White fish authentication by COIBar-RFLP: Toward a common strategy for the rapid identification of species in convenience seafood

The global trade and the increased demand for seafood products have encouraged the common practice of replacement of valuable species with species of lower value worldwide. The species of the genus Merluccius are often subject to fraudulent substitution due to their high commercial interest. The present investigation of labeling accuracy on 54 samples taken from 20 convenience seafood products collected from Southern Italy markets, allowed the identification of four species through DNA barcoding: Gadus chalcogrammus, Merluccius merluccius, Merluccius productus and Merluccius paradoxus. Mislabeling was observed in seven of 20 (35%) products (frozen breaded steaks and fish fingers), six of which (30%) were labeled as hake (M. merluccius). To reduce analysis time of fish species authentication, a COIBar-RFLP, using DNA barcoding in combination with PCR-RFLP methods, was performed for species discrimination. The restriction enzyme HinfI yielded differential digestion patterns suitable for unveiling inconsistencies between product labels and genetic species identification. The COIBar-RFLP represents an effective tool for fish authentication in convenience seafood and responds to the emerging interest in molecular identification technologies that reduce processing time and eliminate the need for lab-based DNA sequencing.     

Larval migration of the zoonotic parasite Anisakis pegreffii (Nematoda: Anisakidae) in European anchovy,Engraulis encrasicolus: Implications to seafood safety

Anisakid nematodes belonging to the species Anisakis pegreffii are distributed in a wide variety of fishes from the Mediterranean Sea and they are known to cause the human zoonosis anisakiasis. The present study investigated, for the first time, the response of A. pegreffii larvae (identified to species level by allozymes and mtDNA cox2 sequence analysis) to the storage temperature of European anchovies, Engraulis encrasicolus. The larval motility of A. pegreffii was studied in 1300 fish specimens, captured from a highly infested area (FAO 37.2.1, 43°8′N, 14°16′E), maintained under different temperatures (2 °C, 5 °C, 7 °C), and examined at different time intervals (immediately after fishing, 24 h, 48 h and 72 h). Parasitological analysis was carried out with the UV-press method. The results showed that the increase of infection values with A. pegreffii in the fillets of anchovies was statistically positively related to the increase of the temperature (at 5 °C and 7 °C) and time of storage (after 24 h, 48 h, and 72 h). Accordingly, a significant statistical correlation between the increasing of the worm burden in the fillets and a decreasing of A. pegreffii in the viscera was observed. In contrast, those fish constantly maintained at 2 °C showed no statistically significant variation in infection either in the viscera or the fillets, after 24, 48 and 72 h. In the same batches of anchovies, larvae of the non-zoonotic nematode parasite Hysterothylacium aduncum (identified to the species level by ITS rDNA sequences analysis) were found, but they were never observed infecting the musculature of the anchovies. Our results suggest that temperature plays an important role in the post-mortem motility of A. pegreffii larvae in anchovies. In addition, the presence of A. pegreffii in the fillets inspected immediately after their capture indicates that intra-vitam migration may also occur. As a consequence, the importance of the adoption of rules to prevent human anisakiasis, as the deep freezing to −20 °C for 24 h, was underlined.     

Traceability technologies for farm animals and their products in China

The history of traceability reveals that nomadic herders as early as 1000 BCE marked livestock with irons and ear incisions in order to protect against thefts. Nowadays, we build traceability systems to document the origin of foods, and in order to ensure safer foods when tracking and recalling products. A holistic traceability system includes, as a minimum, identification elements, databases and an information flow. The animal identification elements refers to body marks, ear tags, Radio-Frequency Identification (RFID) tags, retina image recognition, or DNA fingerprinting. The product identification refers to barcodes (EAN UCC, PLU, and GS1), 2D barcodes (QR, VC, and DM) and RFID or Electronic Product Code (EPC). The present review describes existing and upcoming traceability technologies for farm animals and their products, to update the common methods for information collection and data inquiry, with the view to expound traceability policies and regulations between developed and developing countries. The benefits of the new technologies and their practical limitations are also discussed.     

