Establishment Of Detection Method To Detect Canine Parvovirus And Its Antibody

Canine parvovirus disease is an acute,highly contagious disease of dogs and carnivores caused by canine parvovirus(canine parvovirus,CPV).The main clinical manifestations of the disease include acute hemorrhagic gastroenteritis,vomiting and severe leukopenia.CPV are highly contagious,especially to puppies and can also infect adult dogs.The mortality rate of the disease can be as high as 80%.Adult dogs can be infectious when they are inapparently infected.It is extremly harmful to our country’s dog industry.Currently,there is no specific treatment modality of the disease other than early vaccine as a prevention method.Early detaction and treatment of the disease in early stage has certain efficacy.Therefore,the establishment of a fast and accurate detection method of early canine parvovirus disease is urgent.In this study,we built a fast detection method from the perspective of pathogen and antibody detection.1.Establishment and Application of Pathogen Detection MethodWe designed a pair of primers that amplified 574 bp fragment based on CPV VP2 gene’s conserved sequence and optimize the reaction system and reaction condiion to established a fast and sensitive nanoparticle-assisted PCR(nanoPCR)detection method of caine parvovirus(CPV).NanoPCR is 1000 times more sensitive than conventional PCR,with a minimum detection amount of 10 copies/μL of CPV DNA.No cross-reaction with other viruses was observed through specificity test.Furthermore,the CPV positive rates of 46 clinical samples collected from Beijing,Jilin and Gunagxi were 93.4%(43/46)and 78.3%(36/46)using nanoPCR and conventional PCR respectively.These clinical data indicated that nanoPCR is more sensitive and applicable for detection of low concentrations of CPV clinical samples.Faeces of dogs detected positive with nanoPCR were collected(from rectal swabs of canine suspected to be infected Canine parvovirus).The isolated virus could generate cytopathic effects(CPE)in Feline keyned 81(F81)cells,and was identified by NanoPCR test,hemgglutination test,and immunoperoxidase monolayer assay(IPMA).The results indicated that the isolates had obvious characteristics of pig red cell agglutination,and was positive of IPMA;the specific 564 bp fragment was obtained from sample genomes by NanoPCR test was gene sequencing analysis showed that the virus shared high gene homology with CPV-2c and had two special amino acids mutation of 267(F)→267(Y),324(Y)→324(I),which thus was named CPV-2c-Gungxi23,The results of this study lay a foundation for further understanding of the variation of CPV,the selection of vaccine strains and the effective prevention and control of CPV disease.2.Establishment of antibody detection methodCanine parvovirus antibody positive serum prepared in laboratory was used as detection method of IPMA(immunoperoxidase monolayer assay).The optimal results of IPMA reaction conditions showed that 100 times diluted virus,with 1:100 ratio of positive serum and 1:3000 ratio of HRP-SPA secondary antibody was the best reaction condition.Sensitivity test showed that serum can still react when do the 1:2000 diluted with no cross reaction with other virus positive serum.The positive rate of 46 clinical serums samples was 65.2%(30/46).This method provides a new technique for serological diagnosis of CPV method as a simple and fast supplementary to the epidemiological investigation of canine parvovirus.