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

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...

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Autores principales: Hansen, Sören, Roller, Marco, Alslim, Lamia M. A., Böhlken-Fascher, Susanne, Fechner, Kim, Czerny, Claus-Peter, Abd El Wahed, Ahmed
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627389/
https://www.ncbi.nlm.nih.gov/pubmed/30934956
http://dx.doi.org/10.3390/diagnostics9020036
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author Hansen, Sören
Roller, Marco
Alslim, Lamia M. A.
Böhlken-Fascher, Susanne
Fechner, Kim
Czerny, Claus-Peter
Abd El Wahed, Ahmed
author_facet Hansen, Sören
Roller, Marco
Alslim, Lamia M. A.
Böhlken-Fascher, Susanne
Fechner, Kim
Czerny, Claus-Peter
Abd El Wahed, Ahmed
author_sort Hansen, Sören
collection PubMed
description 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.
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spelling pubmed-66273892019-07-23 Development of Rapid Extraction Method of Mycobacterium avium Subspecies paratuberculosis DNA from Bovine Stool Samples Hansen, Sören Roller, Marco Alslim, Lamia M. A. Böhlken-Fascher, Susanne Fechner, Kim Czerny, Claus-Peter Abd El Wahed, Ahmed Diagnostics (Basel) Article 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. MDPI 2019-03-29 /pmc/articles/PMC6627389/ /pubmed/30934956 http://dx.doi.org/10.3390/diagnostics9020036 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Hansen, Sören
Roller, Marco
Alslim, Lamia M. A.
Böhlken-Fascher, Susanne
Fechner, Kim
Czerny, Claus-Peter
Abd El Wahed, Ahmed
Development of Rapid Extraction Method of Mycobacterium avium Subspecies paratuberculosis DNA from Bovine Stool Samples
title Development of Rapid Extraction Method of Mycobacterium avium Subspecies paratuberculosis DNA from Bovine Stool Samples
title_full Development of Rapid Extraction Method of Mycobacterium avium Subspecies paratuberculosis DNA from Bovine Stool Samples
title_fullStr Development of Rapid Extraction Method of Mycobacterium avium Subspecies paratuberculosis DNA from Bovine Stool Samples
title_full_unstemmed Development of Rapid Extraction Method of Mycobacterium avium Subspecies paratuberculosis DNA from Bovine Stool Samples
title_short Development of Rapid Extraction Method of Mycobacterium avium Subspecies paratuberculosis DNA from Bovine Stool Samples
title_sort development of rapid extraction method of mycobacterium avium subspecies paratuberculosis dna from bovine stool samples
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627389/
https://www.ncbi.nlm.nih.gov/pubmed/30934956
http://dx.doi.org/10.3390/diagnostics9020036
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