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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 蕭仁傑 | |
dc.contributor.author | Meng-I Chen | en |
dc.contributor.author | 陳孟宜 | zh_TW |
dc.date.accessioned | 2021-06-15T05:46:56Z | - |
dc.date.available | 2015-08-31 | |
dc.date.copyright | 2010-08-31 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-08-18 | |
dc.identifier.citation | Banford, H. M., Bermingham, E., Collette, B. B. & McCafferty, S. S. (1999). Phylogenetic systematics of the Scomberomorus regalis (Teleostei : Scombridae) species group: Molecules, morphology and biogeography of Spanish mackerels. Copeia, 596-613.
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G., Perez-Martin, R. I., Pardo, M. A., Perez-Villareal, B., Gilardi, P. & Riese, J. (2007). Comparison of DNA extraction methods from muscle of canned tuna for species identification. Food Control 18, 1211-1215. Chow, S. & Kishino, H. (1995). Phylogenetic relationships between tuna species of the genus Thunnus (Scombridae: Teleostei): Inconsistent implications from morphology, nuclear and mitochondrial genomes. Journal of Molecular Evolution 41, 741-748. Elliott, N. G. & Ward, R. D. (1995). Genetic relationships of eight species of Pacific tunas (Teleostei: Scombridae) inferred from allozyme analysis. Marine and Freshwater Research 46, 1021-1032. Finnerty, J. R. & Block, B. A. (1995). Evolution of Cytochrome-B in the Scombroidei (Teleostei) - Molecular Insights into Billfish (Istiophoridae and Xiphiidae) Relationships. Fishery Bulletin 93, 78-96. Han, K. P. & Ely, B. (2002). Use of AFLP analyses to assess genetic variation in Morone and Thunnus species. Marine Biotechnology 4, 141-145. Hird, H. J., Hold, G. L., Chisholm, J., Reece, P., Russell, V. J., Brown, J., Goodier, R. & MacArthur, R. (2005). Development of a method for the quantification of haddock (Melanogrammus aeglefinus) in commercial products using real-time PCR. European Food Research and Technology 220, 633-637. Infante, C., Catanese, G., Ponce, M. & Manchado, M. (2004). Novel method for the authentication of frigate tunas (Auxis thazard and Auxis rochei) in commercial canned products. Journal of Agricultural and Food Chemistry 52, 7435-7443. Lopez, I. & Pardo, M. A. (2005). Application of relative quantification TaqMan real-time polymerase chain reaction technology for the identification and quantification of Thunnus alalunga and Thunnus albacares. Journal of Agricultural and Food Chemistry 53, 4554-4560. Lowenstein, J. H., Amato, G. & Kolokotronis, S. O. (2009). The real maccoyii: identifying tuna sushi with DNA barcodes--contrasting characteristic attributes and genetic distances. PLoS One 4, e7866. Marko, P. B., Lee, S. C., Rice, A. M., Gramling, J. M., Fitzhenry, T. M., McAlister, J. S., Harper, G. R. & Moran, A. L. (2004). Mislabelling of a depleted reef fish. Nature 430, 309-310. Quinteiro, J., Sotelo, C. G., Rehbein, H., Pryde, S. E., Medina, I., Perez-Martin, R. I., Rey-Mendez, M. & Mackie, I. M. (1998). Use of mtDNA direct polymerase chain reaction (PCR) sequencing and PCR-restriction fragment length polymorphism methodologies in species identification of canned tuna. Journal of Agricultural and Food Chemistry 46, 1662-1669. Ram, J. L., Ram, M. L. & Baidoun, F. F. (1996). Authentication of canned tuna and bonito by sequence and restriction site analysis of polymerase chain reaction products of mitochondrial DNA. Journal of Agricultural and Food Chemistry 44, 2460-2467. Rehbein, H., Kress, G. & Schmidt, T. (1997). Application of PCR-SSCP to species identification of fishery products. Journal of the Science of Food and Agriculture 74, 35-41. Sanchez, A., Quinteiro, J., Rey-Mendez, M., Perez-Martin, R. I. & Sotelo, C. G. (2009). Identification of European Hake Species (Merluccius merluccius) Using Real-Time PCR. Journal of Agricultural and Food Chemistry 57, 3397-3403. Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007). MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24, 1596-1599. Ward, R. D. (1995). Population genetics of tunas. Journal of Fish Biology 47, 259-280. Ward, R. D., Elliott, N. G., Innes, B. H., Smolenski, A. J. & Grewe, P. M. (1997). Global population structure of yellowfin tuna, Thunnus albacares, inferred from allozyme and mitochondrial DNA variation. Fishery Bulletin 95, 566-575. Ward, R. D., Zemlak, T. S., Innes, B. H., Last, P. R. & Hebert, P. D. N. (2005). DNA barcoding Australia's fish species. Philosophical Transactions of the Royal Society B-Biological Sciences 360, 1847-1857. Zilberman, N., Reikhav, S., Hulata, G. & Ron, M.(2006). High-throughput genomic DNA extraction protocol from tilapia's fin tissue. Aquaculture 255, 597-599. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47077 | - |
dc.description.abstract | 鮪魚是高經濟性魚種,不同種鮪魚的價格差異非常大,經由魚片的方式進行貿易與販售的鮪魚,已無法從外觀特徵辨識種類,因此只能依賴基因鑑種。過去利用基因鑑定物種是以DNA定序鑑種技術最為可靠,但需時長達數天到一週。本研究的目的在於利用Real-time PCR技術,發展快速即時鑑定鮪魚物種的方法。此研究目標是以設計具有物種專一性的引子和雜合探針(hybridization probe)用於偵測到單一鹼基多型性(Single Nucleotide Polymorphism, SNP)的變異,藉以區別物種。在此研究中,我們已成功在cytochrome b和control region基因中,針對南方黑鮪(Thunnus maccoyii)、大目鮪(T. obesus)、太平洋黑鮪(T. orientalis)和黃鰭鮪(T. albacares)的SNP設計專一性探針,以及針對大目鮪的物種設計專一性引子,並利用MCA(Melting Curve Analysis)的方法利用Tm值大小將太平洋黑鮪和長鰭鮪與其他物種作區隔。探針和引子皆具高度的鑑別力;探針的準確率皆高達75%以上,南方黑鮪和太平洋黑鮪的探針更高達100%。以觀察員依外型鑑定的大目鮪、黃鰭鮪和長鰭鮪作探針鑑種,其結果大致與型態一致。針對不同濃度的DNA模板(500, 250, 50, 25, 5, 2.5, 0.5, 0.25 ng μl -1)進行與個別探針表現的穩定性和再現性做測試;其結果顯示探針偵測DNA模板的最低濃度為0.25 ng μl -1,且DNA濃度與Ct (Cycle threshold)值呈反比。將探針以旗魚科、劍旗魚科和鯊魚的樣本做物種鑑定測試,四個探針的測試結果皆可判讀為非目標物種,表示其引子和探針具有相當的鑑別度。此方法從DNA萃取到以Real-Time PCR技術鑑定物種只需半個工作天,並且可避免傳統PCR與DNA定序鑑定法可能發生的汙染。 | zh_TW |
dc.description.abstract | Tuna is one of highly valuable seafood, and usually distributed as filleted, broiled and canned products. There are 8 species in Thunnus genus, and each species has very different commercial values, therefore it is important to authenticate tuna species when the morphology characters were removed. DNA based techniques has been extensively used recent years for validation of the species of tuna products. Real-time PCR is one of bio-molecular approaches to identify the quality and quantity of target genes, and can be completed within 2 hours compared to DNA sequencing method, which would take 3 days at least. In this research, we developed a rapid method for species identification by using TaqMan probes and species specific primer to detect single-base variation of the target gene. According to our results, two species specific primers were successfully used to distinguish T. obesus from others by analyzing Ct values and to differentiate T. alalunga and T. orientalis from other species by melting curve analysis. Moreover, we successfully identified T. maccoyii , T. obesus, T. orientalis and T. albacares from other species by SNPs (single nucleotide polymorphisms) probe, designed from mtDNA cytochrome b and control region gene. The correction rates of these probes were higer than 75% and up to 100%. Furthermore, we tested the probes on the samples which were morphologically identified and canned tuna products. The revealed Most results are consistent with morphology. Also, the four probes are tested in sensitivity with eight dilutions of DNA template (500, 250, 50, 25, 5, 2.5, 0.5, 0.25 ng μl -1). The results of sensitivity test suggested that the detection limit was 0.25 ng μl -1, and the Ct value is inversely proportional to the concentration of DNA template. Moreover, we applied tuna specific probes to detection non-Thunnus species, which are Istiophoridae, Xiphiidae, Carcharhiniformes and Lamniformes. The result shows that the primers of the probe systems can only amplify Thunnus genus species, indicating the good species-specificity of the probes. These assays are very useful to rapidly discriminate tuna species in a short time without post-PCR contamination. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T05:46:56Z (GMT). No. of bitstreams: 1 ntu-99-R97241214-1.pdf: 1552922 bytes, checksum: ee0ad859849dc46b50c1d19a6198e660 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 中文摘要….……………………………………………………………….................. ii
英文摘要….………………………………………………………………..................iii 目錄………………………………………………………………………………... v 表目錄……………………………………………………………………………….vii 圖目錄……………………………………………………………………………….ix 壹 前言….…………………………………………………………………………….1 1.1 基因鑑種的需求…………………………………………………………….1 1.2 前人研究概況……………………………………………………………….2 1.3 本研究目的………………………………………………………………….4 貳 材料與方法…………………………………………………………………..……5 2.1樣本採集……………………………………………………………………..5 2.2 去氧核醣核酸的萃取 ……………………………………………………...5 2.3 基因序列資料庫的建立……………………………………………………5 2.4 Real-Time PCR conditions…………………………………………………6 2.5 資料判讀方法………………………………………………………………6 2.6 探針的穩定性和再現性測試…………………………………………….6 2.7交互作用分析………………………………………………………………7 2.8不同比例的探針濃度測試…………………………………………………7 2.9未知樣本的測試………………………………………………………….7 2.10資料分析與物種判讀方法…………………………………………………8 參 結果………………………………………………………………………………9 3.1 DNA萃取與序列分析……………………………………………………9 3.1.1 DNA萃取與定序序列資料庫建立……………………………………9 3.1.2 序列整理與SNP分析…………………………………………………9 3.2 SYBR green試劑的應用…………………………………………………10 3.2.1 針對物種設計的專一引子的鑑別力…………………………………10 3.2.2片段長度的差異……………………………………………………….11 3.3高度專一性的探針………………………………………………………..11 3.3.1 提高溫度增加探針(probe)的專一性鑑別力…………………………11 3.3.2 探針(probe)的專一性鑑別力…………………………………………12 3.3.3 探針的再現性和極限濃度測試………………………………………14 3.3.4 探針對常見經濟魚種但非鮪魚屬的樣本的專一鑑別性……………15 3.3.5 不同比例的探針濃度測試……………………………………………..15 3.3.6 未知樣本的測試 ……………………………………………………..17 3.3.7 罐頭未知物種的鑑別…………………………………………………..18 肆 討論……………………………………………………………………..………..19 4.1 選擇合適組織DNA萃取……………………………………………….. 19 4.2 以專一引子鑑定物種………………………………………………….....19 4.2.1 以MCA鑑定物種…………………………………………..………...20 4.3 以TaqMan Probe 鑑定物種……………………………………………...20 4.3.1提高引子的黏合溫度(Ta) ………………………………………..…….20 4.3.2探針本身的專一性………………………………………………..…….21 4.3.3探針設計與物種親緣關係………………………………………..…….22 4.3.4探針的再現性和極限濃度測試…………………………………..…….23 4.3.5 探針對相近物種但非鮪魚屬的專一鑑別性………………..…..…….24 4.3.6 不同比例的探針濃度測試………………………………………....….24 4.3.7藉探針鑑別只由外型辨識的物種的鑑別性……………………………….….24 4.3.8罐頭未知物種的鑑別……………………………………………..…....25 伍 結論……………………………………………………………………..………..27 參考文獻……………………………………………………………………………28 表 目 錄 Table 1 The primers for traditional PCR of four molecular markers designed for Thunnus species. ……………………………………………………………….…..31 Table 2 The Thunnus species collected during this study. The N represents the individual numbers for each species. ………………………………………….…..32 Table 3 The number of reference sequences of four markers of Thunnus species amplified during this study. ………………………………………….…………….33 Table 4 The diagnostic sites of the positions among 6 species based on cytochrome b gene. ………………………………………….