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Kit for detecting gene mutation of deafness

A kit and sample technology, applied in the medical field, can solve the problems of cumbersome operation, limited wide application, high cost, etc., and achieve the effect of cumbersome operation

Active Publication Date: 2014-12-17
山东省第二人民医院(山东省耳鼻喉医院、山东省耳鼻喉研究所)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] So far, for the existing known sites, the methods for detecting mutation sites include direct sequencing, fluorescent quantitative PCR, gene chip method, etc. Among them, the direct sequencing method is the gold standard for identifying mutations, but the operation is cumbersome and expensive. Expensive limits its wide application

Method used

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  • Kit for detecting gene mutation of deafness
  • Kit for detecting gene mutation of deafness
  • Kit for detecting gene mutation of deafness

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Extraction of Genomic DNA

[0035] 1) Add 2ml blood sample to 5ml Buffer AP1 and mix vigorously;

[0036] 2) Add 1ml Buffer AP2, mix immediately, and centrifuge at 4630g for 15min;

[0037] 3) Transfer the supernatant to the medium preparation tube, turn on the negative pressure, keep the negative pressure, add 7ml Buffer W1, 8ml Buffer

[0038] W2, 4ml Buffer W2;

[0039] 4) Remove the medium volume tube head and centrifuge at 12000g for 2min;

[0040] 5) Add 0.4ml Elution buffer, let stand at room temperature for 5 minutes, centrifuge at 12000g for 2 minutes, and store at -20°C for later use.

Embodiment 2

[0041] Embodiment 2 PCR amplifies target fragment

[0042] Use specific primers included in the kit

[0043] Upstream primer: 5'-AAGTTTGAGATTACCGTGGGC-3', the nucleotide sequence of which is shown in SEQ ID NO.1;

[0044] Downstream primer: 5'-CGACAGGAATGGGAAGAAAA-3', the nucleotide sequence of which is shown in SEQ ID NO.2;

[0045] The target product is 345bp in length;

[0046] Perform PCR amplification according to the following system and procedures:

[0047] PCR reaction system:

[0048]

[0049] PCR reaction program:

[0050]

[0051] Use 1% agarose gel to detect whether the size of the PCR product is correct, and measure the product concentration. The electrophoresis process is as follows:

[0052] 1) Gel preparation (1% agarose): Weigh 3g of agarose and dissolve it in 100ml 1xTBE solution;

[0053] 2) Sol: take it out after heating in a microwave oven until it boils;

[0054] 3) Cool glue: cool to about 40°C after taking it out;

[0055] 4) Laying glue: ...

Embodiment 3

[0060] Example 3. Enzyme digestion identification

[0061] The PCR product obtained in the previous step was digested with restriction endonuclease AatII, and the system was as follows:

[0062]

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Abstract

The invention relates to the field of medicines and particularly relates to a kit for detecting gene mutation of deafness. The kit comprises specific primers, a PCR mixed liquid, an enzyme digestion reaction mixed liquid and the like, wherein corresponding specific primers are specially designed for the deaf gene. Mutation sites from G to A of a WFS1 gene c.2389 in the body of a patient can be quickly detected by using the kit, so that large-scaled screening and preventive inspection of deafness related gene mutation on a national scale, particularly in underdeveloped areas can be better carried out. Moreover, the kit is low in detection cost, simple to operate and high in accuracy.

Description

technical field [0001] The invention relates to the medical field, in particular to a kit for detecting deafness gene mutations. Background technique [0002] Hearing impairment refers to the organic or functional abnormalities of the nerve centers at all levels of sound transmission, sensory sound and comprehensive analysis of sound in the auditory system, resulting in varying degrees of hearing loss, which is commonly called deafness. Deafness not only affects the quality of life of individuals, but also brings a heavy burden to families and society. It is estimated that as many as 278 million people in the world have hearing impairment, and there are about 27.8 million patients in China, accounting for about 10% of the global patients. There are many causes of hearing loss, such as aging, noise exposure, use of ototoxic drugs, and genetic changes. Studies have found that genetics plays an important role in the mechanism of hearing impairment formation. Tremendous progr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q1/6883C12Q2600/156C12Q2531/113C12Q2521/301
Inventor 王海波白晓卉李建峰樊兆民吕怀庆张凤国徐磊
Owner 山东省第二人民医院(山东省耳鼻喉医院、山东省耳鼻喉研究所)