Inactivation of Anisakis pegreffii larvae in anchovies (Engraulis encrasicolus) by salting and quality assessment of finished product

There is an increasing reporting of Anisakiasis in humans at the world level and this disease has become a concern for the public health and for the fish and derived product trade. Humans acquire the infection by the ingestion of live larvae present in raw or almost raw (e.g., marinate, salted) fish products if the processing is insufficient to devitalize the worms. The aim of this study was to asses a dry salting process in killing Anisakis pegreffii larvae in naturally infected European anchovies (Engraulis encrasicolus) and to evaluate the quality assessment. The results show that a dry salting process with a salt concentration of 21% in all parts of the anchovy fillets devitalize A. pegreffii larvae in a 15 day period. The finished product showed a good panel acceptance and anchovies reached a good quality grade.     

A unique specification method for processed unicorn filefish products using a DNA barcode marker

The present study evaluated and compared the utility of the cytochrome b and cytochrome c oxidase I genes as mitochondrial markers for determining the original species and for food regulatory control. Evaluation of a selected DNA barcode region, using a newly designed species-specific primer set, generated a species-specific ‘fingerprint’, and tested the efficacy of this barcode during analysis of simulated canned fish meats, dried fish fillets, fried fish powders, and commercial doubtful products. Results suggest that the cytochrome b gene is an effective gene marker for the purpose of species authentication. When used in conjunction with the newly designed primer set, the selected DNA barcode region was demonstrated to be highly discriminatory, accurate, efficient, and species-specific. Because of current circumstances within trade of fish tissues, mitogenomics-based technology may provide an efficient and reliable means of resolving problems relating to fish meat adulteration and mislabeling.     

DNA barcoding for the identification of common economic aquatic products in Central China and its application for the supervision of the market trade

Common economic aquatic products are important contributors to the human food supply. However, with the rapid globalization of the aquatic products industry, aquatic products market has become increasingly disordered. Therefore, an accurate and convenient method for identifying common economic aquatic products is important and necessary in many areas. DNA barcoding, which constitutes the analysis of a short fragment of the mitochondrial cytochrome c oxidase subunit I (COI) sequence, has been widely used in species identification. To discriminate common economic aquatic species using DNA barcoding, we collected 534 COI barcode sequences of 66 common species consisting of 39 fish, 9 crustaceans and 18 mollusks. The intraspecific genetic distances based on the Kimura 2-parameter (K2P) model were less than 1.37% for fish, 7.32% for crustaceans and 3.40% for mollusks, whereas the intragenus distances ranged from 3.91% to 13.82% for fish, 14.99%–16.17% for crustaceans and 14.82%–16.36% for mollusks. The average intraspecific K2P distance was also compared with the average intragenus distance. The taxonomic resolution ratio values obtained for fish, crustaceans and mollusks were 58.50, 21.59 and 27.63 respectively, which are higher than the threshold of (10×). A neighbor-joining (NJ) tree based on the K2P distance, and a maximum likelihood (ML) tree, based on the GTR + I + G model, were constructed, and all of the species could be identified unambiguously in the trees with several branches exhibiting high bootstrap values. Our results demonstrated high efficiency of DNA barcoding as an efficient molecular tool for the identification of common economic aquatic products, and 8 substitute species were successfully detected in 66 species. Our analyses also indicated that the common aquatic products trade industry could be effectively monitored and managed by DNA barcoding. Therefore, a simple identification database of common economic aquatic products was constructed.     

