Establishment And Application Of Fluorescent Quantitative RT-PCR Method For Canine Astrovirus

Canine astrovirus（CAst V）can cause vomiting,diarrhea,fever and other symptoms in dogs,with threat to the health of dogs,which mainly infects puppies.It is prevalent in different regions of the world and is widely distributed.Current detection methods for CAst V include:electron microscopy,semi-nested RT-PCR,fluorescence quantitative RT-PCR,nested RT-PCR,next-generation sequencing,and RT-PCR etc.For providing a rapid and sensitive method for the diagnosis and epidemiological investigation of CAst V,a fluorescence quantitative RT-PCR detection method for CAst V was established in this study,and the molecular detection of clinical samples of dogs in Chengdu areas was carried out using this method,which could provide a theoretical basis for the prevention and control of this disease.This study achieved the following results:1.A fluorescent quantitative RT-PCR method for detecting CAst V was successfully established,which has good specificity and sensitivity,and the minimum detection limit of CAst V is 8.46×10¹ copies﹒μL^-1 with good reproducibility.2.The experimental samples are 334 fecal samples of canine anal smears of all ages with healthy or intestinal symptoms provided by pet hospitals and shelters in Chengdu from October 2020 to January 2022.Among them,there are intestinal symptoms.185samples and 149 clinical health samples.Molecular detection results of 334 cases of canine anal swabs showed that the total positive rate of CAst V by fluorescence quantitative RT-PCR was 18.56%（62/334）,and the total detection rate of common RT-PCR was 10.48%（35/334）,the difference between the two methods was extremely significant（P<0.01）.The results show that the established quantitative RT-PCR method has a higher detection rate of CAst V than the common RT-PCR method and is more sensitive.3.The analysis of infection-related factors showed that the infection risk of CAst V was related to the health status and age of dogs.The detection rate of CAst V in samples with intestinal symptoms was 27.57%（51/185）,and the detection rate of CAst V in samples without intestinal symptoms was 7.38%（11/149）.The results showed that the detectionrate of CAst V in samples with intestinal symptoms was significantly different from that in samples without intestinal symptoms（P<0.01）,indicating that CAst V infection was closely related to the occurrence of diarrheal diseases in dogs;CAst V infection was defferent among dogs of different ages,the detection rate in puppies was significantly higher than that in adult dogs（P<0.05）,indicating that the risk of CAst V infection was closely related to the age of the dog,with no significant difference among dogs of different breeds,immune status and gender（P>0.05）.A fluorescence quantitative RT-PCR detection method for CAst V established in this paper provides a specific,sensitive and reliable method for the diagnosis and detection of CAst V.Molecular detection of CAst V infection in Chengdu areas will further enrich the epidemiological data of the disease,and provide theoretical and technical support for the diagnosis,prevention and control of this disease.