Research On Susceptibility Test Of M.Tuberculosis Strains To Rifampin By DNA Sequencing ,PCR-SSCP ,and Micro-well Phage Replication Assay

Objective To elucidate the characterization of rpoB mutation in rifampin-resistant clinical isolates of Mycobacterium tuberculosis from China, to evaluate the significance of susceptibility test of M. tuberculosis strains to rifampin by DNA sequencing, PCR-SSCP, and micro-well phage replication assay. Methods (l)588bp DNA fragment of rpoB gene including 81bp code region (Rifampin Resistance Determination RegionfRRDR]) was sequenced.242 strains of Mycobacterium tuberculosis, including 193 rifampin-resistant strains and 46 rifampin-susceptible strains and 3 artificially induced rifampin-resistant strains were sequenced. Analysed the rpoB gene fragment that including RRDR of 41rifampin-reistant strains and 25 rifampin sensitive strains by DNA sequencing and PCR-SSCR (3)40 rifampin resistant strains and 16 rifampin sensitive strains were test its' susceptibility to rifampin by micro-well phage replication assay(MPRA). Results (1)89.1%(172/193) rifampin-resistant strains had rpoB gene mutations in RRDR, no mutation was found in rifampin-susceptible strains. 46.1% rifampin-resistant strains had mutations located at 531ser; 17.1% strains had mutations located at 526-His, combinative mutation rate was 12.4%; 4 strains had synonymous mutations; No deletion or insertion mutation was found among all strains. High level rifampin resistant strains (250 u g/ml rifampin resistant) had higher mutation frequency at 531-Ser than low level rifampin resistant strains (50 P g/ml rifampin resistant) (PO.05). (2)The accordance rate of DNA sequencing and PCR-SSCP was 93.9%(62/66),compared with DNA sequencing, the sensitivity of PCR-SSCP was 92.1%(35/38),the specialty of PCR-SSCP was 96.4%(27/28).(3)The accordance of MPRA with absolute concentration method was 92.9%(52/56),compared with absolute concentration method, the sensitivity of MPRA was 95.0%(38/40),the specialty of MPRA was 87.5%(14/16) Conclusions (l)About90% rifampin resistant clinical strains of Mycobacterium tuberculosis from China had rpoB mutations; 531-Ser and 526-His were the most common positions to be substituted, the added mutation rate was about 46%; High level rifampin resistant strains had a higher frequency of 531-Ser mutation than low level rifampin resistant strains; No insertion or deletion mutation was found in our specimens; DNA sequencing is significant in drug choosing for tuberculosis treatment. (2)PCR-SSCP was of much significance in rifampin susceptibility analysis of clinical isolates of M. tuberculosis. (3)MPRA was a simple, sensitive method in rifampin resistance screening, and it had a good perspective in using.