摘要
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 the simple and rapid examination system for the detection of Glycyrrhiza uralensis. Two detection methods,real-time PCR and LAMP, were developed for Glycyrrhiza uralensis detection, and then their specificity and sensitivity were compared. Methods：Extract the DNA of Glycyrrhiza uralensis and then amplify the special periods with designed primers. Process the re- al-time PCR and LAMP experiments to test the specificity of primers by the contrast of other species which were similar to Glycyrrhiza uralensis. Dilute the template concentration from 0. 67 × 10 ng/μL to 0. 67 × 10^-5 ng/μL to make a contrast of the sensitivity between real-time PCR and LAMP. Results：Real-time PCR could amplify the special periods of Glycyrrhiza uralensis while other similar species was negative. The lowest concentration which real-time PCR could detect was 0. 67 × 10^-4 ng/μL. Similarly,the LAMP could detect the lowest concentration which was 0. 67× 10^-4 ng/p,L in 60 min which show the same sensitivity to the real-time PCR. Besides, the Lamp showed the specificity because other similar species couldn＇t amplify except Glycyrrhiza uralensis. It＇s obvious to observe green when put SYBR Green I into the LAMP reaction product. Conclusion：The real-time PCR and the LAMP are all sensitive, accurate and specific in the detection However, the LAMP is more convenient and cost less time. What＇s more, the expensive equipment is not necessary for the LAMP. In a word, the LAMP is a better choice for the detection of Glycyrrhiza uralensis.
出处
《中药材》
CAS
CSCD
北大核心
2016年第12期2725-2729,共5页
Journal of Chinese Medicinal Materials
基金
教育部新世纪优秀人才项目(NCET-11-0842)
中央民族大学学术团队建设项目(2015MDTD25C
2015MDTD13C)
筹推进一流大学和一流学科建设经费(10301-0150200604)
国家质检总局科技计划项目(2013IK291)
