應用單一步驟巢式複合式聚合酶鏈鎖反應同步鑑定刺足根蟎、羅賓根蟎及長毛根蟎

"The application of single-step nested multiplex polymerase chain reaction for the identification of Rhizoglyphus echinopus, R. robini and R. setosus simultaneously"

刺足根蟎（Rhizoglyphus echinopus（Fumouze＆Robin））、羅賓根蟎（R. robiniClaparede）及長毛根蟎（R. setosus Manson）為世界性重要害蟎，本研究開發單一步驟巢式複合式聚合酶鏈鎖反應（Single-step nested multiplex PCR）技術以同步鑑定此三種根蟎。首先利用一般性引子，增幅放大並解序其核糖體DNA18S-28S片段，根據根蟎種間ITS1片段（the first internal transcribed spacer，ITS1）核酸序列之差異，設計具種專一性引子，以增幅放大此三種根蟎之專一性DNA片段，其片段長度為533、312、404bp，可清楚地於電泳圖上，直接區分此三種根蟎。同時放入據18S及5.8S轉錄區序列設計的一般性引子，可提升此單一步驟巢式複合式聚合酶鏈鎖反應方法的靈敏度至pg程度。增幅放大的專一性DNA片段，若再輔以限制酶（Taq I/ Mfe I/ Ssp I/ Sty I）可更準確鑑定此三種根蟎。

"A specific single-step nested polymerase chain reaction (PCR) assay was developed to identify the three species of worldwide economically important bulb mites: Rhizoglyphus echinopus, R. robini and R. setosus simultaneously. General primers were used to amplify the 18S-28S region of ribosomal DNA by PCR. Sequences of the ITS1 region of the PCR products were used to construct species-specific primers which were then used to amplify the species specific DNA fragments. These fragments which can be visualized readily after electrophoresis were 522, 312 and 404 bp for R. echinopus, R. robini and R. setosus, respectively. The plus strand universal primers derived from the sequence in the 18S and 5.8S coding region increase the sensitivity of this single-step nested multiplex PCR assay to pg degree. Treatment of these species-specific DNA fragment with Taq I, Mfe I, Sty I and Ssp I further increase the accuracy in differencing these three species."