Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the unprecedented global pandemic of coronavirus disease-2019 (COVID-19). Efforts are needed to develop rapid and accurate diagnostic tools for extensive testing, allowing for effective containment of the infection via timely identification and isolation of SARS-CoV-2 carriers. Current gold standard nucleic acid tests require many separate steps that need trained personnel to operate specialist instrumentation in laboratory environments, hampering turnaround time and test accessibility, especially in low-resource settings. We devised an integrated on-chip platform coupling RNA extraction based on immiscible filtration assisted by surface tension (IFAST), with RNA amplification and detection via colorimetric reverse-transcription loop mediated isothermal amplification (RT-LAMP), using two sets of primers targeting open reading frame 1a (ORF1a) and nucleoprotein (N) genes of SARS-CoV-2. Results were identified visually, with a colour change from pink to yellow indicating positive amplification, and further confirmed by DNA gel electrophoresis. The specificity of the assay was tested against HCoV-OC43 and H1N1 RNAs. The assay based on use of gene N primers was 100% specific to SARS-CoV-2 with no cross-reactivity to HCoV-OC43 nor H1N1. Proof-of-concept studies on water and artificial sputum containing genomic SARS-CoV-2 RNA showed our IFAST RT-LAMP device to be capable of extracting and detecting 470 SARS-CoV-2 copies mL-1 within 1 h (from sample-in to answer-out). IFAST RT-LAMP is a simple-to-use, integrated, rapid and accurate COVID-19 diagnostic platform, which could provide an attractive means for extensive screening of SARS-CoV-2 infections at point-of-care, especially in resource-constrained settings.
Keywords: COVID-19; IFAST; LAMP; Point-of-care; SARS-CoV-2.
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