Development And Evaluation Of Droplet Digital PCR Assay For The Detection Of CyHV-2 And Selection Single-Chain Recombinant Antibodies Against CyHV-2

CyHV-2(Cyprinid herpesvirus 2) is a double-stranded DNA virus. It is the second herpesvirus isolated from Cyprinid, is named Cyprinid herpesvirus 2(CyHV-2)according to the rules of International Committee on Taxonomy of Viruses. It is a member of Cyprinivirus, Alloherpesviridae. In this study we established a droplet digital PCR(ddPCR) method which is used for accurate quantification of CyHV-2DNA, and the ddPCR was compared with quantitative real-time PCR(RT-qPCR) on the specificity、practical application、reproducibility and sensitivity. For detecting CyHV-2 DNA, the sensitivity of the ddPCR assays was lowed 20 fold than RT-qPCR.The correlation coefficient(R2) showed a good linearity of amplification for both ddPCR(R2=0.994) and RT-qPCR(R2=0.994) assays. A positive correlation(R2=0.989)was observed between ddPCR and RT-qPCR assays. For determine the same dilution series of CyHV-2 DNA, the expected numbers of DNA copies calculated by RT-qPCR was always raised 10 fold higher than the number of copies determined by ddPCR.CyHV-2 DNA reproducibility determinated by ddPCR were found to be significantly more stable than by RT-qPCR. The assay of ddPCR have no cross-react with other similar fish herpesviruses, including CyHV-3(Cyprinid herpesvirus 3) 、 CCV(Channel catfish virus)、STIV(Soft-shelled turtle iridovirus),and EHNV(Epizootic hematopoietic necrosis virus), and the specificity of ddPCR is consistent with RT-qPCR. Therefore, the ddPCR method proved to be more precise than RT-qPCR.This absolute quantitation new tool may be useful to the standardize quantitative detection of CyHV-2 DNA.In order to obtain scFv antibody to detect CyHV-2, antibody-displaying phage was selected in four panning rounds against cyprinid herpesvirus 2(CyHV-2) by phage display approach, two positive clones which could produce soluble scFv antibody induced by IPTG were obtained. Dot blot results showed that the two scFv antibodies could specifically recognize CyHV-2. The soluble scFv antibodies showed a molecular weight 27 kDa by Western blot. All scFv antibodies could specifically recognize CyHV-2 proteins without cross-reaction with other virus proteins by ELISA.The results of immunohistochemical assay showed that all of scFv antibodies reacted positively with virus in CyHV-2-infected tissues. For ELISA, two scFv antibodies(P1A3, P1A5) showed had not cross-reaction with CyHV-3、CCV、STIV and EHNV.These scFv antibodies will be useful in diagnostic test development and pathogenesis studies for CyHV-2.