Location: Warmwater Aquaculture Research UnitTitle: A multiplex real-time polymerase chain reaction assay differentiates between Bolbphorus damnificus and Bolbophorus type II sp) Author
Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/1/2010
Publication Date: 7/1/2010
Publication URL: http://handle.nal.usda.gov/10113/55483
Citation: Griffin, M.J., Wise, D.J., Yost, M.C., Doffitt, C.M., Pote, L.M., Greenway, T.E., Khoo, L.H. 2010. A multiplex real-time polymerase chain reaction assay differentiates between Bolbphorus damnificus and Bolbophorus type II sp. Journal of Veterinary Diagnostic Investigation. 22:615-622. Interpretive Summary: An assay using quantitative real-time PCR was developed to allow for simultaneous identification of Bolbophorus damnificus and Bolbophorus type II species cercariae. This is an important development as it allows for accurate disease challenges utilizing either Bolbophorus damnificus and Bolbophorus type II species cercariae.
Technical Abstract: 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, Planobella trivolvis. Identification of the 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 transfer hydrolysis probes were designed to amplify the 18S small unit 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 ~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 cercariae by the duplex real-time qPCR was in complete agreement with previously established end-point PCR protocols, demonstrating the assay to be more rapid, quantifiable means of parasite identification.