Development of a Two Triplex Real-Time Polymerase Chain Reaction for Rapid Detection of Six Carbapenemase Genes in Enterobacteriaceae

Abstract

Carbapenem resistance is a serious clinical and public health threat. Carbapenemase can confer carbapenem resistance, and most carbapenemase genes are plasmid encoded so resistance can easily spread. In this study, we aimed to develop a novel system based on the TaqMan platform for the rapid detection of 6 clinically prevalent carbapenemase genes: Klebsiella pneumoniae carbapenemase, New Delhi metallo-β-lactamase, oxacillinase, imipenem-hydrolyzing, Verona integron-encoded metalloβ-lactamase, and Guiana extended-spectrum β-lactamase. Methods: The triplex assay was verified by testing genomic DNA of 6 carbapenemase-producing Klebsiella pneumoniae. It was validated with a blinded panel of 310 Enterobacteriaceae isolates, including 225 carbapenemase-producers and 85 non-producers, by direct colony triplex real-time polymerase chain reaction (PCR). The real-time PCR was performed using the ABI 7500 fast instrument (Applied Biosystems, CA, USA) and specific primers for each carbapenemase target were designed to include modified peptide-nucleic acid oligonucleotides. Results: No amplification was detected among the negative samples. The result showed 100% concordance with the genotypes previously identified. The entire assay, including DNA extraction and real-time PCR, was completed within 2 hours. Conclusion: The newly developed triplex real-time PCR assay was useful for the rapid, accurate and simultaneous detection of 6 carbapenemase genes in Enterobacteriaceae, suggesting its potential to allow an early decision on the appropriate treatment, management, and prevention of the spread of resistant infections in hospitals.

Fast Track Diagnostic Tools for Clinical Management of Sepsis: Paradigm Shift from Conventional to Advanced Methods

“…Furthermore, the results of the diagnosis should be able to provide appropriate decisions and antibiotic stewardship within a key time window of hours in order to limit morbidity and death [14]. The procedures that are sensitive, specific and quick for identifying the pathogen are therefore the major operational instruments for critical care units [18,19].…”