Establishment Of Rapid Detection Methods For Canine Circovirus And Sequence Analysis Of Prevalent Strains In Guangxi

Canine circovirus(Canine CV)is a newly discovered circovirus.Since its first report in the USA in 2012,it was confirmed that Canine CV infection had gradually developed a global epidemic.Dogs are considered as the natural host of Canine CV.Dogs of different ages and genders can be infected and puppies are more susceptible than adult dogs.As the academics and pet clinic industry pay more attention to Canine CV,there is an urgent need to develop new rapid detection methods with better sensitivity,specificity and convenient operation.Recombinase-aid amplification(RAA)can amplify the target fragment rapidly at 37-42℃,which is suitable for instant diagnosis.Digital PCR,the third generation of PCR,enables accurate absolute quantitation of target molecules by partitioning the reaction system into numerous compartments.The RAA and digital PCR methods of Canine CV detection were successfully established.Specificity,repeatability and sensitivity tests were conducted respectively to evaluate the two methods.In addition,in order to investigate the prevalence and variation of Canine CV in Guangxi,500 dog serum samples were collected from several regions of Guangxi in 2022 for pathogen detection.The whole genome sequences were obtained by amplifying positive samples of Canine CV,and phylogenetic analysis was conducted subsequently.The results were as follows:1.The established RAA method of Canine CV can be completed within15～20 min at 39℃.There was no cross-reactivity with canine parvovirus,canine adenovirus,canine parainfluenza virus,rabies virus and canine distempervirus,which indicated good specificity of the method.Sensitivity test showed the minimum detection was 9.06×10~2copies/μL.82 dog fecal swabs were tested by RAA and the positive rate of Canine CV was consistent with PCR results.2.In the study,established digital PCR method of Canine CV had good specificity and there was no cross-reactivity with other canine viruses.Compare with q PCR,digital PCR had high sensitivity with the minimum detection of 6.62 copies/μL,which was 10 times higher than that of q PCR.The quantitative results of digital PCR between standard plasmids had good linearity in the range of 6.62×10~0～6.62×10~5copies/μL with a correlation of 1and the amplification efficiency of 94.4%.Repeatability tests in and between groups showed good repeatability of the methods.96 dog serum samples were tested by digital PCR and the positive rate of Canine CV was 3.13%(3/96),which was higher than that of q PCR.3.A total of 25 Canine CV positive samples were detected in five regions of Guangxi in 2022,with a positive rate of 5.0%.15 whole genome sequences of Canine CV strains were amplified and sequenced.The nucleotide homology of sequenced strains was 85.8%～100%and the homology between sequenced strains and reference strains was 80.7%～96.5%.Phylogenetic tree based on the ORF2 gene showed that Canine CV strains can be divided into three main genotypes.15 strains from Guangxi were classified as Canine CV-1c and Canine CV-2.In conclusion,Canine CV detection methods based on RAA and digital PCR were successfully established.Both of RAA and digital PCR showed high specificity and good sensitivity and they were applicable to the detection of clinical samples.Based on the detection of Canine CV in some regions of Guangxi in 2022 and the whole genome sequencing and analysis of prevalent strains,it was found that prevalent strains in Guangxi currently were Canine CV-1c and Canine CV-2.