Abstract: The COVID-19 pandemic has highlighted bottlenecks in large-scale, frequent testing of populations for infections. PCR-based diagnostic tests are expensive, reliant on expensive centralized labs, can take days to deliver results, and are prone to backlogs and supply shortages. Antigen tests, that bind and detect the surface proteins of a virus, are rapid and inexpensive but suffer from high false negative rates. To address this problem, we have created an inexpensive, simple, and robust 60-minute Do-It-Yourself (DIY) workflow to detect viral RNA from nasal swabs or saliva with high sensitivity (0.1 to 2 viral particles/µl) and specificity (>97% True Negative Rate) utilizing reverse transcription loop-mediated isothermal amplification (RT-LAMP). Our workflow, ALERT (Accessible LAMP-Enabled Rapid Test), incorporates the following features: 1) Increased shelf-life and ambient temperature storage by using wax layers to isolate enzymes from reaction, 2) Improved specificity by using sequence-specific QUASR reporters, 3) Increased sensitivity through use of a magnetic wand to enable pipette-free concentration of sample RNA and cell debris removal, 4) Quality control with a nasopharyngeal-specific mRNA target, and 5) Co-detection of other respiratory viruses, such as Influenza B, by duplexing QUASR-modified RT-LAMP primer sets. The flexible nature of the ALERT workflow allows easy, at-home and point-of-care testing for individuals and higher-throughput processing for centralized labs and hospitals. With minimal effort, SARS-CoV-2-specific primer sets can be swapped out for other targets to repurpose ALERT to detect other viruses, microorganisms or nucleic acid-based markers.
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