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

A single-tube PCR method was developed for efficient identification of nontuberculous mycobacteria (NTM) and their environmental isolates in about 3 111 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 beat 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.