Screening Of New Resistance Mutations In Isoniazid Or Streptomycin Resistant Mycobacterium Tuberculosis

The critical of improve the cure rate and reduce the mortality of TB is detection in early stage and received early treatment. Traditional Lowenstein-Jensen(LJ) diagnostic methods need a long detection period, amost one or two months, so would delay the optimal timing of treatment of patients. In recent years, with the rapid development of molecular biology, emerged a mount of rapid genotype diagnosis methods. But only approximately 60%~80% of INH-resistant or STR-resistant Mycobacterium tuberculosis can be diagnosed by common resistance genes mutations such as katG, inhA, rpsL, rrs. So discovery of new drug resistance mutations not only improve the diagnostic accuracy rate of genotype methods and lay the foundation for the improvement and development of new genotype diagnostic tools, but also have far-reaching significance to comprehensive understanding of the molecular mechanisms underlying Mycobacterium tuberculosis resistant to isoniazid or streptomycin, and also discovery of new drug targets and then discovery of new anti-TB drugs. Based on those, in this study, we evaluated the accuracy of genotype diagnosis methods, followed by identifing new predictied resistance mutations. And finally, by controlling the single factor(drugs) variable method, constructed vitro model of isoniazid or streptomycin resistant Mycobacterium tuberculosis successfully, thus excluding impact of environmental factors on Mycobacterium tuberculosis base mutations, achieved the purpose of screening of new resistance mutations effectively.The main contents and findings of this study are as follows:1、To assess the accuracy and detection limit of genotypic diagnostic methods,and then to provide a reasonable diagnostic route.We used gradiently diluted Mycobacterium tuberculosis which had known their phenotype,as experimental samples,and we found that Xpert MTB/RIF had higher detection sensitivity,the limit of detection for XpertMTB/RIF were equivalent to traditional culture(3×102个数/mL)and lower than gene chip and GenoType?MTBDRplus.In the case of a higher content of MTB(>3×104个数/mL),genotype diagnostic methods can detect rifampicin or isoniazid resistant TB,so it had high accuracy to diagnosis drug-resistant TB by the genotype methods.2、Finding new resistance mutations of isoniazid or streptomycin resistant TB is the basis of improvement the diagnostic accuracy rate of genotype methods. We used one hundred and twenty-nine clinical tuberculosis isolates and were amplified by PCR, and then analyzed of their sequencing results, we found that among the 44 INH-resistant isolates, 8 isolates(17.78%) had the predicted SNP at the Rv1482c~Rv1483 1673425 position, among the 45 STR-resistant isolates, 5 isolates(11.11%) had the predicted SNP at the Rv1194c~Rv1195 1338631 position. Our findings suggested that the intergenic region Rv1482c~Rv1483, Rv1194c~Rv1195 can serve as new resistance gene in isoniazid or streptomycin resistance TB, respectively. And also can improve the accuracy of genotypic methods to diagnosis isoniazid or streptomycin resistant TB.3、In order to find potential new resistance genes effectively in isoniazid or streptomycin resistance TB, and lay the foundation for accurate and efficient genotypic diagnostic methods, so we constructed vitro model of isoniazid or streptomycin resistant Mycobacterium tuberculosis. We discovered seven and eight potential new resistant genes which were associated with isoniazid or streptomycin resistance TB respectively, by analyzing the complete genome sequence of the induced resistant strains and control strain, but they also need to be further verified.In conclusion, the results of this study showed that genotypic methods are clinical indispensable auxiliary techniques for diagnostic of drug-resistant TB. While finding new resistance mutations of isoniazid or streptomycin resistant TB effectively can improve the diagnostic accuracy rate of genotype methods, and have great significance for further improvement and development of new rapid genotype diagnostic methods.