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| 題 名 | 西方花薊馬之分子鑑定:rDNA之單管巢式聚合酵素鏈鎖反應=Molecular Identification of a Plant Quarantine Pest "Frankliniella occidentalis" by One-Tube Nested PCR Targeting Ribosomal DNA Internal Transcribed Spacer Regions |
|---|---|
| 作 者 | 劉逸琪; | 書刊名 | 植物保護學會會刊 |
| 卷 期 | 46:1 2004.03[民93.03] |
| 頁 次 | 頁27-46 |
| 分類號 | 433.3 |
| 關鍵詞 | 西方花薊馬; 巢式聚合酵素鏈鎖反應; 核糖體DNA; ITS區間序列; 鑑定; Frankliniella occidentalis; Nested PCR; rDNA; ITS region sequence; Identification; |
| 語 文 | 中文(Chinese) |
| 中文摘要 | 西方花薊馬(Frankliniella occidentalis (Pergande))為全世界重要經濟作物的害蟲,也是我國主要檢疫害蟲。為建立西方花薊馬的分子診斷鑑定技術,本研究以核糖體DNA (rDNA)中之28S及ITS2區域核酸序列,分析西方花薊馬與已發表於NCBI基因庫之薊馬之親緣關係,結果顯示,除梳缺花薊馬(F. schultzei)與花薊馬屬(Frankliniella spp.)薊馬親緣關係較疏遠外,其餘花薊馬屬薊馬與西方花薊馬的親緣演化樹仍歸為同群。選取ITS1及ITS2 rDNA區域,設計出對西方花薊馬具專一性引子對(FO1/FO2),配合Moritz等人(2000)發表之一般引子對(CS249/ CS250),進行單管巢式聚合酵素鏈鎖反應(one- tube nested PCR),最終得到約900 bp之PCR產物;而單管巢式PCR靈敏度可達1pg程度。增幅放大的目標基因產物,再輔以限制酵素(NaeI/ SacI/ HinfI)切割製作限制酵素圖譜(restriction fragment length polymorphism, RFLP),可更準確鑑定西方花薊馬。本研究顯示,此次開發單管巢式聚合酵素鏈鎖反應-限制酵素圖譜之分子檢測方法,具有高專一性、高靈敏度及高準確性的特性,且操作簡便、快速,操作工具易於取得,可有效率地鑑定西方花薊馬。 |
| 英文摘要 | The western flower thrips, Frankliniella occidentalis (Pergande), is a major worldwide pest of agriculture and horticulture and a quarantine pest in Taiwan. To improve the identification technique for this pest, a species-specific, 1-tube nested PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) detection method was developed. The 28S rDNA and ITS2 region of F. occidentalis and the rDNA sequences of other thrips in the GenBank were analyzed using the Phylip program to reveal the phylogenetic relationship between F. occidentalis and other described thrips species. Thrips belonging to the genus Frankliniella , i.e., F. occidentalis (Pergande), F. intonsa (Trybom), and F. tenuicornis Uzel', are more closely related to F. schultzei (Trybom). The ITS1 and ITS2 sequences of F. occidentalis and its related species were aligned and compared, and species-specific PCR primers for F. occidentalis , FO1 and FO2, were designed. The identification method of F. occidentalis consisted of amplification of the rDNA region by a common primer pair CS249/CS250, followed by a second PCR with a species-specific primer pair FO1/FO2 for F. occidentalis . By using the common primer pair, an amplicon of approximately 1400 bp was yielded from F. occidentalis . Using a species-specific primer pair, a DNA fragment of approximately 900 bp was amplified. The sensitivity of F. occidentalis to the 1-tube nested PCR was found to be 1 pg of DNA in the reaction. These results suggest that the method is rapid, simple, and available as a tool for identification of the quarantine pest, F. occidentalis. |
本系統中英文摘要資訊取自各篇刊載內容。