Development of a Species-Specific PCR Assay for Correct Labeling of Eight Squid Species from Loliginidae and Ommastrephidae Families (Mollusca: Cephalopoda)

Squid is an important seafood resource in Asian and European countries. Due to the different nutritional and market values of various squid species, substitutions between species often occur for higher profits. In this study, a species-specific PCR assay was developed for genetic discrimination of eight commercially important squid species from each other, based on the single nucleotide polymorphism (SNP) sites exploited from mitochondrial cytochrome oxidase subunit I (COI) sequences. The introduction of additional mismatches both in the forward and reverse primers ensured absolute allele specificity despite a moderate annealing temperature. The eight squid species can be clearly discriminated from each other by simplex PCR, and the developed multiplex species-specific PCR assay can detect 1% of intentional adulteration of non-target squid species into target species down to the 0.1 ng level of template DNA without PCR optimization. Therefore, the present study provides a simple and reliable genetic assay for identification of squid origin either in single or mixed species, and the developed assay will be useful in correct labeling and quality control of squid products.     

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.     

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.     

DNA barcoding,species-specific PCR for the identification of three stored-product pest species of genus Palorus (Coleoptera: Tenebrionidae)

Flour beetles of the genus Palorus (Coleoptera: Tenebrionidae) are important secondary storage pests. Correct identification of these insects is urgently required for integrated pest management. However, traditional morphological classification methods cannot identify insect fragments or immature stages and require skilled taxonomists with training and experience. In this study, three Palorus species were distinguished with two molecular techniques; DNA barcoding and species-specific PCR based on the mitochondrial DNA cytochrome oxidase subunit I (COI) gene. Forty-nine individuals of Palorus subdepressus (Wollaston), Palorus ratzeburgi (Wissman) and Palorus cerylonoides (Pascoe) were collected from 19 different locations across rice processing factory, feed factory, flour mill and bulk storages China. The COI sequences were analyzed by K2P distances and neighbor-joining (NJ) tree for DNA barcoding. The results show that these three Palorus species can be successfully identified by DNA barcoding. For the species-specific PCR study, we designed specificity primer pairs for the three Palorus species and evaluated their sensitivity. The results showed that the three Palorus species can be identified by the species-specific PCR method. Our results indicated that the two techniques can be independently used to identify the Palorus species in China for integrated pest management and quarantine. This is the first time Palorus species have been identified using two molecular techniques and the P. ratzeburgi and P. cerylonoides data have filled a gap in BOLD and GenBank.     

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。     

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。     

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。     

Molecular identification of cephalopod species by FINS and PCR-RFLP of a cytochrome b gene fragment

Identification of commercial species is a relevant issue to assure the correct labeling of seafood products. In this work two different molecular techniques, FINS (Forensically Informative Nucleotide Sequencing) and PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) were developed to identify 8 cephalopod species (families Loliginidae and Ommastrephidae) employing a fragment of the cytochrome b gene. DNA amplification for all of the species was carried out with a new set of specific primers designed in this study for cephalopods. FINS is a technique based on DNA sequencing, while PCR-RFLP allows direct species identification by comparing specific DNA restriction patterns. Both techniques are useful for cephalopod species identification. 17 food products (mainly "squid rings") were analyzed and the species employed for their manufacture identified by FINS.     

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.     

乌贼DNA条形码的筛选与验证

目的筛选适于鉴定乌贼的DNA条形码,建立鉴定舟山常见乌贼种类的DNA条形码技术体系。方法用聚合酶链反应(polymerase chain reaction, PCR)对现有5组DNA条形码引物组合进行了筛选,设计一对新的DNA条形码引物以供筛选备用。结果现有引物与部分乌贼的DNA模板存在错配而缺乏通用性,在部分乌贼DNA的扩增受阻。本实验设计的12S rRNA基因引物可以特异性扩增乌贼的DNA,提高了乌贼鉴定的准确度。结论本研究设计的12S rRNA基因引物可以作为现有乌贼DNA条形码鉴定的补充。     

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.     

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。     

4组鱼类DNA条形码引物的筛选与优化

目的通过对DNA条形码氧化酶亚基I基因(cytochrome oxidase subunit I,COI)的4组常用引物进行比较筛选,选择适于舟山鱼类鉴定的最佳引物。方法以舟山主产大黄鱼、小黄鱼、带鱼和乌贼样品DNA为模板,对4组常用DNA条形码引物进行PCR体系优化。通过比较PCR产物量、特异性、灵敏度和测序成功率,综合分析筛选最佳引物组。结果 4组引物均能扩增大黄鱼、小黄鱼和带鱼的DNA,对于乌贼DNA的扩增具有明显差异。COI优化引物具有易于扩增、测序简单等优势,更适于作为海产品鉴定的DNA条形码引物。结论本研究为舟山海产品的市场监管和实验室大量样品检测提供了技术参考。     

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。     

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.     

