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The main sources of natural drugs include various biological species such as plants, animals, and microorganisms. The accurate identification of these species is the bedrock of natural drug development. We propose a novel method of species identification in this paper: analysis of whole-genome (AGE), a molecular diagnostic method used to identify species by finding species-specific sequences from the whole genome and precisely recognizing the specific target sequences. We elaborate that the principle for species identification based on AGE is that the genome sequences of diverse species must differ and divide the implementation strategy of the method into two levels of research and application. Based on our analysis of its characteristics, the method would have the potential advantages of reliable principle, high specificity, and wide applicability. Moreover, three crucial concerns related to building method systems including genome acquisition, bioinformatics analysis, and database construction, are further discussed. In summary, we offer theoretical underpinnings and methodological guidance for the development of bioinformatics software and commercial kits, indicating AGE has great application potential in objects, subjects, and industries.
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Rhei Radix et Rhizoma is one of the most used medicinal materials in China. Its original species are Rheum palmatum, Rh. tanguticum, and Rh. officinale. Rhei Radix et Rhizoma derived from different original species are significantly different in their active ingredients and pharmacological effects. To develop an accurate, rapid, and specific identification method, we obtained the chloroplast genomes of the three original species by Illumina Novaseq sequencing. We designed specific DNA barcodes from the hypervariable regions, which can accurately identify the three original species. The experimental results showed that the total length of the chloroplast genomes of Rh. tanguticum, Rh. officinale and Rh. palmatum were 161 039 bp, 161 093 bp, and 161 136 bp, respectively. All the three genomes were represented as typical quadripartite structures. A total of 131 genes, including 86 protein-coding genes, 37 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes were identified from each chloroplast genome. Five pairs of primers based on the hypervariable regions were designed to efficiently amplify 42 samples. Results confirmed that five hypervariable regions, rps16-trnQ, psaA-ycf3, psbE-petL, ndhF-rpl32, and trnT-trnL, can be used as specific DNA barcodes for the identification of Rh. tanguticum, Rh. officinale, and Rh. palmatum. These results provided genetic information for further species identification of Rhei Radix et Rhizoma, and improve the safety of this clinical medication as well as standardize the market for Rhei Radix et Rhizoma.
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Adulterants and counterfeits were found in some of the commercial traditional Chinese medicine (TCM) decoctions in Hongjin Xiaojie Jiaonang, Hongjin Xiaojie Pian, and Chaihuang Keli during the national drug sampling inspection. However, it was difficult to determine the species of the adulterants and counterfeits by conventional testing methods. Therefore, a total of 184 samples of the TCM decoctions and raw materials belong to the prescriptions of above mentioned traditional Chinese patent medicines, including Bupleuri Radix, Bajiaolian, Heimayi, and Shufuchong, were collected and authenticated by DNA barcoding technology. 111 ITS2 sequences were obtained from 115 commercial TCM decoctions and raw materials of Bupleuri Radix, among which 71 were Bupleurum chinense, three were B. scorzonerifolium, and 31 were closely related species in the same genus. In addition, counterfeits derived from different genera, such as Ailanthus altissima (one sample), Saposhnikovia divaricate (two samples), and Solidago decurrens (three samples), were also detected. 21 ITS2 sequences were obtained from 22 commercial TCM raw materials of Bajiaolian, among which 15 were Diphylleia sinensis and six were Dysosma versipellis and other species in genus Dysosma. For 22 Heimayi samples, PCR amplification of COI sequence was failed due to genomic DNA degradation. Among 38 Shufuchong samples, 24 COI sequences were obtained and only nine of them were the genuine species (Armadillidium vulgare) recorded in the Chinese Pharmacopoeia, 11 were Porcellio laevis, two were Mongoloniscus sinensis, and two samples could not be identified due to the limitation of database. This study demonstrates that DNA barcoding technology is suitable for the species authentication of the decoctions of traditional Chinese patent medicine prescription. It is a conductive way for the establishment of traceability system for the whole TCM industrial chain.
