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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

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1.4kb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0.1ng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。

並列摘要


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.

並列關鍵字

entomopathogenic fungi Nomuraea rileyi RADP-PCR SCAR

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