Detection of Shiga toxin producing Escherichia coli, stx1, stx2 and Salmonella by two high resolution melt curve multiplex real-time PCR

Authors: SINGH, PRASHANT - Florida State University; LIU, YUEJIAO - University Of Missouri; Bosilevac, Joseph - Mick; MUSTAPHA, AZLIN - University Of Missouri

Submitted to: Food Control
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/18/2018
Publication Date: 9/24/2018
Citation: Singh, P., Liu, Y., Bosilevac, J.M., Mustapha, A. 2018. Detection of Shiga toxin producing Escherichia coli, stx1, stx2 and Salmonella by two high resolution melt curve multiplex real-time PCR. Food Control. 96:251-259. https://doi.org/10.1016/j.foodcont.2018.09.024.
DOI: https://doi.org/10.1016/j.foodcont.2018.09.024

Interpretive Summary: During processing, meat such as beef can become contaminated with bacteria like E. coli and Salmonella that can make consumers sick. Therefore, meat processors and government agencies routinely test for these bacteria. There are 7 types of E. coli that must be individually tested for, while a separate test can detect all Salmonella. The most common tests require expensive reagents, 2 separate samples, and 4 separate test kits. This work describes an approach that identifies all 7 E. coli and Salmonella using just 2 tests and 1 sample. The tests do not use expansive reagents like fluorescent DNA probes but instead measures the temperature at which the DNA melts to identify the 8 different targets. The 2 tests were validated using inoculated ground beef and beef trim and on 200 natural beef samples. The inoculated samples were all identified correctly, while the natural samples were more challenging, but with most properly identified. The test described here is effective and economical for the routine screening of E. coli and Salmonella from the same beef sample.

Technical Abstract: In the United States, Shiga toxin-producing Escherichia coli (STEC) O157 and six non-O157 serogroups O26, O45, O103, O111, O121 and O145 are considered adulterants in non-intact beef. Further, Salmonella is responsible for one of the highest numbers of foodborne infections worldwide. Multiple foods, and especially meats, are routinely tested for these pathogens using methods like PCR. However, with such a large group of organisms, even multiplex detection systems are challenged by the number of targets required, not to mention the expense of differently labels oligo probes. The aim of this study was to design multiplex real-time PCR assays for the detection of seven STEC serogroups, stx1, stx2 genes and virulent strains of Salmonella. Two multiplex real-time PCR melt curve assays with internal amplification controls (IAC) were standardized. The first assay detected E. coli O121, E. coli O145, E. coli O157, stx1, and stx2. The second assay targeted E. coli O26, E. coli O111, E. coli O103, E. coli O45, and Salmonella. Ground beef and beef trim inoculated with 5-27 CFU/325 g of STEC and 9-36 CFU/325 g of Salmonella could be detected following an 8-10 h enrichment at 40°C ± 2°C in buffered peptone water containing 8 mg/L vancomycin. The assays showed reproducible results for beef products with different fat contents. These assays do not rely on fluorescent-labeled probes or immunomagnetic beads, yet accurately detect seven STEC serogroups, seven stx gene subtypes and Salmonella, making them suitable for routine testing of STEC and Salmonella in beef.