Multiplex PCR for rapid detection of genes encoding CTX-M extended-spectrum β-lactamases

Abstract

Sir, CTX-M extended-spectrum β-lactamases (ESBLs) are increasingly prevalent worldwide among Escherichia coli and Klebsiella spp., including in the UK, where over half of all microbiology laboratories have encountered producers.1,2 More than 40 CTX-M β-lactamases have been described and are divided into five phylogenetic groups, with different groups prevalent in different countries.3 However, characterization of the blaCTX-M alleles present in clinical isolates is time-consuming, typically requiring multiple PCRs with universal and then group-specific primers.1,2,4 To facilitate monitoring, we sought to develop a multiplex assay to differentiate alleles belonging to these five phylogenetic groups. The sequences of reference blaCTX-M alleles (source: http://www.lahey.org/studies/) were aligned, and group-specific regions were identified. Primers specific for alleles encoding enzymes belonging to groups 1, 2 and 9 were designed in silico using Primer 3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi), whereas those for blaCTX-M-25/-26 and for blaCTX-M-8/-40 were developed and optimized empirically. Primer pairs and predicted amplicon sizes were: group 1, 5′-AAA AAT CAC TGC GCC AGT TC and 5′-AGC TTA TTC ATC GCC ACG TT (415 bp); group 2, 5′- CGA CGC TAC CCC TGC TAT T and 5′-CCA GCG TCA GAT TTT TCA GG (552 bp); group 9, 5′-CAA AGA GAG TGC AAC GGA TG and 5′-ATT GGA AAG CGT TCA TCA CC (205 bp). Fragments of alleles encoding enzymes of groups 8 (666 bp) and 25 (327 bp) were amplified with two specific forward primers and a shared reverse primer: 5′-TCG CGT TAA GCG GAT GAT GC (group 8 forward); 5′-GCA CGA TGA CAT TCG GG (group 25 forward); and 5′-AAC CCA CGA TGT GGG TAG C (groups 8/25 reverse).