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Detection of Mycobacterium Tuberculosis Resistant Genes by Membrane Reverse Dot Blot Hybridization

Author: ChenDanHua
Tutor: CaoYiCheng
School: South China University of Technology
Course: Biochemistry and Molecular Biology
Keywords: M. tuberculosis Resistance katG gene rpoB gene Membrane anti- dot blot hybridization Polymerase chain reaction ( PCR )
CLC: R440
Type: Master's thesis
Year: 2011
Downloads: 23
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Abstract

In the world in recent years, tuberculosis showed a resurgence of the phenomenon, which brought a huge challenge for the prevention and treatment of tuberculosis. Especially resistant (DR-TB) and multidrug-resistant (MDR-TB) TB has a wide distribution, and spread quickly, so TB has been considered to be one of the grim task facing the current global control of infectious diseases. Used first-line anti-TB drugs in the treatment of tuberculosis, isoniazid (INH), rifampicin (RFP), streptomycin (SM), therefore all countries have attached great importance to tuberculosis resistant to these drugs Mycobacterium. In the current specification of Mycobacterium tuberculosis resistance methods and the gold standard is the traditional culture method, but this method in the consumption of the entire detection process is particularly long, and thus can not effectively understand and correct treatment of tuberculosis. Isoniazid (INH) and rifampicin (RFP), two kinds of very effective anti-TB drugs, so has been in play a very important role in the treatment of tuberculosis. Resistant to isoniazid causes this mutation causes this enzyme catalase - peroxidase encoding gene katG gene deletions and mutations causing common 315 serine mutation threonine peroxidase catalase affinity for its substrate INH reduced, making the loss of nearly half of the enzyme activity, in all of the INH-resistant strains, about 50% of the mutations to this form and cause INH highly resistant. Produce its core area of ??rifampin-resistant rpoB gene mutations. 65% -86% of the mutations in this region occurs in the 526th histidine mutation to tyrosine and serine 531 was mutated to leucine, this mutation will lead to highly resistant to rifampicin (MIC gt; 32ug/ml). Therefore, we focus on today's known resistance mutations representative sites, this study selected katG (315) and rpoB (531) mutation site as a research object detected. This study was repeated fumble membrane reverse dot blot hybridization technique in the detection of TB drug resistance optimum conditions, and eventually established using a pore size of 0.45μm nylon membrane, the probe a final concentration of 1.5 pmol / μL, hybridization temperature of 60 ° C. hybridization time of 30 min, washed membrane temperature of 60 ° C, 20 min film wash time to detect the resistance of Mycobacterium tuberculosis. And by the experimental results show that the application of the film under these conditions the reverse dot blot hybridization successfully detect katG gene of Mycobacterium tuberculosis resistant to isoniazid and rifampicin resistance in rpoB gene mutations, and compared with DNA sequencing technology, accurate detection was 100%. Film reverse dot blot hybridization technique is simple, rapid, sensitive and specific direct detection of Mycobacterium tuberculosis katG, rpoB gene mutation for the clinical detection of Mycobacterium tuberculosis resistant to provide supplementary diagnostic tool for the development of the detection of TB drug the sex kit experimental basis.

Detection of Mycobacterium Tuberculosis Resistant Genes by Membrane Reverse Dot Blot Hybridization

Abstract

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