ABSTRACT
Rhei Radix et Rhizoma is a kind of commonly used Chinese medicinal materials. Due to the overharvesting, the wild resource is endangering. Large market demand caused severely adulterant of commercial Rhei Radix et Rhizoma medicinal materials and decoction pieces. This manuscript reviewed the advances of the original species authentication in the industrial chain of Rhei Radix et Rhizoma during the latest decade, including characteristics and microscopic features, phytochemical analysis on anthraquinones, and molecular authentication based on DNA barcoding. Accordingly, an original species authentication route for the industrial chain of Rhei Radix et Rhizoma was summarized:(1)the identification of seeds and seedlings by DNA barcoding;(2) the selection of high variable sites based on the chloroplast genome;(3)biomonitoring of the Rhei Radix et Rhizoma medicinal materials and decoction pieces by two-dimensional DNA barcode;(4)traceability of Chinese patent medicines by third-generation sequencing. In conclusion, the combination of molecular identification and traditional identification methods provides a new idea for the identification of the original species of Rhei Radix et Rhizoma in the industrial chain and a essential guidance for the research of drug safety and efficacy of Rhei Radix et Rhizoma.
Subject(s)
Animals , Anthraquinones , Drugs, Chinese Herbal , Plant Roots , Rheum , RhizomeABSTRACT
Although the guiding principles for molecular identification of traditional Chinese medicines (TCM) using DNA barcoding have been recorded in the Chinese Pharmacopoeia, there is still a lack of systematic research on its application to commercial TCM decoctions. In this study, a total of 212 commercial TCM decoctions derived from different medicinal parts such as root and rhizome, fruit and seed, herb, flower, leaf, cortex, and caulis were collected to verify applicability and accuracy of the method. DNA barcodes were successfully obtained from 75.9% (161/212) of the samples, while other samples failed to be amplified due to genomic DNA degradation. Among the 161 samples, 85.7% of them were identified as recorded species in the Chinese Pharmacopoeia (2020 edition). In addition, 14 samples could be identified as species recorded in the Chinese Pharmacopoeia and their closely related species in the same genus. Morphological identification for the unconfirmed samples showed that eight were genuine species and three were adulterants, while the other three were unidentifiable due to lack of morphological characteristics. Furthermore, the DNA barcodes of seven samples accurately mapped to the sequences of adulterants. Remarkably, counterfeit products were detected in two samples. These results demonstrate that DNA barcoding is suitable for the identification of commercial TCM decoctions. The method can effectively detect adulterants and is appropriate for use throughout the industrial chain of TCM production and distribution, and by the supervisory agencies as well.
ABSTRACT
DNA barcoding technology, a method of identifying biological species through a standard sequence, is widely used in the identification of traditional Chinese medicine (TCM), promoting the renaissance of TCM authentication discipline. The whole industrial chain of TCM includes three sections: the planting and collecting in the upstream chain, the production of TCM in the midstream chain and the circulation in the downstream chain. DNA barcoding technology, which possesses accurate, common, and objective advantages, plays an important role in the whole industrial chain of TCM. In the upstream, it is used to identify the seeds, seedlings and medicinal plants, ensuring the original source is correct. In the middle, it is used to identify Chinese medicinal materials, Chinese herbal slices and Chinese patent medicines, ensuring the materials of enterprises are correct and the clinical medication is safe. In the downstream, it participates in the establishment of traceability system for TCM, achieving the recording, inquiry and traceability of information. Therefore, DNA barcoding technology should help to control the whole production process, to protect the rights and interests of consumers and contribute to the supervision of TCM. Combined with some study cases in recent years, this paper introduces the application of DNA barcoding technology in the whole industrial chain of TCM, which is of great significance to promote the modernization of TCM industry and their internationalization.
