Detection and identification of marine fish mislabeling in Guangzhou's supermarkets and sushi restaurants using DNA barcoding

In this study, DNA barcoding was applied to identify the distinct species of fish products in Guangzhou supermarkets and sushi restaurants in order to confirm whether products were correctly labeled. Samples were analyzed using mitochondrial cytochrome C oxidase subunit I (CO I) gene as the target. Our results showed that the CO I gene of all 139 samples examined was successfully amplified by PCR. When sequenced, 30 samples (21.58%) were mislabeled as the wrong species, 11 samples had insufficient information provided on the label to determine if the labeling was correct (7.91%), and four samples failed sequencing (2.88%). We also found that the use of proper labels for fish products in sushi restaurants was higher than that in supermarkets. As a simple, rapid, and efficient technology, DNA barcoding can be widely used for species identification of fish products. Our work shows that regulation of the labeling of fish products, as we evaluated in Guangzhou and other markets in China, is needed on a global scale.     

Polychlorinated biphenyls in salmon and salmon feed: global differences and bioaccumulation

Concentrations of 160 polychlorinated biphenyl (PCB) congeners or congener groups were determined in approximately 600 farmed Atlantic salmon from around the world and wild (ocean-caught) Pacific salmon from the Northeast Pacific. Concentrations and PCB congener profiles were analyzed to provide insight into the sources and uptake of PCBs in salmon as well as regional differences. Although total PCB concentrations in wild salmon appeared to be correlated to total lipid content, the increased proportion of total lipids in the farmed salmon could not account for the much greater PCB concentrations. We investigated the PCB congener patterns of hundreds of salmon samples using principal component analysis to further illuminate regional and species differences. Three major PCB patterns were observed, in most wild fish (except British Columbia and Oregon chinook), in farmed fish from the Atlantic, and in most farmed fish from the Pacific. The PCB congener profiles of farmed salmon often closely corresponded to a sample of commercial feed purchased in the same region, indicating that the feed is likely to be the major source of PCBs for farmed salmon. In such cases where PCB profiles in fish and feed were similar, a comparison of congener concentrations in fish and the feed showed that the majority of congeners, with some notable exceptions, were bioaccumulative to the same extent, irrespective of physical properties.     

DNA barcoding detects market substitution in North American seafood

Seafood authentication and food safety concerns are a growing issue in today’s global marketplace, although traditional morphology-based identification keys and existing molecular approaches have limitations for species identification. Recently, DNA barcoding has gained support as a rapid, cost-effective and broadly applicable molecular diagnostic technique for this purpose. However, the maturity of the barcode database as a tool for seafood authentication has yet to be tested using real market samples. The present case study was undertaken for this reason. Though the database is undergoing continual development, it was able to provide species matches of >97% sequence similarity for 90 of 91 samples tested. Twenty-five percent of the samples were potentially mislabeled, demonstrating that DNA barcodes are already a powerful tool for the identification of seafood to the species level. We conclude that barcodes have broad applicability for authenticity testing and the phylogeographic patterning of genetic diversity can also inform aspects of traceability.     

Lipid composition and contaminants in farmed and wild salmon

Levels of omega-3 (n-3) and omega-6 (n-6) fatty acids and lipid-adjusted concentrations of polychlorinated biphenyls (PCBs), dioxins, toxaphene, and dieldrin were determined in 459 farmed Atlantic salmon, 135 wild Pacific salmon, and 144 supermarket farmed Atlantic salmon fillets purchased in 16 cities in North America and Europe. These were the same fish previously used for measurement of organohalogen contaminants. Farmed salmon had greater levels of total lipid (average 16.6%) than wild salmon (average 6.4%). The n-3 to n-6 ratio was about 10 in wild salmon and 3-4 in farmed salmon. The supermarket samples were similar to the farmed salmon from the same region. Lipid-adjusted contaminant levels were significantly higher in farmed Atlantic salmon than those in wild Pacific salmon (F = 7.27, P = 0.0089 for toxaphene; F = 15.39, P = 0.0002 for dioxin; F > or = 21.31, P < 0.0001 for dieldrin and PCBs, with df = (1.64) for all). Levels of total lipid were in the range of 30-40% in the fish oil/fish meal that is fed to farmed salmon. Salmon, especially farmed salmon, are a good source of healthy n-3 fatty acids, but they also contain high concentrations of organochlorine compounds such as PCBs, dioxins, and chlorinated pesticides. The presence of these contaminants may reduce the net health benefits derived from the consumption of farmed salmon, despite the presence of the high level of n-3 fatty acids in these fish.     

