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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蕭仁傑(Jen-Chieh Shiao) | |
| dc.contributor.author | Shang-Jung Han | en |
| dc.contributor.author | 韓尚融 | zh_TW |
| dc.date.accessioned | 2021-05-12T09:36:30Z | - |
| dc.date.available | 2019-09-19 | |
| dc.date.available | 2021-05-12T09:36:30Z | - |
| dc.date.copyright | 2018-08-24 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-08-18 | |
| dc.identifier.citation | Backus, R.H., Springer, S. and Arnold Jr., E.L. (1956). A contribution to the natural history of the white-tip shark, Pterolamiops longimanus (Poey). Deep-Sea Research 3: 176–188
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Observations of pelagic fishes of the tropical Atlantic. Copeia 1954: 179–188. Mori, Y., Nagamine, K., Tomita, N., & Notomi, T. (2001). Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochemical and biophysical research communications, 289(1), 150-154. Nagamine, K., Hase, T., & Notomi, T. (2002). Accelerated reaction by loop-mediated isothermal amplification using loop primers. Molecular and cellular probes, 16(3), 223-229. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., & Hase, T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic acids research, 28(12), e63-e63. Oliver, S., Braccini, M., Newman, S. J., & Harvey, E. S. (2015). Global patterns in the bycatch of sharks and rays. Marine Policy, 54, 86-97. Paul, M., Baranzoni, G. M., Albonetti, S., & Brewster, J. D. (2015). Direct, quantitative detection of Listeria monocytogenes in fresh raw whole milk by qPCR. 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E., Heeger, P., Mynatt, R. L., Truett, A. A., Walker, J. A., & Warman, M. L. (2000). Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT). Biotechniques, 29(1), 52-54. Tsai, J. J., Liu, L. T., Lin, P. C., Tsai, C. Y., Chou, P. H., Tsai, Y. L., & Lee, P. Y. A. (2018). Validation of POCKIT™ dengue virus reagent set for rapid detection of dengue virus in human serum on a field-deployable PCR system. Journal of clinical microbiology, JCM-01865. Tsai, Y. L., Wang, H. C., Lo, C. F., Tang-Nelson, K., Lightner, D., Ou, B. R., & Teng, P. H. (2014). Validation of a commercial insulated isothermal PCR-based POCKIT test for rapid and easy detection of white spot syndrome virus infection in Litopenaeus vannamei. PLoS One, 9(3), e90545. Tsen, H. Y., Shih, C. M., Teng, P. H., Chen, H. Y., Lin, C. W., Chiou, C. S., & Chiang, Y. C. (2013). Detection of Salmonella in chicken meat by insulated isothermal PCR. Journal of food protection, 76(8), 1322-1329 Walsh, P. S., Metzger, D. A., & Higuchi, R. (1991). Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques, 10(4), 506-513. Ward, R. D., Holmes, B. H., White, W. T., & Last, P. R. (2008). DNA barcoding Australasian chondrichthyans: results and potential uses in conservation. Marine and Freshwater Research, 59(1), 57-71. Wathne F. 1959. Summary report of exploratory long-line fishing for tuna in Gulf of Mexico and Caribbean sea, 1954–1957. Commercial Fisheries Review 21: 1–26. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/handle/123456789/1329 | - |
| dc.description.abstract | 平滑白眼鮫(Carcharhinus falciformis)與污斑白眼鮫(C. longimanus)主要分布於全球熱帶海洋水域，因為遭受大量的捕撈，族群量已經明顯下降，目前已被漁業署分別列入三大洋區的禁捕物種。物種鑑定是漁業管理的必要條件，有鑑於鯊魚釣獲後經常在漁船上處理過魚身，以至於無法從外觀判定種類，使用生命條碼技術鑑定物種通常需要1至2週才能得知結果，可能緩不濟急，因此本研究針對平滑白眼鮫與污斑白眼鮫開發快速檢驗方法，設計物種專一性引子及探針，透過隔絕式恆溫聚合酶連鎖反應(iiPCR)以及恆溫式圈環形核酸增幅技術(LAMP)開發快篩方法，搭配快速萃取DNA套件，及手持式設備或乾浴槽，在1小時內完成檢測。共81個樣本於實驗室進行iiPCR檢測，真陽性率(目標物種判定為陽性結果)為71%、真陰性率(非目標物種判定為陰性結果)為100%。65個樣本使用凍乾試劑搭配DNA快速萃取套件於實驗室模擬或現場操作進行檢測，真陽性率為89%、真陰性率為93%。70個樣本於實驗室進行LAMP檢測，真陽性率為61%、真陰性率為100%。11個樣本於實驗室模擬現場操作條件搭配DNA快速萃取套件進行LAMP檢測，真陽性率為100%、真陰性率為50%。根據測試結果，本研究開發的iiPCR與LAMP兩項檢測皆具備於現場操作的條件，然而，不論是iiPCR或是LAMP檢測對非目標物種都會有偽陽性的結果出現，兩項檢測皆非常靈敏容易受汙染可能是造成此現象的原因之一，另外是紅肉丫髻鮫(Sphyrna lewini)在iiPCR檢測中因DNA序列與目標物種相似而有極高的機會出現偽陽性。儘管有些許偽陽性的狀況，但iiPCR及LAMP的檢測時間短以及操作簡單等特性很適合漁業管理者作為現場初步的快速篩檢工具，提升執法效率。 | zh_TW |
