Molecuar Epidemiology Of Mycobacterium Tuberculosis And Rapid Detection Of Resistance-associated Genes

The resistant Mycobacterial tuberculosis secondary to the non-standardized treatment, especially the multi-drug-resistant(MDR) M. tuberculosis has become the most serious problem. Exploring the mechanism of resistance and the rapid detection methods now is the focus of tuberculosis control.It is well known that katG gene and rpoB gene are closely associated with the resistance of the M. tuberculosis to isoniazid and rifampin. The mutations in two genes can explain at least 50% resistance to isoniazid and more than 95% resistance to rifampin. The most common mutation of katG gene in INH resistant M. tuberculosis is S315T, while 96% rifampin-resistant M. tuberculosis possesses mutations within a 81bp rifampin resistance-determining region. The genetic alterations are characteristic in different countries and regions, so it is urgent to make clear the characterization of variations in katG and rpoB in the tuberculosis patients in China. Furthermore, detection of .drug resistance associated genotypes should be explored on the basis of local molecular epidemiology.In Part 1, we screened the S315T mutation in clinical isoniazid-resistant M. tuberculosis strains by the method of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) firstly, and then we made direect sequencing in the rest non-S315T mutated strains to search other unknown resistance-associated mutations. The mutations in rpoB gene were simultaneously analyzed by direct sequencing. Meanwhile, we genotyped the M. tuberculosis by a new method named Multiple loci Variable Number of Tandem Repeats Analysis (MLVA). The association between genotyping and the resistance were investigated in a pilot study by this new genotyping method in the eastern region of China. In Part 2, multiplex allele specific polymerase chain reaction (MAS-PCR) method was developed to detect the mutations in katG and rpoB genes, to deduce the resistance to isoniazid and rifampin rapidly.Part 1. Molecular Epidemiology of Resistant Mycobacterium Tuberculosis in eastern region of ChinaA Resistant phenotypes and their associated genotypes in resistant Mycobacterium Tuberculosis StrainsIt is well known that katG and rpoB gene are the most important 2 genes associated with clinical resistance to isoniazid and rifampin. In this study, we investigated katG and rpoB gene in clinical resistant Mycobacterium tuberculosis strains collected from the samples of tuberculosis patients in eastern region of China. Totally, 429 clinical Mycobacterium tuberculosis specimens were included in our research. PCR-RFLP method was applied to screen the S315T mutation firstly. Complete katG gene sequencing was performed in the remaining resistant strains without S315T mutation to explore the unknown mutations associated with resistance The rifampin- resistant determining region of rpoB gene was analyzed by direct sequencing in 96 clinical TB strains. The target fragment of katG gene from 407 strains were amplified and analyzed by RFLP. Ser315 mutations were found in 76.9% (166/216) resistant stains. Complete or part deletion of katG gene was detected in 2 highly resistant specimens. Sequencing results in 48 resistant strains showed that 315, 463 and 234 sites were the most frequent mutation sites. Other sites mutations were also found but distributed dispersedly with low prevalence rate as less than 5%. Besides S315T, S315N were also common in China (8.7%). In the mean time, we found the most common mutation sites in rpoB gene were 531,526 and 516. The results from the study of genotypes associated most common clinical resistant phenotypes can be helpful to develop new methods to detect the resistant M.tuberculosis.B. fingerprint pattern diversity of the resistant Mycobacterium Tuberculosis.The development of new method to discern the molecular fingerprint (genotype) of M.tuberculosis has been revolutionized and can be used to deepen our understanding of the transmission of TB. In this study, we genotyped the clinical M. tuberculosis strains by Multiple Loci Variable Number Tandem Repeat Analysis (MLVA) method, and explore the relationship between the genotyping and the resistance. We amplified DNA from 50 clinical M. tuberculosis by 12 pairs of primers corresponding to 12 VNTR sites. We noticed that 12 loci were all polymorphic and 5 kinds of allele fragments had never been reported. Of 50 M. tuberculosis strains, 56%(28/50) were clustered, with the largest one containing 16 strains. Much high similarity was found among these strains. No significant difference was found between the distribution of clusters in resistant and sensitive M.tuberculosis groups. Meanwhile, the genotype of clustered resistant and sensitive M.tuberculosis was found to be same, suggesting there was active recent transmission of both resistant and sensitive M.tuberculosis strains existing in this region and, resistance was most of the time induced during treatment other than transmission. The genotyping...