A qPCR Assay for the Threatened Pilbara Olive Python (Liasis olivaceus barroni)

Abstract

The Pilbara olive python (Liasis olivaceus barroni) is endemic to the Pilbara region of Western Australia and listed as threatened. Despite being a priority conservation target, they are difficult to monitor as their cryptic nature complicates conventional surveying methods, constraining effective conservation in a region that is heavily impacted by the natural resource industry. As part of ongoing efforts to improve monitoring and conservation of this endangered top predator, we developed a subspecies-specific probe-based qPCR assay to allow for the detection of L. o. barroni DNA in environmental samples. The assay was validated in silico and in vitro against closely related non-target species, with synthetic DNA used for assessing assay sensitivity and the effect of inhibition. Field validation was performed using water samples collected from rock pools across the Pilbara (n = 21) that had previously been investigated with eDNA metabarcoding methods. Our validation showed that the assay is specific to L. o. barroni and highly sensitive, with modeled limits of detection and quantification of 4 copies/reaction and 26 copies/reaction, respectively. We successfully detected L. o. barroni DNA in 10/10 field samples that had previously shown the presence of L. o. barroni based on metabarcoding. Additionally, 2/11 samples reported as negative through metabarcoding were observed to amplify using our qPCR assay, a discrepancy we ascribe to improved sensitivity resulting from the shorter amplicon length of our assay and less competition for primers in the qPCR reactions. This qPCR assay will be a valuable tool for the eDNA biomonitoring of L. o. barroni, furthering our understanding of its distribution, ecology, and conservation status.