點帶石斑魚多病原即時定量檢測系統開發及應用

在過去二十年裡，世界水產養殖業迅速發展，產量及生產面積也相對地在快速擴大中，然而，在人類大量密集式養殖下，導致在養殖過程中迅速地爆發了由病毒所引起的疾病。在臺灣衆多養殖魚類中，石斑魚為其中一種備受養殖業者親睞的魚種，因其具有較高的經濟價值；不幸地，石斑魚也因為在長期密集式的養殖下，通常在養殖期達三到五年時，便開始容易遭到數種病原感染，導致養殖業者的大量虧損，因此，本實驗室以研究並開發可檢測多種病原體的技術為首要防疫方針。研究中所注重的病原體包括神經壞死病病毒 (NNV)、彩虹病毒 (iridovirus) 及弧菌等易感染石斑魚的病原，導致魚隻的高死亡率和巨大的經濟損失，為減少此危害必須儘快發展堪用的病原偵測系統，這三類病原體能引起石斑魚的免疫系統反應，並在短時間引發大量的Mx蛋白表現以對抗病原體，因此我們希望透過檢測石斑魚體內的免疫系統所產生的Mx基因表現量，以及病原體的數量，建立即時性多重檢測系統 (multiplex real-time PCR)，以快速偵查病原感染情形及魚體健康狀況。從實驗結果，我們成功設計不同引子和探針以同時偵測病原及魚體內Mx抗病毒蛋白表現，此外也建立了即時性多重檢測系統的最佳反應引子濃度、溫度及PCR循環數。最後，我們也成功從室外養殖場偵測到17病魚體內含有NNV病毒及魚體Mx抗病毒蛋白有明顯提高。

關鍵字

時性多重檢測系統； Mx抗病毒蛋白；神經壞死病病毒；彩虹病毒；及弧菌

並列摘要

In this world, aquaculture has been developed quickly during last two decades and contributed greatly to the nation economic. However, the massive culturing aquatic products have led to sudden infection outbreak. In Taiwan, grouper (Epinephelus coioides) fish is one of the most popular species for consumption because of its higher commercial value than other fishes. Unfortunately, it has a high disease occurrence rate, which disease usually developed after the starting of farming 3 to 5 year. So in our laboratory, we are developing a technique to detect infectious pathogen at the earlier infection stage. The main pathogens that infect the grouper are including nervous necrosis virus (NNV), iridovirus and Vibrio anguillarum. They can cause a lot of serious diseases for grouper which would lead to high mortality rate, create huge losses to grouper aquaculture industry as well as affecting the nation economy. Therefore, in order to decrease the mortality rate in grouper caused by these pathogens, we need to develop a technique that able to detect pathogens infection in grouper as quick as possible. Thus, we had developed multiplex real-time polymerase chain reaction (multiplex real-time PCR) technique to detect multiple pathogens that infect grouper and detect changes levels of Mx (myxovirus resistance) protein release by the immune system concurrently. In our results, we had successfully designed primers and probes to detect multiple pathogens (NNV, iridovirus and Vibrio anguillarum) and Mx gene concurrently. Next, we also optimized the condition for multiplex real-time PCR by manipulate the concentration of primers and probe, total cycles, time and temperature. Optimization of PCR condition allows the detection and diagnosis of multiple pathogens in grouper can be done in high sensitivity and specificity. Lastly, we had successfully to detect NNV in 17 grouper samples that infected by NNV and also detected the increase of Mx protein concentration in these samples.

並列關鍵字

Multiplex real-time PCR ； Mx protein ； Epinephelus coioides ； Nervous necrosis virus (NNV) ； Iridovirus ； Vibrio anguillarum

參考文獻

Fatima O, Christian L, Deborah G, and Adib R. 2007. Real-time RT-PCR (TaqMan) assays for the detection of Grapevine Leafroll associated viruses 1-5 and 9. J Virol Methods 141:22-29.

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