同時偵測TSWV、WSMoV及IYSV三個血清群病毒多重反轉錄聚合酶連鎖反應技術之開發

"Development of a multiplex RT-PCR method for simultaneous detection of tospoviruses in TSWV, WSMoV and IYSV serogroups"

"Tospovirus為全世界危害經濟作物的植物病毒屬，其中以鳶尾花黃斑病毒（Iris yellow spot virus, IYSV）、番茄斑點萎凋病毒（Tomato spotted wilt virus, TSWV）及西瓜銀斑病毒（Watermelon silver mottle virus, WSMoV）為代表種命名的3個血清群，分別危害百合科、茄科及葫蘆科等重要經濟作物造成嚴重的農損危害。病毒N基因保留區針對病毒三個血清群分別設計型簡併引子，利用增幅出特定DNA片段的聚合酶連鎖反應（polymerase chain reaction, PCR）將包括TSWV血清群內的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）、番茄黃化斑點病毒（Tomato chlorotic spot virus, TCSV）及TSWV等七種病毒可利用引子對Pr-dTS-f/Pr-dTS-r增幅出312 bp片段；WSMoV血清群內的番椒黃化病毒（Capsicum chlorosis virus, CaCV）、彩色海芋黃化斑點病毒（Calla lily chlorotic spot virus, CCSV）、花生頂芽壞疽病毒（Groundnut bud necrosis virus, GBNV）、甜瓜黃斑病毒（Melon yellow spot virus, MYSV）、番茄壞疽斑點相關病毒（Tomato necrotic spot-associated virus, TNSaV）、番茄輪狀斑點病毒（Tomato zonate spot virus, TZSV）、西瓜頂芽壞疽病毒（Watermelon bud necrosis virus, WBNV）及WSMoV等八種病毒可以引子對FWi3/RWi1增幅出475 bp片段；以及IYSV血清群內的IYSV、蓼屬輪斑病毒（Polygonum ringspot virus, PolRSV）和番茄黃環病毒（Tomato yellow ring virus, TYRV）等三種病毒可使用引子對iF6/RWi1增幅出134 bp片段。混合上述簡併引子進行多重反轉錄聚合酶連鎖反應（Multiplex reverse transcription-PCR），成功地同時檢測三個血清群病毒。本研究所開發的檢測技術提供防檢疫快速便捷的檢測工具。"

"Tospovirus is one of important plant virus genera. Serogroups, named Iris yellow spot virus (IYSV), Tomato spotted wilt virus (TSWV) and Watermelon silver mottle virus (WSMoV), cause serious economic losses of Liliaceae, Solanaceae and Cucurbitaceae crops, respectively. The degenerated primers designed from the conserved regions of N genes were used in polymerase chain reaction (PCR) to amplify specific DNA fragments of the N gene of tospoviruses. Using the primer pair iF6/RWi1, a 134-bp DNA fragment was amplified from IYSV, Polygonum ringspot virus (PolRSV) and Tomato yellow ring virus (TYRV) of IYSV serogroup. A 312-bp DNA fragment was amplified from Alstroemeria necrotic streak virus (ANSV), Chrysanthemum stem necrosis virus (CSNV), Groundnut ringspot virus (GRSV), Impatiens necrotic spot virus (INSV), Melon severe mosaic virus (MeSMV), Tomato chlorotic spot virus (TCSV) and TSWV of TSWV serogroup using the primer pair Pr-dTS-f/Pr-dTS-r. A 475-bp DNA fragment could be amplified from 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 of WSMoV serogroup using the primer pair FWi3/RWi1. These degenerate primers were integrated to develop a multiplex reverse transcription (RT)-PCR method successfully to simultaneously detect and distinguish tospoviruses in the three serogroups. The developed multiplex RT-PCR method can be a useful detection tool for prompt and simplified field survey and virus quarantine."