Establishment And Preliminary Application Of Real-time PCR For Detection Of Infectious Bovine Infectious Rhinotracheitis Virus

Infectious bovine rhinotracheitis(IBR)is a disease caused by bovine herpesvirus type I(Bovine herpesvirus 1,BHV-1)that can cause severe respiratory disorders in cattle.IBR is highly contagious and can cause the disease of many animals such as cattle,sheep and goats.The main symptoms include upper respiratory tract infections often accompanied by conjunctivitis,and sometimes genital infections,pustular vulvovaginitis in female animals,and balanitis in aborted male animals.BHV-1 is generally transmitted through respiratory,eye and genital secretions.The virus can remain dormant throughout the life cycle of the host.This virus is self-reactivating and can be reactivated by stress and corticosteroid therapy,leading to recurrence,resulting in perpetuation of the virus and transmission in the environment,severely affecting animal health and productivity.Therefore,it is important to establish a sensitive,stable and rapid IBRV detection method for accurate screening of infected animals and effective control of IBR transmission.The IBRV gB gene is the key gene for the virus to grow and replicate in the host cell.The main function of the protein encoded by IBRV gB gene is to help the virus to absorb and invade the host cell,and to spread and fuse among the host cells.At the same time,the gB gene is highly conserved.Therefore,some of the IBRV gB gene was cut off in this study and its immunogenicity and reactivity were verified by prokaryotic expression system.Finally,a Taq Man real-time PCR assay for the detection of IBRV gB gene was established,and clinical samples were tested to verify the method.1?According to the available sequence of IBRV gB gene in Gen Bank,the dominant antigenic epitopes of the whole sequence were analyzed,and the relative strong antigenicity,hydrophilicity and surface display probability of gB gene were obtained(amino acids at positions 273-393).A pair of specific primers was designed to amplify the truncated gB gene fragment.The recombinant protein gB was obtained by prokaryotic expression system.The polyclonal antibody was prepared by immunizing mice after purification.The immunogenicity of recombinant protein gB was analyzed by Western blot and the titer of polyclonal antibody was determined by indirect ELISA.The results showed that the truncated gB gene of360 bp was successfully cloned,and the molecular weight of the the gene was about 14.3 k Da,without transmembrane region and signal peptide,and it was a hydrophilic protein.Two acylation sites and two phosphorylation sites each.Secondary structure analysis found that?-helix,extended chain,random coil and?-turn accounted for 36.67%,13.33%,45%and 5%respectively.After IPTG induction,the recombinant protein gB was mainly expressed in inclusion bodies,and its molecular weight was about14kda.Western blot confirmed that the recombinant protein gB had strong reactivity.The titer of serum antibody reached 1:25600 after immunization,which proved that the recombinant protein gB could produce the corresponding antibody and had strong immunogenicity.2?In this experiment,specific primers and Taqman-gB probes were designed based on the intercepted IBRV gB gene fragments,and the IBRV Taq Man fluorescent quantitative PCR detection method was established.The results showed that the correlation coefficient(R~2)of the standard curve was 0.999,and there was a good linear relationship within 10~2-10~9copies/?L.The results of sensitivity test showed that the lowest detectable rate was 1.35×10~1copies/?L,and the specific test results showed that this method had not detected other bovine coronaviruses such as bovine coronavirus,bovine viral diarrhea virus and norovirus,which confirmed that the method had strong specificity.The results of repeatability test showed that the coefficient of variation of repeated experiments between groups and in groups was less than 2%,indicating that the repeatability was good.3?The IBRV detection method established in this experiment was used to detect 218 cattle blood samples collected throughout Xinjiang.59 positive samples were detected,and the remaining samples were negative,the positive rate of IBRV was 27.06%,which showed that this method was stable and reliable,and could be used for the detection of clinical ibrv positive samples.