晚疫病菌PCR檢測方法之建立

Late blight caused by the oomyceteous pathogen Phytophthora infestans is a devastating disease of potato and tomato worldwide. The objective of the present study is to develop a method for rapid detection of P. infestans from potato tubers and tomato seeds. Three oligonucleotide primers (Pi1S-1, Pi2A-1, and Pi2A-2) were designed based on the internal transcribed spacer sequences of the fungus, which made two primer sets: Pi1S-1/Pi2A-1 and Pi1S-1/Pi2A-2. PCR analysis demonstrated that DNA fragments of predicted size, using either Pi1S-1/Pi2A-1 or Pi1S-1/Pi2A-2 as the primer set, were amplified from all the P. infestans isolates tested. Moreover, when the amount of template DNA was reduced to 1 pg, PCR primed with Pi1S- 1/Pi2A-2 could still give rise to expected DNA fragment. No signals were detected with DNA from other fungal species analyzed in the study. To detect P. infestans present in the potato tuber, a rapid method for DNA extraction followed by PCR with Pi1S-1/Pi2A-2 was established. This method will provide a useful tool for rapid detection of P. infestans in potatoes and tomatoes. 晚疫病菌Phytophthora infestans (Montagne) de Bary是引起馬鈴薯與蕃茄晚疫病的病原真菌，分佈遍及全世界，旦造成非常嚴重之危害。為了開發快速簡易之晚疫病菌檢測技術，以便應用於健康種薯檢測，我們根據疫病菌(Phytophthora spp.)核糖體核酸ITS序列之並列分析結果設計核酸引子(Pi1S-1, Pi2A-1與Pi2A-2)，並以Pi1S-1/Pi2A-1或Pi1S-1/Pi2A-2為引子對進行PCR。分析結果顯示，不論以Pi1S-1/Pi2A-1或Pi1S-1/Pi2A-2為引子對都可自所測試之晚疫病菌菌株增殖出預期大小之核酸片段，但自其他種類之供試菌株則觀察不到增殖產物，顯示其對晚疫病菌其有非常高之專一性。研究也顯示，將DNA減量綱1Pg時，以Pi1s-1/Pi2A-2為引子對仍可自晚疫病菌增頭出預期之核酸片段。因此，這一項技術應可成為進行晚疫病菌鑑定與檢測之有用工具。此外，我們還建立了自接種馬鈴薯快速抽取DNA，並以Pi1s-1/Pi2A-2為引子對進行晚疫病菌檢測之技術，可應用於輔助馬鈴薯健康種薯之檢測