摘要
蘭花乃目前廣受各方歡迎之觀賞植物，在已知感染蘭花之25種病毒中，齒舌蘭輪斑病毒(odontoglossum ringspot tobamovirus, ORSV)被認為是影響蘭花經濟生產之主要病毒。傳統上採用免疫檢定法偵測ORSV之感染多能獲致良好結果，但近年來發現利用免疫檢定法並無法將部分組織培養中感染ORSV之蘭花幼苗順利檢出，推測此可能與組織培養幼苗體內病毒濃度偏低而超出免疫檢定法之敏感極限有關。本研究嘗試發展敏感度高於免疫檢定法之核酸探針雜配法(DNA probehybridization)與反轉錄聚合瓷連鎖反應(Reverse transcription- polymerase chain reaction, RT-PCR)供ORSV之偵測。試驗中根據已知之ORSV鞘蛋白基因之核酸序列，設計一對可以由ORSV RNA中增幅獲得一段約372 bp之核酸產物之專一性引子對(OS- 1/ OS- 4)，其中OS-4乃對應ORSV鞘蛋白基因核啟酸位置358-375之互補股引子，其序列為5'- TCTTATTGCACAGTTGC- 3'，而OS-1 則為對應核啟酸位置4-21之病毒股引子，序列為5'- TCTTACACTATTACAGAC- 3'。此引子對於95 C變性1分鐘，50 C黏合1分鐘及72 C聚合2分鐘且經26 個循環之反應條件下，確實可由感染ORSV之蝴蝶蘭、文心蘭、嘉得利亞蘭、報歲蘭、及素心蘭等五種蘭花葉片組織所抽取之全量RNA中，增幅獲得與預估值相符之核酸產物，而同樣條件下對於感染CyMV或健康無病毒蘭花之RNA試料則未獲得任何產物。將上述由ORSV RNA增幅所獲之DNA產物選殖於pUC18載體，並轉型於E. coli D H5α 菌株中，利用商業化之DIG標定程序，由轉型株合成出含該DNA片段之非放射性核酸探針。此核酸探針與上述感染ORSV之五種蘭花組織之全量RNA可產生專一性雜配反應，但與健康對照及本省常見之二種tobamoviruses，包括cucumber green mottle mosaic tobamovirus (CGMMV)及tobacco mosaictobamovirus (TMV)並不呈現任何正反應訊號。試驗中利用蘭花汁液稀釋定量之ORSV純化病毒，分別以ELISA、核酸探針雜配及RT-PCR進行偵測並比較三者之敏感度，結果證實三者中以RT-PCR之敏感度最高，其可偵測之最低病毒濃度極限接近1 fg/ml，為ELISA的10^7倍，而為核酸探針雜配法之10^3倍。另外OS-1/OS-4引子對除可偵測ORSV外，並可與CGMMV之RNA =反應，於相同之RTPCR程序下增幅出一接近400 bp之DNA產物，但不與TMV之RNA產生任何反應，顯示此引子對與本實驗所發展之RT-PCR程序未來亦可應用於相關tobamoviruses之鑑別。
並列摘要
Orchids are currently one of the most popular ornamental crops in the world market. There have been at least 25 different viruses documented to infect orchids, however, among them odontoglossum ringspot tobamovirus (ORSV) and cybidium mosaic potexvirus (CyMV), are considered economically important to the orchid cultivation industry. Traditionally, ORSV is detected by serological methods, with a sensitivity generally acceptable. However, recent information indicates that some ORSV-infected orchid seedlings derived from tissue culture escape from ELISA indexing, indicating the need of developing more sensitive detection techniques, especially for tissue culture orchids. In the course of this study, we designed a primer pair (OS-1/OS-4) based on the known nucleotide sequence of ORSV coat protein gene, which could specifically amplify ORSV RNA into a 372-bp DNA product in reverse transcription-polymerase chain reaction (RT-PCR). OS-4 (5'- TCTTATTGCTACAGTTGC-3') was used as anti-viral sense primer, while OS-1 (5'- TCTTACACTATTACAGAC-3') was the viral sense one. The optimum reaction conditions for each cycle of PCR were denaturation at 95 C for 1 min, annealing at 50 C for 1 min and elongation at 72 C for 2 min and 26 cycles were generally used. Five different orchid species commonly grown in Taiwan were tested by RT-PCR and satisfactory results were consistently obtained. We also cloned the PCR product amplified from ORSV RNA into pUC18 plasmid and obtained a non-isotopic DIG-labeled DNA probe for dot-blot hybridization test. The DNA probe specifically hybridized with various ORSV-infected orchids but not with healthy controls and two other tobamoviruses, tobacco mosaic tobamovirus (TMV) and cucumber green mottle mosaic tobamovirus (CGMMV). In comparing the sensitivity of ORSV detection in crude orchid sap, we found that RT-PCR was the most sensitive one which could detect ORSV in a concentration 10^7 times lower than that detectable by ELISA, and 10^3 times lower than that detectable by probe hybridization. Besides, a similar size DNA product could also be amplified from the RNA template of CGMMV but not from TMV RNA by OS-1/OS-4 primers in RT-PCR. The possible application of the OS- 1/OS-4 primer pair in detecting CGMMV by RT-PCR is discussed.