Optimisation of DNA extraction and validation of PCR assays to detect Mycobacterium avium subsp. paratuberculosis

Journal of Microbiological Methods
Verlaine J TimmsBrett A Neilan

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 com...Continue Reading

References

Mar 29, 2003·Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc·Jane Christopher-HenningsEric A Nelson
May 18, 2004·Veterinary Microbiology·Elke VansnickFrançoise Portaels
Sep 3, 2004·Journal of Bacteriology·Makeda SemretMarcel A Behr
Aug 6, 2005·Journal of Clinical Microbiology·Makeda SemretMarcel A Behr
Aug 24, 2005·Proceedings of the National Academy of Sciences of the United States of America·Lingling LiVivek Kapur
Dec 19, 2006·Journal of the American Veterinary Medical Association·Michael T CollinsScott J Wells
Apr 1, 2011·Veterinary Immunology and Immunopathology·Richard J WhittingtonAuriol C Purdie
Nov 7, 2013·PloS One·Nicolas RadomskiMarcel A Behr

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Citations

Dec 28, 2017·Advanced Biomedical Research·Samira MohammadiMahshid Salehi
May 9, 2021·Animal Microbiome·Chloe MatthewsJim O' Mahony

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