| IBRV can cause more system infection of bovine and lead to great economic losses. This experiment according to the IBRV gE gene conservative sequence designed and synthesized a specificity primer, application of the primer, we established PCR and Real-time fluorescent quantitative methods, and use these methods to testing simulation samples. According to IL-2, IFN-y and β-actin gene designed and synthesized a specificity primers, established the standard curve of Real-time fluorescent quantitative PCR and linear regression equation, and application IBRV infection mononuclear macrophage of bovine Oh、6h、12h、24h、48h to analyzed mRNA level of IL-2, IFN-γ and β-actin. The results of two parts are as follows:(1) The specificity of PCR and Real-time fluorescent quantitative detection the gene IBRV gE is good. Application these methods amplification IBRV, BVDV (Bovine Viral Diarrhea Virus), BRV (Bovine Rotavirus), MDBK cells and bovine serum DNA, the result show that only IBRV is positive.(2) The sensitivity of PCR and Real-time fluorescent quantitative PCR detection the gene IBRV gE is higher. The sensitivity of PCR and real-time PCR are7×103copies/μL and70copies/uL, both differ100times.(3) Application these two methods of simulated sample were tested, the contents of PCR and Real-time fluorescent quantitative PCR are3TCID50/reaction and0.03TCID50/reaction.(4) After IBRV infection mononuclear macrophage of bovine can dramatically reduce IL-2and IFN-y transcription (P<0.05).We use the same primer establish PCR Real-time fluorescent quantitative PCR. Two kinds of PCR together have potentially value. At the same time, establish PCR detection method to distinguish wild or vaccine infection. By comparing the expression level dynamic change of IBRV infection mononuclear macrophages, the cells factor IL-2and IFN-y, analysis the role of IBRV in influencing mononuclear macrophages immunological function, to lay the foundation further illustrate the IBRV pathogenesis mechanism. |
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