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感染海芋之芋頭嵌紋病毒檢測方法之研發與比較

Development and Comparison of Three Detection Methods for Calla Lily-infecting Dasheen mosaic virus

摘要


海芋為天南星科、馬蹄蓮屬多年生球根花卉,近年來深受消費者喜愛,栽培面積持續增加,已成為國際間重要的花卉作物。病毒性病害為目前海芋栽培上的主要限制因子之一,國內外曾有16種病毒被報導可感染海芋,其中芋頭嵌紋病毒(Dasheen mosaic virus, DsMV)可危害16屬以上的天南星科植物,其分佈遍及世界各地,具有經濟重要性。本研究針對感染海芋的DsMV研發出三種檢測方法。其一是檢測DsMV鞘蛋白的方法,先依據DsMV彩色海芋分離株(DsMV-ZAN)之序列設計專一性引子對,從其cDNA株中增幅出全長度鞘蛋白基因,選殖至表現載體後,再以大腸桿菌系統表現DsMV重組鞘蛋白,並且製備出DsMV抗血清。以間接式酵素連結抗體免疫吸附法(indirect-ELISA, I-ELISA)測試,結果顯示DsMV抗血清具有極高的力價;而以免疫轉漬分析證明DsMV抗血清不會與其他感染海芋的同屬病毒反應,顯示其具高專一性。其次為檢測DsMV核酸的方法,於DsMV基因體的3'端非轉譯區及鞘蛋白區域,共設計三組引子對,以製備二種長短不同的DNA探針,並以墨點雜合法(dot-blot hybridization, DBH)對罹病植物粗萃取液及植物全RNA進行檢測;結果顯示此三種DNA探針皆具有高專一性,且靈敏度相似;但以純化的全RNA為檢測樣品時,效果較植物粗萃取液為佳。第三種方法則是結合免疫與核酸檢測原理的免疫吸附反轉錄聚合酶連鎖反應(immunocapture RT-PCR, IC-RT-PCR)。當以感染DsMV的罹病植物粗萃取液作為檢測材料,比較I-ELISA、DBH及IC-RT-PCR三者之靈敏度時,結果以IC-RT-PCR靈敏度最佳,比I-ELISA靈敏度高125倍以上,而DBH次之,比I-ELISA靈敏度高4倍。之前未曾有以DBH或IC-RT-PCR檢測DsMV之報告,或這些方法與ELISA比較的相關報導,因此希望本研究所研發的DsMV檢測方法未來可應用於每芋種苗病毒驗證作業,以協助國內業者生產健康海芋種苗。

並列摘要


Calla lilies (Zantedeschia spp.), belonging to the family Araceae, are perennial bulbous flowers. Because calla lilies are very popular among consumers and the cultivation areas continuously increase, they become important flower crops worldwide. The viral disease is one of the limiting factors for growing calla lilies and 16 viruses have been reported. Among these viruses, Dasheen mosaic virus (DsMV), a member of the genus Potyvirus, is an important and widely spread virus which can infect at least 16 genera of aroid plants. In this study, three detection methods were developed for calla lily-infecting DsMV (DsMV-ZAN). The first method was to detect the coat protein of DsMV. Based on the sequence of DsMV-ZAN isolate, specific primers were designed to amplify the frill-length of coat protein gene, and then cloned into the expression vector. Recombinant DsMV coat protein was expressed by Escherichia coli and used as antigen to prepare DsMV antiserum. The result of indirect-ELISA (I-ELISA) indicated DsMV antiserum possessed good titer. Moreover, its specificity was proved by immunoblot analysis since it did not react with other calla-infecting potyviruses. The second method was to detect DsMV RNA. Three primer pairs located in 3'UTR and the coat protein region of DsMV were designed and used to prepare three DNA probes with different length. The results of dot-blot hybridization (DBH) demonstrated that all probes were highly specific and had similar sensitivities in detecting DsMV in crude sap extracts and purified total RNAs although the latter showed better result than the former. The third method, immunocapture-RTPCR (IC-RT-PCR), was developed by combining the immunological and nucleic acid detection methods. When the sensitivity of I-ELISA, DBH and IC-RT-PCR was compared by detecting DsMV in crude sap extracts, the results indicated that IC-RT-PCR was about 125 times more sensitive than I-ELISA, and DBH was about 4 times more sensitive than I-ELISA. There is neither paper about detecting DsMV by DBH or IC-RT-PCR nor report about comparing ELISA with these methods published before. Therefore, the DsMV detection methods developed in this study can be applied to virus certification scheme of calla lily and help to produce healthy seedlings in Taiwan.

被引用紀錄


Hu, W. C. (2009). 海芋potyvirus之基因體序列分析、快速檢測及應用RNAi防治其感染之研究 [doctoral dissertation, National Taiwan University]. Airiti Library. https://doi.org/10.6342%2fNTU.2009.02005
Lin, W. F. (2007). 利用重組鞘蛋白製備可偵測多種海芋病毒之廣效性單株抗體 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342%2fNTU.2007.00490

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