Authentication of raw and processed tuna from Indonesian markets using DNA barcoding,nuclear gene and character-based approach

Establishing seafood authentication methods is an important task for fisheries research laboratories and food control authorities. Nowadays, the extent of fish species substitution is suspected being greater than ever before in commercial markets. In order to provide reliable polymerase chain reaction (PCR)-based authentication systems for tunas, we collected and analyzed authentic tuna reference samples and tuna-food products from Indonesian markets. Our analytical methods mainly relied on identification using the mitochondrial cytochrome c oxidase subunit I (COI) gene, as a genetic marker for “DNA barcoding,” as well as the rhodopsin (RH1) gene as a nuclear marker. Additionally, we identified species-specific nucleotide diagnostic positions (characters) to complete the results obtained basic local alignment search (BLAST) and phylogenetic analysis. Authentication results of tuna-food products showed relatively successful amplification for the COI gene; RH1 acted as an alternative solution for some of the samples, which had failed to react in COI-PCR. Species of the genus Thunnus could not be unambiguously differentiated by BLAST and phylogenetic analysis (neighbor-joining tree) in all cases due to the high similarity of the COI sequences. However, the character-based identification method was found to be helpful for species assignment in case of tuna-food products. Therefore, our findings demonstrated that the COI gene could be more reliable used as a tool for Indonesian commercial tuna products authentication, if the sequencing results were combined with the character-based identification using differences at certain nucleotide positions.     

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.     

Differentiation of fish species by PCR-based DNA analysis of nuclear genes

Food control laboratories are confronted with new challenges in respect to fishery product authentication; examples are identification of hybrids (e.g. in case of catfish, tilapia, sturgeon, snapper) and assignment of fish to certified stocks. Against this background, differentiation of fish species and populations by polymerase chain reaction (PCR)–based DNA analysis of nuclear genes has become of considerable importance as a tool for the completion of mitochondrial gene analysis. Four applications of nuclear gene analysis are presented: (1) fish species identification using the intronless rhodopsin RH1 gene; (2) detection of “fish” as an allergenic foodstuff by means of universal primers amplifying a segment of a parvalbumin gene; (3) differentiation of cod (Gadus morhua) from various fishing grounds by exon-primed intron-crossing PCR of a parvalbumin gene intron; (4) detection of hybridization between North Atlantic redfish species (genus: Sebastes) by restriction fragment length polymorphism analysis of amplicons obtained from the second intron of the 7S ribosomal protein gene.     

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。     

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。     

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.     

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.     

Molecular identification of dried squid products sold in China using DNA barcoding and SYBR green real time PCR

ABSTRACT

Dried squid products are popular in China as a snack food, side dishes, or refreshments, and the market appeal can be reflected by the high price that occasionally reaches 497 RMB per kg. However, the absence of harmonisation around the definition of squid, as well as the problems with visual inspection for processed seafood products, make alternative species substitution for dried squid products a frequent occurrence. The aim of the present study was to apply a DNA barcoding approach for species identification of 48 dried squid products collected from the largest online shopping platform in China. Moreover, we also developed a novel SYBR green real-time PCR assay (simplex and duplex followed by a melting curve analysis) specific for Illex argentinus and Todarodes pacificus based on cytochrome C oxidase subunit I (COI) gene.

Results highlighted the successful DNA extraction and PCR amplification of a 655 bp COI gene fragment from all products. A maximum similarity value in the range of 98-100% was obtained for all readable sequences using the BOLD and BLAST public databases and four species (Dosidicus gigas, Uroteuthis edulis, I. argentinus, and T. pacificus) were identified. The specificity of the designed primer sets was confirmed against 23 non-target species, and the newly developed methods were successfully applied to screen I. argentinus and T. pacificus in dried squid products.

Overall, DNA barcoding is a robust tool for seafood species identification and the novel method is effective in screening I. argentinus and T. pacificus in food products.     

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.     

STABILITY OF FISH MUSCLE PARVALBUMINS AT DIFFERENT pH VALUES

Parvalbumins of herring (Clupea harengus) and carp (Cyprinus carpio) are essential components of the pattern ofsarcoplasmic proteins used for fish species identification by isolelectric focusing. In fishery products oflowpH, like marinated herring (pH ～ 4), parvalbumins were not detectable. It was demonstrated for herring and carp that parvalbumins were not precipitated at pH 4, but were degraded by fish muscle proteases.     

Tetrodotoxin detection and species identification of pufferfish in retail roasted fish fillet by DNA barcoding in China

This study identifies the pufferfish species and detects tetrodotoxin (TTX) in roasted fish fillet samples collected in Beijing, Qingdao and Xiamen, China. The cytochrome c oxidase I (COI) gene was used as the target gene for identification of the pufferfish species in the samples. Enzyme-linked immunosorbent assay (ELISA) screened the TTX levels in samples that had been detected as containing pufferfish by DNA barcode. A total of 125 samples were identified by DNA barcodes; 32 (26%) samples contained pufferfish composition and, among them, 26 (81%) were the highly toxic species Lagocephalus lunaris. All 32 samples containing the pufferfish composition were positive for TTX with levels ranging from 100 to 63 800 ng g^–1. Most of the 32 samples contained the highly toxic L. lunaris. Based on the results, we suggest that the monitoring of roasted fish fillet should be strengthened and the processing procedures should be standardised to minimise TTX poisoning caused by pufferfish.     

