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 emesis-causing 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.

FIG. 1.

(A) Melting curves of SYBR green I real-time PCR products used for the detection of emetic Bacillus cereus (F4810/72) and Staphylococcus aureus (WS2608). DNA was isolated with the NucleoSpin food kit from a duplex enrichment experiment with artificial inoculated rice after 6 h. Cell counts correspond to 10⁸ CFU/g F4810/72 and 10⁷ CFU/g WS2608 (Table 5). The melting point of emetic B. cereus in the simplex reaction is about 80.0°C and shifts down to 79.3°C in the duplex reaction, whereas the melting point for S. aureus is about 83.4°C in both reactions. (B) Melting curves of the duplex SYBR green I real-time PCR products from the food-borne outbreaks (for details on the outbreaks, see Results and Discussion). Only the amplicon specific for emetic B. cereus could be detected in both cases.