Establishment And Application Of Fluorescent Quantitative PCR Method For Canine Bocavirus

Bocavirus clinically causes gastrointestinal symptoms such as vomiting and diarrhea in humans and many animals,as well as respiratory symptoms such as cough and runny nose in humans,pigs and cattle.Canine bocavirus（CBoV）is a novel virus that was discovered in 2012 and has spread widely around the world,having been identified and reported in China,the United States,Germany,Korea,Thailand,Vietnam,Hong Kong and other countries and regions.The major symptom of CBoV infection in dogs is diarrhea.But because of the short time of reporting CBoV,there is a lack of abundant regional epidemiological data at home and abroad.There is no establishment of real-time fluorescent quantitative PCR method for CBoV.The purpose of this experiment was to establish a real-time fluorescent quantitative PCR assay for CBoV,and use the method to conduct a molecular epidemiological investigation of CBoV infection in Chengdu,providing basic support for disease prevention and control and diagnosis.1.By optimizing the reaction system and reaction conditions,a fluorescent quantitative PCR assay for CBoV based on SYBR Green I was established successfully.The test showed that the method has good specificity,sensitivity and reproducibility,and the minimum detection limit was 3.73×10¹ copies·μL^-1.2.A total of 330 fecal samples from dogs collected from Chengdu,Sichuan Province from March to November 2021 were detected by the established fluorescence quantitative PCR method.And the results showed that the total positive rate of CBoV in Chengdu was20.61%（68/330）.3.The analysis of infection-related factors showed that the infection of CBoV was related to hybrid or pure breed of dogs（P<0.01）,and the detection rate was higher in the samples of pure breed dogs.The infection of CBoV was correlated with the age of dogs,the detection rate of adult dogs over 12 months of age was extremely significantly different from that of dogs≤3 months of age and dogs aged 4～12 months,respectively（P<0.01）,and detection rate of dogs≤3 months of age was 44.68%（42/94）higher than that of dogs aged 4～12 months,which was 26.47%（18/68）,but there was no statistical difference between them（P>0.05）.The detection rate of 35.93%（60/167）in dogs with intestinal infection symptoms was significantly higher than that in dogs without intestinal infection symptoms（P<0.01）,rate of 4.91%（8/163）,showing that the infection of CBoV is closely related to intestinal infection in dogs.Infection of dogs with CBoV was related to immune status,and the detection rate of unimmunized dogs was significantly higher than that of immunized dogs（P<0.05）.Infection of CBoV was related to season,and the detection rate in spring was higher than that in other three seasons and there was a very significant difference（P<0.01）.The gender of dogs was not related to infection of CBoV（P>0.05）.In this study,a real-time fluorescent quantitative PCR method for CBoV was established for the first time,providing a fast and sensitive tool for the detection of CBoV,and enriching the molecular epidemiological data of CBoV in dogs in Chengdu area,providing certain reference data for the prevention and control of CBoV.