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目的:建立快速检测中药材甘草的荧光定量PCR和环介导等温核酸扩增(Loop-mediated isothermal amplification,LAMP)两种技术,并对它们的特异性和灵敏性进行比较。方法:以中药材甘草作为实验对象,提取DNA后,用设计的PCR引物及LAMP引物组合分别对甘草进行普通PCR、荧光定量PCR以及LAMP实验,检测引物的特异性,同时建立模板DNA的浓度梯度,对荧光定量PCR及LAMP的灵敏性进行检测。结果:甘草的特异性序列可以通过荧光定量PCR得到有效扩增,而与甘草相近的混伪品结果显示阴性。荧光定量PCR最低可检测到甘草的浓度为0.67×10~(-4)ng/μL。在LAMP特异性检测中,甘草出现浊度值,其他混伪品显示阴性。灵敏度性检测中,LAMP检测在60 min反应时间内同样检测到的甘草的最低DNA浓度为0.67×10~(-4)ng/μL,反应产物加入荧光染料SYBR GreenⅠ后反应液呈现肉眼可观察的明显的亮绿色。结论:LAMP检测和real-time PCR检测具有相似的特异性、灵敏度和精确性,但LAMP检测方法更为简单、方便,所需时间更短,而且不需要昂贵的仪器。因此,LAMP检测中药的方法更适合于对中药材甘草检测的推广使用。
OBJECTIVE: To establish a rapid detection of two kinds of technologies of fluorescence quantitative PCR and loop-mediated isothermal amplification (LAMP) of licorice, and to compare their specificity and sensitivity. Methods: The Chinese herbal medicine Licorice was used as the experimental object. After the DNA was extracted, the PCR primers and LAMP primer combinations were used to detect the specificity of the primers, and the concentration gradient of the template DNA , The detection of fluorescence quantitative PCR and LAMP sensitivity. Results: The specific sequence of Glycyrrhiza glabra can be effectively amplified by fluorescence quantitative PCR, but the result of the mixture with licorice showed negative. Fluorescent quantitative PCR can detect the lowest concentration of licorice 0.67 × 10 ~ (-4) ng / μL. In LAMP-specific assays, the haze value appears for licorice and negative for other dummies. In the sensitivity test, the minimum DNA concentration of the same detected glycyrrhiza was 0.67 × 10 ~ (-4) ng / μL in the LAMP test within 60 min reaction time. After the reaction product was added with the SYBR Green Ⅰ fluorescent dye, the reaction solution was visually observable Obviously bright green. Conclusion: LAMP and real-time PCR assays have similar specificity, sensitivity and accuracy, but the LAMP assay is simpler and easier, requires less time, and does not require costly instrumentation. Therefore, the LAMP method is more suitable for the detection of traditional Chinese medicine licorice detection of the promotion of the use of.