Significance An important limitation of current assays for the detection of SARS-CoV-2 stems from their reliance on time-consuming, labor-intensive, and laboratory-based protocols for viral isolation, lysis, and removal of inhibiting materials. While RT-PCR remains the gold standard for performing clinical diagnostics to amplify the RNA sequences, there is an urgent need for alternative testing platforms that are rapid, accurate, simple, and portable. Here, we demonstrate isothermal RT-LAMP nucleic acid-based detection of SARS-CoV-2 with an additively manufactured cartridge and a smartphone-based instrument for testing that can be performed at the point of sample collection. The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per μL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection.
【저자키워드】 SARS-CoV-2, point-of-care, RT-LAMP, COVID-19 diagnostics, smartphone reader, 【초록키워드】 coronavirus, protocol, COVID-19 pandemic, POC, diagnostic test, Diagnosis, SARS-CoV-2 virus, RT-PCR, virus, diagnostics, Laboratory, RNA extraction, clinical samples, VTM, limit of detection, Isolation, reverse transcription, isothermal amplification, Swab, platform, Alternative pathway, isothermal, POC test, Volume, Viral transport medium, acute respiratory syndrome, gold standard, sequence, primer, while, RNA sequences, cartridge, positive sample, nasal fluid, Complete, controls, negative sample, clinical sample, performed, detect, example, required, amplify, characterized, provide, RNA copy, inhibiting, time-consuming, labor-intensive, Significance, was tested, 【제목키워드】 Rapid, isothermal amplification, detection system,