A duplex real-time polymerase chain reaction assay for differentiation between Bolbophorus damnificus and Bolbophorus type II species cercariae.

A duplex quantitative real-time polymerase chain reaction (qPCR) assay was developed to differentiate between Bolbophorus damnificus and Bolbophorus type II species cercariae. Both trematode species are prevalent throughout the commercial catfish industry, as both infect the ram's horn snail, Planorbella trivolvis, which is commonly found in catfish ponds. Identification of cercaria to species is important in catfish disease challenge experiments, as only B. damnificus has been shown to have negative impacts on channel catfish. Oligonucleotide primers and fluorescence resonance energy transfer hydrolysis probes were designed to amplify the 18S small subunit ribosomal DNA gene of each species. The quantification cycle indicative of the number of cercariae in the sample prep was determined, and standard curves correlating to cercaria numbers were established. For both species, the assay was found to be highly repeatable and reproducible, with a linear dynamic range covering 7 orders of magnitude. The sensitivity limit of the assay was approximately 1/256th of a cercaria, regardless of species, and there was no remarkable interference between the 2 assays when run simultaneously within the same reaction. In a field study, identification of cercaria by the duplex real-time qPCR assay was in complete agreement with previously established end-point PCR protocols, demonstrating the assay to be a more rapid, quantifiable means of parasite identification.