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Triplex PCR‐based detection of enterotoxigenic Bacillus cereus ATCC 14579 in nonfat dry milk
Author(s) -
Gracias Kiev S.,
McKillip John L.
Publication year - 2011
Publication title -
journal of basic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.58
H-Index - 54
eISSN - 1521-4028
pISSN - 0233-111X
DOI - 10.1002/jobm.200900348
Subject(s) - bacillus cereus , cereus , tryptic soy broth , microbiology and biotechnology , amplicon , enterotoxin , food science , biology , multiplex polymerase chain reaction , chemistry , bacteria , polymerase chain reaction , escherichia coli , gene , biochemistry , genetics
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)