DNA Mini-barcodes Research Articles

DNA Mini-Barcodes, a Potential Weapon for Conservation and Combating Illegal Trade of Pangolin

Background Smuggling and illegal trade of pangolins and their scales has drastically reduced the wild population of pangolins. Accurate species identification is currently in urgent need as a powerful weapon for combating pangolin smuggling and trade and conserving the already endangered pangolin species. Aim of the study To develop an efficient method based on DNA mini-barcodes for accurate pangolin species identification and authentication of processed pangolin scales against the non-target species. Materials and methods The primers for amplifying the DNA mini-barcodes were designed based on cytochrome C oxidase subunit I (COI) gene fragments. The mini-barcodes were compared with the two universal barcodes (COI and Cytb) for performance in pangolin species identification by calculating the Kimura-2-parameter (K2P) distance, assessing the clustering dendrogram, and analyzing the BLAST similarity and barcoding gap. The accuracy of the three barcodes was also compared for authentication of pangolin scales against non-target species. Results Comparison of the three barcodes showed that the mini-barcode form COI had the highest amplification success rate (100%) and high variable sites (40.0%), with the ratio of mean inter- to intraspecific distance ratio was 25 and a distinct DNA barcoding gap. In the neighbor-joining (NJ) tree constructed based on the mini-barcode regions, each species of the pangolin family formed an obvious clade respectively, and the clades were all separated from those of the non-target species, indicating that the genetic information in the mini-barcode was sufficient for species identification. Conclusion The DNA mini-barcodes based on COI gene fragments provide an effective and accurate method for identification of pangolin species and authentication of pangolin scale products.

Application of DNA mini-barcoding and infrared spectroscopy for the authentication of the Italian product “bottarga”

Tuna “Bottarga” is an artisanal food speciality prepared from the roe of Bluefin Tuna (Thunnus thynnus). Due to the recent restriction of Tuna catches fixed by EU, cheaper substitutes from yellowfin tuna (Thunnus albacares) are currently available in the mass distribution. Since the product is sold sliced or grated, morphological distinctive features may be lost, and fraudulent substitution is possible. In this study we tested DNA mini-barcoding and infrared spectroscopy as tools to trace its authenticity. A certified chemical standard, fresh (n = 4) and processed (n = 11) ovarian identified by the morphological observation of the tuna catches, commercial bottargasamples (n = 5) and artificial aged aliquots of all these samples (n = 50) were used in the experiments. The results showed that infrared spectroscopy failed in the taxonomic distinction, whereas enabled PLS-DA classification of aged samples (and thus the identification of fresh prepared artisanal products) with model non-error rate in fitting equal to 0.96, and 0.89 in cross-validation. Conversely, DNA mini-barcoding with mtDNA control region, success in species identification (100% maximum identity by the standard comparison BLAST approach against GenBank), regardless the processing state of the product.

Specific DNA mini-barcoding for identification of Gekko gecko and its products

BackgroundThe dry body of the Tokay Gecko (Gekko gecko) is the source of a valuable traditional Chinese medicine, it is therefore listed as a Class II protected animal species in China. Due to increasing market demand and a declining supply of the species, a considerable number of adulterants have emerged in the market. Thus, it is necessary to establish an accurate and rapid method of identification for distinguishing G. gecko from its adulterants and for separating it from highly processed products.MethodsA total of 274 COI sequences were analyzed by using MEGA 5.0 software. Several specific primers were designed to amplify mini-barcode regions and identify G. gecko from its counterfeits and products.Results274 COI sequences of G. gecko and 15 adulterants species were analyzed. G. gecko could be distinguished from its adulterants through BLAST analysis, intra- and inter-specific distance analyses, and an NJ tree based on COI sequences. Two pairs of specific primers designed for this study, COISF2/COISR2 and COISF3/COISR3, amplified 200- and 133-bp fragments of the COI region, respectively, both of which were suitable for the identification of G. gecko and its adulterants. Furthermore, COISF3/COISR3 detected G. gecko in 15 batches of products.ConclusionTherefore, the specific DNA mini-barcoding method developed here may be a powerful tool for the identification of G. gecko and counterfeits, and may also be used to distinguish G. gecko from its highly processed by-products.

