The Application Of Fluorescent Microsphere-Based Technology For Multiplexed Detection Of RpoB Mutations In Rifampin-Resistant Mycobacterium Tuberculosis Isolates

The emergence and spread of drug-resistant M tuberculosis, especially rifampin-resistant M. tuberculosis, is one of important reasons for increasingly severe tuberculosis. It had reported, mutations have been found in the rpoB gene of > 96% of rifampin-resistant clinical M. tuberculosis isolates. A considerable amount of methods have been devoted in the last few years to the detection of rpoB mutations, but most of them have their own limitations.In this study, a fluorescent microsphere-based technology had been introduced to the multiplexed detection of rpoB mutations in clinical M. tuberculosis isolates. And the main research work in this paper was divided into three parts,(l)PCR Amplification of rpoB high variation region;(2)Multiplexed detection of rpoB mutations in clinical rifampin-resistant M. tuberculosis isolates using five microsphere-bound allele specific probes;(3)Comparison of microsphere-based technology, rifampin susceptibility testing, DNA sequencing and PCR-SSCP analysis for detection of rifampin resistance in M. tuberculosis.It was showed in the results, a point mutation in either codon 531 or 526 was observed in 10 of 42 clinical M. tuberculosis isolates, and this result is concordance with phenotypic rifampin susceptibility testing and DNA sequencing results. The comparison of PCR-SSCP analysis indicated, some rifampin-resistant M. tuberculosis had not been detected. These results suggested that fluorescent microsphere-based technology is a more sensitive and veracious tool in multiplexed detection of rpoB mutations. In addition, by DNA sequencing, double mutations in a same codon were observed in two resistant strains; more than double mutations in different codons of one rifampin-susceptible strain were also detected, this may be the effect of suppressor mutation.