2013-01-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/682321摘要：一、植物檢疫nepovirus之鑑定相關資料庫與標準鑑定流程之建立 針對我國所公告的植物檢疫nepovirus收集相關資訊，建立植物檢疫nepovirus鑑定 相關資料庫。對於欠缺檢測相關產品和文獻的植物檢疫nepovirus，則依據序列分析 比對結果，設計nepovirus廣效性引子對，以及病毒的專一性引子對，供後續使用。 必要時可從外國研究機構購買植物檢疫nepovirus乾燥材料，用來測試特定植物檢疫 nepovirus的專一性引子對。建立一套植物檢疫nepovirus之標準鑑定流程，提供給 植物檢疫單位參考使用。 二、具檢疫重要性類病毒有害生物整合性診斷方法之研發與快速檢驗組套之開發 類病毒是具有感染力的RNA並且可以造成多種植物病害，大小大約介於250至400個核 酸，並且為環形單股RNA、形成二級結構。植物與類病毒之間的交互作用至今仍然所 知甚少，即使類病毒構造簡單，但卻可以造成農業損失。柑橘是重要的果樹作物之 一，但遭受類病毒的危害甚廣，除了主要的病原是柑橘鱗砧類病毒(Citrus Exocortis Viroid; CEVd)以及啤酒花矮化類病毒(Hop Stunt Viroid; HSVd)之外。 近來，我們對台灣新竹，斗六，屏東及花蓮的田間柑橘園進行採樣檢測六就已被報 導的柑橘累病毒，發現台灣田間除了CEVd 及HSVd外，也存在著柑橘矮化類病毒 ( Citrus dwarfing viroid; CDVd) 及 柑橘裂皮症類病毒(Citrus bark cracking viroid; CBCVd) 兩種可感染柑橘的類病毒。因此，對於類病毒在台灣田間的偵測及 進口農作物的檢疫，吾人不得不開始正視類病毒病害的嚴重性。而當務之急則是開 發快速極高靈敏的類病毒診斷技術，提供給產官學進行應用。 由於類病毒為僅有RNA的病原。因此，本研究計畫將以單步驟反轉錄聚合脢反應 建立已知六種柑橘類病毒的快速靈敏的偵測技術; 也將進一步開發出相關的診斷組 套，提供給第一線防檢疫人員使用。以期能防止柑橘類病毒在台灣的擴散及危害。 <br> Abstract: To establish an identification information database for plant quarantine nepoviruses, we will collect and organize all required information. For the plant quarantine nepovirus lack of detection methods and related products, nepovirus broad-spectrum primer pairs and the virus-specific primer pairs will be designed according to the results of multiple sequence alignment. These primer pairs will be used in later detection tests. If it is necessary, the selected phytosanitary nepovirus dried material will be purchased from foreign research institutions, and then used to test the specificity of the virus-specific primer pairs. Finally, the standard operation protocol of plant quarantine nepoviruses will be established and provided to the authorities of plant inspection and quarantine. Viroids are the infectious RNA that lead to various plant growth disorder. Ranging from 250 to 400 nt, they are single stranded and closed covalently, forming a secondary structure. In spite of the simplicity of genome composition, viroids is of importance for the economic loss caused by them, although the pathogenesis and interaction between plant and viroids are rarely understood. Citruses are one of the most important fruit in economy. In many place citruses suffer from viroid disease. Citrus Exocortis Viroid (CEVd) and Hop Stunt Viroid (HSVd) are the two major citrus viroids, transmitted by mechanical injury or grafting. CEVd causes stunt, bark-scaling, and leaf curve and makes citrus prone to plant pathogen; HSVd causes stunt and discoloration. Recently, we have surveyed the existence of 6 known citrus viroids in the citrus fields in Taiwan, we found 4 out of the 6 citrus viroids existed in the fields, including CEVd, HSVd, CDVd (citrus dwarfing viroid), and CBCVd (citrus bark cracking viroid). Our finding revealed a serious quarantine and inspection issue of viroid-related diseases to be aware. To resolve this issue, we have to develop and establish techniques for viroid detection. In this current proposal, we proposed to develop onestep RT-PCR for 6 citrus viroids since viroids contain only naked RNA molecules devoid of any other proteins or DNA. Furthermore, we also plan to a citrus detection kit for all 6 citrus viroid in a multiplex PCR based assay. The achievement of our study will be beneficial for firstline inspection and quarantine personnel and avoid the invasion and spread of citrus viroids in Taiwan.植物檢疫Nepovirus屬病毒鑑定柑橘鱗砧類病毒啤酒花矮化類病柑橘矮化類病毒柑橘裂皮症plant quarantinenepovirusidentificationCitrus exocortis viroidHop stunt viroidCitrus dwarfing viroidCitrus bark cracking viroidone step RT-PCR