Isolation And Identification Of An Avian Reovirus And Establishment Of Anti-pollution RT-LAMP Detection Method

Avian reovirus?ARV?belongs to the Reovirdae family of Orthoreovirus,which can cause avian viral arthritis,dwarf syndrome,respiratory diseases,intestinal diseases,immunosuppression and so on.As a result,broilers'feed conversion rate is low,their weight gain decreases and their growth is nonuniform.Besides lameness,layer chickens also cause the decrease of egg production and hatching rate.All of these increase the cost of breeding.Since 1985,when avian reovirus infection was first reported in China,it has been reported in many provinces and municipalities.The infection situation has become increasingly serious,which seriously jeopardizes the development of poultry industry.Deepening researches on this disease are of positive significance to the healthy and stable development of chicken industry.In this study,an ARV strain was isolated from an immuned broiler suspected of ARV infection,and its sequence analysis and physicochemical properties were studied.At the same time,a rapid UDG-RT-LAMP detection method which can eliminate carryover contamination was established,and the reaction conditions of the method were optimized.All the studies have provided theoretical basis and new ideas for the prevention and rapid detection of ARV.In this study,SPF chicken embryos and chicken embryo liver cell?CEL?were used to isolate viral pathogens from immune broiler joint disease materials suspected of ARV infection in Weifang,Shandong Province.The isolate was named WF,and was sequenced and analyzed,virulence was determined,animal regression test,physical and chemical properties were tested.The characteristic lesions of chicken embryos and CEL cells inoculated with the treatment solution were consistent with the reports.RNA was extracted separately,and the band size of amplified products in RT-PCR was as expected.The genetic evolution analysis of?C gene indicated that the isolate belonged to the fourth branch,the nucleotide homology between the isolate and reference strains ranged from 50.3%to 74.7%,and the amino acid homology ranged from 22.7%to 54.3%.The homology with the common vaccine strains was low.The isolate had an ELD₅₀0 of 10^-5.25/0.2 mL and a TCID₅₀0 of 10^-5.29/0.1 mL,which was sensitive to 60?and 70?,insensitive to 50?and chloroform,and stable in the pH range of3.0 to 9.0.Animal regression test showed that the clinical manifestations and necropsy changes of 1-day-old healthy SPF chicks infected with the strain were consistent with naturally occurring cases.A set of specific LAMP amplification primers was designed based on ARV M1 gene,and a UDG-LAMP system for ARV detection was preliminarily established,and the reaction conditions were optimized.The optimum concentration was 1.2 mM for dNTP,8 mM for MgSO₄,320 U/mL for Bst 2.0 WarmStart DNA polymerase and 60 min for amplification at65?.The method has good specificity and can only amplify ARV.The sensitivity is high,and the minimum detection limit is 3×10² copies/?L,which is 100 times higher than that of the PCR method.The effect of anti-carryover contamination is obvious.In the clinical sample test,the positive detection rate of the UDG-RT-LAMP method was 16.7%higher than that of the RT-PCR method,which indicated that the established UDG-RT-LAMP assay is more advantageous in clinical detection applications.