Detection Of Rifampin Resistant Gene Of Mycobacterium Tuberculosis By PCR And Sigle-strand Conformation Polymorphism Analysis

Objective: To establish PCR-SSCP for analyzing rpoB mutation of Mycobacterium tuberculosis and to evaluate its use in clinics.Methods: A rpoB fragment was a amplified from Mycobacterium tuberculosis trains and sputum specimens by PCR with a pair of primers designed according to the RFP resistant determinant of rpoB. The fragments of rpoB were analyzed by single-stranded conformation polymorphism (SSCP). Different PCR-SSCP patterns were obtained from RFP resistant stains and RFP susceptible control.Results:A 258bp fragments of rpoB were amplified from all Mycobacterium tuberculosis strains. The 258bp in rpoB gene of 11 RFP susceptible strains and 37 RFP resistant strains were investigated by single-strand conformation polymorphism analysis(SSCP) with silver stain.H37RV was used as a control,11 RFP susceptible strains and 3 resistance to RFP strains showed same SSCP pattern, however, there is a difference in the SSCP pattern of 34 resistance to RFP. Among the rifampin- resistant (MDR) strains were 78.4%(29/37). The MDR strains,93.1%(27/29) rpoB mutation in SSCP. Conclusion: Rifampin is known to be a fast-acting bactericidal agent against Mycobacterium tuberculosis and has greatly shortened the duration of chemotherapy. However, rifampin-resistant or multidrug-resistant mycobacterium tuberculosis strains have become a newly emerging problem. This PCR-SSCP is an accurate and stable method to identify rpoB showed mutation of Mycobacterium tuberculosis, and it is useful for quickly identifying the Rifampin-resistant or the MDR strains in clinics.