Distinguishing between Artemisia stolonifera and A. argyi by specific PCR of leaves and non-glandular trichomes / 中国中药杂志

Artemisia stolonifera is a relative of A. argyi. The two species are difficult to be distinguished due to the similarity in leaf shape and have even less distinctive features after processing. This study aims to establish a method to quickly distinguish between them. At the same time, we examined the reasonability and applicability of the specific polymerase chain reaction(PCR) method. The C/T single nucleotide polymorphism was detected at the position 202 of the sequence, based on which specific primers were designed to identify these two species. The PCR with the specific primer JNC-F and the universal primer ITS3R produced a specific band at 218 bp for A. argyi and no band for A. stolonifera, which can be used to detect at least 3% of A. argyi samples mixed in A. stolonifera samples. The PCR with the specific primer KY-F and the universal primer ITS3R produced a specific band at 218 bp for A. stolonifera and no band for A. argyi, which can be used to detect at least 5% of A. stolonifera samples mixed with A. argyi. The limit of detection of the established method was 5 ng DNA. The established PCR method can accurately distinguish between A. stolonifera and A. argyi, which provides an experimental basis for the quality control of A. stolonifera and determines whether the herbs are adulterated.

Desenvolvimento de metodologia para identificação de fungos através de metabarcoding aplicada em amostras humanas de adenocarcinoma gástrico e carcinoma epidermoide de pênis / Development of methodology for fungi identification through metabarcoding applied in gastric adenocarcinoma and penile epidermoid carcinoma of human sample

O microbioma humano compreende material genético da microbiota de um local do corpo e tem influência direta ou indireta na manutenção da homeostase. O distúrbio da microbiota pode estar relacionado ao desenvolvimento de doenças. A população fúngica ainda é muito pouco estudada no contexto do microbioma. No presente estudo, foi desenvolvida uma metodologia para identificação de fungos por metabarcoding. A metodologia desenvolvida foi aplicada em mostras de pacientes portadores de adenocarcinoma gástrico ou carcinoma epidermoide de pênis. De modo geral, em ambos os tumores foi verificada a redução de diversidade fúngica conforme a evolução do estadiamento patológico. Também foram verificados resultados não concordantes ao analisar espécies diferencialmente abundantes em dados de sequenciamento da região ITS2 e de WGS nas amostras de lavado gástrico. Este trabalho reforça a importância em se estudar os fungos e sua associação com doenças como o câncer e incentiva próximos estudos através do desenvolvimento de uma metodologia específica para o micobioma.

The human microbiome comprises genetic material from the microbiota of a body site and has a direct or indirect influence on the maintenance of homeostasis. The disturbance of the microbiota may be related to the development of diseases. The fungal population is still very little studied in the context of the microbiome. In this study, a methodology was developed to identify fungi by metabarcoding. The methodology developed was applied to samples from patients with gastric adenocarcinoma or squamous cell carcinoma of the penis. In general, in both tumors, a reduction in fungal diversity was observed according to the evolution of the pathological staging. Discordant results were also found when analyzing differentially abundant species in sequencing data from the ITS2 region and WGS in gastric lavage samples. This work reinforces the importance of studying fungi and their association with diseases such as cancer and encourages further studies through the development of a specific methodology for the mycobiome

Identification of Medicinal Aconitum Species Based on ITS2 Sequences and Analysis of Genetic Relationship / 中国实验方剂学杂志

