本文關鍵詞： 魷魚 種類鑒定 DNA條形碼 熒光定量PCR技術 環(huán)介導等溫擴增技術　出處：《浙江工商大學》2017年碩士論文　論文類型：學位論文

【摘要】：魷魚分布于全世界,包含許多有重要經(jīng)濟價值的品種,它們常被加工成各種風味食品來銷售。近年來,該類產(chǎn)品的捕撈和消費逐漸上升。而由于不同魚種的營養(yǎng)價值和滋味成分有很大不同,其價格差異巨大,因其引發(fā)的魚類產(chǎn)品安全性問題也逐漸成為關注焦點。雖然很多國家都有相應的法規(guī),要求產(chǎn)品外包裝上必須注明產(chǎn)品的學名、產(chǎn)地等信息,但是標簽制度的實施及摻假造假的判定標準都必須建立在.準確、快速的種類鑒定的基礎上。然而,至今仍然沒有針對魷魚,特別是魷魚深加工產(chǎn)品的快速種類鑒定方法。本文以柔魚科魷魚為研究對象,陰性對照選擇與目標品種親緣關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、熒光定量PCR技術和環(huán)介導等溫擴增技術(LAMP)對其進行品種分析和鑒定。主要研究結果如下:1.利用通用引物,成功擴增莖柔魚、柔魚、阿根廷滑柔魚和太平洋褶柔魚的線粒體COI基因片段,獲得長約658 bp的DNA條形碼。結合BOLD和GenBank數(shù)據(jù)庫中17種柔魚科品種的條形碼,進行多重序列比對,發(fā)現(xiàn)序列變異主要集中在堿基第三位點,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)分常見柔魚科魷魚品種�；贙2P模型的統(tǒng)計分析顯示,所有條形碼的平均種間遺傳距離是種內遺傳距離的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,這三個條形碼構建的鄰接樹都能形成置信度較高的單系群。因此DNA條形碼技術能高效鑒定魷魚品種,且微型條形碼技術可用于深加工魷魚商品的種類鑒定。2.使用其他線粒體基因,建立了針對柔魚科的熒光定量PCR探針檢測體系和分別針對四種常見魷魚品種的種類特異性檢測體系。通過分析GenBank數(shù)據(jù)庫中的線粒體基因,選擇線粒體Cytb基因用于檢測柔魚、COI基因用于檢測莖柔魚和太平洋褶柔魚、ATPase 6基因用于檢測阿根廷滑柔魚,并根據(jù)12S rDNA基因設計了針對柔魚科的特異性引物和探針。研究表明:1)四個種類特異性探針體系能夠區(qū)分國內市場里四種最常見的魷魚品種;2)針對柔魚科品種設計的Probe-12S rDNA體系能有效區(qū)分柔魚科魷魚與其它頭足類品種;3)根據(jù)標準曲線的斜率算得所建立檢測體系的擴增效率為95.8-102.6%;4)在混合DNA樣品中,目標DNA也能高效擴增而不受非目標DNA的干擾。因此加工樣品里的魷魚成分也能被檢出,能滿足相關魷魚類品種的檢測需要。3.選擇線粒體COI基因設計針對莖柔魚的LAMP種類特異性引物,分別開發(fā)了基于LAMP擴增的實時熒光檢測法和指示劑檢測法。研究表明:針對莖柔魚設計的引物具有較強的特異性,在反應溫度為65℃時,兩種檢測方法都能有效區(qū)分莖柔魚與其它相似頭足類品種;兩種LAMP檢測方法的絕對檢出限能達到10 pg/反應,相對檢出限能達到0.01%;這兩種方法也能用于頭足類加工產(chǎn)品的檢測,適用于魷魚類商品的現(xiàn)場、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.

【學位授予單位】：浙江工商大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：TS254.7

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