| Mycobacterium tuberculosis (M. tuberculosis) is still the most pathogensin the world. The incidence of tuberculosis was reversed in the mid-1980s mainly due to epidemic human immunodeficiency virus infection and increasing of immigration throughout the world and spreading of drug-resistant M. tuberculosis, this situation is likely to deteriorate in the future. The most important factor of high incidence of tuberculosis is multi-drug resistance of M. tuberculosis. Rifampin(RFP) and Isoniazid(INH) were the most two effective drugs for therapy tuberculosis, but now their effects are very limited because of the spreading of multi-drug-resistant M. tuberculosis(MDRTB). Resistance to drug is mainly due to genomic mutations in specific genes of M. tuberculosis. Up to now, twelve genes were known to be linked to resistance to anti-tuberculosis drugs, eg. rpoB for RFP resistance and katG, inhA, aphC and kasA for INH resistance. Resistance to multiple drugs may be the consequence of an accumulation of mutations. In this study, the rpoB and katG mutations and their relation to multi-drug resistance of M. tuberculosis were analysed. We also tried to set up a rapid,simple andinexpensive method for screening the multi-drug resistance of M. tuberculosis.Part one:Study on the rpoB and katG genes mutations and theirrelation to multi-drug resistance of MycobacteriumtuberculosisTo explore the molecular mechanism of the multi-drug resistance of M. tuberculosis tuberculosis, drug(RFP, INH, streptomycin and ethambutol) susceptibility of 113 clinical isolates of M. tuberculosis was determinated by indirect resistance ratio method . Moreover, DNA sequence of rpoB and katG genes of 30 drug-sensitive and 18 multi-drug-resistant clinical isolates was analysed. Their results showed that 64 isolates were sensitive to REP and INH, 20 isolates were resistant to RFP but sensitive to INH,11 isolates were sensitive to RFP but resistant to INH and 18 isolates were MDRTB. TheTCG(Serine, Ser)-> TTG (Leucine, Leu)mutation at codon 531 in the rpoB occured in 89% (16/18) MDRTB isolates,but only 3% (1/30) drug- sensitive isolates was found the mutation. Furthermore, The CAC(Histidine, His)^>AAC(Asparagine, Asn) mutation at condon 526 and the CTG(Leu)->CCG(Proline, Pro) mutation at condon 533 in the rpoB occurred in 11%(2/18) MDRTB isolates.As for the katG , The AGC(Serine,Ser)->ACC (Threonine, Thr) mutation at condon 315 occured in 44%(8/18) MDRTB isolates. These results showed that the TCG(Ser)~> TTG(Leu) mutation at codon 531 in the rpoB was highly related to the multi-drug resistance of Mycobacterium tuberculosis (P=l. 08 X 10"*), the AGC(Ser) -> ACC(Thr)mutation at cordon 315 in the katG was significantly related to the multi-drug resistance of Mycobacterium tuberculosis(P=l. 16 X 10~4).It revealed that the TCG(Ser) -> TTG(Leu) mutation at codon 531 in the rpoB gene can be the mark for screening the multi-drug resistant of M. tuberculosis. To shorten the time of drug suscepitibility testing of M. tuberculosis and reduce the cost of patients, we tried to set up a rapid,simple and inexpensive method for screening the multi-drug resistance of M.tuberculosis. A special polymerase chain reaction(PCR) were applied to analysis the TCG(Ser) ^>TTG(Leu) mutation at codon 531 in the rpoB gene(multi-drug resistance) in 113 isolates of M. tuberculosis. The correlation between the results of DNA sequence analysis and the PCR was 100%. According to indirect resistance ratio method, the sensitivity and specificity of PCR was 89% and 80%, respectively. We believed that the low-cost assay may have potential applicability for rapid screening of multi-drug resitance in M. tuberculosis.Part two:Establishment the multi-drug-resistant & RFP-resistant & INH-resistant M. tuberculosis strainsTo offer materials for resistance studies of M. tuberculosis, we established 3 drug-resistant M. tuberculosis strains ( 1 for multi-drug-resistant , 1 for RFP-resistant and 1 for INH-resistant). In this study all MDRTB ,RFP-resistant M. tube...
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