A Quantum Dot-Based Integrated Lateral Flow Assay For Multiplex Point-of-Care Detection Of Novel Coronavirus,Influenza A Virus,Influenza B Virus And Adenovirus

ObjectiveIn this study,a quantum dot-based lateral flow assay method with simple operation process and high detection sensitivity was established to realize integrated detection of SARS-CoV-2,influenza A virus(IAV),influenza B(IBV)virus and adenovirus(ADV).Methods1.The fluorescence microspheres made of quantum dots(QBs)are linked to virus antibodies through the activation of N-Hydroxysuccinimide(NHS)and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDC).2.The performance of the test strip on a nitric acid cellulose(NC)membrane is improved by adjusting the content of labeling and capture antibodies,using Goat anti-mouse IgG antibody as a control line and respiratory virus capture antibody as the detection line.3.615 nm QBs are conjugated with four different antibodies(IAV,IBV,ADV,SARS-CoV-2),four test strips are created.The fluorescence signal detection is performed using a multiplex test cartridge and a portable multiplex fluorescent strip reader.Laboratory-developed fluorescence signal detectors are suitable for detecting respiratory virus content in human pharyngeal swab.ResultsThe linear range of antigen detection for SARS-CoV-2,IAV,IBV,and ADV is 0.01-100ng/mL,0.05-100ng/mL,0.5-100ng/mL,and 1-1000ng/mL,respectively.The detection limits are 0.01ng/mL,0.05ng/mL,0.31ng/mL,and0.40ng/mL,which show better sensitivity compared to colloidal gold lateral flow assay.The coefficient of variation within batch were 6.09%,2.24%,7.92%,and12.43%,and coefficient of variation between batches 8.23%,4.52%,11.88% and13.94%,respectively,indicating good repeatability and stability of the method.The recovery rate of high,medium and low concentrations of each respiratory virus antigen was 90.67%-105.85%,which proved that the method had good accuracy.Different combinations of respiratory viruses and other respiratory pathogens were detected to verify the good specificity of the detection system.The simulated throat swab samples were used for clinical verification,and the virus particles were compared by PCR and lateral flow assay,which confirmed the clinical feasibility of this experiment.ConclusionsThe quantum dot lateral flow assay has achieved integrated detection of four respiratory viruses,including SARS-CoV-2,IAV,IBV,and ADV.This method has a simple process,low cost,fast detection speed,and high sensitivity.It can be applied to pharyngeal swab samples and is an ideal screening method for respiratory viruses.