Development Of Duplex Real-time PCR Assay For The Detection Of IBRV

a real-time PCR was developed with 2 pair of primers and Taqman probes targeting the conserved region of IBRV gE and gB gene sto establish a sensitive detection protocol for infectious bovine rhinotracheitis virus(IBRV).At the same time,proceeded to testing with The sensitivity,specificity and clinical test samples.The result showed that the method was of high sensitivity and The minimum detectable concentration approximately was 2 copies/μL;the assay was highly specific for amplification of IBRV,and no amplification with bovine mycoplasma,bovine parainfluenza virus type 3,bovine viral diarrhea virus,pasteurella and burcella;And the method was highly reproducible and a coefficient of variation was less than 2%.With the method of establishing for testing for 54 cattle lung and 18 cattle nasal swab samples,the result showed that positive 17 were IBRV postive in 54 lung samples,including 12 double positive,5 gB postive and gE negative,and 37 double negative;1 IBRV gB postive and gE negative in 18 nasal swabs,and 17 double negative.This research established a real-time PCR detection method that was of the advantages of rapid,sensitive,accurate detection and could be used for IBRV detection.In addition,according to the published GenBank IBRV gB and gE gene sequence,design synthesis 2 with enzyme sites primers constructed eukaryotic expression vector.The result showed that the purpose of this test successfully amplified out fragments and cloned it to pMD-19Tsimple carrier.To identify successful pMD-19t simple-gB and pMD-19t simple-gE recombinant plasmid by BamH I and EcoR I double enzyme,connected to the expression vector pFastBac1,and ovtained recombinant plasmid pFBgB and pFBgE.Via a series of appraisal,obtained the gB and gE gene recombinant grain,for transfection insect Sf9/Sf21 cells laid a foundation.