牛疱疹病毒Ⅰ型二重LUX荧光PCR鉴别检测方法的建立

Establishment of duplex LUX real-time fluorescent PCR assay for detecting bovine herpesvirus 1

采用LUX新型荧光PCR技术原理，建立了快速检测牛疱疹病毒I型（BHV-1）以及鉴别野毒感染和基因缺失疫苗免疫动物的二重实时荧光PCR方法。结果显示，该方法对多株BHV-1病毒均呈典型gE、gC双基因阳性反应，对其他常见动物疱疹病毒以及健康牛基因组DNA等均呈阴性反应；对细胞增殖病毒液的gE、gC双基因鉴别的检测敏感性可达0．4～O．04TCID50比常规PCR方法高100倍以上；对带毒牛血清、抗凝全血、牛新鲜精液和冷冻精液基因鉴别的检测敏感性分别达0．04TCID50、0．4TCID50、0．4TCID50和4TCID50。采用该方法从临床牛血样、鼻拭子样品中检出IBRV阳性样品，检测全程仅需约2h。对单基因克隆质粒的检测进一步证实该方法能特异地鉴定gE、gC基因，检测灵敏度分别达90、30拷贝。结果表明，该方法可应用于临床快速诊断BHV-1病毒感染，鉴别BHV-1病毒感染与对应的基因缺失疫苗免疫动物。

A duplex LUX fluorescent PCR assay was established for detecting gE and gC gene of bo-vine herpesvirus type 1 （BHV-1） using LUX self-quenched primer technology. BLAST analysis showed that the primer＇s sequences were BHV-1 specific and highly conserved. The specificity of the assay was confirmed with BHV-1 strain, other related bovine viruses and herpesvirus, and genomic DNA from control cells and healthy cattle. The assay could detect 0.4--0.04 TCID50 of the cell harvest of Barta Nu/67 viruses. This resulted in 100-fold increase in sensitivity when compared to a gel-based PCR method. The detection limit of the assay was 0.04, 0.4, 0.4 and 4 TCID50 for infected bovine serum, whole blood, fresh and frozen semen respectively. The procedure could be completed within 2 hours. The assay could Identify and differentiate genes specific for gE from gC, as few as 90 and 30 copies, respectively. The above results demonstrated that the method could be used for rapid diagnosis of and differentiation of BHV-1 infection from immunization with the gE-deleted vaccines.