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.     

Isolation and molecular characterization of Vibrio parahaemolyticus from fresh, low-temperature preserved, dried, and salted seafood products in two coastal areas of eastern China

A total of 1293 seafood samples from fishing farm, retail markets, restaurants and cooking rooms of hotels in Jiangsu province and Shanghai city of China were collected and analyzed for the prevalence of Vibrio parahaemolyticus during July to October in 2007. Two hundred and fifty one isolates of V. parahaemolyticus were identified, of which 8 isolates were positive for tdh and 2 were positive for trh gene. Three tdh positive isolates were identified from low-temperature preserved seafood samples and 5 isolates from fresh seafood samples, of these tdh positive isolates, 3 were positive in ORF8-PCR test. The genetic diversity among V. parahaemolyticus isolates was assessed using random amplified polymorphic DNA (RAPD)-PCR and the results showed that there were 33 different genetic patterns that were clustered into nine groups (groups A to I) at 82% similarity level. About 31.9% of the isolates belong to type III9d that were widely distributed in fresh, iced, frozen, dried and salted seafood samples. Seven tdh positive isolates belonged to group A and one belonged to group C, 2 trh positive isolates were type I10d belonging to group F, which was identical to that of reference strains isolated from patients. This study demonstrated genetic variability within V. parahaemolyticus isolates from seafood in Chinese markets and confirmed the presence of toxigenic V. parahaemolyticus not only in fresh but also in iced and frozen seafood products indicating that low-temperature preserved seafood might be also a vehicle for transmitting pathogenic V. parahaemolyticus.     

Authentication of swordfish (<Emphasis Type="Italic">Xiphias gladius</Emphasis>) by RT–PCR and FINS methodologies

In the present study, two methods for the authentication of swordfish (Xiphias gladius) were developed. The first one is based on a TaqMan probe real-time PCR technology using the cytochrome oxidase subunit I (COI), while the second one is based on the phylogenetic analysis of DNA sequences (forensically informative nucleotide sequencing, FINS) using the cytochrome b (cyt b) gene fragment. Both techniques can be applied depending on the laboratory equipment and allow the detection of fraudulent or unintentional mislabelling of this species. The developed methodologies were validated and subsequently were applied to 30 commercial samples labelled as swordfish or X. gladius in order to determinate whether the species used for their manufacturing corresponded to this species. These tools are useful to clarify questions related to the correct labelling of commercial products and to verify the correct traceability in commercial trade and for fisheries control.     

Listeria species in seafood: isolation and characterization of Listeria spp. from seafood in San Luis, Argentina

One hundred seafood samples (fish, squid and mussel) were collected from the Argentine Atlantic coast and screened for Listeria spp. The isolates were characterized by biochemical tests, serotyping, phage typing and macrorestriction enzymes analysis of DNA (pulsed-field gel electrophoresis). The overall frecuency (n=100) ofListeria spp. was 12%. Of the 12 isolated strains, three strains isolated from different squid samples were identified as L. monocytogenes and nine strains from fish, mussels and squid as L. innocua. All three L. monocytogenes strains belonged to serovar 4b; eight strains of L. innocua were serovar 6a and one strain of L. innocua was serovar 6b. All three L. monocytogenes strains, but only one of the nine L. innocua strains, were phage-typeable. One restriction profile was detected with Apa I and Asc I for L. monocytogenes strains and three with the same enzymes for L. innocua strains. The combination of these patterns allowed definition of four distinct groups within the 12 strains. The results of this study showed that phenotypic methods remain appropriate but that pulsed-field gel electrophoresis is useful for epidemiological purposes.     

Recent trends for the employment of jumbo squid (Dosidicus gigas) by-products as a source of bioactive compounds with nutritional,functional and preservative applications: a review

Only 50%–60% of total seafood catch is used for human consumption, seafood processing being considered as one of the main sources of by-products. Among marine species, jumbo squid (JS; Dosidicus gigas) represents the most important squid fishery, showing an increasing economic interest in many countries. As for any other marine species, the regular cleaning, dressing and processing produce high quantities of by-products (skin, head, fins, viscera, tentacles, unclaimed mantle, etc.) that are rich in many nutrients (proteins, lipids, minerals, vitamins, enzymes, biopolymers, etc.). This review compiles information about extraction and employment of JS by-products with the aim of enhancing their economic value and reduce environmental drawbacks. A special emphasis is given to the relevance in developing methods susceptible to transform by-products into useful and profitable products susceptible to be applied in several industries such as food, medicine, agrochemical or pharmaceutical. Future possible trends for widening this profitable use are mentioned.