Stomach Content DNA (scDNA) Detection and Quantification for Predator Diet Assessment Using High-Throughput...

Area of Science:

• Ecology
• Molecular Biology
• Genetics

Background:

• Stomach content DNA (scDNA) analysis is standard for trophic interactions.
• scDNA metabarcoding offers broad diet data but may miss prey due to predator DNA.
• Targeted quantitative PCR (qPCR) allows single-species detection in scDNA mixtures.

Purpose of the Study:

• To develop and validate a panel of single-species qPCR assays for 28 prey fish species.
• To assess the sensitivity, specificity, and interference of these assays using high-throughput qPCR (HT-qPCR).
• To evaluate the potential of HT-qPCR for revolutionizing scDNA and environmental DNA (eDNA) monitoring.

Main Methods:

• Developed 28 single-species qPCR assays targeting the CO1 region of prey fishes.
• Validated assays using high-throughput OpenArray nanofluidic technology.
• Tested sensitivity (0.00503 pg to 0.0221 ng), specificity, and PCR inhibition effects.

Main Results:

• Assays demonstrated high sensitivity with varying detection limits.
• Interference effects were quantified, showing a range of sensitivity reduction.
• Validated OpenArray qPCR assays against standard qPCR, finding comparable sensitivity.

Conclusions:

• HT-qPCR provides a sensitive, targeted, and quantitative method for multispecies detection in scDNA.
• This technology can significantly reduce laboratory effort in diet analysis and eDNA monitoring.
• HT-qPCR has the potential to revolutionize ecological monitoring by enabling simultaneous detection of multiple species.