Simple methods for rapid determination of sulfite in food products

Two simple methods for sulfite determination in food products were proposed. The modified para-rosaniline (PRA)-formaldehyde method showed a much broader linear range (0.05–5.0 mg l⁻¹ as SO₂) than the commonly used procedure for the sulfur dioxide detection in the atmosphere (0.05–1.0 mg l⁻¹ as SO₂). By using a standard reference color card, this method only needed 5 min to complete a test. The 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) method was another good alternative for rapid determination of sulfite since it only needed one main reagent and the method was robust and easy to operate with the linear range of 0.10–4.3 mg l⁻¹ as SO₂. When applying to real food samples, the DTNB method had good recoveries for all the tested samples and the results agreed well with those obtained by the official iodometric titration. The modified PRA-formaldehyde method worked well with all the tested dried food products, though matrix effect of sulfite binding was observed for the beer samples. Thus, the modified PRA-formaldehyde method has advantages of sensitivity and rapidity, but the DTNB method offers a wider range of applications. Both methods provided practical ways for in situ determination of sulfite by non-professional operators with the modified PRA-formaldehyde method suited for food samples with less sulfite binding problems.     

Detection of mislabelled seafood products in Malaysia by DNA barcoding: Improving transparency in food market

Seafood mislabelling is a global issue following the increasing worldwide seafood trade particularly of processed seafood products, as well as a general lack of regulations and tight enforcement in some countries. This study is a pioneering seafood forensics survey conducted in Malaysia. A total of 62 seafood samples, either raw, frozen or variously processed, were collected from commercial sources. Molecular analyses were performed by sequencing Full DNA Barcoding (FDB) with target region of ∼700 bp or Mini Barcoding (MDB) with smaller target region of only ∼150 bp. The DNA barcode sequences obtained were compared with those available on BOLD and GenBank databases. The DNA targets were successfully amplified and sequenced from 81% of seafood samples. Among these samples, 16% were found to have been mislabelled at source. This study supports the view that DNA barcoding can be a powerful tool in seafood forensics.     

Identification of meat and poultry species in food products using DNA barcoding

DNA barcoding is a promising method for the sequencing-based identification of meat and poultry species in food products. However, DNA degradation during processing may limit recovery of the full-length DNA barcode from these foods. The objective of this study was to investigate the ability of DNA barcoding to identify species in meat and poultry products and to compare the results of full-length barcoding (658 bp) and mini-barcoding (127 bp). Sixty meat and poultry products were collected for this study, including deli meats, ground meats, dried meats, and canned meats. Each sample underwent full and mini-barcoding of the cytochrome c oxidase subunit I (COI) gene. The resulting sequences were queried against the Barcode of Life Database (BOLD) and GenBank for species identification. Overall, full-barcoding showed a higher sequencing success rate (68.3%) as compared to mini-barcoding (38.3%). Mini-barcoding out-performed full barcoding for the identification of canned products (23.8% vs. 19.0% success), as well as for turkey and duck products; however, the primer set performed poorly when tested against chicken, beef, and bison/buffalo. Overall, full barcoding was found to be a robust method for the detection of species in meat and poultry products, with the exception of canned products. Mini-barcoding shows high potential to be used for species identification in processed products; however, an improved primer set is needed for this application.     

DNA and Mini-DNA barcoding for the identification of Porgies species (family Sparidae) of commercial interest on the international market

The morphological similarity among Sparidae species, which are characterized by a different market price, represents a serious problem for their trade and for stock management, since it encourages frauds for substitution. The most accredited morphological method for their identification is based on the dental-plate, but this approach is not simple and cannot be used for prepared products. When molecular methods are used the DNA degradation induced by cooking is the main drawback. In this work, we collected 314 reference tissues belonging to 75 Sparidae species and we produced a dataset of full (FDB) and mini-barcode (MDB) reference sequences starting from DNA extracted from fresh and ethanol-preserved tissues using universal primes. Moreover, some fresh samples were cooked. The FDB was successfully amplified in 91% (fresh), 50% (cooked) and 81% (ethanol-preserved) samples, while the amplification rates of the MDB were considerably higher in case of cooked (100%) and ethanol-preserved (94%) samples. The same primers were used for the amplification of the DNA obtained from 58 market samples (MS). All the DNA barcodes were compared with BOLD and GenBank using IDs and BLAST analysis. FDB was able to provide unambiguous species-level identifications for 53 (78%) and 44 (64.7%) reference samples analyzed on BOLD and GenBank, respectively. The Mini-DNA barcode (MDB) showed a lower discriminating power with 32 (45.7%) and 29 (41.4%) sequences unambiguously matched to a species on BOLD and GenBank. However, the MDB allowed to identify all the reference sequences as belonging to the Sparidae family. FDB and MDB showed a similar performance in analyzing the MS, allowing to highlight 21 (38%) mislabeled MS. Our study, while confirming the FDB as a reliable tool for fish authentication, proposes the MDB as a promising tool to recover molecular information in case of cooked products.     

