Species identification of dried scallop adductor muscle (yao zhu) products sold on the market

Dried scallop adductor muscle, known as yao zhu, has been ranked in the list of the eight sea treasures in Chinese cuisine, it has been commonly recommended and consumed in China over many centuries due to its delicious taste and abundant nutrition. However, the true species composition of the dried scallop adductor muscles in the trade is unclear. Indeed, it is difficult to identify the scallop species just based on their morphological characters of processed adductor muscles. In this study, 60 yao zhu products commercialized in Chinese market were analyzed in order to authenticate species. Direct sequencing and BLAST method based on a fragment of the 16S rRNA gene were carried out and results showed that 34 products (56.7%) were scallop species Argopecten irradians, and other 26 products (43.3%) were the scallop species Mizuhopecten yessoensis, indicated the yao zhu market were dominated by the both species, which were the introduced aquaculture scallop species. Moreover, a simple and fast PCR method proved to be capable of unequivocal identification between the two species. Therefore, this work facilitates the identification of yao zhu products in the market, support of food control and protection of consumers’ rights.     

A multiplex PCR assay for species identification of raw and cooked bonito

Attempts are made to establish one-step multiplex PCR assay for distinguishing five species of raw and cooked bonito including Euthynnus pelamis, Euthynnus affinis, Auxis rochei, Auxis thazard, and Sarda orientalis. After constructing the 1141 bp complete mitochondrial cytochrome b genes of five bonito species and other five contrastive Scombridae species, five sets of species-specific primer were designed to amplify different cytochrome b gene fragments in each species individually. The amplified lengths of fragments were respectively 143 bp for A. rochei, 236 bp for E. pelamis, 318 bp for A. thazard, 398 bp for E. affinis and 506 bp for S. orientalis, which could be obviously differentiated from each other on DNA electrophoresis. The five sets of species-specific primer were mixed and applied to simultaneously detect bonito species. All species from 12 commercial raw fish and five species out of eight cooked bonito fillets were successfully identified by the multiplex PCR assay. Experiments carried out demonstrate that the multiplex PCR assay was useful for identifying species of non-overheating fish product.     

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.     

Rapid and reliable species identification of scallops by MALDI-TOF mass spectrometry

Correct identification of scallop species is of crucial importance to the seafood industry to assess marketability of the product, as well as to prevent unintentional mislabeling and to comply with current legislative guidelines. However, various scallop species are virtually indistinguishable and to date there is no fast, reproducible, and inexpensive technique for species identification of scallops. A new promising tool in the identification of various organisms is the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). In this study, we aimed to 1) evaluate the possibility of MALDI-TOF MS as a tool for rapid and reliable species identification of scallops and 2) assess the occurrence of mislabeled scallops sold in Switzerland. We therefore used 16 scallop reference samples, as well as scallops sampled at eight Swiss retail facilities and identified the scallop species both by 16S rRNA PCR and MALDI-TOF MS. For all tested scallop species, MALDI-TOF MS yielded reproducible and unique mass spectra profiles covering a wide molecular mass range of 3000–30,000 Da. Species-specific biomarker protein mass patterns were determined. Species identification of reference scallop samples using MALDI-TOF MS was in accordance with species identification using the established 16S rRNA PCR tool. Identification of the scallop samples purchased in Switzerland also yielded identical results for MALDI-TOF MS and the 16S RNA PCR approach. We were able to show that several of the scallop samples were labeled incorrectly. All mislabeled scallops belonged to the species Placopecten magellanicus, but were marketed as “Jacobsmuschel”, a commercial name reserved for Pecten spp. In this study, we show that MALDI-TOF MS allows for rapid, reliable, and highly accurate species identification of scallops.     

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.     

Italian market fish species identification and commercial frauds revealing by DNA sequencing

To guarantee consumers about the security of what they eat is the main goal of all the institutions working to assure food safety. In seafood field it appears of prime importance to be able to identify fish species throughout the production cycle also when the product has been already processed (breaded fish, fillets etc). In this context the latest system proposed for species identification is the so called “DNA-Barcoding”, made by carrying out the DNA sequencing of a standard barcode region. In the present study we carried out bi-directional sequence analysis of mitochondrial cytochrome b gene (Cytb) and cytochrome oxidase subunit I gene (COI). The main goal of the research was to unambiguously classify the most common fishes traded in Italy, being able to ascertain possible labeling frauds made substituting value species with less precious ones. With both genes we could correctly identify almost all the samples in study: respectively 58/58 with COI and 56/58 with Cytb. This study confirms mitochondrial genes Cytb and COI as good candidates for fish species identification by DNA sequencing. This method appears particularly suitable especially when morphological characterization is difficult, (for very close species), or impossible (for transformed foodstuffs) to carry out. What's more we could efficiently identify many fishes belonging to the Mediterranean fauna, which, to our knowledge, underwent for the first time this kind of analysis. Further development will concern the extension of the analysis to a largest number of local species.     

