鑑別Erwinia carotovora subsp. carotovora及Erwinia chrysanthemi細菌性軟腐病菌專一性引子之閉發

Abstract

利用隨機增幅多型性核酸技術(RAPD)增幅彩色海竽細菌性軟腐病菌菌株Erwinia carotovora subsp. carotovora (Ecc)之基因體DNA，篩選出一11,200 bp具專一性之片段Y20，該片段經選劉安進行轉型質體pY20Ecc-1200 bp核酸序列分析，再依據其序列與NCBI核酸資料庫比對得該序列與E. chrysanthemi (Ech)之rhi N基因有83％相似處，並根據相同序列處設計出專一性引子對Ec3F / Ec4R，可用於區分Ecc及Ech。應用此專一性引子對進行聚合酵素連鎖反應，對於留期式225株Ecc菌株均可增幅出497 bp之片段，供試85株Ech菌株均可增幅出548 bp之片段，但對供試之其他非標的菌株皆無法增幅出任何片段。以該引子對偵測彩色海芋細菌性軟腳肉菌Ecc及蝴蝶蘭軟腐病菌Ech之DNA時，靈敏度同樣為10-50 pg，且與6屬7種其他非標的菌株DNA混合時並不影響其偵測結果。以Ec3F/Ec4R偵測Ecc及Ech菌液之靈敏度時，最低分別可測到1.2 10 1及1.1 10 1個細菌數，且與其他2屬3種其他非標的細菌之菌液混合時並不會影響其偵測結果。以Ec3F / Ec4R建立對於Ecc及Ech之單一菌落決速檢定法，確實可偵測並區分Ecc及Ech菌株，且可應用於田間軟腐病株之偵測，因此，確認本試驗所設計之引子對Ec3F / Ec4R能快速鑑定及偵測軟腐病菌，並能區分Ecc及Ech。 A specific PCR (polymerase chain reaction) primer pair has been developed using RAPD (random amplified polymorphic DNA) to differentially detect Erwinia carotovora subsp. carotovora (Ecc) and E. chrysanthemi (Ech). A specific 1,200 bp DNA fragment was amplified and cloned from Ecc DNA using OPY20 primer, one of the sixty random primers used in RAPD. Nucleotide sequence of this 1,200 bp DNA showed 83% similarity to Ech rhiN gene, where conserved sequences were displayed in two regions. But in Ech rhiN sequence, there are additional 49 bp nucleotides inserted between these two conserved regions. Herein, a set of primers Ec3F / Ec4R was designed and applied to differentially detect Ecc and Ech by PCR. Using bacterial chromosomal DNA as templates, a distinct 497 bp fragment can be amplified from 225 Ecc strains, and a 548 bp fragment was amplified from 85 Ech strains. No fragment can be amplified from the other seven bacterial species in six genera. Sensitivity of PCR for detecting Ecc and Ech was between 10 50 pg for purified DNA and 1.1 101 cfu and 1.2 101 cfu for cultured cells, respectively. It took 3-4 hr to detect Ecc and Ech by colony PCR using Ec3F / Ec4R primer set and the target bacteria in infected tissues of cala lily and phalaenopsis. Results presented here suggest that the primer pair Ec3F / Ec4R applied in PCR can be very useful in rapid identification, detection, and differentiation of the soft rot pathogens Ecc and Ech.