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Journal of Clinical Microbiology, January 2004, p. 453-457, Vol. 42, No. 1
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.1.453-457.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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

Izhar U. H. Khan and Jagjit S. Yadav*

Molecular Toxicology Division, Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0056,

Received 5 May 2003/ Returned for modification 4 June 2003/ Accepted 29 September 2003

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.

* Corresponding author. Mailing address: Molecular Toxicology Division, Department of Environmental Health, University of Cincinnati Medical Center, Kettering Laboratory, 3223 Eden Ave., Room 138, Cincinnati, OH 45267-0056. Phone: (513) 558-4806. Fax: (513) 558-4397. E-mail: Jagjit.Yadav{at}uc.edu.

Journal of Clinical Microbiology, January 2004, p. 453-457, Vol. 42, No. 1
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.1.453-457.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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