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KMID : 1120220200110010053
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Osong Public Health and Research Perspectives
2020 Volume.11 No. 1 p.53 ~ p.59
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Development of a Two Triplex Real-Time Polymerase Chain Reaction for Rapid Detection of Six Carbapenemase Genes in Enterobacteriaceae
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Choi Ji-Ae
Bae Song-Mee
Kim Jung-Wook
Lee Kwang-Jun
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Abstract
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Objectives: 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 metallo-β-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.
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KEYWORD
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carbapenemase, Enterobacteriaceae, real-time PCR, triplex PCR,
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