The design and analysis of unique DNA primer sets and probes to identify and distinguish the bacillus cereus group species: developing a real-time DNA-biosensor

Bacillus cereus from the Bacillus cereus group species, which consist of: Bacillus cereus, Bacillus thuringiensis, Bacillus anthracis, Bacillus weihenstephanensis, Bacillus mycoides and Bacillus pseudomycoides is one of the most frequently isolated bacterial foodborne pathogens. Growth of B. cereus results in production of several highly active toxins therefore, consumption of food containing 105-106 bacteria (spores)/g or toxins, is sufficient to cause emetic and diarrhoeal syndromes. The most common source of this bacterium is milk and mixed food products that include milk powder, thus is of particular concern in the baby formula industry. In this study 138 strains of B. cereus group spp. were characterized based on their phenotypic and genotypic features. The study developed unique DNA primers for use in PCR and these were then tested via real-time PCR (RT-PCR): (i) the motB gene encoding the flagellar motor protein MotB was used as a PCR primer target. (ii) New primers and probes, targeting a hypothetical protein, unique only for B. pseudomycoides strains were then developed. (iii) A RT-PCR assay developed together with species specific TaqMan probes were able to differentiate B. weihenstephanensis and B. pseudomycoides strains. (iv) In addition multiplex PCR with primers targeting motB and a hypothetical protein proved successful in identification of the B. cereus group spp. with differentiation of B. pseudomycoides. This is the first description of a molecular technique able to distinguish B. pseudomycoides from other members belonging to the B. cereus group spp. and the first RT-PCR protocol to use the motB gene as a diagnostic target. The assays performed well with milk samples artificially contaminated with bacteria belonging to the B. cereus group spp. To analyze the ability to detect bacterial spores, fat and nonfat milk was contaminated and treated to destroy spores or allow germination. The design of a hybridization probe for use in a biosensor was then undertaken. The two probes designed were optimised for a hybridisation reaction using dot blot analysis: one for the detection of all species belonging to the B. cereus group spp., and the second for detection only of B. pseudomycoides strains. Currently DNA must be initially extracted for analysis in a biosensor and the study tested the efficiency of two commercial DNA extraction kits. To be able to receive a signal via the biosensor from bacterial spores present in milk, each sample should be pre-treated by incubation of the milk for the proper time and temperature.