【作者】 方绍庆；

【导师】 陆承平；

【作者基本信息】 南京农业大学 ， 兽医（专业学位）， 2011， 博士

【摘要】 随着我国海产品国际贸易的飞速发展,某些贸易商为了追求经济利益,在一些价值高昂的海产品中夹杂价值低廉的海产品,以次充好、鱼目混珠,严重影响了国际贸易的正常秩序。然而,国内外尚无海产品来源成分的检测标准和方法,针对这一现状,本研究以鳕鱼、扇贝、中国鲎为研究对象,建立了鳕鱼DNA特异性检测PCR方法、扇贝SSR物种鉴定方法、中国鲎实时荧光PCR鉴定方法,为海产品物种防伪及保护的分子鉴定进行了有益的探索,也可为海产品贸易的商业打假提供技术支撑。1.鳕鱼防伪为防止海产品贸易中以鲶鱼等廉价鱼冒充鳕鱼产品,根据GenBank中7个鳕属及其他5种鱼类的mtDNA细胞氧化酶Ⅱ (COⅫ)基因序列,设计特异性引物,建立了可对鳕鱼产品进行特异性检测的PCR方法。应用结果表明,所建立的方法适用于鳕鱼物种的特异性检测鉴定,可为鳕鱼贸易提供有力的技术保障。2010年3月至2011年3月期间,应用所建立的鳕鱼PCR检测鉴定方法,在烟台、威海、青岛、荣成、蓬莱、龙口、莱州等山东省内10个口岸城市,广东、辽宁、北京、上海、江苏、福建、四川、广西等9个省市渔业企业、各地出入境检验检疫局、科研院所推广和应用。一年期间检测样品约3256份。截止2011年3月共检出总计345批次掺假可疑的鳕鱼制品,总货值4906万元。2.扇贝种类分子鉴定扇贝种类繁多,不同种类扇贝经济价值差别较大,市场上用廉价扇贝冒充名贵的现象普遍,仅从形态上无法区分各种类扇贝,因此从分子水平上对扇贝种类进行鉴定是十分必要的。本研究建立了栉孔扇贝(Chlamys farreri),虾夷扇贝(Patinopecten yessoensis),海湾扇贝(Argopecten irradians)和华贵栉孔扇贝(Mimachlamys nobilis)的微卫星鉴定方法，该方法具有高度特异性和敏感性,可用于扇贝及其深加工制品的种类鉴定,为扇贝贸易的商业打假提供技术支撑。自2009年以来,先后在山东东方海洋股份有限公司、北京市华都峪口水产有限责任公司等全国主要水产企业推广应用,鉴定其引进的扇贝苗种类。不同扇贝苗养殖条件不同,因为混养造成而经济损失。采用本方法可有效区分扇贝苗种类,分开养殖,最大限度地减少混养造成的损失。近三年,累计经济效益3153万元。上个世纪90年代,我国已发现进口的华贵栉孔扇贝种苗混有有其他种类扇贝,但因缺乏科学证据无法索赔。2010年初通过采用本方法,成功鉴定出从美国进口的华贵栉孔扇贝种苗纯度不高。促使外方公司将纯度最好的华贵栉孔扇贝种苗提供给中国扇贝进口公司,维护了我国水产品公司在国际贸易中的正当权益扇贝物种鉴定方法-SSR法已申请获准制定检验检疫行业标准。3.中国鲎分子鉴定为建立我国特有的珍稀水生动物中国鲎的分子保护方法,根据GenBank中中国鲎及其他17种节肢动物门动物的mtDNA细胞氧化酶Ⅲ(COXⅢ)基因序列,应用DNASTAR7.0软件设计特异性引物,建立了中国鲎SYBR Green I实时荧光PCR鉴定方法,为保护我国珍稀动物进行了有益的探索。2010年,山东省某市查获一批生物制剂,疑似有鲎成分,将样品用本实验方法进行检测,证明该生物制剂确为含有鲎成分。生产商对作案事实供认不讳。中国鲎物种鉴定-荧光PCR法已申请获准制定检验检疫行业标准。更多还原

