Triplex PCR-based detection of enterotoxigenic Bacillus cereus ATCC 14579 in nonfat dry milk



Although many strains of Bacillaceae are considered nonpathogenic, Bacillus cereus is recognized worldwide as a bacterial pathogen in a variety of foods. The ability of B. cereus to cause gastroenteritis following ingestion of contaminated food is due to the production of enterotoxins. The ubiquity of this genus makes it a persistent problem for quality assurance in food processing environments. The primary objective of this study was to develop and apply a multiplex real-time PCR-based assay for rapid and sensitive detection of enterotoxigenic B. cereus. Template DNA was separately extracted from tryptic soy broth (TSB)-grown and 2.5% Nonfat Dry Milk (NFDM)-grown B. cereus using a commercial system. Three enterotoxin gene fragments (hblC, nheA, and hblA) were simultaneously amplified in real-time followed by melting curve analysis to confirm amplicon identity. Resolution of melting curves (characteristic Tm) was achieved for each amplicon (hblC = 74.5 °C; nheA = 78 °C; and hblA = 85.5 °C in TSB and 84 °C in NFDM) with an assay sensitivities of 101 CFU/ml for both TSB and NFDM-grown B. cereus compared to 104 CFU/ml in either matrix using gel electrophoresis. The results demonstrate the potential sensitivity of real-time bacterial detection methods in a heterogenous food matrix using real-time PCR. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)