Descurainiae Semen Lepidii Semen, also known as Tinglizi, originates from Brassicaceae plants Descurainia sophia or Lepidium apetalum. The former is commonly referred to as "Southern Tinglizi(Descurainiae Semen)", while the latter is known as "Northern Tinglizi(Lepidii Semen)". To scientifically and accurately identify the origin of Tinglizi medicinal materials and traditional Chinese medicine products, this study developed a specific DNA mini-barcode based on chloroplast genome sequences. By combining the DNA mini-barcode with DNA metabarcoding technology, a method for the qualitative and quantitative identification of Tinglizi medicinal materials and Chinese patent medicines was established. In this study, chloroplast genomes of Southern Tinglizi and Northern Tinglizi and seven commonly encountered counterfeit products were downloaded from the GenBank database. Suitable polymorphic regions were identified to differentiate these species, enabling the development of the DNA mini-barcode. Using DNA metabarcoding technology, medicinal material mixtures of Southern and Northern Tinglizi, as well as the most common counterfeit product, Capsella bursa-pastoris seeds, were analyzed to validate the qualitative and quantitative capabilities of the mini-barcode and determine its minimum detection limit. Additionally, the mini-barcode was applied to Chinese patent medicines containing Tinglizi to authenticate their botanical origin. The results showed that the developed mini-barcode(psbB) exhibited high accuracy and specificity, effectively distinguishing between the two authentic origins of Tinglizi and commonly encountered counterfeit products. The analysis of mixtures demonstrated that the mini-barcode had excellent qualitative and quantitative capabilities, accurately identifying the composition of Chinese medicinal materials in mixed samples with varying proportions. Furthermore, the analysis of Chinese patent medicines revealed the presence of the adulterant species(Capsella bursa-pastoris) in addition to the authentic species(Southern and Northern Tinglizi), indicating the occurrence of adulteration in commercially available Tinglizi-containing products. This study developed a method for the qualitative and quantitative identification of multi-origin Chinese medicinal materials and related products, providing a model for research on other multi-origin Chinese medicinal materials.
DNA Barcoding, Taxonomic/methods* ; Drugs, Chinese Herbal/chemistry* ; Drug Contamination ; Genome, Chloroplast ; Medicine, Chinese Traditional

Artemisiae Scoporiae Herba is derived from Artemisia scoparia or A. capillaris. The accurate identification of the herbs, particularly when dealing with bulk samples, is critical for ensuring the quality and efficacy of the medicinal product. This study aimed to establish a comprehensive molecular approach by combining multiple markers for the precise identification of Artemisiae Scoporiae Herba. The ITS2 from A. scoparia, A. capillaris, and other common Artemisia species were retrieved from GenBank. MEGA was used to build a phylogenetic tree with these sequences, and the effectiveness of ITS2 in species identification was assessed. The analysis revealed that while ITS2 could distinguish Artemisiae Scoporiae Herba from other closely related species of Artemisia, it was insufficient to differentiate between A. scoparia and A. capillaris. To address this limitation, the chloroplast genome of A. capillaris was assembled and compared with the published chloroplast genomes of A. scoparia and A. capillaris, on the basis of which a DNA mini-barcode was developed. The rpoA-rps11 region was selected as the target for the development of mini-barcode due to its potential for distinguishing between these two species. Specific primers were designed to differentiate A. scoparia from A. capillaris. The ITS2 sequences and the newly developed mini-barcode were used together for Sanger sequencing to identify individual samples of Artemisiae Scoporiae Herba, while DNA metabarcoding was employed for the identification of bulk samples. The identification results of representative individual samples and bulk samples from different regions consistently confirmed A. capillaris. This study established a method that combined ITS2 and mini-barcode to identify bulk samples of Artemisiae Scoporiae Herba from different regions. This approach overcomes the limitations of morphological and chemical methods, enhancing species identification accuracy and supporting a stable supply of medicinal materials.
Artemisia/classification* ; DNA Barcoding, Taxonomic/methods* ; Phylogeny ; DNA, Plant/genetics* ; DNA, Ribosomal Spacer/genetics*

