乌梢蛇实时荧光定量PCR法的建立及与普通PCR鉴别方法的比较研究

The establishment of real-time fluorescent quantitative PCR method for Zaocys dhumnades and its comparison with the common PCR identification method

目的为了进一步优化乌梢蛇药材鉴别的准确性,提高鉴别速度。方法基于CO1片段序列特征,该研究设计了乌梢蛇的特异性引物、探针,通过条件的优化建立了乌梢蛇的实时荧光定量PCR快速鉴别方法。结果利用该方法对正品及伪品15个物种30份样本进行了验证,结果全部8份乌梢蛇正品均能从30个药材样本中准确地鉴别出来。结论该方法与药典方法比较,准确性一致,但所用设备少、检测所需要时间短,仅需40 min至1 h即可得出结果,而药典方法需要4 h左右;灵敏度高,检测限可达到1×10^-3 ng/反应,检测结果在0.001%~100%范围内具有良好的线性关系,相关系数R^2=1,而药典方法仅为1×10^-2 ng/反应。该方法具有特异性强、检测速度快、灵敏度高、所需设备少的特点,适用于乌梢蛇中药材及其饮片真伪快速检测。

Objective In order to further optimize the accuracy and speed of identification of Zaocys dhumnades.Methods In this study,Zaocys dhumnades′specific primers and probes were designed according to the characteristics of CO1 fragment sequence.A real-time quantitative PCR method was established identification for identify Zaocys dhumnade quickly.Results This method was used to verified 30 genuine and pseudo-goods samples coming from 15 species and all the 8 pieces of genuine Zaocys dhumnades samples can correctly distinguished from 30 samples.Conclusion The accuracy of this method was consistent with that of pharmacopoeia.Compared with the pharmacopoeia method,this method needed less devices and only took 40 min to 1 h to get the result,while the pharmacopoeia method spends about 4 hours.Results showed that this method possess very good sensitivity,very good linear relationship.The detection limit can reached 1×10^-3 ng/copies,and the detection result was in the range of 0.001%~100%with correlation coefficient R 2=1;While the pharmacopoeia method was only 1×10^-2 ng/copies.This method had the characteristic of strong specificity,fast detection speed,high sensitivity and less equipment,so it was suitable for the rapid detection of the authenticity of Zaocys dhumnades and its pieces.