The Application Of DNA Barcoding Combined With High Resoltion Melting To Detect Seed Medicinal Materials And Grain Sprout Herbs

Seed, as main reproductive organs of most of herbal plants, is also an important part of traditional Chinese medicine(TCM), and play an important role in the fight against disease. Due to its small volume and similar appearance, it is very difficult to accurately identify the constituent plant species only through naked eye. In parallel with increasing popularity and accessibility of medicinal products, there is also numerous adulterants and unknown original species in market. Resulting in conditions such as impacting on the safety and efficacy of clinical application and TCM. The correct identification of seed herbal medicine is, therefore, essential for their safe use. More recently, a universal,publically available DNA barcoding system for identifying herbal materials has been established, and has a great significant role for discriminating TCM and medicinal plants. Based on the internal transcribed spacer 2(ITS2) and psb A-trn H sequences, this study including 64 species of 51 seed herbal medicines, and 3 grain sprout herbs which had been included in The Chinese Pharmacopoeia(2015edition), as well as their commercial medicinal products. In addition,ITS2 DNA barcode combined with High Resolution Melting(HRM)technology was used to evaluate whether this technique was equally useful for species discrimination of seed medicine materials. The results were shown in following:1. DNA was extracted from seed medicines using the Plant Genomic DNA Extraction kit(Tiangen Biotech Co., China) in accordance with the manufacturer's instructions. PCR amplification was performed using the ITS2 and psb A-trn H universal primers. The Codon Code Aligner V4.2.5(Codon Code Co., USA) was used to proofread and assembled the contigs,and generated consensus sequences. All the sequences were submitted to DNA barcode database of TCM. Finally, seed medicine products collected from markets were detected and authenticated using the same method. The botanical origin was identified by Blast method based on DNA barcode database. Results showed that most of the seed medicinal products were correctly labeled, with only a few were identified as adulterants.2. The genomic DNA were extracted from grain sprout herbs, their ITS2 sequences were amplified, and purified PCR products were sequenced.Sequences were assembled using the Codon Code Aligner. The genetic distances, variable sites and the neighbor-joining(NJ) phylogenetic tree were computed by MEGA 6.0 in accordance with the Kimura2-parameter(K2P) model. The results showed that their intraspecific genetic distances were all much lower than the interspecific ones of themselves and their adulterants. The NJ tree based on ITS2 sequence indicated that Oryza sativa, Setaria italica, and Hordeum vulgar formed one monophyletic clade respectively, in addition any one of the grain sprouts herbs and its adulterants could be distinguished clearly. Therefore,ITS2 sequence was suitable to be as a barcode to identify the grain sprouts herbs and their adulterants.3. The DNA barcode ITS2 combined with High Resolution Melting analysis was used to detect toxic herbal seed including Hyoscyami Semen and its adulterants. The sensitivity of this method in adulterant detection was assessed by analyzing samples Hyoscyami Semen DNA mixed in Astragali Complanati Semen, and Armenicae Semen Amarum mixed in Persicae Semen. Results showed that DNA barcode combined High Resolution Melting(Bar-HRM) successfully identified the adulterants,and detected the proportion of Hyoscyamus niger DNA extract within an admixture, and could detected the proportion of Prunus persica and Prunus armeniaca. In particular, the sensitivity of HRM in detecting Hyoscyamus niger DNA extract in Astragalus complanatus DNA extract,and Prunus armeniaca in Prunus persica were as low as 1%, and presented a good linear relationship(former r=0.9758, later r=0.9685).The authentication of herbal species by Bar-HRM could provide an excellent inspection method for regulatory agencies and promote consumers trust, as well as raising the quality of herbal products.In conclusion, integrated DNA barcoding and High Resolution Melting technology provided an efficient and reliable authentication system to the seed medicinal materials and their adulterants. It also provided new technique to ensure clinical quality and safety in using traditional Chinese medicine.