ObjectiveThe internal transcribed spacer （ITS） 2 region of ribosomal gene， a DNA barcode， was employed to identify 12 medicinal Aconitum species and the genetic relationship among the species was analyzed. MethodA total of 30 samples of the 12 species were collected. The DNA was extracted with spin column plant genomic DNA kit and the universal primers of ITS2 sequence were used for polymerase chain reaction （PCR） amplification， followed by electrophoresis detection and bi-directional sequencing. The yielded sequences were aligned and spliced by CodonCode Aligner 17.0 and sequence variation was analyzed by MEGA 7.0. The secondary structure was predicted by ITS2 Database and the neighbor-joining （NJ） method was applied to generate the phylogenetic tree. ResultThe ITS2 sequences of the 12 species were 220-221 bp， with the average guanine and cytosine （GC） content of 64.09%， 140 variable sites， 137 informative sites， and 81 conservative sites. The intraspecific genetic distance （K2P） was smaller than the interspecific genetic distance. According to the secondary structures of ITS2 sequences and NJ cluster analysis， A. scaposum， A. sinomontanum， and A. barbatum had close genetic relationship， while the rest nine showed close kinship， particularly A. soongaricum and A. yinschanicum. ConclusionITS2 sequence is of great value for the molecular identification and genetic relationship determination of Aconitum， which provides a new method for the study of ethnomedicine.

Phylogenetic and Mutation Points Analysis of Sophora japonica cv. jinhuai Based on DNA Barcoding Sequences / 中国实验方剂学杂志

ObjectiveTo conduct phylogenetic analysis of internal transcribed spacer 2 （ITS2） and chloroplast gene segments including psbA-trnH， rbcL， and matK of Sophora japonica cv. jinhuai resource samples from different geographical sources， and to explore the genetic diversity of S. japonica cv. jinhuai. MethodPolymerase chain reaction （PCR） method was used to amplify the nucleic acid sequences of ITS2， psbA-trnH， rbcL， and matK of S. japonica cv. jinhuai. Neighbor joining （NJ） method was used to construct phylogenetic trees， and Kimura 2-Parameter （K2P） model was used to calculate the genetic distance of different samples. MEGA and BIOEDIT softwares were applied for mutiple alignment and analysis of ITS2， psbA-trnH， rbcL， and matK sequences of S. japonica cv. jinhuai. ResultThe lengths of ITS2 sequence were 278-279 bp. The lengths of psbA-trnH were 289 bp. The lengths of rbcL sequence were 673 bp. The lengths of matK sequences were 786-792 bp. There were 3 mutation points in ITS2 and psbA-trnH， no mutation point in rbcL， and 13 mutation points in matK. The samples of S. japonica cv. jinhuai were clustered into two groups based on the phylogenetic tree constructed by ITS2 sequences. The sample of seedling tree in Baibao was clustered into one group， while the other 25 samples were clustered into another group. For the psbA-trnH sequence， the success rate of PCR amplification of 28 samples of S. japonica cv. jinhuai was 100%. The 28 samples of S. japonica cv. jinhuai were clustered into three groups based on the clustering results of psbA-trnH sequence. The sample of seedling tree in Shaoshui was clustered into one group. The five samples of grafting tree and seedling tree in Miaotou， grafting trees in Jiantang， Wenqiao， and Daxu， and seeding tree in Xianshui were clustered into one group. The other 21 samples were clustered into another group. The 26 samples of S. japonica cv. jinhuai were clustered into two groups based on the phylogenetic tree constructed by matK sequences. The sample of seedling tree in Xianshui was clustered into one group， while the other 25 samples were clustered into another group. The clustering results of the rbcL sequence of S. japonica cv. jinhuai could not distinguish 28 resource samples. The phylogenetic tree constructed by the combined sequence of ITS2+psbA-trnH+rbcL+matK divided S. japonica cv. jinhuai resource samples into 4 groups. The 13 samples of seedling trees in Qiyang， Daoxian， Miaotou， Shaoshui， Shitang， Xianshui， Jiantang， and Xiangli， and grafting trees in Qiyang， Miaotou， Yongsui， Wenqiao， and Yangtang were clustered into one group. The sample of seedling tree in Wenqiao was clustered into one group. The sample of seedling tree in Daxu was clustered into one group. The remaining samples were clustered into another group. ConclusionPhylogenetic and mutation analysis provide the theoretic foundation to investigate the evolution of the resources of S. japonica cv. jinhuai， and evaluate their genuineness. The results of mutation points can be used to identify the related S. japonica cv. jinhuai resources. The findings of this study show that the combination of different gene sequences has an optimal effect on plant identification.

