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Journal of Clinical Microbiology, February 2007, p. 544-547, Vol. 45, No. 2
0095-1137/07/$08.00+0     doi:10.1128/JCM.01728-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Rapid Detection and Identification of Metallo-ß-Lactamase-Encoding Genes by Multiplex Real-Time PCR Assay and Melt Curve Analysis{triangledown}

Rodrigo E. Mendes,1,2* Katia A. Kiyota,2 Jussimara Monteiro,1,2 Mariana Castanheira,1 Soraya S. Andrade,1,2 Ana C. Gales,1 Antonio C. C. Pignatari,1 and Sergio Tufik2,3

Laboratório Especial de Microbiologia Clínica and Laboratório ALERTA, Division of Infectious Disease, Federal University of São Paulo,1 AFIP—Medicina Laboratorial, São Paulo,2 Department of Psychobiology, Federal University of São Paulo, São Paulo, Brazil3

Received 21 August 2006/ Returned for modification 26 September 2006/ Accepted 30 October 2006

Metallo-ß-lactamase enzymes (MßL) are encoded by transferable genes, which appear to spread rapidly among gram-negative bacteria. The objective of this study was to develop a multiplex real-time PCR assay followed by a melt curve step for rapid detection and identification of genes encoding MßL-type enzymes based on the amplicon melting peak. The reference sequences of all genes encoding IMP and VIM types, SPM-1, GIM-1, and SIM-1 were downloaded from GenBank, and primers were designed to obtain amplicons showing different sizes and melting peak temperatures (Tm). The real-time PCR assay was able to detect all MßL-harboring clinical isolates, and the Tm-assigned genotypes were 100% coincident with previous sequencing results. This assay could be suitable for identification of MßL-producing gram-negative bacteria by molecular diagnostic laboratories.

* Corresponding author. Mailing address: Division of Infectious Diseases, Federal University of São Paulo, Rua Leandro Dupret, 188, São Paulo, SP, Brazil CEP 04025-010. Phone: (55-11) 5081-2819 or -2965. Fax: (55-11) 5571-5180. E-mail: rodrigo.mendes{at}lemc.com.br.

{triangledown} Published ahead of print on 8 November 2006.

Journal of Clinical Microbiology, February 2007, p. 544-547, Vol. 45, No. 2
0095-1137/07/$08.00+0     doi:10.1128/JCM.01728-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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