A CRISPR/Cas12a based portable biosensor (Cas12a-PB) was developed to simultaneously visually detect CaMV35S promoter and Lectin gene from genetically modified (GM) soybean powders (Roundup Ready^@). The Cas12a-PB, mainly made of polymethylmethacrylate (PMMA) and PMMA tape, has a connection structure, three channels and three detection chambers. The CRISPR/Cas12a detection reagents were preloaded in detection chambers and the reaction tube was connected to the connection structure by screw threads. After amplification, the amplicons were gone into three detection chambers by swinging the Cas12a-PB to conduct dual detection. Positive samples would produce green fluorescence while negative samples were black under the irradiation of 490 nm LED light. In this study, the Cas12a-PB successively combined with ordinary PCR, rapid PCR and loop-mediated isothermal amplification (LAMP) to achieve dual detection, which made detection process more convenient and portable. As low as 0.1% transgenic ingredients in soybean powders could be detected and the specificity of Cas12a-PB was confirmed with GM maize powders (MON810, GA21), GM soybean powders (DP305423), non-GM peanut and rice as targets. In the end, an amplification chamber combining with Cas12a-PB on a PMMA chip was further designed to eliminate the use of reaction tube and mineral oil, which made operation simpler. The established Cas12a-PB would provide a new reliable solution for multiple targets detection in clinic diagnostics, food safety, etc.

开发了一种基于 CRISPR/Cas12a 的便携式生物传感器 (Cas12a-PB)，用于同时视觉检测转基因 (GM) 大豆粉 (Roundup Ready ^@ ) 中的CaMV35S启动子和凝集素基因。 Cas12a-PB主要由聚甲基丙烯酸甲酯（PMMA）和PMMA胶带制成，具有连接结构、三个通道和三个检测室。检测室中预装CRISPR/Cas12a检测试剂，反应管通过螺纹与连接结构连接。扩增后，通过摇动Cas12a-PB将扩增子分入三个检测室进行双重检测。在490 nm LED灯照射下，阳性样品会产生绿色荧光，而阴性样品则呈黑色。本研究中，Cas12a-PB先后与普通PCR、快速PCR和环介导等温扩增（LAMP）结合，实现双重检测，使检测过程更加方便、便携。以转基因玉米粉（MON810、GA21）、转基因大豆粉（DP305423）、非转基因花生和大米为靶标，可以检测到大豆粉中低至0.1%的转基因成分，并证实了Cas12a-PB的特异性。最终，进一步设计了在PMMA芯片上结合Cas12a-PB的扩增室，消除了反应管和矿物油的使用，使操作更加简单。建立的Cas12a-PB将为临床诊断、食品安全等领域的多目标检测提供新的可靠解决方案。