From Nontargeted to Targeted Analysis: Feature Selection in the Differentiation of Truffle Species (Tuber spp.) Using 1H NMR Spectroscopy and Support Vector Machine.

The price of different truffle types varies according to their culinary value, sometimes by more than a factor of 10. Nonprofessionals can hardly distinguish visually the species within the white or black truffles, making the possibility of food fraud very easy. Therefore, the identification of different truffle species (Tuber spp.) is an analytical task that could be solved in this study. The polar extract from a total of 80 truffle samples was analyzed by 1H NMR spectroscopy in combination with chemometric methods covering five commercially relevant species. All classification models were validated applying a repeated nested cross-validation. In direct comparison, the two very similar looking and closely related black representatives Tuber melanosporum and Tuber indicum could be classified 100% correctly. The most expensive truffle Tuber magnatum could be distinguished 100% from the other relevant white truffle Tuber borchii. In addition, signals for a potential Tuber borchii and a potential Tuber melanosporum marker for targeted approaches could be detected, and the corresponding molecules were identified as betaine and ribonate. A model covering all five truffle species Tuber aestivum, Tuber borchii, Tuber indicum, Tuber magnatum, and Tuber melanosporum was able to correctly discriminate between each of the species.

Mitochondrial Analysis of Sparidae Species to Detect a New DNA Barcoding Marker for Dentex gibbosus to Utilize against Fraud.

Dentex gibbosus (Pink dentex) is a fish species of increasing economic interest in the Mediterranean Sea that is consumed both whole and processed. The growing value of this sparid in European markets is responsible for its substitution with fraudulent species. The distinctive morphologic feature of D. gibbosus is the conspicuous hump on the forehead in the older and larger specimens. However, the head is regularly convex in young individuals, requiring high skills and competencies for correct identification. Authentication becomes even more challenging in the case of prepared and processed products. Therefore, the molecular characterization of Pink dentex plays a crucial role in preventing commercial fraud with species substitution. This paper proposes a comparative mitogenome analysis between 19 sparid species of commercial interest as a tool to accurately design species-specific primers targeting a fragment of the NAD2 gene for the identification of D. gibbosus. We successfully detected Pink dentex DNA both using endpoint and real-time PCR. The findings showed the high specificity of the designed primers, demonstrating this a suitable, fast, and cost-effective method that could be used for the unambiguous identification of Pink dentex. This innovative approach for sparid authentication is expected to contribute to seafood traceability, public health assurance, integrity, and the credibility of the seafood industry.

DNA from herbs can be obtained from air and authenticated by polymerase chain reaction.

DNA barcoding of herbs allows accurate species authentication. However, the DNA of herbs are often not easily PCR amplified due to co-extraction of inhibitors. Methods have been developed to improve DNA extraction to reduce contaminants. These methods usually require toxic chemical treatments or expensive commercial kits and are labor intensive. In this report, we collected the air passed from the herbs and directly amplified the DNA obtained. Results showed that DNA could be obtained, and it was PCR amplifiable. Sequencing of the amplified DNA allowed species authentication. This DNA collection method is applicable to herbs from different plant tissues. It has the advantages of reducing the use of toxic substances and more economical.

Characterization of the natural peptidome of four leeches by integrated proteogenomics and pseudotargeted peptidomics.

