Diagnostic Real-Time PCR Assays for the Detection of Emetic Bacillus cereus Strains in Foods and Recent Food-Borne Outbreaks

Abstract

Cereulide-producing Bacillus cereus can cause an emetic type of food-borne disease that mimics the symptoms provoked by Staphylococcus aureus. Based on the recently discovered genetic background for cereulide formation, a novel 5 nuclease (TaqMan) real-time PCR assay was developed to provide a rapid and sensitive method for the specific detection of emetic B. cereus in food. The TaqMan assay includes an internal amplification control and primers and a probe designed to target a highly specific part of the cereulide synthetase genes. Additionally, a specific SYBR green I assay was developed and extended to create a duplex SYBR green I assay for the one-step identification and discrimination of the two emesiscausing food pathogens B. cereus and S. aureus. The inclusivity and exclusivity of the assay were assessed using a panel of 100 strains, including 23 emetic B. cereus and 14 S. aureus strains. Different methods for DNA isolation from artificially contaminated foods were evaluated, and established real-time assays were used to analyze two recent emetic food poisonings in southern Germany. One of the food-borne outbreaks included 17 children visiting a day care center who vomited after consuming a reheated rice dish, collapsed, and were hospitalized; the other case concerned a single food-poisoning incident occurring after consumption of cauliflower. Within 2 h, the etiological agent of these food poisonings was identified as emetic B. cereus by using the real-time PCR assay.

Detection of Emetic Bacillus cereus by Real-Time PCR in Foods

“…Although Fricker et al 2007 andWehrle et al 2010 used multiplex real-time PCR assays to simultaneously detect different enteropathogenic bacteria, simplex real-time PCR assays based on TaqMan probe and SYBR green I could be used for the rapid and conclusive identification of emetic type strains among B. cereus isolates, as well as some selective agar media normally utilized for isolation of foodborne B. cereus. However, the enrichment of foods contaminated with the level of 10 0 cfu/g was required for the assays.…”

“…Primers and probes Table 1 designed by Fricker et al 2007 , targeting a region of the ces genes, were synthesized by Life Technologies Japan TM and used for conventional PCR and real-time PCR assays.…”

Detection of B. Cereus and Some of Its Virulence Genes in Some Dairy Desserts and Children Diarrhea

“…cereus is undervalued as a foodborne pathogen, despite the fact that, it has been implicated in various foodborne outbreaks worldwide (Bennett et al, 2013) and this could be attributed to the lack of effective surveillance, underreporting of B. cereus linked food poisoning, and cases of B. cereus food poisoning apparently confused with Staphylococcus aureus and Clostridium perfringens food poisoning due to comparable symptoms (Fricker et al, 2007;StenforsArnesen et al, 2008), finally, B. cereus is not taken in account when diarrhea cases are diagnosed even if the fecal samples gave negative results for the presence of Salmonella, Shigella and Entamoeba in the middle east (Organji et al, 2015).…”

The Application of PCR-Based Methods in Food Control Agencies – A Review

“…Fricker et al, (2007) reported on the successful application of real-time PCR in the detection of B. cereus, which together with the closely associated S. aureus are the two most important bacteria responsible for food-associated intoxications. The traditional detection of the emetic toxin associated with these bacteria is often difficult, time consuming and expensive.…”