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.     

Patulin assessment and fungi identification in organic and conventional fruits and derived products

In this study, patulin levels in apples and tomatoes obtained in conventional and organic farms with 0 and 100% of rotten area, were determined by gas chromatography–mass spectrometry (GC–MS) to assess the influence of the production system with patulin content. Additionally, mould species were isolated and identified using classic morphological techniques and a DNA barcode system based on nrITS, β-tubulin and calmodulin genes. The most frequently isolated genera were Rhizopus, Mucor, Alternaria, Cladosporium, Botrytis, Aspergillus and Penicillium. Higher patulin levels were found in samples contaminated by Penicillium expansum. Patulin levels were not significantly different in samples obtained from the two production system (conventional and organic). Among the three studied apple varieties, Golden delicious was the most contaminated with patulin followed by Reineta. Fuji apples with 0 and 100% of rotten area did not present quantifiable patulin levels. A survey for first time realized in commercialized tomato products showed that 35.7% of the samples (10 of 28) contained patulin in levels ranging from 3.22 to 47.72 μg/kg. In apple juices, patulin levels ranged from 1.86 to 45.47 μg/kg, which is below the maximum legal requirement (50 μg/kg).     

Species identification of snapper: A food poisoning incident in Taiwan

A snapper (Lutjanidae fish) is a carnivorous coral reef fish that is distributed in sea areas around Taiwan. In December 2008 in southern Taiwan, a food poisoning incident occurred due to the ingestion of snapper, and the causative residue of ciguatera was investigated using a toxicity assay. To identify the species of the causative sample, six suspected species of Lutjanidae fish commonly found in Taiwan were analyzed using both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. According to the low molecular weight region (<30.0 kD) of species-specific patterns extracted from myofibrillar and sarcoplasmic proteins, the 6 snapper species could be clearly differentiated by the SDS-PAGE method. Furthermore, a consistent 465 bp sequence of the mitochondrial cytochrome b gene from the 6 snapper species was amplified by the PCR method and was rapidly distinguished by the analysis of restriction enzymes. According to both SDS-PAGE and PCR-RFLP methods, the poisonous sample was identified as Lutjanus bohar, which is also a notorious Lutjanidae species containing ciguateric toxins.     

Mislabelling of beef and poultry products sold in Malaysia

Meat species specification is important for consumer protection and increases concern in food labelling regulations enforcement. Although regulations exist for processed meat products, information on the prevalence of meat products mislabelling and regulatory compliance in Malaysia is lacking. In this study, 143 prepacked (beef and poultry) meat products (sausages, cold cut meats, cooked whole muscle meats, breaded products, meatballs and ground meats) were purchased from several national and international supermarket chains in Malaysia. These samples were analysed for the presence of common meat species (buffalo, cattle, chicken, goat, sheep, duck and goose) and meats prohibited by Islamic laws (“Haram”) (cat, dog, monkey, pig and rat) using species-specific primers. The results showed that 112 (78.3%) samples were mislabelled, attributed by the false declaration of species and/or presence of undeclared meat species. The mislabelled products consisted of 17/28, 3/4, 6/8, 19/25, 48/56, and 19/22 of sausage, cold cuts, cooked whole muscle meat, breaded product, ground meat, and meatball samples, respectively. Buffalo DNA was detected in 40 out of the 58 samples labelled as beef. The presence of undeclared chicken and buffalo DNA were detected in 33/58 and 62/84 of beef and chicken products, respectively. The five “Haram” meat sources, however, were not detected in all meat products tested. The presence of chicken or buffalo DNA in these products could be attributed to unintentional cross contamination from food processing equipment, especially meat grinder, and lack of proper cleaning or inadequate hygiene. In conclusion, this study shows that majority of the samples are not legally compliant, signifying that substitution and mislabelling of meat products are commonplace in Malaysia. Strict implementation of the Malaysia Food Regulations 1985 alongside with regular surveillance and monitoring programmes are compulsory to alleviate and deter mislabelling issues.     

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.     

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.     

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.     

