采用多重PCR方法建立一种快速准确鉴别人参、三七和西洋参的检测体系,并将其用于市售样品的检测。利用筛选的人参、三七和西洋参特异的单核苷酸多态性位点设计引物,在引物3′末端进行锁核酸修饰和碱基错配以封闭末端,增强引物特异性。建立多重PCR检测体系,并对反应条件进行优化。对市售样品进行验证。建立的多重PCR检测体系,人参、三七和西洋参特异性条带分别为249、366、212 bp。在59 ℃的退火温度,35个循环,40 ng的DNA,人参、三七、西洋参引物量体积比为0.15 μL∶1.25 μL∶0.3 μL的反应条件最佳。对于不同参类产品进行检测,结果表明,粗加工产品的阳性率高于深加工产品。选取市场上不同来源的粗加工产品进行多重PCR检测阳性率达到80%以上。多重PCR检测体系灵敏度高,重复性好,可用于市售样品的检测。
This study aimed to establish a rapid and accurate detection system for identifying Panax ginseng, Panax notoginseng, and Panax quinquefolius by multiplex PCR and apply it to detect commercial samples. The selected single nucleotide polymorphism (SNP) sites specific to Panax ginseng, Panax notoginseng, and Panax quinquefolius were used to design primers, locked nucleic acid (LNA) modification and base mismatch were carried out at the 3' end of the primer to close the end and enhance the specificity of the primer. The multiplex PCR detection system was established and the reaction conditions were optimized. The commercial samples were verified. The multiplex PCR detection system was established, and the specific bands of ginseng, Panax ginseng, Panax notoginseng, and Panax quinquefolius were 249, 366, and 212 bp respectively. At the annealing temperature of 59 ℃, 35 cycles, 40 ng of DNA, and the primer ratio of Panax ginseng, Panax notoginsen, and Panax quinquefolius was 0.15 μL∶1.25 μL∶0.3 μL, the reaction conditions were the best. The detection of different reference products showed that the positive rate of rough-processed products was higher than that of deep-processed products. Rough products from different sources in the market were selected for multiplex PCR detection, and the positive rate reached over 80%. A multiplex PCR detection system for Panax ginseng, Panax notoginseng, and Panax quinquefolium was established, which could be used for the detection of commercial samples.
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