Analysis Of RpoB Gene Mutations In Rifampin-Resistant Mycobacterium Tuberculosis Isolates

Tuberculosis is an epidemic disease which threats people's health severely. Rifampin is one of the most important anti-tuberculosis medicines and plays an important role in a short-term chemotherapy for tuberculosis. In recent years, the emergence of drug-resistant Mycobacterium tuberculosis, especially rifampin-resistant Mycobacterium tuberculosis, has brought a bad efect to the chemotherapy result. The traditional testing method for rifampin-resistance can no longer satisfy the requirement of the short-term chemotherapy because it takes a long time. As reported, it is about 96% of rifampin-resistant Mycobacterium tuberculosis clinical isolates which resulted from the rpoB gene mutation, for which, several methods for rapid testing of rifampin-resistance have been developed. Among these methods, PCR-SSCP is a widly applied technology.This research began with detecting the rpoB gene mutations of 37 Mycobacterium tuberculosis clinical isolates by polymerase chain reaction - single strand conformation polymorphism(PCR-SSCP) to study the application value of this method as a new molecule testing method for drug susceptibility. Then, to get more knowledge about the rpoB gene mutations and provide some scientific information to study the molecule mechanism of resistance to rifampin in mycobacterium tuberculosis, the rpoB gene sequence of mycobacterium tuberculosis clinical isolates from shannxi was analysed by direct sequencing of PCR products.The analysis of PCR-SSCP shows that, in contrast to Mycobacterium tuberculosis strain H37Rv, 17 out of 18 rifampin-resistant strains were observed that their rpoB genes were with abnormal SSCP patterns, and the sensitivity in the test is 94.4%. 3 out of 19 rifampin-susceptible isolates were observed that their rpoB genes were with abnormal SSCP patterns, and the specificity in the test is 84%. Compared with the results of DMA direct sequencing, PCR-SSCP has the same results as that except for failing to check out the rpoB gene mutation of a rifampin-resistant isolate. It proves that PCR-SSCP is a rapid and sensitive mutation testing method.In addition, the results of DNA direct sequencing reveal that 1 out of 18 rifampin-resistant isolates has deletion in codons 513 and 514 of rpoB, and the other 17 are all singe-point mutation in their rpoB genes. Among the 17 isolates, 8 isolates display TCG-TTG mutation in codon 531, 5 isolates have CAC-TAC or AAC or CGC mutation in condon 526, 3 isolates have GAC-GTC mutation in codon 516, and the last one has CAA- AAA mutation in codon 513. Meanwhile, among 12 rifampin-susceptible isolates, 3 non-rifampin-resistant isolates have mutation relating rifampin-resistance in codon 531, 526 or 511 of rpoB gene respectively. It probably resulted from the incorrect result of drug susceptibility testing.The research results further prove that resistance to rifampin in Mycobacterium tuberculosis resulted from the rpoB gene mutation and PCR-SSCP can quickly detect rifampin resistance in Mycobacterium tuberculosis clinical isolates.