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 T m ) 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 10 1 CFU/ml for both TSB and NFDM‐grown B. cereus compared to 10 4 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)