A multiplex PCR method for the identification of commercially important salmon and trout species (Oncorhynchus and Salmo) in North America

Abstract: The purpose of this study was to develop a species-specific multiplex polymerase chain reaction (PCR) method that allows for the detection of salmon species substitution on the commercial market. Species-specific primers and TaqMan® probes were developed based on a comprehensive collection of mitochondrial 5′ cytochrome c oxidase subunit I (COI) deoxyribonucleic acid (DNA) “barcode” sequences. Primers and probes were combined into multiplex assays and tested for specificity against 112 reference samples representing 25 species. Sensitivity and linearity tests were conducted using 10-fold serial dilutions of target DNA (single-species samples) and DNA admixtures containing the target species at levels of 10%, 1.0%, and 0.1% mixed with a secondary species. The specificity tests showed positive signals for the target DNA in both real-time and conventional PCR systems. Nonspecific amplification in both systems was minimal; however, false positives were detected at low levels (1.2% to 8.3%) in conventional PCR. Detection levels were similar for admixtures and single-species samples based on a 30 PCR cycle cut-off, with limits of 0.25 to 2.5 ng (1% to 10%) in conventional PCR and 0.05 to 5.0 ng (0.1% to 10%) in real-time PCR. A small-scale test with food samples showed promising results, with species identification possible even in heavily processed food items. Overall, this study presents a rapid, specific, and sensitive method for salmon species identification that can be applied to mixed-species and heavily processed samples in either conventional or real-time PCR formats. Practical Application: This study provides a newly developed method for salmon and trout species identification that will assist both industry and regulatory agencies in the detection and prevention of species substitution. This multiplex PCR method allows for rapid, high-throughput species identification even in heavily processed and mixed-species samples. An inter-laboratory study is currently being carried out to assess the ability of this method to identify species in a variety of commercial salmon and trout products.     

Differentiation of the rainbow trout (Oncorhynchus mykiss) from Atlantic salmon (Salmon salar) by the AFLP-derived SCAR

A species-specific SCAR marker for rainbow trout, which was used to detect adulteration and fraudulent labeling in Atlantic salmon products, has been developed based on the AFLP analysis and evaluated in this study. The SCAR marker could be amplified and visualized in 1% agarose gel in all tested rainbow trout samples and absent in all salmon samples. Using DNA admixtures, the detection of 1% (0.5 ng), 10% (5 ng) rainbow trout DNA in Atlantic salmon DNA for fresh and processed samples, respectively was readily achieved. The molecular approach was sensitive and demonstrated to be a rapid and reliable method for identifying frauds in salmon products and could be extended for applications of species identification in food industry.     

