title: Detection of Mycobacterium avium subsp. paratuberculosis by buoyant density centrifugation, sequence capture PCR and dot blot hybridisation.
abstract: 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.
authors.affiliation: National Veterinary Institute, P.O. Box 8156, Dep., NO-0033 Oslo, Norway.
authors.forename: Stefania
authors.initials: S
authors.lastname: Halldórsdóttir
authors.forename: Stina
authors.initials: S
authors.lastname: Englund
authors.forename: Sigrun Fredsvold
authors.initials: SF
authors.lastname: Nilsen
authors.forename: Ingrid
authors.initials: I
authors.lastname: Olsaker
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000382:microbiology*; D002499:Centrifugation, Density Gradient / Q000662:veterinary; D015169:Colony Count, Microbial / Q000662:veterinary; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005243:Feces / Q000382:microbiology; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics / Q000302:isolation & purification*; D009693:Nucleic Acid Hybridization; D010283:Paratuberculosis / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D011205:Povidone; D012680:Sensitivity and Specificity; D012822:Silicon Dioxide; D013548:Sweden
chemical_list: D004251:DNA Transposable Elements; D004269:DNA, Bacterial; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone