Objective: To establish a method for rapid identification of Liriopes Radix adulterated in Ophiopogon Radix medicinal materials and decoction pieces by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Methods: By comparison ITS sequences restriction site of Ophiopogonis Radix and Liriopes Radix, the specific restriction site of Liriopes Radix were selected, and the primers for PCR-RFLP reaction were designed. The different Ophiopogonis Radix and Liriopes Radix were amplified by PCR, PCR-RFLP reaction system annealing temperature, primer concentration, cycle number, enzyme digestion reaction time were and restriction endonuclease type optimized, and accuracy of the method were investigated. In addition, the PCR-RFLP method which had established was used to investigate the applicability of Ophiopogonis Radix, Liriopes Radix and adulterated sample from different sources. Results: A PCR-RFLP method for identifying Ophiopogonis Radix mixed with Liriopes Radix was established with the annealing temperature at 58 ℃ and the number of cycles of 30. Liriopes Radix and Liriopes Radix adulterated in Ophiopogonis Radix could be digested by PmlI restriction endonuclease after amplification with specific primers, the two single DNA bands were detected between 100-250 bp, which were not found in Ophiopogonis Radix. This method could be used to identify Ophiopogonis Radix, Liriopes Radix and the adulterated sample, and the minimum detection limit of this method for Liriopes Radix adulterated in Ophiopogonis Radix was 5%. Conclusion: The established PCR-RFLP method is specific and good stable, can be applied to the qualitatively identify Ophiopogonis Radix, Liriopes Radix and its adulterated sample, which can be used to establish supplem entary method.
[1] 中华人民共和国药典 2020年版. 一部[S].2020: 162
ChP 2020. Vol Ⅰ[S].2020: 162
[2] 张萍, 李明华, 石岩, 等. 2017年全国中药材及饮片质量概况[J].中国中药杂志, 2018, 43(21): 4198
ZHANG P, LI MH, SHI Y, et al. Overview of the quality of Chinese herbal medicines and decoction pieces in 2017[J].China J Chin Mater Med, 2018, 43(21): 4198
[3] 曹晖, 刘玉萍. 分子标记技术在中药种鉴别中的应用[J].中国药学杂志, 1998, 33(5): 269
CAO H, LIU YP. Application of molecular marker technology in identification of traditional Chinese medicine species[J].Chin Pharm J, 1998, 33(5): 269
[4] 袁媛, 蒋超, 黄璐琦. 中药材分子鉴别现场运用的策略与实践[J].中国中药杂志, 2013, 38(16): 2553
YUAN Y, JIANG C, HUANG LQ. Strategy and practice of field application of molecular identification of Chinese medicinal materials[J].China J Chin Mater Med, 2013, 38(16): 2553
[5] 陈士林, 吴问广, 王彩霞, 等. 药用植物分子遗传学研究[J].中国中药杂志, 2019, 44(12): 2421
CHEN SL, WU WG, WANG CX, et al. Study on molecular genetics of medicinal plants[J].China J Chin Mater Med, 2019, 44(12): 2421
[6] 张丹, 王颖莉, 杜晨晖, 等. 生物学技术在药用植物鉴定中的研究进展[J].中国实验方剂学杂志, 2021, 27(1): 214
ZHANG D, WANG YL, DU CH, et al. Research progress of biotechnology in identification of medicinal plants[J].Chin J Exp Tradit Med Form, 2021, 27(1): 214
[7] WANG CZ, LI P, DING JY, et al. Simultaneous identification of Bulbus Fritillariae Cirrhosae using PCR-RFLP analysis[J].Phytomedicine, 2007, 14(9): 628
[8] 郑传进, 生书晶, 赵树进. 基于trnL-trnF序列分析的何首乌PCR-RFLP分子鉴别[J].中药材, 2012, 35(4): 543
ZHENG CJ, SHENG SJ, ZHAO SJ. PCR-RFLP molecular identification of Polygonum multiflorum Thunb. based on trnL TRNF sequence analysis[J].J Chin Med Mater, 2012, 35(4): 543
[9] 杜明凤, 李明军, 陈庆富. 淫羊藿属植物PCR-RFLP遗传多样性研究[J].中草药, 2012, 43(3): 562
DU MF, LI MJ, CHEN QF. Genetic diversity of Epimedium by PCR-RFLP[J].Chin Tradit Herb Drugs, 2012, 43(3): 562
[10] 王翀, 张媛媛, 杨娟, 等. 绞股蓝属植物及其混淆品乌蔹莓的PCR-RFLP鉴别研究[J].中国中药杂志, 2008, 33(19): 2167
WANG C, ZHANG YY, YANG J, et al. Identification of Gynostemma pentaphyllum and its adulterant Cayratia japonica by PCR-RFLP[J].China J Chin Mater Med, 2008, 33(19): 2167
[11] 陈霞, 张永文, 周刚. 中药补充检验方法和检验项目相关问题探讨[J].中国新药杂志, 2021, 30(21): 1949
CHEN X, ZHANG YW, ZHOU G. Discussion on problems related to supplementary test methods and test items of traditional Chinese medicine[J].Chin J New Drugs, 2021, 30(21): 1949
[12] 史中飞, 赖晶, 宋平顺, 等. PCR-RFLP鉴别当归药材及饮片中掺混伪品——欧当归的方法[J].中国实验方剂学杂志, 2021, 27(9): 168
SHI ZF, LAI J, SONG PS, et al. Identification of Levisticum officinale Adulterated in Angelica sinensis by PCR-RFLP[J].Chin J Exp Tradit Med Form, 2021, 27(9): 168
[13] 赖晶, 史中飞, 宋平顺, 等. 基于ITS序列位点特异性PCR鉴别北柴胡掺伪藏柴胡[J].现代中药研究与实际, 2021, 35(2): 18
LAI J, SHI ZF, SONG PS, et al. Identification of Bupleurum chinense adulterated with Bupleurum chinense by site-specific PCR based on its sequence[J].Res Pract Chin Med, 2021, 35(2): 18
[14] 张文娟, 刘薇, 魏锋, 等. 聚合酶链式反应-限制性片段长度多态性法用于检定川贝母掺伪情况的研究[J].药物分析杂志, 2014, 34(10): 1830
ZHANG WJ, LIU W, WEI F, et al. Detection of adulteration in Fritillaria thunbergii by polymerase chain reaction restriction fragment length polymorphism[J].Chin J Pharm Anal, 2014, 34(10): 1830
[15] 韩凤梅, 李紫, 蔡敏, 等. 山麦冬的HPLC/ESI-MS特征指纹图谱研究[J].中草药, 2005, 36(9): 119
HAN FM, LI Z, CAI M, et al. HPLC/ESI-MS fingerprint of Radix Liriopes[J].Chin Tradit Herb Drugs, 2005, 36(9): 119
[16] 李敏, 茅学群, 黄龙妹, 等. 川麦冬聚合酶链式反应鉴定方法的研究[J].中国药学杂志, 2017, 52(11): 936
LI M, MAO XQ, HUANG LM, et al. Study on the identification method of Ophiopogon japonicus by polymerase chain reaction[J].Chin Pharm J, 2017, 52(11): 936
[17] 刘阳, 杨淑霞, 李敏惠, 等. 引物浓度与退火温度不当导致巢式PCR非特异性扩增[J].成都医学院学报, 2008, 3(2): 111
LIU Y, YANG SX, LI MH, et al. Improper primer concentration and annealing temperature lead to nonspecific amplification of nested PCR[J].J Chengdu Med Coll, 2008, 3(2): 111