Establishment Of Nested PCR And Real-Time PCR Methods To Detect Infectious Bovine Rhinotracheitis Virus

Bovine infectious rhinotracheitis virus (IBRV) is an important pathogen of bovine infectious rhinotracheitis(IBR) and is wildly distributed in the whole world. The virus infection could cause enormous economic lost on cattle industry. At present, main measure to control the IBR is to prevent cattle from IBRV infection by vaccination and the method for eradicating the IBR is to kill infected cattle after detection. Thereefore, it is very important to establish a sensitive and rapid detection method. IBRV was reported to have only one serum type. The gB gene is necessary for virus replication. The glycoprotein E(gE) deleted marker vaccine is wildly used in many countries , especially those experiencing a high seroprevalence. It is also necessary to establish a method to distinguish wild-type virus from virus lacking glycoprotein E. To develop a method which can rapid detection of infectious bovine rhinotracheitis virus and discriminate between wild-type virus and virus lacking glycoprotein E, we establish 2 nested PCR assays and 2 real-time PCR assays.1. Establishment of Nested PCR Method to Detect Bovine Infectious Rhinotracheitis VirusAccording to the sequences of gB and gE genes of bovine infectious rhinotracheitis virus in GenBank, two pairs of nested PCR primers were designed by the primer premier 5.0 and the nested PCR methods were developed for IBRV detection. The result of electrophoresis showed that all primers had the specific bands. The negative results were achieved from the other Herpesvirus such as Pseudorabies virus(PRV), Marek's disease virus(MDV) and Duck plague virus(DPV). The detection limit of the primers gB/BN was 0.01 TCID₅₀ and the detection limit of the primers gE/EN was 0.1 TCID₅₀.2. Establishment of Real-time PCR Method to Detect Bovine Infectious Rhinotracheitis VirusBy the gB and gE gene of IBRV, two set of primers and Taqman probes were designed, respectively. After the quantitative curve of the assay was established using tenfold serial...