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DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...
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Optimizing Nucleic Acid Extraction from Extended Bovine Semen for Endemic and High-Consequence Pathogens.

Amanda Zimmerman1, Anne Vandenburg-Carroll1, Douglas G Marthaler2

  • 1Wisconsin Veterinary Diagnostic Laboratory, University of Wisconsin-Madison, Madison, WI 53706, USA.

Animals : an Open Access Journal From MDPI
|December 11, 2025
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Summary

Accurate detection of bovine semen pathogens requires optimized nucleic acid extraction methods to overcome PCR inhibitors. Tailored protocols are essential for reliable surveillance and international trade in animal germplasm.

Keywords:
IndiMag Pathogen KitMagMAX CORENAHLNPCR inhibitionextenderextractioninfluenza A virussemen

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Area of Science:

  • Veterinary Microbiology
  • Molecular Diagnostics
  • Animal Reproduction

Background:

  • Accurate pathogen detection in bovine semen is vital for animal health surveillance and international trade.
  • Bovine semen contains PCR inhibitors that reduce nucleic acid extraction efficiency and assay sensitivity.
  • Existing extraction platforms require evaluation for their effectiveness with bovine semen matrices.

Purpose of the Study:

  • To evaluate two National Animal Health Laboratory Network-approved extraction platforms for pathogen detection in bovine semen.
  • To assess the impact of different input volumes and pretreatment strategies on PCR inhibition.
  • To determine the optimal extraction method for reliable pathogen surveillance in bovine germplasm.

Main Methods:

  • Two extraction platforms (MagMAX CORE and IndiMag Pathogen Kits) were tested with 88 negative extended semen samples.
  • Influenza A virus (IAV) PCR assays with different exogenous internal controls (ICs) were used to assess PCR inhibition.
  • Validation included naturally infected semen and limit of detection studies with *Mycoplasma bovis*.

Main Results:

  • Internal controls showed variable passing rates (31.8%–100.0%) dependent on extender and extraction protocol.
  • The IndiMag Pathogen 100-na was selected for further diagnostic evaluation.
  • Tailored extraction methods are crucial for overcoming semen-associated inhibition.

Conclusions:

  • Optimized nucleic acid extraction is critical for accurate pathogen detection in bovine semen.
  • Method selection significantly impacts PCR inhibition and assay sensitivity.
  • Effective protocols are essential for reliable animal health surveillance and international trade.