Molecular Authentication Research Articles

Molecular authentication discloses the species composition of whitebait products in the Taiwanese market

Molecular authentication discloses the species composition of whitebait products in the Taiwanese market

Molecular authentication of Paeonia species for paeonia radix production using plastid and nuclear DNA markers

Paeonia lactiflora is a perennial herbaceous plant popular in oriental medicine and as an ornamental plant. Two related species, P. veitchii and P. obovata, share several medicinal uses with P. lactiflora. To develop a molecular authentication system for these three Paeonia species, which are used to produce the Chinese herbal medicine Paeonia Radix, we generated super-barcode information. This included complete plastid genomes (plastomes) and 45S nuclear ribosomal DNA (45S nrDNA) from two accessions of P. lactiflora and one accession each of P. veitchii and P. obovata, using whole-genome sequencing data. The four plastomes range from 152,519 to 152,731 base pairs (bp) in length and have the same gene content and order. The 45S nrDNA transcriptional units range from 5879 to 6315 bp in length and show 33 SNPs among the three species. Phylogenetic analyses based on plastome and 45S nrDNA sequences clearly identified each species as a monophyletic group. Inter- and intraspecific polymorphisms were discovered among these three Paeonia species and Paeonia suffruticosa. Four molecular markers were developed to discriminate between P. lactiflora, P. veitchii, P. obovata, and P. suffruticosa, the main species used to produce Paeonia Radix in Korea and China. These markers successfully authenticated the species when applied to multiple herbal samples collected from various origins.

DNA barcoding: An effective molecular tool for species identification, molecular authentication and phylogeny studies in plant science research

DNA barcoding is a technique for identifying specimens using brief, standardised DNA segments. In a variety of fields, including phylogeny, ecology, population genetics and biodiversity, DNA barcoding has become a successful method for precisely distinguishing species. The method is straightforward, efficient in both time and money and accurate. The key to successful DNA barcoding is choosing the right DNA marker. Since the idea of a quick approach for species identification was first put up in 2003, the scientific community has been keen to realise the potential of DNA barcodes. Cytochrome c oxidase, I (COI) region of the mitochondrial genome is mostly recognised as a standard barcoding region in animals. Later, rbcL + matK pairing, with a 70 % discriminating efficiency, was suggested by the Plant Working Group (PWG) of the Consortium for the Barcoding of Life (CBOL) as the standard barcode in plants. Three conditions must be met for a gene region to be an efficient DNA barcode: it must have sufficient species-level genetic divergence and variability, it must have conserved flanking regions for the widest taxonomic use and for generating universal PCR primers and it should be long enough to facilitate current capability for sequencing and extracting DNA. Different combinations of plastid coding, non-coding and nuclear markers are utilised as supplemental markers to boost the degree of plant species differentiation. The reliability of different barcodes in distinguishing species varies among different groups of plants. As DNA barcoding approaches its twentieth anniversary, technologies are still being developed that make use of this resource, which is constantly expanding in a variety of biological disciplines. Plant DNA barcoding, which became a scientific advance during the last ten years, is frequently employed as a taxonomical aid in identifying species. It is a way of choosing genetic loci that identifies and distinguishes an organism's membership from specific species, variations or even intervarieties. It varies from molecular phylogeny, which identifies an unknown sample from an existing classification rather than identifying patterns of association.

Cyt b gene as a valid molecular authentication marker of cow leathers

Authentication of true (genuine) cow leathers is in high demand to promote merchandise and economic growth. The present study employs RT-PCR-based TaqMan assay to facilitate the identification. Species-specific primers and probes were designed utilizing the existing NCBI data on mitochondrial DNA (mtDNA) genes, particularly the cytochrome b region (Cyt b). Mitochondrial DNA extracted from leather samples of both Bos taurus and Bos indicus and analyzed following the appropriate procedures. The RT-PCR results showed the designed primers and probes are exceptionally precise for cow leather samples. The established detection limit for the assay is estimated as 0.1 ng of DNA. In summary, the amplifiable mtDNA extracted from finished leather enables the identification of authentic cow leathers using the RT-PCR TaqMan assay, representing a pioneering report in this field.

Overcoming Challenges in DNA Extractions from Triphala Ingredients: A Way Forward for Optimization of Conventional and Digital PCR Assays for Molecular Authentication

Overcoming Challenges in DNA Extractions from Triphala Ingredients: A Way Forward for Optimization of Conventional and Digital PCR Assays for Molecular Authentication

Molecular authentication of commercial "Qian-hu" through the integration of nrDNA internal transcribed spacer 2 and nucleotide signature.

