Development of a set of multiplex PCR assays for the detection of genes encoding important β-lactamases in Enterobacteriaceae
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Open Access
- 12 January 2010
- journal article
- research article
- Published by Oxford University Press (OUP) in Journal of Antimicrobial Chemotherapy
- Vol. 65 (3), 490-495
- https://doi.org/10.1093/jac/dkp498
Abstract
To develop a rapid and reliable tool to detect by multiplex PCR assays the most frequently widespread β-lactamase genes encoding the OXA-1-like broad-spectrum β-lactamases, extended-spectrum β-lactamases (ESBLs), plasmid-mediated AmpC β-lactamases and class A, B and D carbapenemases. Following the design of a specific group of primers and optimization using control strains, a set of six multiplex PCRs and one simplex PCR was created. An evaluation of the set was performed using a collection of 31 Enterobacteriaceae strains isolated from clinical specimens showing a resistance phenotype towards broad-spectrum cephalosporins and/or cephamycins and/or carbapenems. Direct sequencing from PCR products was subsequently carried out to identify β-lactamase genes. Under optimized conditions, all positive controls confirmed the specificity of group-specific PCR primers. Except for the detection of carbapenemase genes, multiplex and simplex PCR assays were carried out using the same PCR conditions, allowing assays to be performed in a single run. Out of 31 isolates selected, 22 strains produced an ESBL, mostly CTX-M-15 but also CTX-M-1 and CTX-M-9, SHV-12, SHV-5, SHV-2, TEM-21, TEM-52 and a VEB-type ESBL, 6 strains produced a plasmid-mediated AmpC β-lactamase (five DHA-1 and one CMY-2) and 3 strains produced both an ESBL (two SHV-12, one CTX-M-15) and a plasmid-mediated AmpC β-lactamase (DHA-1). We report here the development of a useful method composed of a set of six multiplex PCRs and one simplex PCR for the rapid screening of the most frequently encountered β-lactamases. This method allowed direct sequencing from the PCR products.Keywords
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