Optimization of DNA extraction to detect Bacillus cereus from food using a PCR technique-

Bacillus species are extremely widespread in nature and may be isolated from soil, water, dust, air and many foods. The genus Bacillus is characterized by the formation of heat resistant endospores. B. cereus has been recently considered as an ‘emerging’ food-borne pathogen. A couple of new primers (BCFW1 and Bcrevnew) directed to identify B. cereus is described as well as protocols for optimization of DNA extraction for basic groups of food products which are particularly prone to contain this microorganism. To avoid inhibitory effects exerted by particular food matrices, specific pre-extraction steps are recommended in this work: a treatment with α-amylase from Aspergillus oryzae E.C. 3.2.1.1. (Sigma, Germany) for boiled rice or washing the pellet with EDTA solution for minced meat, pasteurized milk and salads. The lowest possible level of detection was obtained for pasteurized milk (20 cells/g) and salad (30 cells/g) and it was comparable with the results of classical microbiological analyses. Generally, for milk, salad and rice samples the limit of detection was close to 50 cells/g, whereas for minced meat it was definitively higher (about 500 cells/g) and did not reflect results of performed standard plate counts. As the result of the studies, promising, handy protocols for (i) an efficient DNA extraction from food matrices and (ii) fast, species-specific PCR identification were prepared and presented.