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Updated: Mar 27, 2026

Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications
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Validating COI eDNA Metabarcoding Primers for Detection of Subterranean Fauna.

Mieke van der Heyde1,2, Michael Curran3,4, Stephanie Floeckner3

  • 1Subterranean Research and Groundwater Ecology (SuRGE), trace and Environmental DNA (TrEnD) Laboratory, School of Molecular and Life Sciences, Curtin University, Bentley, Western Australia, Australia.

Molecular Ecology Resources
|March 24, 2026
PubMed
Summary

Selecting the right primers is crucial for environmental DNA (eDNA) studies of subterranean fauna. The fwhF2/fwhR2n primer set showed the best performance in detecting cave biodiversity using eDNA.

Keywords:
environmental DNAinvertebratesmetabarcodingstygofaunasubterranean fauna

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Area of Science:

  • Ecology
  • Molecular Biology
  • Conservation Biology

Background:

  • Subterranean ecosystems harbor unique and ancient fauna, but are difficult to sample.
  • Environmental DNA (eDNA) metabarcoding is a promising tool for subterranean biodiversity assessment.
  • Primer bias and non-target amplification can limit the effectiveness of eDNA metabarcoding.

Purpose of the Study:

  • To validate COI metabarcoding primers for assessing subterranean faunal diversity.
  • To evaluate primer performance across various subterranean taxa using in silico, in vitro, and in situ analyses.

Main Methods:

  • In silico curation of COI sequences from the Barcode of Life Database (BOLD).
  • In vitro testing of 14 primer combinations using mock communities with varying DNA concentrations.
  • In situ field validation using haul net samples from subterranean environments.

Main Results:

  • Primer performance varied significantly, with different efficiencies and detection limits across taxa.
  • The BF1/jgHCO2198 primer combination detected 82% of taxa but only at high DNA concentrations.
  • The fwhF2/fwhR2n primer combination detected the highest proportion of subterranean taxa (68%) in diluted mock communities and performed well in situ, identifying 13 of 16 known taxa plus four new ones.

Conclusions:

  • COI metabarcoding is a valuable tool for subterranean biodiversity assessment.
  • Primer selection is critical for accurate and comprehensive eDNA studies in subterranean ecosystems.
  • The fwhF2/fwhR2n primer set shows significant potential for subterranean eDNA research and conservation.