Authentication and traceability of fish maw products from the market using DNA sequencing

Fish maws (dried swimbladders of fishes) are regarded as traditional luxurious delicacies, medicine and tonics, which have been recommended and consumed in Asia over many centuries. At the commercial level, they are ranked as different values based on species. However, fish maw species and the trade are still unknown and undocumented. The processing treatments make them difficult for species identification based on morphological characterization. In the present study, the genetic identification of the main commercialized species of fish maws has been carried out, based on the amplification of a fragment of mitochondrial 16S rRNA gene and subsequent BLAST analysis. The applicability of all kinds of processed products was verified, including dried, water soaked, salt fried and salt fried plus water soaked forms. The result indicated this method was applicable to all of them, showed that 53.3% of the products were incorrectly labeled and 58.3% of “croaker” products were substituted with catfish or perch species. Moreover, results indicated that besides traditional trade in Asia, the fish maw trade has been globally expanded to meet the growing demand of market. Therefore, this method can be useful in normative control of processed products, particularly in the authenticity of imported species, verifying the correct traceability in commercial trade, the correct labeling, and also for fisheries control of endangered species to conserve stocks biodiversity.     

Molecular identification of grouper species using PCR-RFLP technique

A PCR-RFLP method was used to identify five species of raw and processed groupers belonging to the genus, Epinephelus viz. E. areolatus, E. bleekeri, E. faveatus, E. longispinis and E. undulosus. DNA extracted from all the species using phenol-chloroform method amplified the mt 16S rRNA gene, unique for the genus, Epinephelus, using GenF and EpiR primers at 300 bp. This fragment did not amplify the DNA of even closely related grouper genus, Cephalopholis, making the method more specific the grouper genus, Epinephelus. The specificity was further ascertained by performing the internal control that targeted 18S rRNA, which was eukaryotic specific. Digestion of PCR products with three selected restriction enzymes viz. SduI, BciVI, and Sau3AI followed by PAGE yielded species specific patterns that enabled identification of three grouper species viz. E. bleekeri, E. faveatus and E. longispinis, while the other two species viz. E. areolatus and E. undulosus gave similar pattern in the gel, although minor variations in the molecular sizes of digested products were noticed. Chilled, frozen and cooked groupers also gave similar PCR-RFLP pattern to that of raw groupers making this method suitable even for processed products.     

Species identification of commercial jerky products in food and feed using direct pentaplex PCR assay

The direct pentaplex PCR assay was developed for simultaneous identification using species-specific primer sets and a universal eukaryotic primer set in processed jerky products without DNA extraction. The specific primer sets of target meat species amplified the expected 83-, 133-, 166-, and 204-bp PCR products for pork, chicken, beef, and duck, respectively, and obtained no cross-reactivity against a total of sixteen animal species. A universal eukaryotic primer set amplified a 99-bp conserved fragment in all meat species. To evaluate the sensitivity of this assay, the different percentages of jerky samples were prepared with the meat species having the possibility to be mixed. Adulterated beef jerky samples contaminated with 0.1, 0.5, 1, 5, 10, and 50% pork and adulterated duck jerky samples contaminated with 0.1, 0.5, 1, 5, 10, and 50% chicken were prepared in a laboratory. The detection level of direct pentaplex PCR was below 0.1% pork in adulterated beef jerky and 0.1% chicken in adulterated duck jerky. The optimized assay was also applied to the analysis of commercial food and feed jerky products. The meat species in commercial jerky products were successfully identified without DNA extraction.     

