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Rapid detection and identification of metallo-beta-lactamase-encoding genes by multiplex real-time PCR assay and melt curve analysis.

ABSTRACT: Metallo-beta-lactamase enzymes (MbetaL) 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 MbetaL-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 MbetaL-harboring clinical isolates, and the Tm-assigned genotypes were 100% coincident with previous sequencing results. This assay could be suitable for identification of MbetaL-producing gram-negative bacteria by molecular diagnostic laboratories.

SUBMITTER: Mendes RE 

PROVIDER: S-EPMC1829038 | biostudies-literature | 2007 Feb

REPOSITORIES: biostudies-literature

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Rapid detection and identification of metallo-beta-lactamase-encoding genes by multiplex real-time PCR assay and melt curve analysis.

Mendes Rodrigo E RE   Kiyota Katia A KA   Monteiro Jussimara J   Castanheira Mariana M   Andrade Soraya S SS   Gales Ana C AC   Pignatari Antonio C C AC   Tufik Sergio S  

Journal of clinical microbiology 20061108 2

Metallo-beta-lactamase enzymes (MbetaL) 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 MbetaL-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 t  ...[more]

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