Animal-derived drugs are an indispensable part of folk medicine worldwide. However, their chemical constituents are poorly approached, which leads to the low level of the quality standard system of animal-derived drugs and further causes a chaotic market. Natural peptides are ubiquitous throughout the organism, especially in animal-derived drugs. Thus, in this study, we used multi-source leeches, including Hirudo nipponica (HN), Whitmania pigra (WP), Whitmania acranulata (WA), and Poecilobdella manillensis (PM), as a model. A strategy integrating proteogenomics and novel pseudotargeted peptidomics was developed to characterize the natural peptide phenotype and screen for signature peptides of four leech species. First, natural peptides were sequenced against an in-house annotated protein database of closely related species constructed from RNA-seq data from the Sequence Read Archive (SRA) website, which is an open-sourced public archive resource. Second, a novel pseudotargeted peptidomics integrating peptide ion pair extraction and retention time transfer was established to achieve high coverage and quantitative accuracy of the natural peptides and to screen for signature peptides for species authentication. In all, 2323 natural peptides were identified from four leech species whose databases were poorly annotated. The strategy was shown to significantly improve peptide identification. In addition, 36 of 167 differential peptides screened by pseudotargeted proteomics were identified, and about one-third of them came from the leucine-rich repeat domain (LRR) proteins, which are widely distributed in organisms. Furthermore, six signature peptides were screened with good specificity and stability, and four of them were validated by synthetic standards. Finally, a dynamic multiple reaction monitoring (dMRM) method based on these signature peptides was established and revealed that one-half of the commercial samples and all of the Tongxinluo capsules were derived from WP. All in all, the strategy developed in this study was effective for natural peptide characterization and signature peptide screening, which could also be applied to other animal-derived drugs, especially for modelless species that are less studied in protein database annotation.

Lateral flow immunoassay-based absolute point-of-care technique for authentication of meat and commercial meat products.

Point-of-care (POC) assay is an emerging technique for rapid initial screening of meat fraud incidents in a resource-limited environment. To achieve this goal, a simple extraction protocol is proposed for efficient recovery of meat proteins from raw, heat-processed, and commercial samples as well as meat offals without utilizing sophisticated laboratory settings. A sandwich-format lateral flow immunoassay (LFIA) was developed based on gold nanoparticles as labels and immunoglobulins (IgG and IgY) as biomarkers for meat species identification in raw and cooked meat mixes. The test system showed a sensitivity of 10 ng/mL allowing the detection of as low as 0.063% pork and chicken meat and 0.125% sheep meat (lamb) in meat mixes within 15 min including sample preparation. Reproducibility of the assay was confirmed by the fully consistent intra- and inter-laboratory tests and RT-PCR method. The current study developed a field-deployable extraction technique and highly-specific, sensitive, reproducible, cost-effective, and user-friendly LFIA-based assay for rapid species authentication in raw, cooked, and commercial meat samples and meat offals. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05663-2.

Identification of Pinelliae Rhizoma and its counterfeit species based on enzymatic signature peptides from toxic proteins.

BACKGROUND: Pinelliae Rhizoma (PR), a toxic medication, with long history, is commonly used for eliminating phlegm. Due to the shortage of wild resources and the relative lacking of cultivation technology, it is often confused with its counterfeit species in the market, such as Typhonii Rhizoma (TR), Arisaematis Rhizoma (AR) and tubers of Typhonium flagelliforme (TF) and Pinellia pedatisecta (PP). PURPOSE: It was aimed to screen signature enzymatic peptides from toxic proteins to identify PR and its four counterfeit species. STUDY DESIGN: A comparative proteogenomics strategy based on open-source transcriptome data was applied for screening signature peptides from toxic proteins, which were applied for species authentication of PR and its counterfeit species. METHODS: Firstly, the open-source transcriptome data was used for constructing the annotated protein database, which was used for peptides identification. Secondly, the toxicity of different fractions of PR were evaluated by the rat peritoneal inflammation model. Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to profile the main proteins bands of five species, whose sequences were identified based on the in-gel digestion experiment by using ultra-high-performance liquid chromatography/quadrupole-Orbitrap mass spectrometry. Finally, the label-free proteomic analysis was performed to character the proteins and screen the signature peptides of five species, which were validated in commercially available products by dynamic multi reaction monitor (DMRM). RESULTS: The results in this study confirmed that protein was the main toxic components of PR. Both Pinellia ternata agglutinin (PTA) and trypsin inhibitor (TI) like proteins are the main proteins, which were characterized by proteomic analysis based on four annotated protein database. Meanwhile, seven signature peptides from toxic proteins were screened and validated with good repeatability and specificity in commercial products. CONCLUSION: Seven signature enzymatic peptides from toxic protein screened by the comparative proteogenomics strategy based on open-source transcriptome data achieved good identification ability of PR and its four counterfeit species.

