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標題: 茄科細菌性斑點病菌檢測生物晶片之研發
Development of DNA Array for the Detection and Identification of Solanaceous Bacterial Spot-causing Xanthomonads
作者: Miao-Fang Ho
關鍵字: 細菌性斑點病;聚合酵素連鎖反應;毛地黃素;DNA生物晶片;bacterial spot;polymerase chain reaction;digoxigenin;DNA array
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茄科細菌性斑點病菌 (bacterial spot-causing xanthomonads, BSX)為國際重要之植物檢疫病原,於茄科作物上可造成嚴重的經濟損失。在台灣已被報導之BSX有Xanthomonas euvesicatoria、X. perforans及X. vesicatoria。應用X. euvesicatoria、X. perforans及X. vesicatoria之專一性引子對RST13/RST14、HpaF-f/HpaF-r及Xv1f/Xv1r進行多重引子聚合酵素連鎖反應 (multiplex polymerase chain reaction, multiplex PCR),可將台灣現有之BSX進行鑑別。由於BSX可藉由種子傳播,開發快速準確之檢測技術更顯得重要。DNA生物晶片檢測技術具有高靈敏度及專一性,近年來已被應用於多種植物病原之檢測,然目前尚未有可供檢測BSX之生物晶片,因此本研究擬進行BSX檢測生物晶片之研發。本研究分別利用專一性引子對RST13/RST14、HpaF-f/HpaF-r及Xv1f/Xv1r對三種BSX菌株進行PCR,以獲得專一性DNA片段,利用此些DNA片段進行DNA生物晶片之製作,經雜合反應測試後,顯示此生物晶片具有專一性。進一步測試DNA生物晶片之雜合反應時間及呈色反應時間與靈敏度關聯,結果顯示,於雜合1小時呈色30分鐘之條件下,其檢測靈敏度可達10 ρg。此外,應用multiplex PCR技術,配合BSX生物晶片測試,結果顯示可同時檢測鑑定此三種BSX菌株。進行最適化之測試結果顯示,以1 μM毛地黃素配合multiplex PCR,所獲得之探針專一性最佳。此BSX生物晶片實際應用於人工帶菌種子及人工接種之罹病番茄植株檢測上,可有效將此三種BSX檢測並鑑定。本研究研發出可同時檢測並鑑定之BSX生物晶片,未來可提供防檢疫人員進行BSX之檢測,並擬訂防治策略。

Bacterial spot is an important disease of tomato and pepper in Taiwan. This disease was caused by the solanaceous bacterial spot-causing xanthomonads (BSX) that including Xanthomonas euvesicatoria, X. vesicatoria, X. perforans and X.gardneri. BSX are considered as important quarantine phytopathogenic bacteria because of they can be transmitted by seeds and seedlings. In Taiwan, three BSX, X.euvesicatoria, X. perforans and X. vesicatoria were occurred, and they were detected and distinguished by multiplex polymerase chain reaction (multiplex PCR). In this study, the highly specific and efficient DNA array technique was used to developed the detection technique for BSX. The BSX DNA array was made by a nylon membrane and fixed the specific DNA fragments amplified by PCR with primer pairs HpaF-f/r, RST13/14 and Xv1f/1r, respectively. The result of specificity test of the DNA array showed that the specific signals were emerged on the BSX strains. The detection limit of the DNA array developed in this study was 10 ρg with 1 hr hybridization and 30 min colorization. The probe labeled with 1 μM digoxigenin in multiplex PCR could detect and distinguish BSX by hybridized with BSX DNA array. The BSX DNA array could detection and distinguish BSX from artificially infested tomato seeds and artificially infested tomato tissue. The BSX DNA array developed in this study will be used for plant quarantine to detect and distinguish BSX from plant samples and developing control strategies of BSX.
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