東亞蘭嵌紋病毒快速偵測方法之建立

Abstract

蝴蝶蘭為目前廣受歡迎的觀賞用植物，而臺灣是蝴蝶蘭之最大出口國。蝴蝶蘭之病毒病害是影響其外銷品質的主因。東亞蘭嵌紋病毒（Cymbidium Mosaic virus, CymMV）則是重要蘭花病毒之一。此病毒感染蝴蝶蘭之後，對於蝴蝶蘭之產量及品質都會造成嚴重之影響，更凸顯了偵測此病毒之重要性。為解決蝴蝶蘭病毒病害，需建立無病毒之種原及加強蘭園病毒病害之管理，但二者皆有賴於正確的病毒偵測。目前常用於CymMV的偵測技術是以抗體為主的酵素連結免疫抗體法(ELISA)，此方法能夠大量檢測樣品，但其偵測準確度與敏感度有其限制；另有利用反轉錄聚合酵素鍊鎖反應（RT-PCR）之偵測方法，其準確度較高，不過在成本及技術層面上則高過以抗體為主的偵測技術。本論文首先探討酵素連結免疫抗體法準確度的限制因子，發現病毒含量過低或是鞘蛋白胺基酸序列有變異，皆為造成ELISA偵測準確度不高之原因。因此，本論文針對現行RT-PCR偵測方法進行改進，首先對五種核酸萃取法進行比較，比較所需成本、萃取時間，且經過核酸定量後，比較五種萃取法利用RT-PCR偵測CymMV之靈敏度，發現一種只需研磨植物組織便能進行RT-PCR之萃取方法，符合低成本、快速且高偵測靈敏度之目標，使RT-PCR能夠更有效的應用於CymMV之高通量快速偵測。 關鍵字：蝴蝶蘭、東亞蘭嵌紋病毒、酵素連結免疫抗體法、多元抗體、反轉錄聚合酵素鍊鎖反應

Phalaenopsis sp., an ornamental crop belonging to the Orchidacea, is an important commercial crop in the floral industry. Taiwan is one of the biggest exporters of Phalaenopsis orchid in the world market. Cymbidium Mosaic virus（CymMV）is one of the most economically important virus of Phalaenopsis orchid. The yield and quality of orchid production are influenced by this virus. To solve this problem, the virus-free orchids that used for breeding and the management of viral disease in orchid nurseries need to be developed. Therefore, a reliable and effective detection method of CymMV are needed. Enzyme-linked immunosorbent assay （ELISA）is a common method to detect CymMV in the orchid industry in Taiwan; however, high ratio of false positive detection are reported. Alternatively, reverse transcription polymerase chain reaction（RT-PCR）can be applied to the detection of CymMV, however, it is costy and highly trained technician are required for its accuracy. In this study, we found that low accumulation of some isolates of CymMV in orchids contribute partly to the high ratio of false positive detection of CymMV. Surprisingly, some isolates accumulate to high levels but still can’t be detected by ELISA. Sequence analysis revealed distinctive amino acids changed in the coat protein ORF of these isolates. To overcome the problems that applying RT-PCR in commercial CymMV detection, we alleviated the efforts of nucleic acids purification that commonly used in RT-PCR detection and develop a RT-PCR based CymMV rapid detection methods. The sensitivity of this method was similar to 3 commonly used RNA extraction methods in CymMV detection when same amounts of purified nucleic acid are used as template. Because the commonly used nucleic acid purification methods used for RT-PCR detection will cause nucleic acid loss (the yield loss range from 40.8% ~ 96%) during the purification, the rapid detection methods is the most sensitive methods in CymMV detection.