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.

FIG. 1.

(a) Characteristic melting peaks (colored lines) of the amplicons generated by primers targeting the five MβL types so far described when MβL-harboring clinical isolates were submitted to the real-time PCR assay. Colors and genes targeted, from left to right, are as follows: blue, bla_GIM-1 (T_m, 72.0°C); red, bla_IMP-type genes (T_m, 76.5°C); green, bla_SIM-1 (T_m, 80.5°C); pink, bla_SPM-1 (T_m, 83.5°C); orange, bla_VIM-type genes (T_m, 89.0°C). (b) Amplicons generated by primers targeting the five MβL types and the internal-control gene (16S rRNA). Visualization was performed in a UV light box after electrophoresis on a 1.5% agarose gel containing 0.5 μg/ml ethidium bromide. Lane 1, SPM-1 amplicon (798 bp; strain 48-1997A); lane 2, SIM-1 amplicon (569 bp; strain 03-9-T104); lane 3, VIM-type amplicon (382-bp; strain 7-406); lane 4, IMP-type amplicon (188 bp; strain 48-696D); lane 5, GIM-1 amplicon (72 bp; strain 73-5671); lanes 6 to 9, the internal-control amplicon (1,499 bp; strains A. calcoaceticus ATCC 33305, P. aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 700603, and Enterobacter aerogenes ATCC 13048, respectively); lane 10, negative control; lanes M, molecular size markers (50-bp DNA ladder; Invitrogen).