DNA条形码技术在深圳鱼肉制品鉴定中的应用

以线粒体细胞色素氧化酶亚基Ⅰ（COⅠ ）基因为目标基因,应用DNA条形码技术鉴别深圳批发市场和超市零售鱼肉制品的种类来源,判别其产品标签是否正确。本研究调查的77 份鱼肉制品均能扩增出特异性条带,28 份样品与产品标签标示不符,“错贴”率高达36.36%,其中所有标示“龙俐鱼”的商品都是低价的“巴丁鱼”（Pangasianodon hypophthalmus）。DNA条形码技术可用于鱼肉制品的来源物种鉴定。     

DNA条形码技术在肉品防欺诈鉴别中的应用

以DNA条形码技术鉴别进出口监督抽检的鱼肉等水产制品的种类来源,用以判别其与申报或产品标签是否相符.分别提取鱼肉等样品的基因组DNA,以目前国际上比较公认的动物线粒体细胞色素氧化酶COⅠ基因通用引物进行PCR扩增.PCR产物经测序分析后,将得到的扩增片段序列与Genbank数据库进行序列比对,同时提交Barcoding Life DNA条形码数据库(BOLD)进行鉴定分析.本批次监督抽检的16份鱼肉、鱼丸等水产制品中除1份样品未能成功获得鱼肉COⅠPCR扩增外,其余15份样品均顺利得到种类来源鉴定,鉴定结果约有31.25％的样品与产品标签标示不符.作为一种简单、快速、有效的分子鉴定技术,DNA条形码可以直接应用于鱼肉等动物源性食品的种类鉴定.     

线粒体细胞色素b基因在鱼唇制品物种鉴定中适用性的研究

目的 探讨线粒体细胞色素b基因(Cytochrome b, Cyt b)在鱼唇物种鉴定的适用性,为鲨鱼类物种鉴定增加新的DNA条形码。方法 利用Cyt b作为DNA条形码对从全国31个城市购买252份鱼唇样品进行PCR扩增,测序、利用BLAST功能进行近源种同源基因比较分析,鉴定鱼唇制品鱼种,对鲨鱼物种进行濒危评价分析。结果 成功扩增出250个样品,找到的一致性鲨鱼物种基因序列相似性均在99%以上。涉及8个鲨鱼种,最多样品为大青鲨,占样品65.5%,其余还有镰形真鲨、路氏双髻鲨、锤头双髻鲨、灰鲭鲨、远洋白鳍鲨、沙拉真鲨和黑边鳍真鲨。结论 应用Cyt b作为DNA条形码对鲨鱼种鉴定结果与COI DNA条形码结果一致,在鲨鱼种鉴定时可以使用Cyt b 基因及COI基因联合鉴定条形码,为深加工海产品物种鉴定提供更多的技术支撑。     

Microbiological quality of sushi from sushi bars and retailers

Sushi is a traditional Japanese food, mostly consisting of rice and raw fish. Fish is considered a healthy food, but as with other animal products, consumption of raw muscle incurs potential health risks such as ingestion of pathogenic bacteria or parasites. In this study, 250 sushi samples were analyzed for their microbiological status and the prevalence of pathogenic bacteria. A comparison was made between frozen sushi from supermarkets and fresh sushi from sushi bars. Aerobic mesophilic bacteria counts differed for sushi from these two sources, with means of 2.7 log CFU/g for frozen sushi and 6.3 log CFU/g for fresh sushi. The prevalence of Escherichia coli and Staphylococcus aureus was higher in the fresh samples. Salmonella was found in four (1.6%) of the sushi samples, and Listeria monocytogenes was found in three (1.2%) of the samples. These results indicate that the microbiological quality of industrially processed sushi is higher than that of freshly prepared sushi. The quality of freshly prepared sushi strongly depends on the skills and habits of the preparation cooks, which may vary.     

Marketplace substitution of Atlantic salmon for Pacific salmon in Washington State detected by DNA barcoding

Accurate identification of seafood in the marketplace is an issue of international concern, due to high rates of market substitution of cheaper or more widely available species for expensive or high-demand species. Salmon samples from stores and restaurants throughout western Washington, USA were tested using DNA sequencing of a short section of the mitochondrial cytochrome c oxidase I (COI) gene (DNA barcoding) to identify Atlantic salmon substituted for Pacific salmon. Of 99 salmon samples, 11 (11%) were Atlantic salmon sold as Pacific salmon. More than 38% of restaurant samples were mislabeled to species, while only 7% of store samples were mislabeled. Market substitution rates were significantly greater in restaurants compared to stores, and consistently greater in winter compared to spring, although not significantly. The high market substitution rate in restaurants documents a pressing need for more monitoring and enforcement specifically in restaurants. DNA barcoding is a valuable tool for rapid and definitive authentication of salmon in the marketplace, and should be more widely adopted to discourage market substitution.     

