Timely and large-scale screening of pathogen-caused infection is highly important to prevent the latent epidemic. With its high amplification efficiency and less requirement on the instrument, the isothermal nucleic acids amplification (INAA) technique has shown great potential in realizing point-of-care (POC) detection of pathogens. However, the ability to realize a high-order multiplexity in one tube, which is significant in the screening of a wide array of pathogens, is still a tricky task for INAA. Herein, we develop a high-order one-pot multiplexing detection method using recombinase-assisted loop-mediated isothermal amplification (RALA). The method shows a high-level multiplexity that could bear over 40 primer sequences in one pot, capable of screening 11 target pathogens with high sensitivity. Furthermore, a simple and portable device was developed in this study to facilitate POC detection. Coupled with this miniaturized device, the strategy was successfully applied to the highly efficient screening of contrived real-life samples. Taken together, this study provides a new but facile choice for rapid screening of infected samples, showing the potential in controlling the transmission of infectious pathogens.

及时、大规模地筛查病原体引起的感染对于预防潜伏性流行非常重要。等温核酸扩增（INAA）技术具有扩增效率高、对仪器要求低等优点，在实现病原菌即时检测（POC）方面显示出巨大潜力。然而，在一个试管中实现高阶多重性的能力，这对于筛选多种病原体具有重要意义，对于 INAA 来说仍然是一项棘手的任务。在此，我们开发了一种使用重组酶辅助环介导等温扩增 (RALA) 的高阶一锅多路复用检测方法。该方法具有高水平的多重性，一锅可承载40多个引物序列，能够以高灵敏度筛选11个目标病原体。此外，本研究开发了一种简单便携的设备，以方便 POC 检测。再加上这种小型化设备，该策略成功地应用于人为的真实样本的高效筛选。总而言之，这项研究为快速筛查受感染样本提供了一种新的但简便的选择，显示了控制传染性病原体传播的潜力。