Establishment And Preliminary Application Of Assays For Detection Of Bovine Coronavirus

Bovine coronavirus(BCoV)is one of the important pathogens causing diarrhea in newborn calves.It is widely distributed worldwide and seriously affects the healthy development of the cattle industry.All the time,the harm caused by BCoV is increasing in cattle farms of Hebei Province.These viral diarrhea diseases are not only very similar in clinical symptoms,but also very similar to those caused by Escherichia coli,Salmonella,coccidia,etc.,which are difficult to distinguish.As a result,cattle farms cannot make rapid and effective diagnosis and take effective prevention and control measures.Therefore,the development of a detection method for BCoV has become an urgent problem to be solved.This research takes BCoV as the research object,to determine the genetic evolution characteristics of BCoV epidemic strains in Hebei Province,a BCoV epidemic strain(BCoV/CH/HB-BD/2019)identified from the feces and intestinal contents of a calf with diarrhea in a cattle farm in Hebei Province was amplified with the full sequence of S,N,and HE genes and determination,and the homology and genetic evolution analysis of each gene was carried out with 51 bovine coronavirus reference strains in GenBank.Comparedwith the BCoV reference strain,BCoV/CH/HB-BD/2019 has amino acid mutation sites in both S gene and HE gene.Although in the gene recombination analysis,the HE gene sequence of the HB-BD strain did not detect the gene recombination signal,we found that the HE gene sequence of the HB-BD strain was the main parental virus inferred from the Xinjiang strain(KU886219.1).Evolutionary analysis found that BCoV HB-BD strain and human coronavirus(HCoV-OC43)belong to the β-coronavirus genus 2a subgroup,and the homology is as high as 96.0%,while the SARS-CoV-2 that is spreading worldwide It belongs to the 2b subgroup of β-coronavirus.BCoV and SARS-CoV-2 belong to different subgroups of the same coronavirus.This study provides a basis for the prevention and control of bovine coronavirus disease and further reveals the relationship between corona viruses.This study uses the recombinant protein pET32a-N with good reactogenicity as the antigen protein,compose a detection matrix with bovine serum diluted in different multiples to determine its protein coating concentration and serum dilution multiples,and conduct a series of optimization of its reaction conditions.Using HRP-rabbit anti-bovine IgG as the secondary antibody,the enzyme-labeled antibody dilution factor was optimized and serially diluted.It was determined that the enzyme-labeled antibody was the best when the enzyme-labeled antibody was diluted 1:10000 in the ELISA antibody detection method in this study.This ELISA detection method has good repeatability and specificity.The coefficient of variation within and between repeated trials within and between groups should not exceed 10%;The results of the specificity test showed that there was no antigenic cross-reactivity with BRV,BVDV and BNoV positive sera;The results show that the BCoV IgG antibody indirect ELISA detection method established in this study can be used as a technical means for BCoV serological diagnosis.In this study,a pair of specific primers were designed according to the conservative region of BCoV N gene,and the conventional RT-PCR detection method of BCoV N gene and the SYBR Green I fluorescent quantitative PCR detection method were established respectively.Through the establishment of conventional RT-PCR detection methods,the specific primers of this study were amplified,showing that their specificity and sensitivity are good,the primer can be used as the amplification primer of the fluorescent quantitative PCR detection method to react,and establish the SYBR Green I fluorescent quantitative PCR detection method.The established BCoV Quantitative Real-time PCR detection method shows a good linear relationship;Its melting curve shows a narrow,sharp and single peak with good specificity;For sensitive detection,the minimum detection limit is 22.4 copies/μL,while the minimum detection limit of conventional PCR sensitivity detection is 2.24×102 copies/μL;For Quantitative Real-time PCR detection method,it was found that the coefficient of variation within and between groups did not exceed 5%.It shows that the detection method has good stability.From 2018 to 2019,57 diarrhea samples collected from cattle farms with diarrheal diseases in Hebei Province were tested.The positive rate of BCoV diarrhea detected by the Quantitative Real-time PCR detection method established in this research was 71.9%(41/57),the positive rate of BCoV diarrhea detected by conventional PCR detection method was 59.6%(34/57).It shows that the Quantitative Real-time PCR detection method has higher sensitivity and accuracy,and plays an important role in the comprehensive prevention and control of BCoV.