水稻縞葉枯病偵測技術之發展

The Development of Immunological Detection Techniques of Rice Stripe Virus

陳慶忠(Ching-Chung Chen) ； 趙佳鴻(Chia-Hung Chao)

《臺中區農業改良場研究彙報》 64期 (1999/09) Pp. 1-11

https://doi.org/10.6956/BTDAIS.199909.0001

摘要

應用酵素聯結法、組織轉漬法及西方浸漬法等技術偵測感染水稻縞葉枯病毒（RSV）之水稻組織及媒介昆蟲斑飛蝨之帶毒情形。利用夾層免疫酵素聯結法（DAS ELISA）及間接免疫酵素聯結法，縞葉枯病毒抗血清稀釋至32768倍，純化鞘蛋白及粗汁液抗原分別稀釋至10^(-5)及10^(-6)均能產生專一性免疫反應。以DAS ELISA分析吸毒後且完成潛伏期之斑飛蝨帶毒蟲其A405讀值介於0.754～1.301間，未吸毒對照蟲則為0.021～0.069。西方墨點法偵測吸毒後個別斑飛蝨，帶毒蟲在RSV純化鞘蛋白（34.7 K）相同位置產生專一性免疫反應。以組織轉漬法在硝化纖維膜擠壓罹病水稻根、莖、葉或斑飛蝨蟲體後，處理RSV多元抗體及山羊白兔膽鹼酵素均能產生紫色反應，健康對照則無反應。試驗中斑飛蝨蟲體組織擠壓後會粘附於硝化纖維膜上而產生非專一性反應，造成對帶毒蟲個體判定之困擾。

關鍵字

水稻縞葉枯病毒 ； 媒介昆虫 ； 血清偵測技術

並列摘要

Rice stripe virus (RSV) disease has been recognized as the economically important disease on rice crop in Taiwan. In this study, some serological methods, including double-antibody sandwith (DAS) enzyme-linked immunosorbent assay (ELISA), indirect ELISA, tissue blotting on nitrocellulose membranes and western blotting were applied to detect RSV in RSV infected rice plant and its insect vector, Laodelphax striatellus. In DAS and indirect ELISA test, antiserum against RSV diluted at 32,768 fold showed positive reaction with purified coat protein and crude sap of RSV infected leaf at the dilution of 10^(-5) and 10^(-6), respectively. When using DAS ELISA to detect the individual viruliferous L. striatellus, the absorbance value at 405 nm were between 0.754 and 1.305, while those of the healthy insect were between 0.021 and 0.069. The western blot result showed that the viruliferous insect also reacted with antiserum. After direct tissue blotting and treated with polyclonal antibodies of RSV and goat-rabbit chondroitinase, the root, stem and leaf of infected rice plant or insect bodies all revealed the purple color. However, pressing the individual insect on the nitrocellulose membrane, it produced non-specific reaction, leading probably to a confusion for the detection.

並列關鍵字

rice stripe virus ； insect vector ； serologocal detection method