Scutellaria baicalensis is a commonly used Chinese medicinal herb. In this study, we identified the germplasm resources of commercial S. baicalensis samples based on trnH-psbA, petA-psbJ, and ycf4-cemA sequences according to the available chloroplast genome sequencing results, and measured the content of baicalin by HPLC. Through the above means we determined the best DNA barcode that can be used to detect the germplasm resources and evaluate the quality of commercial S. baicalensis samples. A total of 104 samples were collected from 24 provinces, from which DNA was extracted for PCR amplification. The amplification efficiencies of trnH-psbA, petA-psbJ, and ycf4-cemA sequences were 100%, 59.62%, and 25.96%, respectively. The results of sequence analysis showed that 5, 4, and 2 haplotypes were identified based on trnH-psbA, petA-psbJ, and ycf4-cemA sequences, respectively. However, the sequences of haplotypes in commercial samples were different from that of the wild type, and the joint analysis of three fragments of S. baicalensis only identified 6 haplotypes. Furthermore, the phylogenetic analysis and genetic distance analysis indicated that trnH-psbA could be used to identify S. baicalensis from adulterants. The above analysis showed that trnH-psbA was the best fragment for identifying the germplasm resources of commercial S. baicalensis samples. We then analyzed the haplotypes(THap1-THap5) of commercial S. baicalensis samples based on trnH-psbA and found that THap2 was the main circulating haplotype of the commercial samples, accounting for 86.55% of the total samples, which indicated the scarce germplasm resources of commercial S. baicalensis samples. The content of baicalin in all the collected commercial S. baicalensis samples exceeded the standard in Chinese Pharmacopoeia and had significant differences(maximum of 12.21%) among samples, suggesting that the quality of commercial S. baicalensis samples varied considerably. However, there was no significant difference in baicalin content between different provinces or between different haplotypes. This study facilitates the establishment of the standard identification system for S. baicalensis, and can guide the commercial circulation and reasonable medication of S. baicalensis.
Chromatography, High Pressure Liquid ; DNA Barcoding, Taxonomic/methods* ; DNA, Plant/genetics* ; Phylogeny ; Scutellaria baicalensis/genetics*

OBJECTIVETo provide a feasible and cost-effective next-generation sequencing (NGS) method for accurate identification of viral pathogens in clinical specimens, because enormous limitations impede the clinical use of common NGS, such as high cost, complicated procedures, tremendous data analysis, and high background noise in clinical samples.

METHODSViruses from cell culture materials or clinical specimens were identified following an improved NGS procedure: reduction of background noise by sample preprocessing, viral enrichment by barcoded oligonucleotide (random hexamer or non-ribosomal hexanucleotide) primer-based amplification, fragmentation-free library construction and sequencing of one-tube mixtures, as well as rapid data analysis using an in-house pipeline.

RESULTSNGS data demonstrated that both barcoded primer sets were useful to simultaneously capture multiple viral pathogens in cell culture materials or clinical specimens and verified that hexanucleotide primers captured as many viral sequences as hexamers did. Moreover, direct testing of clinical specimens using this improved hexanucleotide primer-based NGS approach provided further detailed genotypes of enteroviruses causing hand, foot, and mouth disease (HFMD) and identified other potential viruses or differentiated misdiagnosis events.

CONCLUSIONThe improved barcoded oligonucleotide primer-based NGS approach is simplified, time saving, cost effective, and appropriate for direct identification of viral pathogens in clinical practice.

Clinical Laboratory Techniques ; DNA Barcoding, Taxonomic ; DNA Primers ; Enterovirus ; classification ; genetics ; isolation & purification ; Herpesvirus 4, Human ; genetics ; isolation & purification ; Humans ; Influenza B virus ; genetics ; isolation & purification ; Real-Time Polymerase Chain Reaction ; Sequence Analysis, DNA ; methods ; Sequence Analysis, RNA ; methods

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, Taxonomic ; methods ; DNA, Plant ; genetics ; Discriminant Analysis ; Drugs, Chinese Herbal ; chemistry ; classification ; Introns ; Plant Proteins ; genetics ; Plants, Medicinal ; chemistry ; classification ; genetics

Hyoscyami Semen, the mature dried seed of Hyoscyamus niger L., has long been used as a traditional Chinese medicine to treat human diseases. Hyoscyami Semen is found in local markets in China. In markets, sellers and buyers commonly inadvertently mix the seeds of H. niger with the seeds of related species such as Hygrophila salicifolia (Vahl) Nees, Astragalus complanatus R. Br., Cuscuta australis R. Br., Cuscuta chinensis Lam., and Impatiens balsamina L. because of their similar morphologies or similar names. Thus, developing a reliable method for discriminating H. niger seeds from its adulterants is necessary to reduce confusion and ensure the safe use of Hyoscyami Semen. The present study was designed to evaluate the efficiency of high-resolution melting analysis combined with DNA barcoding (Bar-HRM) with internal transcribed spacer 2 to discriminate H. niger. Our results show that Bar-HRM successfully identified the adulterants and detected the proportion of H. niger DNA extract within an admixture. In particular, HRM detected H. niger DNA extract in A. complanatus DNA extract at concentrations as low as 1%. In conclusion, the Bar-HRM method developed in the present study for authenticating H. niger is rapid and cost-effective. It can be used in the future to guarantee the purity of Hyoscyami Semen for the clinical use.
China ; DNA Barcoding, Taxonomic ; methods ; DNA, Intergenic ; chemistry ; genetics ; DNA, Plant ; chemistry ; genetics ; Discriminant Analysis ; Drug Contamination ; Drugs, Chinese Herbal ; chemistry ; Hyoscyamus ; genetics ; growth & development ; Seeds ; genetics ; growth & development ; Transition Temperature