Identification of species in ground meat products sold on the U.S. commercial market using DNA-based methods

The objective of this study was to test a variety of ground meat products sold on the U.S. commercial market for the presence of potential mislabeling. Forty-eight ground meat samples were purchased from online and retail sources, including both supermarkets and specialty meat retailers. DNA was extracted from each sample in duplicate and tested using DNA barcoding of the cytochrome c oxidase I (COI) gene. The resulting sequences were identified at the species level using the Barcode of Life Database (BOLD). Any samples that failed DNA barcoding went through repeat extraction and sequencing, and due to the possibility of a species mixture, they were tested with real-time polymerase chain reaction (PCR) targeting beef, chicken, lamb, turkey, pork and horse. Of the 48 samples analyzed in this study, 38 were labeled correctly and 10 were found to be mislabeled. Nine of the mislabeled samples were found to contain additional meat species based on real-time PCR, and one sample was mislabeled in its entirety. Interestingly, meat samples ordered from online specialty meat distributors had a higher rate of being mislabeled (35%) compared to samples purchased from a local butcher (18%) and samples purchased at local supermarkets (5.8%). Horsemeat, which is illegal to sell on the U.S. commercial market, was detected in two of the samples acquired from online specialty meat distributors. Overall, the mislabeling detected in this study appears to be due to either intentional mixing of lower-cost meat species into higher cost products or unintentional mixing of meat species due to cross-contamination during processing.     

Risk analysis and rapid detection of the genus Thermoascus,food spoilage fungi

Recently the numbers of spoilage incidents in food industry by the species of Thermoascus are increasing, but the risk of food spoilage have never been evaluated.It became obvious that their heat-resistances were higher than those of other heat-resist fungi, Byssochlamys, Hamigera and Neosartorya by our analyses. On the other hand, Thermoascus aurantiacus and Byssochlamys verrucosa had the idh gene, but they showed no patulin production in Potato dextrose broth or Czapek-glucose medium. Therefore, Thermoascus must be discriminated from other fungi in the food industry. We developed a rapid and highly-sensitive method of detecting Thermoascus in the genus level by using PCR. This method is expected to be extremely beneficial for the surveillance of raw materials in the food production process.     

Optimization and evaluation of the qPCR-based pooling strategy DEP-pooling in dairy production for the detection of Listeria monocytogenes

Diagnostic strategies to detect foodborne pathogens such as L. monocytogenes in food processing environments are cost and time consuming necessities to ensure safe food products. While two-step pooling diagnostic strategies incorporating PCR have been successfully introduced in recent years, such strategies to date have not been employed for food and hygiene monitoring. The objective of this study was to develop and evaluate the applicability of a cost-effective PCR pooling approach called direct enrichment PCR (DEP)-pooling based on an existing L. monocytogenes monitoring setup. The proposed pooling strategy is based on a first enrichment step, subsequent PCR-pooling and re-evaluation of PCR-positive pools with the VIDAS DUO. Overall, more than 3000 individual routine samples from a European cheese production facility were tested with the proposed DEP-pooling and compared in parallel with existing L. monocytogenes monitoring. In this approach equivalent results were obtained. Proposed DEP-pooling utilizes the advantages of both microbiological enrichment and PCR to identify negative samples faster in a cost efficient way with an overall cost reduction of ∼60% based on 1% positive samples.     

