环介导等温扩增法鉴定检测冬虫夏草  

Identification and detection of Cordyceps sinensis with LAMP

作　　者：李奎[1] 李琦[1] 袁媛[2] 陈江源[1] 黄璐琦[2] 刘志国[1] 

机构地区：[1]武汉工业学院生物与制药工程学院,湖北武汉430023 [2]中国中医科学院中药研究所,北京100700

出　　处：《中草药》2011年第8期1605-1608,共4页Chinese Traditional and Herbal Drugs

基　　金：湖北省楚天学者计划项目;武汉市科技局对外科技合作与交流计划项目(201070934341)

摘　　要：目的建立冬虫夏草的环介导等温扩增(loop-mediated isothermal amplification,LAMP)方法,实现对冬虫夏草的快速检测。方法针对冬虫夏草的CS2 serine protease(csp2)基因设计LAMP内外引物对;利用CTAB法提取冬虫夏草DNA;优化扩增反应条件;采用包括冬虫夏草在内的6种不同虫草进行LAMP扩增,并对阳性产物酶切鉴定以检测其特异性,通过对模板DNA的10倍梯度稀释检测其灵敏度;紫外灯下观察凝胶电泳或加入荧光染料SYBR Green I的扩增产物。结果设计的LAMP引物可以特异地针对冬虫夏草的csp2基因进行LAMP扩增,产物可被限制性内切酶Taq1酶切,且有较高的灵敏度,检出限达6 pg/mL。结论 LAMP法可以实现对冬虫夏草的快速鉴定检测,在中药鉴定中有着广阔的应用前景。Objective The method of loop-mediated isothermal amplification （LAMP） was employed to detect and identify Cordyceps sinensis rapidly. Methods Specific LAMP primers were designed according to the CS2 serine protease （csp2） gene of Cordyceps sinensis. C. sinensis DNA was extracted using CTAB method. The reaction conditions of LAMP were optimized. Specificity of LAMP reaction was validated by six different strains and using restriction enzyme Taql digested the LAMP products. Sensitivity of LAMP was tested with diluted C. sinensis solution with 10-fold gradient. LAMP products were shown by gel electrophoresis or adding SYBR Green I. Results The method of LAMP for detecting C. sinensis was effective and specific. The detection limit of LAMP assay was up to 6 pg/mL. Conclusion LAMP protocol is a promising method for the identification and detection of C. sinensis and Chinese materia mediea as well.

关 键 词：冬虫夏草 环介导等温扩增 鉴定 检测 检出限 

分 类 号：R282.5[医药卫生—中药学]