maternally inherited drug-induced deafness and a use method thereof. Background technique [0002] Mitochondrial DNA (Mitochondria DNA, mtDNA) mutation is one of the important causes of sensorineural deafness, and these mutations are mainly located in mitochondrial 12S rRNA and tRNA genes. The homogeneous A1555G and C1494T mutations located in the 12S rRNA gene are associated with non-syndromic deafness caused by aminoglycoside antibiotics (Aminoglycoside Antibiotics, AmAn) (Prezant TR et al., Nat Genet, 1993, 4:259-294; Guan MX, Ann N Y Acad Sci, 2004, 1011: 259-271; Fischel Ghodsian, Pharmacogenomics, 2005, 6: 27-36). Although the mtDNA A1555G or C1494T mutation itself can cause deafness, the vast majority of A1555G or C1494T mutation carriers are treated with AmAn (such as streptomycin, gentamicin, kanamycin, tobramycin, gnomecin, et...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 管敏鑫吕建新李智渊朱翌杨爱芬郑静唐霄雯王金丹
Owner WENZHOU MEDICAL UNIV
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