Peucedani Radix, also known as "Qian-hu" is a traditional Chinese medicine derived from Peucedanum praeruptorum Dunn. It is widely utilized for treating wind-heat colds and coughs accompanied by excessive phlegm. However, due to morphological similarities, limited resources, and heightened market demand, numerous substitutes and adulterants of Peucedani Radix have emerged within the herbal medicine market. Moreover, Peucedani Radix is typically dried and sliced for sale, rendering traditional identification methods challenging. We initially examined and compared 104 commercial "Qian-hu" samples from various Chinese medicinal markets and 44 species representing genuine, adulterants or substitutes, utilizing the mini barcode ITS2 region to elucidate the botanical origins of the commercial "Qian-hu". The nucleotide signature specific to Peucedani Radix was subsequently developed by analyzing the polymorphic sites within the aligned ITS2 sequences. The results demonstrated a success rate of 100% and 93.3% for DNA extraction and PCR amplification, respectively. Forty-five samples were authentic "Qian-hu", while the remaining samples were all adulterants, originating from nine distinct species. Peucedani Radix, its substitutes, and adulterants were successfully identified based on the neighbor-joining tree. The 24-bp nucleotide signature (5'-ATTGTCGTACGAATCCTCGTCGTC-3') revealed distinct differences between Peucedani Radix and its common substitutes and adulterants. The newly designed specific primers (PR-F/PR-R) can amplify the nucleotide signature region from commercial samples and processed materials with severe DNA degradation. We advocate for the utilization of ITS2 and nucleotide signature for the rapid and precise identification of herbal medicines and their adulterants to regulate the Chinese herbal medicine industry.

Time-Controlled Authentication Strategies for Molecular Information Transfer.

Modern cryptography based on computational complexity theory is mainly constructed with silicon-based circuits. As DNA nanotechnology penetrates the molecular domain, utilizing molecular cryptography for data access protection in the biomolecular domain becomes a unique approach to information security. However, building security devices and strategies with robust security and compatibility is still challenging. Here, this study reports a time-controlled molecular authentication strategy using DNAzyme and DNA strand displacement as the basic framework. A time limit exists for authorization and access, and this spontaneous shutdown design further protects secure access. Multiple hierarchical authentications, temporal Boolean logic authentication, and enzyme authentication strategies are constructed based on DNA networks'good compatibility and programmability. This study gives proof of concept for the detection and protection of bioinformation about single nucleotide variants and miRNA, highlighting their potential in biosensing and security protection.

Systematic review on fingerprinting development to determine adulteration of Chinese herbal medicines

BackgroundIt has been a current research hospots using fingerprinting technology for quality control of Chinese herbal medicines (CHMs), which provides a scientific basis for establishment of overall quality control in accordance with the characteristics of CHMs. The fingerprinting technology for CHMs is diverse, and the research field covers many disciplines, such as analytical chemistry, pharmacology, pharmaceutics, biochemistry, and molecular biology. PurposeTo effectively understand the key areas and future directions of research regarding the fingerprint and adulteration of CHMs. Methods/resultsthis paper analyzed 879 articles in this field in the Web of Science Core Collection from 2000 to 2023 with CiteSpace and VOSviewer, and systematically assessed the research process, hotspots, topic distribution among disciplines, etc. The most prominent contributors of fingerprint and adulteration of CHMs research are mainly from China, India, the United States, England, and Brazil. The knowledge domains of fingerprint and adulteration of CHMs research focus mainly on the topics of molecular authentication, DNA barcoding, HPLC, near-infrared spectroscopy, manage data, chemometrics, and electrochemical fingerprinting. Most countries have recognized the pharmaceutical potential of natural products, and have paid more attention to the fingerprint and adulteration of CHMs in the past decade. Future the research tends to focus more on molecular identification and authentication, and electrochemical and chromatographic fingerprinting in controlling the adulteration of CHMs. ConclusionThis research provides a valuable reference for scholars in related fields to analyze existing research results, understand the development trend, and explore new research directions.

