A new multiplex PCR for the detection of hbl genes in strains of the 'Bacillus cereus group'.

Abstract

The Bacillus cereus group includes the species B. cereus , B. thuringensis , B. anthracis , B. mycoides , B. pseudomycoides and B. weihenstephanensis which are characterized by remarkable genetic affinities (Ash et al ., 1991; Carlson et al ., 1994; Henderson et al ., 1995; Stephan, 1996; Borin et al ., 1997; Nilsson et al ., 1998; Shangkuan et al ., 2000). These micro-organisms are responsible for foodborne disease through the production of several enterotoxins (Prus et al ., 1999) of which haemolysin BL (HBL) is the most studied. HBL is a three-component protein, including two lytic (L 1 and L 2 ) and one binding protein (B) and all three components must be simultaneously present to consider B. cereus group strains as potential pathogens (in’t Veld et al ., 2001). HBL has haemolytic and dermonecrotic activity which is responsible for the increase in vascular permeability and it is considered the primary virulence factor in the diarrhoic syndrome (Beecher and Wong, 1994). The transcription of complex HBL is codified from the operone hbl . Traditional PCR, by means of specific primers (Hansen and Hendriksen, 2001), can only detect single genes ( hblA , hblD , hblC ) encoding the components of HBL (B, L 1 e L 2 ). The identification of the genes could be faster using only one PCR reaction. In the present study we developed a multiplex PCR for the detection of operon hbl genes, both on reference and wild strains isolated from ovine milk and ‘ricotta’ cheese.