- 期刊
利用核糖體內轉錄區間序列及聚合酵素連鎖反應檢測作物根瘤線蟲
PCR-mediated Detection of Meloidogyne spp. Based on Ribosomal Internal Transcribed Spacer Sequences
摘要
本研究主要目的為比對分析台灣地區作物根瘤線蟲的核糖體DNA序列，並藉以設計對根瘤線蟲具有專一及靈敏特性之引子對，配合聚合酵素連鎖反應(PCR)增幅技術，研發作物根瘤線蟲之分子檢測系統。利用過去相關研究中所得之植物病原線蟲通用性引子，自南方根瘤線蟲、爪哇根瘤線蟲、花生根瘤線蟲以及水稻根瘤線蟲DNA增幅含內轉錄區間及5.8S之核糖體DNA序列。經選殖解序後發現南方、爪哇及花生等根瘤線蟲彼此間之序列相同度可達99%，而與水稻根瘤線蟲間之相同度則僅有85%。經以得自南方根瘤線蟲DNA序列設計檢測用PCR引子對Mi148/Mi452，利用此引子對可自根瘤線蟲DNA獲得一約342bp的專一性增幅產物，而測試其它屬之植物病原線蟲或腐生性線蟲則無類似之PCR產物。利用此引子對可以由病株及土壤中順利偵測到根瘤線蟲之存在。此外，經測試發現此引子對敏感度可達10pg，並可在僅有5隻二齡幼蟲存在的土壤中檢測出根瘤線蟲，顯示此專一性引子對可實際應用於田間作物根瘤線蟲檢測之所需。
並列摘要
The main objective of the study is to design a pair of specific primers for the detection of Meloidogyne spp. by PCR. The internal transcribed spacer (ITS) region of the ribosomal DNA from the representative isolates of M. incognita, M. javanica, M. arenaria, and M. graminicola were amplified by universal primers. The entire ITS region, including 5.8S subunit, was cloned and sequenced. The aligned results of obtained sequences showed that a high level of sequence identity was found among M. incognita, M. javanica, and M. arenaria. Two PCR primers, Mil48 and Mi452, were designed based on rDNA sequence of M. incognita. The primer pair was subsequently shown to amplify a 342 bp fragment from the DNA of Meloidogyne spp. On the contrary, this primer pair failed to amplify any fragments from the DNA from other tested nematodes. Using this primer pair, Meloidogyne spp. could be successfully detected from diseased plants and soil. The detection limit of the primer pair was as few as 10 pg template DNA, or 5 juveniles in soil.
延伸閱讀
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