基于环介导等温扩增技术快速鉴定宽体金线蛭

Rapid authentication of Whitmania Pigra Whitman based on loop-mediated isothermal amplification technology

关键词:  宽体金线蛭  环介导等温扩增技术  分子鉴定  快速检测

Key words:  Whitmania Pigra Whitman  loop-mediated isothermal amplification technology  molecular identification  rapid authentication

摘要:

[目的] 建立中药材宽体金线蛭的环介导等温扩增（LAMP）检测技术，实现对宽体金线蛭及其常见混伪品的快速检测。[方法] 针对宽体金线蛭线粒体细胞色素氧化酶I（COI）的基因序列设计LAMP引物。在64℃恒温条件下，用设计的LAMP引物组对水蛭进行LAMP扩增。[结果] LAMP反应结束后，宽体金线蛭均扩增出梯状条带，对照组无条带产生。加入0.2 μL荧光染料10 000×SYBR Green Ⅰ后，宽体金线蛭反应液在自然光下呈绿色（阳性），对照组呈橙色（阴性）。将加入了0.2μL 10 000×SYBR Green Ⅰ的扩增产物置于紫外灯下观察，结果宽体金线蛭在紫外光下呈绿色荧光（阳性），对照组无绿色荧光产生（阴性）。[结论] 利用LAMP技术可实现宽体金线蛭及其常见混伪品的快速鉴别，为水蛭中药材的现场鉴别提供技术支撑。

Abstract:

[Objective] We aim to establish a loop-mediated isothermal amplification(LAMP) technique for the rapid differentiate of Whitmania Pigra Whitman from other closely related species.[Methods] We designed LAMP primers based on the mitochondrial cytochrome c oxidase I(COI) gene sequences of Whitmania Pigra Whitman. Subsequently, LAMP amplification of leech was performed at a constant temperature of 64℃ using the newly designed LAMP primer sets.[Results] After the reaction, ladder-like bands were observed in positive samples by electrophoresis, whereas control groups are produced without bands. After adding 0.2 μL 10 000×SYBR GreenⅠdye, the positive samples changed color from orange to green under both ultraviolet and natural light, while the control groups remained orange.[Conclusion] LAMP technology can be used for the rapid authentication of Whitmania Pigra Whitman, and provides technical support for the on-site identification of leeches in herbal market.