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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 #381346

Research Project: Mitigation Approaches for Foodborne Pathogens in Cattle and Swine for Use During Production and Processing

Location: Meat Safety and Quality

Title: High-resolution melt assay for detection of virulent lineages of Shiga toxin-producing Escherichia coli O26 and O111

item VELEZ, FRANK - Florida State University
item Bosilevac, Joseph - Mick
item SINGH, PRASHANT - Florida State University

Submitted to: Journal of Food Protection
Publication Type: Abstract Only
Publication Acceptance Date: 5/1/2021
Publication Date: 7/18/2021
Citation: Velez, F.J., Bosilevac, J.M., Singh, P. 2021. High-resolution melt assay for detection of virulent lineages of Shiga toxin-producing Escherichia coli O26 and O111. [Abstract]. Journal of Food Protection. 84(Suppl A): P. 151-152.

Interpretive Summary:

Technical Abstract: Introduction: In 2016, 5,441 culture-confirm cases of Shiga toxin-producing Escherichia coli (STEC) infection were reported in the United States. Infection by STEC strains results in bloody diarrhea, hemolytic uremic syndrome, and renal failure. According to the CDC, national enteric disease surveillance report STEC-O26 and -O111 are responsible for 16% and 10.7% of STEC cases, respectively. Presence of avirulent O26 and O111 E. coli in red meat can result in product hold up and financial losses. Previously, single nucleotide polymorphisms (SNP) in O-antigen genes were identified that associated with virulent STEC lineages. Purpose: This study aimed to develop a real-time PCR assay that identified E. coli O26 and E. coli O111 while determining if they were of a virulent STEC lineage. Methods: Serogroup-specific primers targeting the O26 fnl1 and O111 wbdK genes were designed. High-resolution melting real-time PCR assays were developed for specific identification of O26 and O111 serogroups and differentiation of virulent STEC lineages. The assay was validated using DNA from 101 bacterial strains, DNA samples from a federal regulatory surveillance program, and inoculated beef and spinach samples. Results: Serogroup-specific O26 and O111 primers showed 100% specificity. Virulent STEC-O26 and O111 formed distinct melt profiles in the normalized and differential melting curves plots from those of avirulent E. coli O26 and O111 strains. Overall the O26 and O111 HRM assays showed greater than 90% sensitivity and specificity. The assays were able to accurately detect all inoculated strains following a 15 h enrichment period. Significance: The assay developed in this study specificaly identifies O26 and O111 serogroups and differentiates the positive results into virulent STEC and avirulent E. coli lineages. It can be used to reduce the number of potential positive results caused by the presence of mixed avirulent E. coli in red meat.