基于LNA-TaqMan探针实时荧光定量PCR检测技术的药用黄精掺伪研究  

Adulteration Detection of Polygonati Rhizoma Based on Real-time Fluorescence Quantitative PCR Technology with LNA-TaqMan Probe

作　　者：王多梅 胡冲 蒲婧哲 陈灵丽 杨建波 张亚中[1,2] 张文娟 WANG Duo-mei;HU Chong;PU Jing-zhe;CHEN Ling-li;YANG Jian-bo;ZHANG Ya-zhong;ZHANG Wen-juan(Anhui Institute of Food and Drug Control,Hefei 230051,China;NMPA Key Laboratory for Quality Research and Evaluation of Traditional Chinese Medicine,Hefei 230051,China;National Institutes for Food and Drug Control,Beijing 102629,China)

机构地区：[1]安徽省食品药品检验研究院药品检验研究所,安徽合肥230051 [2]国家药品监督管理局中药质量研究与评价重点实验室,安徽合肥230051 [3]中国食品药品检定研究院,北京102629

出　　处：《中国现代中药》2024年第3期457-462,共6页Modern Chinese Medicine

基　　金：安徽省药品监督管理局监管科学研究重点项目(AHYJ-KJ-202209);常见与重要中药材及饮片DNA分子鉴定研究项目(TCM2021-YJ07)。

摘　　要：目的:建立一种快速、灵敏、有效的实时荧光定量聚合酶链式反应(PCR)方法,用于检测湖北黄精掺伪药用黄精。方法:基于锁核酸(LNA)-TaqMan探针实时荧光定量PCR技术,利用不同基原药用黄精样品的叶绿体DNA中trnC-petN基因序列差异,根据常见混伪品湖北黄精特异性差异位点设计筛选探针引物,并对引物及LNA-TaqMan探针的特异性进行验证。根据扩增曲线临界循环数(Ct)值的差值计算湖北黄精掺伪比例。结果:基于LNA-TaqMan探针能够特异性地检测出湖北黄精并确定掺伪比例,在湖北黄精掺伪1%时,仍可稳定检出。结论:该方法简便准确、稳定可靠,可以用于药用黄精掺伪湖北黄精定量检测。Objective:A rapid,sensitive,and efficient real-time polymerase chain reaction(PCR)approach was developed in this work in order to detect the adulteration of Polygonatum zanlanscianense Pamp with Polygonati Rhizoma.Methods:Based on Locked Nucleic Acid(LNA)-TaqMan probe real-time fluorescence quantitative PCR technology,the sequence differences of the trnC-petN gene of chloroplast DNA from various original samples of Polygonati Rhizoma was employed in this study to designe and screen specific primers and probes of specific differential sites for common adulterants.The specificity of the primers and LNA-TaqMan probes(Locked nucleic acid probes)were validated.The adulteration ratio of P.zanlanscianense with Polygonati Rhizoma was calculated according to the difference in Ct values of the amplification curve.Results:The results indicated that the real-time fluorescence quantitative PCR technology with LNA-TaqMan probe detection method can specifically detect the adulteration of P.zanlanscianense with Polygonati Rhizoma and determine the adulteration ratio.Stable detection was achieved even at 1%adulteration level.Conclusion:The method is simple,accurate,reproducible and stable,and can be used for the quantitative detection of Polygonati Rhizoma adulterated with P.zanlanscianense.

关 键 词：湖北黄精 锁核酸-TaqMan探针 实时荧光定量聚合酶链式反应 掺伪 鉴定 

分 类 号：R252.5[医药卫生—中医内科学]