| Canine coronavirus(CCo V)is a single-stranded positive-sense RNA virus with a genome length of approximately 30 kb,which can cause highly contagious diseases.The characteristic of CCo V infection is high morbidity but low mortality,with clinical symptoms including gastroenteritis,anorexia,vomiting,diarrhea,and dehydration.Canine parvovirus(CPV)is the most common canine pathogen,widely distributed worldwide,and is a highly contagious disease with a high incidence and mortality rate.Acute enteritis is currently the most common symptom of CPV,with clinical symptoms including anorexia,depression,lethargy,fever,vomiting,and severe diarrhea.CCoV and CPV are common canine enteric viruses,and due to their highly contagious nature,both viruses are widely prevalent.When CCo V and CPV are co-infected,the symptoms are particularly severe.Infection with CCo V or CPV can cause gastroenteritis and other symptoms such as diarrhea.The clinical manifestations of the two viruses are similar,making it difficult to clinically distinguish between CCo V and CPV.Therefore,this study established a method that can rapidly detect both viruses,providing a powerful tool for the clinical diagnosis of CCo V and CPV infections.This study successfully isolated two strains of CCoV(ML24 and ML26)from 34 anal swab samples of diarrheal dogs from a pet hospital in Shanghai.After continuous passage,obvious cytopathic effects were observed.The typing identification of the two strains of CCo V showed that both were type II and were not co-infected with type I.The TCID50 of ML24 strain was 10-5.34/100μl.Through PCR amplification and gene sequencing,the N gene sequence of the ML26 strain was successfully obtained.Genetic evolution analysis showed that its relationship with the Heilongjiang strain was the closest.Compared with other CCo V strains,the nucleotide homology was 95.2%-97.9%.Six anal swab samples were collected from diarrheal dogs in a breeding farm in Qingdao,Shandong Province,and six strains of CPV were successfully isolated.After continuous passage,obvious cytopathic effects were observed in CRFK cells.The results of ordinary PCR showed that QD1,QD2,QD3,and QD6 strains were infected with CPV alone,and QD4 and QD5 strains were co-infected with CCo V.Through gene sequencing,it was determined that all six strains of CPV were CPV-2c.The TCID50 of QD3 strain was 10-6.67/100μl.Through PCR amplification and gene sequencing,the full-length gene sequence of QD3 strain CPV was successfully obtained.The genetic evolution tree showed that its relationship with the Anhui strain was the closest.Compared with other CPV strains,the nucleotide homology was 91.4%-98.2%.According to the gene sequences of CCo V and CPV in Gen Bank,corresponding primers were designed,and a dual-fluorescence quantitative PCR detection method for CCo V and CPV was established by optimizing the reaction system and amplification program.The sensitivity,specificity,and repeatability were evaluated.The results showed that the optimal annealing temperature was 60℃,and the primer concentrations were CCo V 1.0 μmol/L and CPV 0.1μmol/L,with a detection limit of 102 copies/μL for both viruses.The intra-and inter-batch variation coefficients were less than 2%.The method was used to detect Canine adenovirus(CAV),Canine circovirus(Ca CV),Canine distemper virus(CDV),Canine influenza virus(CIV),and Canine parainfluenza virus(CPIV),with no specific amplification.The results of testing 68 clinical samples showed that the positive rates of CCo V and CPV were 22.0% and 61.8%,respectively.Compared with the results of ordinary PCR detection,the method established in this study was more sensitive.This study successfully established a dual-fluorescence quantitative PCR method for CCo V and CPV,which has the advantages of strong specificity,high sensitivity,and good repeatability,and can be used for the differential diagnosis of CCo V and CPV,providing technical support for the detection of these two viruses. |
PDF Full Text Request