Method for detecting 20 mutation sites of deaf genes

A detection method and mutation site technology, which is applied in biochemical equipment and methods, microbe determination/inspection, etc., can solve the problem of low carrying rate of pathogenic mutation alleles

Inactive Publication Date: 2016-01-20
GUANGZHOU DARUI BIOTECH +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Dai Pu et al. screened 1680 cases of non-syndromic deafness cases in deaf schools in 18 provinces in China, and found that the carrier rate of 235delC allele was the highest, followed by 299-300delAT, 176dell6bp, and other pathogenic deafness. Carriage of the mutant allele is low

Method used

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  • Method for detecting 20 mutation sites of deaf genes
  • Method for detecting 20 mutation sites of deaf genes
  • Method for detecting 20 mutation sites of deaf genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0136] Example 1: Use of the detection method in normal people

[0137] 1. Reagents used in this method: OMEGAE.Z.N.A. TM BloodDNAKit, AgenaComplete GoldGenotypingReagentSet10x384, PrimerP1P2Mix (500uMeach), PrimeP3Mix, absolute ethanol.

[0138] 2. Specimen collection, transportation and preservation:

[0139] (1) Specimen collection: The specimen is whole blood. The blood is routinely taken 5ml of venous blood, treated with EDTA for anticoagulation.

[0140] (2) Storage: It can be tested immediately and stored at 4°C for one week.

[0141] (3) Transportation: 0℃ curling should be used for specimen transportation.

[0142] 3. Detection steps and result analysis:

[0143] (1) Extract genomic DNA from whole blood:

[0144] 1) Transfer ≤250μL of blood to the EP tube. If the blood is less than 250μL, use ElutionBuffer to make up 250μL.

[0145] 2) Add 25μLOB protease and 250μL BufferBL, vortex at high speed for 25s to mix.

[0146] 3) Water bath at 65°C for 10 minutes. During the water bath...

Embodiment 2

[0176] Example 2: Application of this method to detect abnormal GJB2SNP locus

[0177] A blood sample was selected, and the patient was suspected of deafness. The kit of Example 1 was used to extract DNA according to the above-mentioned standard procedure, and then PCR was performed to amplify chromosome-specific SNP sites, and the SAP enzyme treatment was used to make the previous PCR step The dNTP in the dNTP loses its activity, and then undergoes a single-base extension reaction, which is finally detected and analyzed by a mass spectrometer. There is a loss of heterozygosity at this site of GJB2 (GJB2_176-191del16). This method can realize the rapid detection of GJB2SNP locus abnormalities. among them Figure 1B GJB2 (GJB2_176-191del16) abnormal mass spectrum analysis chart.

Embodiment 3

[0178] Example 3: Application of this method to detect abnormal GJB3SNP locus

[0179] A blood sample was selected, and the patient was suspected of deafness. The kit of Example 1 was used to extract DNA according to the above-mentioned standard procedure, and then PCR was performed to amplify chromosome-specific SNP sites, and the SAP enzyme treatment was used to make the previous PCR step The dNTP in the dNTP loses its activity, and then undergoes a single-base extension reaction, which is finally detected and analyzed by a mass spectrometer. There is a heterozygous mutation in this position (rs74315318) of GJB3. This method can realize rapid detection of GJB3SNP site abnormalities. among them Figure 2A GJB3 (rs74315318) abnormal mass spectrometry analysis chart.

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Abstract

The invention relates to a method for detecting 20 hot mutation sites of four deaf genes such as GJB2, SLC26A4, GJB3 and 12S rRNA in a clinical sample, designing amplimers and single base extension primers of different sites aiming at abnormal SNP gene sites of the four genes by a matrix-assistant laser desorption / ionization flight time mass spectrum detection method and then carrying out mass spectrometry of a specific SNP site genotype.

Description

Technical field [0001] The present invention relates to a detection method for detecting 20 hot-spot mutation sites of deafness in clinical samples, in particular to a multiplex PCR technology combined with matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOFMS) technology through mass spectrometry and multi-primer extension The combined use of technology and MassARRAY technology is a detection method for SNP genotype analysis. technical background [0002] Deafness is one of the most common genetic diseases clinically. According to the results of the second national sample survey of persons with disabilities in 2006, there are currently 27.8 million people with hearing and speech disabilities in my country, accounting for 33.5% of the 82.96 million people with disabilities in the country. Among them, 20.04 million people with hearing disabilities, ranking first among all types of disabilities. And it is increasing at a rate of 30,000-100,000 de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2537/143C12Q2535/125C12Q2565/627
Inventor 李明高旭年李粉霞李尔华刘启祥何丹林紫慧曾莉
Owner GUANGZHOU DARUI BIOTECH
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