Rapid detection of IMP, NDM, VIM, KPC and OXA-48-like carbapenemases from Enterobacteriales and Gram-negative non-fermenter bacteria by real-time PCR and melt-curve analysis

European Journal of Clinical Microbiology & Infectious Diseases : Official Publication of the European Society of Clinical Microbiology
Massimo MentastiMandy Wootton

Abstract

Carbapenemase-producing microorganisms are increasingly isolated and often associated with treatment failures and outbreaks. The need for reliable and timely detection and/or confirmation of carbapenemase production is paramount; therefore, a real-time PCR assay targeting IMP, NDM, VIM, KPC and OXA-48-like carbapenemases was designed and validated. All available allele variants of the above carbapenemases were downloaded from the Beta-Lactamase DataBase ( http://bldb.eu/ ), aligned with Clustal Omega and primers designed using Primer-BLAST. Real-time PCR monoplexes were optimized for the QuantStudio 6-Flex (Applied Biosystems) using the PowerUp SYBR Green Master Mix (Life Technologies) and validated using a panel of 204 characterised strains carrying a wide range of beta-lactamases, sometimes in combination. Melt-curve analysis was used to confirm positive results. The in silico approach allowed primers to be designed in conserved regions of the KPC and NDM alignments, while three primer sets for IMP and two for VIM were necessary to ensure amplification of the different variants. One primer set was designed for OXA-48-like; however, it is unlikely to detect all variants. Expected results were obtained for all 204 tested strain...Continue Reading

References

Jan 30, 2008·The Journal of Antimicrobial Chemotherapy·Orjan SamuelsenArnfinn Sundsfjord
Sep 4, 2014·The Journal of Antimicrobial Chemotherapy·Laura L WrightNeil Woodford
Jan 27, 2017·Journal of Clinical Microbiology·Laurent Dortet, Thierry Naas
Apr 22, 2017·Nucleic Acids Research·Szymon ChojnackiRodrigo Lopez
Jul 20, 2017·Journal of Enzyme Inhibition and Medicinal Chemistry·Thierry NaasBogdan I Iorga
Nov 23, 2017·Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America·Robert A BonomoLars F Westblade
May 20, 2018·Diagnostic Microbiology and Infectious Disease·Marianne LundMikala Wang
May 28, 2019·The Journal of Antimicrobial Chemotherapy·Saoussen OueslatiThierry Naas

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Datasets Mentioned

BETA
M62653

Methods Mentioned

BETA
PCR
electrophoresis
the

Software Mentioned

generated
Clustal Omega
Primer
BLAST
Clustal

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