DNA barcoding is an effective technique in species identification. To determine the candidate sequences which can be used as DNA barcode to identify in Papaver genus, five potential sequences (ITS, matK, psbA-trnH, rbcL, trnL-trnF) were screened. 69 sequences were downloaded from Genbank, including 21 ITS sequences, 10 matK sequences, 8 psbA-trnH sequences, 14 rbcL sequences and 16 trnL-trnF sequences. Mega 6.0 was used to analysis the comparison of sequences. By the methods of calculating the distances in intraspecific and interspecific divergences, evaluating DNA barcoding gap and constructing NJ and UPMGA phylogenetic trees. The sequence trnL-trnF performed best. In conclusion, trnL-trnF can be considered as a novel DNA barcode in Papaver genus, other four sequences can be as combination barcode for identification.
DNA Barcoding, Taxonomic ; methods ; Papaver ; classification ; genetics

Both market research and literature reports both found that the ootheca of mantodea was all used as medicine. However, Chinese Pharmacopoeia only records the ootheca of three mantis species. The clinical use of ootheca unrecorded in Chinese Pharmacopoeia, will pose potential risks to drug safety. It's urgent to identify the origin of Mantidis Oötheca. The current researches about original animal in Mantidis Oötheca are based on morphology and unanimous. DNA barcoding fill gaps of the traditional morphological identification, which is widely used in animal classification studies. This study first use DNA barcoding to analyze genetic distance among different Mantidis Oötheca types, align COI sequences between mantis and Mantidis Oötheca and construct the phylogeny tree. The result confirmed that Tenodera sinensis and Hierodula patellifera were the origin insects of Tuanpiaoxiao and Heipiaoxiao, respectively, and Statilia maculate and Mantis religiosa were the origin insects of Changpiaoxiao.
Animals ; DNA ; genetics ; DNA Barcoding, Taxonomic ; methods ; Electron Transport Complex IV ; genetics ; Insect Proteins ; genetics ; Mantodea ; classification ; genetics ; Medicine, Chinese Traditional ; Phylogeny

The study is aimed to ensure the quality and safety of medicinal plants by using ITS2 DNA barcode technology to identify Corydalis boweri, Meconopsis horridula and their close related species. The DNA of 13 herb samples including C. boweri and M. horridula from Lhasa of Tibet was extracted, ITS PCR were amplified and sequenced. Both assembled and web downloaded 71 ITS2 sequences were removed of 5. 8S and 28S. Multiple sequence alignment was completed and the intraspecific and interspecific genetic distances were calculated by MEGA 5.0, while the neighbor-joining phylogenetic trees were constructed. We also predicted the ITS2 secondary structure of C. boweri, M. horridula and their close related species. The results showed that ITS2 as DNA barcode was able to identify C. boweri, M. horridula as well as well as their close related species effectively. The established based on ITS2 barcode method provides the regular and safe detection technology for identification of C. boweri, M. horridula and their close related species, adulterants and counterfeits, in order to ensure their quality control, safe medication, reasonable development and utilization.
Base Sequence ; China ; Corydalis ; chemistry ; classification ; genetics ; DNA Barcoding, Taxonomic ; methods ; DNA, Plant ; chemistry ; genetics ; DNA, Ribosomal Spacer ; chemistry ; genetics ; Molecular Sequence Data ; Nucleic Acid Conformation ; Papaveraceae ; chemistry ; classification ; genetics ; Phylogeny ; Plants, Medicinal ; chemistry ; classification ; genetics

To optimize indices of molecular identification for authentication of Ginseng Radix et Rhizoma and Panacis Quinquefolii Radix, four indices, including sequence similarity, specific positions, genetic distance and phylogenetic tree, were compared based on trnL-trnF sequences. Total DNA was extracted from Ginseng Radix et Rhizoma and Panacis Quinquefolii Radix, and trL-trnF sequences were amplified and sequenced. Sequence similarity was calculated by BLAST analysis. Specific positions were compared by DNAman software. Genetic distance and phylogenetic tree were analyzed by Mega software. The results showed that the inter-specific and intra-specific similarity of P. ginseng and P. quinquefolius respectively was 100% and 99. 6%. There were four specific positions at G153A, T463A, C732G and T818C. The inter-specific genetic distance (0) of trL-trnF sequences was lower than intra-specific genetic distance (0. 004). P. ginseng can be distinguished from P. quinquefolius based on the phylogenetic tree. It is concluded that Ginseng Radix et Rhizoma and Panacis Quinquefolii Radix can be authenticated by identification indices of sequence similarity, specific positions, genetic distance and phylogenetic tree. Index of specific positions based on trnL-trnF sequences is the most efficient index to authenticate Ginseng Radix et Rhizoma and Panacis Quinquefolii Radix.
Chloroplasts ; genetics ; DNA Barcoding, Taxonomic ; methods ; Panax ; classification ; genetics ; Phylogeny ; Plant Proteins ; genetics ; Rhizome ; classification ; genetics