Development and Evaluation of Droplet Digital PCR Assay for the Detection of CyHV-2 and Comparative Analysis

Abstract:

CyHV-2 (Cyprinid herpesvirus 2) is a double-stranded DNA virus first isolated from goldfish, Carassius auratus (L). It is classified as a member of the genus Cyprinivirus, which includes carp pox (CyHV-1), koi herpesvirus (CyHV-3) and anguillid herpesvirus-1 (AngHV-1). It has an icosahedral shape with an average diameter of 100–110 nm. CyHV-2 can cause approximately 50% to 100% mortalities in cultured goldfish when water temperature is between 15℃ and 25℃. Therefore, rapid and precise detection of CyHV-2 is needed to prevent and control disease outbreak. In this study we established a droplet digital PCR (ddPCR) method used for accurate quantification of CyHV-2 DNA. The ddPCR method was compared with the quantitative real-time PCR (qPCR) in the aspect of the sensitivity, reproducibility, specificity and practical application. In terms of detecting CyHV-2 DNA, the sensitivity of the ddPCR assays was 20-fold lower than qPCR. The correlation coefficient (R2) obtained from linear regression analysis showed a good linearity of amplification for both ddPCR (R2=0.994) and qPCR (R2=0.994) assays. A positive correlation (R2=0.989) was observed between ddPCR and qPCR assays. To determine the same dilution series of CyHV-2 DNA, the expected numbers of DNA copies calculated by qPCR was 10-fold higher than the number of copies determined by ddPCR. CyHV-2 DNA reproducibility determined by ddPCR was found to be significantly more stable than by qPCR. The ddPCR assay had no cross-reaction with other similar fish herpesviruses, including CyHV-3, CCV (Channel catfish virus)、STIV (Soft-shelled turtle iridovirus) and EHNV (Epizootic hematopoietic necrosis virus). By contrast, the specificity of ddPCR was consistent with qPCR. Therefore, the ddPCR method was proven to be more precise than qPCR. This new absolute quantitation tool will be useful to standardize quantitative detection of CyHV-2 DNA.