DNA-based one-pot rapid biosensing platform integrating turn-back loop primer-accelerated LAMP and Pyrococcus furiosus Argonaute for meat visual authentication.

内容摘要

Meat adulteration is a significant global food safety challenge, creating a pressing need for rapid and on-site detection technologies. Herein, we present an intelligent one-pot biosensing platform termed one-pot TLAMP-PfAgo assay (OTPA) that integrates the rapid amplification of turn-back loop primer-accelerated loop-mediated isothermal amplification (LAMP) (TLAMP) with the sequence-specific detection of Pyrococcus furiosus Argonaute (PfAgo). This system features a clever heat-activatable design using microcrystalline wax to spatially separate reactions within a single tube, enabling contamination-free and streamlined operation. The OTPA assay achieves sensitive and specific detection, with limits of detection as low as 3×10-4 ng/μL for pork DNA and 2×10-4 ng/μL for beef DNA within 30 min. It successfully enables duplex target identification and has been validated with commercial meat products, showing perfect concordance with standard polymerase chain reaction (PCR)-based qualitative detection. Notably, the result can be directly visualized under blue light, underscoring the substantial potential of this cost-effective and simple platform for point-of-care testing (POCT) and intelligent biosensing in food safety surveillance. 肉类掺假是全球食品安全面临的一项重大挑战，因而迫切需要开发快速的现场检测技术。在此，本研究提出了一种名为OTPA（一锅法TLAMP-PfAgo分析）的智能一锅法生物传感平台。该平台将基于折返环引物加速的环介导等温扩增技术（LAMP）（TLAMP）与激烈火球菌（Pyrococcus furiosus）的Argonaute蛋白（PfAgo）的序列特异性检测功能完美融合，采用一种巧妙的热激活设计，利用微晶蜡在单管内实现反应的空间隔离，从而确保了操作的无污染与流程的简化。OTPA分析法具备极高的灵敏度与特异性，在30分钟内对猪肉DNA和牛肉DNA的检测限分别低至3×10-4 ng/μL和2×10-4 ng/μL。此外，它成功实现了双靶标鉴定，并在市售肉制品中得到了验证，其结果与标准的PCR定性检测高度一致。值得注意的是，检测结果可在蓝光下直接通过肉眼可视化，这充分凸显了这种经济高效且简便的平台在食品安全监测的即时检测（POCT）与智能生物传感领域中所具备的巨大潜力。.