Rapid authentication of Chaenomeles species by visual volatile components fingerprints based on headspace gas chromatography-ion mobility spectrometry combined with chemometric analysis.

INTRODUCTION: Chaenomeles, including Chaenomeles speciosa (ZP), Chaenomeles sinensis (GP), Chaenomeles tibetica (XZ), and Chaenomeles japonica (RB), has been widely used as food in China for thousands of years. However, only ZP, was recorded to be the authentic medicinal Chaenomeles. Therefore, the rapid and accurate method for the authenticity identification of Chaenomeles species is urgently needed. OBJECTIVE: To develop a method for rapid differentiation of Chaenomeles species. METHODS: The visual volatile components fingerprints based on headspace gas chromatography-ion mobility spectrometry (HS-GC-IMS) combined with chemometric analysis, including principal component analysis (PCA), linear discriminant analysis (LDA) and partial least-squares discriminant analysis (PLS-DA), were utilised for the authentication of Chaenomeles species. RESULTS: The visual volatile components fingerprints by the GC-IMS intuitively showed the distribution features of the volatile components for different Chaenomeles samples. The LDA and PLS-DA models successfully discriminated Chaenomeles species with original discrimination accuracy of 100%. Fifteen volatile compounds (VOCs) (peaks 9, 12, 13, 19, 23, 24, 35, 48, 57, 65, 67, 76, 79, 80, 83) were selected as the potential species-specific markers of Chaenomeles via variable importance of projection (VIP > 1.2) and one-way analysis of variance (P < 0.05). CONCLUSIONS: This study showed that the visual volatile components fingerprints by HS-GC-IMS combined with chemometric analysis is a meaningful method in the Chaenomeles species authentication.

COIBar-RFLP Molecular Strategy Discriminates Species and Unveils Commercial Frauds in Fishery Products.

The DNA analysis is the best approach to authenticate species in seafood products and to unveil frauds based on species substitution. In this study, a molecular strategy coupling Cytochrome Oxidase I (COI) DNA barcoding with the consolidated methodology of Restriction Fragment Length Polymorphisms (RFLPs), named COIBar-RFLP, was applied for searching pattern of restriction enzyme digestion, useful to discriminate seven different fish species (juveniles of Engraulis encrasicolus and Sardina pilchardus sold in Italy as "bianchetto" and Aphia minuta sold as "rossetto"; icefish Neosalanx tangkahkeii; European perch, Perca fluviatilis and the Nile Perch, Lates niloticus; striped catfish, Pangasianodon hypophthalmus). A total of 30 fresh and frozen samples were processed for DNA barcoding, analyzed against a barcode library of COI sequences retrieved from GenBank, and validated for COIBar-RFLP analysis. Cases of misdescription were detected: 3 samples labeled as "bianchetto" were substituted by N. tangkahkeii (2 samples) and A. minuta (1 sample); 3 samples labeled as "persico reale" (P. fluviatilis) were substituted by L. niloticus and P. hypophthalmus. All species were simultaneously discriminated through the restriction pattern obtained with MspI enzyme. The results highlighted that the COIBar-RFLP could be an effective tool to authenticate fish in seafood products by responding to the emerging interest in molecular identification technologies.

Rapid identification of Takifugu genus using visual loop-mediated isothermal amplification.

Some Takifugu species are commonly found in the coastal areas of China, Japan, Thailand, and Korea and cause pufferfish poisoning, which is toxic and even lethal to humans. From 2010 to 2015, there were 430 cases of pufferfish poisoning worldwide, resulting in 52 deaths. Identification of Takifugu species is imperative to reduce financial losses and ensure food safety. Here, visual loop-mediated isothermal amplification (LAMP) was applied to identify Takifugu species. Conserved regions within the mitochondrial DNA among different Takifugu species were selected to design LAMP primers. In 55 min of amplification, sufficient DNA was obtained to observe the results with the naked eye, without the need for complicated instruments. The method was highly specific, with no cross-detection of 17 other fish species, namely, 7 Tetraodontiformes species and 10 commercially important fish. The method showed a detection limit of 0.1 ng Takifugu DNA and was successfully validated to detect Takifugu in cooked fish and the vomitus of poisoned patients. This rapid and visual LAMP method is a useful tool to prevent false labeling, protect consumer rights, and reduce the risk of pufferfish poisoning. PRACTICAL APPLICATION: The loop-mediated isothermal amplification method established in this study can identify cooked or digested fish products containing 1% or more of Takifugu. Therefore, it can be used for the visual detection of Takifugu products and the medical diagnosis of Takifugu poisoning.

