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Submitted to: American Society for Microbiology
Publication Type: Abstract Only Publication Acceptance Date: 2/17/2006 Publication Date: 5/22/2006 Citation: Cook, K.L. 2006. Targeting mycobacterium avium subsp. paratuberculosis in the environment. American Society for Microbiology.(ISBN 1-55581-389-5) Interpretive Summary: Technical Abstract: Diagnosis of Johne’s disease, an enteric infection of cattle caused by Mycobacterium avium subsp. paratuberculosis (MAP), has been impeded by the lack of rapid, reliable detection methods. The goal of this study was to optimize detection of MAP in environmental samples. Experiments were conducted to determine the best method for extracting MAP DNA (four methods tested) and to evaluate sampling protocols for obtaining (direct sampling or sample pooling) and processing (direct extraction or pre-treatment) environmental samples. The MAP IS900 sequence was targeted in DNA extracts using quantitative, real-time PCR (QRT-PCR) or regular PCR. Extraction of DNA from manure from a MAP infected dairy cow was most effective with Qbiogene’s Fast® DNA Soil kit; it gave the highest yield of DNA (116 ng µl-1) and the highest copy number of the targeted MAP IS900 sequence (4.8 X 106 copies IS900 g-1). Serial dilutions of a pure culture of MAP (ATCC 700535) were mixed with topsoil (2.0 kg) to determine the concentration of MAP that could be detected following extraction by the Fast® DNA Soil kit. Sample collection was evaluated by either directly removing samples (0.5g) from the soil/MAP mix or by pooling 10 separate one gram samples, mixing and then using a sub-sample (0.5g) for DNA extraction. The limit of detection of MAP was better when samples were pooled (4.8 X 104 copies IS900 g-1 soil or 103 copies IS900 mL-1 original inoculum) than by direct sampling (1.1 X 105 copies IS900 g -1 or 104 copies IS900 mL-1 original inoculum). Three different pre-treatments were tested to increase removal of bound cells prior to DNA extraction. Pre-treatment actually reduced (by 63%) detection of MAP IS900 sequences as compared to sampling without pre-treatment. Results from these experiments suggest that the method of DNA extraction significantly affects the yield and quality of target DNA. Pre-treating samples to improve cell removal has limited use, but pooling samples may improve detection when target organisms are not evenly distributed or are present in low concentrations. |
