Laboratory of Plant Physiology, Faculty of Biotechnology, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece.
J Virol Methods. 2011 Oct;177(1):94-9. doi: 10.1016/j.jviromet.2011.06.024. Epub 2011 Jul 12.
Biosensors based on living cells are characterized by high sensitivity, selectivity and rapid response times. A novel portable cell biosensor system for the detection of plant viruses, based on immobilized 'Vero' cells carrying on their membrane virus specific antibodies was developed and was designated as High Throughput Bioelectric Recognition Assay (BERA-HTP). BERA-HTP was tested for the detection of purified Potato virus Y (PVY), Cucumber mosaic virus (CMV) and Tobacco rattle virus (TRV) and of CMV and TRV in single, as well as in mixed infections in two different plant host species. The sensor was based on live, mammalian cells, the membrane of which has been artificially saturated with antibodies specific to different plant viruses. The attachment of PVY, CMV or TRV viral particles to the homologous electroinserted antibodies caused a virus-specific change of the cell membrane electric potential that was not observed with virus-free samples or with heterologous viruses. Fluorescence microscopy observations showed that attachment of virus particles to the cell membrane bearing the homologous antibody, was associated with a decrease of [Ca(2+)]cyt. The perspective for the development of BERA-HTP as a portable, reliable and rapid (duration of assay for 96 samples: ∼70 min) detection method of plant viruses in the field is discussed.
基于活细胞的生物传感器具有高灵敏度、选择性和快速响应时间的特点。本研究开发了一种新型便携式细胞生物传感器系统，用于检测植物病毒，该系统基于固定在膜上带有病毒特异性抗体的“Vero”细胞，被命名为高通量生物电识别分析（BERA-HTP）。BERA-HTP 用于检测纯化的马铃薯 Y 病毒（PVY）、黄瓜花叶病毒（CMV）和烟草脆裂病毒（TRV），以及在两种不同植物宿主中单种和混合感染时 CMV 和 TRV 的检测。该传感器基于活的哺乳动物细胞，其膜已被人工饱和了针对不同植物病毒的抗体。PVY、CMV 或 TRV 病毒颗粒与同源电插入抗体的附着引起了细胞膜电势的病毒特异性变化，而在无病毒样本或异源病毒中未观察到这种变化。荧光显微镜观察表明，病毒颗粒与携带同源抗体的细胞膜的附着与 [Ca(2+)]cyt 的减少有关。讨论了 BERA-HTP 作为一种便携式、可靠和快速（96 个样本的检测时间：约 70 分钟）的田间植物病毒检测方法的发展前景。
