DNA metabarcoding analysis of fungal community on surface of four root herbs / 中草药·英文版

OBJECTIVE@#Angelicae Sinensis Radix (ASR, Danggui in Chinese), Cistanches Herba (CH, Roucongrong in Chinese), Ginseng Radix et Rhizoma (PG, Renshen in Chinese), and Panacis Quinquefolii Radix (PQ, Xiyangshen in Chinese), widely used as medicine and dietary supplement around the world, are susceptible to fungal and mycotoxin contamination. In this study, we aim to analyze their fungal community by DNA metabarcoding.@*METHODS@#A total of 12 root samples were collected from three main production areas in China. The samples were divided into four groups based on herb species, including ASR, CH, PG, and PQ groups. The fungal community on the surface of four root groups was investigated through DNA metabarcoding via targeting the internal transcribed spacer 2 region (ITS2).@*RESULTS@#All the 12 samples were detected with fungal contamination. Rhizopus (13.04%-74.03%), Aspergillus (1.76%-23.92%), and Fusarium (0.26%-15.27%) were the predominant genera. Ten important fungi were identified at the species level, including two potential toxigenic fungi (Penicillium citrinum and P. oxalicum) and eight human pathogenic fungi (Alternaria infectoria, Candida sake, Hyphopichia burtonii, Malassezia globosa, M. restricta, Rhizopus arrhizus, Rhodotorula mucilaginosa, and Ochroconis tshawytschae). Fungal community in ASR and CH groups was significantly different from other groups, while fungal community in PG and PQ groups was relatively similar.@*CONCLUSION@#DNA metabarcoding revealed the fungal community in four important root herbs. This study provided an important reference for preventing root herbs against fungal and mycotoxin contamination.

DNA barcoding in herbal medicine:Retrospective and prospective / 药物分析学报

DNA barcoding has been widely used for herb identification in recent decades,enabling safety and innovation in the field of herbal medicine.In this article,we summarize recent progress in DNA bar-coding for herbal medicine to provide ideas for the further development and application of this tech-nology.Most importantly,the standard DNA barcode has been extended in two ways.First,while conventional DNA barcodes have been widely promoted for their versatility in the identification of fresh or well-preserved samples,super-barcodes based on plastid genomes have rapidly developed and have shown advantages in species identification at low taxonomic levels.Second,mini-barcodes are attractive because they perform better in cases of degraded DNA from herbal materials.In addition,some mo-lecular techniques,such as high-throughput sequencing and isothermal amplification,are combined with DNA barcodes for species identification,which has expanded the applications of herb identification based on DNA barcoding and brought about the post-DNA-barcoding era.Furthermore,standard and high-species coverage DNA barcode reference libraries have been constructed to provide reference se-quences for species identification,which increases the accuracy and credibility of species discrimination based on DNA barcodes.In summary,DNA barcoding should play a key role in the quality control of traditional herbal medicine and in the international herb trade.

A novel biological sources consistency evaluation method reveals high level of biodiversity within wild natural medicine: A case study of Amynthas earthworms as "Guang Dilong"

For wild natural medicine, unanticipated biodiversity as species or varieties with similar morphological characteristics and sympatric distribution may co-exist in a single batch of medical materials, which affects the efficacy and safety of clinical medication. DNA barcoding as an effective species identification tool is limited by its low sample throughput nature. In this study, combining DNA mini-barcode, DNA metabarcoding and species delimitation method, a novel biological sources consistency evaluation strategy was proposed, and high level of interspecific and intraspecific variations were observed and validated among 5376 Amynthas samples from 19 sampling points regarded as "Guang Dilong" and 25 batches of proprietary Chinese medicines. Besides Amynthas aspergillum as the authentic source, 8 other Molecular Operational Taxonomic Units (MOTUs) were elucidated. Significantly, even the subgroups within A. aspergillum revealed here differ significantly on chemical compositions and biological activity. Fortunately, this biodiversity could be controlled when the collection was limited to designated areas, as proved by 2796 "decoction pieces" samples. This batch biological identification method should be introduced as a novel concept regarding natural medicine quality control, and to offer guidelines for in-situ conservation and breeding bases construction of wild natural medicine.

Application and progress in high-throughput sequencing technology for meat adulteration detection / 生物工程学报

Adulteration in meat products is a widespread issue that could lead to serious threats to public health and religious violations. Technology that offers rapid, sensitive, accurate and reliable detection of meat species is the key to an effectual monitoring and control against meat adulteration. In recent years, high-throughput sequencing-based DNA metabarcoding technology has developed rapidly. With the characteristics of being high-throughput, highly precise and high-speed, this technology can simultaneously identify multiple species in complex samples, thus offering pronounced advantages in the surveillance of adulteration in meat and meat products. Starting with an introduction of the major developments in the high-throughput sequencing technology in the past two decades, this review provides an overview of the technical characteristics and research methods of DNA metabarcoding, summarizes the application of DNA metabarcoding technology in meat adulteration detection over the last few years, discusses the challenges of using DNA metabarcoding technology in the detection of meat adulteration, and provides future prospects on the development of this technology.