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OBJECTIVE@#To retrospectively evaluate clinical and radiographic records of chronic periodontitis patients who underwent extraction-orthodontic treatment, in order to determine the effect of the treatment on probing depth, alveolar bone height of teeth adjacent to the extraction sites.@*METHODS@#In the study, 33 chronic periodontitis patients who had finished extraction-orthodontic treatment were selected, the periodontal examination system tables and panoramic tomography were recorded before treatment (T0) and after treatment (T1), and the periodontal probing depth (PD), residual alveolar bone height (RBH) of the teeth adjacent to extraction sites (TAES) and the non-teeth adjacent to extraction sites (NTAES) were measured at T0 and T1.@*RESULTS@#There was insignificant difference in PD of TAES and NTAES at T0 [(2.40±0.51) mm vs. (2.42±0.55) mm,P>0.05], neither was that at T1 [(2.70±0.67) mm vs. (2.67±0.64) mm, P>0.05]; From T0 to T1, PD of TAES and NTAES had mean increases of 0.3 mm [(2.40±0.51) mm vs. (2.70±0.67) mm,P<0.01] and 0.25 mm [(2.42±0.55 mm vs. (2.67±0.64) mm, P<0.01], respectively. And PD of TAES and NTAES increased from T0 to T1 statistically in the same degree [(0.30±0.64) mm vs. (0.25±0.58) mm,P>0.05]; at T0, RBH of TAES was 0.024 smaller than that of NTAES (0.74±0.16 vs. 0.76±0.16,P<0.05), but there was no difference in RBH between the TAES and NTAES at T1 (0.78±0.14 vs. 0.79±0.12,P>0.05); From T0 to T1, RBH of TAES and NTAES had mean increases of 0.04 (0.74±0.16 vs.0.78±0.14,P<0.05) and 0.02 (0.76±0.16 vs. 0.79±0.12,P<0.05), respectively. And the change of RBH between TAES and NTAES from T0 to T1 had no statistical difference (0.04±0.11 vs. 0.02±0.08,P>0.05)RBH of TAES in the side close to extraction sites was as the same as that of TAES in the side away from the extraction sites at T0 (0.73±0.17 vs. 0.74±0.16,P>0.05). From T0 to T1, RBH of both sides of TAES had mean increases of 0.04 (0.73±0.11 vs. 0.77±0.11,P<0.05) and 0.04 (0.74±0.11 vs. 0.78±0.11,P<0.05), respectively. But for both sides of TAES, from T0 to T1, there was no significant difference in change of RBH (0.04±0.11 vs. 0.04±0.11,P>0.05).@*CONCLUSIONS@#With strict control of periodontal inflammation and maintenance of oral hygiene, orthodontic treatment preserves the periodontal conditions in patients with chronic periodontitis, and the extraction-orthodontic treatment can preserve the bone height of the teeth adjacent to extraction sites.
Subject(s)
Humans , Oral Hygiene , Periodontal Diseases/therapy , Retrospective Studies , Tooth , Tooth Extraction , Treatment OutcomeABSTRACT
In order to authenticate the components of antler powder in the market, DNA barcoding technology coupled with cloning method were used. Cytochrome c oxidase subunit I (COI) sequences were obtained according to the DNA barcoding standard operation procedure (SOP). For antler powder with possible mixed components, the cloning method was used to get each COI sequence. 65 COI sequences were successfully obtained from commercial antler powders via sequencing PCR products. The results indicates that only 38% of these samples were derived from Cervus nippon Temminck or Cervus elaphus Linnaeus which is recorded in the 2010 edition of "Chinese Pharmacopoeia", while 62% of them were derived from other species. Rangifer tarandus Linnaeus was the most frequent species among the adulterants. Further analysis showed that some samples collected from different regions, companies and prices, contained adulterants. Analysis of 36 COI sequences obtained by the cloning method showed that C. elaphus and C. nippon were main components. In addition, some samples were marked clearly as antler powder on the label, however, C. elaphus or R. tarandus were their main components. In summary, DNA barcoding can accurately and efficiently distinguish the exact content in the commercial antler powder, which provides a new technique to ensure clinical safety and improve quality control of Chinese traditional medicine
Subject(s)
Animals , Antlers , DNA Barcoding, Taxonomic , Deer , Medicine, Chinese Traditional , Polymerase Chain Reaction , Powders , Quality ControlABSTRACT
OBJECTIVE: To identify the commercial medicinal materials and decoction pieces of Lycii Cortex and its adulterants using DNA barcoding technology. METHODS: A total of 137 samples, including 105 voucher samples belonging to nine species, seven GenBank sequences, and 25 test samples were involved in this study. Experiments were performed in accordance with the DNA barcoding standard operating procedures (DNA barcoding SOP) to get the ITS2 sequences. A DNA barcode database of Lycii Cortex and its adulterants were successfully constructed using 112 ITS2 sequences, which were amplified from the voucher samples and downloaded from the GenBank. This database was used to identify the commercial medicinal materials and decoction pieces of Lycii Cortex. RESULTS: The lengths of the ITS2 regions of the Lycii Cortex were 212-230 bp. The ITS2 sequences could clearly distinguish Lycii Cortex and its adulterants. Fifty percent of the commercial samples gained the ideal genomic DNA for the sequence amplification. Using the established database, the above-mentioned sequences were authenticated as Lycium chinense. CONCLUSION: ITS2 Sequence may be a suitable marker for the identification of Lycii Cortex and its adulterants. The DNA barcode databaseof Lycii Cortex and its adulterants constructed in this study are able to successfully identify the raw materials of the commercial medicinal materials and decoction pieces of Lycii Cortex that are currently available in the market.
