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 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.
Khan
Izhar U H
IU
Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0056, USA.
Yadav
Jagjit S
JS
D001433:Bacteriolysis; D001483:Base Sequence; D006801:Humans; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA