Multiplexed on-site sample-in-result-out test through microfluidic real-time PCR (MONITOR) for the detection of multiple pathogens causing influenza-like illness

ABSTRACT The global COVID-19 pandemic and frequent outbreaks of other infectious diseases, such as influenza and monkeypox, often initially manifest with non-specific influenza-like symptoms. Portability to the outbreak site and accurate identification of the various pathogens causing influenza-like illness are crucial for the rapid implementation of effective control measures. Utilizing real-time PCR and microfluidic technology, a multiplexed on-site sample-in-result-out test through microfluidic real-time PCR (MONITOR) has been developed for detecting the pathogens responsible for influenza-like illness. The operator simply needs to introduce the sample to a highly integrated microfluidic chip (requiring ≤1 minute), and the system can autonomously execute sample pre-processing, nucleic acid extraction, and PCR amplification and deliver results for eight pathogens within 85 minutes. The detection limit of MONITOR for the eight pathogens ranges from 0.78 to 6.25 copies/µL. Standard curves demonstrate notable linearity and amplification efficiency. A simulated clinical sample test reveals MONITOR’s sensitivity, specificity, and accuracy at 97.5%, 100%, and 98%, respectively. Bland-Altman analysis demonstrates strong agreement between the cycle threshold of positive MONITOR samples and quantitative polymerase chain reaction (qPCR) ( R 2 = 0.952), suggesting MONITOR’s ability to indirectly quantify pathogen load. The fully enclosed structure of the system chip minimizes sample and environmental cross-contamination, rendering the assay independent of a specialized laboratory. The portable, fully automated MONITOR facilitates on-site, comprehensive detection of multiple pathogens, even non-specialized operators with minimal training. This presents a promising approach for the rapid and precise diagnosis of influenza-like illness in grassroots communities and underserved rural areas lacking centralized laboratories. IMPORTANCE This study combines quantitative polymerase chain reaction (qPCR) and microfluidics to introduce MONITOR, a portable field detection system for multiple pathogens causing influenza-like illness. MONITOR can be rapidly deployed to enable simultaneous sample-in-result-out detection of eight common influenza-like illness (ILI) pathogens with heightened sensitivity and specificity. It is particularly well suited for communities and regions without centralized laboratories, offering robust technical support for the prompt and accurate monitoring and detection of ILI. It holds the potential to be a potent tool in the early detection and prevention of infectious diseases.