M.tuberculosis drug resistant gene detection reagent kit and process for preparation

Abstract

The invention relates to a tuberculosis mycobacterium drug resistance gene detection kit and its preparation method. The kit is prepared by the following steps: designing and synthesizing corresponding oligonucleotide probes according to different mycobacteriums and tuberculosis mycobacterium complex 16S ribosome ribonucleic acids (rRNA), dotting the oligonucleotide probes according to certain sequence on the pretreated nitrate fiber film by dot plot method, then obtaining drug resistance gene detection identification film sheet, and hybridizing with the PCR amplification products having biotin marks. According to the bluish purple signal strength of the probes hybridization to interpret the result with naked eyes, the invention can be used for detecting five drug resistance genes of tuberculosis mycobacterium of clinical specimens or clinical separation strains.

Description

technical field [0001] The invention relates to the preparation and application of a Mycobacterium tuberculosis drug-resistant gene detection kit, which belongs to the technical field of medical molecular biology diagnosis, in particular to the preparation of the Mycobacterium tuberculosis drug-resistant gene detection kit and its application in tuberculosis diagnosis Applications. Background technique [0002] Tuberculosis is still one of the major infectious diseases that endanger human health in the world. Since 1985, the prevalence of AIDS, tuberculosis-infected immigrants and some people living in poverty have caused the incidence of tuberculosis to rise in the United States and other developed countries in Europe and the United States, especially tuberculosis. The problem of bacterial drug resistance and the incidence of non-tuberculous mycobacterial disease are increasing year by year, which makes the treatment of tuberculosis even worse. At present, there are about ...

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Problems solved by technology

[0005] Among the established molecular drug susceptibility testing methods, although the PCR-SSCP technique is simple, it can only determine whether there is a mutation in the gene, but cannot detect the location and nature of the mutation. Some drug resistance gene loci are naturally polymorphic or some Gene mutation has nothing to do with drug resistance, so PCR-SSCP detection may produce false positives; PCR-RFLP technology uses PCR to amplify a DNA fragment containing a specific restriction site, and the fragment is digested by a restriction endonuclease and electrophoresis can show two If the gene is mutated and the enzyme cleavage site disappears, the electrophoresis of the fragment after enzyme digestion still shows a PCR amplified fragment, which has the advantage of high specificity, but it can only be used to analyze known Gene point mutation at a specific site in the sequence; PCR-direct sequence method refers to the purification of the PCR amplification product of the drug-resistant gene, which is directly used for DNA sequence analysis. This method can not only detect gene mutations, but also determine the location and nature of mutations , which can discover new mutation sites, is a decisive method for detecting gene mutations, but the technical requirements are high, specialized technicians are required, and the required sequencing instruments are expensive and costly, making it difficult to routinely carry out in clinical laboratories; PCR-gene chip The analysis method uses gene chip technology to simultaneously analyze multiple drug-resistant genes of Mycobacterium tuberculosis. This method is simple, fast, and can be carried out on a large scale, but the technical requirements are high, and the required spotting system and fluorescence analysis system are expensive. But it is difficult to popularize and apply; the principle of single-strand probe reverse hybridization test (reversehybridization-based line probe assay, referred to as LiPA) is to use biotin-modified specific primers to amplify DNA, so that the PCR product has a biotin label, and the PCR After denaturation, the product is hybridized with a specific oligonucleotide probe immobilized on a membrane, and the result is displayed by enzyme immunochromogenic method

Belgian Innogenetics Company launched the INNO-LiPA Rif. Mycobacterium tuberculosis detection kit and the matching automatic detection instrument (Auto-LiPA) according to this principle, which contains 5 probes for the wild-type rpoB gene sequence and 4 probes for specific The probe for site mutation is only used to detect RFP-resistant genotypes of Mycobacterium tuberculosis, which is simple, fast, and easy to promote, but the types of gene mutations detected are limited

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