Alanine racemase gene fragments as probes for detecting Bacillus stearothermophilus and Bacillus psychrosaccharolyticus in foods

Alanine racemase gene fragments containing non-conserved regions of the gene were evaluated as probes for detecting Bacillus stearothermophilus and Bacillus psychrosaccharolyticus in foods. The gene fragments were amplified from genomic DNA of each bacterium by polymerase chain reaction with degenerate oligonucleotide primers, and labeled with digoxigenin as probes for detecting each bacterium. Foods and bacteria were treated at 25°C for 10 min in 0.1 N NaOH containing 0.5% SDS before being directly spotted onto nylon membranes for DNA hybridization. When the specificities of the probes were analyzed using a total of 86 strains (23 genera and 65 species) of bacteria including 29 Bacillus strains (20 species), each probe was specific for the respective target bacteria. A variety of foods inoculated with B. stearothermophilus or B. psychrosaccharolyticus were positive as determined by hybridization with the respective probe, whereas uninoculated foods were negative. The alanine racemase gene fragments could be used as specific probes for detecting B. stearothermophilus and B. psychrosaccharolyticus in foods.