樹木疫病檢測套組商品化 The establishment of detection kit for phytophthora diseases

疫病菌屬(Phytophthora)包含100多種疫病菌，其中許多為植物重要病原菌。傳統上以形態觀察為基礎之檢測方法所需時間長，且需要專業植物病理人員進行操作，而多數分子鑑定法雖然鑑定時間較短，但通常涉及繁複的檢測步驟及昂貴儀器的使用。為掌握檢測及防治先機，本研究應用恆溫環狀擴增法(LAMP, Loop-mediatedisothermal amplification) 開發新的快速病害檢測技術，以期提高植物疫病菌的診斷效率。本研究針對Phytophthora parasitica、P. cambivora、P. cinnamomi、P. nicotianae、P. capsici、P. citricola、P. citrophthora 、P. cryptogea
、P. heveae、P. palmivora 及P. infestans 等病原菌的ITS核酸序列之保守性序列區域，設計三個LAMP引子組(命名為Phyto-spp1、2與3)，進行LAMP反應增幅測試結果顯示，三組引子組皆能對十種疫病病原核酸有良好增幅反應，專一性測試顯示， 當反應溫度為62.5℃，三組引子組除了可對十種疫病病原核酸增幅，同時也都會對與疫病菌同科之腐霉菌(Pythium sp.)產生增幅反應；而當反應溫度拉高到67℃時，第二及第三組具有良好專一性。本研究所開發的Phyto-spp2及Phyto-spp3引子組，可對Phytophthora spp. 進行LAMP專一性增幅，而反應溫度為62.5℃時，靈敏度較先前研發的PCR檢測法可提升100倍，可做為另一種植物疫病菌之快速分子檢測法。 此技術經由調整其最佳反應條件與標準檢測流程後，已與生技業者合作並完成技術轉移，開發商業化檢測套組。未來在防治樹木疫病上，可應用於新植樹木之檢測及健康苗木的篩檢，有效降低防治成本。本技術商品量產化之後可行銷推廣至國際市場，創造無限商機。

The Genus Phytophthora contains more than 100 species, and many of them are important plant pathogens. Traditional process for Phytophthora identification mostly based on morphology observation is time and professional labor consuming. On the contrary, molecular technique requires less time, but it usually needs complicated experimental procedures and expensive instruments. For increasing accuracy and efficiency of Phytophthora diagnosis, a new method based on the loopmediated isothermal amplification (LAMP) was developed. In this study, three LAMP primer sets (named Phyto-spp1, 2, and 3) were designed from the conserved sequence region of the ITS sequence of the pathogens, Phytophthora parasitica, P. cambivora, P. cinnamomi, P. nicotianae, P. capsici, P. citricola, P. citrophthora, P. cryptogea, P. heveae, P. palmestra and P. infestans. The experiment results of the LAMP reaction showed that all three primer sets could all detect the DNA of ten Phytophthora species. And the specificity tests showed that when the reaction temperature is 62.5℃, the three primer sets could also detect Pythium sp., which shares the same family, Pythiaceae, with Phytophthora spp. Primer set2 and set3 showed good specificity when the reaction temperature was rised to 67℃. The two sets of primers (Phyto-spp2 and Phyto-spp3) can specifically detect Phytophthora spp., and the detection sensitivity at LAMP reaction temperature 62.5℃ is 100 times higher than the previously developed Polymerase Chain Reaction (PCR) method. In our study, LAMP could be another rapid molecular diagnosis technique for the detection of Phytophthora spp. After adjustment of optimal condition
tests and Standard Operating Procedure, the technique has been used to develop commercial detection kit and the technique transfer with a biotechnology company has been accomplished. The detection kit can not only be used on health check for new planted trees and seedlings, but also reduced the cost of disease control. After mass production, the commercial detection kit can be promoted to international market to create further more opportunities.