Detection of Escherichia coli O157:H7 in Food Using Real-Time Multiplex PCR Assays Targeting the stx 1, stx 2, wzy O157, and the fliC h7 or eae Genes

Abstract

Escherichia coli O157:H7 is an important foodborne pathogen, and foods of bovine origin and fresh produce have been linked to outbreaks. Real-time multiplex PCR assays were developed to detect E. coli O157:H7 in different foods. Apple cider and raw milk (25 ml) and ground beef and lettuce (25 g) were inoculated with 2 or 20 colony-forming units (CFU) of E. coli O157:H7 380-94 and subjected to enrichment in RapidChek E. coli O157:H7 broth at 42°C. One milliliter of the enrichments was removed at 8 and 20 h, and following DNA extraction, real-time multiplex PCR assays targeting the stx 1 , stx 2 , and wzy O157 genes in combination with probes and primers targeting either the fliC h7 or the eae genes were performed using OmniMix HS beads and the SmartCycler. The sensitivity of the real-time multiplex PCR assay was about 225 CFU/PCR. E. coli O157:H7 was detected (fluorescent signal generated for all gene targets) in apple cider, raw milk, lettuce and ground beef samples inoculated with 2 or 20 CFU/g or 25 ml after both 8 and 20 h of enrichment. Enrichments of uninoculated food samples were negative using the multiplex PCR targeting the stx 1 , stx 2 , wzy O157 , and eae genes; however, using the assay targeting the stx 1 , stx 2 , wzy O157 , and fliC h7 gene combination, a positive result was always obtained for the fliC h7 gene using uninoculated ground beef enrichments. Use of other primer sets targeting the fliC h7 gene gave similar results. The real-time multiplex PCR assays targeting the stx 1 , stx 2 , eae, and wzy O157 or the fliC h7 genes are sensitive and specific and can be used for the detection of E. coli O157:H7 in food, except that the fliC h7 gene may not be a suitable target for the detection of E. coli O157:H7 in ground beef.

Evaluation of a Loop-Mediated Isothermal Amplification Suite for the Rapid, Reliable, and Robust Detection of Shiga Toxin-Producing Escherichia coli in Produce

“…Upon initial development, LAMP assays targeting STEC virulence genes (32) and seven O serogroups (33) were shown to be 100% specific by testing 90 and 120 strains, respectively. However, the specificity of qPCR assays evaluated in this study was not reported when initially developed (35), except that the O157 qPCR targeting the wzy gene was reported to be 100% specific (34). Such high specificity also agreed with that for other LAMP assays for STEC and E. coli O157 (20,24,29,30).…”

“…As a comparison, qPCR assays developed by USDA scientists (34,35) were performed. Similarly, 10 sets of primers/probes were used; 3 targeted common STEC virulence genes (stx 1 , stx 2 , and eae) and 7 targeted the wzx or wzy gene on the O-antigen gene clusters of the seven adulterant STEC serogroups.…”

“…The qPCR assays tested were recently developed by scientists from the U.S. Department of Agriculture (USDA) (34,35).…”

Rapid and Specific Detection of Escherichia coli Serogroups O26, O45, O103, O111, O121, O145, and O157 in Ground Beef, Beef Trim, and Produce by Loop-Mediated Isothermal Amplification

“…In addition, the findings of this study corroborated those of two others (8,47) that suggested comparable sensitivities of LAMP and qPCR. It is notable that LAMP assays took less time to run (50 min) than qPCR assays (at least 1.5 h) recently developed by USDA scientists (13,15), therefore markedly shortening the total assay time.…”

Detection of foodborne pathogens by qPCR: A practical approach for food industry applications

“…For this purpose, genes stx1, stx2, rfbE (lipopolysaccharide O-antigen synthesis gene for O157), wbdI (O111), wzx (flippase gene for O26), wzy (O-antigen polymerase specific for O113), wzy (O91), wbgN (glycosyltransferase gene specific for O55), ihpl (based on the polymorphism between the genes encoding the putative adhesin from "O-island 29" of STEC O157 and STEC O145 O145), eae (adherence intimin protein for O103), and fliC H7 (flagellar antigen H7) were successfully applied (DebRoy et al, 2004;Gonzales et al, 2011;Kagkli et al, 2011;Perelle, Dilasser, Grout, & Fach, 2003, 2004Samuel, Hogbin, Wang, & Reeves, 2004). Out of these, genes stx1 and stx2 (Shiga-toxin genes 1 and 2) are considered two major virulence factors for E. coli O157 and other STEC (Bonetta et al, 2011;Prendergast et al, 2011) and the eae gene has also been applied for serotype O157 detection by other authors (Adiguzel et al, 2012;Franz, Klerks, De Vos, Termorshuizen, & van Bruggen, 2007;Fratamico & DebRoy, 2010;Kawasaki et al, 2010;Zhang et al, 2009). Different combinations of several genes have been published and alternative targets have been selected, like hlyA (hemolysin) in combination with fliC, stx1, stx2, eae, and rfbE (Bai, Shi, & Nagaraja, 2010).…”