A rapid and simple signature peptides-based method for species authentication of three main commercial Pheretima.

Pheretima with various activities is a commonly used animal-derived traditional medicine in Asia countries. However, almost half of them are non-pharmacopoeia species in the market due to the similar morphological characteristics between medicinal and non-medicinal species. This study aims to establish an effective method based on signature peptides for species authentication of three main commercial Pheretima, including two major Pheretima species (Amynthas aspergillum, Metaphire vulgaris) and one main adulteration (Metaphire magna). Firstly, the species of 52 batches of commercial Pheretima were authenticated based on DNA barcodes. Secondly, proteomic analysis was performed for protein characterization of three main commercial Pheretima. Furthermore, their signature peptides were screened and validated using ultra-high performance liquid chromatography coupled with mass spectrometry (UPLC-MS/MS) in multiple reaction monitoring (MRM) mode. Moreover, a simplified sample processing method was developed. Finally, large quantities of commercial Pheretima samples were analyzed for further verifying the feasibility of the signature peptides-based method. The result showed that the established method had a great application potential for authenticity identification of commercial Pheretima. SIGNIFICANCE: The authenticity assessment of medicinal materials is a main issue in the quality control process as deceptive practices could imply severe health risks. In this study, a rapid and simple method based on signature peptides was established for species authentication of three main commercial Pheretima, which can be an effective alternative to complex DNA barcoding and difficult morphological identification, and provided a reference for improvement of Pheretima quality standards.

Authentication of three main commercial Pheretima based on amino acids analysis.

Pheretima has been used as an animal-derived traditional Chinese medicine for thousands of years in Asian countries due to its multi-activities. However, more than half of the commercial Pheretima are adulterants according to the previous research. Besides, the standards of Pheretima are still inadequate in the identification of Pheretima species. In this study, an amino acids (AAs) analytical method established based on the ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-QqQ-MS) in multiple reaction monitoring (MRM) mode through derivatization with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) was used for qualitative and quantitative analysis of the total AAs of three main commercial Pheretima (two major Pheretima species, Amynthas aspergillum, Metaphire vulgaris, and one main counterfeit, M. magna). As a result, 16 AAs were detected and quantitated in their hydrolyzed samples. Then, multivariate statistical analysis was applied to distinguish the three commercial Pheretima based on their AAs level. Finally, four AAs (Thr, Glu, Asp, and Arg) were screened as species-differential AAs, which may be used as chemical markers to distinguish the three commercial Pheretima. This study deeply described the outline of AAs in Pheretima and offered a good reference for its species authentication.

Phylogenetic analysis and development of molecular markers for five medicinal Alpinia species based on complete plastome sequences.

