本文關(guān)鍵詞： 魷魚(yú) 種類鑒定 DNA條形碼 熒光定量PCR技術(shù) 環(huán)介導(dǎo)等溫?cái)U(kuò)增技術(shù)　出處：《浙江工商大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：魷魚(yú)分布于全世界,包含許多有重要經(jīng)濟(jì)價(jià)值的品種,它們常被加工成各種風(fēng)味食品來(lái)銷售。近年來(lái),該類產(chǎn)品的捕撈和消費(fèi)逐漸上升。而由于不同魚(yú)種的營(yíng)養(yǎng)價(jià)值和滋味成分有很大不同,其價(jià)格差異巨大,因其引發(fā)的魚(yú)類產(chǎn)品安全性問(wèn)題也逐漸成為關(guān)注焦點(diǎn)。雖然很多國(guó)家都有相應(yīng)的法規(guī),要求產(chǎn)品外包裝上必須注明產(chǎn)品的學(xué)名、產(chǎn)地等信息,但是標(biāo)簽制度的實(shí)施及摻假造假的判定標(biāo)準(zhǔn)都必須建立在.準(zhǔn)確、快速的種類鑒定的基礎(chǔ)上。然而,至今仍然沒(méi)有針對(duì)魷魚(yú),特別是魷魚(yú)深加工產(chǎn)品的快速種類鑒定方法。本文以柔魚(yú)科魷魚(yú)為研究對(duì)象,陰性對(duì)照選擇與目標(biāo)品種親緣關(guān)系相近的頭足類品種(如真蛸和虎斑烏賊等),采用DNA條形碼技術(shù)、熒光定量PCR技術(shù)和環(huán)介導(dǎo)等溫?cái)U(kuò)增技術(shù)(LAMP)對(duì)其進(jìn)行品種分析和鑒定。主要研究結(jié)果如下:1.利用通用引物,成功擴(kuò)增莖柔魚(yú)、柔魚(yú)、阿根廷滑柔魚(yú)和太平洋褶柔魚(yú)的線粒體COI基因片段,獲得長(zhǎng)約658 bp的DNA條形碼。結(jié)合BOLD和GenBank數(shù)據(jù)庫(kù)中17種柔魚(yú)科品種的條形碼,進(jìn)行多重序列比對(duì),發(fā)現(xiàn)序列變異主要集中在堿基第三位點(diǎn),且序列突變未飽和,故適用于系統(tǒng)發(fā)育分析。將完整條形碼拆分為9塊區(qū)域,初步探討了利用DNA微型條形碼區(qū)分常見(jiàn)柔魚(yú)科魷魚(yú)品種�；贙2P模型的統(tǒng)計(jì)分析顯示,所有條形碼的平均種間遺傳距離是種內(nèi)遺傳距離的18-41倍,其中完整條形碼、微型條形碼-204-2和102-2中存在明顯的條形碼間隙;在聚類分析中,這三個(gè)條形碼構(gòu)建的鄰接樹(shù)都能形成置信度較高的單系群。因此DNA條形碼技術(shù)能高效鑒定魷魚(yú)品種,且微型條形碼技術(shù)可用于深加工魷魚(yú)商品的種類鑒定。2.使用其他線粒體基因,建立了針對(duì)柔魚(yú)科的熒光定量PCR探針檢測(cè)體系和分別針對(duì)四種常見(jiàn)魷魚(yú)品種的種類特異性檢測(cè)體系。通過(guò)分析GenBank數(shù)據(jù)庫(kù)中的線粒體基因,選擇線粒體Cytb基因用于檢測(cè)柔魚(yú)、COI基因用于檢測(cè)莖柔魚(yú)和太平洋褶柔魚(yú)、ATPase 6基因用于檢測(cè)阿根廷滑柔魚(yú),并根據(jù)12S rDNA基因設(shè)計(jì)了針對(duì)柔魚(yú)科的特異性引物和探針。研究表明:1)四個(gè)種類特異性探針體系能夠區(qū)分國(guó)內(nèi)市場(chǎng)里四種最常見(jiàn)的魷魚(yú)品種;2)針對(duì)柔魚(yú)科品種設(shè)計(jì)的Probe-12S rDNA體系能有效區(qū)分柔魚(yú)科魷魚(yú)與其它頭足類品種;3)根據(jù)標(biāo)準(zhǔn)曲線的斜率算得所建立檢測(cè)體系的擴(kuò)增效率為95.8-102.6%;4)在混合DNA樣品中,目標(biāo)DNA也能高效擴(kuò)增而不受非目標(biāo)DNA的干擾。因此加工樣品里的魷魚(yú)成分也能被檢出,能滿足相關(guān)魷魚(yú)類品種的檢測(cè)需要。3.選擇線粒體COI基因設(shè)計(jì)針對(duì)莖柔魚(yú)的LAMP種類特異性引物,分別開(kāi)發(fā)了基于LAMP擴(kuò)增的實(shí)時(shí)熒光檢測(cè)法和指示劑檢測(cè)法。研究表明:針對(duì)莖柔魚(yú)設(shè)計(jì)的引物具有較強(qiáng)的特異性,在反應(yīng)溫度為65℃時(shí),兩種檢測(cè)方法都能有效區(qū)分莖柔魚(yú)與其它相似頭足類品種;兩種LAMP檢測(cè)方法的絕對(duì)檢出限能達(dá)到10 pg/反應(yīng),相對(duì)檢出限能達(dá)到0.01%;這兩種方法也能用于頭足類加工產(chǎn)品的檢測(cè),適用于魷魚(yú)類商品的現(xiàn)場(chǎng)、快速種類鑒定。
[Abstract]:Squid are distributed throughout the world, including many economically important species, they are often processed into a variety of food to sell. In recent years, the products of fishing and consumption gradually increased. But because of the nutritional value and taste compounds of different species are very different, the price difference is huge, because fish product safety issue the lead has gradually become the focus of attention. Although many countries have the corresponding laws and regulations, requirements of product packaging must indicate the product name, origin and other information, but the label system implementation and determination of adulteration standard must be based on accurate, rapid identification of the species on the basis. However, still not for squid, especially the fast identification method of squid deep processing products. In this paper, squid squid as the research object, the relationship between negative control and genetic close head Foot varieties (such as octopus and squid and