Study Of TCM Nucleotide Signature Based On Mini-barcoding

Herbal products(HPs)are important material basis of the development of Traditional Chinese Medicine industrial chain.A series of processing technology is necessary for the preparation molding of HPs,which leads to the morphological character deficiency,chemical profile change and serious DNA degradation of raw herbal materials.Hence,traditional identification methods at morphological and chemical level or commonly-used molecular methods are sometimes difficult to be applied in the identification of raw materials in processed HPs.Due to the lack of efficient and accurate identification method;there is a very serious adulteration problem in HPs,which dramatically impede the further development of TCM industry.So it is necessary to develop an efficient molecular method for the establishment of a standard quality control system for HPs.Based on Mini-barcoding technique,we initially proposed the conception of TCM nucleotide signature.It is an extremely short(20-50bp)and species-specific DNA sequence.By shortening amplification length of targeted sequences,the nucleotide signature methods significantly improve the identification efficiency and can rapidly identify species of TCM in HPs.The main research contents are as follows:(1)Firstly,database for screen of the nucleotide signature sequences was built based on DNA barcode sequences of targeted species.After sequences alignment,short and species-specific sequences were searched based on the SNP sites.(2)Secondly,genome DNA was extracted from HPs samples using optimized DNA extraction methods.After the design of specific primers,short sequences containing the nucleotide signature were amplified using PCR or RPA technique.(3)Finally,different downstream detection methods,like agarose gel electrophoresis,pyrosequencing and lateral flow stripes,were used to detect amplification results.And the identification results of HPs were obtained.In this study,we established the nucleotide signature for Panax quinquefolius,Ophiocordyceps sinensis,and Ginkgo biloba.Using this method,we succeeded in identifying these species within decoctions,Chinese Patent Medicines and diet supplementary.According to the experiment results of P.quinquefolius and O.sinensis decoctions,it was concluded that serious DNA degradation indeed happened in processed herbal materials.And the nucleotide signature method could overcome the DNA degradation problem.According to the identification results of Chinese patent medicines containing P.quinquefolius and herbal health products containing G.biloba,the high adulteration rate was found.In the detection results,5 samples and 2 samples of 24 different batches of P.ginseng Chinese patent medicines were respectively replaced and adulterated by unlisted P.quinquefolius;9 samples of 28 different batches of G.biloba HPs were adulterated with unlisted S.japonica ingredients.O.sinensis and its five common adulterates can be identified using PCR methods with species-specific primers.This study established nucleotide signature for P.quinquefolius,O.sinensis,and G.biloba.Combined with different sorts of downstream detection technique,this method can realize the rapid,accurate efficient and low-cost identification method for herbal HPs.It has been proved that the nucleotide signature method can be developed as one fast on-site identification technique to detect the key ingredients and unlisted fillers in the raw materials of HPs and further help complete the safety control system of HPs.