BACKGROUND: Alpinia species are widely used as medicinal herbs. To understand the taxonomic classification and plastome evolution of the medicinal Alpinia species and correctly identify medicinal products derived from Alpinia species, we systematically analyzed the plastome sequences from five Alpinia species. Four of the Alpinia species: Alpinia galanga (L.) Willd., Alpinia hainanensis K.Schum., Alpinia officinarum Hance, and Alpinia oxyphylla Miq., are listed in the Chinese pharmacopeia. The other one, Alpinia nigra (Gaertn.) Burtt, is well known for its medicinal values. RESULTS: The four Alpinia species: A. galanga, A. nigra, A. officinarum, and A. oxyphylla, were sequenced using the Next-generation sequencing technology. The plastomes were assembled using Novoplasty and annotated using CPGAVAS2. The sizes of the four plastomes range from 160,590 bp for A. galanga to 164,294 bp for A. nigra, and display a conserved quadripartite structure. Each of the plastomes encodes a total of 111 unique genes, including 79 protein-coding, 28 tRNA, and four rRNA genes. In addition, 293-296 SSRs were detected in the four plastomes, of which the majority are mononucleotides Adenine/Thymine and are found in the noncoding regions. The long repeat analysis shows all types of repeats are contained in the plastomes, of which palindromic repeats occur most frequently. The comparative genomic analyses revealed that the pair of the inverted repeats were less divergent than the single-copy region. Analysis of sequence divergence on protein-coding genes showed that two genes (accD and ycf1) had undergone positive selection. Phylogenetic analysis based on coding sequence of 77 shared plastome genes resolves the molecular phylogeny of 20 species from Zingiberaceae. In particular, molecular phylogeny of four sequenced Alpinia species (A. galanga, A. nigra, A. officinarum, and A. oxyphylla) based on the plastome and nuclear sequences showed congruency. Furthermore, a comparison of the four newly sequenced Alpinia plastomes and one previously reported Alpinia plastomes (accession number: NC_048461) reveals 59 highly divergent intergenic spacer regions. We developed and validated two molecular markers Alpp and Alpr, based on two regions: petN-psbM and psaJ-rpl33, respectively. The discrimination success rate was 100 % in validation experiments. CONCLUSIONS: The results from this study will be invaluable for ensuring the effective and safe uses of Alpinia medicinal products and for the exploration of novel Alpinia species to improve human health.

Wound healing potential of licorice extract in rat model: Antioxidants, histopathological, immunohistochemical and gene expression evidences.

Wound healing is a public health concern. Licorice gained a great attention for its antioxidant and anti-inflammatory properties which expand its valuable effects as a herbal medicine. In this study, we pointed out to the wound healing potential and the mechanism by which licorice alcoholic extract can modulate cutaneous wound healing through immune, antioxidant, histopathological, immunohistochemical (IHC) and molecular studies. 24 Wister rats were assigned into 3 groups (n = 8 each); control group, topical and oral supplied groups. Licorice extract administration significantly increased total and differential leucocyte counts, phagocytic activity of neutrophils, antioxidant biomarkers as superoxide dismutase (SOD), glutathione peroxidase activities (GPx) and reduced glutathione (GSH) content with a notable reduction in oxidative stress marker malondialdehyde (MDA). Moreover, histopathological findings detected complete re-epithelialization with increasing collagen synthesis while IHC results revealed a significant enhancement in the expression of α-SMA, PDGFR-α, FGFR1 and Cytokeratin 14 in licorice treated groups compared with the control group. Licorice extract supplementation accelerated wound healing by increasing angiogenesis and collagen deposition through up-regulation of bFGF, VEGF and TGF-ß gene expression levels compared with the control group. UPLC-PDA-MS/MS aided to authenticate the studied Glycyrrihza species and recognized 101 potential constituents that may be responsible for licorice-exhibited potentials. Based on our observations we concluded that licorice enhanced cutaneous wound healing via its free radical-scavenging potential, potent antioxidant activities, and anti-inflammatory actions. Therefore, licorice could be used as a potential alternative therapy for wound injury which could overcome the associated limitations of modern therapeutic products.

Single nucleotide polymorphism-based methodology for authentication of bovine, caprine, ovine, camel, and donkey meat cuts.

To guarantee food safetyand sustainability, it is necessary to verify meat authenticity. This study focused on the development of single nucleotide polymorphism-based polymerase chain reaction-restriction fragment length polymorphism (SNP-based PCR-RFLP) and forensically informative nucleotide sequence (FINS) methodologies based on PCR amplification of the mitochondrial 12S rRNA gene for discrimination of six red meat species, that is, cattle, buffalo, goat, sheep, camel, and donkey. FINS allowed the unambiguous identification of all species analyzed. In addition, six SNPs, where a restriction site for TasI could be localized using a preliminary in silico analysis, gave a unique RFLP pattern for each species. The results revealed a low level of species substitution (8%) in the tested meat samples. In particular, one buffalo and goat samples have been substituted with cow and sheep, respectively. Finally, the developed techniques herein showed high potentials to be routinely used as reliable and fast tools to avoid meat species substitutions. PRACTICAL APPLICATION: This research deals with genetic techniques to trace meats. This kind of research helps the concerned agencies to build capacity to safeguard consumer sentiments as well as providing better market access and better food price and quality for the consumer.