Comparative study of DNA extraction methods for soybean derived food products

The present work describes the comparison of four DNA extraction methods applied to a wide range of soybean derived food products. The methods included the commercial kits NucleoSpin and GeneSpin, the CTAB, and the Wizard methods. The protocols have been compared for their extraction efficiency, evaluated by the determination of yield and purity of DNA extracts, as well as amplifiability. All the methods produced DNA suitable for PCR amplification for the majority of analysed foods, with the exception of soybean sauces and some processed foods. The NucleoSpin and GeneSpin methods showed the best results for DNA yield and purity, when applied to soybean flours and protein isolates. The CTAB and Wizard methods were generally well suited to all kind the food matrices tested. The extraction of processed foods, such as drinks, desserts or vegetarian foods was better achieved with the CTAB or the Wizard methods as both produced high number of amplifiable extracts. The four methods showed similar performances for real-time PCR amplification.     

A multiplex nested PCR assay for the simultaneous detection of genetically modified soybean,maize and rice in highly processed products

The use of genetically modified organisms (GMOs) as food products becomes more and more widespread. The European Union has implemented a set of very strict procedures for the approval to grow, import and/or utilize GMOs as food or food ingredients. Thus, analytical methods for detection of the GMOs are necessary in order to verify compliance with labeling requirements. There are few effective screening methods for highly processed GM (genetically modified) products. Four genes (CP4-EPSPS, Cry1A(b), BAR, and, PAT) are common exogenous genes used in commercialized transgenic soybean, maize, and rice. In the present study, a multiplex nested polymerase chain reaction (PCR) method was developed to simultaneously detect the four exogenous genes and one endogenous gene in two runs. We tested eleven representative highly processed products samples (soya lecithin, soya protein powder, chocolate beverage, infant rice cereal, soybean refine oil, soybean salad oil, maize oil, maize protein powder, maize starch, maize jam) using the developed method, and amplicons of endogenous gene and transgenic fragments were obtained from all the processed products except for soybean refined oil, soybean salad oil and maize oil, and the sensitivity was 0.005%. These results indicate that multiplex nested PCR is appropriate for qualitative detection of transgenic soybean, maize and rice in highly processed products except for refined oil.     

Comparison of real-time PCR and ELISA-based methods for the detection of beef and pork in processed meat products

Two commonly used methodologies for species detection within processed meat products are real-time polymerase chain reaction (PCR), a DNA-based method, and enzyme-linked immunosorbent assay (ELISA), a protein-based method. In this study, a real-time PCR assay was compared to a commercial ELISA kit based on sensitivity, specificity, agreement among duplicate samples, cost, time, and ease of use. Fifteen reference samples containing known percentages (0.1–99.9%, w/w) of pork and beef were analyzed in duplicate using both methods. Thirty commercial products, including sausages, pet treats, and canned meats, were also tested in duplicate with each method. Reference sample analysis showed real-time PCR was able to detect pork in duplicate samples at 0.10% and beef at 0.50% in the binary mixtures. ELISA detected pork in duplicate samples at 10.0% and beef at 1.00% in the binary mixtures. When the results of reference and commercial samples were combined, real-time PCR demonstrated the greatest agreement among duplicate samples, at 96.7%, compared to 95.6% agreement for ELISA. The real-time PCR assay used in this study was found to be less expensive, while ELISA was less time-consuming and easier to perform. Both methods were successful at identifying species in ground meats, sausage, and deli meat samples; however, pet treats and canned meats proved more challenging. Overall, it was determined that the real-time PCR assay was optimal for species identification in processed meat products when a low detection limit is required; however, the ELISA kit may be advantageous in other situations due to its ease of use.     

