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Journal of Clinical Microbiology, August 2001, p. 2799-2806, Vol. 39, No. 8
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.8.2799-2806.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Identification of 54 Mycobacterial Species by PCR-Restriction Fragment Length Polymorphism Analysis of the hsp65 Gene

Francesca Brunello,1 Marco Ligozzi,1 Emanuela Cristelli,1 Stefano Bonora,2 Enrico Tortoli,3 and Roberta Fontana1,*

Dipartimento di Patologia, Sezione di Microbiologia, Università di Verona and Servizio di Microbiologia dell'Azienda Ospedaliera di Verona, Verona,1 Clinica delle Malattie Infettive, Università di Torino, Turin,2 and Servizio di Microbiologia, Ospedale Careggi Firenze e Centro di Riferimento Nazionale per i Micobatteri, Florence,3 Italy

Received 19 March 2001/Returned for modification 29 April 2001/Accepted 28 May 2001

A total of 121 reference and clinical strains of both slowly and rapidly growing mycobacteria belonging to 54 species were studied for restriction fragment length polymorphism of a PCR-amplified 439-bp segment of the gene encoding the 65-kDa heat shock protein. Restriction digests were separated by 10% polyacrylamide gel electrophoresis (PAGE). By including a size standard in each sample, the restriction fragment profile was calculated using a computer-aided comparison program. An algorithm describing these 54 species (including 22 species not previously described) is proposed. We found that this assay based on 10% PAGE provided a more precise estimate than that based on agarose gel electrophoresis of the real size of restriction fragments as deduced from the sequence analysis and allowed identification of mycobacteria whose PCR-restriction fragment length polymorphism analysis patterns were unequivocally identified by fragments shorter than 60 bp.

* Corresponding author. Mailing address: Università di Verona, Dipartimento di Patologia, Sezione di Microbiologia; Strada le Grazie 8, 37100 Verona, Italy. Phone: 0039-45-8028191. Fax: 0039-45-584606. E-mail: roberta.fontana{at}univr.it.

Journal of Clinical Microbiology, August 2001, p. 2799-2806, Vol. 39, No. 8
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.8.2799-2806.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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