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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.
Abstract:
Accurate pathogen detection in bovine semen is crucial for animal health surveillance and international trade. Semen presents unique challenges due to the presence of PCR inhibitors from seminal plasma and extender components, reducing nucleic acid extraction efficiency and sensitivity. The two National Animal Health Laboratory Network-approved extraction platforms (MagMAX CORE and IndiMag Pathogen Kits) were evaluated using 88 negative extended semen samples at 200 µL input volume, reduced input volumes, and pretreatment strategies with two influenza A virus (IAV) PCR assays, containing different exogenous internal controls (ICs) to assess PCR inhibition. The ICs yielded overall passing rates from 31.8% to 100.0% and varied greatly based on the extender formulation and extraction protocol. Validation continued with naturally infected semen containing Mycoplasma bovis, bovine viral diarrhea virus, bovine herpesvirus-1, and the limit of detection using Mycoplasma bovis. The IndiMag Pathogen 100-na was then selected for evaluation of diagnostic sensitivity and specificity, reproducibility, and detection limits with IAV-spiked samples, using the two IAV PCR assays and their ICs. Selected archived semen samples used in this study were screened and were negative for IAV by both PCR assays. These findings underscore the importance of tailored extraction methods in overcoming semen-associated inhibition and facilitating reliable pathogen surveillance in bovine germplasm.
Insights
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.
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.
