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題 名 | PCR for Direct Detection of Edwardsiella Tarda from Infected Fish and Environmental Water by Application of the Hemolysin Gene=利用聚合酶連鎖反應法直接檢測愛德華氏菌溶血基因應用於愛德華氏菌感染病魚及養殖池水之迅速診斷 |
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作 者 | 陳昭德; 賴碩彥; | 書刊名 | 動物研究學刊 |
卷 期 | 37:3 1998.07[民87.07] |
頁 次 | 頁169-176 |
分類號 | 387.102 |
關鍵詞 | 愛德華氏菌症; 聚合酶連鎖反應法直接檢測; Edwardsiellosis; PCR diagnosis; |
語 文 | 英文(English) |
中文摘要 | 愛德華氏菌的溶血基因核��酸序列,依其上轉譯之遺傳密碼,可解讀出三個開放 讀碼區,其中第三開放讀碼區已經由 SDS-PAGE 証實帶有 34 kDa 的溶血基因。當以帶有該 菌的溶血基因 Hindlll 片段用作探針, 與臺灣近十年來由養殖池感染病魚所採集到的 39 株愛德華氏病原菌作核��酸雜交時,不論菌株是否釋出溶血外毒素至菌體外,或產出溶血內 毒素保存於菌體內,皆可被愛德華氏菌溶血基因探針雜交檢測到。又以第二開放讀碼區的 5' 端及第三開放讀碼區的 3' 端,設計一對引子,以愛德華氏菌體直接加熱釋出核��酸作 模板,利用聚合�t連鎖反應法,可很敏銳的於電泳分離過的洋菜膠上觀察到形成預測的 1109 bp PCR 片段。 因此更進一步嘗試以聚合�t連鎖反應法快速診斷愛德華氏病原菌存在 於感染病魚體否時,於模擬愛德華氏病原菌注射感染吳郭魚試驗中,很快速的利用此法可檢 測到愛德華氏菌進入感染魚的肝腎腸及血液中,顯示利用溶血基因普遍存在於本土分離愛德 華氏菌之特性,以本實驗設計的引子用於快速診斷愛德華氏菌感染魚確屬快速可行的良好方 法之一。 |
英文摘要 | A DNA fragment associated with hemolysin production in the Edwardsiella tarda strain ET16 was labeled with nonradioactive DIG and used to probe chromosomal DNA from 40 E. tarda strains. The resulting hybridization patterns were classified into 2 groups, matching the group of strains which secreted or didn't secrete hemolysin, except for anomalous patterns produced by strain ET83 and reference strain ATCC 15947. Those strains yielding a 5.3-kb hybridization band, which corresponds to the original hindlll cloned fragment from the chromosome of ET16, were assigned to group Ⅰ, while the presence of other hybridized Hindlll bands from strains producing endo-hemolysin were characteristic of group Ⅱ. When 2 oligomers selected from the beginning region of ORFⅡ and the end region of ORF Ⅲ were used as primers for PCR assay, a 1109-bp PCR product was generated by all 40 examined E. tarda strains. Thus, the hemolysin gene sequence spanning the region of ORF Ⅱ and ORF Ⅲ was concluded to be conservative. Live bacterial cells from the visceral organs and blood of infected fish, as well as bacteria in environmental water were detected by a direct PCR assay which yielded the 1109-bp fragment. |
本系統中英文摘要資訊取自各篇刊載內容。