Development of a single-tube, cell lysis-based, genus-specific PCR method for rapid identification of mycobacteria: optimization of cell lysis, PCR primers and conditions, and restriction pattern analysis |
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Authors: | Khan Izhar U H Yadav Jagjit S |
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Affiliation: | Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0056, USA. |
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Abstract: | A single-tube PCR method was developed for efficient identification of nontuberculous mycobacteria (NTM) and their environmental isolates in about 3 h without conventional DNA isolation. The following three steps were optimized or developed: (i). a simple, 6-min direct cell lysis protocol as a PCR prestep for generation of DNA-template, (ii). an improved Mycobacterium-specific PCR amplification protocol with a broader species specificity using newly designed primers targeting a 228-bp region of the 65-kDa heat shock protein (hsp) gene and optimal PCR amplification conditions, and (iii). a genus-specific restriction analysis of the PCR product for conclusive identification of the unknown NTM isolates. |
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