開發甜瓜萎凋病菌帶菌種子之快速檢測流程

甜瓜 (Cucumis melo L.) 是高經濟價值作物，甜瓜萎凋病菌 (Fusarium oxysporum f. sp. melonis, Fom) 能汙染種子並進行病原菌傳播引起甜瓜萎凋病 (Fusarium wilt of melon)，為全世界甜瓜產業主要的限制因子。目前國際間之慣用法主要利用吸濕紙法及培養皿檢查法進行帶菌種子檢測。由於此些方法需要花費較多的時間及人力成本，且不易以型態鑑別區分出甜瓜種子是否帶有甜瓜萎凋病菌，本論文希望能開發出能快速檢測出甜瓜萎凋病菌帶菌種子之分子檢測技術。以聚合酶連鎖反應 (polymerase chain reaction, PCR) 為基礎之分子檢測技術，常被用於檢測鐮孢菌 (Fusarium) 及其他植物病原菌，其檢測流程依序為取樣、核酸萃取及分生技術檢測。本試驗擬利用聚合酶鏈鎖反應技術、即時聚合酶連鎖反應 (real-time PCR) 及配合2套萃取系統，針對甜瓜帶菌種子進行檢測分析，參考國際上針對甜瓜萎凋病菌之專一性引子對Fa15F/Fa15R所增幅之核酸片段Fa15301，設計出專一性引子TDCP1F/TDCP1R、TDCP2F/TDCP2R及探針TDCpr1，並確認這些引子對Fom具專一性。以標準DNA、基因組DNA及菌絲為檢體進行PCR靈敏度測試，結果顯示：Fa15F/Fa15R、TDCP1F/TDCP1R及TDCP2F/TDCP2R的靈敏度相當，在SYBR green-based real-time PCR系統中也獲得相似的結果，另在TaqMan probe-based real-time PCR系統中，TDCP2F/TDCP2R引子組之靈敏度較高。針對人工模擬帶菌種子樣本，利用管柱核酸萃取法進行核酸萃取，搭配PCR、SYBR green-based real-time PCR及TaqMan probe-based real-time PCR，皆可檢出帶菌率為 0.25% 的帶菌種子，此外，本研究也開發出快速核酸萃取法，能對帶菌種子進行核酸萃取，搭配SYBR green-based real-time PCR及TaqMan probe-based real-time PCR進行分析，結果顯示，TDCP1F/TDCP1R、TDC2F/TDCP2R的靈敏度高於Fa15F/Fa15R，可檢出0.25% 帶菌率的種子。本研究也進行同日間與異日間之帶菌種子檢測再現性測試，結果發現以TDCP2F/TDCP2R引子組搭配TaqMan probe-based real-time PCR之再現性結果最佳，同日間與異日間之變異率分別為0.78%與1.5%。

關鍵字

快速核酸萃取法；聚合酶連鎖反應； SYBR green-based real-time PCR ； TaqMan probe-based real-time PCR

並列摘要

Melon (Cucumis melo L.) is an economically important crop in the world. Fusarium oxysporum f. sp. melonis (Fom) is a destructive phytopathogen that causes a seed-borne wilt disease on melons. The blotter and agar-plate methods are simple and inexpensive ways to detect Fom-contaminated seeds. Such methods need to spend a lot of time and labor costs, and untrained people may be incapable of Fom identification based on morphological observations. Development of a specific and efficiently tool for Fom detection is important. PCR-based techniques have been frequently applied for detection of Fusarium and other plant pathogens. A standard procedure for molecular detection includes sampling, DNA extraction, and molecular identification. For the Fom molecular detection purpose, we have developed the reliable molecular methods to detect Fom-contaminated seeds by using PCR and real-time PCR combined with the two DNA extraction methods. The data indicated that the novel primer sets TDCP1F/TDCP1R and TDCP2F/TDCP2R and TaqMan probe TDCpr1 derived from the amplified sequence of the race 2-specific primers Fa15F/Fa15R published by Luongo et al. (2012) were specific to Fom. The results of the sensitivity evaluation showed that the TDCP2 real-time PCR assay was the most sensitive method for the detections of standard template, genomic DNA, and mycelium of Fom. High detection reproducibility in Fom-contaminated seed detection by the TDCP2 real-time PCR assay was also observed among the results generated from intraday-/interday-assays. Intraday-assay variation was 0.78% and interday-assay variation was 1.5% for the TDCP2 TaqMan probe-based real-time PCR assays.