Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/98156
標題: 茄科作物多種目標類病毒快速檢測系統之研發
Development of multiplex viroid rapid detection system for Solanaceae plants
作者: 吳建甫
Chien-Fu Wu
關鍵字: 茄科植物
類病毒
反轉錄聚合酶鏈鎖反應
快速檢測系統
Solanacea plants
viroids
reverse transcription polymerase chain reaction
rapid detection system
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摘要: 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.
URI: http://hdl.handle.net/11455/98156
文章公開時間: 10000-01-01
Appears in Collections:植物病理學系

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