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.     

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.     

Potential use of DNA barcodes in regulatory science: applications of the Regulatory Fish Encyclopedia

The use of a DNA-based identification system (DNA barcoding) founded on the mitochondrial gene cytochrome c oxidase subunit I (COI) was investigated for updating the U.S. Food and Drug Administration Regulatory Fish Encyclopedia (RFE; http://www.cfsan.fda.gov/-frf/rfe0.html). The RFE is a compilation of data used to identify fish species. It was compiled to help regulators identify species substitution that could result in potential adverse health consequences or could be a source of economic fraud. For each of many aquatic species commonly sold in the United States, the RFE includes high-resolution photographs of whole fish and their marketed product forms and species-specific biochemical patterns for authenticated fish species. These patterns currently include data from isoelectric focusing studies. In this article, we describe the generation of DNA barcodes for 172 individual authenticated fish representing 72 species from 27 families contained in the RFE. These barcode sequences can be used as an additional identification resource. In a blind study, 60 unknown fish muscle samples were barcoded, and the results were compared with the RFE barcode reference library. All 60 samples were correctly identified to species based on the barcoding data. Our study indicates that DNA barcoding can be a powerful tool for species identification and has broad potential applications.     

高通量二代测序基因条形码技术在动物源性制品物种鉴定中的应用

该研究利用二代测序和DNA条形码技术对生物制品中动物源性成分鉴定方法进行探索性研究。首先对常见的哺乳动物、禽类、鱼类物种以及多种混合样本进行了核酸提取,并利用PCR技术对其线粒体基因16S rRNA区域354 bp的检测位点进行扩增。使用Illumina Miseq二代测序技术获取所有序列信息,并与Gen Bank数据库比对分析样本中的物种组成。结果显示,混合样品中所有动物源性成分均得到正确鉴定;鹅和鸭核酸的最低检测下限为0.5ng/μL;市售商业化动物源性制品经检测发现2起标签不符问题。该检测方法在一个高通量测序和结果比对分析实验周期内,实现了混合DNA片段序列信息的一次性获取。检测结果准确,适用范围广,有望为动物制品标识符合性检查、打击走私等方面提供技术保障。     

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.     

Molecular identification of five commercial flatfish species by PCR–RFLP analysis of a 12S rRNA gene fragment

Refrigerated or frozen fillets of commercial flatfish species are sometimes mislabelled, and identification of those products is needed to avoid fraudulent substitution. Molecular identification of five commercial flatfish species (order Pleuronectiformes), ie Lepidorhombus whiffiagonis (megrim), Platichthys flesus (flounder), Reinhardtius hippoglossoides (Greenland halibut), Scophthalmus maximus (turbot) and Solea vulgaris (= S solea) (sole), has been carried out on the basis of the amplification of an approximately 433 bp segment from the mitochondrial 12S rRNA gene using the polymerase chain reaction (PCR) and universal primers. Direct DNA sequencing from two PCR products for each flatfish species was carried out, and sequences were used to select six restriction enzymes. PCR products of 15 individuals of each species were cut with each enzyme, resulting in species‐specific restriction fragment length polymorphism (RFLP). The five flatfish species could be identified by application of the restriction enzyme AluI as well as by using different combinations of a pair of enzymes, ie DdeI and either AciI or MwoI. No intraspecific genetic polymorphism was found for any of the six enzymes. Results confirmed the usefulness of this technique to distinguish and genetically characterise refrigerated or frozen pieces of these five flatfish species. Copyright © 2003 Society of Chemical Industry     

Identification of meat species by PCR-RFLP of the mitochondrial COI gene

Meat authenticity verification is pertinent for economical, religious or public health concerns. The present study investigates the use of PCR-RFLP of a part of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene for identification of species origin of raw meat samples of cow, chicken, turkey, sheep, pig, buffalo, camel and donkey. PCR yielded a 710-bp fragment in all species. The amplicons were digested with seven restriction endonucleases (Hind II, Ava II, Rsa I, Taq I, Hpa II, Tru 1I and Xba I) that were selected based on the preliminary in silico analysis. Different levels of polymorphism were detected among samples. The level of COI variation revealed using only Hpa II was sufficient to generate easily analyzable species-specific restriction profiles that could distinguish unambiguously all targeted species. Compared to previously published reports for the determination of meat origin at the molecular level, the approach developed here is much cheaper and faster for routine identification of meats in food control laboratories.     

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。     

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。