……………. ………. ………. …….34 Table 5 The diagnostic sites of the positions on d-loop among 6 species…………..35 Table 6 The diagnostic sites of the positions on ATPase gene among 6 species……36 Table 7 The diagnostic sites of the positions on COI among 6 species……………..37 Table 8The probes were designed based on the SNPs (Single Nucleotide Polymorphisms) of four molecular markers for 5 tuna species…………………….38 Table 9 The primers designed based on ATPase marker for Thunnus albacores (YFT) and Thunnus obesus (BET)………………………………………………………….39 Table 10 The Ct value results of BET species-specific primer on ATPase region between 5 species…………………………………………………………….…….40 Table 11. The deletion nucleic acid fragment on control region for Thunnus orientalis (PBT) and Thunnus alalunga (ALB). ……………………………………….…….41 Table 12. The primer sequences are designed for amplifying the fragment based on d-loop (control region) contains deletion for Thunnus orientalis and some Thunnus alalunga species. …………………………………………………………….…….42 Table 13. The correction rate of four probes test on five species of Thunnus genus.. 43 Table 14. The four pairs of primers for four probe systems………………………….44 Table 15. The four target species probes designed in this study……………………..45 Table 16 The species-specific primers for Thunnus albacores from reference and this study………………………………………………………………………………….46 Table 17 The Ct values (mean ± standard variation) of four probes tests on target and non-target species……………………………………………………………………47 Table 18 The endpoints of fluorescence (mean ± standard deviation) of four probes tests on target and non-target species……………………………………………….48 Table 19 The individual numbers and the average Ct values of four probes test on non-Thunnus species……………………………………………………………….49 Table 20 The individual numbers and the average fluorescence values of four probes test on non-Thunnus species………………………………………………………..50 Table 21 The two-way ANOVA table on the Ct value of PBT probe between probe volume treatments and target or non-target species………………………………….51 Table 22. The two-way ANOVA table on the fluorescence of PBT probe between probe volume treatments and target or non-target species…………………………52 Table 23 The two-way ANOVA table on the Ct value of SBT probe between probe volume treatments and target or non-target species………………………………….53 Table 24. The two-way ANOVA table on the fluorescence of SBT probe between probe volume treatments and target or non-target species…………………………54 Table 25 The average Ct values and standard deviation of four probes tested on unknown samples……………………………………………………………………55 Table 26 The average fluorescence values and standard deviation of four probes tested on unknown samples. ………………………………………………………………56 圖 目 錄 Figure 1 The standard operation process using the probe systems on tuna species identification. ………………………………………………………………………...57 Figure 2 Comparison of primers and probe of the Thunnus species detection TaqMan system (T. maccoyii) with different tuna species including the alignment with the genus of thunnus of 141 bp consensus. …………………………………………58 Figure 3 Comparison of primers and probe of the Thunnus species detection TaqMan system (T. obesus) with different tuna species including the alignment with the genus of thunnus of 133 bp consensus. …………………………………………59 Figure 4 Comparison of primers and probe of the Thunnus species detection TaqMan system (T. albacares) with different tuna species including the alignment with the genus of thunnus of 82 bp consensus. ……………………………………60 Figure 5 Comparison of primers and probe of the Thunnus species detection TaqMan system (T. orientalis) with different tuna species including the alignment with the genus of thunnus of 133 bp consensus. ……………………………………61 Figure 6 Phylogenetic tree constructed using neighbor-joining method based on genetic distances estimated from mitochondrial control region gene……………….62. Figure 7 Neighbor-joining tree of cytochrome b sequences using K2P substitution model. ……………………………………………………………………………..