Pyrazinamide susceptibility testing of Mycobacterium tuberculosis by high resolution melt analysis

2014 
Summary Pyrazinamide (PZA) plays the important role in shortening the tuberculosis treatment period and in treating MDR-TB. Phenotypic PZA susceptibility methods are limited because they require specialized acidified media, which increases costs and complexity. In this study we developed a genotypic high resolution melt (HRM) analysis technique to detect pncA mutations associated with PZA resistant Mycobacterium tuberculosis . Seven overlapping primer pairs were designed to cover the entire pncA gene and upstream regions. Each gene segment was individually amplified by real-time PCR followed by HRM analysis. The assay was evaluated on 98 clinical M. tuberculosis isolates (41 PZA susceptible by MGIT method, 55 PZA resistant, 2 undetermined). HRM was 94% concordant to full-length sequencing results, with most discrepancies attributable to mixed populations per HRM or transversions. Sequencing and HRM yielded 82% and 84% concordance, respectively, to phenotypic PZA susceptibilities by MGIT, with most discrepancies attributable to isolates with wild-type pncA but phenotypic PZA resistance. This HRM technique is a simple and high-throughput method for screening clinical M. tuberculosis samples for PZA resistance.
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