Establishment And Preliminary Application Of Real-time Fluorescence Quantitative RT-PCR Detection Method For Canine Coronaviru

Canine coronavirus disease is an infectious disease of dogs that can cause acute enteritis in dogs.Clinically,canine coronavirus(CCoV)is often co-infected with other pathogens of intestinal infectious diseases,and sick dogs may have systemic clinical symptoms.In recent years,there have been reports of CCoV variant strains that can infect dogs of different ages with high morbidity and mortality.Great harm to pet dogs.At present,CCoV detection methods include ordinary RT-PCR,colloidal gold test paper.When the virus content is low and other strict detection conditions,these detection methods will lead to misdiagnosis and missed diagnosis,which will affect the diagnosis and treatment of the disease.Fluorescence quantitative RT-PCR detection method has the advantages of high sensitivity and specificity.Therefore,this study aimed to establish a real-time quantitative RT-PCR detection method for CCoV,and then provide technical support for the clinical detection and epidemiological investigation of CCoV.Download different types of CCoV gene sequences from the NCBI gene bank,and design primers and probes at the highly homologous 3’UTR through DNA star comparison.By optimizing the amplification system and reaction conditions for annealing temperature,enzyme concentration,primer concentration,probe concentration,draw a standard curve.CCoV positive plasmid,canine parvovirus,canine distemper virus,canine infectious hepatitis and canine parainfluenza virus were detected to evaluate the specificity of the detection method;the plasmid standard was diluted into 9 different concentration gradients as templates,and fluorescence quantitative RT was performed-PCR amplification to evaluate the sensitivity of the detection method;plasmid standards with different concentrations were selected,the number of templates was 3,and fluorescence quantitative RT-PCR amplification was performed to evaluate the repeatability of the detection method.100 clinical samples were collected from 4 pet hospitals in Daqing City,and were amplified by ordinary RT-PCR,commercial kits and the detection method established in this experiment to evaluate the application effect of the detection method.The results showed that the correlation coefficient of the standard curve was 0.996,and it had a good linear relationship in the range of 2.61×10~1～2.61×10~8copies/μL.In addition to the positive test results for canine coronavirus positive plasmid,the nucleic acid amplification results for canine parvovirus,canine distemper virus,canine infectious hepatitis and canine parainfluenza virus were all negative with strong specificity.The lower limit of detection was2.61×101 copies/μL,and the sensitivity was good;the inter-assay and intra-assay coefficients of variation were both less than 2%,and the repeatability was good.Among the 100 clinical samples,7 positive samples were detected by this test method,and 3 positive sample was detected by ordinary RT-PCR.The coincidence rate with this test method was 96%.The commercial kit detected 4 positive samples,and the coincidence rate with this test method was97%.In conclusion,this experiment successfully established the CCoV Taq Man real-time fluorescence quantitative RT-PCR detection method.The method has the characteristics of strong specificity,high sensitivity and good repeatability.Can provide technical support for clinical detection and epidemiological investigation of CCoV.