ABSTRACT
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 oftreptomycin and ethambutol resistance in MTB.
ABSTRACT
<p><b>OBJECTIVE</b>To evaluate the clinical performance of a probe melting analysis (PMA)-based real-time PCR detection kit in rapid detection of rifampin-resistant mutations in Mycobacterium tuberculosis (MTB).</p><p><b>METHODS</b>The specificity of the assay was evaluated by detecting 37 non-tuberculous mycobacteria (NTM), and the detection limit of the method was evaluated by genomic DNA of a standard strain H37Rv. Finally, 962 clinical isolates were analyzed with the PMA assay by detecting mutations in rifampin resistance-determining region (RRDR) of rpoB gene, and results were verified with DNA sequencing.</p><p><b>RESULTS</b>Among 37 NTM strains, three strains showed drug resistant mutation signals. The PMA method could detect down to 30 bacteria per reaction. Sample analysis showed that 186 of 962 isolates were mutants, 751 isolates were wild type and 25 isolates failed to give amplification signals. Among the mutant samples detected, 112 samples from November 2009 to April 2010 were further analyzed by sequencing, as well as 200 wild-type samples. The results showed a complete agreement with the PMA assay except for 5 samples failed in sequence analysis.</p><p><b>CONCLUSION</b>The PMA assay is rapid, accurate and easy-to-use, and thus can be used for detection of rifampin-resistant in clinical isolate samples.</p>