Genetic And Evolutionary Analysis Of Feline Bocavirus In Some Areas Of Anhui Province And Establishment Of Quantitative PCR Detection Method

Feline Bocavirus(FBo V)is a virus associated with Feline diarrhoea and has three genotypes.FBo V-1 was first identified in 2012 in Hong Kong,China,which is the world first discovered the existence of the cat Bocavirus.Then in 2014,American researchers discovered two new genotypes,named FBo V-2 and FBo V-3.FBo V was also found for the first time in Northeast China.However,there is no report on whether FBo V exists in Anhui,and its genetic evolution information has not been studied.In addition,the current detection method for FBo V-1 is only PCR,but the low detection sensitivity is likely to lead to missed diagnosis.Therefore,it is imperative to develop a highly efficient and sensitive method.In this study,the full genome sequences of 5 FBo V strains were successfully amplified from 58 Feline feces samples collected in Anhui Province.Sequence analysis showed that the 5 FBo V strains contained 2 FBo V-1 strains,1 FBo V-2strains and 2 FBo V-3 strains.Phylogenetic analysis of the complete gene sequence and NS1 gene also verified that the five FBo V strains detected formed three different clusters with their genotype reference strains,respectively.Genetic recombination analysis showed that there were two recombinant strains,namely MASCY1 and HFTH-22,which were FBo V-1 and FBo V-2,respectively.According to the analysis of Sim Plot,the recombination regions were mainly on NP1,VP1 and VP2.Selection pressure showed that both FBo V-1 and FBo V-3 had positive mutation sites and tended to mutate,while FBo V-2 had no positive mutation sites and tended to be conserved.This study is the first to report the prevalence of FBo V in Anhui Province,which is helpful to further understand the phylogenetic and molecular characteristics of FBo V,and provide reference for the prevention of FBo V.In this study,a SYBR Green I-based quantitative polymerase chain reaction(q PCR)assay was established to detect FBo V-1.It is the first time to create a fluorescent quantitative detection method for the detection of FBo V-1,which has the advantages of high sensitivity,rapidity,and strong specificity.Successfully constructed q PCR method based on conservative NS1,The melting curve showed a single melting peak at 83.0?.The results of sensitivity showed that the detection limit of the q PCR was 3.87×10~1copies/?L.Of note,the detection limit of the polymerase chain reaction(PCR)was 3.87×10~3copies/?L.The highest intra-assay and inter-assay coefficients of variation(CV%)were 0.98%and 1.42%,respectively.The positive detection rate of 128 clinical samples using the q PCR and the PCR was7.0%(9/128)and 4.7%(6/128),respectively.Taken together,these results indicated that the established q PCR assay has good sensitivity,high specificity,and good reproducibility.Therefore,it could provide support for the rapid and efficient clinical detection of FBo V-1.