應用複合式 PCR 同時偵測並區分三種蝴蝶蘭

Abstract

臺灣為蝴蝶蘭重要種苗輸出國，因此提供健康的種苗是最重要的課題。危害蝴蝶蘭的病害中細菌性病害目前有Acidovorax avenae subsp. cattleyae (AAC) 引起之褐斑病、Burkholderia gladioli (BG) 引起之葉斑病及Pectobacterium chrysanthemi (PCH) 引起之軟腐病等三種，其中褐斑及葉斑兩者初期病徵不易區分，與真菌性病害的病徵也不易區分。本研究為達快速且明確區分不同病原引起的病害，因而研發複合式(multiplex) PCR，並應用於標的物之檢測，不但可快速且同時偵測到AAC、BG 及 PCH 三種病原細菌，且不論樣品中標的物僅存在單一菌或存在兩種菌或存在三種菌，均可針對標的物分別產生463 bp (AAC)、350 bp (BG) 及548 bp (PCH) 的產物，除可確認外又能區分三種病原細菌，並與供試6 屬11 種非標的菌無任何產物產生，靈敏性測試結果顯示可偵測到細菌數之最低量約為1.8~1.9 × 101 cfu/ml，可偵測到細菌全DNA 之最低量約為50~100 pg，顯示其專一性及靈敏性均高。人工接種或田間罹病之蝴蝶蘭組織經由簡易萃取法後即可進行複合式聚合酵素連鎖反應，並得到專一且可明確區分之片段。由試驗結果確知所使用的複合式多引子組確可應用於蝴蝶蘭三種細菌性病害(褐斑病、葉斑病及軟腐病) 之快速且準確之診斷，鑑定及偵測。 Exportation of Phaelanopsis seedlings is an important industry in Taiwan; therefore, seedling health is one of the most concerned issues. Of the diseases in Phaelanopsis, brown spot caused by Acidovorax avenae subsp. cattleyae (AAC), leaf spot caused by Burkholderia gladioli (BG), and soft rot caused by Pectobacterium chrysanthemi (PCH) are important bacterial diseases. Among them, symptoms of brown spot and leaf spot are not easy to distinguish and might be misdiagnosed with some other fungal diseases at the initial stage of infection. For the purpose of rapid and differentiate these different diseases, this study was conducted to develop a multiplex polymerase chain reaction technique (multiplex PCR) to detect, distinguish, and identify AAC, BG and PCH with high sensitivity and specificity. Diseased tissues of Phaelanopsis obtained from artificial inoculation or natural infection could be subjected to the multiplex PCR after a simple DNA extraction procedure. After the multiplex PCR, DNA fragment could be specifically amplified from the samples of AAC, BG, and PCH with the length of 463 bp, 350 bp, and 548 bp, respectively. On the contrary, no DNA fragment was obtained from the multiplex PCR using a total of 32 non-target bacterial strains which belong to 6 genera 11 species. Sensitivity of PCR for detecting AAC, BG, and PCH was between 50 and 100 pg for purified DNA and 1.8 - 1.9×101 cfu/ml for cultured cells, respectively. It took 3-4 hr to detect target bacterium from colony or infected tissues of phalaenopsis. These results revealed the multiplex PCR technique could be applied for the detection, diagnosis, and identification of the three bacterial diseases in Phaelanopsis seedlings rapidly and accurately.