Molecular authentication and discrimination between banana shrimp and Pacific white shrimp by duplex PCR-lateral flow dipstick (dPCR-LFD) system

Banana shrimp is easily substituted by Pacific white shrimp due to its relatively similar morphology and appearance. The duplex PCR-lateral flow dipstick (dPCR-LFD) system was developed to authenticate these two species based on the mitochondrial cytochrome b (Cytb) gene. Two pairs of species-specific primers were designed for dPCR amplification. The specificity was also tested toward nine marine species. The sensitivity of the method was also evaluated at the DNA range of 0.02 %-60 % of adulterated Pacific white shrimp DNA in DNA from banana shrimp. Several types of cooked samples under various conditions were also tested. The developed dPCR-LFD system could specifically differentiate banana shrimp from Pacific white shrimp. No cross-amplification was observed in the specificity test. The sensitivity test showed the detection limit of 0.02 % (0.01 ng) of Pacific white shrimp DNA. The cooking processes including boiling, frying, and steaming did not show any effect on the efficiency of the dPCR-LFD system. This technique could be therefore implemented for the authentication of banana shrimp and Pacific white shrimp products as well as further use for other seafood frauds.

Comparative plastomes of eight subgenus Chamaesyce plants and system authentication of Euphorbiae Humifusae Herba

Euphorbiae Humifusae Herba (EHH) was provided with medicinal and edible uses, but frequently was adulterated with its closely related species. Hence, this study sought to identify EHH via an integrated approach comprising data from its morphological evaluation, HPLC analysis, comparative plastomes analysis and allele-specific PCR identification. First, the morphological characteristics of 8 subgenus Chamaesyce plants were summarized. Then, HPLC analysis showed that 18 batches of EHH were adulterated or unqualified. Furthermore, the plastomes of the 8 subg. Chamaesyce species were analyzed. Phylogenetic analysis revealed a sister relationship among the 8 subg. Chamaesyce species. The allele-specific PCR authentication was developed by the nucleotide polymorphisms (SNPs) and insertions or deletions (InDels) analysis. The results of allele-specific PCR showed that 27 batches of EHH were adulterated, indicating that the superior sensitivity of molecular authentication over the other methods used. This study provided a reference for rational use and phylogenetic research of EHH.

Ethnopharmacology of five flowers herbal tea, a popular traditional beverage in Hong Kong and South China.

It has been a long-standing tradition of using herbal tea for preventive and therapeutic healthcare in Hong Kong and South China and Five Flowers Tea is one of the most popular herbal teas. Based on the principle of traditional Chinese medicine, the pharmacological functions are to clear heat and dispel dampness in the body. Heat and dampness are thought to contribute to a range of health problems, especially during the hot and humid season in South China and Hong Kong. The most prevalent herbs in the formula contain bioactive compounds including flavonoids, alkaloids and terpenoids, which have a wide range of pharmacological properties including anti-inflammation, antivirus, antidiarrhoea, antibacteria, and antioxidation. However, with the composition varies widely, the ethnopharmacological benefits described may not be delivered uniformly. This study is to provide a comprehensive analysis on the composition of the Five Flowers Tea sold inHong Kong and investigate the rationale behind the selection of herbs used in the formula. This study also provides information on the variation and quality ofthe Five Flowers Tea in the market. Thirty-three Five Flowers Tea samples were collected from various locations in Hong Kong. The size, texture, colour and organoleptic properties were documented. Macroscopic and molecular authentication methods were employed to identify the individual components. Macroscopic identificationrevealed there were 23 herbs belonging to 18 plant families. The most prevalent herb was Bombax ceiba L., followed by Chrysanthemum morifolium. Ten adulterants and the existence of insect Lasioderma serricorne were confirmed byDNA barcoding techniques. This study employed a comprehensive approach to authenticate the herbs in FiveFlowers Tea samplescollected from various locations in Hong Kong. Macroscopic andmolecular methodswere used to identify the herbs and adulterants. The findings revealed the varied composition in Five Flowers Tea and the occurrence of adulterants in some samples. This shows that quality assurance of Five Flowers Tea is essential for the effective use of this popular folk medicine.

Molecular authentication, metabolite profiling and in silico-in vitro cytotoxicity screening of endophytic Penicillium ramusculum from Withania somnifera for breast cancer therapeutics.

The online version contains supplementary material available at 10.1007/s13205-023-03906-3.

