RESUMEN

Detection of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) by polymerase chain reaction (PCR) is often hampered by the lack of efficient methods for sample treatment. We report a protocol for analysis of faecal samples based on buoyant density centrifugation in Percoll and IS900 sequence capture PCR combined with a dot blot assay for detection of low-grade infection of M. paratuberculosis. Serial dilutions of M. paratuberculosis genomic DNA and M. paratuberculosis bacteria were used to assess the sensitivity of the method. The final evaluation was performed with spiked faecal samples, which also were analysed by culture. The presence of PCR inhibitory substances in processed faecal samples was evaluated by including a PCR internal control. By using buoyant density centrifugation, sequence capture PCR, and dot blot hybridisation, we achieved a sensitivity of 10(3)CFU (colony forming units)/g of faeces. The detection limit by culture was assessed to 10(2)CFU/g of faeces. We conclude that the described protocol is a fast and sensitive alternative to bacterial culture of faecal samples.

Asunto(s)

Enfermedades de los Bovinos/microbiología , Mycobacterium avium subsp. paratuberculosis/aislamiento & purificación , Paratuberculosis/microbiología , Reacción en Cadena de la Polimerasa/veterinaria , Animales , Bovinos , Centrifugación por Gradiente de Densidad/veterinaria , Recuento de Colonia Microbiana/veterinaria , Elementos Transponibles de ADN/genética , ADN Bacteriano/química , ADN Bacteriano/genética , Heces/microbiología , Mycobacterium avium subsp. paratuberculosis/genética , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa/métodos , Povidona , Sensibilidad y Especificidad , Dióxido de Silicio , Suecia