Applicability of DNA barcode for identification of fish species

DNA barcoding is a species identification technique, which uses a very short DNA sequence from a region of approximately 650 base-pairs in the 5'-end of the mitochondrial cytochrome c oxidase subunit I gene as a marker to identify species of mammals and fishes. The applicability of DNA barcoding for identification of fish species consumed in Japan was studied. Among thirty-one fresh or processed fishes were obtained from the market, two samples could not be identified due to lack of data in the Barcode of Life Data (BOLD) database. However, BLAST-search of 16S rRNA genes in the National Center for Biotechnology Information (NCBI) database and the PCR-RFLP method published by the Food and Agricultural Materials Inspection Center (FAMIC) were found to be applicable to identify these 2 fishes. The results show that the DNA barcoding technique is potentially useful as a tool for confirming the proper labeling of fish species in the Japanese market.     

DNA barcoding for the species identification of commercially important fishery products in Indonesian markets

The DNA barcoding approach was used for the species identification of 44 Indonesian commercial fishery products. Additionally, the intronless nuclear rhodopsin gene fragment (RH1) was added to the analysis to enable the identification of species not yet barcoded and possible hybrids. The 655‐bp cytochrome C oxidase subunit I (COI) gene fragment marker was successfully amplified and used to identify 86% of the total fish samples at the species level using the BOLD and BLAST public databases. Moreover, the RH1 marker was used to complete COI analysis. For a number of fish species, the COI sequences (six species) and RH1 sequences (eight species) were the first entries submitted to GenBank. This study demonstrated that COI barcoding is a promising tool for Indonesian fishery products and confirmed that it could be adopted in the future for regular seafood control as part of the Indonesian integrated food traceability system.     

DNA barcoding reveals high substitution rate and mislabeling in croaker fillets (Sciaenidae) marketed in Brazil: The case of “pescada branca” (Cynoscion leiarchus and Plagioscion squamosissimus)

The removal of morphological features during fish processing hinders identification to the species level, increasing the chances of species substitution and the mislabeling of marketed products. We used DNA barcoding to assess whether species substitutions occur in croaker (Sciaenidae) fillets labeled as “pescada branca” sold in the Brazilian Amazon, where two species are known under this vernacular name (Cynoscion leiarchus and Plagioscion squamosissimus). A 577-bp cytochrome C oxidase subunit I (COI) sequence was obtained from 137 fillets and compared with the sequences of whole Sciaenidae fish that were identified based on their morphology and the reference sequences of the BOLD and GenBank public databases. DNA barcoding was able to identify 90% of the samples analyzed to the species level, and the results showed a high rate of species substitution in the fillets labeled as “pescada branca”. The substitution rate was 100% if using the criterion that the fillets should be C. leiarchus and 76.6% if using the criterion that they should be P. squamosissimus. Additionally, the results show that “pescada branca” was replaced in most cases by species of lower commercial value, which clearly demonstrates economic fraud aimed at increased profits. Our data confirm that DNA barcoding is a sensitive and reliable tool that can be applied to authenticate processed fish.     

Applying COI Barcode to Identify Animal Origin of Food

DNA barcoding possesses advantages of high resolution, high sensitivity, and capability in capturing as much identity information as possible. However, highly varying sources of food materials and a complicated supply chain bring about challenge to the application of barcoding methods. In this study, different barcode systems were compared to establish a robust method for tracing animal species in food. Experiments on food samples from mammal, poultry, and fish proved that a mini barcode system targeting a 192 bp COI gene fragment was able to accurately identify both raw and highly processed animal food. In order to distinguish species in a mixed food sample, cloning technique was used by which as low as 10% target animal ingredient could be detected. Testing of marketed food products verified the capability of the mini barcoding method in identifying illegally claimed product.     

