A Colony Blot Immunoassay for the Rapid Identification of Bacillus cereus

Abstract

A colony blot immunoassay was developed for the rapid identification of Bacillus cereus using antibodies against the 28.5-kDa cell-surface antigen of B. cereus. Suspect colonies from plates were blotted onto a Whatman #541 membrane, dried, and fixed by UV irradiation. The membrane was then immersed in an anti-B. cereus antibody-horseradish peroxidase conjugate for 60 min. After washing and reacting with 4-chloro-1-naphthol and H2O2, the appearance of purple spots indicated the presence of B. cereus. This assay effectively identified 61 of 62 B. cereus strains tested. Among 38 non-B. cereus strains, which were other Bacillus spp. (19 genera), 36 gave true-negative results, and 2 showed false-positive results. The sensitivity and specificity for B. cereus were 98.4 and 94.7%, respectively. The present assay is easy to use, and the rapid identification of B. cereus can be completed in 2.5 h.