All-in-one multiplex isothermal reaction combining miniaturized device enables rapid screening of infected samples

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. • A one-pot high-level multiplexing isothermal amplification was achieved. • A new screening strategy was provided for the efficient screening of infections. • A miniaturized device and a flat-bottom chip were developed.