鹿茸物种来源DNA条形码鉴定技术研究

目的 受经济利益驱动,鹿茸标签不符情况时有发生,损害消费者利益的同时,也给产业的发展带来了负面影响,探究鹿茸的鹿种鉴定方法为鹿茸市场监管提供技术支持。方法 本研究以线粒体细胞色素氧化酶I基因（Cytochrome oxidase I gene, COI）和线粒体细胞色素b基因（Cytochrome b gene , Cytb）为靶基因对鹿茸样品进行鉴定,并对两种基因的鉴别能力进行了比较。结果 发现COI存在无法鉴别梅花鹿和马鹿的情况,而Cytb可以将所有鹿茸鉴定至种水平。并将Cytb作为目标片段,建立了鹿茸中物种来源鉴定的DNA条形码方法。并利用该方法对市场上销售的53份鹿茸样品进行标签符合性鉴定。结论 进一步证实了使用Cytb的DNA条形码方法可以有效鉴定出市售鹿茸样品的物种来源。收集到的53份市售鹿茸样品中,仅有21份样品与标签标识物种相符;25份样品存在将低价鹿茸标为高价鹿茸的现象;7份样品缺少明确的物种信息。本研究结果可以为监管部门规范鹿茸产品标签标识提供技术支撑。     

Molecular barcoding reveals mislabelling of commercial fish products in Italy

In this work, we present molecular barcoding results obtained in 69 processed fish products belonging to 27 teleost species traded in Italian commercial markets during 2008. DNA barcoding using direct sequencing of about 900 bp of mitochondrial genes cytochrome oxidase subunit I (COI) and cytochrome b (Cyt b) revealed uncorrect labelling in 22 samples (32%). Among substituted species, 18 (26%) were serious frauds under both economic and nutritional points of view. In some cases, frauds concerned species of major conservation regard and reported in the IUCN and CITES directories. Results add further concern on the trading of processed fish products in Italy from both health and conservation points of view.     

DNA条形码COI序列在常见肉类鉴别中的应用研究

为了对常见的4种肉类及相关肉制品进行掺假鉴定,判别与产品标签是否相符,本研究以COI基因为靶基因,建立了4种动物源性食品DNA条形码鉴别技术。分别提取牛、羊、猪、鸭四大物种的基因组DNA为模板,以其COI基因的保守序列区设计6对通用引物,结合文献报道及数据库提供的7对通用引物进行PCR扩增,并将测序结果提交Gen Bank数据库Blast比对,评价不同DNA条形码的检测鉴别能力。筛选出COI-A为最优序列,在4个物种中扩增效率100%。对抽检的20个批次的肉加工品样品进行检测,鉴定结果约有90%的样品与产品标签标示的成分相符。其中1个批次的牛丸制品因肉类成分含量低未扩增成功,1个批次的牛丸制品检出鸭源成分,判定掺假。DNA条形码技术快速有效,本研究筛选的COI-A序列可直接用于牛、羊、猪、鸭及其肉制品的鉴定,并为其它常见动物源性食品的种类鉴定提供一定参考依据。     

基于DNA条形码技术的海参物种鉴定

DNA条形码技术(DNA barcoding)是一种新型高效的物种鉴别方法。本研究基于DNA条形码技术,以线粒体细胞色素氧化酶I基因(COI)和16S核糖体RNA(16S rRNA)基因作为靶基因对海参物种进行鉴别,结果表明COI基因或16S rRNA基因均能实现大部分海参的物种鉴定,部分样品需结合两个靶基因鉴定出来。将所建立的DNA条形码方法用于市售海参样品的物种鉴定,24份市售海参样品中10份市售海参样品的物种鉴定结果与标签名称相符,6份样品与标签名称不符,存在将低价海参品种标为高价海参的现象;其余8份样品的标签只有商品名但没有明确的物种信息,利用DNA条形码技术对其鉴定可得到明确的海参种名。本研究结果证实DNA条形码技术可应用于市售海参的物种鉴定,为海参的监管提供技术支撑。     

Novel multiplex PCR-RFLP assay discriminates bovine,porcine and fish gelatin substitution in Asian pharmaceuticals capsule shells

