Establishment And Application Of Indirect ELISA Detection Method For Novel Duck Reovirus

Novel duck reovirus（NDRV）infection is an avian infectious disease caused by novel duck reovirus（NDRV）,which is characterized by necrosis of duck spleen.Clinically,there are a large number of pinhead-sized white necrotic foci and a large number of hemorrhagic spots on the liver surface.The spleen is enlarged with dark red bleeding symptoms,and there are many gray and white necrotic spots on the surface,some of which converge to form flower spots.Sometimes bleeding lesions can be observed in the heart,kidney,bursa of fabricius,etc.The pathogen can infect the alimentary tract and respiratory tract,the disease progresses rapidly and the mortality rate is high.The study showed that the new duck reovirus is an RNA virus,which is classified as Orthoreovirus in Reovirdae.As the virus is a multi-segment double-stranded RNA,it is easy to produce gene mutations,which greatly increases the possibility of producing new pathogenic strains,and then bring new harm to the waterfowl breeding industry.Therefore,detection is of great significance for the prevention,control and purification of NDRV.ELISA detection method has become a fast and accurate detection method commonly used at present because of its high sensitivity,convenient and simple operation.In this study,a recombinant protein was obtained by prokaryotic expression of σC gene of NDRV,and an indirect ELISA method was established to detect NDRV.The indirect ELISA method was then applied for serological investigation.The results were as follows: Total RNA of NDRV was extracted from diseased ducks with necrosis of spleen.The σC gene sequence of NDRV was amplified by PCR and cloned into p MD18-T vector,named p MD18T-σC.Then,the σC gene sequence of NDRV on p MD18T-σC vector was digested and connected to p ET-32 A expression vector,which was named p ET-32a-σC.BL21 cells were transformed with PET-32A-σC expression vector to increase the expression of recombinant protein.The purified protein was analyzed by Western blot.Using i ELISA technique,the recombinant σ C protein was used as envelope antigen to detect NDRV antibody in duck serum.Then the conditions of the established i ELISA method were optimized to determine the best conditions of the established i ELISA detection method.The established i ELISA method was used to detect goose parvovirus（GPV）,new goose parvovirus（NGPV）,Muscovy duck parvovirus（MDPV）,duck hepatitis virus type I（DHAV-1）,duck hepatitis virus type iii（DHAV-3）and NDRV positive serum.The results showed that the detection values of other serum were much smaller than those of NDRV positive serum,which proved that the established i ELISA method had high specificity for detecting NDRV serum antibodies.The established i ELISA method was used to detect serum samples of artificially challenged ducks,ducks immunized with inactivated vaccine for 10 days and ducks resistant to NDRV for 30 days.The results showed that the positive rate of ELISA in challenged ducks was up to 100%.After 10 days of immunization with inactivated vaccine,the positive rate of ELISA was 100%.ELISA results showed that the positive rate of 30 days after challenge was consistent with that of 10 days after immunization.These results indicate that the established i ELISA method is feasible for serological investigation of NDRV antibodies.The feasibility of the established i ELISA was further verified by the detection of clinical samples.The positive rate of σC-ELISA was more than 80% in 100 sera from clinically healthy ducks.The results showed that the positive rate of ELISA method was very high.The positive rate of σC-ELISA in 100 sera from clinically healthy ducks was up to 78%.The positive rate of NDRV was as high as 100%.The positive rate of σC-ELISA was 69.5% in 200 samples of duck serum randomly sent from two farms in Shandong Province.The above data indicated that the infection rate of NDRV was high in Chinese ducks,which could easily cause immunosuppression and large-scale secondary infection.The research on the new duck reovirus is still urgent.This study made a certain contribution to further understand the epidemic status of duck reovirus disease,provided a rapid and simple method for the detection of duck reovirus,and laid a certain technical foundation for the subsequent research.