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In order to provide a new method for the identification of Placenta hominis, the COI barcode has been employed to identify the P. hominis medicinal materials and its adulterants. Genomic DNA was extracted from the experimental samples. The COI sequences were amplified and sequenced bi-directionally. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner. NJ tree was constructed by MEGA6.0 software. COI sequences can be successfully obtained from all experimental samples. The intra-specific variation and inter-specific divergence were calculated. The average intra-specific K2P distance of P. hominis was 0.001 and the maximum intra-specific distance was 0.008. The cluster dendrogram constructed can be seen that the same genus is together, and distinguished from its adulterants. It is concluded that P. hominis and its adulterants can be correctly identified by DNA barcoding method.
Subject(s)
Animals , Cattle , Female , Humans , Pregnancy , DNA Barcoding, Taxonomic , Methods , Drug Contamination , Electron Transport Complex IV , Genetics , Medicine, Chinese Traditional , Molecular Sequence Data , Phylogeny , Placenta , Chemistry , Quality Control , Sheep , SwineABSTRACT
In order to identify Peucedani Radix, Peucedani Decursivi Radix and their adulterants, the internal transcribed spacer 2 (ITS2) regions of Peucedani Radix, Peucedani Decursivi Radix and their adulterants were amplified and bidirectionally sequenced based on the Principles for Molecular Identification of Traditional Chinese Materia Medica Using DNA Barcoding, which has been promulgated by Chinese Pharmacopoeia Commission. Sequences were analyzed and assembled by Codon Code Aligner V3. 7.1. The relevant data were analyzed by MEGA 5. 0. Species identification analyses were performed by using the nearest distance methods and neighbor-joining (NJ) methods. The result showed that the ITS2 sequence lengths of Peucedani Radix were 229-230 bp and the average intra-specific genetic distances were 0.005. The ITS2 sequence lengths of Peucedani Decursivi Radix were 227 bp and the sequences contained no variation site. The average inter-specific K2P genetic distance of Peucedani Radix, Peucedani Decursivi Radix and their adulterants species were 0.044 and 0.065 respectively. The minimum inter-specific divergence is larger than the maximum intra-specific divergence of Peucedani Decursivi Radix. The nearest distance methods and NJ trees results indicated that Peucedani Radix, Peucedani Decursivi Radix and their adulterants species could be identification clearly. The ITS2 regions can stably and accurately distinguish Peucedani Radix, Peucedani Decursivi Radix and their adulterants.
Subject(s)
Apiaceae , Classification , Genetics , DNA Barcoding, Taxonomic , Methods , DNA, Ribosomal Spacer , Drug ContaminationABSTRACT
Since the research of molecular identification of Chinese Materia Medica (CMM) using DNA barcode is rapidly developing and popularizing, the principle of this method is approved to be listed in the Supplement of the Pharmacopoeia of the People's Republic of China. Based on the study on comprehensive samples, the DNA barcoding systems have been established to identify CMM, i.e. ITS2 as a core barcode and psbA-trnH as a complementary locus for identification of planta medica, and COI as a core barcode and ITS2 as a complementary locus for identification of animal medica. This article introduced the principle of molecular identification of CMM using DNA barcoding and its drafting instructions. Furthermore, its application perspective was discussed.
Subject(s)
Animals , China , DNA , Genetics , DNA Barcoding, Taxonomic , Methods , DNA, Ribosomal Spacer , Genetics , Drugs, Chinese Herbal , Classification , Electron Transport Complex IV , Genetics , Materia Medica , Classification , Medicine, Chinese Traditional , Plant Proteins , Genetics , Plants, MedicinalABSTRACT
DNA barcoding is a rapidly developing frontier technology in the world and will be useful in promoting the quality control and standardization of traditional Chinese medicine. Until now, many studies concerning DNA barcoding have focused on leaf samples but rarely on Chinese herbal medicine. There are three issues involved in DNA barcoding for traditional Chinese medicinal materials: (1) the extraction methods for total DNA of the rhizomes of the medicinal materials; (2) intra-specific variation among samples from different places of origin; (3) accuracy and stability of this method. In this study, Gentianae Macrophyllae Radix was used to verify the stability and accuracy of DNA barcoding technology. Five regions (ITS2, psbA-trnH, matK, rbcL, and ITS) were tested for their ability to identify 86 samples of Gentianae Macrophyllae Radix and their adulterants. After improving the DNA extraction method, genomic DNA from all samples was successfully obtained. To evaluate each barcode's utility for species authentication, PCR amplification efficiency, genetic divergence, and species authentication were assessed. Among all tested regions only ITS2 locus showed 100% of PCR amplification and identification efficiencies. Based on the established method, we successfully identified two samples of Gentianae Macrophyllae Radix bought in pharmacy to the original species.