Gelatin is widely used in pharmaceuticals as a protective coating, such as soft and hard capsule shells. However, the animal source of gelatin is a sensitive issue because certain gelatins such as porcine and bovine gelatins are not welcome in Halal, Kosher and Hindus’ consumer goods. Recently, we have documented DNA barcoding and multiplex PCR platforms for discriminating porcine, bovine and fish gelatins in various fish and confectionary products; but those assays were not self-authenticating and also not tested in highly refined pharmaceutical products. To address this knowledge gap, here we report a self-authenticating multiplex PCR-restriction fragment length polymorphism (RFLP) assay to identify animal sources of various gelatin in pharmaceutical capsules. Three different restriction enzymes, BsaAI, Hpy188I and BcoDI were used to yield distinctive RFLP patterns for gelatin-based bovine (26, 94 bp), fish (97, 198 bp) and porcine (17, 70 bp) DNA in control experiments. The specificity was cross-tested against 16 non-target species and the optimised assay was used to screen gelatin sources in 30 halal-branded pharmaceuticals capsule shells. Bovine and porcine DNA was found in 27 and 3 of the 30 different capsules products. The assay was suitable for detecting 0.1 to 0.01 ng total DNA extracted from pure and mixed gelatins. The study might be useful to authenticate and monitor halal, kosher, vegetarian and Hindu compliant pharmaceuticals, foods and cosmetics.     

DNA条形码技术对鱼子酱物种溯源及鉴定

为探讨DNA条形码技术在鱼子酱物种鉴定中的适用性，利用细胞色素b（Cytochrome b，Cyt b）和细胞色素氧化酶I亚单位I（Cytochrome Oxidase I，COI）作为DNA条形码对鱼子酱样品进行DNA提取、聚合酶链式反应（Polymerase Chain Reaction，PCR）、测序、利用NCBI网站和BOLD鉴定系统进行基因比较分析，构建系统发育树，鉴定鱼子酱物种，对我国鱼子酱产品物种标签符合性情况进行检查。购买的40份样品，一致性鲟鱼物种基因序列相似性均在99%以上，涉及5个鲟鱼种，其中杂交种占比75%、西伯利亚鲟、施氏鲟、欧鳇、俄罗斯鲟占25%。说明Cyt b、COI作为DNA条形码可以对鱼子酱进行物种鉴定，检测的鱼子酱产品均为鲟鱼子酱，无造假，但是45%产品标签物种替代或物种标识不清。加强对产品物种标识重视及鉴定技术的开发，有助于我国鱼子酱对外贸易发展，保障我国鲟鱼产业可持续性健康发展。     

Establishment of a standard reference material (SRM) herbal DNA barcode library of Vitex negundo L. (lagundi) for quality control measures

The majority of the population in the Philippines relies on herbal products as their primary source for their healthcare needs. After the recognition of Vitex negundo L. (lagundi) as an important and effective alternative medicine for cough, sore throat, asthma and fever by the Philippine Department of Health (DOH), there was an increase in the production of lagundi-based herbal products in the form of teas, capsules and syrups. The efficiency of these products is greatly reliant on the use of authentic plant material, and to this day no standard protocol has been established to authenticate plant materials. DNA barcoding offers a quick and reliable species authentication tool, but its application to plant material has been less successful due to (1) lack of a standard DNA barcoding loci in plants and (2) poor DNA yield from powderised plant products. This study reports the successful application of DNA barcoding in the authentication of five V. negundo herbal products sold in the Philippines. Also, the first standard reference material (SRM) herbal library for the recognition of authentic V. negundo samples was established using 42 gene accessions of ITS, psbA-trnH and matK barcoding loci. Authentication of the herbal products utilised the SRM following the BLASTn and maximum-likelihood (ML) tree construction criterion. Barcode sequences were retrieved for ITS and psbA-trnH of all products tested and the results of the study revealed that only one out of five herbal products satisfied both BLASTn and ML criterion and was considered to contain authentic V. negundo. The results prompt the urgent need to utilise DNA barcoding in authenticating herbal products available in the Philippine market. Authentication of these products will secure consumer health by preventing the negative effects of adulteration, substitution and contamination.     

基于DNA条形码技术对常见石首鱼科鱼胶的物种鉴定

摘　要：目的　运用DNA条形码技术对常见石首鱼鱼胶进行物种鉴定。方法　通过对26份鱼胶样品基因组DNA提取，PCR扩增COI基因、测序，用BOLD物种鉴定系统，与数据库中已有鱼类序列进行比对分析，鉴定出各鱼胶的物种；根据Kimura双参数模型计算样品序列遗传距离，并将所得序列构建NJ和MP系统发育树，进行聚类分析。结果　26份鱼胶样品通过鉴定引物“Fish-F”、“Fish-R”均可实现扩增，条带清晰单一，扩增和测序成功率均为100%；BOLD鉴定结果显示，26份鱼胶样品中23份能够确定物种来源（相似性达98%以上），包括石首鱼科12属15种鱼类，且多数为外来物种，另外3份鱼胶可推测其近缘物种。此外，系统发育树聚类分析结果与物种鉴定结果一致。结论　目前石首鱼类鱼胶来源物种较多，且多为外来基原鱼种。DNA条形码技术与BOLD鉴定系统相结合，可对大部分鱼胶进行准确的物种鉴定。     

