Design and validation of a PCR protocol to specifically detect the clade of Philaster sp. associated with Diadema antillarum scuticociliatosis

Diadema antillarum scuticociliatosis (DaSc), caused by a scuticociliate closely related to Philaster apodigitiformis, has affected Caribbean long-spined urchins since at least January 2022. Quantitative PCR (qPCR) is currently the standard method for detection of this ciliate in tissue and coelomic fluid samples, yet this method requires specialized equipment and is more expensive than standard PCR methods. The DaSc scuticociliate occurs against a backdrop of endo- and ecto-symbiotic ciliates which complicate detection using universal or pan-phylum PCR primer sets. To overcome these limitations, we designed and validated a sensitive and specific PCR primer (scutico-634F) and nested two-step PCR protocol to detect this taxon, which excludes other ciliates associated with D. antillarum and has poor affinity for other related ciliates. This primer and protocol for the DaSc-associated Philaster clade (DaScPc) allow for widely-accessible investigation of this pathogen in new regions and within environmental reservoirs.