Development of multiplex viroid rapid detection system for Solanaceae plants

茄科植物於全球分布廣泛，其中包含許多重要的經濟作物，如番茄 (tomato, Solanum lycopersicum)、馬鈴薯 (potato, Solanum tuberosum)、茄子 (eggplant, Solanum melongena) 及菸草 (tobacco, Nicotiana tabacum) 等。絕對寄生性病原的病毒及類病毒對於茄科植物的影響很大，可造成嚴重的經濟損失。類病毒為一環形單股RNA小分子，可經由機械接種或是種子傳播。目前國際上茄科種子進出口檢疫重要的茄科類病毒主要有6種，分別為馬鈴薯紡錘形塊莖類病毒 (potato spindle tuber viroid, PSTVd)、鯨魚藤潛伏類病毒 (columnea latent viroid, CLVd)、番茄黃色矮化類病毒 (tomato chlorotic dwarf viroid, TCDVd)、番茄莖頂矮化類病毒 (tomato apical stunt viroid, TASVd)、番茄植株雄化類病毒 (tomato planta macho viroid, TPMVd) 及辣椒小果類病毒 (pepper chat fruit viroid, PCFVd)。臺灣目前已有PSTVd及PCFVd的相關報導，但目前並沒有可同時針對上述6種檢疫類病毒感染之快速檢測方法與標準流程。因此，本研究的目的為開發可用於植株及種子上，同時檢測6種目標類病毒的反轉錄聚合酶鏈鎖反應快速檢測系統。首先，構築6種檢疫類病毒的感染性選殖株，並將其生體外轉錄之RNA接種至聖女番茄植株後，可引發類病毒感染病徵，如矮化(stunting)、莖部壞疽 (stem necrosis) 及葉片褪色 (chlorosis) 等。利用所設計之6種檢疫類病毒專一性引子對，可分別於樣本中有效的增幅出專一性條帶，而所設計的簡併式引子對，可同時於樣本中增幅出目標條帶，並結合市售的one-step RT-PCR套組使用後，可大幅減少檢測流程所需之步驟與時間，所設計之引子對亦可應用於番茄種子樣本檢測。本研究所開發之茄科作物檢疫類病毒快速檢測系統未來可用於進出口或田間植株樣本及種子之類病毒檢測，以確保國內茄科作物產業可以穩定發展。Solanaceae plants such as tomato, potato, eggplant, and tobacco, are commercially important crops and widely planting around the world. The obligatory parasitic viruses and viroids could infect and cause severe yield loss on Solanaceae plants. Viroids are circular single stranded RNA which could be transmitted by mechanical operation or seeds. Several seed-borne viroids including tomato spindle tuber viroid (PSTVd), columnea latent viroid (CLVd), tomato chlorotic dwarf viroid (TCDVd), tomato apical stunt viroid (TASVd), tomato planta macho viroid (TPMVd) and pepper chat fruit viroid (PCFVd) are important quarantine pathogens in Taiwan as well as other countries. Currently PSTVd and PCFVd were reportedly occurred in Taiwan. However, there is no avaliable detection measurement for these 6 quaratined target viroids. The objective of this study is to develop a rapid one-step reverse transcription polymerase chain reaction (RT-PCR) system for simultaneously detecting these 6 viroids in seeds and infected plants. The infectious DNA clones of the 6 viroids were successfully constructed and inoculated into tomato plants and causing stunting, stem necrosis, and chlorosis symptoms. Degenerate primer pairs were designed and could simultaneously amplify expected fragments from these 6 target viroids by one-step