Study On Rapid Detecting Method Of Multi-drug Resistant Gene Of Mycobacterium Tuberculosis

The epidemic of tuberculosis got a recovery from 1980s in last century, because of the drug resistance of mycobacterium tuberculosis. The epidemic situation and chemotherapy effect experienced a great change result from multidrug resistant and high resistant strains occurred or transmitted. WHO estimate 2 billion individuals or so infected with mycobacterium tuberculosis in the world and about 50 million in them caused by resistant strains now. Most resistant strains related to the gene mutation or deletion of some enzymes in mycobacterium tuberculosis owing to unreasonable chemotherapy. Those strains, especially the increase of multi-drug resistant organisms, led negative impact on chemotherapy effect and brought trouble to control tuberculosis. The traditional drug sensitive test, which based on the mechanism of microorganism metabolism cost time usually from 1 to 2 mouths and can not meet the requirement from modern short course chemotherapy, so the rapid and accurate detecting of drug resistance in mycobacterium tuberculosis will be provide gist for establish the best chemotherapy project and effective lower the diffusion of drug-resistant strains in crowed.Along with the development of molecular biology recently, themechanism of drug resistance of the basic drugs to treat TB were established step by step, and develop some detections of mycobacterium tuberculosis using of gene technique, which opened a new way for the test of resistant strains rapidly. Now some better methods includes direct sequencing, hybridization and Polymerase Chain Reaction-Single- Strand conformation polymorphism but the last one is more optional for clinic application because of convenience, low cost and sentivity.Multi-drug resistance tuberculosis, which is resistant to more than two anti- tuberculosis drugs, threw the shadow on our determination of elimination tuberculosis. The resistant of the most commonly used anti-tuberculosis drugs, mostly includeing rifampin, streptomycin and isoniazid, brought much trouble for patient. It is urgent for clinic to establish a sensitive and sepecific protocol aimed at multi-drug resistant detection at the same time.Although there were reports about the PCR-SSCP detection of drug- resistant genes now, failing to detect the majority of the drug-resistant genes at the time. This leads an ineffective treatment with standard chemotherapy. For this, the main research objective of the theme is as follows: to develop a method in order to test RFP, SM and IHN resistant gene mutation named rpoB, rpsL and katG respectively at the same time, and provide rapid , nicety and trusty means for early of clinic theraty.First, to develop the multi-PCR technique. It has been verified that RFP resistance result from the mutation of rpoB which encode subunit in RNA polymerase and about 90%~95% mutation occurred at the 81bp region (507~533 site 27 amino acid codons) in the center of rpoB gene. The mutation of rrs gene encode 16SrRNA and rpsL gene encode S12 protein lead resistant to streptomycin. The mutation of rpsL, 60%~70% occupied SM-resistant strains, usually happened at the site of 43 and 88, is the major molecular mechanism for SM resistant. The katG gene of the encoded hydrogen oxide - peroxidase is the main reason that leads to the resistance of mycobacterium tuberculosis to isoniazid, and associated with the mutation of genes of the inhA and aphC promoter. KatG gene mutation 50% ~70% occupied INH-resistant strains, mostly occurred at the site of 315 and 304. For this purpose, the experiment designed the specific primers to cover as much as possible the mutation sites for the drug-resistant genes. The specific primer of the rpoB gene according to the Genbank: L27989, P1 5'-GAT CAA GAG TCA GAC GGT GTT C-3'; P2 5'-ACG GTG TTG TCC TTC TCC AG-3', the extended section was 365bp; The specific primer of the rpsL gene according to the Genbank: L08011, P, 5'-ACA CCA CCA CTC CGA AGA AG-3'; P25'-TGC GTA TCC AGC GAA CCG-3', the extended section was 201bp; The specific primer of the katG gene was self-designed...