基于环介导等温扩增技术快速检测疣孢褐盘菌

Rapid detection of Peziza badia based on loop-mediated isothermal amplification method

目的基于环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)建立一种常见的有毒蘑菇(疣孢褐盘菌Peziza badia)的快速检测方法。方法使用疣孢褐盘菌的内部转录间隔区(internal transcribed spacer,ITS)作为目的基因设计特异性的LAMP引物,使用可视化法和实时荧光法分别对LAMP扩增结果进行判断,可视化法通过颜色即可判断结果,实时荧光法观察是否有扩增曲线,两种技术相互验证。结果本研究设计的引物特异性强,与其他阴性样品无交叉反应,可视化法和实时荧光法的检测结果一致。本方法的检出限为0.8 ng/μL;将阴性和阳性样品按比例混合,并经过水煮和人工胃液消化后提取DNA,依然能检测到含量为1%的阳性样品。结论本方法对仪器要求低,检测速度快,操作和判断结果简单,有利于方法的传播,可用于疣孢褐盘菌的快速检测。

Objective To establish a rapid method for the detection of common poisonous mushroom(Peziza badia) by loop-mediated isothermal amplification(LAMP). Methods The internal transcribed spacer(ITS) of Peziza badia was used as the target gene to design specific LAMP primers. The LAMP amplification result was judged by visual method and real-time fluorescence method, and the visual method determined the result by color.Real-time fluorescence was used to observe whether there was an amplification curve, and the 2 kinds of techniques verified each other. Results The designed Peziza badia primers showed good specificity and no cross-reaction with other negative samples. The detection results of visualization method and real-time fluorescence method were consistent.The limit of detection of this method was 0.8 ng/μL. By mixing the negative and positive samples in proportion and extracting DNA after boiling and artificial gastric juice digestion, 1% positive samples could still be detected.Conclusion This method has low requirements for instruments, fast detection speed, simple operation and judgment results, which is conducive to the spread of the method, and can be used for the rapid detection of Peziza badia.