[Rapid detection of rifampin- and isoniazid-resistant Mycobacterium tuberculosis using real-time PCR and melting curve analysis].

OBJECTIVE

To evaluate the application of a real-time PCR and melting curve analysis assay for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis (M. tuberculosis).

METHODS

A total of 311 clinical isolates of M. tuberculosis obtained from Shenzhen Center for Chronic Disease Control (SZCCC) were included in the study. These isolates were collected originally from national baseline survey on drug-resistant tuberculosis, project for drug resistance surveillance in Shenzhen and clinical patients in SZCCC between 2007 and 2009. rpoB gene resistance-determining region, ahpC promoter (-44 to -30 and -15 to -3), inhA promoter (-17 to -8), inhA 94 and katG 315 were detected by melting curve analysis after real-time PCR, and the results were compared with that of proportion method and DNA sequencing. The performances of the assay in detecting the resistance of rifampin and isoniazid were compared to that of reference proportion method drug susceptibility test.

RESULTS

Real-time PCR and melting analysis was a closed-tube assay that could be completed within 2 - 3 h. Compared to the results of the proportion method, the sensitivity, specificity and accuracy of the assay for rifampin resistance were 97.8%, 97.1% and 97.4% respectively, and for isoniazid resistance were 86.6%, 98.7% and 92.6% respectively.

CONCLUSIONS

Real-time PCR and melting analysis is a rapid, accurate and closed-tube method that can be used as a screening test for rapid identification of Multidrug-resistant tuberculosis.