Establishment of real-time PCR for direct detection of IBRV in semen and relation between semen carrying virus and serum antibody to IBRV

【Objective】 The study was to develop a real-time PCR for direct detection of infectious bovine rhinotracheitis virus(IBRV) inbovine semen and analyze the relation between semen carrying virus and serum antibody to IBRV,providing technical support for IBRV detection and determination in bovine semen.【Method】 The semen was treated with Sephacryl S-400 chromatography to remove seminal inhibitors or the Proteinase K to liberate IBRV prior to DNA extraction.The primers and probe were designed,Mg2+ concentration,primers concentration and probe concentration were optimized,the real-time PCR method for direct detection of IBRV in bovine semen was developed.120 bovine raw semen samples and 40 extended semen samples were detected with the real-time PCR,traditional PCR and virus isoaltion method respectively.Semen samples and nose swabs samples of 10 bulls were detected regularly with the real-time PCR,and the serum antibody against IBRV were detected at the same time.【Result】 The detective limit was about 0.002 TCID50 to fresh semen samples and 0.02 TCID50 to extended semen samples.Compared with OIE reference method,the real-time PCR was 40 to 400 folds more sensitive in the detection of IBRV in semen samples,and that was 100 folds compared with conventional PCR and virus isolation.120 bovine fresh semen samples and 40 extended semen samples were detected with the real-time PCR,25 semen samples were positive,15 of them were positive by PCR and 12 positive samples by virus isolation.And the relation between bovine semen carrying virus and serum antibody against IBRV was not clear.【Conclusion】 A real-time PCR was developed for direct detection of IBRV in bovine semen and the relation between semen carrying virus and serum antibody against IBRV was analyzed.The results showed that the bull can not be confirmed whether the semen carrying virus through serum antibody.The nose swabs carried virus was intermittent.