Rapid detection and identification of metallo-beta-lactamase-encoding genes by multiplex real-time PCR assay and melt curve analysis |
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| Authors: | Mendes Rodrigo E Kiyota Katia A Monteiro Jussimara Castanheira Mariana Andrade Soraya S Gales Ana C Pignatari Antonio C C Tufik Sergio |
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| Affiliation: | Division of Infectious Diseases, Federal University of S?o Paulo, Rua Leandro Dupret, 188, S?o Paulo, SP, Brazil CEP 04025-010. rodrigo.mendes@lemc.com.br |
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| 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. |
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