Establishment Of Method For Detection Of Woodchuck Hepatitis Virus

Objective To establish a Real-time PCR method for detection of woodchuck hepatitis virus (WHV) DNA and a modified double antibody sandwich ELISA for detection of woodchuck hepatitis surface antigen (WHsAg) in serum of WHV-infected woodchuck. Methods For WHV DNA detection:13pairs of primers were designed according to the coding sequence of woodchuck hepatitis virus core antigen (WHcAg) and woodchuck hepatitis virus surface antigen (WHsAg). The primers demonstrated high sensitivity and specificity were used for Real-time PCR detection of WHV DNA in woodchuck serum. Then, the method was applied in detection of WHV DNA in serum of woodchucks. For WHsAg detection:To establish Methods N-terminal of tandem WHsAg was expressed by E. coli and then purified by Ni-NTA His·Bind Resins. Rabbits and chicken were immunized with purified WHsAg to prepare antiserum and then purify polyclonal antibodies. A modified double antibody sandwich ELISA method was developed to detect WHsAg in serum of woodchuck.Results For WHV DNA detection:The selected primers pair (WHVSF1, WHVSR1), which designed based on5’end of WHsAg gene showed a detection limitation of1×101copies/μL. The R2value of standard curve of viral copy number and the Real-time PCR Ct value was0.997, and no unspecific bands were observed during electrophoresis. For WHsAg detection:The purity of purified WHsAg antigen was95%. The titer of purified rabbit polyclonal anti-WHsAg and chicken polyclonal anti-WHsAg reached1:1,024,000and1:64,000respectively. The limitation of detection by ELISA was10ng/mL.Conclusion A real-time PCR method for detection of WHV DNA in serum of WHV-infected woodchuck and a modified double antibody sandwich ELISA for detection of WHsAg in serum of WHV-infected woodchuck was established. These methods set up a basis tool for WHV model development.