The New Technique Platforms For Screening And Detecting The Mutational Hot Spots Of Genes Associated With M.tuberculosis Drug Resistance

The emergence of drug resistance, especially multiple drug resistant M. tuberculosis Strains enhanced the difficulty of treatment and prevention of TB, and the cases of M. tuberculosis began to rise worldwide. The recent studies show that gene mutation in the genome of M. tuberculosis is one of the main reasons for TB drug resistance. Screening and analyzing the gene mutation types associated with TB drug resistance and its distribution in Chinese population systemically, will help us understand the mechanism that M. tuberculosis can survive from antimicrobial agent, as well as help us develop genetic diagnosis methods for MDR-TB. Nowadays, drug susceptibility testing (DST) in clinic is mainly based on culture systems. Because of the time consuming of these methods, many patients become serious for lacking timely diagnoses and effective therapy. Some molecular techniques, such as PCR- SSCP, PCR-RFLP and DNA sequencing, are used in labs forMDR-TB investigation, but all of them have many kinds of limits, and are not fit for use in clinic. Therefore, it is very necessary to develop a new molecular diagnostic method which can be used in clinic.In order to develop one kind of high-though, low cost, fast and convenience technique platform to investigate the mutation of M. tuberculosis, we have constructed four plasmids with wild type of the rpoB, rpsL, embB and katG gene fragments. Then we optimized the conditions for PCR amplification, preparation for single strand DNA template, and optimized the parameters for Pyrosequencing. Using Pyrosequencing technique we analyzed katG315 , rpoB526 , rpoB531, embB306, rpsL43 and rpsL88 gene sites of 82 MDR-TB clinic isolates. The results showed that the Pyrosequencing technique platform can be used in investigating the hot mutation gene regions of MDR-TB; and the results of our investigation indicate that different gene mutations exist in all of the drug resistant M. tuberculosis isolates to some extent. The mutation rate of katG315 is 54.1%; rpoB526 and rpoB531 is 52.2% and embB306 is 39.6%, however we didn't find rpsL43 mutation, and the mutation rate of rpsL88 is a little higher compared with other reports. Through the experiment, we find a new mutate site: rpoB517 CAG-TAG. and a new type of mutation: embB306 ATG-AGG. These results suggest that the mutations in rpoB, rpsL, embB and katG gene are the essential reason for M. tuberculosis developing RFP, SM, EMB and INH resistance, and these findings are very useful for the diagnosis of MDR-TB.In order to develop the drug resistant gene diagnosis method that can be used in clinic, katG315, rpoB526, rpoB531, embB306 andrpsL88 of M. tuberculosis, based on the analysis results by Pysoquencing, were choose as the target gene sites for investigation. We constructed recombinant plasmids of wild type as well as mutation type of all of these gene sites, and then designed and optimized the experiment parameters and conditions for TDI-FP analysis. Using recombinant plasmids as control samples, we investigated the wild type and mutation type clinic isolates conformed by Pyrosequencing with TDI-FP technology. As a result, we find that TDI-FP technique is a reliable, convenient and low cost method to detect gene mutation of MDR-TB, and it will be a useful technique for drug-resistance diagnosis in clinic in the future.