Multiplexed detection of respiratory pathogens with a portable analyzer in a “raw-sample-in and answer-out” manner

Coronavirus disease 2019(COVID-19)has emerged,rapidly spread and caused significant morbidity and mortality worldwide.There is an urgent public health need for rapid,sensitive,specific,and on-site diagnostic tests for severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection.In this study,a fully integrated and portable analyzer was developed to detect SARS-CoV-2 from swab samples based on solid-phase nucleic acid extraction and reverse transcription loop-mediated isothermal amplification(RT-LAMP).The swab can be directly inserted into a cassette for multiplexed detection of respiratory pathogens without pre-preparation.The overall detection process,including swab rinsing,magnetic bead-based nucleic acid extraction,and 8-plex real-time RT-LAMP,can be automatically performed in the cassette within 80 min.The functionality of the cassette was validated by detecting the presence of a SARS-CoV2 pseudovirus and three other respiratory pathogens,i.e.,Klebsiella pneumoniae,Pseudomonas aeruginosa,and Stenotrophomonas maltophilia.The limit of detection(LoD)for the SARS-CoV-2 pseudovirus was 2.5 copies/μL with both primer sets(N gene and ORF1ab gene),and the three bacterial species were successfully detected with an LoD of 2.5 colony-forming units(CFU)/μL in 800μL of swab rinse.Thus,the analyzer developed in this study has the potential to rapidly detect SARS-CoV-2 and other respiratory pathogens on site in a“raw-sample-in and answer-out”manner.