Establishment And Application Of Multiplex Equantitative Real-Time PCR Detection Method For BVDV,IBRV And AKAV

Bovine viral diarrhea,bovine infectious rhinotracheitis,and Akabane disease can all cause reproductive obstacles in pregnant cattle,resulting in stillbirths and deformed fetuses,causing huge losses to the cattle industry.Since these three diseases are often invisibly infected or mixed with similar symptoms,it is difficult to make a differential diagnosis based on clinical symptoms.With the development of molecular biology,especially the rapid popularization of fluorescence quantitative PCR detection technology,some scholars have successively established Single-plex fluorescence quantitative PCR detection method,but the multiplex fluorescence PCR detection method of bovine viral diarrhea virus,bovine infectious rhinotracheitis virus,and Akaban virus is rarely reported.Therefore,the establishment of multiple fluorescent PCR detection methods for bovine viral diarrhea virus,akaban virus and bovine infectious rhinotracheitis virus to carry out rapid and efficient laboratory differential diagnosis is of great significance for the prevention and treatment of bovine diseases.In this study,based on the conserved gene sequences of bovine viral diarrhea virus,akaban virus,and bovine infectious rhinotracheitis virus,the Geneious Prime software was used to design multiple fluorescent PCR-specific primers and probes.Optimization,the optimal annealing temperature and primer-probe concentration were determined.The BVDV,AKAV,and IBRV nucleic acids that were calibrated by digital PCR were diluted 10 times for a total of 10 concentration gradients,and this method was used for sensitivity test and repeatability verification.The results showed that the minimum detection limit of bovine viral diarrhea virus was 3.5 copies/μL,the minimum detection limit of bovine infectious rhinotracheitis virus was 5.2copies/μL,and the minimum detection limit of akaban virus was 6.7copies/μL,and the coefficient of variation between each data group was below 5.0%.Paratuberculosis,Salmonella,respiratory syncytial virus,rotavirus and coronavirus were used for specificity verification,and the results confirmed that this method would not cross-react with other common bovine pathogens.This method was applied to the beef cattle farms in nine regions(Jiaohe City,Gongzhuling City,Meihekou City,Yushu City,Dunhua City,Liuhe County,Nong’an County,Tongyu County,Lishu County)in Jilin Province from 2017 to 2019.1800 bovine whole blood samples collected by dairy farms,slaughtering enterprises and free-range farmers were tested.The results showed that the detection rate of BVDV was 1.83%(33/1800),and the detection rate of IBRV was 6.5%(117/1800).,The detection rate of AKAV was 0%.The detection rate of 252 bovine whole blood samples with clinical symptoms of miscarriage and fetal malformation collected recently showed that the detection rate of BVDV was 1.19%(3/252),and the detection rate of IBRV was 0.4%(1/252).,The detection rate of AKAV was 68.25%(172/252).In conclusion,the multiplex fluorescence quantitative PCR detection method successfully established in this study has high sensitivity and specificity,and strong stability,and can be widely used in the laboratory differential diagnosis of BVDV,IBRV and AKAV.,providing technical support for the prevention and treatment of bovine viral diarrhea,bovine infectious rhinotracheitis and Akabane disease.