藉由PCR方法快速且具有專一性的診斷鰻魚重要疾病

• Main Authors: 張雅棻, Chang, Ya-Fen
• Other Authors: 指導教授：韓玉山, 臺灣大學：漁業科學研究所
• Format: Thesis
• Language: Chinese
• Published: 2014
• Subjects: 鰻鱺屬; 養殖疾病; PCR檢測; 專一性; 敏感性; Anguilla anguilla
• Online Access: http://ntur.lib.ntu.edu.tw/handle/246246/263809; http://ntur.lib.ntu.edu.tw/bitstream/246246/263809/1/ntu-103-R01b45005-1.pdf

鰻鱺屬的日本鰻以及歐洲鰻在東亞及歐洲地區都是非常重要的養殖產業 (Joh et al., 2013)；然而，自1970年起，這兩種鰻魚的資源量下降且其玻璃鰻價格不斷的上升(Han et al., 2010)，部分東亞地區的鰻魚養殖戶轉而養殖其他鰻魚種類。相較於研究透徹的日本鰻及歐洲鰻來說，其他種類的鰻魚養殖資訊則少了許多。更進一步的來說，在養殖產業上最怕遇到的問題就是疾病問題。因此，本篇研究的目的為藉由PCR發展快速且具有專一性的診斷方法。設計具有專一性且具有敏感性的primers之後，再經過in vitro的測試之後，將此方法實際運用到鰻病檢測上。在專一性測試方面，僅有在含有目標病原菌DNA的組別會出現預期產物；而在敏感性測試方面，其敏感度除了V. anguillarum的組別敏感度為10 ng/μl ，其他組別 (A. hydrophila, E. tarda, S. iniae, P. anguilliseptica, F. columnare, Y. ruckeri, and C. freundii)皆可偵測到1 ng/μl。在實際檢體檢測上，確實在帶有目標病原菌的檢體才會有預期產物的出現。因此，本篇研究所發展具有專一性且敏感性的檢測方法，確實可以被用來快速檢測這些感染了特定病原菌的病鰻。 The anguillid eels are the most important freshwater fish in the aquaculture industry in East Asia and Europe (Joh et al., 2013). Since 1970s, because of the high price of glass eel and decrease of stock size (Han et al., 2010), some eel farmers have tried to culture other eel species. Unlike Anguilla japonica and Anguilla anguilla, there are a few aquaculture information about other anguillid eel. Moreover, diseases, include bacterial and parasites infection, are a major cause of economic loss in the aquaculture industry. For this purpose, the aim of this study is to develop a rapid and specific detection of important diseases of the eel. After designed specific primer sets, the optimal PCR conditions were determined. In addition, sensitivities were estimated to be as low as 10~1 ng for the genomic samples. Moreover, the PCR method was used for identified diseased sample and showed the diagnosis of accuracy. In conclusion, the assay described in this study is a sensitive and specific method for simultaneous detection of eight pathogens in eels. Content 致謝 i 中文摘要 ii Abstract iii Introduction 11 Major Kinds of Bacterial Diseases of Eel 12 1) Red disease caused by Aeromonas hydrophila 12 2) Edwaedsiellosis caused by Edwardsiella tarda 12 3) Red spot disease caused by Pseudomonas anguilliseptica 13 4) Gill disease caused by Flavobacterium columnare (=Flexibacter/Cytophaga columnaris) 13 5) Vibriosis caused by Vibrio anguillarum 14 6) Streptococcosis caused by Streptococcus sp. 15 Other Potential Bacterial Pathogen 15 1) Hagerman redmouth disease caused by Yersinia ruckeri 15 2) The disease caused by Citrobacter freudii 16 Other diseases 16 1) Parasites 16 2) Saprolegniasis 16 Diagnosis of bacterial diseases 17 Materials and methods 19 Source of the bacteria strain and growth conditions 19 Genomic DNA extraction 19 Oligonulcleotide primers 20 Universal primer 20 Specific primer 20 Optimize PCR conditions 21 Specificity and Sensitivity analysis 22 Field diagnosis of diseased eel samples 22 Results 23 PCR optimal conditions 23 Determination of thermal optima for primer annealing. 23 Primer specificity 23 Fish tissue simulation 24 Sensitivity detection 24 Template combinations 24 Field diagnosis of diseased samples 25 1) Aeromonas hydrophila 25 2) Flavobacterium columnare 25 3) Edwardsiella tarda 26 4) Acinetobacter spp. 26 5) Moraxella spp. 27 6) Other diseases 27 Discussion 28 Comparison of PCR with biochemical methods 28 Risk of PCR method 29 Target gene selection 30 Sensitivity 32 Multiplex PCR 35 Conclusion 37 References 38