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Development of Rapid Extraction Method of Mycobacterium avium Subspecies paratuberculosis DNA from Bovine Stool Samples

1
Division of Microbiology and Animal Hygiene, Department of Animal Sciences, Faculty of Agricultural Sciences, University of Goettingen, D-37077 Goettingen, Germany
2
Zoological and Botanical Garden Wilhelma, D-70376 Stuttgart, Germany
*
Author to whom correspondence should be addressed.
Diagnostics 2019, 9(2), 36; https://doi.org/10.3390/diagnostics9020036
Received: 29 January 2019 / Revised: 18 March 2019 / Accepted: 27 March 2019 / Published: 29 March 2019
(This article belongs to the Special Issue Diagnosis of Bacterial Pathogens)
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Abstract

The rapid identification of Mycobacterium avium subspecies paratuberculosis (MAP) infected animals within the herd is essential for preventing the spread of the disease as well as avoiding human exposure. Although culture is seen as the gold standard, there are various molecular assays available i.e., polymerase chain reaction (PCR) or isothermal amplification technique (recombinase polymerase amplification (RPA)) for the detection of MAP. The accuracy of the molecular assays is highly dependent on the DNA extraction method. In order to establish a rapid point of need system for the detection of MAP DNA from stool samples, we developed a rapid DNA extraction protocol (MAP DNA SpeedXtract) specified for use in combination with the RPA. The whole procedure from “sample in” to “result out” was conducted in a mobile suitcase laboratory. The DNA extraction is based on reverse purification by magnetic beads, which reduces the required technical demand. The MAP DNA SpeedXtract was performed within 25 min and only three pipetting steps were needed. The amplification and detection time were 20 min in RPA. The sensitivity and specificity of the developed protocol in comparison with the lab-based silica membrane column extraction and real-time PCR were 90.9% (n = 22) and 100% (n = 23), respectively. In conclusion, we established a rapid and reliable protocol for the extraction and detection of MAP DNA. All reagents are cold chain independent. The entire setup is ideal for point of need identification of MAP infected cases. View Full-Text
Keywords: Mycobacterium avium subsp. paratuberculosis; rapid extraction; mobile suitcase laboratory; SpeedXtract; point of need extraction Mycobacterium avium subsp. paratuberculosis; rapid extraction; mobile suitcase laboratory; SpeedXtract; point of need extraction
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Hansen, S.; Roller, M.; Alslim, L.M.A.; Böhlken-Fascher, S.; Fechner, K.; Czerny, C.-P.; Abd El Wahed, A. Development of Rapid Extraction Method of Mycobacterium avium Subspecies paratuberculosis DNA from Bovine Stool Samples. Diagnostics 2019, 9, 36.

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