An ultra-sensitive, multiplexed, and cost-effective POCT system for the detection of co-infecting respiratory viruses, including SARS-CoV-2, Flu A, Flu B, and RSV, within 30 min.

The co-circulation of respiratory viruses, including SARS-CoV-2, Influenza A (Flu A), Influenza B (Flu B), and respiratory syncytial virus (RSV), poses a significant public health threat. Timely recognition of these viruses allows healthcare professionals to implement effective infection control measures, allocate medical resources properly, and prevent complications from incorrect treatments. Multiplex nucleic acid testing Point-of-care test (mNAT-POCT) circumvents issues of traditional tests, such as high demands on laboratory environments, personnel, and equipment, and limited target analyses, allowing its use in point-of-care settings. However, challenges include primer-primer interactions during fast amplification, high automation requirements, configuring multiple fluorescence channels to avoid spectral overlap, and balancing rapid thermal cycling with sensitive fluorescence signal collection. To address these issues, we developed the multiplexed reverse transcription-quantitative PCR (RT-qPCR) POCT system iNAT SARS-CoV-2/Flu A/Flu B/RSV Assay. This assay enables quick, automatic, and accurate detection of multiple pathogens, improving diagnostic and treatment efficiency for syndromic infectious diseases. The limit of detection (LoD) is 45 copies/mL for SARS-CoV-2, 133 copies/mL for Flu A, 57 copies/mL for Flu B, and 212.5 copies/mL for RSV, with a turnaround time (TAT) of 30 min. Clinical sample analysis showed a 99.36 % agreement with National Medical Products Administration (NMPA) approved reference tests. In conclusion, the iNAT SARS-CoV-2/Flu A/Flu B/RSV Assay performs excellently in detecting and differentiating SARS-CoV-2, Flu A, Flu B, and RSV in respiratory infections, which is crucial for accurate diagnoses.