Establishment And Preli Minary Application Of Taqman Multiplex Fluorescence Quantitative PCR And RAA Rapid Detection Methods For Three Goose Viruses

Goose Parvovirus(GPV)is the pathogen of Goose Plague.The main feature of GPV is its fast transmission,wide range and high fatality rate.GPV is one of the important prevention viruses in goose breeding industry.Goose Paramyxovirus Disease(GPMVD)is a seasonal disease caused by Goose Paramyxovirus(GPMV).An acute infectious viral disease that infects geese regardless of age.GPMV is a variant branch of Newcastle Disease Virus(NDV)and can cause NDV disease in waterfowl.After infection by geese,it can cause lesions in the digestive system,immune system,and nervous system.The spread of GPMV has caused enormous economic losses to the goose industry in China.Goose Circovirus(Go CV)can cause apparent phenomena such as slow growth and weight loss in geese,while damaging the immune system of geese,attacking immune organs,causing lymphocyte atrophy and death,leading to secondary infections of other diseases.Increasing mortality can also cause slow development of the goose industry.In this study,starting with the precision of the detection method,a rapid and efficient detection method was established,followed by sample collection in captive geese farms located in some parts of Jilin Province,using an established method for sample collection of the three viruses,to determine the prevalence of the three viruses in some parts of Jilin Province,and to provide data support and basic methods for subsequent disease prevention and treatment.The details are as follows:(1)Establish a Taqman multiple fluorescence quantitative PCR detection method.Compare the VP3,F,and Rep replication regions of the gene sequences of GPV,GPMV,and Go CV,design specific primers and probes based on the conserved regions,establish a Taqman multiple fluorescence quantitative PCR detection method,optimize the ratio of primers and probes in the detection method,and obtain the optimal reaction conditions.Then,measure the standard curve of the detection method,and conduct anti-interference experiments,The results show that the detection method has good specificity and will not cross react with other viruses or bacteria.The sensitivity test results show that the minimum detection limit for GPV,Go CV,and GPMV is 6.59×10~1 copies/μL、5.57×10~1 copies/μL and 4.87×10~1copies/μL.The sensitivity is 10~4 and 10~2 times higher than that of conventional PCR,and the coefficient of variation of the repeatability test results is less than 2%,indicating the stability of the detection method.(2)This research aim to developed a rapid detection method for GPV,GPMV,and Go CV Recombinase Aided Amplification(RAA)combined with a test strip.RAA specific primers were designed for the conserved regions of GPV VP3,GPMV F,and Go CV Rep genes.After primer selection,reaction temperature,and reaction time optimization,the RAA detection methods for three viruses were successfully established.The RAA assay developed in this study requires only 20 min of reaction at 35°C to complete the amplification,and the products were visually inspected in a universal nucleic acid test strip,with a detection limit of 10~2 copies/μL.At the same time,the results in repetitive experiments are good,indicating that this method can be rapid,simple and reliable in the detection of clinical samples,and can also visually observe the characteristics of the results.(3)Based on the Taqman multiplex fluorescent quantitative PCR detection method and RAA recombinase aided amplification detection method,samples were collected from large-scale breeding farms in some areas of Jilin Province to detect the prevalence of three viruses.Conventional PCR was used as a verification method,80swabs collected from 12 regions were tested,and a total of 8 strains of GPV,6 strains of GPMV,21 strains of Go CV,and 3 strains of GPV mixed Go CV were detected.At the same time,the test results were verified using RAA test results and conventional PCR results,and the results were the same.The sample testing results in some areas of Jilin Province indicate that the likelihood of outbreaks of goose parvovirus and goose Paramyxovirus is low,but the possibility of such outbreaks cannot be completely ruled out.Therefore,further sample testing is required to investigate the three types of viral infections in Jilin Province.Additionally,the infection rate of goose circovirus is relatively high,and extensive sample testing is needed to validate this.As a result,greater attention should be paid to goose circovirus.