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

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.

Advances in identification of traditional Chinese medicinal materials seeds using DNA barcoding technology / 中草药

Seeds are the source of the Chinese medicine industry. The accurate identification of seeds is not only related to the genuineness of Chinese medicinal materials, but also related to the safety and effectiveness of traditional Chinese medicine. Since a large proportion of Chinese medicinal material seeds are small in size, rich in inclusions, and their morphological characters are easy to be changed by the grade of maturity and environmental conditions, it is difficult to identify seeds through traditional methods. DNA barcoding is a novel genetic method designed for accurate species identification using a standard DNA locus, and it has gradually become a research hotspot for the seed authentication of Chinese medicinal materials. In this paper, we briefly summarized the recent research developments of the DNA barcoding identification for Chinese medicinal material seeds, demonstrate the technical characteristics of using DNA barcoding to authenticate the seeds of Chinese medicinal materials, and pointed out that different protocols should be employed according to the types of seeds. We further proposed a research method for the identification of commercially available Chinese medicinal materials seed mixtures using the DNA metabarcoding, which provides new ideas for the identification of Chinese medicinal material seeds.

Application of DNA metabarcoding technology in identification of Chinese patent medicines / 中国中药杂志

Metabarcoding technology is a research method derived from the combination of traditional DNA barcodes and highthroughput sequencing technologies. It can quickly,easily and efficiently identify and restore biological samples from multiple species.Biological species are currently widely used in environmental biology research. In the market of traditional Chinese medicines,adulteration and quality instability have severely restricted the sustainable development of the related industries. This article introduced the background of the metabarcoding technology and its preliminary application in the identification of Chinese patent medicines. It also outlined the possible problems in the research process and prospected to the development of the DNA metabarcoding technology.

Application of DNA metabarcoding in species identification of Chinese herbal medicines / 中国中药杂志

DNA metabarcoding,one rapid and robust method using specific standard DNA fragments,has been widely used for rapid species identification of a bulk sample through high-throughput sequencing technologies.While it has been widely used in the studies of metagenomics,animal and plant biodiversity,it has gradually come to be used as a profitable method in species identification of mixed Chinese herbal medicines.In this paper,we mainly summarize the current studies of the application of DNA metabarcoding in species identification of mixed Chinese herbal medicines.Moreover,high-throughput sequencing technologies adopted in those studies,such as Sanger,the next-generation,and third-generation sequencing technologies,are discussed.It is conducted to provide a theoretical guidance for the application of DNA metabarcoding in species identification of mixed Chinese herbal medicines and in more other biodiversity studies.

Biomonitoring for traditional herbal medicinal products using DNA metabarcoding and single molecule, real-time sequencing

Global concerns have been paid to the potential hazard of traditional herbal medicinal products (THMPs). Substandard and counterfeit THMPs, including traditional Chinese patent medicine, health foods, dietary supplements, etc. are potential threats to public health. Recent marketplace studies using DNA barcoding have determined that the current quality control methods are not sufficient for ensuring the presence of authentic herbal ingredients and detection of contaminants/adulterants. An efficient biomonitoring method for THMPs is of great needed. Herein, metabarcoding and single-molecule, real-time (SMRT) sequencing were used to detect the multiple ingredients in Jiuwei Qianghuo Wan (JWQHW), a classical herbal prescription widely used in China for the last 800 years. Reference experimental mixtures and commercial JWQHW products from the marketplace were used to confirm the method. Successful SMRT sequencing results recovered 5416 and 4342 circular-consensus sequencing (CCS) reads belonging to the ITS2 and regions. The results suggest that with the combination of metabarcoding and SMRT sequencing, it is repeatable, reliable, and sensitive enough to detect species in the THMPs, and the error in SMRT sequencing did not affect the ability to identify multiple prescribed species and several adulterants/contaminants. It has the potential for becoming a valuable tool for the biomonitoring of multi-ingredient THMPs.

Exploring the environmental diversity of kinetoplastid flagellates in the high-throughput DNA sequencing era

The class Kinetoplastea encompasses both free-living and parasitic species from a wide range of hosts. Several representatives of this group are responsible for severe human diseases and for economic losses in agriculture and livestock. While this group encompasses over 30 genera, most of the available information has been derived from the vertebrate pathogenic genera Leishmaniaand Trypanosoma. Recent studies of the previously neglected groups of Kinetoplastea indicated that the actual diversity is much higher than previously thought. This article discusses the known segment of kinetoplastid diversity and how gene-directed Sanger sequencing and next-generation sequencing methods can help to deepen our knowledge of these interesting protists.