Abstract
DNA barcoding has been used for decades, although it has mostly been applied to some single-species. Traditional Chinese medicine (TCM), which is mainly used in the form of combination-one type of the multi-species, identification is crucial for clinical usage. Next-generation Sequencing (NGS) has been used to address this authentication issue for the past few years, but conventional NGS technology is hampered in application due to its short sequencing reads and systematic errors. Here, a novel method, Full-length multi-barcoding (FLMB) via long-read sequencing, is employed for the identification of biological compositions in herbal compound formulas in adequate and well controlled studies. By directly sequencing the full-length amplicons of ITS2 and psbA-trnH through single-molecule real-time (SMRT) technology, the biological composition of a classical prescription Sheng-Mai-San (SMS) was analyzed. At the same time, clone-dependent Sanger sequencing was carried out as a parallel control. Further, another formula—Sanwei-Jili-San (SJS)—was analyzed with genes of ITS2 and CO1. All the ingredients in the samples of SMS and SJS were successfully authenticated at the species level, and 11 exogenous species were also checked, some of which were considered as common contaminations in these products. Methodology analysis demonstrated that this method was sensitive, accurate and reliable. FLMB, a superior but feasible approach for the identification of biological complex mixture, was established and elucidated, which shows perfect interpretation for DNA barcoding that could lead its application in multi-species mixtures.
Highlights
As DNA barcoding, a dominant method for species identification and discovery [1], emerges as a cost-effective standardized approach for rapid species identification [2,3], it has been widely used in almost all types of organisms
ITS2 and psbA-trnH regions of SMS and relevant independent raw materials were successfully amplified. Sanger sequencing of these PCR products demonstrated that all the raw materials were from correct original species
The raw materials of SJS was tested with ITS2 and CO1
Summary
As DNA barcoding, a dominant method for species identification and discovery [1], emerges as a cost-effective standardized approach for rapid species identification [2,3], it has been widely used in almost all types of organisms. For traditional Chinese medicine (TCM), whose identification is crucial for its safety and effectiveness [4,5,6,7] in clinical practice, various methods have been applied, such as microscopic analysis, chromatography, spectroscopic methodology and molecular biology. Especially DNA barcoding, are relatively more precise and sensitive in general [8,9,10]. The DNA barcoding system for identifying herbal medicine (TcmBarcode system, http://www.tcmbarcode.cn/en/) [11] has been successfully established and widely used [12].
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