RT-PCR. Furthermore, this multiplex system in conjunction with the modified methods for rapid total RNA extraction could be used for detection of viroids in seeds and field-collected samples. The specific primer pairs for these 6 target viroids were designed and could amplify specific bands in respective viroid-infected samples. The degenerate primer pair with commercial one-step RT-PCR kit could amplify target bands in 6 target viroid-infected samples which could be time and labor saving and cost effective. The degenerate and specific primer pairs were also suitable for seed detection. In conclusion, the rapid detection system can be used for detecting the 6 quarantine viroids of Solanacea plants while international seed trading or plants in the field.中文摘要 i 英文摘要 ii 目次 iv 表目次 vii 圖目次 viii 前言 1 前人研究 4 一、類病毒 (viroid) 之基本特性 4 二、類病毒分類現況 4 (一)馬鈴薯紡錘形類病毒科 (Pospiviroidae) 的特性及其分類 5 (二)鱷梨日斑類病毒科 (Avsunviroidae) 的特性及其分類 6 三、感染茄科植物之類病毒種類 6 (一)我國茄科種子出口檢疫之類病毒 7 (1)馬鈴薯紡錘形塊莖類病毒 (potato spindle tuber viroid, PSTVd) 7 (2)鯨魚藤潛伏類病毒 (columnea latent viroid, CLVd) 8 (3)番茄植株雄化類病毒 (tomato planta macho viroid, TPMVd)8 (4)番茄莖頂矮化類病毒 (tomato apical stunt viroid, TASVd)9 (5)番茄黃色矮化類病毒 (tomato chlorotic dwarf viroid,TCDVd)9 (6)辣椒小果類病毒 (pepper chat fruit viroid, PCFVd) 10 (二)其他可感染茄科植物之類病毒 10 (1)菊花矮化類病毒 (chrysanthemum stunt viroid, CSVd) 10 (2)柑橘鱗砧類病毒 (citrus exocortis viroid, CEVd) 11 (3)茄潛隱類病毒 (eggplant latent viroid, ELVd) 12 四、類病毒之傳播途徑 12 五、類病毒病害之檢疫 (quarantine) 及防治 (control) 13 六、類病毒之檢測技術 14 (一)反轉錄聚合酶鏈鎖反應 (RT-PCR) 15 (二)即時定量反轉錄聚合酶鏈鎖反應 (qRT-PCR) 15 (三)反轉錄環形恆溫核酸增幅法 (RT-LAMP) 16 材料與方法 17 一、目標類病毒感染性選植株之建立 17 (一)輸入感染目標類病毒之葉片 17 (二)總量RNA萃取 (傳統法) 17 (三)全長引子對設計、反轉錄聚合酶連鎖反應(RT-PCR)及定序 18 (四)類病毒感染性選殖株構築 (infectious clone) 19 (五)生體外轉錄、RNA接種及病徵之觀察 20 二、六種目標類病毒專一性引子對設計及反轉錄聚合酶連鎖反應測試 21 三、六種目標類病毒簡併式引子對設計及反轉錄聚合酶連鎖反應測試 21 四、總量RNA快速萃取法及One-step RT-PCR套組之測試 22 (一)總量RNA快速萃取 (TPS1緩衝液) 及RT-PCR測試 22 (二)兩種市售One-step RT-PCR套組比較 22 (三)One-step RT-PCR快速檢測流程測試 23 五、六種目標類病毒簡併式及專一性引子對在種子檢測之應用 24 (一)種子總量RNA之萃取 24 (二)簡併式及專一性引子對測試及目標條帶解序 25 結果 26 一、生體外轉錄之類病毒RNA接種番茄植株之病徵觀察與RT-PCR確認 26 二、六種目標類病毒專一性引子對設計及反轉錄聚合酶連鎖反應測試 26 三、六種目標類病毒簡併式引子對設計及反轉錄聚合酶連鎖反應測試 27 四、總量RNA快速萃取法及One-step RT-PCR套組之測試 27 (一)總量RNA快速萃取 (TPS1緩衝液) 及RT-PCR測試 27 (二)市售one-step RT-PCR套組測試比較 27 (三)One-step RT-PCR快速檢測流程測試 28 五、六種目標類病毒簡併式及專一性引子對在種子檢測之應用 28 (一)簡併式引子對檢測及目標條帶解序 28 (二)專一性引子對檢測結果 29 討論 30 參考文獻 34 表 46 圖 5