A New Rapid Method for the Authentication of Common Octopus (Octopus vulgaris) in Seafood Products Using Recombinase Polymerase Amplification (RPA) and Lateral Flow Assay (LFA).

The common octopus (Octopus vulgaris) is a highly valued cephalopod species which is marketed with different grades of processing, such as frozen, cooked or even canned, and is likely to be mislabeled. Some molecular methods have been developed for the authentication of these products, but they are either labor-intensive and/or require specialized equipment and personnel. This work describes a newly designed rapid, sensitive and easy-to-use method for the detection of Octopus vulgaris in food products, based on Recombinase Polymerase Amplification (RPA) and a detection using a Lateral Flow assay (LFA). After studying several gene markers, a system of primers and nfo-probe was designed in the COI (Cytochrome Oxidase I) region and was successfully tested in 32 reference samples (covering 14 species) and 32 commercial products, after optimization. The method was also validated in a ring trial with eight European laboratories and represents a useful tool for food authenticity control at all levels of the value chain.

Integration of Untargeted and Pseudotargeted Metabolomics for Authentication of Three Shrimp Species Using UHPLC-Q-Orbitrap.

In this work, an untargeted and pseudotargeted metabolomics combination approach was used for authentication of three shrimp species (Litopenaeus vanmamei, Penaeus japonicus, and Penaeus monodon). The monophasic extraction-based untargeted metabolomics approach enabled comprehensive-coverage and high-throughput analysis of shrimp tissue and revealed 26 potential markers. The pseudotargeted metabolomics approach confirmed 21 markers (including 9 key markers), which realized at least putative identification. The 21 confirmed markers, as well as 9 key markers, were used to develop PLS-DA models, correctly classifying 60/60 testing samples. Furthermore, DD-SIMCA and PLS-DA models were integrated based on the 9 key markers, with 59/60 and 20/20 samples of the species that were involved and uninvolved in model training correctly classified. The results demonstrated the potential of this untargeted and pseudotargeted metabolomics combination approach for shrimp species authentication.

High performance liquid chromatography fingerprint and headspace gas chromatography-mass spectrometry combined with chemometrics for the species authentication of Curcumae Rhizoma.

Curcumae Rhizoma (Ezhu), a multi-origin Chinese medicine, originates from the dry rhizomes of C. kwangsiensis, C. phaeocaulis and C. wenyujin. The three species have great variation in chemical components and therapeutic effects. To improve safety and effectiveness in clinical use, a strategy integrating chromatographic analysis and chemometrics for the species authentication of Ezhu was proposed. Firstly, systematic analysis of chemical compositions in Ezhu was achieved using high performance liquid chromatography (HPLC) fingerprint and headspace gas chromatography-mass spectrometry (HS-GC-MS). HPLC fingerprints showed that seventeen peaks in common for C. kwangsiensis and eleven peaks in common for C. wenyujin both presented a good similarity (> 0.9, only several samples < 0.8). Eleven common peaks in C. phaeocaulis and the similarity values of most samples were higher than 0.700. Additionally, there were ten common peaks in all Ezhu samples and they had relatively poor similarity with the correlation coefficients ranging from 0.364 to 0.881. For HS-GC-MS, thirty-six volatile components were identified in the three species of Ezhu, mainly monoterpenes and sesquiterpenes. Subsequently, chemometrics including unsupervised principal component analysis (PCA), supervised linear discriminant analysis (LDA), K-nearest neighbors (KNN), back propagation neural network (BP-NN) and orthogonal partial least squares-discrimination analysis (OPLS-DA) was applied to extract useful information from chromatographic profiles. Based on HPLC fingerprint data, PCA could hardly differentiate Ezhu with the three species, and LDA, KNN and BP-NN models provided more than 85 % correct identification. With HS-GC-MS data, PCA could only distinguish C. wenyujin from the other two species, and LDA, KNN, BP-NN and OPLS-DA models achieved excellent classification with 100 % accuracy. Finally, five volatile components (eucalyptol, humulene, ß-elemene, (+)-2-bornanone and linalool) with variable importance for the projection (VIP) values higher than 1 in the OPLS-DA model were selected as potential chemical markers for the species authentication of Ezhu. And the constructed OPLS-DA model using these markers obtained 100 % accuracy. Consequently, a rapid, precise and feasible strategy was established for the discrimination and quality control of Ezhu with different species.