Molecular Identification of Tibetan Codonopsis Plants Based on ITS2 and psbA-trnH Barcode / 中国实验方剂学杂志

ObjectiveTo identify the molecular biology of various species of Tibetan Codonopsis plants based on internal transcribed spacer（ITS）2 and psbA-trnH sequence barcode technology. MethodThe genomic DNA of 28 Tibetan Codonopsis plant samples from four species （Codonopsis canescens，C. foetens subsp. nervosa，C. pilosula， and C. thalictrifolia var. mollis） were extracted，and the ITS2 and psbA-trnH sequences were amplified and sequenced. The related sequences of 81 Tibetan Codonopsis plant samples belonging to 15 species were downloaded from GenBank， and MEGA 6.0 was used for sequence comparison and mutation site analysis. The GC content and genetic distance within and between species were calculated. Additionally， phylogenetic trees were constructed by maximum likelihood （ML） method， neighbor-joining （NJ） method，and unweighted pair-group method with arithmetic means （UPGMA） . ResultAccording to the mutation site，C. canescens， C. pilosula，C. pilosula subsp. tangshen， C. pilosula var. modesta，C. bhutanica，C. clematidea，C. lanceolata，C. subglobosa and C. foetens were distinguished. In the phylogenetic trees，the optimal clustering effects for ITS2 and psbA-trnH sequences were obtained using the ML method and the UPGMA method， respectively， and 12 species were effectively clustered. ConclusionITS2 and psbA-trnH sequences have a high identification rate for species of single origin，but there are still some limitations in identifying variants and original variants. This study provides basis for the identification of affinity relationship and clinical safety of Tibetan Codonopsis plants.

Integrative taxonomy: combining molecular and morphological characteristics to identify Lymnaea (Galba) cubensis, intermediate host of Fasciola hepatica / Taxonomia integrativa: combinando características moleculares e morfológicas para identificar Lymnaea (Galba) cubensis, hospedeiro intermediário de Fasciola hepatica

Abstract Despite the epidemiological importance of the Lymnaeidae family regarding transmission of Fasciola hepatica, knowledge about the diversity and distribution of these molluscs and the role of each species in the expansion of fasciolosis remains sparse. Classical morphological (n=10) identification was performed in lymneids from Lagoa Santa, a municipality in the state of Minas Gerais, Brazil, along with molecular and phylogenetic analysis (n=05) based on the partial nucleotide sequences of the mitochondrial cytochrome c oxidase subunit I gene (COI mtDNA) and ribosomal internal transcribed spacer II (ITS-2 rDNA). The shell morphology made it possible to distinguish the lymneids of Lagoa Santa from Pseudosuccinea columella. Differences found in the penile complex and prostate shape allowed this species to be distinguished from Galba truncatula. However, the homogeneity of reproductive tract characteristics among Lymnaea (Galba) cubensis, L. viator and L. neotropica confirmed that these characteristics show low taxonomic reliability for identifying cryptic species. Genetic divergence analysis for the COI mtDNA gene and ITS-2 region of rDNA revealed greater similarity to Lymnaea (Galba) cubensis. Thus, correct species differentiation is important for monitoring the epidemiological risk of fasciolosis in the state of Minas Gerais, where cases of the disease have increased over recent years.

