A DIRECT PCR DETECTION METHOD FOR CLOSTRIDIUM-TYROBUTYRICUM SPORES INUP TO 100 MILLILITERS OF RAW-MILK

A direct detection method for Clostridium tyrobutyricum spores in up t o 100 ml of raw milk is presented. The bacterial spores are concentrat ed by centrifugation after chemical extraction of the milk components. The vegetative cells are selectively lysed, and their DNA is digested and,washed away. Afterwards, the DNA is liberated from the spores by microwave treatment. For the identification of the C. tyrobutyricum DN A, a two-step PCR method with two nested pairs of primers is used. The primers were derived from the 16S-23S rRNA spacer region of C. tyrobu tyricum, and the specificity of each of them for C. tyrobutyricum is d emonstrated. The detection limit can be estimated to be between 3 and 30 spores in 100 ml of raw milk.