Improving DNA detection sensitivity for the white-bellied pangolin (Phataginus tricuspis) via digital PCR

Pangolins are widely regarded as the world's most trafficked mammals, with their populations plummeting due to intense poaching to supplement the illegal trade in their scales, meat, and other derivatives. A persistent limitation in accurate trade tracing and successful criminal prosecution is the poor quality of seized specimens available for analysis, requiring the development of sensitive detection methodologies. Here, we developed a specific mtDNA-based digital PCR (dPCR) assay for the highly trafficked African white-bellied pangolin (Phataginus tricuspis) and applied it to a wide range of sample types. DNA extract concentration and quality, as assessed via fluorometry and 260/230-260/280 nm absorbance ratios, were highly heterogeneous among sample types due to differences in their inherent biological characteristics. To maximise the sensitivity of the dPCR assay, we used a ten-fold dilution series, resulting in an optimal dilution factor of 1/1000 applicable to any sample type. Due to cross-reactivity in the sister-species samples (P. tetradactyla), we developed a two-fold strategy to define assay detection limits based on the proportion of positive partitions (PPP). This resulted in a conservative threshold (PPP > 0.00012, approximately 0.16 copies/µL) for Phataginus DNA detection, appropriate for general trade investigations, and a species-specific threshold (PPP > 0.004, approximately 4.7 copies/µL) for the specific detection of P. tricuspis. Our success rate in identifying analysed P. tricuspis samples based on these thresholds was 80-70%, respectively. Negative dPCR outputs were significantly associated with lower DNA concentrations and poor-quality samples deviating from ideal absorbance ratio ranges, hence we recommend assessment of DNA quality indicators prior to dPCR testing to ensure optimal resource allocation. Levels of target DNA were variable among sample types. The positive correlation between DNA concentration and PPP indicated high levels of host-specific DNA in (i) rectal swabs, which were overall the best quality sample assessed, and (ii) bone and scale samples, which are the main materials available for trade scenarios. We present, to our knowledge, the first dPCR assay developed for pangolins, thus adding to the forensic toolkit available to curb their illegal trade. Despite our precautionary dPCR threshold estimation, we recommend that future studies reevaluate detection limits using available sample types and dilution series testing, while in the near future a multiplexing assay for all species seems reachable.