Using a species-specific assay to improve detection of an elusive stygobitic vertebrate, Ophisternon candidum, through environmental DNA

Abstract Environmental DNA has become a crucial tool for monitoring the distribution of the elusive Robe River blind cave eel ( Ophisternon candidum) population. Here we document the development of a new metabarcoding assay and qPCR probe-based assay to improve sensitivity and cost-effectiveness of monitoring this species. Using available sequence data, we developed new primers and a probe that could be used for both metabarcoding and qPCR analyses, amplifying a 124 bp fragment of the 16S gene region. These primers were tested in-silico and, after optimisation using a synthetic oligo fragment, were tested on historical and newly collected samples. Sanger sequencing was used to confirm that the qPCR probe was on-target. In the in-silico analysis, other species were found to bind with the metabarcoding assay, however no species other than the target bound with the probe. Testing the new metabarcoding assay and probe on historical samples revealed inconsistent results, possibly due to degradation of DNA extractions. However, using newly collected samples, the new metabarcoding assay and qPCR probe performed well, with higher sensitivity observed with the probe. This study demonstrates the potential for probe-based assays to be applied in subterranean habitats in the detection of rare or cryptic species, leading to more robust management decisions. While a Sanger verification step is recommended to mitigate false positives, even with these steps the probe-based assay provides a significant cost reduction compared to metabarcoding and has a higher sensitivity and specificity to the target species.