title: Optimisation of DNA extraction and validation of PCR assays to detect Mycobacterium avium subsp. paratuberculosis.
abstract: The aim of this study was to investigate DNA extraction methods and PCR assays suitable for the detection of Mycobacterium paratuberculosis in bovine tissue. The majority of methods currently used to detect M. paratuberculosis have been developed using bovine samples, such as faeces, blood or tissue and, in many cases, have been based on detection from pooled samples from a herd. However most studies have not compared PCR results to culture results. In order to address this problem, four DNA extraction protocols and three PCR assays were employed to detect M. paratuberculosis in bovine tissue. Given that culture is reliable from cows, the results were then compared with the known M. paratuberculosis culture status. The following DNA extractions were included, two commercial kits, a boiling method, an in house extraction based on a published method and enrichment by sonication. The three PCR assays used included single round IS900 and f57 assays and a nested IS900 assay. In addition, another PCR assay was validated for the detection of any Mycobacterial species and a universal bacterial 16S rRNA gene assay was used to detect sample inhibition. The in-house DNA extraction was the most consistent in extracting good quality DNA compared to all other methods. The use of two PCR markers, IS900 and f57, and a universal PCR enabled the correct samples to be identified as M. paratuberculosis positive. In addition, when compared to the culture result, false-positives did not occur and PCR inhibition was readily identified. Using an in house DNA extraction coupled with the IS900 and f57 PCR markers, this study provides a reliable and simple method to detect M. paratuberculosis in both veterinary and spill over infections.
authors: School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney 2052, Australia. Electronic address: verlaine.timms@unswalumni.com.
authors: Verlaine J
authors: VJ
authors: Timms
authors: School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney 2052, Australia. Electronic address: h.mitchell@unsw.edu.au.
authors: Hazel M
authors: HM
authors: Mitchell
authors: School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney 2052, Australia. Electronic address: b.neilan@unsw.edu.au.
authors: Brett A
authors: BA
authors: Neilan
mesh_terms: D000818:Animals; D002417:Cattle; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D005189:False Positive Reactions; D020459:Genes, rRNA; D008828:Microbiological Techniques / Q000379:methods; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics / Q000302:isolation & purification*; D010283:Paratuberculosis / Q000175:diagnosis* / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D013048:Specimen Handling / Q000379:methods*
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S
keywords: Crohn's disease; Inflammatory bowel disease; M. paratuberculosis; MAP; Mycobacterium paratuberculosis; Paratuberculosis