Resumo Apesar da importância epidemiológica da família Lymnaeidae na transmissão de Fasciola hepatica, o conhecimento sobre a diversidade e a distribuição desses moluscos e o papel de cada espécie, na expansão da fasciolose, ainda é escasso. Realizou-se a identificação morfológica clássica (n=10) em limneídeos de Lagoa Santa, município do estado de Minas Gerais, Brasil, juntamente com a análise molecular e filogenética (n=05), baseada nas sequências parciais de nucleotídeos do gene mitocondrial da subunidade I do citocromo c oxidase (COI mtDNA) e espaçador interno, transcrito do DNA ribossomal II (ITS-2 rDNA). A morfologia da concha possibilitou distinguir os limneídeos de Lagoa Santa de Pseudosuccinea columella. As diferenças encontradas no complexo peniano e na forma da próstata permitiram que essa espécie fosse distinta de Galba truncatula. No entanto, a homogeneidade das características do trato reprodutivo entre Lymnaea (Galba) cubensis, L. viator e L. neotropica confirmou que essas características apresentam baixa confiabilidade taxonômica para a identificação de espécies crípticas. A análise da divergência genética para o gene COI mtDNA e região ITS-2 do rDNA revelou maior similaridade entre os limneídeos de Lagoa Santa com Lymnaea (Galba) cubensis.

Application of DNA barcoding technology to national drug sampling inspection / 药学学报

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.

Analysis of fungal diversity on the surface of Armeniacae Semen Amarum using a DNA metabarcoding technique / 药学学报

Medicinal and edible Armeniacae Semen Amarum (ASA) is susceptible to fungal contamination because it is rich in oil and other nutrients. In this study, the fungal community diversity in ASA samples was analyzed based on a DNA metabarcoding technique to provide evidence for its safe use. Twelve batches of ASA samples samples from four medicinal material markets and three processing approaches were collected. Total DNA was extracted, the ITS2 sequences were amplified, and high-throughput sequencing was performed using the Illumina MiSeq PE300 platform. The results show that Ascomycota was the most dominant fungus in ASA samples. The predominant genus in sample SW1_P was Diutina, whereas the most predominant genus in the other samples was Aspergillus. Three harmful fungi were identified, namely, Aspergillus flavus, Wallemia sebi, and Rhizopus arrhizus. In addition, significant differences were observed in the relative abundance of Botryosphaeriales and Alternaria in ASA samples from different collection sites. Meanwhile, there were significant differences in the relative abundance of Hypocreales and Cladosporium in ASA samples from different processing approaches. In summary, the DNA metabarcoding technique can effectively clarify the fungal community diversity and quickly detect potential toxigenic fungi in ASA samples, thus providing a warning for mycotoxin contamination.

Molecular authentication of the medicinal species of Rhizoma et Radix Heraclei, Radix Angelicae Sinensis, Radix Angelicae Pubescentis and Rhizoma et Radix Notopterygii by integrating ITS2 and its secondary structure / 药学学报

This study is to identify Chinese medicinal materials Rhizoma et Radix Heraclei, Angelicae Sinensis, Radix Angelicae Pubescentis and Rhizoma et Radix Notopterygii based on ITS2 and its secondary structure. Total 26 ITS sequences of 7 species were downloaded from GenBank, the ITS2 sequences were annotated by HMMer method. The NJ phylogenetic tree was built by MEGA software, the intraspecific and interspecific K2P genetic distance were analyzed by MEGA as well. The ITS2 secondary structures of all taxa were predicted by ITS2 database. Sequence matrix of primary structure and secondary structure was aligned by 4Sale software. And the profile neighbor joining (PNJ) phylogenetic tree was constructed via the the ProfDistS software based on the distance method. The results show that, the average interspecific genetic distance was far greater than the average intraspecific genetic distance, an obvious barcoding gap was noted among all taxa; NJ tree showed that all species were clustered into seperate branches; each species had different secondary structures; the PNJ tree showed higher resolution than NJ tree. Therefore, ITS2 is suggested to be used as a barcode for distinguishing the original plants of Rhizoma et Radix Heraclei, Radix Angelicae Sinensis, Radix Angelicae Pubescentis and Rhizoma et Radix Notopterygii in this study, this provides some scientific basis for classification and accurate identification of these Chinese medicinal materials.

DNA barcoding identification of commercial decoctions in traditional Chinese medicine / 药学学报

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.

