猪瘟病毒IPMA检测方法的建立

Establishment of immunoperoxidase monolayer assay detecting classical swine fever virus

为建立一种可定位和直观判断的猪瘟病毒检测方法,本研究应用猪瘟单克隆抗体作为一抗,建立了猪瘟病毒免疫过氧化物酶单层细胞试验检测体系,并对其相关的反应条件进行优化,对其特异性、灵敏性、重复性和可靠性进行了检测。结果显示,最佳病毒接种量为0.5个TCID（50）（TCID（50）为1×10^（-5）/mL）,固定前最佳的病毒孵育时间为48 h,一抗的最佳稀释度为1∶300,二抗的最佳稀释度为1∶2 000。所建立的猪瘟病毒免疫过氧化物酶单层细胞试验与其他几种常见猪病毒无交叉反应。在对300份来自不同地区的临床血样检测中,IPMA与RT-PCR和ELISA阳性检测结果符合率可达92%以上。研究结果说明,该方法具有良好的特异性、敏感性和可靠性。本研究为高效和便捷的猪瘟临床检测方法的研发奠定了基础。

To establish a localizable and visible method for detecting classical swine fever virus（CSFV）,this study was designed to establish an immunoperoxidase monolayer assay（IPMA） with monoclonal antibody against CSFV.We optimized the reaction conditions and examined the specificity,sensitivity,reproducibility and reliability.The optimized conditions included that virus inoculation amount was 50 L,virus incubation time was 48 h,the ratio of diluted first antibody is 1 ∶ 300,the ratio of diluted second antibody was 1 ∶ 2000;no cross reactivity was detected with specific serum of several other common pig viruses.The coincidence of IPMA,RT-PCR and ELISA was over 92% in detecting 300 samples collected from different areas.These results indicated that IPMA has good specificity,sensitivity and reliability.The present study has laid the foundation for development of efficient and convenient methods for basically clinical detection of swine fever.