title: Enterotoxigenic Bacillus spp. DNA fingerprint revealed in naturally contaminated nonfat dry milk powder using rep-PCR.
abstract: Dry milk powders and functional ingredients frequently contain high levels of viable bacterial spores, some of which may result in growth of toxigenic Bacillus spp. in reconstituted and temperature-abused foods. Samples from nonfat dry milk (NFDM), infant milk formula (IMF), coffee creamer, lecithin, and cocoa powder were subjected to a short heat treatment followed by enrichment in tryptone phosphate glucose yeast extract (TPGY) broth at 32 degrees C for 12-25 hours to obtain cell densities of 10(6) CFU ml(-1). DNA was extracted using a modification of established protocol, leading to the development of an optimized method for each food system. Purified DNA was amplified by rep-PCR using extragenic sequence-targeting primers and optimized for each food. PCR fingerprints from each food were analyzed electrophoretically for banding patterns earlier correlated to that of enterotoxigenic Bacillus spp. and Bacillus cereus positive control DNA fingerprints. Reverse passive latex agglutination (RPLA) and Bacillus Diarrhoeal Enterotoxin Enzyme Linked Immunosorbent Assay (Tecra Diagnostics) confirmed the presence of HBL and NHE enterotoxin production in NFDM, Coffee creamer, infant milk formula, and two lecithin samples but not in cocoa powder. These results demonstrate the utility of rep-PCR not only as a tool for bacterial genotyping, but a unique means of quality control and hygiene monitoring in food microbiology.
authors.affiliation: Roche Diagnostics Corporation, Indianapolis, IN 46256, USA.
authors.forename: Robin M
authors.initials: RM
authors.lastname: Cooper
authors.forename: John L
authors.initials: JL
authors.lastname: McKillip
mesh_terms: D000818:Animals; D001407:Bacillus / Q000235:genetics / Q000302:isolation & purification*; D016172:DNA Fingerprinting; D004768:Enterotoxins / Q000032:analysis*; D005516:Food Microbiology; D008892:Milk / Q000737:chemistry / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013171:Spores, Bacterial / Q000302:isolation & purification
chemical_list: D004768:Enterotoxins