Evaluation of mercury,cadmium and lead levels in fish and fishery products imported by air in North Italy from extra-European Union Countries

The levels of cadmium (Cd), mercury (Hg) and lead (Pb) were evaluated in 251 samples of fish and fishery products randomly collected between January 2010 and December 2013 from products imported through the airport of Milan Malpensa from extra-European Union (extra-EU) Countries and inspected at the Border Inspection Post by official public veterinarians. In this study, products were classified in four categories: predatory fish (larger predatory fishes like tunafish and swordfish, and spiny dogfish), non-predatory fish (other species of fishes of lower dimensions and different feeding behavior, mollusc (cephalopods and bivalves) and crustacean). Analyses were performed by accredited laboratory. All the concentrations were below the levels expressed in Regulation (EC) No 1881/2006 except for two samples of swordfish that exceeded in Hg content. Predatory fish had a significantly higher content of Hg (median value 375 μg kg⁻¹) while mollusc had a significantly higher content of Cd (median value 66.5 μg kg⁻¹) compared to the other groups of seafood included in this study. No relationship was found between the individual concentration of metals.     

A practical test system for sensitive, rapid screening and authentication of peanut allergens in imported and exported food products in Chinese Customs

Proper food allergen labeling protects consumers with serious food allergies, such as peanut (Arachis hypogaea) allergies. Currently, there is no widely accepted standard peanut allergen detection protocol for use by Customs agencies, which could avoid trade disputes caused by improper labeling. Herein we developed a peanut allergen screening and confirmation system for sensitive, rapid identification of peanut allergen ingredients which used gold immunochromatography assay strips followed by confirmatory western blotting. Gold immunochromatography assay strips were prepared with polyclonal antibodies against total peanut proteins for preliminary peanut allergen screening in foods labeled peanut-free. Western blotting with Ara h1-specific monoclonal antibodies was performed to confirm the results to exclude false positive results. Of 285 food samples tested, 164 were labeled as containing peanut allergens. The gold immunochromatography assay determined that 116 were negative, in accordance with their original labels. Five samples were positive, which was not consistent with their labels. These 5 positive samples were subjected to western blotting confirmatory tests. Only one was confirmed to be positive. We reported this result to the manufacturer and suggested they change the product label. This system, which includes sequential classification, screening, confirmation and reporting steps, was useful for monitoring peanut contamination in imported and exported foods by Chinese Customs.     

Development of triplex PCR for simultaneous detection of maize,wheat and soybean

A reliable and fast detection of important food plants, such as maize (Zea mays L.), wheat (Triticum aestivum L.), and soybean (Glycine max L.) is of particular interest for food authenticity and safety assessment. In this study, the novel multiplex polymerase chain reaction (PCR) method was developed for the rapid qualitative detection of soybean, maize and wheat. To this purpose, new soybean-specific and maize-specific PCR primers were designed. Their specificity was assayed by uniplex PCRs with different plant species, namely maize, soybean, wheat, oats (Avena sativa), and barley (Hordeum vulgare L). Gel electrophoresis of the amplification products demonstrated high specificity of both primer pairs for identification of relevant species. Subsequently, based on the developed DNA markers, the species-specific triplex PCR targeting maize invertase gene, soybean lectin gene and wheat low-molecular-weight glutenin subunit was developed and optimized for simultaneous identification of these three plant species. The developed PCR method enables specific, effective and rapid detection of maize, wheat and soybean and may be used for food analysis.     

Novel nuclear barcode regions for the identification of flatfish species

The development of an efficient seafood traceability framework is crucial for the management of sustainable fisheries and the monitoring of potential substitution fraud across the food chain. Recent studies have shown the potential of DNA barcoding methods in this framework, with most of the efforts focusing on using mitochondrial targets such as the cytochrome oxidase 1 and cytochrome b genes. In this article, we show the identification of novel targets in the nuclear genome, and their associated primers, to be used for the efficient identification of flatfishes of the Pleuronectidae family. In addition, different in silico methods are described to generate a dataset of barcode reference sequences from the ever-growing wealth of publicly available sequence information, replacing, where possible, labour-intensive laboratory work. The short amplicon lengths render the analysis of these new barcode target regions ideally suited to next-generation sequencing techniques, allowing characterisation of multiple fish species in mixed and processed samples. Their location in the nucleus also improves currently used methods by allowing the identification of hybrid individuals.