利用多重连接探针扩增结合熔解曲线法鉴别人参与西洋参中掺杂的研究

Identification of mutual adulteration of Panax ginseng and Panax quinquefolius by multiplex ligation-dependent probe amplification combined with melting curve method

目的:针对人参和西洋参饮片中相互掺杂问题建立快速检测鉴别方法。方法:依据多重连接探针扩增(multiplex ligation-dependent probe amplification, MLPA)技术检测序列单核苷酸多态性(SNP)和点突变的原理,针对人参与西洋参的18S序列寻找SNP位点;设计特异性探针进行MLPA扩增,并由熔解曲线形成的熔解曲线峰得出熔解温度(melting temperature,T_(m)),根据T_(m)值的差异来鉴别人参与西洋参,并进行特异性、灵敏度、掺伪比实验,验证方法的可行性。结果:人参探针的特异性熔解曲线T值为(79.3±0.2)℃,西洋参探针的熔解曲线T_(m)值为(81.2±0.2)℃。当西洋参探针与人参探针比例为0.6∶1.0时,人参掺杂10%的西洋参,或者西洋参中掺杂10%的人参均可被有效检出。结论:本研究建立了稳定的人参与西洋参掺伪鉴别方法,该方法具有特异性强,灵敏度高的特点。

Objective: To establish a rapid detection and identification method for mutual adulteration in prepared slices of Panax ginseng and Panax quinquefolius. Methods: Based on the principle of using multiplex ligation-dependent probe amplification(MLPA) to detect single nucleotide polymorphism(SNP) and point mutation in sequences, a specific probe was designed to find SNP sites in the 18 S sequences of Panax ginseng and Panax quinquefolius for MLPA amplification. The melting temperature(T_(m)) was derived from the peak of the melting curve, and the differences in T_(m) values were used to identify Panax ginseng and Panax quinquefolius. Specificity, sensitivity and adulteration ratio experiments were performed to verify the feasibility of the method. Results: The specific melting curve Tm value of Panax ginseng probe was(79.3±0.2)℃, and that of Panax quinquefolius probe was(81.2±0.2)℃. When the ratio of Panax quinquefolius probe to Panax ginseng probe was 0.6∶1.0.Panax ginseng adulterated with 10% Panax quinquefolius, or Panax quinquefolius adulterated with 10% ginseng could be effectively detected. Conclusion: This study establishesa stable method for the identification of Panax ginseng adulterated with Panax quinquefolius, which is specific and sensitive.