| dc.description.abstract | Identifing shark species at the landing site has been difficult when the sharks were dissected with key diagnostic features being removed. Molecular approaches such as DNA barcoding and qPCR methods can identify shark species but the experimental process is time consuming and have to be carried out in molecular labs. Therefore, more rapid and reay-to-use method is warranted in order to simplify the process of identifying certain shark species on-site. Insulated isothermal PCR (iiPCR) and loop-mediated isothermal amplification (LAMP) were used in this study to identify silky shark (Carcharhinus falciformis) and oceanic whitetip shark (C. longimanus) by designing species-specific TaqMan probes and primers. Shark DNA were simply extracted for the amplification of mitochondrial NADH dehydrogenase subunit 2 (ND2) gene within 30-60 min in either an iiPCR portable device (Micro POCKITTM) or a dry bath. 81 samples were tested by iiPCR assay in the laboratory with sensitivity (true positive rate) 71% and specificity (true negative rate) 100% , respectively while 65 samples were tested in the harbor using lyophilized reagent with sensitivity 89% and specificity 93%. 70 samples were tested by LAMP assay in the laboratory with sensitivity 61% and specificity 100%, while 11 samples were tested by LAMP assay using fast DNA extraction kit to stimulate operating in the harbor with sensitivity 61% and specificity 100%. According to the result, iiPCR and LAMP assay developed in this study are suitable to operate on-site. Although some misjudgment might because of two assays are too sensitive that little cross containmination will lead to false positive result, characteristics such as time-saving, portable, user friendly make iiPCR and LAMP assays useful tools for fishery manager to check shark species on-site. | en |
| dc.description.provenance | Made available in DSpace on 2021-05-12T09:36:30Z (GMT). No. of bitstreams: 1 ntu-107-R05b45015-1.pdf: 3458089 bytes, checksum: bf495c448341adca04dc4726df5fcd90 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 論文審定書 i
致謝 ii 摘要 iii Abstract iv 目錄 v 圖目錄 vi 表目錄 x 附錄目錄 xii 壹、前言 1 1.1 研究對象 1 1.2 鯊魚漁獲物之鑑種困難度 3 1.3 利用分子生物技術鑑種 4 1.4 分子生物學物種專一快速篩檢技術 5 1.5 研究目的 7 貳、材料方法 9 2.1 鯊魚樣本採集與處理DNA萃取 9 2.2樣本之鑑種 11 2.3 隔絕式恆溫聚合酶連鎖反應(iiPCR)物種專一引子及螢光探針之設計 12 2.4隔絕式恆溫聚合酶連鎖反應(iiPCR)條件 13 2.5驗證引子與TaqMan Probe之物種專一性 13 2.6 iiPCR凍乾試劑測試 14 2.7 偵測極限濃度測試 14 2.8 恆溫式圈環型核酸增幅反應(LAMP)物種專一引子之設計 14 2.9恆溫式圈環形核酸增幅反應(LAMP)條件 15 叁、結果 16 3.1樣本採集狀況以及DNA序列資料 16 3.2 iiPCR物種專一性引子與探針設計 17 3.3 iiPCR物種專一性引子與探針測試 18 3.4 iiPCR反應條件測試 18 3.5 iiPCR凍乾試劑測試 20 3.6 iiPCR偵測極限濃度測試 20 3.7 LAMP物種專一性引子設計 21 3.8 LAMP反應條件測試 21 肆、討論 22 4.1 iiPCR探針與引子的設計與檢測 22 4.2利用Micro POCKIT裝置進行iiPCR鯊魚物種快篩 23 4.3 利用iiPCR凍乾試劑進行物種快篩 25 4.4 iiPCR凍乾試劑現場操作評估 26 4.5 LAMP反應條件測試 27 4.6恆溫式圈環形核酸增幅反應(LAMP)檢測現場操作評估 28 4.7 iiPCR檢測、LAMP檢測與現行方式比較 28 五、結論 30 陸、參考文獻 31 | |
| dc.language.iso | zh-TW | |
| dc.subject | 隔絕式恆溫聚合?連鎖反應 | zh_TW |
| dc.subject | 鯊魚 | zh_TW |
| dc.subject | 恆溫式圈環形核酸增幅技術 | zh_TW |
| dc.subject | 分子生物檢測 | zh_TW |
| dc.subject | insulated isothermal PCR | en |
| dc.subject | molecular assay | en |
| dc.subject | loop-mediated isothermal amplification | en |
| dc.subject | shark | en |
| dc.title | 利用隔絕式恆溫聚合酶連鎖反應及恆溫式圈環形核酸增幅技術快速鑑定平滑白眼鮫及污斑白眼鮫 | zh_TW |
| dc.title | Fast identification of Carcharhinus falciformis and C. longimanus by insulated isothermal PCR and loop-mediated isothermal amplification | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 韓玉山(Yu-San Han),陳韋仁(Wei-Jen Chen),莊守正(Shoou-Jeng Joung) | |
| dc.subject.keyword | 鯊魚,分子生物檢測,隔絕式恆溫聚合?連鎖反應,恆溫式圈環形核酸增幅技術, | zh_TW |
| dc.subject.keyword | shark,molecular assay,insulated isothermal PCR,loop-mediated isothermal amplification, | en |
| dc.relation.page | 120 | |
| dc.identifier.doi | 10.6342/NTU201803608 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2018-08-19 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 漁業科學研究所 | zh_TW |
| 顯示於系所單位： | 漁業科學研究所 | |
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