IDENTIFICATION AND DETECTION OF BACILLUS SPOROTHERMODURANS SPORES IN 1, 10, AND 100 MILLILITERS OF RAW-MILK BY PCR

A PCR method was developed to detect spores of Bacillus sporothermodur ans in 1, 10, and 100 ml of raw milk. Two primers were derived from a unique sequence after subtractive hybridization of B. sporothermoduran s DNA with DNA of MB 397, a not yet identified spore-forming bacterium isolated from raw milk, closely related to B. sporothermodurans. Spec ific identification was proven on a large collection of Bacillus strai ns and on strains from relevant taxa. The detection of B. sporothermod urans in raw milk is based on activation, germination, and outgrowth o f the spores, followed by PCR identification. Spores from 10 and 100 m l were concentrated by centrifugation after chemical extraction of the milk components. The total test takes 28 h. The detection limits are 9, 0.4, and 0.22 CFU/ml for 1, 10, and 100 ml, respectively.