Abstract：Abstract: The purpose of this study is to establish a TaqMan-MGB real-time PCR assay for rapidly, specificly, sensitively detecting the katG gene mutation of Mycobacterium tuberculosis (M. tuberculosis). To the design of TaqMan-MGB specific probes and primers, katG315 in M. tuberculosis was analyzed and compared. Through optimization of reaction conditions, the rapid assay to detection of M. tuberculosis resistant to isoniazid was founded; katG gene was cloned into PMD 18 -T vector, these positive standard strains and different strains evaluated the specificity, sensitivity and repeatability; M. tuberculosis which were known sequencing results were detected the specificity and sensitivity of this assay. The minimum detection limit of the real-time PCR method in this study was 10 target genes copies/μl, which was 100 times lower than that of conventional PCR. As the results of non-tuberculosis mycobacterium (NTM) and non-mycobacterium bacterium control strains tested with this method were negative, the specificity were 100%. The coefficients of variation for both intra-and inter-experimental were less than 1%, indicating remarkable repeatability. Compared with the results of sequencing, the sensitivity and specificity of wild-type and mutant-type probes were 100% respectively. The whole process which is rapid detecting katG 315G→C in M. tuberculosis is sensitive, specific, easy, and short time.
2012, Vol. 28 
