Abstract
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.
Highlights
The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs
The swab is transported to viral transport medium (VTM) solution and gently agitated to transfer the viruses from the swab into the VTM
The lysed sample and the reverse transcription loop mediated isothermal amplification (RT-loop-mediated isothermal amplification (LAMP)) reagents are loaded in 1- and 5 mL-syringes, respectively, the syringes are attached to the microfluidic cartridge, and the lysed sample and RT-LAMP reagents are simultaneously injected into the cartridge
Summary
The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. The Abbott ID isothermal amplification technology claims the delivery of positive results in less than 15 min while offering a device with portable size and weight This test requires a specialized instrument with welldocumented availability issues [24,25,26]. There may be issues with accuracy of this test, claims about accuracy remain under review [27] Another isothermal technology with good performance is the SARS-CoV-2 LAMP Diagnostic Assay from Color Genomics, with a limit of detection (LOD) of around. 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. 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
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