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Journal of Clinical Microbiology, November 2006, p. 3855-3862, Vol. 44, No. 11
0095-1137/06/$08.00+0     doi:10.1128/JCM.01214-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Differentiation of Mycobacterial Species by hsp65 Duplex PCR Followed by Duplex-PCR-Based Restriction Analysis and Direct Sequencing

Department of Microbiology and Immunology, Cancer Research Institute and Liver Research Institute, College of Medicine, Seoul National University, Seoul 110-799,¹ Mogam Biotechnology Research Institute Diagnostic Lab, Yongin 449-903,² The Korean Institute of Tuberculosis, The Korean National Tuberculosis Association, Seoul 137-140, South Korea³

Received 13 June 2006/ Returned for modification 6 August 2006/ Accepted 15 August 2006

Here we describe a novel duplex PCR method which can differentiate Mycobacterium tuberculosis and nontuberculosis mycobacteria (NTM) strains by amplifying hsp65 DNAs of different sizes (195 and 515 bp, respectively). The devised technique was applied to 54 reference and 170 clinical isolates and differentiated all strains into their respective groups with 100% sensitivity and specificity. Furthermore, a duplex PCR-restriction analysis (duplex PRA) and a direct sequencing protocol were developed to differentiate NTM strains at the species and subspecies levels based on previously reported hsp65 DNA sequences (H. Kim et al., Int. J. Syst. Evol. Microbiol. 55:1649-1656, 2005) and then applied to 105 NTM clinical isolates. All NTM isolates were clearly differentiated at the species and subspecies levels by subsequent procedures (PRA or direct sequencing) targeting 515-bp NTM duplex PCR amplicons. Our results suggest that novel duplex PCR-based methods are sensitive and specific for identifying mycobacterial culture isolates at the species level.

Published ahead of print on 23 August 2006.

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