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頁籤選單縮合
題 名 | Development of a Novel Species-Specific DNA Marker for Rapid Detection of the Entomopathogenic Fungus, Nomuraea rileyi, in Infected Insects=快速偵測受感染蟲體內綠殭菌之專一性脫氧核糖核酸標記之開發 |
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作 者 | 曾羽凱; 呂家芸; 吳孟學; 侯豐男; | 書刊名 | 臺灣昆蟲 |
卷 期 | 30:3 2010.09[民99.09] |
頁 次 | 頁179-191 |
分類號 | 433.3 |
關鍵詞 | 蟲生真菌; 綠殭菌; 機增幅多型性技術; 特徵性序列增幅區域; Entomopathogenic fungi; Nomuraea rileyi; RADP-PCR; SCAR; |
語 文 | 英文(English) |
中文摘要 | 藉由隨機增幅多型性技術 (RAPD) 分析而產生的特徵性序列增幅區域 (SCAR),從中發展具種專一性之脫氧核糖核酸標記 (DNA marker),以偵測蟲生真 菌綠殭菌 (Nomuraea rileyi)。在台灣地區親緣關係近似的綠殭菌分離株中,增幅出 一條共同的 1.4 kb 長度的 DNA 序列,但在白殭菌 (Beauveria bassiana) 與黑殭 菌 (Metarhizium anisopliae) 並無此一增幅片段。此 DNA 片段序列被應用於設計 一組長度在 20 個核苷酸的脫氧核糖核酸引子對,即 NS1/NS2,可增幅出一條長度 在 284 個核甘酸的 DNA 片段。利用 NS1/NS2 引子對可成功的在本地的綠殭菌 17 株分離株及 2 株美國的綠殭菌品系中增幅出預期長度的 DNA 片段,但在白殭菌與 黑殭菌品系則無法增福出任何片段。在靈敏度與干擾試驗,僅 0.1 ng 的 DNA 板模 即可增幅出預期長度的 DNA 片段,而且不受宿主昆蟲斜紋夜蛾 (Spodoptera litura) 的 DNA 干擾,能維持相同高靈敏度。同時也可在受綠殭菌感染的斜紋夜蛾之活幼蟲 及殭蟲中,增幅出 284 個核苷酸長度的 DNA 片段,但受白殭菌及黑殭菌感染的殭 蟲則無法增幅出此一 DNA 片段。另外,本結果亦顯示受綠殭菌感染一天後的幼蟲也 能利用此 NS1/NS2 引子偵測出蟲體有綠殭菌存在。因此,本方法具有快速偵測蟲體 受綠殭菌感染之潛力,同時亦可應用於調查綠殭菌在田間之分佈。 |
英文摘要 | A species-specific DNA marker for detection of the entomopathogenic fungus, Nomuraea rileyi, was developed from sequence-characterized amplified regions (SCARs) derived from a random amplification of polymorphic DNA (RAPD) analysis. A common 1.4 kb DNA fragment was amplified in closely related N. rileyi isolates from Taiwan but not in Metarhizium anisopliae or Beauveria bassiana. This fragment was used for designing a pair of 20-mer oligonucleotide primers which were amplified to be a single band of the 284 bp DNA fragment. The predicted size of the DNA fragment was also amplified from 17 domestic N. rileyi isolates and from two other isolates from the United States using a NS1/NS2 primers pair, but they were not from B. bassiana or M. anisopliae. In the sensitivity and interference assays, a predicted amplicon DNA could be detected using a DNA template as low as 0.1 ng, without disturbing it with DNA from the host insect, Spodoptera litura. A 284 bp amplicon DNA was detected from live and mummified S. litura larvae infected with N. rileyi isolates but was not detected with B. bassiana and M. anisopliae isolates using this specific primers pair. These results indicate that detection of N. rileyi in infected larvae is possible one day after inoculation using a NS1/NS2 primer pair. Therefore, this method has the potential for rapidly detecting N. rileyi in infected insects, and is also useful for surveying the distribution of this fungus in the field. |
本系統中英文摘要資訊取自各篇刊載內容。