Application of DNA barcoding to detect mislabeling of fish fillet products from Jabodetabek’s market

Mislabeling of fish fillet product is one of the key issues in food safety and sustainability. Species identification is an important step of fish fillet traceability and DNA barcoding has been proved as a standard method. Fourty seven 47 fish fillet products were collected from modern and traditional markets in the Jabodetabek area. DNA barcoding was used to analyze the compliance of the product label. This research also highlighted that near threatened (NT), Vulnerable (VU), endangered (EN), and critically endangered (CR) species considered to be facing a high risk of extinction have been used as a substitution in fish fillet product. The application of DNA mini-barcoding gives better resolution in species identification for commercial species. From this research, we found that there are some mislabeled fish fillet products, including blue shark meat that is listed as Near Threatened in IUCN Red List in one of the products.

DNA mini-barcoding of leporids using noninvasive fecal DNA samples and its significance for monitoring an invasive species.

Introduced in South America at the end of the 19th century, the European hare population has expanded dramatically and now represents a risk to native Brazilian forest rabbits. Monitoring the invasive Lepus europaeus and its coexistence with native Sylvilagus brasiliensis is a challenge that can be efficiently addressed by the use of molecular tools. This work describes a set of primers useful for amplifying three mini‐barcodes for the molecular identification of both invasive and native leporid species using degraded fecal DNA. In addition, tests in silico indicate that these mini‐barcodes can successfully amplify the DNA sequences of a number of leporids. These mini‐barcodes constitute a powerful tool for the monitoring and management of the invasive L. europaeus and the conservation of native rabbits.

Development of a DNA mini-barcoding protocol targeting COI for the identification of elasmobranch species in shark cartilage pills

Many elasmobranch (shark and ray) species are considered threatened and their identification in processed products is important for conservation and authentication purposes. However, identification of elasmobranch species in shark cartilage pills has proven difficult using existing methodologies. The objective of this study was to develop a DNA mini-barcoding protocol using a ~130 bp region of the cytochrome c oxidase subunit I (COI) gene for species identification in shark cartilage pills. A total of 22 shark cartilage products underwent DNA extraction in duplicate using the DNeasy Blood and Tissue Kit (Qiagen). The effectiveness of a clean-up step following DNA extraction was analyzed by comparing DNA purity values and polymerase chain reaction (PCR) amplification rates. Next, five different mini-barcode primer sets were compared based on amplification rates, and the three top-performing primer sets were used in DNA sequencing. The incorporation of a clean-up step following DNA extraction showed a slight advantage over DNA extraction alone, with a higher amplification rate (52.3% vs. 47.8%) and A260/A230 value (3.3 vs. 0.6). The three primer sets selected for DNA mini-barcoding showed DNA sequencing rates of 54.5–65.9% among the 44 duplicate samples. When the results for all three primer sets were combined, 18 of the 22 shark cartilage products were identified to the species or genus level. On an individual basis, the best-performing primer set identified 16 of the 22 products to the species or genus level. Overall, the protocol developed in this study increased the identification rate for elasmobranches in cartilage products by more than 2-fold as compared to previous research.

DNA Mini-Barcoding: A Derived Barcoding Method for Herbal Molecular Identification.

In recent years, the demand for natural herbal products (NHP) has increased; however, the quality of these products is difficult to confirm due to the lack of a comprehensive quality control system. Traditional methods are not effective in detecting processed ingredients. DNA barcoding is an established technique that has been used for more than 10 years. This technique uses short standard sequences (generally 200–600 bp) to identify species. While a complete DNA barcode is difficult to obtain from NHP due to DNA degradation, mini-barcoding is a complementary tool to identify species in NHP. DNA mini-barcoding uses smaller DNA segments for polymerase chain reaction amplification and can be applied to identify species rapidly. The present review summarizes the development and application of DNA mini-barcodes over recent years and discusses the limitations of this technique. This review also compares mini-barcoding and meta-barcoding, a technique using universal polymerase chain reaction primers to simultaneously amplify multiple DNA barcodes and identify many species in a single environmental sample. Additionally, other detection methods that can be combined with mini-barcodes, such as nucleotide signatures, high-resolution DNA melting analysis, and gold nanoparticles, are discussed. DNA mini-barcoding can fill the gaps left by other methods in the field of herbal molecular identification.

