[A rapid screening program on the resistance to streptomycin and ethambutol in Mycobacterium tuberculosis isolates by PCR melting curve analysis].

OBJECTIVE: To evaluate the effects of PCR melting curve analysis assay on a rapid screening program regarding the resistance of Mycobacterium tuberculosis (MTB) clinical isolates to streptomycin and ethambutol. METHODS: A total of 331 clinical isolates of MTB had been collected since 2007-2009 in Shenzhen. Mutations at codon 306, 378-380, 406 and 497 of embB gene, codon 43, 88 of rpsL gene, and 513-517, 905-908 region of rrs gene were detected by PCR melting curve analysis. Results were compared with that of conventional drug susceptibility test. RESULTS: Compared to drug susceptibility test, sensitivity, specificity and accuracy for streptomycin resistance were 78.6%, 90.1% and 86.7%, respectively while 83.0%, 93.3% and 91.8%, respectively for ethambutol resistance detected by PCR melting curve analysis. PCR melting curve method was in good agreement with drug susceptibility test. CONCLUSION: PCR melting curve analysis on genetic regions associated with resistance to streptomycin and ethambutol seemed to be a rapid, specific and closed-tube method so it could be used for detection of streptomycin and ethambutol resistance in MTB.

[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.