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Appl. Environ. Microbiol., Dec 1995, 4141-4146, Vol 61, No. 12
Copyright © 1995, American Society for Microbiology

A direct PCR detection method for Clostridium tyrobutyricum spores in up to 100 milliliters of raw milk

LM Herman, JH De Block and GM Waes
Government Dairy Research Station, Agricultural Research Centre Ghent, Melle, Belgium.

A direct detection method for Clostridium tyrobutyricum spores in up to 100 ml of raw milk is presented. The bacterial spores are concentrated 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 DNA, 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. tyrobutyricum, and the specificity of each of them for C. tyrobutyricum is demonstrated. The detection limit can be estimated to be between 3 and 30 spores in 100 ml of raw milk.

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