中药地龙中参环毛蚓环介导等温扩增快速检测方法

Rapid identification of Pheretima aspergillum in earthworms using loop-mediated isothermal amplification technology

关键词:  参环毛蚓  环介导等温扩增技术  分子鉴定  快速检测

Key words:  Pheretima aspergillum  loop-mediated isothermal amplification  molecular identification  rapid authentication

摘要:

[目的] 建立中药材地龙中参环毛蚓的环介导等温扩增（LAMP）检测技术，实现对参环毛蚓的快速检测。[方法] 采集不同产地地龙，提取总DNA后将所提取的总DNA进行线粒体细胞色素氧化酶Ⅰ亚基（COI）基因片段扩增、测序、比对，判断各产地地龙物种。用设计的LAMP引物组进行地龙LAMP实验，反应结束后向扩增产物中加入核酸染料SYBR GreenⅠ，分别在自然光、紫外光下直接通过肉眼来观察颜色变化及利用琼脂糖凝胶电泳来检测其引物的特异性。[结果] 利用新设计的LAMP引物对参环毛蚓实现了特异性扩增，且检出限达40 fg/μL，具有极高的灵敏度。[结论] 利用LAMP技术可实现参环毛蚓的现场快速检测。

Abstract:

[Objective] Establishing the technology of loop-mediated isothermal amplification (LAMP) to realize the rapid detection of Pheretima aspergillum in earthworms.[Methods] We collected earthworms in different areas and extracted DNA of these specimens.Subsequently,PCR amplification,sequencing and alignment were performed based on the mitochondrial cytochrome c oxidase subunit I (COI) genes to identify thespecies of samples. We performed LAMP experiment using the newly designed primers. After reaction,the SYBR Green I dye was added in amplification product,and the specificity of primers were detected by observing the color changes under natural and ultraviolet light respectively,or analyzed by agarose gel electrophoresis.[Results] Pheretima aspergillum were successfully specific amplified using the newly designed LAMP primer setsand the detection limitup to 40 fg/μL,with highly sensitivity.[Conclusion] LAMP technology can be utilized to realize the field rapid detection of Pheretima aspergillum.