Design and validation of a PCR protocol to specifically detect the clade ofPhilastersp. associated withDiadema antillarumscuticociliatosis

Abstract

ABSTRACTDiadema antillarumscuticociliatosis (DaSc), caused by a scuticociliate closely related toPhilaster 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 withD. antillarumand 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.