用於鑑定番茄斑點萎凋病毒（TSWV）血清群病毒種類引子對組之開發

Development of primer sets for identification of the member species of Tomato spotted wilt virus (TSWV) serogroup

"Tospovirus病毒屬寄主範圍廣泛，為世界性危害重要經濟作物的植物病毒之一。根據核鞘蛋白（nucleocapsid protein, NP）的血清學親緣關係，tospoviruses可進一步分類為數個血清群（serogroups），大部分的病毒分屬於以西瓜銀斑病毒（Watermelon silver mottle virus, WSMoV）及番茄斑點萎凋病毒（Tomato spotted wilt virus, TSWV）命名之兩大血清群。WSMoV血清群病毒發生於亞洲，而TSWV血清群病毒則主要在歐美地區。TSWV血清群的成員尚有水仙百合壞疽條斑病毒（Alstroemeria necrotic streak virus, ANSV）、菊花莖部壞疽病毒（Chrysanthemum stem necrosis virus, CSNV）、花生輪斑病毒（Groundnut ringspot virus, GRSV）、鳳仙花壞疽斑點病毒（Impatiens necrotic spot virus, INSV）、甜瓜嚴重嵌紋病毒（Melon severe mosaic virus, MeSMV）、辣椒壞疽斑點病毒（Pepper necrotic spot virus, PNSV）、番茄黃化斑點病毒（Tomato chlorotic spot virus, TCSV）及矮南瓜致死黃化病毒（Zucchini lethal chlorosis virus, ZLCV）等。本研究利用分析TSWV血清群病毒之N基因序列設計多組上游引子（forward primers：FT1、FT2、FT3、FT4、FT5）及下游引子（reverse primers：RT1、RT2、RT3、RT4、RT5），彼此互相交叉配對為25組引子對，以ANSV、CSNV、GRSV、INSV、MeSMV、TCSV及TSWV等七種病毒的N基因選殖質體進行聚合酶連鎖反應（polymerase chain reaction, PCR）測試。除了編號05: FT1/RT5、10: FT2/RT5、15: FT3/RT5、20: FT4/RT5及25: FT5/RT5等引子，對所有病毒N基因選殖質體完全沒有任何反應外，其他引子對則藉由擴增片段有無的多形性（polymorphism）可鑑定出不同的病毒種類。使用不同引子對組合：編號02：FT1/RT2、03: FT1/RT3、04: FT1/RT4、06: FT2/RT1及07: FT2/RT3等5個引子對，可成功地自感染病毒的菸草（Nicotiana benthamiana）葉片組織總量RNA中，以反轉錄聚合酶連鎖反應（reverse transcription-polymerase chain reaction, RT-PCR）鑑定出TSWV及INSV。研究結果顯示，所設計之引子對組具有鑑定TSWV血清群病毒種類之潛力。"

"Tospovirus is one of important plant virus genera that has a wide host rang infecting more than 1090 species of 82 plant families and causes symptoms of spots, chlorosis, mottle, stunt, wilt and necrosis. According to the serological relationship of nucleocapsid protein (NP), tospoviruses are divided into serogroups. Most of tospoviruses are classified in two major serogroups named from Watermelon silver mottle virus (WSMoV) and Tomato spotted wilt virus (TSWV). The WSMoV-serogroup tospoviruses are prevailing in Asia while the TSWV-serogroup tospoviruses are mainly distributed in Europe and America. The members of TSWV serogroup, such as Alstroemeria necrotic streak virus (ANSV), Chrysanthemum stem necrosis virus (CSNV), Groundnut ringspot virus (GRSV), Impatiens necrotic spot virus (INSV), Melon severe mosaic virus (MeSMV), Pepper necrotic spot virus (PNSV), Tomato chlorotic spot virus (TCSV), Tomato spotted wilt virus (TSWV) and Zucchini lethal chlorosis virus (ZLCV), are important quarantine viruses in Taiwan. In this study, the nucleotide sequences of N genes of TSWV-serogroup tospoviruses were analyzed to design forward primers FT1, FT2, FT3, FT4 and FT5 and reverse primers RT1, RT2, RT3, RT4 and RT5. The forward and reverse primers were cross-mated to form 25 primer pairs that were used to test the accessibility for amplifying signals from the cloned plasmids carrying the N genes of ANSV, CSNV, GRSV, INSV, MeSMV, TCSV and TSWV in polymerase chain reaction (PCR). Except the primer pairs 05: FT1/RT5, 10: FT2/RT5, 15: FT3/RT5, 20: FT4/RT5 and 25: FT5/RT5, most primer pairs could be used to amplify certain DNA fragments, ranging from 200 bp to 800 bp, from different N gene clones. Moreover, tospovirus species could be identified from the polymorphisms of amplicons. The primer sets 02: FT1/RT2, 03: FT1/RT3, 04: FT1/RT4, 06: FT2/RT1 and 07: FT2/RT3 were successfully used to identify TSWV and INSV from total RNAs of virus-infected Nicotiana benthamiana leaf tissues by reverse transcription-PCR. Our results showed that the primer sets have a great potential for identification of TSWV-serogroup tospoviruses."