Development of rapid diagnostic tests for multiplexed detection of COVID-19 and Influenza diseases

Abstract Objectives This study investigates diagnosis of upper respiratory diseases with similar clinical symptoms, specifically COVID-19, originated from the SARS-CoV-2 virus, and Influenza infections associated with Influenza viruses. Effective treatment relies on rapid and accurate diagnosis, especially due to pandemic potential. This research aims to develop a multiplex lateral flow assay (LFA) based diagnosis method for detecting SARS-CoV-2 and Influenza A H1N1 viruses simultaneously. Methods Lateral flow assay utilizing gold nanoparticles were developed with specific monoclonal antibodies targeting the nucleocapsid (NP) protein of SARS-CoV-2 and the hemagglutinin (HA) protein of Influenza A H1N1. Multiplexing was achieved by immobilizing two different capture antibodies for both targets on the same test. Recombinant antigens were employed to perform analytical assessments of specificity and cross-reactivity. Results The limit of detection (LOD) of the developed system in both single and multiplex formats was found to be: 1 ng/mL for SARS-CoV-2 NP, 1 × 10 3 PFU/mL for active viruses and 5 ng/mL for Influenza A H1N1 HA. Negative control experiments confirmed no cross-reactivity among target antigens, demonstrating high assay specificity. The low LOD values and absence of cross-reactivity indicate excellent analytical sensitivity and specificity. Simultaneous detection of both pathogens offers significant advantages for rapid clinical decision-making and infection control. Conclusions Our research presents a potential diagnostic approach utilizing a singular LFA platform for the early and accurate detection of COVID-19 and Influenza infections. The proposed multiplex strategy could improve public health outcomes by facilitating rapid therapeutic intervention and successful disease management during respiratory infection epidemics.