Molecular authentication of surimi-based products (fish cakes, 鱼糕) sold on the Chinese e-commerce: Traditional (DNA barcoding) and innovative techniques (metabarcoding) to tackle seafood fraud

Molecular authentication of surimi-based products (fish cakes, 鱼糕) sold on the Chinese e-commerce: Traditional (DNA barcoding) and innovative techniques (metabarcoding) to tackle seafood fraud

Labelling compliance assessment and molecular authentication of Grilled Fish Products Sold on Chinese e-commerce: Traceability issues related to the use of umbrella trade names

Labelling compliance assessment and molecular authentication of Grilled Fish Products Sold on Chinese e-commerce: Traceability issues related to the use of umbrella trade names

Molecular Authentication and Phytochemical Evaluation of Indigenous Germplasm of Genus Physalis for Sustainable Utilization

Physalis species are used as an indigenous food and medicine in Kenya. However, species identification and an analysis of the health-promoting bioactive compounds and antioxidant properties are lacking. In this study, we report the molecular identification and mineral and phytochemical profiling of wild Physalis accessions. Leaf samples of 10 Physalis accessions were collected and used for species identification using nuclear ITS2 and plastid rbcL barcodes. Ripe fruits were collected from the same accessions and analyzed for mineral, total phenolic, tannin, and flavonoid contents, and antioxidant activities. The Physalis species were discriminated based on the ITS2 barcode and identified as Physalis purpurea. The genetic diversity, distance, and polymorphism of the ITS2 region of Physalis accessions were high due to the high rate of singleton and parsimony mutations. No genetic diversity, distance, or polymorphism was observed based on the rbcL barcode. The mineral content was significantly different (p < 0.05) for calcium, zinc, nickel, copper, and lithium among the Physalis accessions. No significant variation (p > 0.05) was found for phenolic acids or flavonoids, but the tannic acid content varied significantly (p < 0.05). DPPH free radical scavenging varied significantly (p < 0.05) among Physalis accessions. In conclusion, nuclear ITS2 was used to successfully identify the Physalis species of all the accessions as Physalis purpurea. The present study confirmed that Physalis purpurea has a significantly high mineral and phytochemical content and antioxidant activity. The findings from this study can be used to facilitate exploitation of Physalis purpurea in genetic breeding, their application in pharmaceutical, cosmetic, and nutritional value as well as conservation and sustainable use.

Tracing tiger nut (C. esculentus L.): functional food from the colossal Cyperus genus

Tiger nuts (also called earth almonds or chufa) are the stolons of the sedge Cyperus esculentus L. and have been traditionally used as food or as an additive to drinks such as horchata de chufa. Recently, chufa experienced a hype as functional food because of its richness in dietary fiber and as a source of unsaturated fatty acids. Its close relative, nutgrass (Cyperus rotundus L.) has similarly long history of human use, but rather for medical purposes, due to a high content of secondary compounds. Both species are invasive weeds responsible for high yield losses in crop plants, especially in Southeast Asia, Africa, and subtropical America. They are not easily discerned, occur together, and are both traded under similar or identical vernacular names. For the purpose of consumer protection, it is important to discriminate them in commercial samples, which is not trivial by morphological markers and often impossible since they are mostly used in highly processed form. Therefore, a molecular authentication assay was developed using taxonomically authenticated plants as reference material. The plastidic marker psbA-trnH igs (intergenic spacer) and the nuclear marker Internally Transcribed Spacer were used for barcoding. To develop a sequencing-free fingerprinting assay, a single-nucleotide polymorphism in the ITS2 region could be employed to discriminate C. esculentus from its possible adulterant C. rotundus by a tailored Amplificatory Refractory Mutation System PCR. This approach was successful both, in reference to plants, as well as in highly processed commercial products such as tiger nut flour. We show that by a single PCR we can detect surrogation by nutgrass down to a threshold of 10% in tiger nuts as contribution to safeguard product authenticity against possible adulterations in a growing global market.

DNA barcodes, ISSR, RAPD and SCAR markers as potential quality control tools for molecular authentication of black and white mulberry

