Fully Integrated Microfluidic Platform for Multiplexed Detection of Hunov by a Dynamic Confined-Space-Implemented One-Pot Rpa-Lamp System

Human norovirus (HuNoV) is the leading cause of nonbacterial acute gastroenteritis, which is highly infectious, rapidly evolving, and easily transmitted through feces. The accurate and early detection of HuNoV subtypes is essential for effective treatment, early surveillance, risk assessment, and disease prevention. In this study, a portable multiplex HuNoV detection platform that combines integrated microfluidics and cascade isothermal amplification, using a streamlined protocol for clinical fecal-based diagnosis is presented. To overcome the problems of carryover contamination and the incompatibility between recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP), a Dynamic confined-space-implemented One-pot RPA-LAMP colorimetric detection system (DORLA) is developed by creating a hydrogen bond network. The DORLA system exhibits excellent sensitivity, with detection limits of 10 copies µL^-1 and 1 copy µL^-1 for HuNoV GI and GII, respectively. In addition, a portable diagnostic platform consisting of a thermostatic control module and an integrated 3D-printed microfluidic chip for specific HuNoV capture, nucleic acid pretreatment, and DORLA detection, which enables simultaneous diagnosis of HuNoV GI and GII is developed. A DORLA-based microfluidic platform exhibits satisfactory performance with high sensitivity and portability, and has high potential for the rapid point-of-care detection of HuNoV in clinical fecal samples, particularly in resource-limited settings.