Detection Of Pyrazinamide-Resist Ant Gene PncA In Clinical Isolates Of Mycobacterium Tuberculosis In Qingdao District

Objective To investigate the mutations of pneA gene in clinical isolates of Myeobae-terium tuberculosis and the relationship between pncA gene and pyrazinamide resistance strains in Qingdao district, and to provide a rapid and reliable method for detecting gene mutation, which will make up the madequaleness of routine drug-sensitivity test.Methods In 76 clinical isolates of Mycobacteriutn tuberculosis , Mycobacterium tuberculosis were directly detected by sputum smear, the drug resistance were detected by BACTEC 460 system. With the DNA Star softwares, two pair of PCR primers were created from the whole sequences of H37Rv standard strain in Gene Bank . one pair for com served sequence of 1S6110. the other for pncA gene, and the target genes were amplified by PCR technique . The mutations of pncA gene were detected by PCR-SSCP, with a positive reference of Mycobacterium tuberculosis H37Rv strain.Results In the 76 clinical isolates . 20 strains were positive by sputum smear. With the BACTEC-460 system 44 strains were pyrazinamide sensitive and 32 were pyrazinamide resistant , which including 24 low pyrazinamide-resislant strains and 8 high pyrazinamide resistant strains. The whole rate of pyrazinamide-resistance was 42. 1 % (.32/76.). With the PCR technique, the IS6110 amplification products (245bp) were successfully detected in 76 strains, the pncA amplification products ( 254bp) were detected in 69 strains, and 7 srains were negative, which including 5 pyrazinamide-resistant strains and 2 pyrazinamide sensitive strains. With PCR-SSCP, 18 strains displayed abnormal pncA SSCP profile, in which 13 were pyrazinamide-resistant strains including 8 low pyrazinamide resistant strains and 5 high, and the mutation rate of pncA gene was 48. l%( 13/27). The. other 5 strains were pyrazinamide sensitive and the muctation rate of pncA gene was 11.9% ( 5/42). 51 strains displayed normal pncA SSCP profile, in which 14 were pyrazinamide resistant strains including 13 low pyrazinamide resistant strains and 1 high, and 37 were pyrazi namide-sensitive stmingConclusion The mutation o: pncA gene is one of the major molecular mechanism in in Mycohactetium tuberculosis pyrazinamide-resistance clinical isolates in Qingdao. PCR-SS CP appears to be a promising method for the rapid detection of pyrazinamide- resistant My cobacterium tuberculosis, making up the inadequateness of routine drug-sensitivity test.