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Development of a Novel Real-Time PCR Assay with High-Resolution Melt Analysis To Detect and Differentiate OXA-48-Like β-Lactamases in Carbapenem-Resistant Enterobacteriaceae

ABSTRACT The rapid global spread of carbapenem-resistant Enterobacteriaceae (CRE) poses an urgent threat to public health. More than 250 class D β-lactamases (OXAs) have been described in recent years, with variations in hydrolytic activity for β-lactams. The plasmid-borne OXA-48 β-lactamase and its variants are identified only sporadically in the United States but are common in Europe. Recognition of these OXA-48-like carbapenemases is vital in order to control their dissemination. We developed a real-time PCR assay based on high-resolution melt analysis, using blaOXA-48-like-specific primers coupled with an unlabeled 3′-phosphorylated oligonucleotide probe (LunaProbe) homologous to OXA-48-like carbapenemase genes. The assay was validated using genomic DNA from 48 clinical isolates carrying a variety of carbapenemase genes, including blaKPC, blaSME, blaIMP, blaNDM-1, blaVIM, blaOXA-48, blaOXA-162, blaOXA-181, blaOXA-204, blaOXA-244, blaOXA-245, and blaOXA-232. Our assay identified the presence of blaOXA-48-like β-lactamase genes and clearly distinguished between blaOXA-48 and its variants in control strains, including between blaOXA-181 and blaOXA-232, which differ by only a single base pair in the assay target region. This approach has potential for use in epidemiological investigations and continuous surveillance to help control the spread of CRE strains producing OXA-48-like enzymes.