Letters in Applied Microbiology 1998, 27, 168–172 A RAPD-PCR method for large-scale typing of Bacillus cereus J. Nilsson*, B. Svensson, K. Ekelund and A. Christiansson Swedish Dairies’ Association, Research and Development Department, Lund, Sweden 1750/98: received and accepted 28 May 1998 J. NILSSON, B. SVENSSON, K. EKELUND AND A. CHRISTIANSSON. 1998. A robust RAPD- PCR procedure for large-scale typing of Bacillus cereus was developed. It is based on a simple DNA preparation, involving only freezing and boiling of cells in water with active carbon. By using a computerized system for data collection and processing, an efficient system for handling RAPD patterns for large-scale investigations was achieved. The procedure was highly discriminatory for Bacillus cereus strains and was found to give reproducible classification of RAPD fingerprints for five reference strains. INTRODUCTION Bacillus cereus is a spore-forming bacterium that limits the keeping quality of pasteurized milk. In the absence of Gram- negative contaminants, spore-forming bacteria, in particular B. cereus, will grow and subsequently spoil the milk (Griffiths 1992). Contamination of milk by spores of Bacillus cereus may occur at the farm or at the dairy plant (So rgaard 1975; Van Heddeghem and Vlaemynck 1992; Crielly et al. 1994). Several sources of farm contamination have been identified, but most investigations have not been able to rank their importance (Van Heddeghem and Vlaemynck 1992). There are different opinions about the relative importance of contamination at the farm or dairy. In order to resolve these questions and clarify the routes of contamination, methods to differentiate between strains of B. cereus are needed. Bacillus cereus is also a food poisoning organism which may produce one or several enterotoxins in food and probably also in the intestine (Granum et al. 1996). Recently, psychro- trophic strains have been demonstrated to be toxin producers, including isolates from milk (Buchanan and Schulz 1994). This adds a new dimension to the risk analysis that is needed concerning the contamination of food with B. cereus. Strains of B. cereus can be differentiated by serotyping (Shinagawa 1993). Biotyping using API was able to dis- criminate some dairy plant-related strains in an investigation at two dairies (Te Giffel et al. 1996). Phage typing is also possible, but has not been used extensively (Va ¨sisa ¨nen et al. 1991; Ahmed et al. 1995). Some classification with respect to contamination sources has been made using careful deter- Correspondence to: Dr A. Christiansson, Swedish Dairies’ Association, Research and Development Department S-223 70 Lund, Sweden (e- mail: anders@smr.se). *Present address: Procordia Food AB, Research and Development, S-241 81 Eslo ¨v, Sweden. © 1998 The Society for Applied Microbiology mination of minimum growth temperatures for different iso- lates from milk products and the dairy environment (Va ¨sisa ¨nen et al. 1991). Plasmid profiling has been used in investigations of food poisoning, but not all strains of B. cereus contain plasmids (DeBuono et al. 1988). Cellular fatty acid profiling, as well as PCR directed against a fragment of the haemolysin AB gene of B. cereus in combination with restriction enzyme cleavage of the PCR product, has been used for typing of food isolates of B. cereus (Schraft et al. 1996). RAPD-PCR can be used to amplify certain segments of a genome by using a short arbitrary primer (Welsh and McClel- land 1990). This method has proved useful for obtaining fingerprints of organisms for epidemiological investigations in medicine or food science. By this method, strains of B. thuringiensis, used commercially as biological insecticides and closely related to B. cereus, could be differentiated from each other (Brousseau et al. 1993). However, care must be taken when using RAPD-PCR, as several factors have been reported to affect the reproducibility of the method (Hilton et al. 1997). The aim of the present investigation was to develop an RAPD-PCR method for differentiation of strains of B. cereus. In order to be able to process a large number of strains, a simple but reproducible sample preparation method, a robust and discriminative RAPD-PCR method, and a data handling procedure to process the band patterns in a simple manner, were sought. MATERIALS AND METHODS Bacterial strains and primers The bacterial strains used were Bacillus cereus ATCC 7064, ATCC 11986 var. mycoides, ATCC 14579, ATCC 27877,