利用TiiPCR偵測技術於西瓜果斑病種子檢測之應用

西瓜為葫蘆科作物，亦為全世界之蔬菜類中產量最高的作物。在其生產過程中，常因Acidovorax citrulli所引起的西瓜果斑病(BFB)發生而造成經濟上的重大損失並降低產品價值。BFB為重要且在種子上零容忍之國際檢疫性病害，主要的原因為帶菌的種子可成為田間初次感染源，也是最重要的傳染途徑。此病原菌除可造成果實危害外，亦可感染西瓜子葉和真葉而影響產量。因此，對於瓜類種子是否帶有果斑病菌的檢測為防止病害發生的重要課題。在此需求下，快速且靈敏的偵測技術有開發的價值。其中，TaqMan探針結合隔絕式恆溫聚合酶連鎖反應技術(TiiPCR) 可利用螢光訊號的增加直接判讀結果，目前已有商品化利用於魚蝦病害快速檢測的應用。本研究試圖以此技術為基礎開發西瓜種子上果斑病菌之檢測技術。首先，以A. citrulli之專一性片段設計專一性引子對及螢光探針，經調整反應條件下，每個反應中含質體於DNA 10 copies及每個反應中含有10個以下細菌細胞之稀釋液均可達到80%以上之偵測率，最小之螢光訊號的S/N ratio比值亦超過1.8。此外，本研究亦證明所開發之條件對果斑病菌具有高度專一性，對其他多種不同病原菌均無法測得，推測發生交叉汙染機率低。並且進一步分別將10%-1%人工帶菌的種子培養於不同培養基中進行評估，結果顯示於King’ s B培養液中培養12小時，對1%帶菌種子之偵測率仍達66%，若延長培養時間至24小時即可達到100％之檢測率；然而，在0.2%帶菌種子培養於100 ml King’ s B中卻無法有效測得，進一步將培養24小時之培養液經離心濃縮再稀釋後對0.2%帶菌種子的偵測率即可達100％。由上述結果，本研究將所發展之TiiPCR技術結合KB培養基建立一標準流程於檢測帶有西瓜果斑病菌的種子，為一快速、靈敏度且專一性高之檢測工具。

關鍵字

細菌性果斑病；檢測技術； TiiPCR ；種子偵測

並列摘要

Watermelon is an important vegetable crop of the Cucurbitaceae family worldwide. During its production, Acidovorax citrulli is the causal agent of bacterial fruit blotch (BFB). BFB is an important international quarantine disease with zero tolerance on seed. The infested seeds can be a primary source of inoculum in the field. Hence, a rapid and sensitive method for detecting A. citrulli infested seeds is important to manage BFB. Under this demand, rapid and sensitive detection method such as TaqMan probe-based insulated isothermal PCR (TiiPCR) could be developed for further application. TiiPCR was directly uses fluorescence signal interpretation results and developed to obviate the need of post-amplification processing to avoid cross-contamination. In this study, we sought to develop a method to detect infested seeds with A. citrulli based on TiiPCR. First, specific primers and probes were designed based on the DNA fragment of A. citrulli genome. Under the adjusted reaction conditions, results revealed that 10 copies of plasmid DNA and less than 10 bacterial cells in each reaction tube were detectable over 80 % detection rate with intensification on fluorescent signal over 1.8. In addition, the condition is highly specific to A. citrulli and suggested the probability of cross-contamination is low. Furthermore, the adjusted conditions in combination with the incubation in various media were applied to detect their efficacy on 10%-1% of infested seeds. Results showed that the incubation with King’s B broth (KB) for 24 hr was superior to enhance detection rate in all condtions. While 1% of 100 infested seeds were incubated in 100 ml of KB for 24 hr, the detection rate reached 100%. However, it was undetectable with 0.2% of 500 infested seeds in 100 ml of KB. We further demonstrated that 100% of detection rate could be achieved by removing the cultural medium following 10 folds dilution. In this study, we demonstrated that a rapid and sensitive standard protocol for seed detection method of Acidovorax citrulli was established. Application of this method to manage watermelon seeds for reducing BFB would be promising in the future.