基于鸟枪蛋白组学与质谱多反应监测技术的三文鱼物种鉴别研究

目的 三文鱼肉质鲜美, 营养丰富, 深受广大消费者喜爱,在经济利益驱动下, 不少商家用低值三文鱼如大马哈鱼和虹鳟冒充高值三文鱼大西洋鲑, 严重扰乱了水产市场秩序。方法 以市场上常见的大西洋鲑、大马哈鱼和虹鳟3个物种为研究对象,基于鸟枪蛋白组学分析流程,样品经蛋白质提取、胰蛋白酶消化和超高效液相色谱串联三重四级杆飞行时间质谱仪分离鉴定, 得到的总离子流图谱与Uniprot蛋白质数据库对比分析, 筛选得到3个物种的特征肽段,利用Skyline软件构建特征肽的多反应监测（MRM）离子对,然后经三重四极杆质谱仪进行鉴定。结果 最终共筛选出7条特征肽段, 包括在大西洋鲑肌肉中找到的4条特征肽段, 在大马哈肌肉中找到的1条特征肽段, 在虹鳟肌肉中找到的2条特征肽段。结论 基于鸟枪蛋白组学,结合MRM技术,筛选得到了大西洋鲑、大马哈鱼和虹鳟3个物种中的特征标记肽段,建立了不同种类三文鱼的物种精准定性鉴别方法。     

三文鱼水产品掺假情况调查

目的调查广东省市售三文鱼水产品掺假情况。方法应用实时荧光PCR技术检测大西洋鲑鱼和虹鳟鱼的方法,对广东省内批发市场、超市、餐饮寿司店和网络平台等场所的三文鱼掺假情况进行调查。结果98批次样品中,有6批次未检出大西洋鲑鱼,未检出率为6.12%。通过PCR测序结果鉴定,这6批次未检出大西洋鲑鱼的样品中,4批次为虹鳟鱼,1批次为王鲑,1批次为银鲑。结论三文鱼水产品掺假情况较少,但掺假风险仍然存在,建议监管部门加强监管。     

Use of terrestrial based lipids in aquaculture feeds and the effects on flesh organohalogen and fatty acid concentrations in farmed Atlantic salmon

Consumption of salmon, wild or farmed, has been encouraged by many scientists and by national and international health organizations due to the potential health benefits associated with their high contents of omega-3 (n-3) highly unsaturated fatty acids (n-3 HUFAs). In 2004, there was increased public concern regarding the safety of farmed Atlantic salmon following the publication of several studies that indicated higher levels of organohalogens in their flesh relative to those noted in the flesh of wild Pacific salmon. Farmed salmon obtain most of these contaminants from the consumption of marine fish oil (MFO) present in salmon feed. In both a laboratory feeding trial and an on-farm field study, partial replacement of MFO in aquaculture feeds with economical and abundant lipids of terrestrial origin resulted in farmed Atlantic salmon with reduced flesh polychlorinated biphenyl and polychlorinated dibenzodioxin and furan concentrations. Flesh levels of n-3 HUFAs (g/(100 g serving)) were lower in farmed Atlantic salmon fed diets with alternative lipids relative to farmed salmon fed more traditional MFO-based diets. However, the former salmon were found to have higher flesh levels of n-3 HUFAs and also similar or lower flesh levels of organic contaminants than some species of market-size wild Pacific salmon. These findings showthat consumption of either farmed Atlantic salmon or wild Pacific salmon can meet recommended weekly n-3 HUFA levels with minimal concurrent intake of flesh organohalogens.     

Development of real-time PCR assays for the detection of Atlantic cod (Gadus morhua), Atlantic salmon (Salmo salar) and European plaice (Pleuronectes platessa) in complex food samples

We have developed species-specific real-time PCR assays for the identification of Atlantic cod (Gadus morhua), Atlantic salmon (Salmo salar) and European plaice (Pleuronectes platessa) in food products. The species-specific assays, comprising a set of primers and probe for each species, were designed using genomic genes (pantophysin for Atlantic cod, growth hormone for Atlantic salmon and parvalbumin for European plaice) which were then optimised for specificity and selectivity. The sensitivity and the effect of heat and pressure on amplification efficiency were then determined for each assay. These assays were then used to analyse DNA extracted from commercial fish products and model food samples spiked with each of the fish species. The target species was successfully identified in all samples analysed, demonstrating the applicability of these assays to the analysis of food products.     

