[关键词]
[摘要]
目的 为研究DNA条形码技术在中成药鉴定中的应用,以三七片为研究对象,对方法的适用性、专属性与精密度进行考察。方法 收集15批次市售三七片样品,考察三七片DNA提取条件,并对“中药材DNA条形码分子鉴定法指导原则”中PCR扩增、序列获得、结果判定等方法适用性进行确认;收集三七、人参、西洋参,制作三七片及其掺伪品,考察方法的专属性和重现性。结果 三七片取样量100 mg,56 ℃水浴8 h所获得DNA的质量浓度平均值为60.7 ng/μL,PCR扩增、序列获得和结果判定均可获成功;三七、人参和西洋参的ITS2序列长度均为230 bp,三七与人参、三七与西洋参的序列间均存在7个稳定的SNP位点,自制三七片和掺伪三七片均可成功获得ITS2序列,不同比例三七与人参、三七与西洋参的测序峰图在SNP位点处呈现相应峰高比的SNP套峰具备专属性;重复性、中间精密度和重现性考察符合《中国药典》2015年版(通则9101)相关要求。结论 ITS2序列作为DNA条形码能够稳定、准确鉴定三七片的原料药材,具备良好的专属性和精密度,三七片DNA条形码分子鉴定法将为保障三七片临床用药安全提供新的技术手段,并对《中国药典》其他收载单方制剂的鉴定提供参考。
[Key word]
[Abstract]
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.
[中图分类号]
R282.12
[基金项目]
国家自然科学基金项目(81703659);国家自然科学基金项目(81503189);中国博士后科学基金资助项目(2017M610815);北京市自然科学基金资助项目(7202109)