Phylogenetic Of CanineCV And Established Of RT-qPCR Detection Method With CaAstV

Canine circovirus(Canine CV)was first reported in China in 2016.However,little is known about the prevalence of Canine CV in Anhui and Jiangsu provinces in China.In this study,a total of 128 dog fecal samples were collected from the two provinces of Anhui and Jiangsu in central and eastern China for testing.The complete genome were amplified for Canine CV positive samples,and phylogenetic and bioinformatics analysis were performed on them.At the same time,the clinical symptoms caused by Canine CV and Canine Astrovirus(CaAstV)infection are very similar,which brings a certain degree of difficulty to clinical diagnosis.In this study,the Rep gene of Canine CV and the Cap gene of CaAstV were used as the target fragments,two pairs of specific primers were designed respectively,and a dual RT-q PCR detection method based on SYBR Green I was established.The results show that:Firstly,genetic study and bioinformatics analysis.Three Canine CV positive samples were detected in 128 dog stool samples collected in Anhui and Jiangsu provinces,China.The whole genome sequence of these 3 Canine CV positive samples was successfully amplified by PCR and confirmed by sequencing.After the sequence was assembled,the complete genome sequence was uploaded to the NCBI Gen Bank database,and the accession numbers of the three strains were:MN863535,MN863536,and MN863537.According to the analysis of the phylogenetic analysis of the whole genome,it is found that Canine CV has a high similarity with the fox circovirus in mammals.Among all the Canine CVs in the Gen Bank database,they are closely related to the isolates from Guangxi and Heilongjiang,China.Secondly,we found four potential recombination events that were confirmed by more than three different methods in RDP 4.0.The homology of the 3 Canien CV strain at the nucleotide level is 91.0%-91.7%,and the nucleotide homology of the other 17 Canien CV strains is between 84.2%-96.3%.The homology of Canine CV Cap protein at nucleotide and amino acid levels is 80.6%-96.3%and 90.7%-97.8%,respectively.The results of bioinformatics analysis show that the Cap protein of Canine CV is a hydrophilic and stable protein,without signal peptide and transmembrane domain.There are 33 phosphorylation sites,and there may be 7 potential B cell epitopes and 1Th cell epitope.Secondly,establishment of a duplex SYBR green I-based real-time PCR assay for the rapid detection of Canine CV and CaAstV.In the assay that we established in this study,Canine CV has a Tm value of 79°C and CaAstV has a Tm value of 86°C,which has high specificity and can be based on the Tm value of the two viruses to distinguish these two viruses.This assay is highly specific and has no cross-reactivity with other common canine viruses.This assay has high sensitivity,and its detection limits are 9.25×10~1 copies/?L and 6.15×10~1 copies/?L,which are 100 times that of traditional PCR detection methods.At the same time,the method has good repeatability and reliability.The positive detection rate of this method in clinical sample detection is also higher than that of traditional PCR detection methods.In summary,this study successfully amplified the complete genome sequence of three Canine CV strains,and successfully constructed a duplex SYBR green I-based real-time PCR assay with high sensitivity,strong specificity and good reproducibility for the Rep gene of Canine CV and the Cap gene of CaAstV.This study expands the genetics data of Canine CV and provides an effective,economical and easy-to-promote detection method for the clinical detection of Canine CV and CaAstV.