Identification Of Chinese Patent Medicines Based On DNA Barcode:from Mini-barcode To Meta-barcode

The quality and safety of Chinese patent medicines are the basis of the development of traditional Chinese medicines(TCMs).In the light of Chinese Pharmacopeia,the traditional detection methods for CPMs comprise micro-examination and chemical measurement.There are many kinds of CPMs with complex and diverse manufacturing techniques.Traditional identification methods cannot fully conduct qualitative analysis on the ingredients of CPMs.Therefore,CPMs can be easily adulterated or counterfeited by illegal manufacturers,and it will exert bad influences on consumers safety and society.The research is exampled by three types of CPMs and starts from multi-molecular diagnostic on the foundation of DNA mini-barcode and meta-barcode.This work is designed to focus on the CPMs quality control,associating with comparison of different sequencing techniques and data analysis.It can contribute to the establishment of standard methodology for CPMs quality assurance.Methodology:1.Firstly,we improved the DNA extraction methods to obtain high quality DNA from CPMs.2.Secondly,we established the database of DNA barcode from medicinal plants,screening high conservative regions or Single Nucleotide Polymorphisms(SNP)sites through MEGA and design specific primer pairs to amplify the target DNA mini-barcode,then identified the results through electrophoresis and sanger sequencing.3.Thirdly,we combined clone analysis with next generation sequencing technology Meanwhile,the bioinformatics will be used for meta-barcode analysis in single base pair specifically.Results:1.A unique nucleotide signature from ITS2 region of Eucommiae Folium and stable SNPs from Lonicera species were developed based on 76 original plants.Species primer pairs were designed to amplify the DNA mini-barcode.Mixtures of powdered Lonicerae japonicae Flos and Lonicerae Flos resulted in double peaks at the expected SNP positions,of which the peak height were roughly indicative of the species’ ratio in the mixed powder.In the identification of 47 CPMs we found out that 78%were adulterated with Eucommiae Folium or Lonicera Flos,which proved DNA mini-barcode can be effective for CPMs identification.2.20 CPMs of Fritillariae cirrhosae were selected for cloning experiments,and three batches were also analyzed using next generation sequencing(NGS)and meta-barcoding data were obtained.In the detection results of 20 CPMs,unlisted species such as Fritillariae pallidiflorae and Fritillariae ussuriensis were contained and merely 2 batches of CPMs had Fritillariae cirrhosae.3.NGS were used to investigate the complicated composition of Renshenjianpi pills(RSJP).11 herbal materials from RSJP’s prescription were used to establish a control model and sequencing results were analyzed.The bioinformatics depth analysis based on single base pair corresponding from meta-barcode can successfully identified 9/11 species from the prescription and filter out the related species.This work can provide a ameliorating methodology for CPMs’ quality control based on the DNA mini-barcode and meta-barcode.It will widen the range of the traditional identification methods and let the identification more accurate.