Selected polycyclic aromatic hydrocarbons in smoked tuna, swordfish and Atlantic salmon fillets

The presence of polycyclic aromatic hydrocarbons (PAHs) in commercial smoked fillets of tuna, swordfish and Atlantic salmon was studied. In Europe, the smoking technique is widely used in fish processing and these species are the most representative of the taste of consumers for smoked products. Samples were purchased on the Italian market and analysed by HPLC. Mean concentrations (ng g⁻¹) of acenaphthene (4.4, 6.2, 11.2), phenanthrene (11.5, 18.5, 8.9), anthracene (2.4, 5.2, 1.8), fluoranthene (17.0, 9.4, 4.7), benzo( k )fluoranthene (0.3, 0.1, 0.2) and benzo( a )pyrene (1.3, 0.1, 0.4) were found in Atlantic salmon, tuna and swordfish respectively. Benzo( b )fluoranthene (1.2 ng g⁻¹) was detected only in Atlantic salmon whereas dibenz( a,h )anthracene was never detected in this species, but only in tuna (0.5 ng g⁻¹) and swordfish (1.1 ng g⁻¹). Atlantic salmon contained the highest level of benzo( a )pyrene (2.8 ng g⁻¹) which is below the European regulatory level of 5 ng g⁻¹.     

SPECIES SUBSTITUTION OF RETAIL SNAPPER FILLETS

A total of 121 retail market snapper fillets were collected throughout Florida and tested for compliance with labeling regulations. Samples were identified as to species by isoelectric focusing techniques against 12 authentic snapper species. Of the 81 red snapper samples, 24 (30%) were confirmed as real red snapper (Lutjanus campechanus), while 57 (70%) were mislabeled. Most of the other snapper species were correctly labeled. The major substitute for red snapper was scarlet snapper (Lutjanus sanguineus), an imported red-skin Pacific snapper species. Nomenclature of fish sold in the market is confusing since the same species of fish is frequently sold by several different names.     

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。     

Microbial communities on Australian modified atmosphere packaged Atlantic salmon

The role of specific spoilage organisms (SSO) in products such as Atlantic salmon has been well documented. However, little is known about what other micro-organisms are present and these organisms may indirectly influence spoilage by their interactions with the SS0. We used a combination of culture-based and DNA-based methods to explore the microbial communities found on Atlantic salmon fillets packed in a modified atmosphere of carbon dioxide and nitrogen. After 15 days the communities were dominated by Shewanella spp. or Carnobacterium spp. and a variety of other genera were present in smaller numbers. Variability in the microbial community composition in packages processed on the same day was also observed. This was mostly due to differences in the presence of minor members of the community including species from genera such as Iodobacter, Serratia, Morganella and Yersinia. The combination of culture-based and culture-independent methods provided greater insight into the development of microbial communities on Atlantic salmon than would have been possible using only one method. This work highlights the potential importance of lactic acid bacteria (LAB) in fresh Atlantic salmon stored under modified atmosphere conditions.     

Flesh quality of market-size farmed and wild British Columbia salmon

This study compared the flesh quality of farmed and wild sources of British Columbia (BC) salmon with respect to concentrations of polychlorinated biphenyl compounds, polychlorinated dibenzodioxins/dibenzofurans and their associated toxic equivalents, total mercury (THg), methylmercury (MeHg), and selected fatty acids of known importance for human health viz., omega-3 (n-3) highly unsaturated fatty acids (n-3 HUFAs) and (n-6) fatty acids. Skinned fillets from known sources of farmed Atlantic, coho, and chinook salmon (n = 110) and wild coho, chinook, chum, sockeye, and pink salmon (n = 91) were examined. Atlantic salmon contained higher PCB concentrations (means, 28-38 ng/g) than farmed coho or chinook salmon, and levels in these latter species were similar to those in wild counterparts (means, 2.8-13.7 ng/g). PCB levels in Atlantic salmon flesh were, nevertheless, 53-71-fold less than the level of concern for human consumption of fish, i.e., 2000 ng/g as established by Health Canada and the U.S. Food and Drug Administration (US-FDA). Similarly, THg and MeHg levels in all samples were well below the Health Canada guideline (0.5 microg/g) and the US-FDA action level (1.0 microg/g). On average, THg in farmed salmon (0.021 microg/g) was similar to or lower than wild salmon (0.013-0.077 microg/g). Atlantic salmon were a richer source (mean, 2.34 g/100 g fillet) of n-3 HUFAs than the other farmed and wild sources of salmon examined (means, 0.39-1.17 g/100 g). The present findings support the recommended weekly consumption guidelines for oily fish species (includes all BC salmon sources) for cardio-protective benefits as made by the American Heart Association and the UK Food Standards Agency.     

