Multiplex high resolution melt curve real-time PCR for detection of Shiga-toxin producing and blaCTX-M-harboring E. coli in beef products

Abstract

This study aimed to develop a rapid multiplex high resolution melt-curve (HRM) qPCR with an internal amplification control (IAC) for the simultaneous detection of Shiga-toxin producing Escherichia coli (STEC) and blaCTX-M-carrying E. coli in beef. Specific primer pairs targeting eaeA, uidA, stx1, stx2 and blaCTX-M genes were designed. The specificity of the optimized assay was validated using genomic DNA from Gram-positive, Gram-negative, and STEC (non-O157 and O157:H7) strains. The accuracy of the assay for detecting blaCTX-M was confirmed using genomic DNA from bacteria previously reported to carry this gene. Beef products were inoculated with a cocktail of E. coli O157:H7 and E. coli M-1, an isolate reported to harbor blaCTX-M, at concentrations of 10, 100, and 1000 CFU/mL. Among non-STEC DNA samples (n = 45) tested, no target genes were detected in 32 isolates. However, the assay was able to detect uidA and blaCTX-M in seven and six bacterial isolates, respectively. Among the STEC bacterial isolates (n = 57), at least two or more target genes were detected in 50 STEC isolates. All isolates carried the eaeA gene, 55 had stx1, 26 had stx2, and 26 had both stx genes. The uidA gene was detected in all non-O157:H7 isolates, whereas no E. coli O157:H7 isolates was found to be positive for uidA. The blaCTX-M gene was not detected in any of the STEC isolates tested but it was detected in 20 of 23 DNA samples from isolates previously verified as positive for the gene, thus demonstrating the technique's accuracy. Enrichment times ranging from 4 to 8 h were required to achieve a detection limit of 10 CFU/mL in 325 g of inoculated beef. The multiplex HRM real-time PCR assay can be used as a rapid, yet specific, tool for routine examination of STEC and blaCTX-M-harboring bacteria that are present in low concentrations in beef products.