Subject(s)
DNA Barcoding, Taxonomic , Methods , DNA, Plant , Genetics , Drug Contamination , Genetic Variation , Genome, Plant , Gentiana , Classification , Genetics , Plant Roots , Genetics , Plants, Medicinal , Genetics , Polymerase Chain Reaction , Methods , Quality Control , Sequence Analysis, DNA , Species SpecificityABSTRACT
In this study, Notopterygii Rhizoma et Radix was used to verify the stability and accuracy of DNA barcodes in identification of Chinese materia medica for the first time. All genomic DNAs from thirty one samples were extracted. The ITS (internal transcribed spacer) regions were amplified and sequenced bi-directionally. Obtained sequences were assembled using the CodonCode Aligner. And the sequences of the ITS regions were aligned through Clustal-W and the genetic distances were computed using MEGA 5.0 in accordance with the kimura 2-parameter (K2P) model. The neighbor-joining (NJ) phylogenetic trees were constructed. The ITS2 regions were obtained by using the hidden Markov model (HMM)-based annotation methods from the ITS sequences. Results indicated that the lengths of ITS regions of Notopterygii Rhizoma et Radix were 603-604 bp, while the lengths of ITS2 regions were 228 bp. The haplotypes of ITS/ITS2 regions of Notopterygii Rhizoma et Radix were the same as those of the original plant leaves. The intra-specific genetic distances were smaller than inter-specific ones in ITS/ITS2 regions of Notopterygium incisum and N. franchetii. The NJ trees showed that N. incisum, N. franchetii and its adulterants can be easily differentiated according to their monophyly. Therefore, ITS/ITS2 regions as DNA barcodes can stably and accurately distinguish Notopterygii Rhizoma et Radix from its adulterants and could provide a new technique to ensure clinical safety in utilization of traditional Chinese medicines.
Subject(s)
Apiaceae , Classification , Genetics , DNA Barcoding, Taxonomic , Methods , DNA, Plant , Genetics , DNA, Ribosomal Spacer , Genetics , Phylogeny , Plant Roots , Genetics , Plants, Medicinal , Genetics , Rhizome , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To study on the compliance antiretroviral(ARV) drugs and interrelated factors of HIV/AIDS patients undertaking highly active antiretroviral therapy(HAART) to improve clinical treatment.</p><p><b>METHODS</b>3 counties in Henan province were selected including 2 counties from China Cares Program points and one county where HIV/AIDS was serious. All cases studied had already received antiretroviral therapy (ART) for 2-12 months. Several indicators through questionnaire were studied including drug adherence, side effect, symptoms status before and after treatment and ART measures etc. At the same time, blood was collected to analyze CD4+ T-lymphocyte, the virus loads of HIV and drug genotype resistance by reverse transcription-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>In the therapy group, most patients had taken ARV drugs for 4-8 months, which accounted for 78.24% of the total number, with the adherence rate above 90% as 67.51%. The main reason for the patients not listening to their doctor's advice was due to ART drug's side effects (66.95%), including queasiness, vomiting and tetter. In the therapy group, 82.57% of patients' symptoms were obvious. Adherence had a great impact on the improvement (P < 0.05). After therapy, the total count of patients' CD4+ T-lymphocyte's kept stable or improved with slow speed. At the time of 3 months and 6 months after ART, the rates of improvement were 55.1% and 50.8%. However CD4+ count did not show much difference between the twe group. The prevalence rate of HIV drug resistance strain roise from 13.9% in non-remedial group to 45.4% at the time of 3 months after therapy and 62.7% at the time of 6 months after therapy. The resistance against non-nucleoside reverse transcriptase inhibitors (NNRTI) had improved obviously.</p><p><b>CONCLUSION</b>Patients with HIV/AIDS receiving HAART, compliance seemed directly affect the curative effect and the implementation of therapy schedule and should be improved to avoid drug resistance.</p>