臺灣博碩士論文加值系統

寄生蟲是水產養殖常見病原，目前業界一般都是魚隻有異狀或死亡時才進行魚體鏡檢，確定有寄生蟲後再投藥治療，但已造成經濟損失。在傳統鏡檢方法中需要對魚隻進行解剖，也需要專業人員進行寄生蟲型態鑑定，且可能因為在卵或幼蟲各階段型態改變無法明確判斷為何種寄生蟲。本論文開發高靈敏性、高特異性、定量檢測之TaqMan Real-time PCR方法來檢測養殖水體中指環蟲屬(Dactylogyrus spp.)或車輪蟲屬(Trichodina spp.) 18S rRNA基因之DNA，其檢測靈敏性：指環蟲可達5個標的基因/反應，車輪蟲可達2個標的基因/反應；特異性佳，對養殖魚類常見病原菌：鏈球菌、奴卡氏菌、螢光假單胞菌皆無交叉反應，且不會檢測到非指環蟲屬或非車輪蟲屬18S rRNA基因；定量範圍為7個十級數，R² = 1；另外我們在養殖水體中檢測到寄生蟲DNA時，後續在魚體的鰓也能鏡檢到寄生蟲。指環蟲與車輪蟲TaqMan Real-time PCR方法可在魚體有寄生蟲症狀前就能從養殖水體中檢測到寄生蟲eDNA，並即時投藥，以達到預防的效果。養殖水體寄生蟲eDNA檢驗，是種非侵入及預警式檢測方法，且DNA不會隨著寄生蟲生活史發育階段的型態而改變，較傳統鏡檢不易有偽陰性結果，適用於水產養殖之寄生蟲風險管理。

Parasites are common pathogens in aquaculture. At present, in the industry, fish body microscopy is usually performed only when it is abnormal or dead, and it is determined that parasites are present before treatment, but it has caused economic losses. In traditional microscopy methods, fish need to be dissected and performed parasite type identification required by profession further, and it may be impossible to determine which parasite is due to the type at each stage of the egg or larva. This study aimed to develop a high-sensitivity, specificity, and quantitative analytical method by using TaqMan Real-time PCR to detect Dactylogyrus spp. or Trichodina spp. 18S rRNA in aquaculture water. High detection sensitivity: Dactylogyrus can be as low as 5 target genes/reaction, and Trichodina can be as low as 2 target genes/ reaction, with high specificity, no interaction to common pathogens in cultured fish : Lactococcus garvieae, Nocardia seriolae and Pseudomonas fluorescens. In addition, no detection non-Dactylogyrus spp or non-Trichodina spp. 18S rRNA. The quantitative ranges of 7 ten levels, R² = 1. In addition, we also found the Parasites from the fish's gills by microscopy when the Parasites DNA was detected in the culture water. The TaqMan Real-time PCR assays would be able to detect Parasites eDNA from culture water to achieve the preventive effect through drug administration before the fish has symptoms. eDNA detecting of parasites in aquaculture water is a non-invasive and early warning detection method. Otherwise, the pseudo-negative results of TaqMan Real-time PCR assays are less than traditional microscopy because Parasite DNA does not change as the development of the life cycle, which is applicable for aquaculture risk management.