Dentex dentex Frauds: Establishment of a New DNA Barcoding Marker.

The common dentex (Dentex dentex (Linnaeus, 1758)) is an iconic fish in the Mediterranean diet. Due to its commercial and organoleptic importance, this sparid is highly appreciated in European markets and is often subjected to species substitution frauds. Comparative mitogenomics is a suitable approach for identifying new and effective barcode markers. This study aimed to find a molecular tag useful for unequivocally discriminating the sparid species D. dentex. The comparison of the complete mitochondrial DNA (mtDNA) sequences of 16 sparid species allowed us to highlight the potential of the NAD2 gene for direct identification purposes. Common dentex-specific primers were created and successfully evaluated by end-point and real-rime PCR (Polymerase Chain Reaction) for several fish species, achieving amplification only in the D. dentex. The method proposed in this study appears fast, simple, and inexpensive and requires affordable instrumentation. This approach provides unambiguous results for the common dentex authentication without the sequencing step. The presence/absence assay for D. dentex can be executed in a few hours of lab work. Therefore, national authorities responsible for food safety and traceability could apply and make full use of DNA-testing methods for deterring operators from false seafood declarations.

Detecting mislabelling in meat products using PCR-FINS.

Economically motivated adulteration (EMA) or misrepresentation of meat products is of concern, especially in developing countries, due to obvious health hazards and religious sensitivities. As Indian cooking involves prolonged heat treatments and addition of spices and condiments, species authentication of food, especially meat products, may be challenging. This study evaluated the efficacy of Polymerase Chain Reaction-Forensically Informative Sequencing (PCR-FINS) in meat speciation of highly processed meat. Further the prevalence of mislabelling in processed and deeply cooked meat products being sold in supermarkets and restaurants in a south Indian city was investigated. FINS targeting the mitochondrial cytochrome b gene and the ATP synthase gene was applied to identify meat species of 106 meat products labelled as chicken, beef, carabeef, mutton and pork. Mislabelling was detected in more than half of mutton (52.3%) and carabeef (55.5%), and in under a third (27.2%) of beef products. PCR-FINS is a reliable method for meat species identification even in highly processed food but there is a need for appropriate universal primers which can target all common species used in meat products. This study is the first of its kind from the South Indian state of Kerala.

A Rapid Method for the Identification of Fresh and Processed Pagellus erythrinus Species against Frauds.

The commercialization of porgies or seabreams of the family Sparidae has greatly increased in the last decade, and some valuable species have become subject to seafood substitution. DNA regions currently used for fish species identification in fresh and processed products belong to the mitochondrial (mt) genes cytochrome b (Cytb), cytochrome c oxidase I (COI), 16S and 12S. However, these markers amplify for fragments with lower divergence within and between some species, failing to provide informative barcodes. We adopted comparative mitogenomics, through the analysis of complete mtDNA sequences, as a compatible approach toward studying new barcoding markers. The intent is to develop a specific and rapid assay for the identification of the common pandora Pagellus erythrinus, a sparid species frequently subject to fraudulent replacement. The genetic diversity analysis (Hamming distance, p-genetic distance, gene-by-gene sequence variability) between 16 sparid mtDNA genomes highlighted the discriminating potential of a 291 bp NAD2 gene fragment. A pair of species-specific primers were successfully designed and tested by end-point and real-time PCR, achieving amplification only in P. erythrinus among several fish species. The use of the NAD2 barcoding marker provides a rapid presence/absence method for the identification of P. erythrinus.