Rapid detection and identification of metallo-beta-lactamase-encoding genes by multiplex real-time PCR assay and melt curve analysis.
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.
Mendes
Rodrigo E
RE
Division of Infectious Diseases, Federal University of São Paulo, Rua Leandro Dupret, 188, São Paulo, SP, Brazil CEP 04025-010. rodrigo.mendes@lemc.com.br
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
D015373:Bacterial Typing Techniques; D017931:DNA Primers; D006090:Gram-Negative Bacteria / Q000145:classification* / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011550:Pseudomonas aeruginosa / Q000145:classification / Q000201:enzymology / Q000235:genetics; D013997:Time Factors; D044366:Transition Temperature; D001618:beta-Lactamases / Q000096:biosynthesis / Q000235:genetics*
D017931:DNA Primers; D001618:beta-Lactamases