中文摘要--------------------------------------------------------------------------------- i
英文摘要--------------------------------------------------------------------------------- ii
致謝--------------------------------------------------------------------------------------- iii
目錄--------------------------------------------------------------------------------------- iv
表目錄------------------------------------------------------------------------------------ ix
圖目錄------------------------------------------------------------------------------------ xi
附錄目錄--------------------------------------------------------------------------------- xiv
前言--------------------------------------------------------------------------------------- 1
一、魚類寄生蟲檢查方法------------------------------------------------------------ 1
二、環境DNA(environmental DNA, eDNA)檢驗現況------------------------- 1
三、指環蟲與車輪蟲------------------------------------------------------------------ 4
四、即時聚合酶鏈鎖反應(Real-time PCR) ------------------------------------- 11
五、研究目的--------------------------------------------------------------------------- 12
研究架構--------------------------------------------------------------------------------- 14
材料與方法------------------------------------------------------------------------------ 15
一、實驗材料--------------------------------------------------------------------------- 15
二、培養基及緩衝液配製------------------------------------------------------------ 18
三、實驗儀器設備--------------------------------------------------------------------- 19
四、研究方法--------------------------------------------------------------------------- 20
(一) 指環蟲與車輪蟲TaqMan Real-time PCR方法之開發-------------------- 20
1. 指環蟲試劑系統--------------------------------------------------------------------- 20
(1) 引子對設計及評估----------------------------------------------------------------- 20
(2) 標準品DNA建立-------------------------------------------------------------------- 20
2. 車輪蟲試劑系統--------------------------------------------------------------------- 23
(1) 引子對設計及評估----------------------------------------------------------------- 23
(2) 標準品DNA建立-------------------------------------------------------------------- 23
3. DNA萃取方法------------------------------------------------------------------------ 24
(1) 水體樣品前處理-------------------------------------------------------------------- 24
(2) 水體沉澱物DNA萃取方法-------------------------------------------------------- 24
(3) 魚鰓樣品前處理-------------------------------------------------------------------- 26
(4) 魚鰓洗出之沉澱物DNA萃取方法----------------------------------------------- 27
(二) 指環蟲與車輪蟲TaqMan Real-time PCR方法之確效-------------------- 28
1. 指環蟲PCR反應條件--------------------------------------------------------------- 28
2. 車輪蟲PCR反應條件--------------------------------------------------------------- 28
(三) 田間試驗---------------------------------------------------------------------------- 29
1. 樣品採集------------------------------------------------------------------------------- 29
研究結果---------------------------------------------------------------------------------- 30
一、指環蟲TaqMan Real-time PCR方法之開發-------------------------------- 30
(一) 試劑系統---------------------------------------------------------------------------- 30
1. 引子對與探針設計及評估--------------------------------------------------------- 30
2. 標準品DNA建立--------------------------------------------------------------------- 30
(二) 水體沉澱物DNA萃取方法評估------------------------------------------------ 30
二、指環蟲TaqMan Real-time PCR方法之確效-------------------------------- 31
(一) 樣品結果判讀---------------------------------------------------------------------- 31
(二) 定量線性區間---------------------------------------------------------------------- 32
(三) 靈敏性------------------------------------------------------------------------------- 32
(四) 重複性------------------------------------------------------------------------------- 33
(五) 特異性------------------------------------------------------------------------------- 33
1. 指環蟲TaqMan Real-time PCR特異性測試----------------------------------- 33
2. 陽性養殖池水體樣品特異性分析------------------------------------------------- 34
三、指環蟲田間試驗------------------------------------------------------------------- 34
(一) 指環蟲eDNA陽性養殖池之魚隻指環蟲的檢驗----------------------------- 34
(二) 單一指環蟲18S rRNA基因數估算--------------------------------------------- 35
(三) 病魚指環蟲鰓鏡檢與TaqMan Real-time PCR檢測之比較--------------- 36
四、車輪蟲TaqMan Real-time PCR方法之開發--------------------------------- 36
(一) 試劑系統----------------------------------------------------------------------------- 36
1. 引子對與探針設計及評估---------------------------------------------------------- 36
2. 標準品DNA建立--------------------------------------------------------------------- 39
(二) 水體沉澱物DNA萃取方法評估------------------------------------------------- 39
五、車輪蟲TaqMan Real-time PCR方法之確效-------------------------------- 40
(一) 樣品結果判讀---------------------------------------------------------------------- 40
(二) 定量線性區間---------------------------------------------------------------------- 40
(三) 靈敏性------------------------------------------------------------------------------- 41
(四) 重複性------------------------------------------------------------------------------- 41
(五) 特異性------------------------------------------------------------------------------- 42
1. 車輪蟲TaqMan Real-time PCR特異性測試----------------------------------- 42
2. 陽性養殖池水體樣品特異性分析------------------------------------------------- 43
六、車輪蟲田間試驗------------------------------------------------------------------- 43
(一) 車輪蟲eDNA陽性養殖池之魚隻車輪蟲的檢驗----------------------------- 43
(二) 單一車輪蟲18S rRNA基因數估算-------------------------------------------- 44
(三) 病魚車輪蟲之鰓鏡檢與TaqMan Real-time PCR檢測比較-------------- 45
七、田間應用---------------------------------------------------------------------------- 45
(一) 養殖池寄生蟲eDNA盛行率調查----------------------------------------------- 45
(二) 水產養殖之寄生蟲風險管理---------------------------------------------------- 47
討論--------------------------------------------------------------------------------------- 55
一、水體沉澱物DNA萃取方法------------------------------------------------------ 55
二、指環蟲TaqMan Real-time PCR方法----------------------------------------- 56
三、車輪蟲TaqMan Real-time PCR方法----------------------------------------- 59
四、指環蟲及車輪蟲TaqMan Real-time PCR田間應用----------------------- 63
結論---------------------------------------------------------------------------------------- 65
參考文獻---------------------------------------------------------------------------------- 67
表------------------------------------------------------------------------------------------- 73
圖------------------------------------------------------------------------------------------- 98
附錄---------------------------------------------------------------------------------------- 121

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