Establishment And Application Of Phage-mediated Avian Infectious Bronchitis Virus ELISA Detection Method

Avian infectious bronchitis(IB)is an acute and highly contagious respiratory infectious disease distributed globally,and it is one of the main infectious diseases that endangers the domestic poultry industry.Avian infectious bronchitis virus(IBV)is the pathogen of IB.The S1 protein of IBV is the target of host's immune response,which can induce the host to produce corresponding neutralizing antibodies and hemagglutination inhibitory antibodies.Due to the characteristics of IBV,it has important practical value to explore the pathogenicity of different IBV strains and develop a rapid and effective detection method that can directly detect antigens to monitor IBV infection in poultry.In this research,the phage with highest affinity to IBV was screened from five phages which bear high affinity peptides with IBV S1 protein,and an indirect ELISA detection method mediated by IBV high affinity phage for IBV pathogen was established.Then the conditions were optimized.The final ELISA reaction conditions are as follows:the affinity phage with the concentration of 10⁷ pfu/m L was coated overnight at 4?,5%skim milk sealed at room temperature(RT)for 3 h,IBV polyclonal antibodies were incubated at RT for 1 h at a dilution of 1:1000,the horseradish peroxidase labeled secondary antibody was incubated at RT with dilution of 1:2000 for 1 h.The established indirect ELISA detection method has high sensitivity and specificity and good reproducibility.The sensitivity of this method can detect the virus concentration of 10^-2 pfu/m L.This method has no cross-reactivity with other poultry-derived infectious agents which demonstrate similar clinical symptoms.At the same time,we infected the 10-day-old chickens with IBV MD strain,and the pathogenicity of this strain was preliminarily explored.The IBV-infected chicks began to show obvious clinical symptoms and pathological changes 3 days after virus challenge,among which,it became the most obvious 5 to 7 days post infection.Autopsy showed typical spotted kidney lesion.With RT-PCR method,IBV was able to be detected with the appearance of positive band 3 days post infection,afterwards with the brightest band 5 to 7 days and disappeared 21 days post infection.At the same time,the established indirect ELISA method was used to detect the pathogen,and the result was nearly the same as those of RT-PCR method.