三七染料法实时荧光聚合酶链式反应鉴别方法的建立

Establishment of a real-time fluorescent polymerase chain reaction method for identification of Panax notoginseng based on fluorescent dye

中文关键词:  三七  实时荧光聚合酶链式反应  染料法  鉴别

英文关键词:Panax notoginseng  real-time fluorescent polymerase chain reaction  dye method  identification

中文摘要:

      目的 采用TB Green染料法实时荧光聚合酶链式反应(polymerase chain reaction, PCR)技术, 基于叶绿体基因psbA-trnH序列建立鉴别三七的实时荧光PCR方法。方法 通过比较三七及其同属的人参、西洋参和竹节参的psbA-trnH序列, 设计出一对三七特异性引物, 优化退火温度, 进行灵敏度实验, 并验证方法的特异性、适用性和抗干扰性, 最后对市售三七样品进行检测。同时以18S rDNA引物为内参质控, 对DNA提取及反应体系进行质控。结果 所建立的三七染料法实时荧光PCR方法灵敏度为0.001 ng/μL; 特异性较好, 与18种近缘及常见中药材均无交叉反应; 适用性和抗干扰性也较好; 对28份不同加工方式的市售三七样品检测的结果与测序结果一致。结论 建立的三七染料法实时荧光PCR方法具有快速、准确、灵敏的特点, 可用于三七产品中三七成分的鉴别。

英文摘要:

      Objective To establish a real-time fluorescent polymerase chain reaction (PCR) method for the identification of Panax notoginseng based on the psbA-trnH sequence of chloroplast gene using fluorescent PCR technique by TB Green dye method. Methods By comparing the psbA-trnH sequences of Panax notoginseng and Panax ginseng, Panax quinquefolius and Panax japonicus, a pair of Panax notoginseng-specific primers was designed, the annealing temperature was optimized, sensitivity experiments were performed, and the specificity, applicability and anti-interference of the method were verified, and finally, commercial samples of Panax notoginseng were detected. The 18S rDNA primer was used as the internal reference for quality control of DNA extraction and reaction system. Results The sensitivity of the established real-time fluorescence PCR method with Panax notoginseng dye method was 0.001 ng/μL; the specificity was good, and there was no cross-reactivity with 18 kinds of closely related and common Chinese herbal medicinal materials; the applicability and anti-interference were also good; the results of the detection of 28 commercially available Panax notoginseng samples with different processing methods were consistent with the sequencing results. Conclusion The established real-time fluorescence PCR method of Panax notoginseng dye method is rapid, accurate and sensitive, and can be used to identify Panax notoginseng components in Panax notoginseng products.