Cuticular ridge patterns applied for identifying adult female worms of Haemonchus species with various vulvar morphological types in infected native goats in Laos

@#Species of the blood sucking nematode Haemonchus are a main problem in the small ruminant industry worldwide. Haemonchus worms were taken from 68 infected native goats slaughtered in three provinces of Laos in June and July 2019. Cuticular ridge patterns were used for the first time to identify adult female Haemonchus spp. and their vulvar morphs were characterized. The results showed that the variations in vulvar morphology of female Haemonchus spp. presented a knobbed morph as the dominant morphotype and predominant linguiform B subtype was also detected. In total, 270 selected female worms from each vulvar morph type were examined based on their cuticular ridge patterns in cross sections at positions of the esophageal-intestinal junction (EI), the 4 mm region from the anterior end (4 mm), and the mid-body (MB). Only Haemonchus contortus was identified and most worms had constant numbers of ridges at EI, 4 mm, and MB, namely 30, 26, and 22 ridges, respectively, accounting for 99.26%, 97.41%, and 97.04%, respectively, of worms detected, while the lowest variation in the number of ridges was at region EI which is recommended as the single best position. Based on synlophe and ITS2 sequence analysis, it was assumed that H. contortus might dominate in the sample areas with the possible numbers of ridges of H. contortus females in the ranges 29-30, 25-27, and 21-23 for positions EI, 4 mm, and MB, respectively. The cuticular ridge pattern was a useful character for identifying female Haemonchus species in this study and could be utilized as an affordable alternative method for epidemiological studies and as part of parasite control management in native goats of Laos.

ESTUDIOS MORFO-ANATÓMICOS, CITOGENÉTICOS Y MOLECULARES EN Talinum (TALINACEAE): UNA NUEVA ENTIDAD PARA EL NOROESTE ARGENTINO / Morfo-anatomical, cytogenetic and molecular studies in Talinum (TALINACEAE): a new entity from the northwest of Argentina

RESUMEN Talinum paniculatum es una hierba adventicia ampliamente distribuida en Argentina, que tiene importancia económica como maleza de cultivos resistente a herbicidas. Esta especie se presenta en el campo con dos morfotipos y ellos se distinguen por la forma, tamaño y color de sus flores, frutos y hojas. El objetivo de este trabajo fue determinar las características morfo-anatómicas (de hoja y tallo), citogenéticas y moleculares en dos morfotipos de la provincia de Tucumán (Argentina), y establecer diferencias entre ellos. Se utilizaron técnicas morfo-anatómicas y citogenéticas clásicas y se realizaron análisis moleculares con el marcador ITS2. Los resultados evidencian que las características morfológicas, anatómicas, citogenéticas y moleculares de Talinum paniculatum permitieron diferenciar los morfotipos MT1 y MT2. Se concluye que el MT1 corresponde a T. paniculatum y el MT2 a un taxón diferente que aún no se mencionó para la flora de Argentina.

ABSTRACT Talinum paniculatum is an adventitious herb widely distributed in the Argentina. This plant is considered as an economically important herbicide-resistant weed. This species shows two morphotypes in the field which are differentiated by shape, size and colour of their flowers leaves and fruits. The aims of this work were to determinate morpho-anatomical (leave and stem), cytogenetic and molecular traits of two morphotypes from Tucumán province (Argentina) and to establish differences between them. Classical morpho-anatomical and cytogenetic techniques were used, molecular analysis based on the ITS2 marker were performed. The results showed that morphological, anatomical, cytogenetic and molecular traits of T. paniculatum allow us to differentiate the MT1 and MT2 morphotypes. We concluded that MT1 match with T. paniculatum and MT2 is a different taxon still not described for the flora of Argentina.

Postpartum histoplasmosis in an HIV-negative woman: a case report and phylogenetic characterization by internal transcribed spacer region analysis

Abstract The present report describes the first case of postpartum disseminated histoplasmosis in a 24-year-old HIV-negative woman. On the tenth day after vaginal delivery, the patient presented with dyspnea, fever, hypotension, tachycardia, and painful hepatomegaly. Yeast-like Histoplasma capsulatum features were isolated in the buffy coat. The phylogenetic analysis demonstrated that the fungal isolate was similar to other H. capsulatum isolates identified in HIV patients from Ceará and Latin America. Thus, histoplasmosis development in individuals with transitory immunosuppression or during the period of immunological recovery should be carefully examined.

