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

Sören Hansen1, Marco Roller2, Lamia M A Alslim3

  • 1Division of Microbiology and Animal Hygiene, Department of Animal Sciences, Faculty of Agricultural Sciences, University of Goettingen, D-37077 Goettingen, Germany. oeren.hansen@uni-goettingen.de.

Diagnostics (Basel, Switzerland)
|April 3, 2019
PubMed

Insights

Rapid identification of Mycobacterium avium subspecies paratuberculosis (MAP) is crucial. A new DNA extraction protocol, MAP DNA SpeedXtract, combined with RPA, offers a fast, reliable point-of-need detection system for MAP infections.

Area of Science:

  • Veterinary Microbiology
  • Molecular Diagnostics
  • Public Health

Background:

  • Mycobacterium avium subspecies paratuberculosis (MAP) causes significant disease in livestock, necessitating rapid detection methods.
  • Current diagnostic methods like culture are slow, while molecular assays (PCR, RPA) require efficient DNA extraction.
  • Accurate DNA extraction is critical for the sensitivity and specificity of molecular detection of MAP.

Purpose of the Study:

  • To develop a rapid, point-of-need DNA extraction protocol for MAP detection from stool samples.
  • To integrate this protocol with Recombinase Polymerase Amplification (RPA) for a complete diagnostic system.
  • To evaluate the performance of the developed system in a mobile laboratory setting.

Main Methods:

  • Development of MAP DNA SpeedXtract, a magnetic bead-based DNA extraction method.
  • Optimization for speed (25 min) and simplicity (3 pipetting steps).
  • Integration with RPA for amplification and detection (20 min) within a suitcase laboratory.

Main Results:

  • The MAP DNA SpeedXtract protocol demonstrated high sensitivity (90.9%) and specificity (100%) compared to lab-based methods.
  • The entire process, from sample to result, was completed rapidly in a mobile unit.
  • All reagents were cold chain independent, enhancing field applicability.

Conclusions:

  • A rapid and reliable protocol for MAP DNA extraction and detection was successfully established.
  • The developed system is suitable for point-of-need identification of MAP-infected animals.
  • This technology aids in controlling disease spread and preventing human exposure to MAP.
Keywords:
Mycobacterium avium subsp. paratuberculosisSpeedXtractmobile suitcase laboratorypoint of need extractionrapid extraction

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