目的 為研究DNA條形碼技術(shù)在中成藥鑒定中的應(yīng)用，以三七片為研究對象，對方法的適用性、專屬性與精密度進行考察。方法 收集15批次市售三七片樣品，考察三七片DNA提取條件，并對“中藥材DNA條形碼分子鑒定法指導(dǎo)原則”中PCR擴增、序列獲得、結(jié)果判定等方法適用性進行確認；收集三七、人參、西洋參，制作三七片及其摻偽品，考察方法的專屬性和重現(xiàn)性。結(jié)果 三七片取樣量100 mg，56 ℃水浴8 h所獲得DNA的質(zhì)量濃度平均值為60.7 ng/μL，PCR擴增、序列獲得和結(jié)果判定均可獲成功；三七、人參和西洋參的ITS2序列長度均為230 bp，三七與人參、三七與西洋參的序列間均存在7個穩(wěn)定的SNP位點，自制三七片和摻偽三七片均可成功獲得ITS2序列，不同比例三七與人參、三七與西洋參的測序峰圖在SNP位點處呈現(xiàn)相應(yīng)峰高比的SNP套峰具備專屬性；重復(fù)性、中間精密度和重現(xiàn)性考察符合《中國藥典》2015年版（通則9101）相關(guān)要求。結(jié)論 ITS2序列作為DNA條形碼能夠穩(wěn)定、準(zhǔn)確鑒定三七片的原料藥材，具備良好的專屬性和精密度，三七片DNA條形碼分子鑒定法將為保障三七片臨床用藥安全提供新的技術(shù)手段，并對《中國藥典》其他收載單方制劑的鑒定提供參考。

Objective In order to study the application of DNA barcoding in the authentication of Chinese patent medicines, Sanqi Tablets were used as the object to investigate the applicability, specificity and precision of this method. Methods Fifteen batches of commercially available Sanqi Tablet samples were collected. The conditions of DNA extraction for Sanqi Tablet had been first investigated, and the DNA was used for testing the applicability of the methods such as PCR amplification, sequence acquisition, and species authentication in the principles for molecular identification of traditional Chinese materia medica using DNA barcoding. The specificity and reproducibility of DNA barcoding in identification of Sanqi Tablets and its adulterations from the roots of Panax notoginseng, P. ginseng and P. quinquefolius were also studied. Results The Sanqi Tablet sample with an amount of sampling to be 100 mg and a water bath at 56℃ for 8 h gave an average concentration of 60.7 ng/μL and then the PCR amplification, sequence acquisition and species assignment were all successful. The ITS2 sequences of P. notoginseng, P. ginseng and P. quinquefolius were all 230 bp in length, and there were seven stable SNP loci between P. notoginseng and P. ginseng, P. notoginseng and P. quinquefolius. ITS2 sequences could be successfully obtained from lab-made and the adulterated Sanqi Tablets, and the Sanger sequencing chromatograms of different ratios of P. notoginseng and P. ginseng mixtures, P. notoginseng and P. quinquefolius mixtures had heterozygous peaks with corresponding peak height ratio at SNP positions. The repeatability, intermediate precision and reproducibility were all in line with the requirements of "General Regulation 9101" in the Chinese Pharmacopoeia. Conclusion The ITS2 sequence can stably and accurately authenticate the raw materials of Sanqi Tablets with substantial specificity and precision. The DNA barcoding identification method of Sanqi Tablets will provide a new technical tool for ensuring the safety of Sanqi Tablets in clinical medications, and provide reference for the identification of other single-herb products documented in the Chinese Pharmacopoeia.

R282.12

國家自然科學(xué)基金項目（81703659）；國家自然科學(xué)基金項目（81503189）；中國博士后科學(xué)基金資助項目（2017M610815）；北京市自然科學(xué)基金資助項目（7202109）