Chemical characterisation, biogenic amines contents, and identification of fish species in cod and escolar steaks, and salted escolar roe products

Forty-one cod steaks, fifteen escolar steaks, and fifteen salted escolar roe products sold in Taiwan market were purchased and tested to determine the biogenic amine, histamine-forming bacteria, and identification of fish species. The levels of pH value, salt content, Aw, TVBN and APC in all samples ranged from 5.3 to 7.0, 0.7 to 5.6%, 0.80 to 0.99, 0.8 to 59.9 mg/100 g and 2.5 to 7.3 log CFU/g, respectively. None of these samples contained coliform bacteria and Escherichia coli. The average content of histamine in all samples was less than 5 mg/100 g US Food and Drug Administration guideline value. Nine histamine-forming bacterial strains isolated from cod, escolar, and salted escolar roe samples produced 2.0-62.3 ppm of histamine in trypticase soy broth (TSB) supplemented with 1.0% l-histidine (TSBH). Assay of DNA direct sequence and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) revealed that mislabeling rate of 41 cod steaks was 31.6% (13/41). Among them, 7 samples (17%) and 6 samples (14.6%) were identified as Ruvettus pretiosus (oilfish) and Reinhardtius hippoglossoides (Greenland halibut), respectively. In addition, most of escolar steaks and salted escolar roe products were identified as Lepidocybium flavobrunneum (escolar), while other samples were identified as R. pretiosus.     

Genetic identification of the caviar-producing Amur and Kaluga sturgeons revealed a high level of concealed hybridization

China has recently become the leader country for sturgeon aquaculture and caviar production, deeply changing the traditional geography of this market in few years. As a consequence, some species originating from the Far East Asia increased their economic relevance, joining the ones traditionally harvested for caviar. In this context, the possibility to reliably and promptly identify these species on the market has increasing importance for the enforcement of control actions against illegal trade or commercial frauds. The present study focuses on two commercially relevant species, massively reared in China not only as pure species but also as reciprocal hybrids: the Amur (Acipenser schrenckii) and Kaluga (Huso dauricus) sturgeons. We assess the identification power of two putatively diagnostic markers isolated from two predicted introns of the nuclear coding gene Ribosomal Protein L8. The markers were tested on tissue or caviar of 508 individuals of the two species and 31 hybrids. In order to compare results across loci, most individuals were also checked at two already published microsatellite markers, with a good, even if incomplete, identification efficiency for the two species. No marker showed fixed alternative alleles between Amur and Kaluga sturgeons, confirming the difficulty of distinguishing these two sympatric species in spite of the marked morphological differences and the consequent classification into different genera. So far, the multi-locus panel here used represents the more effective tool for the genetic identification of pure Amur and Kaluga sturgeons and resulted to be fully efficient to validate caviar and tissues obtained from hybrids between the two species.     

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.     

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.     

A novel quantitative real-time PCR method for identification and quantification of mammalian and poultry species in foods

Meat adulteration has posed considerable risks to public health. In this study, we developed a novel real-time quantitative PCR method for the detection of some mammalian and poultry species that are used as meat products or meat adulterants. The method was based on the detection of the single-copy nuclear gene myostatin. The specificity, heterogeneity, and copy number of myostatin were evaluated. Additionally, we determined the sensitivity and precision of the method. The results revealed that myostatin had high specificity and low heterogeneity among different mammalian and poultry species. The limit of detection was 5 pg of animal genomic DNA or 0.001% meat ingredient, and the limit of quantification was 10 pg of animal genomic DNA or 0.01% meat ingredient. The quantification results of 12 blind samples showed that the biases between the measured and true values were <25%. Therefore, the developed quantitative real-time PCR method for mammalian and poultry species is suitable for identification and quantification of different meat ingredients as a reference gene.     

基于CaAl-LDHs前体的蒽醌降解物再生固体碱催化剂

以尿素为沉淀剂,采用水热合成法在球形γ-Al_2O_3载体表面原位合成Ca Al-NO_3-LDHs(LDHs:层状复合金属氢氧化物),焙烧后得到Ca Al-LDO/γ-Al_2O_3固体碱催化剂(LDO:层状双金属复合氧化物)。采用XRD,SEM,BET,CO_2-TPD方法对催化剂进行了表征,并考察了催化剂对蒽醌降解物的再生性能。表征结果显示,当Ca O负载量低于20%(w)时,催化剂仍保持较高的比表面积和丰富的孔结构,且表面碱量随Ca O负载量的增加而增加;当Ca O负载量超过20%(w)时,催化剂的比表面积减小,孔道被堵塞,表面碱量降低。实验结果表明,Ca O负载量为20%(w)的0.20-Ca Al-LDO/γ-Al_2O_3催化剂对蒽醌降解物的再生性能最佳,与浸渍法制备的0.20-Ca O/γ-Al_2O_3和水热合成法制备的0.20-Mg Al-LDO/γ-Al_2O_3催化剂相比,0.20-Ca AlLDO/γ-Al_2O_3催化剂的蒽醌降解物再生活性分别提高了76.9%和18.0%。