High-specificity kit for detecting deafness predisposing genes

A deafness susceptibility gene and detection kit technology, which can be used in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of many false negatives and false positives, difficult interpretation of results, and complicated operations, so as to prevent false positives. The effect of positive and false negative, increasing Tm value, shortening primer length

Inactive Publication Date: 2012-07-04
万戈江
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have different defects, such as cumbersome operation, difficult interpretation of results, poor repeatability, many false negatives and false positives, etc.

Method used

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  • High-specificity kit for detecting deafness predisposing genes
  • High-specificity kit for detecting deafness predisposing genes
  • High-specificity kit for detecting deafness predisposing genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] The kit of the present invention detects mutations and DNA samples of normal individuals. The primers used for the detection of mutation hotspots 2168A>G, IVS7-2A>G, 1174A>T, 1226G>A, 1975G>C, 2027T>A of the deaf susceptibility gene SLC26A4 were labeled with blue fluorescent dyes, GJB2 176del16bp, 235delC, The primers for 299delAT detection were labeled with green fluorescent dye, the primers for Amelogenin locus and 12S rRNA 1494C>T, 1555A>G mutation detection were labeled with yellow fluorescent dye, and the internal standard was labeled with red fluorescent dye.

[0070] 1. The 1,000 samples to be tested have all been sequenced and detected by using the technical method of "DNA extraction-PCR amplification-sequencing". Among them, 10 samples of IVS7-2A>G mutation, 10 samples of GJB2 235delC mutation, 10 samples of 12SrRNA 1555A>G mutation, 1 sample of 1494C>T mutation, 1 sample of 2168A>G mutation, 1 sample of 1174A>T mutation 1 sample of 1226G>A mutation, 1 sample ...

Embodiment 2

[0096] The unmodified primers were used to detect DNA samples from normal individual blood spots at the same concentration (5.0ng / 25uL system). The labeled primers are the same as in Example 1. The non-labeled primers used for the detection of deaf disease susceptibility loci were the common primers corresponding to the primers in Example 1 without LNA modification.

[0097] 1. The sample to be tested is the same as the normal individual blood spot in Example 1.

[0098] 2. Genomic DNA extraction from samples

[0099] Genomic DNA was extracted by Chelex method.

[0100] 3. Detection and analysis of amplification and amplification products

[0101] 3.1PCR amplification system:

[0102]

[0103] 3.2 PCR amplification procedure: Same as Example 1.

[0104] 4. Fluorescent detection of the amplified product on a genetic analyzer

[0105] With embodiment 1.

[0106] 5 Conclusion

[0107] The result is as Figure 13 As shown, for male normal individuals, the unmodified pr...

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Abstract

The invention discloses a fluorescent detection kit for detecting 12 deafness predisposing genes simultaneously. The kit can detect 12 mutational hotspots in the most common deafness associated genes of the Chinese in 3 hours. The kit comprises reagents before amplification and reagents after amplification, wherein the reagents before amplification comprise a polymerase chain reaction (PCR) buffer solution, a reaction mixture of MgCl2 and deoxyribonucleoside triphosphates (DNTPs), Taq DNA polymerase, ultrapure water, and a primer mixture for amplifying loci of detection sites at high specificity; and the reagents after amplification comprise a genotyping standard and an internal standard. Deafness gene loci are simultaneously detected at high sensitivity and high specificity by combining a fluorescent labeling technology, a linolenic acid (LNA) nucleoside monomer doping-primer modification technology and a capillary electrophoresis technology for the first time, manpower and material resources and time are greatly saved, and pollution due to multi-step operation is prevented.

Description

technical field [0001] The invention relates to a fluorescent detection kit for simultaneously detecting 12 deaf susceptibility genes, belonging to the technical field of biological detection. Background technique [0002] Worldwide studies on the pathogenic factors of hearing and speech disabilities show that about 60-80% of the patients are caused by genetic factors, and clinical research data from developed countries show that hereditary deafness accounts for about 80% of deafness patients. Therefore, in the past ten years, the research on the pathogenesis and molecular epidemiology of hereditary deafness has become one of the most important contents of deaf research. With the completion of the Human Genome Project, great progress has been made in the positioning and cloning of deafness genes. The molecular genetics research and molecular epidemiological data of deafness have enabled researchers to gradually realize that mutations in susceptibility genes for deafness play...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 万戈江魏宏泉步讯夏子芳
Owner 万戈江
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