双重实时荧光PCR快速鉴别人参和西洋参

Rapid Identification of Panax ginseng and P. quinquefolium by Duplex Real-time PCR

目的建立一种能够快速鉴定人参和西洋参的双重实时荧光PCR方法。方法通过对人参属及其近似种基因序列的分析比对,设计特异性的引物探针,建立双重实时荧光PCR检测方法。PCR反应体系为Premix Ex Taq(Probe qPCR)10μL,人参上下游引物各0.5μL,西洋参上下游引物各0.3μL,人参与西洋参特异性探针各0.4μL,DNA模板2μL,灭菌去离子水补至20μL。反应条件为95℃预变性30 s;然后以95℃变性5 s,60℃退火延伸30 s进行40个循环。应用该方法测试了27份DNA样品,包括7份人参、6份西洋参、4份人参与西洋参混合样、10份其他人参属样品及其他常见中药材样品的DNA,以确定该方法的特异性。将人参与西洋参样品DNA混合后10倍稀释成不同浓度后进行检测,用以确定该方法的灵敏度。结果人参及西洋参样品均在特定的荧光通道下出现典型的阳性扩增曲线,人参与西洋参混合样品均同时出现两条阳性扩增曲线,其它对照样品及空白对照均没有出现阳性扩增曲线。灵敏度检测结果显示该方法检测人参及西洋参的最低检测限均为2 pg DNA/反应。结论本实验建立的双重实时荧光PCR方法能够同时快速、准确、灵敏地鉴别出人参和西洋参。

Objective To established a duplex real-time fluorescence PCR method for rapid identification of Panax ginseng and P.quinquefolium.Methods A duplex real-time PCR method for identification of P.ginseng and P.quinquefolium was developed with specific primers and probe designed by analyzing and comparing the gene sequences of Panax sp.and their similar species.The reaction system was Premix Ex Taq(Probe qPCR)of 10μL,each of forward primer and reverse primer of P.ginseng of 0.5μL,each of forward primer and reverse primer of P.quinquefolium of0.3μL,probe of each species of 0.4μL,template DNA of 2μL,sterilization of deionized water of 5.6μL.The reactions were performed under the following conditions:95℃for 30 s,followed by 40 cycles of denaturation at 95℃for 5 s,annealing and extension at 60℃for 30 s.To detect the specificity of the probe and primers for duplex real-time PCR,a total of 27 DNA samples include 7 P.ginseng samples,6 P.quinquefolium samples,4 P.ginseng and P.quinquefolium mixed samples and 10 other samples were tested.The 10X diluted series templates DNA of P.ginseng and P.quinquefolium mixed samples were tested by the method for sensitivity analysis.Result Results showed that typical amplification curves could be obtained for all the P.ginseng,P.quinquefolium and the mixed samples,but not for the control samples and the blank control.Sensitivity of this method to detect P.ginseng and P.quinquefolium reached to2 pg DNA per reaction similarly.Conclusion The duplex real-time PCR method established in this study enables simultaneous,accurate and sensitive identification of P.ginseng and P.quinquefolium.