Han Jung-Heon, Shin Jun-Sung, Kim Young Ho, Kim Byung-Dong
Center for Plant Molecular Genetics and Breeding Research, Seoul National University, Seoul 151-921, Korea.
J Microbiol Biotechnol. 2007 Nov;17(11):1885-9.
The tissue blot immunobinding assay (TBIA) is widely used for the detection and localization of plant viruses in various plant tissues. The basic experimental procedures of TBIA sampling and blotting were simplified using commercially available micropipette tips. This method was termed the ring-blot immunobinding assay (R-BIA), as the blot on the membrane forms a ring shape. The detection efficacy of R-BIA was tested for two chili pepper viruses, pepper mild mottle tobamovirus (PMMoV) and pepper mottle potyvirus (PepMoV), following the optimized serological procedures of TBIA (length of the incubation period and BSA concentration, and primary and secondary antibodies). Sensitivity of the R-BIA was about 1 ng/ml of purified PMMoV in pepper leaf sap from a healthy pepper plant. R-BIA also showed high specificity in the detection of PMMoV and PepMoV. Moreover, the modified sampling and blotting procedures were simpler and more reliable than other TBIA methods (such as whole-leaf blotting and crushed-leaf blotting), suggesting that the R-BIA may be used for medium- to large-scale detection of plant viruses in laboratories with minimal facilities.
组织印迹免疫结合测定（TBIA）广泛用于检测和定位各种植物组织中的植物病毒。使用市售微量移液器吸头简化了TBIA采样和印迹的基本实验程序。由于膜上的印迹形成环形，该方法被称为环形印迹免疫结合测定（R-BIA）。按照TBIA的优化血清学程序（潜伏期长度、牛血清白蛋白浓度以及一抗和二抗），对两种辣椒病毒，即辣椒轻斑驳烟草花叶病毒（PMMoV）和辣椒斑驳马铃薯Y病毒（PepMoV），测试了R-BIA的检测效果。R-BIA对健康辣椒植株辣椒叶汁中纯化的PMMoV的灵敏度约为1 ng/ml。R-BIA在检测PMMoV和PepMoV时也表现出高特异性。此外，改进后的采样和印迹程序比其他TBIA方法（如全叶印迹和碎叶印迹）更简单、更可靠，这表明R-BIA可用于设施最少的实验室中对植物病毒进行中大规模检测。