other tiger), using DNA barcode technology, fluorescence quantitative PCR and loop mediated isothermal amplification (LAMP) for the analysis and identification of the varieties. The main results are as follows: 1. using universal primers successfully amplified, squid, squid, mitochondrial COI gene fragment of Argentina slide squid and pacificus, DNA bar code length of about 658 BP. The combination of 17 kinds of Ommastrephidae varieties BOLD and GenBank database in the bar code, multiple sequence alignment, sequence variations were found mainly in the base of third loci, and sequence mutation was not saturated, it is suitable for the phylogenetic analysis of the complete code is split into 9. Block area, discussed the use of DNA to distinguish common squid squid mini bar display varieties. Statistical analysis based on K2P model, the average genetic distance among all the bar code is a genetic distance in 18- 41 times, of which the complete bar code, bar code and -204-2 in 102-2 micro bar code gap is obvious; in cluster analysis, the three adjacent tree barcodes can form a higher confidence monophyletic group. So DNA barcode technology can efficiently identify squid species, species identification and micro.2. deep processing of squid bar code the use of other techniques for mitochondrial genes, established quantitative fluorescent PCR probe detection system for Ommastrephidae and respectively for four kinds of common squid species specific detection system. Through the analysis of mitochondrial genes in the GenBank database, select the mitochondrial Cytb gene for the detection of squid, squid Todarodes pacificus and detection for COI gene. The ATPase 6 gene for the detection of Argentina and designed for Illex squid, the specific primers and probe according to the 12S rDNA gene. The study showed that: 1) Four kinds of specific probe system can distinguish the domestic market in the four most common squid species; 2) according to the Ommastrephidae variety design Probe-12S rDNA system can effectively distinguish between squid squid and other cephalopods varieties; 3) according to the standard curve slope is established by measuring the amplification efficiency of 95.8-102.6% system; 4 DNA) in the mixed samples, the target DNA can expand without interference from non target DNA. Therefore the squid component processing in a sample can be detected, can meet the testing needs of.3. squid species selection of mitochondrial COI gene for Dosidicus gigas LAMP type specific primers were developed by LAMP the real-time detection method and detection method based on the indicator. The study shows that has strong specificity for primer design of squid, the reaction temperature is 65 DEG C, two detection methods can effectively distinguish Squid and other similar cephalopod species; two kinds of LAMP method for detection of absolute detection limit can reach 10 pg/, the relative detection limit can reach 0.01%; the two methods can also be used for the detection of cephalopod processing products, applicable to field squid products, rapid identification of species.

【學(xué)位授予單位】：浙江工商大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TS254.7

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