Multiple PCR-LDR (polymerase chain reaction-ligase detection reaction) detection kit for multiple deaf susceptibility genes with high specificity at 14 sites

A PCR-LDR, detection kit technology, applied in the field of DNA molecular detection, can solve problems such as non-functional proteins, gap junction defects, frameshift mutations, etc.

Active Publication Date: 2015-01-28
步迅
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Pathological 235delC mutations lead to frameshift mutations, resulting in non-functional proteins and gap junction defects, thereby affecting the normal opening and closing of ion channels
The 299-300delAT mutation leads to the deletion of most of the CL region of the Cx26 polypeptide, and th...

Method used

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  • Multiple PCR-LDR (polymerase chain reaction-ligase detection reaction) detection kit for multiple deaf susceptibility genes with high specificity at 14 sites
  • Multiple PCR-LDR (polymerase chain reaction-ligase detection reaction) detection kit for multiple deaf susceptibility genes with high specificity at 14 sites
  • Multiple PCR-LDR (polymerase chain reaction-ligase detection reaction) detection kit for multiple deaf susceptibility genes with high specificity at 14 sites

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0130] Example 1: PCR-LDR detection kit for 14 deafness susceptibility loci and its use

[0131] 1. Kit composition: PCR amplification reagents include: PCR buffer, MgCl2, dNTPs mixture Remix, EX-Taq enzyme, 2 sets of primer mixtures (Primer1 contains 4 pairs of primers for GJB2, GJB3, mitochondrial 12SrDNA and WSF1 genes; Primer2 Contains 5 pairs of primers for 2168, 1975-2027, IVS7-2, 1174-1226 and WSF1 gene loci) and ultrapure water; LDR ligation reaction reagents include: 2 sets of universal fluorescent probes and 14 sets of detection probe mixtures, DNA Ligase, 10X buffer and ultrapure water; genotyping reagents include: ROX-300 internal standard, genotyping standards corresponding to 14 deafness gene mutation sites used for genotyping; quality control products include: negative The quality control product is sterilized double-distilled water. The wild-type quality control product is genomic DNA with 14 wild-type sites identified by sequencing. The positive quality contro...

Embodiment 2

[0152] Embodiment 2 determines optimal primer and multiplex PCR system

[0153] First, a series of primer pairs were designed for screening, and finally the optimal primer pair was selected to establish a single-plex PCR reaction system. The results showed that the designed primers could correctly amplify the target bands, and each band was brighter. Singleplex PCR products were verified to be correct by sequencing. Such as Figure 17-1 shown. From left to right, Marker, amplification products corresponding to primers of GJB2, mitochondrial 12SrDNA, 2168, 1975-2027, IVS7-2, 1174-1226, WSF1, and GJB3 genes, Marker1000bp. Secondly establish multiple PCR system, by adjusting different concentration ratio between primers, determine optimal multiple PCR system, multiple PCR system band that the present invention establishes is clear, without non-specific band amplification, such as Figure 17-2 .

Embodiment 3

[0154] Embodiment 3: specificity test

[0155] Materials: 14 known positive DNA samples of deafness loci, positive plasmid samples, and 5 known negative samples were taken. All the above samples were sequenced and tested with this kit at the same time. The results were analyzed and verified: 14 loci The detection results of the deafness susceptibility gene PCR-LDR detection kit are completely consistent with the sequencing results, and the kit has strong specificity.

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Abstract

The invention provides a PCR-LDR (polymerase chain reaction-ligase detection reaction) detection kit for simultaneously detecting 14 deaf susceptibility genes. The kit comprises a packaging box body (1) and PCR amplification and LDR reaction reagents (2)-(5), wherein eight pairs of PCR primers and 16 groups of probes are contained, wherein two groups of universal probes and 14 groups of detection probes are contained. The method is high in sensitivity and clear and accurate to judge. Compared with the conventional sequencing technique, the kit can realize high throughput screening of 14 key deaf gene mutant diagnosing results at one time, so that the kit has the advantages of being economical, efficient, high in accuracy and the like.

Description

technical field [0001] The invention relates to the technical field of DNA molecular detection, in particular to a multiple PCR-LDR detection kit for 14 deafness susceptibility genes. Background technique [0002] Deafness is a common congenital disease that seriously affects the quality of human life. It can be caused by a single gene mutation or a compound mutation of different genes, or by environmental factors (such as medical factors, environmental exposure, trauma, drugs, etc.) or genetic and environmental factors. Both work together. There are 27.8 million people with hearing disabilities in my country, ranking first among all kinds of disabilities (33%). Studies have found that GJB2, SLC26A4, and mitochondrial genes are the three most common pathogenic genes that cause most non-syndromic deafness in China. [0003] The GJB2 gene is the first hereditary deafness gene cloned and identified, and it is also the most common cause of non-synthetic deafness. About 20% of...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6876C12Q1/6883C12Q2600/16
Inventor 步迅张全芳刘艳艳
Owner 步迅
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