Simple identification of some Bacillus species and related genera in food by RAPD analysis combined with morphological observation

Template DNA from Brevibacillus brevis, Geobacillus stearothermophilus, Paenibacillus macerans, P. polymyxa and 10 species of Bacillus was used with the commercially available RAPD Analysis Primers to generate DNA fragments by PCR. Different RAPD band patterns were generated from the DNA of 14 different Bacillus species and related genera with Primer 1 or 4. The result suggested that the single use of Primer 1 or 4 is effective for the simple identification of these strains of Bacillus and related genera by the RAPD analysis. By the RAPD analyses of the template DNA prepared from bacterial colonies formed on nutrient agar plates after cultivation at 35°C for 24 to 48 h, B. brevis, B. circulans, B. firmus, B. licheniformis, P. macerans, P. polymyxa, B. pumilus, B. sphaericus, B. subtilis and B. thuringiensis were identified by the combination of the present RAPD analysis using Primer 4 and morphological observations of colonies, vegetative cells and spores. For identification of B. coagulans and G. stearothermophilus, the RAPD analyses of the template DNA prepared from colonies formed on standard plate agar after cultivation at 55°C for 24 to 48 h might be effective though further improvements of the assay are required for identification of these Bacillus species. For simple identification of B. cereus and B. megaterium by the PCR-based method, the use of other RAPD primers or a specific PCR primer set should be developed in addition to morphological observations of colonies, vegetative cells and spores.