Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/89359
標題: 兩種台灣十字花科蔬菜豆嵌病毒屬病毒鑑別方法之開發及其應用
Development and Application of the Methods for Differentiating Two Cruciferous Comoviruses in Taiwan
作者: Hung-Yu Chao
趙鴻宇
關鍵字: 十字花科蔬菜
豇豆嵌紋屬病毒
蘿蔔嵌紋病毒
蕪菁輪斑病毒
鑑別
單株抗體
Cruciferous vegetables
Comovirus
Radish mosaic virus
Turnip ringspot virus
Differentiation
monoclonal antibody
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摘要: 十字花科(Family Cruciferae or Brassicaceae)蔬菜遭病毒感染後出現嵌紋、贅脈畸生、黃化及矮化等病徵,常造成產量上的損失。目前台灣曾報導發生於十字花科蔬菜的病毒包括花椰菜嵌紋病毒(Cauliflower mosaic virus, CaMV)、胡瓜嵌紋病毒(Cucumber mosaic virus, CMV)、蕪菁嵌紋病毒(Turnip mosaic virus, TuMV)、甜菜西方黃化病毒(Beet western yellows virus, BWYV)、蘿蔔嵌紋病毒(Radish mosaic virus, RaMV)、以及近期發現的蕪菁輪斑病毒(Turnip ringspot virus, TuRSV)。RaMV和TuRSV皆屬於豇豆嵌紋病毒屬(Genus Comovirus),兩者的性狀極為近似, 包括寄主範圍、外觀形態、病毒外鞘組成、媒介昆蟲、血清類緣關係,以及基因體組成和基因體核酸序列等均非常相近,造成鑑別上的困難。本研究以尋求診斷寄主、應用病毒抗血清和單株抗體於不同的血清學技術,以及開發專一性引子對和簡併式引子對配合限制片段長度多型性(restriction fragment length polymorphism, RFLP)等方法,開發出可有效鑑別RaMV與TuRSV的平台。研究發現蘿蔔可作為區分RaMV和TuRSV的診斷寄主(diagnostic host),RaMV感染蘿蔔引起明顯的嵌紋或贅脈畸生病徵;而TuRSV則不論感染與否均不會在蘿蔔引起病徵。以RaMV-TW與TuRSV-TW的單株抗體和含有多元抗體的病毒抗血清應用於不同的血清學技術,可有效鑑別兩病毒。研究發現病毒的抗血清應用於免疫轉漬(immunoblotting)可有效區別兩病毒,但應於瓊脂雙向擴散反應(double diffusion)和間接酵素連結免疫吸附分析(ELISA)時則否;而研究開發的病毒專一性單株抗體則可應用於ELISA,但未能有效用於免疫轉漬。RaMV-TW與TuRSV-TW亦可應用分生技術鑑別之,以兩病毒的RNA 2序列設計專一性引子對,經由自身專一性引子增幅,RaMV可增幅1.1 kb大小之片段,TuRSV則可增幅0.6 kb的片段。而以RNA 1序列為模板設計簡併式引子對增幅出可利用限制酵素BamHI 酶切的RFLP檢測區,該區段的cDNA經BamHI處理後,增幅自RaMV的片段能被酶切成兩段,而TuRSV者則否,顯示專一性引子對及RFLP的方法皆能有效應用於區別RaMV及TuRSV。應用本研究開發的專一性單株抗體並配合TuMV和CMV抗血清於田間檢測,發現目前十字花科蔬菜田間的病毒病害以TuRSV較為普遍,但未發現RaMV感染的病例,TuMV及CMV則為零星發生,且偶有複合感染之情形。
Viruses infect cruciferous vegetables (Family Cruciferae or Brassicaceae) causing various symptoms such as mosaic, enation, yellows, and stunt, and, thus, remarkable losses. Several viral diseases of cruciferous crops have been identified or detected in Taiwan during past decades, including Cauliflower mosaic virus (CaMV), Cucumber mosaic virus (CMV), Turnip mosaic virus (TuMV), Beet western yellows virus (BWYV), Radish mosaic virus (RaMV) and, recently, Turnip ringspot virus (TuRSV). Both RaMV and TuRSV are comoviruses that share remarkable similarities on their host ranges, transmission vectors and modes, serological relationships, as well as the organization and nucleotide and amino acid sequences of their genomes. It is not easy to differentiate TuRSV and RaMV from each other based on their host ranges or serological relatedness in the past. Nonetheless, we have developed some useful approaches based on diagnostic hosts, serological techniques using polyclonal antisera or virus species-specific monoclonal antibodies, species-specific primers, and restriction fragment length polymorphism (RFLP) of specific RT-PCR fragments, to discriminate both viruses. In host range tests, we have found that some cultivars of radish (Raphanus sativus) can be a useful diagnostic host for distinguishing RaMV from TuRSV. Radish shows conspicuous symptoms of mosaic and/or enation after inoculated with RaMV but no symptom at all whether infected by TuRSV or not. In addition, applying virus specific monoclonal antibodies and polyclonal antisera in combined with serological techniques are also applicable to differentiate RaMV and TuRSV. Polyclonal antisera of RaMV and TuRSV can be used to seperate both viruses on immunoblots as the polyclonal antisera detect the large coat protein (LCP) and small coat protein (SCP) of homologous virus, and only the LCP, but not the SCP, of heterologous virus. However, polyclonal antisera are not useful for applying on the double diffusion and ELISA tests. On the other hand, virus species-specific monoclonal antibodies produced in this study could detect homologous virus specifically and discern viruses accurately when applied on ELISA tests but not onto immunoblotting tests. Specific primer sets for RaMV and TuRSV have been designed based on the nucleotide sequences of RNA 2 of both viruses. Virus-specific primer set can be used to amplify a 1.1 kb cDNA fragment of expecting size from RaMV while a 0.6 kb fragment from TuRSV. In addition, the RFLP patters of a cDNA fragment derived from RNA 1 of both RaMV and TuRSV with designed degenerate primer set can be useful for differentiating both viruses after BamHI digestion. Both specific primer sets and RFLP are effective for differentiating RaMV from TuRSV. In addition to those polyclonal antisera of Cucumber mosaic virus (CMV) and Turnip mosaic virus (TuMV), monoclonal antibodies against RaMV and TuRSV produced in this study have been used in field survey of cruciferous viruses. The preliminary field survey showed TuRSV was the virus most detected among cruciferous vegetables with sporadic occurrence of TuMV, CMV, but without RaMV.
URI: http://hdl.handle.net/11455/89359
文章公開時間: 2018-07-16
Appears in Collections:植物病理學系

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