Molecular authentication of Sarcandra glabra and its adulterants using three DNA molecular markers / 中草药

Objective: To identify Sarcandra glabra and its adulterants using three DNA molecular markers including 18 S rRNA gene, ITS2 sequence and SCAR marker, and then provide the basis for its molecular authentication. Methods: 18 S rRNA gene sequence of S. glabra was obtained by PCR amplification, cloning and sequencing, and then Blast comparison was made in NCBI. The ITS2 sequence of S. glabra was obtained by PCR amplification, sequencing and annotation in ITS2 Database. In the meanwhile, the ITS2 sequences of adulterants and other plants were collected from GenBank. Using MEGA5.5, the genetic distance was calculated between species and then the ITS2 sequences were aligned to construct a phylogenetic clustering tree. SCAR molecular marker of S. glabra was obtained by RAPD. After cloning and sequencing, specific primers were designed to amplify S. glabra and its adulterants. Results: The length of 18 S rRNA obtained in our research was 1 820 bp. Blast comparison revealed that there was 99% homology between S. glabra and Chloranthaceae, which proved to be 18 S rRNA gene of S. glabra. The length of the ITS2 sequence in our research was 500 bp. Genetic distance between S.glabra and its adulterants ranged from 0.190 to 0.219, which was far more than genetic distance among adulterants (0.000-0.074). Cluster analysis showed that S. glabra and its adulterants respectively clustered into a different branch, which was far away from other plants. In our research, we obtained SCAR molecular marker of S. glabra and then a pair of specific primers were designed. Using the pair of specific primers, specific products were amplified from genomic DNA of S. glabra, but no specific products were obtained from that of its adulterants. Conclusion: We could authenticate S. glabra and its adulterants effectively with the combination of three molecular markers for establishing a novel method to identify S. glabra and its adulterants, which provides a new idea for the authentication of S. glabra.

Molecular identification of Sanqi Tablets using DNA barcoding and its methodological evaluation / 中草药

Objective: In order to study the application of DNA barcoding in the authentication of Chinese patent medicines, Sanqi Tablets were used as the object to investigate the applicability, specificity and precision of this method. Methods: Fifteen batches of commercially available Sanqi Tablet samples were collected. The conditions of DNA extraction for Sanqi Tablet had been first investigated, and the DNA was used for testing the applicability of the methods such as PCR amplification, sequence acquisition, and species authentication in the principles for molecular identification of traditional Chinese materia medica using DNA barcoding. The specificity and reproducibility of DNA barcoding in identification of Sanqi Tablets and its adulterations from the roots of Panax notoginseng, P. ginseng and P. quinquefolius were also studied. Results: The Sanqi Tablet sample with an amount of sampling to be 100 mg and a water bath at 56 ℃ for 8 h gave an average concentration of 60.7 ng/μL and then the PCR amplification, sequence acquisition and species assignment were all successful. The ITS2 sequences of P. notoginseng, P. ginseng and P. quinquefolius were all 230 bp in length, and there were seven stable SNP loci between P. notoginseng and P. ginseng, P. notoginseng and P. quinquefolius. ITS2 sequences could be successfully obtained from lab-made and the adulterated Sanqi Tablets, and the Sanger sequencing chromatograms of different ratios of P. notoginseng and P. ginseng mixtures, P. notoginseng and P. quinquefolius mixtures had heterozygous peaks with corresponding peak height ratio at SNP positions. The repeatability, intermediate precision and reproducibility were all in line with the requirements of “General Regulation 9101” in the Chinese Pharmacopoeia. Conclusion: The ITS2 sequence can stably and accurately authenticate the raw materials of Sanqi Tablets with substantial specificity and precision. The DNA barcoding identification method of Sanqi Tablets will provide a new technical tool for ensuring the safety of Sanqi Tablets in clinical medications, and provide reference for the identification of other single-herb products documented in the Chinese Pharmacopoeia.