DNA barcoding in authentication of herbal raw materials, extracts and dietary supplements: a perspective

Cultivation, supply, and availability of unadulterated plant raw material in botanical or herbal industry are a challenging task. Plant species that are either in short supply or in high demand are prone to adulteration and substitution. Such unscrupulous activity results in availability of substandard products with declining quality standards ultimately posing safety concerns and health of consumers. Raw materials which are in the form of dried, fragmented or powdered forms are major victims of adulteration. We present here DNA-based marker approach to authenticate botanicals. Recent research advances such as 2D-electrophoresis, microarray, next generation sequencing, high-resolution melting analysis, and PCR-based methods have been reviewed and their role in authentication of raw material, botanicals in herbal extracts, finished formulations, and ingredients in dietary supplements has been presented. Due to extraction processes such as steaming, extraction at extreme conditions and presence of secondary metabolites original DNA integrity are not preserved which lead to interference with DNA extraction and PCR amplification, thus making DNA unsuitable for further molecular analysis. There is a scope to develop and validate DNA mini-barcodes for finished formulations. DNA barcode along with chromatographic methods, metabolomics, proteomics, and genomics has to be used to resolve substitution and quantity of raw material in ambiguity. Emerging regulatory scenario on DNA barcode in various Pharmacopoeia is presented. Though several advanced methods of botanical authentication are available, an industry always expects simple, cost efficient, and quick method to resolve authentication issues.

DNA Mini-Barcodes as A Molecular Marker for Various Hairtail Fish Products Traceability

Hairtail fishery is an important fishery commodity with high economic value as export commodity. Authentication based on DNA mini-barcodes can be applied to overcome seafood fraud and to support Indonesian traceability system. This study was aimed to design species-specific primers of DNA minibarcodes and authentication of various processed hairtail fish products such as smoked, baked, fried, canned, and boiled. Analytical method applied PCR-based of cytochrome c oxidase 1 (COI) full length and<br />mini-barcodes. Mini-barcode primer specific for hairtail (Trichiurus lepturus) successfully amplified DNA of processed hairtail fish. The BLAST analysis showed the samples were identified as Trichiurus sp. and T. lepturus with homology level of 91% to 100%. Phylogenetic analysis showed the processed sample (LC dan LD) were in the same group with T. lepturus.

DNA barcoding to trace medicinal and aromatic plants from the field to the food supplement

The global market of food supplements is growing, along with consumers demand for high-quality herbal products. Nevertheless, substitution fraud, and adulteration cases remain a common safety problem of global concern. In the last years, the DNA barcoding approach has been proposed as a valid identification method and it is now commonly used in the authentication of herbal and food products. The objective of this study was to evaluate whether DNA barcoding can be applied to trace the plant species from the starting raw material to the finished commercial products. We selected a panel of 28 phytoextracts obtained through three different extraction methods (i.e., maceration, percolation and sonication) with different solvents (i.e., ethanol, deionized water and glycerol). Furthermore, we chose six plant species for which we collected and analysed all the intermediates of the industrial production. We sequenced and analyzed the sequence variability at DNA barcoding (psbA-trnH, ITS) and minibarcoding (rbcL 1-B) marker regions. Phytoextracts obtained through hydroalcoholic treatment, with the lower percentage of ethanol (<40%), and aqueous processing, at the lowest temperature, had major rate of sequencing and identification success. This study proves that DNA barcoding is a useful tool for Medicinal and Aromatic Plants (MAPs) traceability, which would provide consumers with safe and high-quality herbal products.

