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

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.