应用点免疫结合法和组织印迹法检测烟草组织中的TMV和CMV

Detection of TMV and CMV in Tobacco Tissue by Dot-ELISA and Tissue Blot-ELISA

报道了应用酶联免疫测定法中的点免疫结合法 (Dot immunoblots ,Dot ELISA)和组织印迹法(Tissueblots ELISA)检测烟草组织中烟草花叶病毒 (TMV)和黄瓜花叶病毒 (CMV)的方法及效果。Dot ELISA检测提纯TMV ,兔抗TMV血清稀释 10 0 0倍 ,最低检出率可达 1.2 5ng/ml;检测TMV病叶汁液 ,最低检出稀释度为 1∶2 560。检测CMV病叶汁液 ,兔抗CMV血清稀释 50 0倍 ,最低检出稀释度为 1∶2 560。Dot ELISA检测低稀释度健康烟叶粗汁液时存在假阳性 ,提高稀释度可消除假阳性。应用Dot ELISA ,选择反应强度高、健康烟叶粗汁液无假阳性的抗血清稀释 10 0 0倍 (TMV)、50 0倍 (CMV) ,样品粗汁液稀释30 0倍 ,以及应用组织印迹法对采自云南曲靖市的花叶样品进行了TMV和CMV检测 ,并与电镜负染法的检测结果验证。表明两种方法都可以用来检测烟叶样品。组织印迹法比Dot ELISA法的操作简单 ,灵敏度高 。

Tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) were detected in tobacco tissue by dot\|immunoblots enzyme\|linked immunosorbent assay (ELISA) and tissue blot\|ELISA.1000\|dilution rabbit\|anti\|TMV antibody could be used to detect purified TMV with limitation of 1.25ng/ml, and for TMV infected tobacco sap extracts with limitation of 1∶2560. 500\|dilution rabbit\|anti\|CMV antibody could be used to detect CMV infected tobacco sap extracts with limitation of 1∶2560. Detection of health tobacco sap extracts with dot\|ELISA in low dilution exhilbited false positive results, high dilution could eliminate false positive phenomena. Dot\|ELISA with rabbit\|anti\|TMV antibody in 1000 dilution, rabbit\|anti\|CMV antibody in 500 dilution, tobacco sample sap extracts in 300 dilution and tissue blots were applied for detected TMV and CMV in mosaic tobacco sample collected from field in Qujing, Yunnan. The samples were detected by electrical microscopy in the same time. The results showed that dot\|ELISA and tissue blot\|ELISA performed well in detection of TMV and CMV in tobacco sample. The tissue blot\|ELISA is more simple and sensitive than dot\|ELISA, and suit for large amount samples routine detection.