A real-time quantitative PCR assay using hydrolysis probes for monitoring scuticociliate parasites in seawater

Abstract

Scuticociliatosis is a serious disease that affects flatfish during culture and against which no effective control measures have yet been developed. Monitoring parasite levels in the water may be a valuable way of establishing the risk of infection and enabling appropriate control measures to be taken, thus representing an advance in controlling the disease. To achieve this objective, we have designed a quantitative real-time PCR (qPCR) assay using primers (f / r ITS2) and a hydrolysis probe that specifically amplify a region of the internal transcribed spacer 2 (ITS2) of the main aetiological agents of scuticociliatosis: Philasterides dicentrarchi and Miamiensis avidus. The slope (m), efficiency (E) and linearity (R2) determined from the standard curves generated are within the optimal values for qPCR. The high analytical sensitivity of the qPCR assay enables quantification of less than 120 pg of DNA per μL of reaction and detection of 1 ciliate per assay. The qPCR assay also exhibits high precision, with intra- and inter-assay coefficients of variation (CV) of respectively 0.27 and 7.57%. The protocol developed for isolating and quantifying ciliates seawater samples it has a recovery efficiency greater than 75% when the ciliate levels are between 103 and 2 × 103 ciliates/L and the turbidity of the water does not exceed one nephelometric turbidity unit (NTU). The real-time qPCR assay developed is a useful and appropriate tool for the specific and sensitive monitoring of scuticociliates in the water used in flatfish farms, enabling the establishment of effective prevention and control programmes.