Development of a real-time PCR assay for the identification of Gyrodactylus parasites infecting salmonids in northern Europe

摘要

ABSTRACT: Gyrodactylus salaris is a monogenean freshwater parasite that causes high mortality in wild Atlantic salmon, and a number of countries employ monitoring programmes for its presence. A TaqMan®-MGB (minor groove binding) probe real-time multiplex assay targeting the internal transcribed spacer ribosomal DNA (ITS rDNA) was developed to simultaneously identify G. salaris/ G. thymalli and 2 other commonly occurring Gyrodactylus species infecting salmonids in northern Europe: G. derjavinoides and G. truttae. In addition, a Gyrodactylus genus-level assay was developed to assess parasite DNA quality. The species-specific real-time PCR method correctly identified target species from a wide geographical range and from a number of salmonid hosts. It did not amplify G. lucii or G. teuchis. These species were successfully amplified using the Gyrodactylus genus real-time assay. The species-specific real-time assay proved to be significantly faster than the currently employed molecular screening method of ITS rDNA PCR amplification followed by restriction fragment length polymorphism analyses (RFLP). However, as with ITS RFLP, the real-time method did not distinguish between G. salaris and the non-pathogenic G. thymalli, its principle advantage being a significant reduction in time to achieve an initial diagnostic screen before the employment of more in-depth analyses for those specimens giving a positive G. salaris/G. thymalli real-time result.