常见柔鱼科鱿鱼品种的分子鉴定技术研究

发布时间：2018-02-03 18:58

本文关键词： 鱿鱼种类鉴定 DNA条形码荧光定量PCR技术环介导等温扩增技术　出处：《浙江工商大学》2017年硕士论文　论文类型：学位论文

【摘要】：鱿鱼分布于全世界,包含许多有重要经济价值的品种,它们常被加工成各种风味食品来销售。近年来,该类产品的捕捞和消费逐渐上升。而由于不同鱼种的营养价值和滋味成分有很大不同,其价格差异巨大,因其引发的鱼类产品安全性问题也逐渐成为关注焦点。虽然很多国家都有相应的法规,要求产品外包装上必须注明产品的学名、产地等信息,但是标签制度的实施及掺假造假的判定标准都必须建立在.准确、快速的种类鉴定的基础上。然而,至今仍然没有针对鱿鱼,特别是鱿鱼深加工产品的快速种类鉴定方法。本文以柔鱼科鱿鱼为研究对象,阴性对照选择与目标品种亲缘关系相近的头足类品种(如真蛸和虎斑乌贼等),采用DNA条形码技术、荧光定量PCR技术和环介导等温扩增技术(LAMP)对其进行品种分析和鉴定。主要研究结果如下:1.利用通用引物,成功扩增茎柔鱼、柔鱼、阿根廷滑柔鱼和太平洋褶柔鱼的线粒体COI基因片段,获得长约658 bp的DNA条形码。结合BOLD和GenBank数据库中17种柔鱼科品种的条形码,进行多重序列比对,发现序列变异主要集中在碱基第三位点,且序列突变未饱和,故适用于系统发育分析。将完整条形码拆分为9块区域,初步探讨了利用DNA微型条形码区分常见柔鱼科鱿鱼品种。基于K2P模型的统计分析显示,所有条形码的平均种间遗传距离是种内遗传距离的18-41倍,其中完整条形码、微型条形码-204-2和102-2中存在明显的条形码间隙;在聚类分析中,这三个条形码构建的邻接树都能形成置信度较高的单系群。因此DNA条形码技术能高效鉴定鱿鱼品种,且微型条形码技术可用于深加工鱿鱼商品的种类鉴定。2.使用其他线粒体基因,建立了针对柔鱼科的荧光定量PCR探针检测体系和分别针对四种常见鱿鱼品种的种类特异性检测体系。通过分析GenBank数据库中的线粒体基因,选择线粒体Cytb基因用于检测柔鱼、COI基因用于检测茎柔鱼和太平洋褶柔鱼、ATPase 6基因用于检测阿根廷滑柔鱼,并根据12S rDNA基因设计了针对柔鱼科的特异性引物和探针。研究表明:1)四个种类特异性探针体系能够区分国内市场里四种最常见的鱿鱼品种;2)针对柔鱼科品种设计的Probe-12S rDNA体系能有效区分柔鱼科鱿鱼与其它头足类品种;3)根据标准曲线的斜率算得所建立检测体系的扩增效率为95.8-102.6%;4)在混合DNA样品中,目标DNA也能高效扩增而不受非目标DNA的干扰。因此加工样品里的鱿鱼成分也能被检出,能满足相关鱿鱼类品种的检测需要。3.选择线粒体COI基因设计针对茎柔鱼的LAMP种类特异性引物,分别开发了基于LAMP扩增的实时荧光检测法和指示剂检测法。研究表明:针对茎柔鱼设计的引物具有较强的特异性,在反应温度为65℃时,两种检测方法都能有效区分茎柔鱼与其它相似头足类品种;两种LAMP检测方法的绝对检出限能达到10 pg/反应,相对检出限能达到0.01%;这两种方法也能用于头足类加工产品的检测,适用于鱿鱼类商品的现场、快速种类鉴定。
[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.

【学位授予单位】：浙江工商大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS254.7

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