Kit and primer pair group for detecting drug-induced deafness susceptibility gene mutation site

A technology for drug-induced deafness and mutation sites, applied in recombinant DNA technology, microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of false positive sensitivity, cumbersome detection process, cross-contamination, etc. Efficiency, low price, low instrument requirements

Inactive Publication Date: 2015-10-07
智海生物工程(北京)股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the detection techniques for the above-mentioned two site mutations related to drug-induced deafness mainly include direct sequencing method, gene chip method, enzyme digestion

Method used

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  • Kit and primer pair group for detecting drug-induced deafness susceptibility gene mutation site
  • Kit and primer pair group for detecting drug-induced deafness susceptibility gene mutation site
  • Kit and primer pair group for detecting drug-induced deafness susceptibility gene mutation site

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Detection of Two Mutation Sites in Human Mitochondrial Genome 12S rRNA Gene

[0049] 1. Primer design

[0050] According to the principle of site-specific primer design, the mutation site was set as the 3' end of the primer, and the mutation site mutation was introduced near the 3' end to improve specificity. The primer 5.0 software was used to design the C1494T and A1555G mutation sites respectively. primers. The design of the primers needs to ensure that only the mutant template can be amplified in a certain amplification system, but not the normal template; and ensure that the Tm values ​​of the PCR products of the C1494T and A1555G mutant templates can be separated during melting curve analysis (such as Tm The difference is above 2°C); while avoiding non-specific Tm peaks caused by by-products.

[0051] Taking the C1494T and A1555G mutations in mitochondrial 12S rRNA as an example, according to the human mitochondrial genome nucleic acid sequence (NC_012...

Embodiment 2

[0080] Example 2 Detection of C1494T and A1555G mutations in clinical samples using primer pairs of the present invention

[0081] Using the primer pair set of Example 1 of the present invention, 200 samples from the general population were detected, and the C1494T and A1555G mutations in the samples were detected. All samples were sequenced and verified by the gold standard sequencing method. 2 cases of positive samples C1494CT, 4 cases of positive samples A1555AG, and 194 cases of negative samples were detected. The kit of the present invention detected 200 cases of clinical samples compared with the gold standard sequencing results. The detection results were consistent, 0 cases were missed, and the accuracy was 100%; For other genotype positive and negative samples that are not within the detection range of this kit, the detection results of this kit are all negative, with a sensitivity of 100% and a specificity of 100%.

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Abstract

The invention relates to the field of gene detection, in particular to a kit and a primer pair group for detecting drug-induced deafness susceptibility gene mutation sites, wherein the kit and the primer pair group can be used for detecting aminoglycoside drug susceptibility genes in mitochondria. A specific primer amplification and real-time fluorescence PCR technology is adopted, a new primer pair group is designed aiming at mutant nucleic acid to be detected, the primer is extended to product a specific PCR product when the mutant nucleic acid exists, a fluorescent substance is added into each reaction system, the fluorescent substance is bonded to the PCR product after amplification is completed, the produced melting peak is analyzed by adopting a melting curve, whether a corresponding mutant type exists on the nucleic acid or not is judged through the existence of the specific melting peak, and the specificity is high; the detection of two mutation sites can be completed by using a single tube and a single channel only, the requirements on instruments are low and the operation is simple to perform.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a kit and a special primer pair for detecting drug-induced deafness susceptibility gene mutation sites by using fluorescent PCR. Background technique [0002] Aminoglycoside antibiotics (streptomycin, gentamicin, kanamycin, sisomicin, tobramycin, etc.) cause protein translation errors by directly binding to 16S rRNA of the 30S subunit in bacterial ribosomes Or the premature termination of protein synthesis produces antibacterial effect. And because of its high-efficiency and broad-spectrum antibacterial effect, it is widely used clinically for the treatment of Gram-negative and positive bacterial infections, but this type of drug has serious ototoxic side effects, and can cause irreversible damage to hearing when used, and even " A needle for deafness". [0003] In recent years, it has been found that this susceptibility is usually maternally inherited and is associated with the A...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q1/6858C12Q2600/156C12Q2563/107C12Q2545/101C12Q2547/101
Inventor 冯东杨海艳沈东艳
Owner 智海生物工程(北京)股份有限公司
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