Saving DNA from museum specimens: The success of DNA mini-barcodes in haplotype reconstruction in the genus Anastrepha (Diptera: Tephritidae)

The fragmentation of DNA in historical specimens is very common, so obtaining sequences that allow molecular identification and the study of diversity is quite challenging. In this study, we used preserved and fresh specimens of the fruit fly genus Anastrepha, a genus of economic impact of fruit crops of the Neotropic. From these specimens, we evaluated: (1) the success PCR amplification rates of mini-barcodes fragments of the cytochrome c oxidase subunit I (COI) gene, and (2) the usefulness of mini-barcodes in the reconstruction of haplotypes for the identification of species and the diversity analysis. We used 93 specimens from 12 species, which had been preserved in 70% ethanol for more than 20 years. Internal primers were designed in the COI region and primers available in the literature were also evaluated. We obtained amplifications for 62.36% of the samples processed, and reconstructed haplotypes between 171 bp and 632 bp. Variable amplification rates between combinations of primers and between species were obtained, and molecular identification of some museum specimens was achieved. It was also possible to compare the haplotypes obtained in four species from which both fresh and museum samples were available. Our results also show the importance of the adjustment of the primers for the amplification, allowing to amplify fragments of up to 400 bp. The use available resources in biological collections is key to increasing knowledge of species of interest, and by means of the amplification of mini-barcodes, short sequences can be obtained that allow the molecular identification of specimens and the reconstruction of haplotypes with multiple purposes.

Identification of Species in the Aromatic Spice Family Apiaceae Using DNA Mini-barcodes.

The species of the aromatic plant family Apiaceae are mainly used as spices and foods, but the family also includes medicinal and some poisonous plant species. Due to the similar chemical compounds or aroma and morphology, the poisonous species are often mistaken for the edible aromatic species. It is therefore imperative to correctly identify the species present at the initial raw stage samples to ensure product safety and efficacy. At the molecular level, plant species can be identified using DNA loci either from nuclear or plastid genome with easily available universal oligonucleotides, a technique called DNA barcoding. However, this is possible when single-species plant material is present but may not work on a mixture of plants species. Another disadvantage is that using universal oligonucleotides is of limited help, especially if the adulterating material is present in low quantities. On the other hand, if using the species-specific oligonucleotides, only single specific adulterating plant material could be detected and, consequently, the unexpected adulterants may go undetected. Therefore, in the current work, four degenerated oligonucleotides from ITS1 and ITS2 regions of the nuclear genome were designed that can bind to a variety of Apiaceae genera only and not to other genera belonging to different plant families. These family-specific oligonucleotides were able to amplify a diagnostic PCR product from 16 Apiaceae species that, upon sequencing, revealed the identity of the plant it was derived from. The size of these products is around 140 bp for ITS1 and approximately 80 bp for the ITS2 region. The size range of the amplified products falls in the category of a desired mini-barcode size to be used for damaged/fragmented DNA and next generation sequencing.

Identification of Plastrum Testudinis used in traditional medicine with DNA mini-barcodes

The carapace of the tortoise Chinemys reevesii is an ingredient of “Guijia”, a traditional Chinese medicine. However, C. reevesii is difficult to raise in aquaculture and is rare in the wild. Counterfeit tablets are made from carapaces of other species. In addition to C. reevesii, other species including Mauremys sinensis, Indotestudo elongate and Trachemys scripta have been used in Plastrum Testudinis as well. After processing, these carapaces are difficult to identify on the basis of morphological characteristics, which impedes law enforcement. Our study used DNA barcoding technology to identify C. reevesii and its substitutes. We extracted concentrated genomic DNA for PCR amplification. Based on the analysis of 61 full-length COI sequences, we designed four pairs of mini-barcode primers: Tu-A, Tu-B, Tu-C and Tu-D. The Tu-B primers sequenced genomic DNA with a success rate of 76.47%, and the Tu-D primers sequenced genomic DNA with a success rate of 88.24%. The identification efficiency of these two mini-barcodes was 70.59% and 64.71%, and the overall identification efficiency was approximately 76.47%. Similarly, a set of mini barcode systems was generated, which may provide an effective and low-cost method for the identification of authentic tortoise shells.

