Development Of Visual Cross-priming Isothermal Amplification Detection And Epidemiological Investigation For Fur Animal Amdoparvovirus

To prevent and control the fur animals(minks,foxes and raccoon dogs)infect with amdoparvovirus.In this study,the primers were designed and synthesized.It was located at the conserved segment of VP2 hypervariable region within most amdoparvoviral genome sequences published in GenBank.Development a rapid quantitative detection and identification method,SYBR Green I qPCR for fur animals amdoparvovirus,and a detection of fur animals amdoparvovirus visual cross-priming isothermal amplification(CPA).The result show that qPCR and visual CPA had a good sensitivity and specificity,and the minimum concentrations of qPCR and visual detection for Aleutian mink disease virus(AMDV)and raccoon dog and arctic fox amdoparvovirus(RFAV)were 5.38×102 copies/?L and 5.93×102 copies/?L,respectively.The qPCR method had a high sensitivity and specific detection of fur animal amdoparvovirus,which can be used to clearly distinguish different amdoparvoviruses by measuring Tm of viral amplicon;The CPA method had a high sensitivity and specific detection of fur animal amdoparvovirus,which was visually detected by fluorochrome.It can provide a widely applicable technique for rapid quarantine,which is more suitable,especially in resource limited laboratories and farms as well as in field conditions.To survey the infection situation of minks,raccoon dogs and blue foxes in Jilin and Liaoning provinces,we used IAT,CIEP and PCR to test the collection 5,135 samples in a total of eight farms from Jilin and Liaoning provinces.The result show that,except blue foxes,a high prevalence of amdoparvovirus both minks and raccoon dogs in these farms.Remarkably,we detected all minks in two farms from Liaoning province and found that different progress after infection with AMDV from two mink farms with distinct AMDV prevalence rates.Futher analysis show,in the farm with a low infection rate,the IAT-CIEP-group was the largest group at 72 %.The PCR test indicated that there were early infections in experimental mink herds.The qPCR result showed that,the low infection rate farms,which continue to eliminate infected minks,are able to do so at a higher sensitivity than high infection rate farms and a diff erence of two orders of magnitude between the numbers of copies of the viral DNA in the two farms.In contrast,for the high infection rate farms,the CIEP+ herd was at 98% infection rate,while for the IAT-CIEP+ group it was only 41 %.It indicated that herds had a mixture of tolerant and resistant mink.Then PCR test further demonstrated that some minks could develop into tolerant and resistant mink.This suggests that farms should annually use IAT,CIEP and PCR to screen for tolerant and resistant mink.Phylogenetic analysis was performed using the partial VP2 hypervariable region sequence from some PCR positive samples.Based on the evolutionary tree,the AMDV sequences fell into five gene groups,designated I–V,with relatively high diversity,however the RFAV sequences were only in group VI.The two provinces AMDV sequences were major present in group I which contained the reference sequences AMDV-Harbin and AMDV-Beijing.But two sequences which named JZ-SD-32 and LZ-ZCSD1-B were felled into group II and V,respectively.AMDV and RFAV in the tree showed in natural infection,the two viruses were not found cross-infection.In a word,we selected eight farms in Jilin and Liaoning provinces for epidemiology survey and use combination quarantine to analyze different infection rate farms.In order to provide a theoretical basis for the prevalent characteristic of AMDV and RFAV and introduction,quarantine of different infection rate farms to eliminate minks.