Detection on detecting katGmutation for rapidly diagnosis of Mycobacterium tuberculosiswith TaqMan-MGB real-time PCR

The purpose of this study was to establish a TaqMan-MGB real-time PCR assay for rapidly,specificly,sensitively detecting the katG gene mutation of Mycobacterium tuberculosis(M.tuberculosis).To conduct 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,and these positive standard strains and different strains was used to 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 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 rapidly detecting katG 315G→C in M.tuberculosis is sensitive,specific,easy,and short time.