Study On The Rapid Differential Detection For The Variant Strain Of Fowl Adenovirus Serotype 4

Fowl adenovirus serotype 4（FAdV-4）infection can cause hydropericardium-hepatitis syndrome（HHS）in poultry and was first reported in Pakistan in 1987.Since 2015,the disease has erupted in Shandong,Henan and other places and spread to many provinces,mainly infecting 3 to6-week-old broilers.The FAdV-4 gene sequence of this epidemic has been changed,and the pathogenicity is significantly increased,causing serious economic losses to the poultry industry in China.Based on the characteristics of the FAdV-4 variant strain,three rapid detection methods for the variant strain of fowl adenovirus serotype 4 were established:sandwich ELISA,duplex fluorescence quantitative PCR and droplet digital PCR.Based on the monoclonal antibody of the FAdV-4 variant strain Penton protein,a sandwich ELISA method for rapid identification of the FAdV-4variant strain was established by optimizing the ELISA reaction conditions,such as the concentration of antigen and antibody and the action time.The specificity of this method was proved to be good and could be used for specific detection of the FAdV-4 variant strain.No cross reaction with other serotypes of avian adenovirus and common avian disease pathogens.With high sensitivity,the minimum detection amount was 9.7×10²TCID₅₀/m L.The coefficient of variation of inter-batch and intra-batch repeatability tests was less than 5%.216 clinical samples were tested by sandwich ELISA with the variant strain of fowl adenovirus serotype 4,8 of which were positive.In 216clinical samples tested by this method,8 variant strain of FAdV-4 were positive.Two sets of primers and probes were designed for specific detection of the FAdV-4 variant strain and all strains of FAdV-4,aiming at the deletion of1966bp at about 35000-37000bp at the right end of the genome sequence of the FAdV-4 variant strain and Hexon gene conserved sequence.A duplex fluorescence quantitative PCR method was established for rapid identification of the FAdV-4 variant strain,and its specificity,sensitivity,repeatability and clinical samples were tested.The results showed that the method had strong specificity,and the variant strain of FAdV-4 was infected when both fluorescence channels showed amplification curves.If only the ROX channel showed amplification curve,it indicated that there was no mutant strain,suggesting the presence of other FAdV-4 strains.Both fluorescent channels did not cross-react to other serotypes of avian adenovirus and a variety of common avian disease pathogens.Sensitivity results showed that the variant strain of FAdV-4 could be detected when the template concentration was 20 copies/μL.Repeatability results showed that the coefficient of variation was less than0.8%in both the inter-batch and intra-batch tests.A total of 216 clinical samples collected from live poultry markets were tested by this method,and a total of 8 FAdV-4 variants were detected positive.The duplex fluorescence quantitative PCR method established in this study was able to double verify the variant strain of FAdV-4 by double fluorescence channel detection.When only the universal detection channel was positive,it indicated that other FAdV-4strains were infected,which provided technical support for the prevention and control of FAdV-4.Specific primers and probes were designed according to the gene sequence difference of the FAdV-4 variant strain.By optimizing the reaction conditions such as the concentration and temperature of primers and probes,a rapid detection method for the variant strain of FAdV-4 by droplet digital PCR was established,and its specificity,sensitivity and repeatability were tested.100 clinical samples were tested by this method and q PCR simultaneously.The results showed that the method had strong specificity and could specifically detect the variant strain of FAdV-4.The results were negative for other serotypes of avian adenovirus and common avian disease pathogens.The minimum detection of FAdV-4 mutant was 2.0 copies/μL.The coefficient of variation of inter-batch and intra-batch repeatability tests was less than 2%.The results of this method were positive in 8 out of 100 clinical samples,which was consistent with the results of fluorescence q PCR.The rapid differential detection method of the FAdV-4 variant strain by droplet digital PCR established in this study has the characteristics of strong specificity,high sensitivity and good repeatability,which can achieve absolute quantification of detection results and can be used for rapid quantitative diagnosis of the FAdV-4 variant strain.In this study,the sandwich ELISA,duplex fluorescence quantitative PCR and droplet digital PCR were established for rapid identification and detection of the FAdV-4 variant strain,which were proved to have strong specificity,high sensitivity and good reproducibility,providing technical support for the prevention and control of the FAdV-4 variant strain.