Identifying Plant Parasitic Nematodes from Imported Plant Products Using Pyrosequencing Approach

我國法規明訂數種植物寄生性線蟲為檢疫有害生物，惟輸入植物產品中檢出的植物寄生性線蟲，通常數量有限或僅包含無法明確鑑定種類的幼蟲個體，導致不易判斷種類並可能造成輸入業者相關損失。本研究除利用外部形態將線蟲成蟲個體鑑定至「屬」級分類地位外，並發展以焦磷酸定序法 (Pyrosequencing) 快速鑑定多種植物寄生性線蟲，提升物種鑑定之效率及正確性。焦磷酸定序法增幅片段不需螢光標記，加上序列判讀長度僅約 300 bp，可應用於快速定序。本試驗利用通用引子對 rDNA2-M/rDNA1.58S 對數種線蟲樣本可增幅出長 500~700 bp 之部分 18S、ITS1 以及 5.8S 之 rDNA 片段，惟長度大於預期，因此修正後改採用 Aph、Dit 及 Xip 作為反置引子，與前置引子 rDNA2-M 共同作為焦磷酸定序專用引子對，分別對葉芽線蟲屬、莖線蟲屬及劍線蟲屬線蟲樣本進行增幅，可成功增幅得到長 200~300 bp 之部分 18S rDNA 片段，將該片段與 NCBI GenBank 進行比對，收集檢疫有害線蟲及其他種類的相似序列片段，建立序列資料庫；另也持續收集線蟲樣本經焦磷酸定序所得之序列建構序列比對資料庫。本試驗線蟲樣本經焦磷酸定序結果可信度偏低，未來若於定序過程中改依推測的可能序列依次加入 dNTPs，或許可解決問題。利用焦磷酸定序法可建立快速鑑定的平台，且所建立的序列資料庫均可無限擴充，是一適合用於快速鑑定檢疫有害生物的技術。Several plant parasitic nematodes are quarantine pests listed in the ‘Quarantine Requirements for The Importation of Plants or Plant Products into The Republic of China’. The plant parasitic nematodes intercepted in imported plants or plant products are usually only with limited number, and are short of identifiable characteristics resulting from non-female individuals or larval stage. With such difficulty in identification of potential nematode pests, additional cost may apply to consignee as the responsible agency may have to implement quarantine treatment. In this study, nematode samples are classified into genus level based on morphological characters, and furthermore into species level via pyrosequencing. Pyrosequencing method does not require fluorescent DNA sequence analyses, and sequence products with 200-300 bp can be readily read for data analysis, which enables rapid quantification of sequence variations, thus successful identification. In this study, universal primers rDNA2-M/rDNA1.58S were used to amplify partial 18S, ITS1 and partial 5.8S rDNA fragments (500-700 bp) of various nematodes, but amplicons are too long to be utilized for pyrosequencing. Therefore, three modified primers including Aph, Dit, and Xip, coupling with rDNA2-M, are used as pyrosequencing specific primers for Aphelenchoides spp., Ditylenchus spp., and Xiphinema spp., respectively, and approximately 200-300 bp partial 18S rDNA fragments were successfully amplified. These sequences are blasted on NCBI GenBank to obtain similar aligned sequences of quarantine nematode pests and other potential organisms, and these data were integrated as database. The sequences obtained from various nematode samples via pyrosequencing were also used to construct another database. Results of sequence alignment indicate that the resolution of pyrosequencing in plant parasitic nematodes seems limited. In near future, the resolution can be elevated by adding dNTPs according to predicted sequences during pyrosequencing, instead of adding in a random fashion. This study demonstrates the utility of pyrosequening technique especially for establishment of a rapid identification platform of quarantine purpose. Also, the expandable nature of the database allows pyrosequening technology even more suitable for quarantine pest identification and database development