兔出血症病毒胶体金免疫层析试纸条诊断方法的建立及初步应用

摘要

To establish a rapid, simple assay for rabbit hemorrhagic disease virus (RHDV) , purified polyclone antibody against RHDV was labeled with colloidal gold particle. Monoclonal antibody (McAb) A3C against RHDV was prepared with recombinant RHDV VP60 protein expressed by baculovirus expression system. The detection zone and quality control zone of the nitrocellulose membrane was made by A3C and goat anti-rabbit IgG. The immune colloidal gold test strip was developed by optimizing the experiment conditions. The sensitive test showed that the test strip can detect the RHDV which erythrocyte agglutination test (HA) titer was 1 : 10which is negative result if the RHDV was detected by HA. It means that the test strip is more sensitive than erythrocyte agglutination test. The crossing test showed that the test strip could not react with other rabbit common pathogens. 127 clinical liver samples of rabbit were detected by the test strip, while a parallel experiment was conducted using HA, and the positive consistent rate of both was 100%. All of the results showed that the test strip is a rapid, sensitive and specific method for detecting RHDV infection. The method has provided a effective tool for diagnosing RHDV, particularly in clinical diagnosis%为了建立一种快速、简便的兔出血症病毒(RHDV)的检测方法,用胶体金标记纯化的RHDV多克隆抗体,以杆状病毒表达系统表达的重组RHDV VP60蛋白为免疫原制备RHDV的单克隆抗体(McAh,简称单抗)A3C,将RHDV McAb和羊抗兔IgG抗体包被在硝酸纤维素膜上,分别作为检测线和质控线,经条件优化研制成RHDV免疫胶体金试纸条.该试纸条可以检出红细胞凝集试验(HA)效价为1:10的RHDV悬液,即HA检测为阴性的样品,该试纸条检测为阳性,其敏感性高于HA;特异性试验结果显示,该试纸条不与家兔其他常见病菌发生交叉反应.应用该试纸条对127份疑似兔出血症家兔肝脏样品进行初步检测,同时用HA做平行试验,阳性符合率为100％.因此,该试纸条是一种快速、灵敏、特异的兔出血症病毒检测方法,为兔出血症的现场诊断提供了有效的方法,显示出很好的临床应用前景.