RESUMO
A PCR method was developed to detect spores of Bacillus sporothermodurans in 1, 10, and 100 ml of raw milk. Two primers were derived from a unique sequence after subtractive hybridization of B. sporothermodurans DNA with DNA of MB 397, a not yet identified spore-forming bacterium isolated from raw milk, closely related to B. sporothermodurans. Specific identification was proven on a large collection of Bacillus strains and on strains from relevant taxa. The detection of B. sporothermodurans in raw milk is based on activation, germination, and outgrowth of the spores, followed by PCR identification. Spores from 10 and 100 ml 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.
Assuntos
Bacillus/isolamento & purificação , Leite/microbiologia , Reação em Cadeia da Polimerase/métodos , Esporos/isolamento & purificação , Animais , Bacillus/genética , Bacillus/crescimento & desenvolvimento , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , DNA Bacteriano/análise , DNA Bacteriano/genética , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Plasmídeos/genética , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade , Análise de Sequência de DNA , Esporos/genéticaRESUMO
A method for direct detection of Listeria monocytogenes in 25 ml of raw milk is presented. The detection limit can be situated between 10 and 5 CFU. The detection method is based on chemical extraction of the milk components and PCR amplification with two nested pairs of primers specific for Listeria monocytogenes.
