Stomach Content DNA (scDNA) Detection and Quantification for Predator Diet Assessment Using High-Throughput Nanofluidic Chip Technology: Species-Specific qPCR Assay Panel Development and Validation.

Stomach content DNA (scDNA) analyses have become the standard practice for measuring trophic interactions. scDNA metabarcoding has provided broadscale diet composition data but can potentially underestimate certain prey species, as many of the recovered sequence reads come from predator-derived DNA, potentially resulting in incomplete diet information. Targeted detection (quantitative real-time PCR-qPCR) strategies allow for single-species detection from complex multispecies scDNA mixtures. A recent advancement in qPCR technology, high-throughput qPCR (HT-qPCR), allows simultaneous multispecies targeted detection and quantification of candidate species. Here, we describe the development and validation of a panel of single-species qPCR assays targeting the CO1 region of 28 prey fishes from the Great Lakes. We performed a three-step validation procedure for all assays using high-throughput OpenArray nanofluidic technology, measuring assay sensitivity, specificity and interference. Specifically, all assays were measured against dilution series of both target and non-target species DNA with detection limits ranging from 0.00503 pg to 0.0221 ng template DNA per reaction. Assays were tested for interference (e.g., PCR inhibitor) effects by creating artificial scDNA samples spiked with serially diluted target species DNA, resulting in a range of reduction in sensitivity (range = 0.0-125x fold). We validated the OpenArray qPCR assays using individual full-reaction TaqMan qPCR for nine of the assays, finding similar sensitivity despite expectations for the loss of sensitivity in the nanoscale reactions. HT-qPCR targeted detection has the potential to revolutionise scDNA (and eDNA) monitoring by significantly reducing laboratory effort to provide sensitive, targeted and quantitative detection data for multiple species simultaneously.