A new set of 16S rRNA universal primers for identification of animal species

In this study, bioinformatics were used to specifically design universal primers within 16S rRNA gene according to the following criteria: the priming sites needed to be sufficiently conserved to permit a reliable amplification (pooled samples) and the genetic marker needed to (a) be sufficiently variable to discriminate among most species and sufficiently conserved within than between species, (b) be short enough to allow also accurate amplification from processed samples (food) and non-invasive approaches (fur, feathers, faeces, etc.) (c) convey sufficient information to assign samples to species and (d) be amplified under variable lab conditions and protocols. Furthermore, short sequences allow the accurate massive inter- and intra-species identification of point mutations by the SSCP technique. The size of the amplified segment ranged from 222 to 252 bp. Amplification and identification success were 100% with all kinds of tissue tested in both raw and processed samples in a wind range of species, mammals (n = 27), fishes (n = 32) birds (n = 19), coleoptera (n = 23), reptiles (n = 5), crustaceans (n = 5) and cephalopods (n = 2), including almost all European mammal and avian game species. In addition, no intra-specific polymorphism was detected. Finally, gene fragments, homologous to those amplified by the primers used herein and retrieved from the GenBank for three animal sets [mammals (n = 248), birds (n = 231) and fishes (n = 644)] showed a particular precise percentage of correct identifications. Therefore, this short segment of the 16S rRNA mitochondrial gene could be a good candidate for a rapid, accurate, low-cost and easy-to-apply and interpret method to identify mammal and avian game species by PCR amplification and sequencing that can be easily incorporated in integrated conservation and forensic programmes.     

Development and validation of fast Real-Time PCR assays for species identification in raw and cooked meat mixtures

Five species-specific Real-Time PCR protocols were developed, standardised and validated for the identification of turkey, chicken, beef, pork and sheep meat in complex food products. Specific primers and probes were designed for each assay. 16S rRNA and cyt-b target genes located in the mitochondrial DNA were used for this purpose. The limit of detection of the Real-Time PCR methods ranged between 0.02 pg and 0.80 pg of template DNA, with an efficiency between 95% and 100%. All methods were able to detect the target species when spiked at 1% in any other species and no relevant difference was observed between the Ct values of raw and cooked samples. An Internal Amplification Control was used to detect possible false negatives due to inhibitory substances eventually present in the sample matrix. The assays were tested on meat mixtures to evaluate the diagnostic sensitivity and specificity.     

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.     

Authentication of meat and commercial meat products from common pigeon (Columba livia) woodpigeon (Columba palumbus) and stock pigeon (Columba oenas) using a TaqMan real-time PCR assay

A species-specific real-time polymerase chain reaction (PCR) assay using TaqMan^® probes has been developed for the identification of meat and meat products from common pigeon (Columba livia), woodpigeon (Columba palumbus) and stock pigeon (Columba oenas). The method combines the use of species-specific primers and TaqMan^® probes that amplify small fragments (amplicons < 200 base pairs) of the mitochondrial 12S rRNA gene, and an endogenous control primer pair that amplifies a 141 bp fragment of the nuclear 18S rRNA gene from eukaryotic DNA. Analysis of experimental raw and heat-treated binary mixtures as well as of commercial meat products from the target species, demonstrated the suitability of the assay for the detection of the target DNAs. The PCR assay reported in this work could be useful in inspection programs to verify the correct labelling of raw and heat-treated pigeon meat products.     

DNA barcoding reveals chaotic labeling and misrepresentation of cod (鳕, Xue) products sold on the Chinese market

The increasing rate of seafood frauds, especially in the case of highly priced species, highlights the need of verifying the identity of fish products. This paper describes the application of DNA barcoding to the identification of 52 products commercialized with the Chinese term 鳕 (Xue, Cod) in supermarkets (Nanjing and Shanghai) and in the online market. Considering the lack of harmonization around the definition of Cod, the mislabeling rate was assessed according to three increasingly stringent definitions: Cod meaning Gadiformes species; Cod meaning Gadus spp.; Cod not meaning any specific species, since a qualifier (“Atlantic”, “Pacific” or “Greenland”) should be added in order to refer to Gadus morhua, Gadus macrocephalus or Gadus ogac, respectively.Results highlighted a very high mislabeling rate, which exceeded 60% even with the less stringent definition. Interestingly, only 42.3% of the samples were Gadiformes, while the others were Perciformes, Pleuronectiformes or toxic Tetraodontiformes species. Economic, ecological and health issues arising from the misuse of the term Cod are discussed in the light of the leading role of China in the seafood worldwide industry and of the increased national consumption of marine species.     

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.