Development of a TaqMan-Based Real-Time PCR Assay for the Specific Detection of Turbot Circovirus.

Abstract

Turbot (Scophthalmus maximus ) is an economically important farming fish in China. Turbot circovirus (TCV), a recently discovered member of the Circovirus genus within the Circoviridae family, has emerged as a significant threat to the turbot aquaculture industry in China. This virus is the causative agent of turbot acute hemorrhagic disease (TAHD), resulting in substantial economic losses in recent years. As a novel pathogen, the development of more accurate and efficient detection methods for TCV remains an urgent need. In this study, the specific probe and primers were designed based on the consensus sequence of the cap gene of TCV to develop a TaqMan probe-based real-time PCR method for virus detection. The specificity of the assay was validated using turbot reddish body iridovirus (TRBIV), viral nervous necrosis virus (VNNV), the cap gene of European catfish (Silurus glanis ) circovirus (CfCV) and European eel (Anguilla anguilla ) circovirus (EeCV), and no cross-reaction was observed, indicating the high specificity of the established method. The detection limit of this assay was 10copies per reaction, with an efficiency of 93.7%, a regression squared value of 0.999, and a linear range from 10copies to 10copies of TCV DNA. The assay demonstrated high specificity, sensitivity and reproducibility, with both intra- and inter-assay coefficients of variation less than 2.0%. Then 390 clinical samples suspected of TAHD collected from 2019 to 2022 were tested using this method, and all the samples tested positive for TCV. In this study, we established a TaqMan qPCR-based method for TCV detection, which could be applied to epidemiological investigations and pathogenic studies of TCV.