猪伪狂犬病病毒直接免疫荧光检测方法的建立及初步应用

摘要

To develop a rapid,sensitive,and accurate direct-immunofluorescence method (DFM) detecting porcine pseudorabies virus (PRV),a fluorescein isothiocyanate-marked monoclonal antibody (FITC-MAb) against the gE protein of PRV was prepared and tested in this study.The results showed that the F/P ratio of the FITC-MAb was 2.578±0.012,which corresponds to the standard requirement.The best working conditions for the method were that the cells were immobilized with 60％ acetone and 40％ ethanol at-20 ℃ for 20 min,marked with 15 μg/mL FITC-MAb,and incubated at 37 ℃ for 1 h.Sensitivity tests showed that the method we established could detect PRV at 107-fold of dilution (1 ×TCID50).The FITC-MAb we prepared only displayed positive reaction with PRV wild type strains Ea and CH/SMX/2012,but had no cross reaction with PRV vaccine strain Bartha-K61,and other pathogenic viruses including classical swine fewer virus (CSFV),porcine parvovirus (PPV),porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV),suggested the method had good specificity.Preliminary application of the method detecting 83 clinical samples showed that the accordance rate of DFM and PCR was 74.7％.Particularly,the accordance rate between the two methods detecting the samples of lymph node and tonsil was 85.7％ and 93.3％,respectively.Our results indicated that the PRV direct immunofluorescence method established in this study layed the foundation for clinical PRV detection and complex diagnosis,which was suitable for clinical application.%为建立一种快速、敏感、准确的猪伪狂犬病病毒(PRV)直接免疫荧光检测方法(DFM),本研究通过制备并纯化PRV gE蛋白单克隆抗体(MAb),将其标记异硫氰酸荧光素(FITC)后对荧光抗体的最佳工作条件进行摸索;并对荧光抗体的特异性、敏感性、重复性和稳定性进行测定;应用DFM对从临床收集的83份疑似病料进行检测,同时用PCR检测作为对照,计算符合率.结果显示,本研究制备FITC标记抗体的F/P比值为2.578±0.012,符合标准;荧光抗体的最佳工作条件为:使用固定剂(60％丙酮+40％乙醇)于-20℃固定20 min,抗体工作浓度为15μg/mL,抗体孵育时间为1h;荧光抗体能在107病毒稀释度(1×TCID50)检出阳性信号,与PCV2、PPV、PRRSV、CSFV、PRV Bartha-K61均无交叉反应;批内和批间重复性良好,-20℃保存5个月染色后荧光强度仍稳定;83份临床样品检测结果显示,DFM与PCR的符合率为74.7％,其中淋巴结和扁桃体的符合率较高,分别为85.7％和93.3％.本研究建立的PRV直接免疫荧光检测方法为临床PRV检测和复合诊断奠定基础,适用于临床推广.