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. 2020 Feb;11(1):53-59.
doi: 10.24171/j.phrp.2020.11.1.08.

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

Ji Ae Choi  1 , Song Mee Bae  1 , Jung Wook Kim  1 , Kwang Jun Lee  1
Affiliations

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

Ji Ae Choi et al. Osong Public Health Res Perspect. 2020 Feb.

Abstract

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.

Keywords: Enterobacteriaceae; carbapenemase; real-time PCR; triplex PCR.

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Conflict of interest statement

Conflicts of Interest The authors have no conflicts of interest declared.

Figures

Figure 1
Figure 1
Triplex real-time PCR efficiencies of 6 carbapenemases. KPC = Klebsiella pneumoniae carbapenemase; NDM = New Delhi metallo-β-lactamase; IMP = imipenem-hydrolyzing; VIM = Verona integron-encoded metallo-β-lactamase; GES = Guiana extended-spectrum β-lactamase; PCR = polymerase chain reaction.
See this image and copyright information in PMC

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