First records of Geodia demosponges from the New England seamounts, an opportunity to test the use of DNA mini-barcodes on museum specimens

We report the first records of the sponge genus Geodia (Demospongiae, Tetractinellida, Geodiidae) from the New England Seamounts and Muir Seamount, at lower bathyal depths. Nine specimens collected between 2000 and 2005 belong to two boreal species (Geodia macandrewii and Geodia barretti) and a temperate species (Geodia megastrella). These records extend the distributions of these deep-sea amphi-Atlantic species to the west. Most of these specimens were originally fixed in formalin, which substantially degraded the DNA. We nonetheless managed to sequence two cytochrome c oxidase subunit I (COI) mini-barcodes: the universal mini-barcode at the 5′ end of the Folmer barcode (130 bp) and a newly proposed mini-barcode at the 3′ end of the Folmer barcode (296 bp). These mini-barcodes unambiguously confirmed our identifications. As an additional test, we also successfully sequenced these two mini-barcodes from the holotype of G. barretti, collected in 1855. We conclude by advocating the use of mini-barcodes on formalin-fixed or old specimens with degraded DNA.

Identification of processed Chinese medicinal materials using DNA mini-barcoding

Most of Chinese medicinal herbs are subjected to traditional processing procedures, including stir-frying, charring, steaming, boiling, and calcining before they are released into dispensaries. The marketing and identification of processed medicinal materials is a growing issue in the marketplace. However, conventional methods of identification have limitations, while DNA mini-barcoding, based on the sequencing of a short-standardized region, has received considerable attention as a new potential means to identify processed medicinal materials. In the present study, six DNA barcode loci including ITS2, psbA-trnH, rbcL, matK, trnL (UAA) intron and its P6 loop, were employed for the authentication of 45 processed samples belonging to 15 species. We evaluated the amplification efficiency of each locus. We also examined the identification accuracy of the potential mini-barcode locus, of trnL (UAA) intron P6 loop. Our results showed that the five primary barcode loci were successfully amplified in only 8.89%–20% of the processed samples, while the amplification rates of the trnL (UAA) intron P6 loop were higher, at 75.56% successful amplification. We compared the mini-barcode sequences with Genbank using the Blast program. The analysis showed that 45.23% samples could be identified to genus level, while only one sample could be identified to the species level. We conclude that trnL (UAA) p6 loop is a candidate mini-barcode that has shown its potential and may become a universal mini-barcode as complementary barcode for authenticity testing and will play an important role in medicinal materials control.

DNA barcoding coupled to HRM analysis as a new and simple tool for the authentication of Gadidae fish species.

This work aimed to exploit the use of DNA mini-barcodes combined with high resolution melting (HRM) for the authentication of gadoid species: Atlantic cod (Gadus morhua), Pacific cod (Gadus macrocephalus), Alaska pollock (Theragra chalcogramma) and saithe (Pollachius virens). Two DNA barcode regions, namely cytochrome c oxidase subunit I (COI) and cytochrome b (cytb), were analysed in silico to identify genetic variability among the four species and used, subsequently, to develop a real-time PCR method coupled with HRM analysis. The cytb mini-barcode enabled best discrimination of the target species with a high level of confidence (99.3%). The approach was applied successfully to identify gadoid species in 30 fish-containing foods, 30% of which were not as declared on the label. Herein, a novel approach for rapid, simple and cost-effective discrimination/clustering, as a tool to authenticate Gadidae fish species, according to their genetic relationship, is proposed.

