Array-based dynamic allele specific hybridization (Array-DASH): Optimization-free microarray processing for multiple simultaneous genomic assays.

We report proof-of-principle experiments regarding a dynamic microarray protocol enabling accurate and semi-quantitative DNA analysis for re-sequencing, fingerprinting and genotyping. Single-stranded target molecules hybridise to surface-bound probes during initial gradual cooling with high-fidelity. Real-time tracking of target denaturation (via fluorescence) during a 'dynamic' gradual heating phase permits 'melt-curve' analysis. The probe most closely matching the target sequence is identified based on the highest melting temperature. We demonstrated a >99% re-sequencing accuracy and a potential detection rate of 1% for SNPs. Experiments employing Hypericum ribosomal ITS regions and HIV genomes illustrated a reliable detection level of 5% plus simultaneous re-sequencing and genotyping. Such performance suggests a range of potential real-world applications involving rapid sequence interrogation, for example, in the Covid-19 pandemic. Guidance is offered towards the development of a commercial platform and dedicated software required to bring this technique into mainstream science.

Health care professionals' personal and professional views of herbal medicines in the United Kingdom.

Health care professionals (HCPs) have a pivotal role in optimizing patient care and should be familiar with complementary and alternative medicines. The aim of the study was to explore UK-based HCP personal and professional opinions and experiences of herbal medicines (HMs). An online questionnaire was distributed via social media to recruit (n = 112) a range of HCPs from across the United Kingdom. HCPs from primary and secondary care, the private sector, and academia took part. A large proportion of participants (62%) said they did not personally use any HMs, and 38% did use HMs. HCPs who had personally used HMs had a positive impression of HMs and were more likely to recommend HMs to patients than those who had not used HMs themselves. Participants were given the opportunity to share their perceptions on the safety and efficacy of HMs and their experiences with patients reporting adverse drug reactions to HMs and herb-drug interactions. HCPs identified their lack of knowledge on HMs and insufficient training, which made them unable to advise patients on the safe use of HMs. More education on HMs would help improve HCP knowledge of HMs and help them make better informed decisions when considering patient pharmaceutical care plans.

DNA Barcoding for Industrial Quality Assurance.

DNA barcoding methods originally developed for the identification of plant specimens have been applied to the authentication of herbal drug materials for industrial quality assurance. These methods are intended to be complementary to current morphological and chemical methods of identification. The adoption of these methods by industry will be accelerated by the introduction of DNA-based identification techniques into regulatory standards and monographs. The introduction of DNA methods into the British Pharmacopoeia is described, along with a reference standard for use as a positive control for DNA extraction and polymerase chain reaction (PCR). A general troubleshooting chart is provided to guide the user through the problems that may be encountered during this process. Nevertheless, the nature of the plant materials and the demands of industrial quality control procedures mean that conventional DNA barcoding is not the method of choice for industrial quality control. The design of DNA barcode-targeted quantitative PCR and high resolution melt curve tests is one strategy for developing rapid, robust, and reliable protocols for high-throughput screening of raw materials. The development of authentication tests for wild-harvested Rhodiola rosea L. is used as a case study to exemplify these relatively simple tests. By way of contrast, the application of next-generation sequencing to create a complete profile of all the biological entities in a mixed herbal drug is described and its potential for industrial quality assurance discussed.

The Use of Traditional Herbal Medicines Amongst South Asian Diasporic Communities in the UK.

Migrant South Asian communities in the UK have brought with them their own traditional forms of medicine, yet little is known about their current use of herbal medicines (HMs) in the UK. The aim of the study was to explore the origins, use and transmission of knowledge of traditional HMs used by diasporic South Asian communities in the UK. A researcher-administered questionnaire was used for data collection (n = 192). An opportunity sampling technique was used to recruit participants across several locations in Birmingham and Leicester. Two thirds of participants (n = 126) stated they used HMs to maintain their health and to treat various health conditions such as digestive problems, skin conditions and diabetes. Almost 2000 actively used HMs were documented including 123 plant species that were identified. Participants imported HMs from abroad as well as sourcing them locally and even growing some of their own plants. Up to 82% (n = 87) of participants who took prescription medicines did not tell their healthcare professionals about any HMs they consumed; this raises concerns about people's knowledge of herb-drug interactions, compliance and effect on prescribed medicine regimens. Similar studies to explore the use of HMs by other ethnic groups are imperative to help optimise pharmaceutical care of patients. Copyright © 2017 John Wiley & Sons, Ltd.

