A rapid, sample-to-answer strategy for the multiplex detection of intact respiratory pathogens

Abstract

Respiratory pathogens have long represented a significant cause of global pandemics, highlighting the critical need for reliable tools to evaluate their infectivity. However, conventional nucleic acid- or antigen-based detection methods often fail to accurately reflect the actual infection risk posed by the intact pathogens present in clinical samples. To address this limitation, this study developed a detection strategy for six-plex intact respiratory pathogens in swabs, coupling a broad-spectrum capture and viability dye treatment with an automated microfluidic platform. Specifically, recombinant human mannan-binding lectin-functionalized magnetic beads (rhMBL-MBs) are used to capture intact pathogens, while improved propidium monoazide (PMAxx) prevents amplification of compromised ones. Following this pretreatment, the microfluidic platform automates the processes of nucleic acid extraction, recombinase aided amplification (RAA), fluorescence detection, and wireless data transmission. This sample-to-answer workflow delivers results within 45 min, achieving the lowest detection limits of 5 50 % tissue culture infectious dose per milliliter (TCID₅₀/mL) for intact pathogens. The clinical utility of this strategy was validated using 40 swabs collected at multiple time points post-diagnosis (confirmed by RT-qPCR/qPCR) and 10 healthy controls. Results showed a 100 % detection rate for samples collected 2–3 days post-diagnosis and 40 % for those collected 7–10 days post-diagnosis, demonstrating its effective differentiation of intact, potentially infectious pathogens in clinical specimens. With high specificity, rapid turnaround, and design flexibility, this approach shows strong potential for assessing transmission risks and informing evidence-based infection control strategies, including decisions on isolation duration.