【Abstract】 With the rapid development of our country’s marine product international trade, some merchants in pursuit of profit adulterated some inexpensive marine products in costly producst, who sold shoddy and masqueraded, which seriously effected the normal order in the international trade. However, there is not a standard or method of detecting original components of marine product in domestic and foreign country. We study ling, callop and horseshoe crab, set up the PCR method specific for ling identification, the species-specifc microsatellite markers method specific for scallop identification, and the real-time PCR method specific for horseshoe crab identification, which effectually probe into the molecular identification for anti-counterfeiting and protecting the marine species.1. Anti-counterfeiting of ling. Avoiding ling and its product was imitated with cheap catfish etc. in the marine product trade, we set up a PCR method specifically amplifying DNA of ling species, mtDNA COXII sequences of7ling subspecies and5other fish species were download from Genbank, and compared by DNASTAR7.0software, designed PCR primers,.The applied results indicated that this method was specific to identification of ling species, which offered the powerful technical guarantee in the ling trade.From March2010to March2011, the PCR method of identifying ling species has been popularized and applied to Yantai, Weihai, Qingdao, Rongcheng, Penglai, Longkou, Laizhou and so on,10port cities of Shandong province, fishery corporations, every city’s entry exit inspection and quarantine bureaus, and scientific research centers in9provinces, Guangdong, Liaoning, Beijing, Shanghai, Jiangsu, Fujian, Sichuan, Guangxi and so on.3,256samples have been inspected in one year, of which345shipments of fake and dubious ling in total have been detected at the end of march2011. The total value was 49,060,000RMB.2. Molecular identification of scallop species. Scallops,which comprise hundreds of species, have very different price, and then the cheap one is usually imitated the rare one for sale in the market. Identification of commercial scallops to the species level is an important issue owing to the difficulty of distinguishing the species by morphological characters. In the present study, we established the species-specific microsatellite markers for Zhikong scallop, Yesso scallop, Noble scallop and Bay scallop to identify commercial scallop..This method well demonstrated high precision and high sensitivity, and more competence in the identification of heavily processed product, which provides a technical support for anti-fake commercial scallops in the trade.From2009, this method has been popularized and applied to identify the importing scallop larvas in Shandong Dongfang Ocean Ltd, Co., Beijing Huadu Yukou Aquatic Products Ltd, Co..and so on, the main aquatic products corporations of our country. The polyculture of different scallop larva which should be in different clutured conditions may cause profit losses. This method well demontrated more competence in the identification of scallop larva species. Separately culturing scallop larvas can reduce the losses caused by polyculture to minimum. The total benefit has been31.53million RMB in this3years.In the1990s, we have found that the importing Noble scallop larvas have been adulterated the other species, but we couldn’t counterclaim for lack of scientific proof. With our method, the low purity of Noble scallop larvas imported from an American company have been detected in the early2010, which urged the company to provide the purest Noble scallop larvas for the Chinese imported company, and maintained the rights and interests of our country’s aquatic product company in the international trade.The method of species-specific microsatellite markers for scallop species identifi-cation has been formed a standard of inspection and quarantine industry.3. Molecular identification of horseshoe crab. We set up a SYBR Green I real-time PCR method specific for horseshoe crab identification for protecting the rare aquatic animal existed only in our country. mtDNA COXIII sequences of horseshoe crab and17other arfhropoda species were download from Genbank, and compared by DNASTAR7.0software. The established method probed into the protection of our country’s rare animals. In2010, a passel of bio-reagent which may comprise some dubious horseshoe crab elements was captured in a city of Shandong province. It was proved that the bio-reagent contained horseshoe crab elements with our method. When confronted the result, the manufacturer confessed.更多还原