Hypothetical biosynthetic pathways of pharmaceutically potential hallucinogenic metabolites in Myristicaceae, mechanistic convergence and co-evolutionary trends in plants and humans.

The family Myristicaceae harbour mind-altering phenylpropanoids like myristicin, elemicin, safrole, tryptamine derivatives such as N,N-dimethyltryptamine (DMT) and 5-methoxy N,N-dimethyltryptamine (5-MeO-DMT) and ß-carbolines such as 1-methyl-6-methoxy-dihydro-ß-carboline and 2-methyl-6-methoxy-1,2,3,4-tetrahydro-ß-carboline. This study aimed to systematically review and propose the hypothetical biosynthetic pathways of hallucinogenic metabolites of Myristicaceae which have the potential to be used pharmaceutically. Relevant publications were retrieved from online databases, including Google Scholar, PubMed Central, Science Direct and the distribution of the hallucinogens among the family was compiled. The review revealed that the biosynthesis of serotonin in plants was catalysed by tryptamine 5-hydroxylase (T5H) and tryptophan 5-hydroxylase (TPH), whereas in invertebrates and vertebrates only by tryptophan 5-hydroxylase (TPH). Indolethylamine-N-methyltransferase catalyses the biosynthesis of DMT in plants and the brains of humans and other mammals. Caffeic acid 3-O-methyltransferase catalyses the biosynthesis of both phenylpropanoids and tryptamines in plants. All the hallucinogenic markers exhibited neuropsychiatric effects in humans as mechanistic convergence. The review noted that DMT, 5-MeO-DMT, and ß-carbolines were natural protectants against both plant stress and neurodegenerative human ailments. The protein sequence data of tryptophan 5-hydroxylase and tryptamine 5-hydroxylase retrieved from NCBI showed a co-evolutionary relationship in between animals and plants on the phylogenetic framework of a Maximum Parsimony tree. The review also demonstrates that the biosynthesis of serotonin, DMT, 5-MeO-DMT, 5-hydroxy dimethyltryptamine, and ß-carbolines in plants, as well as endogenous secretion of these compounds in the brain and blood of humans and rodents, reflects co-evolutionary mutualism in plants and humans.

Challenges in Medicinal and Aromatic Plants DNA Barcoding-Lessons from the Lamiaceae.

The potential value of DNA barcoding for the identification of medicinal plants and authentication of traded plant materials has been widely recognized; however, a number of challenges remain before DNA methods are fully accepted as an essential quality control method by industry and regulatory authorities. The successes and limitations of conventional DNA barcoding are considered in relation to important members of the Lamiaceae. The mint family (Lamiaceae) contains over one thousand species recorded as having a medicinal use, with many more exploited in food and cosmetics for their aromatic properties. The family is characterized by a diversity of secondary products, most notably the essential oils (EOs) produced in external glandular structures on the aerial parts of the plant that typify well-known plants of the basil (Ocimum), lavender (Lavandula), mint (Mentha), thyme (Thymus), sage (Salvia) and related genera. This complex, species-rich family includes widely cultivated commercial hybrids and endangered wild-harvested traditional medicines, and examples of potential toxic adulterants within the family are explored in detail. The opportunities provided by next generation sequencing technologies to whole plastome barcoding and nuclear genome sequencing are also discussed with relevant examples.

The Cultural and Commercial Value of Tulsi (Ocimum tenuiflorum L.): Multidisciplinary Approaches Focusing on Species Authentication.

Tulsi (Holy basil, Ocimum tenuiflorum L., Lamiaceae), native to Asia, has become globalised as the cultural, cosmetic, and medicinal uses of the herb have been popularised. DNA barcoding, a molecular technique used to identify species based on short regions of DNA, can discriminate between different species and identify contaminants and adulterants. This study aimed to explore the values associated with Tulsi in the United Kingdom (UK) and authenticate samples using DNA barcoding. A mixed methods approach was used, incorporating social research (i.e., structured interviews) and DNA barcoding of Ocimum samples using the ITS and trnH-psbA barcode regions. Interviews revealed the cultural significance of Tulsi: including origins, knowledge exchange, religious connotations, and medicinal uses. With migration, sharing of plants and seeds has been seen as Tulsi plants are widely grown in South Asian (SA) households across the UK. Vouchered Ocimum specimens (n = 33) were obtained to create reference DNA barcodes which were not available in databases. A potential species substitution of O. gratissimum instead of O. tenuiflorum amongst SA participants was uncovered. Commercial samples (n = 47) were difficult to authenticate, potentially due to DNA degradation during manufacturing processes. This study highlights the cultural significance of Tulsi, despite a potential species substitution, the plant holds a prestigious place amongst SA families in the UK. DNA barcoding was a reliable way to authenticate Ocimum species.

Applied Barcoding: The Practicalities of DNA Testing for Herbals.

DNA barcoding is a widely accepted technique for the identification of plant materials, and its application to the authentication of commercial medicinal plants has attracted significant attention. The incorporation of DNA-based technologies into the quality testing protocols of international pharmacopoeias represents a step-change in status, requiring the establishment of standardized, reliable and reproducible methods. The process by which this can be achieved for any herbal medicine is described, using Hypericum perforatum L. (St John's Wort) and potential adulterant Hypericum species as a case study. A range of practical issues are considered including quality control of DNA sequences from public repositories and the construction of individual curated databases, choice of DNA barcode region(s) and the identification of informative polymorphic nucleotide sequences. A decision tree informs the structure of the manuscript and provides a template to guide the development of future DNA barcode tests for herbals.

Molecular Verification of the UK National Collection of Cultivated Liriope and Ophiopogon Plants.

A collection of cultivated Liriope and Ophiopogon plants was established in 1996-1998 and subsequently hosted at a horticultural college. Uncertainties about the identification of the accessions, compounded by potential errors in propagation and labelling have led to waning confidence in the identities of the plants in the collection. The potential for using DNA barcoding to determine the species identities of the accessions was investigated. The DNA barcode regions of the plastid ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene (rbcL) and nuclear ribosomal internal transcribed spacer (nrITS) were amplified. DNA sequence analysis allowed the sequences of the accessions to be compared to reference sequences in public databases. A simple haplotype map of the characteristic polymorphic positions in the rbcL regions was used to clearly distinguish between the two genera and assign Ophiopogon accessions to individual species or sub-groups of species. The ITS sequence data confirmed these genus and species assignations and provided greater resolution to distinguish between closely related species. The combination of two DNA barcodes allowed most of the accessions to be assigned to individual species. This molecular verification confirmed the identity of about 70% of the accessions, with the remaining 30% demonstrating a range of mistaken identities at the species and genus levels.

Author Correction: Character-based DNA barcoding for authentication and conservation of IUCN Red listed threatened species of genus Decalepis (Apocynaceae).

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Molecular identification of Hypericum perforatum by PCR amplification of the ITS and 5.8S rDNA region.

The nuclear ribosomal internal transcribed spacer (ITS) sequences of eight Hypericum species were used to design H. perforatum-specific PCR primers by identification of short "microcode" sequences characteristic of the target species. These were tested with three vouchered H. perforatum DNA samples and eight samples from other species within the Hypericum genus. The most efficient primer combination, FO2 and HRI-S, amplified the genomic DNA from all three H. perforatum samples but not from any of the others apart from H. delphicum. The primer pairing was then tested against seven commercially available ornamental varieties of Hypericum; a positive result was obtained only with the H. perforatum sample. Three consumer products retailed as "St. John's wort" herbal remedies were sampled, two of which gave a positive result for H. perforatum. The assay was sensitive enough to detect 0.75 ng H. perforatum present as just 0.1 % of the total DNA. This method has the potential to be replicated in other plant species and presents a novel use for DNA barcoding data.

DNA Authentication of St John's Wort (Hypericum perforatum L.) Commercial Products Targeting the ITS Region.

There is considerable potential for the use of DNA barcoding methods to authenticate raw medicinal plant materials, but their application to testing commercial products has been controversial. A simple PCR test targeting species-specific sequences within the nuclear ribosomal internal transcribed spacer (ITS) region was adapted to screen commercial products for the presence of Hypericum perforatum L. material. DNA differing widely in amount and extent of fragmentation was detected in a number of product types. Two assays were designed to further analyse this DNA using a curated database of selected Hypericum ITS sequences: A qPCR assay based on a species-specific primer pair spanning the ITS1 and ITS2 regions, using synthetic DNA reference standards for DNA quantitation and a Next Generation Sequencing (NGS) assay separately targeting the ITS1 and ITS2 regions. The ability of the assays to detect H. perforatum DNA sequences in processed medicines was investigated. Out of twenty different matrices tested, both assays detected H. perforatum DNA in five samples with more than 10³ ITS copies µL-1 DNA extract, whilst the qPCR assay was also able to detect lower levels of DNA in two further samples. The NGS assay confirmed that H. perforatum was the major species in all five positive samples, though trace contaminants were also detected.

Sequence-Specific Detection of Aristolochia DNA - A Simple Test for Contamination of Herbal Products.