…63 Figure 8 The Ct values versus amount of fluorescence between 5 species using species-specific primer set on ATPase gene for Thunnus obesus (BET)…………….64 Figure 9 The Ct values versus amount of fluorescence between 5 species using species-specific primer set on ATPase gene for Thunnus albacares. …………….65 Figure 10 The melting curve result of D-loop for 5 species. ……………………….66 Figure 11 The melting curve result of two types of fragment lengths in Thunnus alalunga on d-loop gene. …………………………………………………………….67 Figure 12 The result of Ct values versus amount of fluorescence of LNA probe for Thunnus maccoyii with 5 species at annealing temperature 58℃………………….68 Figure 13 The result of Ct values versus amount of fluorescence of LNA probe for Thunnus maccoyii with 5 species at annealing temperature 62.6℃. ……………….68 Figure 14 The result of Ct values versus amount of fluorescence of TaqMan probe for Thunnus maccoyii (SBT) with 5 species at annealing temperature 60℃. ………….69 Figure 15The result of Ct values versus amount of fluorescence of TaqMan probe for Thunnus albacares (YFT) with 5 species at 60 ℃. …………………………….….70 Figure 16 The result of Ct values versus amount of fluorescence of TaqMan probe for Thunnus obesus(BET) with 5 species at 65℃. …………………………….……….71 Figure 17 The result of Ct values versus amount of fluorescence of TaqMan probe for Thunnus orientalis (PBT) with 5 species at 60℃. …………………………...….….72 Figure 18 The linearity test with DNA template of T. albacores probe system…….73 Figure 19 The linearity test with DNA template of T. maccoyii probe system……...75 Figure 20 The linearity test with DNA template of T. orientalis probe system……...77 Figure 21 The Average Ct values and amounts of fluorescence for T. orientalis probe system using T. orientalis as template. . …………………………...….……………79 Figure 22 The Average Ct values and amounts of fluorescence for T. maccoyii probe system using T. maccoyii as template. . …………………………...….…………….79 Figure 23 The Average Ct values and amounts of fluorescence for T. orientalis probe system using other four species as template. ……………………...….…………….80 Figure 24 The Average Ct values and amounts of fluorescence for T. maccoyii probe system using other four species as template. ……………………...….…………….80 Figure 25The YFT probe test result on canned tuna samples. ...….………………...81 Figure 26The PBT probe test result on canned tuna samples. ...….………………...82 Figure 27The SBT probe test result on canned tuna samples. ...….………………...83 Figure 28The BET probe test result on canned tuna samples. ...….………………...84 Figure 29 The result of Ct values versus amount of fluorescence of TaqMan probe for Thunnus maccoyii (SBT) with 5 species at annealing temperature 61℃…………...85 Figure 30 The linearity test with DNA template of T. obesus probe system………..86 Figure 31 The overlapping region of T. albacores probe system and T. maccoyii probe system. ……………………...….……………………………………………………88 | |
dc.language.iso | zh-TW | |
dc.title | 以即時聚合連鎖反應快速鑑定鮪魚種類 | zh_TW |
dc.title | Rapid identification of tuna species by Real-Time PCR | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 張學偉,陳韋仁,曾美珍 | |
dc.subject.keyword | 鮪魚,物種鑑定,即時聚合連鎖反應, | zh_TW |
dc.subject.keyword | tuna,species identification,real-time PCR, | en |
dc.relation.page | 88 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-08-19 | |
dc.contributor.author-college | 理學院 | zh_TW |
dc.contributor.author-dept | 海洋研究所 | zh_TW |
顯示於系所單位: | 海洋研究所 |
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