title: Quality control and optimized procedure of hybridization capture-PCR for the identification of Mycobacterium avium subsp. paratuberculosis in faeces.
abstract: Nucleic acid sequence capture techniques are used to improve both the sensitivity and specificity of PCR for the diagnosis of plant, animal and human diseases. Hybridization capture-PCR (HC-PCR) was first reported as a method for the detection of Mycobacterium avium subsp. paratuberculosis in 1995 and was successfully trialed on a small number of faecal samples from cattle with Johne's disease. A locally optimized HC-PCR method was evaluated on faeces from infected and non-infected animals. However, sample to sample cross contamination during the DNA purification step highlighted that the original format of the test was unsuitable for routine diagnostic use. Here, we report modifications and optimization of HC-PCR, particularly with respect to DNA purification from faeces, hybridization and capture steps. We also identified procedurally sensitive critical points in the test during capture and washing of magnetic beads. Southern blotting was omitted from the protocol to preserve specificity but this resulted in analytical sensitivity of 5000 organisms per 200 mg faecal sample. Nevertheless, HC-PCR detected M.paratuberculosis in pellets from infected sheep diluted at rates of up to 1 in 100 in normal faeces, suggesting that the technique should be evaluated further for low-cost diagnosis in flocks/herds using pooled samples.
authors.affiliation: Elizabeth Macarthur Agricultural Institute, NSW Agriculture, Menangle, New South Wales, Australia. ian.marsh@agric.nsw.gov.au
authors.forename: I
authors.initials: I
authors.lastname: Marsh
authors.forename: R
authors.initials: R
authors.lastname: Whittington
authors.forename: D
authors.initials: D
authors.lastname: Millar
mesh_terms: D000818:Animals; D002161:Camelids, New World; D004247:DNA / Q000302:isolation & purification; D005243:Feces / Q000382:microbiology*; D017403:In Situ Hybridization / Q000379:methods; D009162:Mycobacterium avium / Q000235:genetics / Q000302:isolation & purification; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics / Q000302:isolation & purification*; D010283:Paratuberculosis / Q000175:diagnosis / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011786:Quality Control; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis / Q000382:microbiology*; D013696:Temperature
chemical_list: D004247:DNA