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ARS Home » Plains Area » Clay Center, Nebraska » U.S. Meat Animal Research Center » Meat Safety and Quality » Research » Publications at this Location » Publication #402196

Research Project: Holistic Tactics to Advance the Microbiological Safety and Quality of the Red Meat Continuum

Location: Meat Safety and Quality

Title: Multiplex high resolution melt curve real-time PCR assay for detection of extended-spectrum Beta-lactam-resistant Shiga toxin-producing E. coli

item DHITAL, RAJIV - University Of Missouri
item Bosilevac, Joseph - Mick
item Schmidt, John
item MUSTAPHA, AZLIN - University Of Missouri

Submitted to: Food Control
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/24/2023
Publication Date: N/A
Citation: N/A

Interpretive Summary: Various Escherichia coli can contaminate beef. Shiga toxin-producing E. coli (STEC) are severe pathogens that can present a food safety problem, while antibiotic resistant E. coli can be an indicator of microbiological quality. A new test was developed and validated that identifies several genes of STEC and antibiotic resistant E. coli at the same time. The new assay can be used as a rapid, specific, and cost-effective tool for routine examination of beef for these E. coli, even when present at low numbers.

Technical 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 extended-spectrum ß-lactamase (ESBL)-producing 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 4-8 h were required to achieve 10 CFU/325g detection limit in inoculated beef. The multiplex HRM real-time PCR assay can be used as a rapid, specific, and cost-effective tool for routine examination of STEC and ESBL producing bacteria that are present in low concentrations in beef products.