Molecular identification of Spatholobi Caulis and its adulterants based on DNA barcoding / 中草药

Objective: Molecular biology identification technology was used to screen the appropriate DNA barcoding to establish a fast and accurate method for identifying Spatholobi Caulis. Methods: A total of 72 samples of Spatholobi Caulis and its adulterants were collected, the sample DNA was extracted, the ITS2, matK, psbA-trnH, ITS, and rbcL sequences were amplified and sequenced, and the amplification success rate and sequencing success rate of each sequence were calculated. The alignment of all sequences was determined by MEGA 7.0 software, and the interspecies and intraspecific genetic distance of them were analyzed to evaluate the Barcoding gap, based on the Kimura-2-Parameter (K2P) two-parameter model. Phylosuite software was used to construct the ITS2, matK and psbA-trnH and multi-gene (I-M-P) phylogenetic tree. Results: The amplification success rate and sequencing success rate of ITS2 were the highest (100%), and the sequencing success rates of matK and psbA-trnH were 94.4% and 91.7% respectively, while rbcL and ITS were only 69.4% and 61.1%. Compared with other barcoding, ITS2 has obvious Barcoding gap, and there was less overlap tree showed that ITS2 and psbA-trnH can obviously cluster Spatholobi Caulis and its adulterants into different branches, while matK cannot separate Kadsurae Caulis and Schisandrae Sphenantherae Fructus. I-M-P phylogenetic tree had the same result as ITS2 and psbA-trnH. between species. Conclusion: The identification method based on ITS2 and supplemented by the psbA-trnH sequence can quickly and accurately identify S. Caulis and its adulterants, which can provide the basis for the safety and the accuracy of the clinical application.

Phylogenetic and mutation point analysis of DNA barcoding sequences in Polygonatum cyrtonema / 中草药

Objective: In this work, phylogenetic analysis was used to compare the ITS2 and psbA-trnH sequences of Polygonatum cyrtonema samples from different geographical sources, so as to explore the genetic diversity and genetic relationship of these resources. Methods: PCR method was used to amplify the regions of ITS and psbA-trnH, and the sequences of ITS2 and psbA-trnH were obtained after the amplified fragment sequences were blasted in NCBI database. The neighbor joining (NJ) and maximum parsimony (MP) methods were used to construct phylogenetic trees and Kimura two-parameter (K2-P) model was used to calculate the genetic distance of different samples. Mega and DNAman softwares were applied for mutiple alignment of ITS2 and psbA-trnH sequences of 25 samples of P. cyrtonema. Results: The lengths of ITS2 and psbA-trnH sequences of Anhui Qingyang and Fujian Taining samples of P. cyrtonema were 224 bp and 620 bp, respectively. The lengths of ITS2 and psbA-trnH of the remaining 24 samples were 225 bp and 621 bp, respectively. ITS2 and psbA-trnH had seven and four mutation points, respectively. These 25 samples were clustered into two groups based on ITS2 sequences. Five samples in Hunan and Guizhou were clustered into one group, while the other 20 samples were clustered into another group. The genetic distance showed that the samples from Huaxi and Jianhe in Guizhou Province and Jianyang in Fujian Province had the largest genetic distance. Phylogenetic tree constructed by psbA-trnH sequences were unable to distinguish 25 samples from different geographical sources. Conclusion: Phylogenetic and mutation analysis will provide the theoretic foundation to utilize the resources of P. cyrtonema, investigate their evolution, and evaluate their genuineness. The results of mutation point will also be used in the identification of related P. cyrtonema resources.