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。     

应用DNA条形码技术对深圳市售花胶鱼种鉴定与分析

目的 采用基于DNA条形码技术对深圳市售花胶鱼种进行鉴定与分析。方法 以细胞色素C氧化酶Ⅰ(cytochrome c oxidase, COⅠ)基因为目标基因,应用DNA条形码技术鉴别深圳各药房和超市零售花胶的种类来源。结果 94份花胶样品均扩增出特异性条带。根据BOLD系统鉴定结果统计,深圳地区市售花胶94份样品中,按来源鱼种区分,基原鱼种(鉴定到属)为尼罗尖吻鲈和尖吻鲈属占比29.79%,其次为苏里南犬牙石首鱼占比15.96%,以及双棘原黄姑鱼/褐毛鲿占比8.5%。按来源鱼种所属的科区分,石首鱼科共计41个,占比43.62%;尖吻鲈科共计28个,占比29.79%;鳕科共计7个,占比7.45%。结论 目前我国花胶基原鱼种以石首鱼科为主,外来基原鱼种增多。深圳市售花胶存在真伪混淆现象,DNA条形码技术可用于花胶的来源物种鉴定。     

Detection of Transgenic Atlantic and Coho Salmon by Real-time PCR

Genetic modifications (GM) have been applied to salmon to generate fast-growing strains for potential use in aquaculture. In November 2015, the first transgenic salmon (AquAdvantage® Atlantic salmon) was accepted for commercialization in the USA under defined conditions. The presence of GM food products in the marketplace stimulates the need for detection methods to allow screening for the presence of genetic modifications in seafood products. This paper first shows that it is possible to obtain amplifiable DNA from raw and processed products containing salmon. Detection methods by real-time PCR are proposed in this work. An endogenous gene target was designed to detect salmonid species DNA in samples. In addition, detection methods using real-time PCR were developed for two GM salmon possessing growth hormone transgenes: the AquAdvantage® Atlantic salmon (Salmo salar) developed by AquaBounty for commercial purposes, and the coho salmon (Oncorhynchus kisutch) developed for research purposes by Fisheries and Oceans Canada. The methods are able to detect at least 20 copies of the target. It was found however that one of the construct-specific methods for the AquAdvantage® salmon detection did not work on AquAdvantage® genomic DNA even though it works on the sequence published in GenBank. The other assay however was found to reliably detect AquAdvantage® transgenic sequences in genomic DNA.     

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.     

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)作为DNA条形码在鱼唇制品物种鉴定中的适用性。方法 对全国31个城市购买的252份鱼唇样品进行聚合酶链式反应(polymerase chain reaction, PCR)测序,同源基因比较分析,构建系统发育树,鉴定制作鱼唇产品的鱼种,并对其进行濒危评价分析。结果 成功鉴定250个样品,一致性物种基因序列相似性在99%以上,涉及8个鲨鱼物种,最多样品为大青鲨(Prionace glauca),占样品65.5%,其余还有镰形真鲨(Carcharhinus falciformis)、路氏双髻鲨(Sphyrna lewini)、锤头双髻鲨(Sphyrna zygaena)等7类鲨鱼物种。结论 Cyt b可以作为对鲨鱼物种进行鉴定的一种DNA条形码,在对鲨鱼种鉴定时可以使用Cyt b基因及细胞色素氧化酶亚基I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。