A PCR assay for the detection of Bacillus cereus strains able to produce an
emetic toxin (
cereulide) was developed in this study based on a sequence-characterized amplified region (
SCAR) derived from a random amplified polymorphic
DNA (RAPD) fragment. One of the RAPD fragments generated was selected, cloned, and sequenced. A set of PCR primers was newly designed from the
SCAR obtained (the sequence of the cloned RAPD fragment) and used in this assay. To determine the specificity of the assay, 30 different B. cereus strains, 8 other Bacillus strains (of six species), and 16 other non-Bacillus strains (from 16 genera) were tested. Results were positive for every
emetic B. cereus strain and for only one nonemetic B. cereus strain. For all other bacterial strains, results were negative.
Bacterial DNA for PCR was prepared by a simple procedure using
Chelex 100 resin from the bacterial colony on the
agar plate or from culture after growth in brain heart infusion medium. This PCR assay enabled us to detect the bacteria of
emetic B. cereus grown on
agar plates but not the bacteria of nonemetic B. cereus. To test this PCR assay for the monitoring of the
emetic bacteria, 10 to 70 CFU of B. cereus DSM 4312 (
emetic) per g of food was inoculated into several foods as an
indicator, followed by a 7-h enrichment culture step. Because this PCR assay based on the
SCAR derived from the RAPD fragment was able to detect bacterial cells, this assay should be useful for rapid and specific detection of
emetic B. cereus.