Method for detecting GJB2 gene mutation by fluorescence quantitative PCR technology, and kit thereof

A kit and gene technology, applied in fluorescence/phosphorescence, material excitation analysis, etc., can solve the problems of polluted environment, complicated operation, unsuitable for popularization, etc.

Inactive Publication Date: 2012-05-23
王宝恒
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Direct sequencing of PCR products is currently the most widely used and more accurate method, but this method requires expensive instruments and professional personnel to operate, time-consuming and laborious, and it is difficult to promote in clinical practice
The gene chip method has the advantages of fast and high throughput, but the operation is complicated, there are many steps, the cost is high and it is not easy to popularize
The method of detecting gene mutation with restriction fragment length polymorphism (RFLP) has the advanta

Method used

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  • Method for detecting GJB2 gene mutation by fluorescence quantitative PCR technology, and kit thereof
  • Method for detecting GJB2 gene mutation by fluorescence quantitative PCR technology, and kit thereof
  • Method for detecting GJB2 gene mutation by fluorescence quantitative PCR technology, and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: GJB2 gene 235delC mutation detection kit and its use

[0045] Composition of the kit

[0046] 1 tube of wild type PCR reaction solution (500ul), 1 tube of mutant PCR reaction solution (500ul), 1 tube of positive quality control product (20ul), 1 tube of negative quality control product (20ul), 1 tube of DNA extraction solution (3ml) , 1 bottle (15ml) of erythrocyte lysate.

[0047] Wild-type PCR reaction solution includes: 1XPCR Buffer, 0.2mM dNTPs, Taq enzyme 25U / ml, 0.2uM primer 1 (SEQ ID NO: 1), 0.2uM primer 3 (SEQ ID NO: 3), 0.2uM fluorescent probe 7 (SEQ ID NO: 7).

[0048] Mutant PCR reaction solution includes: 1XPCR Buffer, 0.2mM dNTPs, Taq enzyme 25U / ml, 0.2uM primer 1 (SEQ ID NO: 1), 0.2uM primer 2 (SEQ ID NO: 2), 0.2uM fluorescent probe 7 (SEQ ID NO: 7).

[0049] Steps:

[0050] 1) Gene extraction: Take 100ul EDTA anticoagulated peripheral blood, add 600ul red blood cell lysate, place at room temperature for 5-10 minutes, mix several times duri...

Embodiment 2

[0056] Example 2: GJB2 gene 235delC mutation detection kit and its use

[0057] Composition of the kit

[0058] 1 tube of wild type PCR reaction solution (500ul), 1 tube of mutant PCR reaction solution (500ul), 1 tube of positive quality control product (20ul), 1 tube of negative quality control product (20ul), 1 tube of DNA extraction solution (3ml) , 1 bottle (15ml) of erythrocyte lysate.

[0059] The wild-type PCR reaction solution includes: 1XPCR Buffer, 0.2mM dNTPs, Taq enzyme 25U / ml, 0.2uM primer 1 (SEQ ID NO: 1), 0.2uM primer 3 (SEQ ID NO: 3), 0.5X SYBR GREEN I.

[0060] The mutant PCR reaction solution includes: 1XPCR Buffer, 0.2mM dNTPs, Taq enzyme 25U / ml, 0.2uM primer 1 (SEQ ID NO: 1), 0.2uM primer 3 (SEQ ID NO: 2), 0.5X SYBR GREENI.

[0061] Operation steps and result analysis: with embodiment 1.

Embodiment 3

[0062] Example 3: GJB2 gene 235delC mutation detection kit and its use

[0063] Composition of the kit

[0064] 1 tube of wild type PCR reaction solution (500ul), 1 tube of mutant PCR reaction solution (500ul), 1 tube of positive quality control product (20ul), 1 tube of negative quality control product (20ul), 1 tube of DNA extraction solution (3ml) , 1 bottle (15ml) of erythrocyte lysate.

[0065] Wild-type PCR reaction solution includes: 1XPCR Buffer, 0.2mM dNTPs, Taq enzyme 25U / ml, 0.2uM primer 4 (SEQ ID NO: 4), 0.2uM primer 6 (SEQ ID NO: 6), 0.2uM fluorescent probe 7 (SEQ ID NO: 7).

[0066] The mutant PCR reaction solution includes: 1XPCR Buffer, 0.2mM dNTPs, Taq enzyme 25U / ml, 0.2uM primer 5 (SEQ ID NO: 5), 0.2uM primer 6 (SEQ ID NO: 6), 0.2uM fluorescent probe 7 (SEQ ID NO: 7).

[0067] Operation steps and result analysis: with embodiment 1.

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PUM

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Abstract

The invention provides a method for detecting 235delC mutation of deafness related gene GJB2 by a fluorescence quantitative PCR technology, and a kit thereof. According to the kit, specific oligonucleotide primers are designed according to the 235delC mutational site of the GJB2 gene; a fluorescent substance is adopted, and a sample requiring detection is detected respectively by a wild type fluorescent PCR system and a mutant type fluorescent PCR system; the Ct values of the two reactions and the different value (DeltaCt) of the two Ct values are compared to judge whether the 235delC mutation of the GJB2 gene exists. With adopting the method and the kit of the present invention, the valuable clinical reference information can be provided for the etiological diagnosis of the deafness patient, and the pregnant couple with the possibility of deafness carriers can be screened so as to provide assistance for prenatal diagnosis of the deafness. The kit of the present invention adopts the special PCR reaction system and the special reaction conditions, and specifically detects the 235delC mutational site of the GJB2 gene, such that the kit has advantages of high throughput and automatic tube closing operation, and is applicable for the clinical laboratories.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and particularly relates to a method for detecting the mutation of deafness-related gene GJB2 235delC by primer-specific real-time quantitative PCR technology and a kit thereof. Background technique [0002] The incidence of deafness is high. It is currently confirmed that about 60% of deafness is caused by genetic factors. Deafness caused by genetic factors, 30% of patients belong to syndromic deafness (SHI), and 70% of patients belong to non-syndromic deafness (NSHI) . Autosomal recessive inheritance accounts for 75%-80% of NSHI, that is, both parents of the child have normal hearing, but they are both positive carriers of the disease-causing gene mutation, mostly sporadic. Currently, it is believed that about 50% of autosomal recessive NSHI is inherited It is caused by GJB2 gene mutation, and there are different hotspot pathogenic sites in people of different races and regions. For Asia...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 王宝恒
Owner 王宝恒
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