Use of Phylogenetically Based Hybridization Probes for Rapid Discrimination of Aeromonas hydrophila and Edwardsiella tarda from Aquaculture Environment

應用類緣依據的雜合性探針快速由水產養殖池檢出嗜水性產氣單胞菌及愛德華氏菌

陳昭德(Jau-Der Chen) ； 陳孝昭(Hsiao-Chao Chen) ； 蔡耀欣(Yao-Shin Tsai) ； 黃聖慈(Sheng-Cih Huang)

《臺灣水產學會刊》 33卷2期 (2006/06) Pp. 147-155

https://doi.org/10.29822/JFST.200606.0006

摘要

小次單位核醣體已被認可用於細菌鑑定分類至屬甚或種的層次，依據已完整整理的核醣核酸資料庫的16S rRNA序列，新建構的探針ET996雜合愛德華氏菌的16S rRNA序列，落點位於第37~p37-1螺旋位置，相當於大腸桿菌996到1015的序列。探針AH460和AH642雜合嗜水性產氣單胞菌的16S rRNA序列，落點分別位於第18~18'及p23-1'~24螺旋位置，相當於大腸桿菌460到479的序列及642到661的序列。最適當的雜合溫度依據參考菌做測試，範圍落在42℃到46℃之間。並以94株水產養殖池分離出的環境微生物的小次單位核醣體，做為探針的標的，驗證探針雜合的正確性。嗜水性產氣單胞菌及愛德華氏菌，如預期般的被探針雜合到，相對的，養殖池水環境微生物諸如益生菌、Achromobacter sp., Alcaligenes sp., Flavobacterium sp., Moraxella sp., Pasterurella haemolytica, Pseudomonasaeruginosa, V. alginolyticus, V. carchariae, V. harveyi, V. parahaemolyticus, V. tubiashii,及V. vulnificus等，皆被排除雜合的可能性，因此探針ET996、AH460及AH642相當有效用，可應用於從水產養殖池採集病魚檢體，直接雜合檢定病原菌。

關鍵字

嗜水性產氣單胞菌專－性探針 ； 愛德華氏菌專－性探針 ； 類緣依據的雜合性探針 ； 小次單位核醣體探針

並列摘要

Small subunit RNA has been acknowledged that it could be used in identification of bacteria lineage to genus or species. Based on bacterial 16S rRNA sequences that well documented in RNA database, probe ET996 which targeting positions 996 through 1015 (in E. coli numbering) which forming part of helix 37~p37-1 to hybridize E. tarda was established. In addition, probe AH460 and AH642 crossing positions 460-479 and 642-661 which forming part of helix 18~18' and helix p23-1'~24, respectively, also show promising potential for the development of species-specific probes to A. hydrophila. The optimum hybridization temperature ranging from 42℃ to 46℃ were determined by the use of reference strains as standard. Ninety-four environment strains isolated from aquaculture ponds were used as targets for accuracy testing. E. tarda and A. hydrophila were hybridized by the established probes as expected. In contrast, aquaculture environment strains such as probiotics, Achromobacter sp., Alcaligenes sp., Flavobacterium sp., Moraxella sp., Pasterurella haemolytica, Pseudomonas aeruginosa, V. alginolyticus, V. carchariae, V. harveyi, V. parahaemolyticus, V. tubiashii, and V. vulnificus all were excluded to be blotted by these probes. Thus, probes AH460, AH642 and ET996 are useful and applicable for bacteria identification of samples that directly isolated from diseased-fishes in aquaculture environment.

並列關鍵字

A. hydrophila specific probe ； E. tarda specific probe ； phylogenetic hybridization probe ； SSU probe