title: Multiplex real-time PCR assay for the detection of extended-spectrum β-lactamase and carbapenemase genes using melting curve analysis.
abstract: Real-time PCR melt curve assays for the detection of β-lactamase, extended-spectrum β-lactamase and carbapenemase genes in Gram-negative bacteria were developed. Two multiplex real-time PCR melt curve assays were developed for the detection of ten common β-lactamase genes: blaKPC-like, blaOXA-48-like, blaNDM-like, blaVIM-like, blaIMP-like, blaCTX-M-1+2-group, blaCMY-like, blaACC-like, blaSHV-like and blaTEM-like. The assays were evaluated using 25 bacterial strains and 31 DNA samples (total n=56) comprising different Enterobacteriaceae genera and Pseudomonas spp. These strains were previously characterized at five research institutes. Each resistance gene targeted in this study generated a non-overlapping and distinct melt curve peak. The assay worked effectively and detected the presence of additional resistance genes in 23 samples. The assays developed in this study offer a simple, low cost method for the detection of prevalent β-lactamase, ESBL and carbapenemase genes among Gram-negative pathogens.
authors.affiliation: Food Science Program, University of Missouri, Columbia, USA.
authors.forename: Prashant
authors.initials: P
authors.lastname: Singh
authors.affiliation: Nosocomial Pathogens and Antibiotic Resistance, Robert Koch Institute, 38855 Wernigerode, Germany.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Pfeifer
authors.affiliation: Food Science Program, University of Missouri, Columbia, USA. Electronic address: MustaphaA@missouri.edu.
authors.forename: Azlin
authors.initials: A
authors.lastname: Mustapha
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D015780:Carbapenems / Q000494:pharmacology; D006090:Gram-Negative Bacteria / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification*; D016905:Gram-Negative Bacterial Infections / Q000382:microbiology; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D015780:Carbapenems; D001618:beta-Lactamases; C063912:carbapenemase
keywords: Antibiotic resistance; ESBL; Enterobacteriaceae