Herbal medicines are used globally for their health benefits as an alternative therapy method to modern medicines. The market for herbal products has increased rapidly over the last few decades, but this has in turn increased the opportunities for malpractices such as contamination or substitution of products with alternative plant species. In the 1990s, a series of severe renal disease cases were reported in Belgium associated with weight loss treatment, in which the active species Stephania tetrandra was found to be substituted with Aristolochia fangchi. A. fangchi contains toxic aristolochic acids, which have been linked to kidney failure, as well as cancers of the urinary tract. Because of these known toxicities, herbal medicines containing these compounds, or potentially contaminated by these plants, have been restricted or banned in some countries, but they are still available via the internet and in alternate formulations. In this study, a DNA based method based on quantitative real-time PCR (qPCR) was tested to detect and distinguish Aristolochia subg. Siphisia (Duch.) O.C.Schmidt species from a range of medicinal plants that could potentially be contaminated with Aristolochia material. Specific primers were designed to confirm that Aristolochia subg. Siphisia can be detected, even in small amounts, if it is present in the products, fulfilling the aim of offering a simple, cheaper and faster solution than the chemical methods. A synthetic gBlock template containing the primer sequences was used as a reference standard to calibrate the qPCR assay and to estimate the copy number of a target gene per sample. Generic primers covering the conserved 5.8S rRNA coding region were used as internal control to verify DNA quality and also as a reference gene for relative quantitation. To cope with potentially degraded DNA, all qPCR primer sets were designed to generate PCR products of under 100 bp allowing detection and quantification of A. fangchi gBlock even when mixed with S. tetrandra gBlock in different ratios. All proportions of Aristolochia, from 100 to 2%, were detected. Using standards, associating the copy number to each start quantity, the detection limit was calculated and set to about 50 copies.

Product authenticity versus globalisation-The Tulsi case.

Using the Indian medicinal plant Tulsi (Holy Basil) as a case study, we have tested to what extent the discrepancy between vernacular and scientific nomenclature can be resolved, whether the presumed chemical diversity underlying the medicinal use of Tulsi has a genetic component, and whether it is possible to detect this genetic component using genetic barcoding markers. Based on four plastidic markers, we can define several haplotypes within Ocimum that are consistent across these markers. Haplotype II is congruent with O. tenuiflorum, while haplotype I extends over several members of the genus and cannot be resolved into genetically separate subclades. The vernacular subdivision of Tulsi into three types (Rama, Krishna, Vana) can only be partially linked with genetic differences-whereby Rama and Krishna Tulsi can be assigned to O. tenuiflorum, while Vana Tulsi belongs to haplotype I. This genetic difference is mirrored by differences in the profiles of secondary compounds. While developmental state and light quality modulate the amplitude to which the chemical profile is expressed, the profile itself seems to be linked with genetic differences. We finally develop an authentication assay that makes use of a characteristic single nucleotide polymorphism in one of the barcoding markers, establishing a differential restriction pattern that can be used to discriminate Vana Tulsi.

Genus-Specific Real-Time PCR and HRM Assays to Distinguish Liriope from Ophiopogon Samples.

Liriope and Ophiopogon species have a long history of use as traditional medicines across East Asia. They have also become widely used around the world for ornamental and landscaping purposes. The morphological similarities between Liriope and Ophiopogon taxa have made the taxonomy of the two genera problematic and caused confusion about the identification of individual specimens. Molecular approaches could be a useful tool for the discrimination of these two genera in combination with traditional methods. Seventy-five Liriope and Ophiopogon samples from the UK National Plant Collections of Ophiopogon and Liriope were analyzed. The 5' end of the DNA barcode region of the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcLa) was used for the discrimination of the two genera. A single nucleotide polymorphism (SNP) between the two genera allowed the development of discriminatory tests for genus-level identification based on specific PCR and high-resolution melt curve (HRM) assays. The study highlights the advantage of incorporating DNA barcoding methods into plant identification protocols and provides simple assays that could be used for the quality assurance of commercially traded plants and herbal drugs.

Character-based DNA barcoding for authentication and conservation of IUCN Red listed threatened species of genus Decalepis (Apocynaceae).

