Establishment Of Canine Circovirus Detection Method And Molecular Epidemiological Investigation

Canine circovirus(Canine CV),belonging to the Circovirus of Circoviridae,is a new mammalian circovirus which can cause hemorrhagic gastroenteritis,necrotizing vasculitis,granulomatous inflammation and lymph necrotic then seriously harm the health of canines.In order to understand the prevalence and genetic diversity of Canine CV in China,this test first established the SYBR Green?real-time PCR detection method for the virus Rep gene of Canine CV before a total of 1666 canine serum samples from Guangxi Zhuang Autonomous Region,Guangdong Province,Sichuan Province,Chongqing City,Shanghai,Jiangsu Province,Shandong Province,Jilin Province,Inner Mongolia Autonomous Region,and Beijing;88 fecal samples of other canine animals(fox 37 and raccoon 13,from Liaoning Province;cat 38,from Guangxi Zhuang Autonomous Region)were tested.Then whole-genome amplification on Canine CV positive samples DNA was performed.Homology analysis was finished using bioinformatics software and a phylogenetic tree was built to analyze the genetic evolution of Canine CV in mainland China.Finally,in order to deepen the function of Rep protein,the Canine CV Rep gene was cloned into the prokaryotic expression vector p ET28a to construct a recombinant expression plasmid,and the prokaryotic expression of the Rep protein was tried.The results showed that the Ct value of the Canine CV SYBR Green?real-time PCR method established in this experiment had a good linear relationship with the standard products in the range of6.18×10~9?6.18×10~2 copies/?L,the correlation was 0.998,and the slope was-3.671;the detection sensitivity was 6.18×10~1 copies/?L,and porcine circovirus type 2,porcine circovirus type 3,canine distemper virus,and canine infectious hepatitis virus,canine parvovirus,canine parainfluenza virus,canine adenovirus type 2,rabies virus,etc.were not specifically amplified;all dilution standard templates were at specific melting peak appeared at 86.45?.The above results showed that the SYBR Green?real-time PCR detection method established in this experiment had good sensitivity and specificity,and could met the needs of rapid clinical detection.dog serum samples and feces samples of other canine animals/cat from 10 provinces in the country,were detected.The results showed that the positive rate of dog serum samples was 5.82%(97/1666),and Canine CV was not detected in other animal tissue samples.The genome sequence of 64 Canine CV Chinese epidemic strains was successfully obtained.The homology between the strains was 85.6%?100%,and the sequence homology with 77 reference strains was 78.3%?100%.According to ORF2,Canine CV could be divided into two main genotypes(Canine CV-1 and Canine CV-2)and 8 genotypes(1a,1b,1c,1d and 2a,2b,2c,2d),of which 2c and 2d were newly discovered genes Subtype.Using RDP4 software to perform recombination analysis on the viral genome,it could be seen that recombination events were distributed throughout the Canine CV genome.The prokaryotic expression vector(plasmid p ET-28a-Rep)was successfully constructed and transformed into E.coli BL21 induced by IPTG.Bacterial proteins were collected for SDS-PAGE gel electrophoresis detection.And the target band could be detected at 35 Ku,which indicated that the recombinant protein is mainly expressed in the form of inclusion bodies.The Rep recombinant protein could be recognized by the primary antibody using western blotting while the control group had no specific reaction,indicating that the Rep recombinant protein had been correctly expressed.In this experiment,the SYBR Green?real-time PCR method for Canine CV Rep gene was successfully established,which can achieve rapid,sensitive and accurate diagnosis of Canine CV.The molecular epidemiological survey and genetic evolution analysis of Canine CV in10 provinces of China were completed.It enriched the local and genetic variation data of Canine CV epidemic.In addition,the successful prokaryotic expression of Canine CV Rep protein laid the foundation for the establishment of new serological detection methods and the development of subunit vaccines.