Morus species, especially black mulberry (Morus nigra L.), are well known for their nutritive and economic values. Black mulberry is often a subject for deliberate and undeliberate substitution with the morphologically similar white mulberry (Morus alba L.). In the current study, the genetic diversity of Morus nigra L. and Morus alba L. was assessed using different genetic profiling and fingerprinting techniques namely namely DNA barcoding, Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR). DNA barcoding was implemented using 2 plastid loci (matK, and rbcL-1) and 2 nuclear spacers (ITS and ITS2) aiming at accurate authentication of the studied species. DNA barcoding, targeting the selected loci succeeded to identify and authenticate the studies Morus species but failed to differentiate them, owing to their close genetic resemblance. Out of 52 screened RAPD primers, 27 were able to produce reproducible and clear profiles with 115 amplified fragments of which 37 were polymorphic accounting for 32.17% polymorphism. The number of amplified bands varied from 2 to 7 ranging in size from 100 to 700 bp. Moreover, 55 ISSR primers were evaluated, 108 fragments were produced, using 20 primers, of which 28 were polymorphic accounting for 25.92% polymorphism with size range of 200–800 bp. Sequence Characterized Amplified Region (SCAR) marker was developed based on reproducible banding pattern obtained by RAPD primer (OPG-03), for the purpose of accelerating the initial screening of Morus batches and enabling the preventative rejection of adulterated samples. The polymorphic band obtained was sequenced and specific primers were designed to develop a SCAR marker of approximately 558 bp exclusively for M. nigra, allowing detection of down to 1 ng, of M. nigra. The SCAR marker showed 100% success in differentiating authentic M. nigra and M. alba samples. In addition, the application of the developed SCAR marker was extended to distinguish M. nigra from its four common adulterants namely Ficus carica L., Fragaria vesca L., Ficus racemosa L. and Vitis vinifera L.. The proposed protocol was successfully implemented for the evaluation of 47 commercial M. nigra samples collected from different Egyptian locations.

Molecular authentication of mushroom products: First survey on the Italian market

Molecular authentication of mushroom products: First survey on the Italian market

Genome skimming as an efficient tool for authenticating commercial products of the pharmaceutically important Paris yunnanensis (Melanthiaceae)

BackgroundParis yunnanensis (Melanthiaceae) is a traditional Chinese medicinal plant of significant pharmaceutical importance. Due to previous taxonomic confusion, a congeneric species, Paris liiana, has been mistaken for P. yunnanensis and cultivated on a large scale, leading to the mixing of commercial products (i.e., seedlings and processed rhizomes) of P. yunnanensis with those of P. liiana. This may have adverse effects on quality control in the standardization of P. yunnanensis productions. As the lack of PCR amplifiable genomic DNA within processed rhizomes is an intractable obstacle to the authentication of P. yunnanensis products using PCR-based diagnostic tools, this study aimed to develop a PCR-free method to authenticate commercial P. yunnanensis products, by applying genome skimming to generate complete plastomes and nrDNA arrays for use as the molecular tags.ResultsBased on a dense intraspecies sampling of P. liiana and P. yunnanensis, the robustness of the proposed authentication systems was evaluated by phylogenetic inferences and experimental authentication of commercial seedling and processed rhizome samples. The results indicate that the genetic criteria of both complete plastomes and nrDNA arrays were consistent with the species boundaries to achieve accurate discrimination of P. yunnanensis and P. liinna. Owing to its desirable accuracy and sensitivity, genome skimming can serve as an effective and sensitive tool for monitoring and controlling the trade of P. yunnanensis products.ConclusionThis study provides a new way to solve the long-standing problem of the molecular authentication of processed plant products due to the lack of PCR amplifiable genomic DNA. The proposed authentication system will support quality control in the standardization of P. yunnanensis products in cultivation and drug production. This study also provides molecular evidence to clarify the long-standing taxonomic confusion regarding the species delimitation of P. yunnanensis, which will contribute to the rational exploration and conservation of the species.

Molecular authentication of some rare Iris (Iridaceae) species from Uzbekistan

The determination of nucleotide sequences in DNA, sequencing of chloroplast genomes techniques, based on the use of combinations a significant number genetic markers, as well as the nuclear genome are widely use for inventory of rare plant species, ecological monitoring and species diversity assessment. The results of our molecular analysis are used to examine the taxonomic identity and phylogenetic relationship of the studied Iris species collected in Uzbekistan. The genotyping of three-four functional loci of nrDNA and cpDNA was performed for efficient species identification and confident results. The nuclear ITS gene sequences and chloroplast trnL-trnF, rbcL, matK gene sequences were obtained from 10 Iris species and have proven to be useful as molecular markers for species identification. Sequences of cpDNA regions, have been used to assess interspecific relationships of Iris. Hierarchical clustering were constructed using 13 consensus matK gene sequences. In this study, we consider the possibility of successful application the DNA barcoding as a tool for assessment of wild Iris species diversity in Uzbekistan.