Comparison of DNA Extraction and PCR Setup Methods for Use in High-Throughput DNA Barcoding of Fish Species

DNA barcoding is a sequencing-based method that can be used for the identification of fish species in a regulatory setting. The objective of this study was to compare modified versions of three DNA extraction kits (i.e., Qiagen DNeasy Blood and Tissue Kit, Sigma-Aldrich Extract-N-Amp Kit; and Life Technologies MagMax-96 DNA Multi-Sample Kit) and two polymerase chain reaction (PCR) setup methods (manual vs. automated) for use in DNA barcoding, with a focus on minimizing time, costs, and labor. DNA was extracted from 83 fish products using each of the three kits and the results were compared based on sequencing success and sequencing quality parameters. A subset of 14 fish products was also tested in triplicate to compare PCR setup methods. Initially, reduced sequencing success was observed with the MagMax Kit (88 %) compared to the other two kits (95–96 %); however, after PCR and sequencing were repeated for DNA samples that initially failed, all three methods showed very high sequencing success (98–99 %). Overall, the modified Extract-N-Amp Kit offered the greatest reduction in time and costs, while the DNeasy Blood and Tissue Kit produced sequences with the highest quality and highest initial success rates. Automation of the PCR setup process resulted in slightly greater success (100 %) compared to manual PCR setup (98 %), and reduced the potential for human error that may result from manual pipetting. The results of this study demonstrate the advantages of incorporating rapid and/or automated methods into the DNA barcoding workflow, especially with regard to high-throughput operations.     

13 种河豚鱼CO I基因部分DNA序列分析及在物种鉴定中的应用

应用CO I基因聚合酶链式反应（polymerase chain reaction,PCR）扩增通用引物,对福建省和海南省搜集的3?属13?种河豚鱼样品与9?个未知种名的河豚鱼样品的CO?I基因靶序列片段进行PCR扩增和测序,13?个已知物种样品的DNA序列提交基因库（GenBank）,取得相应的登录号。各物种CO I基因靶序列长度均为681 bp。应用DNAMAN V6软件对样品的DNA序列进行同源性比对分析,建立样品间系统关系同源树。基于CO I基因序列,供试13 个样品被划分为4?个类群组。群间的同源率为84%,群内同源率为90%～100%。根据序列同源性分析结果,9?个未知种名的样品被归类到2?个类群组中,判定这9?个样品为东方鲀属或腹刺鲀属,其中1?个样品（HNW2）为月腹刺鲀,4?个样品（HNW3、HNW4、FJW2、FJW5）为棕斑腹刺鲀,1?个样品（HNW1）为暗鳍腹刺鲀;3?个样品（FJW1、FJW3、FJW4）为横纹东方鲀。探讨基于DNA条形码技术的CO I基因靶序列片段在河豚鱼种属鉴别中应用的可能性。     

Universal mitochondrial 16s rRNA biomarker for mini-barcode to identify fish species in Malaysian fish products

Mislabelling in fish products is a highly significant emerging issue in world fish trade in terms of health and economic concerns. DNA barcoding is an efficient sequencing-based tool for detecting fish species substitution but due to DNA degradation, it is in many cases difficult to amplify PCR products of the full-length barcode marker (~650 bp), especially in severely processed products. In the present study, a pair of universal primers targeting a 198 bp sequence of the mitochondrial 16s rRNA gene was designed for identification of fish species in the processed fish products commonly consumed in Malaysia. The specificity of the universal primers was tested by both in-silico studies using bioinformatics software and through cross-reaction assessment by practical PCR experiments against the DNA from 38 fish species and 22 other non-target species (animals and plants) and found to be specific for all the tested fish species. To eliminate the possibility of any false-negative detection, eukaryotic endogenous control was used during specificity evaluation. The developed primer set was validated with various heat-treated (boiled, autoclaved and microwaved) fish samples and was found to show high stability under all processing conditions. The newly developed marker successfully identified 92% of the tested commercial fish products with 96–100% sequence similarities. This study reveals a considerable degree of species mislabelling (20.8%); 5 out of 24 fish products were found to be mislabelled. The new marker developed in this work is a reliable tool to identify fish species even in highly processed products and might be useful in detecting fish species substitution thus protecting consumers’ health and economic interests.