建立具鑑別WSMoV及IYSV血清群之簡併引子對組與特異性電泳圖譜

Design of degenerated primer sets for establishment of specific electrophoresis patterns to identify virus species within the WSMoV and IYSV serogroups

"Tospovirus屬為全世界重要危害經濟作物的植物病毒之一，而危害我國的則以WSMoV血清群為主，造成茄科及葫蘆科等作物嚴重的經濟損失，本研究擬利用比對WSMoV及IYSV兩個血清群病毒之核鞘基因（nucleocapsid gene, N gene）序列，透過病毒N基因序列內保留區域（conserved region）的分析，設計簡併引子（degenerate primer），分別針對番椒黃化病毒（Capsicum chlorosis virus, CaCV）、彩色海芋黃化斑點病毒（Callalily chlorotic spot virus, CCSV）、花生頂芽壞疽病毒（Groundnutbud necrosis virus, GBNV）、甜瓜黃斑病毒（Melon yellowspot virus, MYSV）、番茄壞疽斑點病毒（Tomato necrotic spotassociatedvirus, TNSaV）、番茄輪狀斑點病毒（Tomato zonatespot virus, TZSV）、西瓜頂芽壞疽病毒（Watermelon bud necrosisvirus, WBNV）及WSMoV等八種WSMoV血清群病毒及IYSV、蓼屬輪斑病毒（Polygonum ringspot virus, PolRSV）、番茄黃環病毒（Tomato yellow ring virus, TYRV）等三種IYSV血清群的病毒N基因選殖質體DNA進行聚合酶連鎖反應（polymerase chain reaction, PCR）測試，其增幅出的PCR產物片段大小分別在140-800 bp區間，挑選出對各種病毒呈現特異電泳圖譜的引子對組，與分別感染菸草台灣現有病毒CaCV、CCSV、MYSV及WSMoV之總量RNA進行反轉錄聚合酶連鎖反應（reverse transcriptionpolymerase chain reaction, RT-PCR）測試，結果與病毒N基因質體DNA所呈現的病毒特異性電泳圖譜一致，說明此引子對組合具有應用於田間病毒診斷鑑定之潛力。而針對各病毒種建構病毒N基因保留區引子對組之特異性電泳圖譜資料庫，未來可供作Tospovirus防疫檢疫工作之參考。"

"Tospovirus is one of important plant virus genera in the world. In Taiwan, the members of WSMoV serogroup cause severe economic losses on solanaceous and cucurbitaceous crops. In this study, the nucleotide sequences of nucleocapsid (N) genes of tospoviruses belonging to the WSMoV and IYSV serogroups were compared. The designed degenerate primer pairs were used for assays in polymerase chain reaction (PCR) amplification using the recombinant plasmids carrying the N gene sequences of eight WSMoV-serogroup tospoviruses, Capsicum chlorosis virus (CaCV), Calla lily chlorotic spot virus (CCSV), Groundnut bud necrosis virus (GBNV), Melon yellow spot virus (MYSV), Tomato necrotic spot-associated virus (TNSaV), Tomato zonate spot virus (TZSV), Watermelon bud necrosis virus (WBNV) and WSMoV, and three IYSV-serogroup tospoviruses, IYSV, Polygonum ringspot virus (PolRSV) and Tomato yellow ring virus (TYRV), as templates. The specific amplicon polymorphism including the presence or absence of signals and the divergent sizes (140-800 bp) of PCR products was obtained when most of primer pairs were used. The degenerate primer pairs were selected to apply for detection of occurring in Taiwan viruses, which are CaCV, CCSV, MYSV and WSMoV from total RNAs of virus-infected Nicotiana benthamiana tissues in reverse transcription-polymerase chain reaction (RT-PCR). The same results as well as the N gene-containing plasmids demonstrated that the developed method can be potentially applied to diagnose and identify tospoviruses in field. Construction of specific amplicon polymorphisms of tospoviruses using the degenerate primer pairs, which designed from the consensus sequences of N genes can be used as a reference for inspection and quarantine mission to prevent invasions from foreign tospoviruses in future."