The steno-endemic species of genus Decalepis are highly threatened by destructive wild harvesting. The medicinally important fleshy tuberous roots of Decalepis hamiltonii are traded as substitute, to meet the international market demand of Hemidesmus indicus. In addition, the tuberous roots of all three species of Decalepis possess similar exudates and texture, which challenges the ability of conventional techniques alone to perform accurate species authentication. This study was undertaken to generate DNA barcodes that could be utilized in monitoring and curtailing the illegal trade of these endangered species. The DNA barcode reference library was developed in BOLD database platform for candidate barcodes rbcL, matK, psbA-trnH, ITS and ITS2. The average intra-specific variations (0-0.27%) were less than the distance to nearest neighbour (0.4-11.67%) with matK and ITS. Anchoring the coding region rbcL in multigene tiered approach, the combination rbcL + matK + ITS yielded 100% species resolution, using the least number of loci combinations either with PAUP or BLOG methods to support a character-based approach. Species-specific SNP position (230 bp) in the matK region that is characteristic of D. hamiltonii could be used to design specific assays, enhancing its applicability for direct use in CITES enforcement for distinguishing it from H. indicus.

Induction of cell division-related genes in quiescent (G0 ) sugar beet cells.

Sugar beet cells maintained in the stationary phase of the batch culture cycle for 2 or more days have been shown to exhibit many of the characteristics of quiescent (G0 ) cells. When such cells were subcultured into fresh medium they progressed through a period of DNA synthesis to a highly synchronised first division, 6 days after subculture. The onset of DNA synthesis and cell division were each delayed by 2 days relative to the timing of the events when the cells were subcultured immediately before entry into the stationary phase. The regulation of gene expression during this extended transition from the G0 phase back to the cell division cycle was investigated. The cell division cycle-related genes Bvcdc2, Betvu;CycA2, Arath;CycB1;1 histone H4 and Bvcrk1 (a novel cdc2-like gene) showed widely differing patterns of expression. Bvcdc2 transcripts were present at low levels in quiescent cells whereas crk1, cyclin and histone transcripts were not detectable. Expression of both Bvcrk1 and Betvu; CycA2 was induced within 1 h after subculture into fresh medium, whereas histone H4 gene expression was not detectable for 24 h and showed a marked increase between 24 and 48 h. B-type cyclin transcripts were not detectable until more than 48 h after subculture. The addition of either sucrose or MS macronutrients to quiescent sugar beet cells was not sufficient to re-initiate cell division but both medium components were able to stimulate the expression of the two 'early' genes (Betvu;CycA2 and Bvcrk1) within 6 h. Furthermore, although the sugar beet cells were habituated, i.e. they were routinely grown without added plant growth regulators, treatment of quiescent cells with IAA and kinetin also induced expression of Betvu;CycA2 and Bvcrk1 without subsequent cell division.

The application of a DNA-based identification technique to over-the-counter herbal medicines.

Reliable methods to identify medicinal plant material are becoming more important in an increasingly regulated market place. DNA-based methods have been recognised as a valuable tool in this area with benefits such as being unaffected by the age of the plant material, growth conditions and harvesting techniques. It is possible that the methods of production used for medicinal plant products will degrade or remove DNA. So how applicable are these techniques to processed medicinal plant products? A simple PCR-based identification technique has been developed for St. John's Wort, Hypericum perforatum L. Thirteen St. John's Wort products were purchased including capsules, tablets and tinctures. DNA was extracted from each product, and the species specific PCR test conducted. DNA was successfully extracted from all thirteen products, using a fast and efficient modified method for extracting DNA from tinctures. Only four products yielded the full length ITS region (850 bp) due to the quality of the DNA. All of the products tested positive for H. perforatum DNA. DNA-based identification methods can complement existing methods of authentication. This paper shows that these methods are applicable to a wide range of processed products, provided that they are designed to account for the possibility of DNA degradation.

PlantID - DNA-based identification of multiple medicinal plants in complex mixtures.

BACKGROUND: An efficient method for the identification of medicinal plant products is now a priority as the global demand increases. This study aims to develop a DNA-based method for the identification and authentication of plant species that can be implemented in the industry to aid compliance with regulations, based upon the economically important Hypericum perforatum L. (St John's Wort or Guan ye Lian Qiao). METHODS: The ITS regions of several Hypericum species were analysed to identify the most divergent regions and PCR primers were designed to anneal specifically to these regions in the different Hypericum species. Candidate primers were selected such that the amplicon produced by each species-specific reaction differed in size. The use of fluorescently labelled primers enabled these products to be resolved by capillary electrophoresis. RESULTS: Four closely related Hypericum species were detected simultaneously and independently in one reaction. Each species could be identified individually and in any combination. The introduction of three more closely related species to the test had no effect on the results. Highly processed commercial plant material was identified, despite the potential complications of DNA degradation in such samples. CONCLUSION: This technique can detect the presence of an expected plant material and adulterant materials in one reaction. The method could be simply applied to other medicinal plants and their problem adulterants.