Establishment And Application Of Microdrop Droplet Digital PCR For Detection Of Aleutian Mink Disease Virus

Aleutian mink disease(AMD)is an infectious disease caused by Aleutian mink disease virus(AMDV),which is mainly characterized by chronic persistent infection and plasma cell infiltration.AMDV can be transmitted in two ways,horizontally and vertically.Once infected,it will cause serious economic losses.At present,there is no effective vaccine for the prevention and control of the disease.The best measure to prevent Aleutian disease in mink is to purify the population in time.A variety of AMDV detection methods have been established,such as ELISA,convective immunoelectrophoresis,fluorescent quantitative PCR,etc.,but they still cannot meet the requirements of"early diagnosis and early detection"for the prevention and control of the disease.In this study,AMDV droplet digital PCR(droplet digital PCR,dd-PCR)quantitative detection method was established and this method has the characteristics of high sensitivity,simple operation,easy quantification.After the detecting application of clinical samples and environmental samples in farms,we found this method could realize the early detection of pathogens,and provided technical support for the introduction of mink,aleutian disease purification and germplasm resource protection.1.Establishment of a dd-PCR method for detection of AMDVAccording to the full length of the gene of the classic AMDV strain ADV-G strain,the PCR primers were designed,and the VP2 gene was successfully amplified,which was then constructed into p MD18-T vector to prepare the AMDV plasmid.According to the primer probes of the real time fluorescent quantitative PCR(RT-qPCR)method in the local standard of Shandong Province(standard number DB/T4204-2020),the concentration of primer probes,annealing temperature,and heating and cooling rate were optimized;the sensitivity test,specificity test and repeatability test for droplet digital PCR were also carried out.Finally a dd-PCR method was established for detection of AMDV.Using plasmid standards as samples,compared with the RT-qPCR detection method of AMDV published in Shandong local standards,the linear relationship between the two test methods were dd-PCR(R~2=1)and RT-qPCR(R~2=0.994),indicating both of them own a good linear relationship.But,the sensitivity test shows that the minimum amount of RT-qPCR detection is 140 copies/?L,and the minimum detection limit of PCR is about 1.4 copies/?L.The characteristic test shows that the established AMDV dd-PCR detection method detects AMDV,mink enteritis parvovirus(MEV),canine distemper virus(CDV),mink influenza virus(MIV),and the results show that AMDV is positive,and other viruses are all Negative,strong specificity;repeatability test shows that the coefficient of variation(CV%)of the experiment within the group and the experiment between groups is less than 3%.Therefore,the detection method has the advantages of high sensitivity,strong specificity and good stability.2.Preliminary application of dd-PCR detection method for mink Aleutian virusThe dd-PCR established in this experimental study was used to detect 507 samples from mink farms with suspected disease,and the established dd-PCR and RT-qPCR were used for nucleic acid detection.The positive rates were 39.0%and 24.6%,respectively.Dd-PCR and RT-qPCR were used for nucleic acid detection.The positive rates of 261 tissue samples were42.1 and 34.9%,and the positive rates of 246 environmental samples were 32.2%and 17.8%,indicating dd-PCR has higher sensitivity and can effectively detect samples with low virus content such as fecal cotton swabs and environmental samples.Analysis of the detection res?Lts of dd-PC and RT-qPCR by Kappa method showed that the Kappa coefficient was0.88,indicating that the two had a good coincidence rate.3.Genetic evolution analysis of mink Aleutian virus VP2 geneIn this study,VP2 gene sequencing was performed on the positive samples detected by dd-PCR,and a total of 17 gene sequences from different samples from different regions and samples in Shandong were obtained.The neighbor-joining method was used to construct a phylogenetic tree of 17 AMDV VP2 genes.Genetic evolution analysis found that the VP2gene sequence of AMDV Shandong isolates is divided into three branches,A,B,C,and environmental samples W-SD11 and W-SD12 from the same farm in Weifang are in the A/B branch respectively,indicating that AMDV has different genes Type can coexist in the same mink farm.There are 11 AMDV Shandong strains VP2 gene sequences gathered in the A and B branches,and are closely genetically related to AMDV foreign isolates(ADV-Far East,AMDV-Utah,AMDV-TR),suggesting that the mink AMDV in Shandong Province may have a relationship Introduction related;the VP2 gene sequence of the 4 AMDV Shandong strains (W-SD19,W-SD20,W-SD21,W-SD27)in the C branch is closely related to the AMDV gene in mink farms in Jilin and Dalian.The domestic introduction of mink in Shandong Province is mostly from the northeast,further indicating that the distribution of AMDV is closely related to the introduction of mink.