Discrimination of gastrointestinal nematode eggs from crude fecal egg preparations by inhibitor-resistant conventional and real-time PCR.
Authors: Demeler Janina, Ramünke Sabrina, Wolken Sonja, Ianiello Davide, Rinaldi Laura, Gahutu Jean Bosco, Cringoli Giuseppe, von Samson-Himmelstjerna Georg, Krücken Jürgen
Journal: PloS one
Summary
# Editorial Summary Accurate species-level identification of gastrointestinal nematodes in equine practice remains challenging; whilst FLOTAC provides precise egg counts, morphological differentiation is often limited to genus level, and existing molecular methods are costly and require substantial faecal samples. Demeler and colleagues developed a direct PCR protocol compatible with FLOTAC that bypasses conventional DNA extraction by lysing eggs recovered from the flotation solution through repeated boiling and freezing cycles, then using these crude lysates directly as PCR template with Phusion polymerase—an inhibitor-resistant enzyme suitable for downstream quantitative real-time PCR. The technique demonstrated sensitivity comparable to traditional DNA extraction (reliably detecting five eggs per gram) with the added advantage of dramatically reduced costs and processing time, and could distinguish species using post-PCR analysis methods including high-resolution melt analysis. For equine practitioners, this approach offers a practical pathway towards definitive nematode species identification from routine faecal samples without requiring expensive isolation kits or outsourcing to specialised laboratories—a significant advance for detecting anthelmintic resistance patterns and tailoring targeted parasite management strategies.
Read the full abstract on PubMed
Practical Takeaways
- •This protocol enables faster and cheaper species-level diagnosis of gastrointestinal nematodes directly from fecal flotation samples, improving treatment targeting and resistance monitoring
- •The method works reliably across multiple host species including horses, cattle, and goats, making it adaptable to mixed-species practices without requiring expensive equipment or lengthy sample preparation
- •Real-time PCR quantification integrated with FLOTAC gives you both egg counts and species identification in one workflow, reducing turnaround time and overall diagnostic costs
Key Findings
- •Direct PCR from crude egg preparations combined with FLOTAC allows accurate quantification of eggs per gram feces and species identification without costly DNA isolation steps
- •Phusion DNA polymerase resists PCR inhibitors in fecal samples, with sensitivity comparable to standard DNA isolation protocols and reliable detection at five epg or below
- •High resolution melt (HRM) analysis and RFLP can distinguish closely related nematode species such as Toxocara cati from T. canis and Necator from Ancylostoma in single reactions