Identification of Bupleurum marginatum var. Wall. ex DC stenophyllum (Wolff) Shan et Li and its analogues based on ITS2 barcode / 中草药

Objective: DNA barcoding technology, a molecular identification method, is applied to distinguish Bupleurum marginatum var. stenophyllum from its analogues in order to ensure the quality and clinical curative effect. Methods: In this study, the internal transcribed spacer 2 (ITS2) regions of 50 samples were amplified by PCR and sequenced bi-directionally. Obtained sequences were assembled using CodonCode Aligner. The genetic distances were computed by MEGA 6.0 in accordance with the kimura 2-parameter (K2P) model and the phylogenetic tree was constructed by Neighbor-joining (NJ) method. Moreover, the secondary structure of ITS2 was predicted using ITS2 database websites. Results: The intra-specific genetic distances were smaller than inter-specific ones in ITS2 regions of B. marginatum var. stenophyllum. The NJ tree and secondary structure results could distinctly differentiate B. marginatum var. stenophyllum and its analogues. Conclusion: ITS2 sequence can scientifically and reliably identify the authenticity of B. marginatum var. stenophyllum and could provide more new and reliable techniques to ensure clinical safety of this traditional Chinese medicine.

Identification of medicinal plants within the Apocynaceae family using ITS2 and psbA-trnH barcodes / 中国天然药物

To ensure the safety of medications, it is vital to accurately authenticate species of the Apocynaceae family, which is rich in poisonous medicinal plants. We identified Apocynaceae species by using nuclear internal transcribed spacer 2 (ITS2) and psbA-trnH based on experimental data. The identification ability of ITS2 and psbA-trnH was assessed using specific genetic divergence, BLAST1, and neighbor-joining trees. For DNA barcoding, ITS2 and psbA-trnH regions of 122 plant samples of 31 species from 19 genera in the Apocynaceae family were amplified. The PCR amplification for ITS2 and psbA-trnH sequences was 100%. The sequencing success rates for ITS2 and psbA-trnH sequences were 81% and 61%, respectively. Additional data involved 53 sequences of the ITS2 region and 38 sequences of the psbA-trnH region were downloaded from GenBank. Moreover, the analysis showed that the inter-specific divergence of Apocynaceae species was greater than its intra-specific variations. The results indicated that, using the BLAST1 method, ITS2 showed a high identification efficiency of 97% and 100% of the samples at the species and genus levels, respectively, via BLAST1, and psbA-trnH successfully identified 95% and 100% of the samples at the species and genus levels, respectively. The barcode combination of ITS2/psbA-trnH successfully identified 98% and 100% of samples at the species and genus levels, respectively. Subsequently, the neighbor joining tree method also showed that barcode ITS2 and psbA-trnH could distinguish among the species within the Apocynaceae family. ITS2 is a core barcode and psbA-trnH is a supplementary barcode for identifying species in the Apocynaceae family. These results will help to improve DNA barcoding reference databases for herbal drugs and other herbal raw materials.

Use of ITS2 and psbA-trnH DNA barcode sequences of wild Phytolacca resources in Guanzhong area of Shaanxi Province / 药学学报

In order to explore the use of DNA barcode in the identification of wild Phytolacca resources in the Shaanxi Guanzhong area, 29 DNA samples were amplified and sequenced by using the universal primers ITS2 and psbA-trnH. The sequences were spliced and proof-read by Codon CodeA aligner V3.0, followed by blast comparison and identification analysis; mega 6.0 was used to analyze sequence characteristics, Kimura 2-Parameter (K2P) was used to analyze distance and intraspecific or interspecific variation, and Neighbor-Joining trees were established to evaluate the ability of two pairs of candidate sequences to distinguish Phytolaccae Radix from its adulterants. The results showed that the success rate of PCR amplification and sequencing of ITS2 and psbA-trnH was 100%; the NJ tree showed that both ITS2 and psbA-trnH sequences could separate P. acinosa, P. americana, other species of the same genus like P. japonica, P. exiensis and two adulterant species into a single clade; primer ITS2 had an advantage over psbA-trnH in determining interspecific genetic distances. Therefore, both ITS2 and psbA-trnH sequences can be used for identification of Phytolacca and their adulterants, which provides a theoretical basis for the distribution of wild Phytolacca resources and their rational development and utilization.