DNA Mini-barcoding for wildlife trade control: a case study on identification of highly processed animal materials

Although DNA barcoding is an efficient tool for species identification, however, its efficiency is uncertain for samples having degraded DNA and incomplete isolation/amplification of COI gene fragment (>500 bp). DNA mini-barcoding is a solution to this problem because small DNA fragment of COI genes is used for species identification. Twelve highly processed, chemically treated and finished animal skin (coats, tanned skins) and fur (mufflers) samples, received from the Sindh Wildlife Department, Pakistan, were subjected to DNA mini-barcoding. Eight mufflers belonged to Vulpes vulpes, one coat to Ursus thibetanus, one tanned skin to Lutra sumatrana, and one muffler to Vulpes sp. Origin of only one coat sample remained unidentified, success rate of 92% indicative of the fact that the mini barcoding technique can be used as a substitute of conventional barcoding where full length barcode (∼650 bp Folmer region) cannot be generated.

Use of the Mitochondrial Control Region as a Potential DNA Mini-Barcoding Target for the Identification of Canned Tuna Species

In this study, a DNA mini-barcoding methodology was developed for the differentiation of species commonly found in canned tuna. Primers were designed to target a 236-base pair (bp) fragment of the mitochondrial control region (CR) and a 179-bp fragment of the first internal transcribed spacer region (ITS1). Phylogenetic analysis revealed the ability to differentiate 13 tuna species on the basis of the CR mini-barcode, except in a few cases of species introgression. Supplementary use of ITS1 allowed for differentiation of introgressed Atlantic bluefin tuna (Thunnus thynnus) and albacore tuna (Thunnus alalunga), while differentiation of introgressed Atlantic bluefin tuna and Pacific bluefin tuna (Thunnus orientalis) requires a longer stretch of the CR. After primer design, a market sample of 53 commercially canned tuna products was collected for testing. This mini-barcoding system was able to successfully identify species in 23 of the products, including albacore tuna, yellowfin tuna (Thunnus albacares), and skipjack tuna (Katsuwonus pelamis). One instance of mislabeling was detected, in which striped bonito (Sarda orientalis) was identified in a product labeled as tongol tuna (Thunnus tonggol). PCR amplification and sequencing was unsuccessful in a number of products, likely due to factors such as the presence of PCR inhibitors and DNA fragmentation during the canning process. Overall, CR and ITS1 show high potential for use in identification of canned tuna products; however, further optimization of the assay may be necessary in order to improve amplification and sequencing success rates.

Exploiting DNA mini-barcodes as molecular markers to authenticate saffron (Crocus sativus L.)

Saffron (Crocus sativus) is the most expensive spice in the world. As a consequence, saffron has frequently been associated with an unrivalled degree of adulteration. The aim of this work was searching for DNA-barcode candidates as molecular markers to differentiate C. sativus from allied species and assessing the authenticity of saffron. New primers were specifically designed targeting three DNA barcode loci, namely ITS1, ITS2 and matK. A real-time PCR assay combined with high resolution melting (HRM) analysis was carried out in order to discriminate different Crocus spp., being further confirmed by sequencing. Data from HRM analysis showed that ITS1 and matK loci were Crocus genus specific, while ITS2 locus was species-specific for C. sativus and Crocus cartwrightianus. This study revealed that genomic tools, and specifically DNA mini-barcodes combined with HRM analysis, can provide simple, fast and highly reliable methods for saffron authentication.

Challenges to a molecular approach to prey identification in the Burmese python, Python molurus bivittatus.

Molecular approaches to prey identification are increasingly useful in elucidating predator–prey relationships, and we aimed to investigate the feasibility of these methods to document the species identities of prey consumed by invasive Burmese pythons in Florida. We were particularly interested in the diet of young snakes, because visual identification of prey from this size class has proven difficult. We successfully extracted DNA from the gastrointestinal contents of 43 young pythons, as well as from several control samples, and attempted amplification of DNA mini-barcodes, a 130-bp region of COX1. Using a PNA clamp to exclude python DNA, we found that prey DNA was not present in sufficient quality for amplification of this locus in 86% of our samples. All samples from the GI tracts of young pythons contained only hair, and the six samples we were able to identify to species were hispid cotton rats. This suggests that young Burmese pythons prey predominantly on small mammals and that prey diversity among snakes of this size class is low. We discuss prolonged gastrointestinal transit times and extreme gastric breakdown as possible causes of DNA degradation that limit the success of a molecular approach to prey identification in Burmese pythons.