Accessible LAMP-Enabled Rapid Test (ALERT) for detecting SARS-CoV-2

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.

Detection of SARS-CoV-2 RNA using RT-LAMP and molecular beacons

“…However, conventional LAMP typically detects only the presence of amplified bulk DNA, and thus, assays can be complicated by nonspecific amplification. Improved specificity can be achieved by sequence-specific detection, and multiple methods have been proposed [10][11][12][13][14][15]. Here, we introduce a particularly convenient and effective method for sequence-specific detection of SARS-CoV-2 RNA in unpurified saliva using molecular beacons-LAMP-BEAC-that does not require manipulation of reaction products, can be carried out in a multiplex format in a "single tube," greatly reduces the potential for false positives, and allows increased sensitivity.…”

“…These methods are promising, but as presently designed they typically require opening of RT-LAMP tubes and secondary manipulation of reaction products, which has the potential to result in contamination of subsequent reactions with amplification products from previous assays. In another detection method, a quencher-fluorophore duplex can be created by adding a short oligonucleotide complementary to a standard LAMP primer which is then displaced upon amplification [12][13][14][15]. These methods are more specific than bulk reporters but are still potentially vulnerable to false positives from spurious amplification.…”

Loop-Mediated Isothermal Amplification (LAMP): The Better Sibling of PCR?

“…Another example of a DIY assay is the LAMP-enabled rapid test (ALERT) [115]. According to its designers, the test can easily be performed at home and care centers, as well as in laboratories conducting large-scale analyses.…”

“…The ALERT is exceptional in that it implements QUASR reporting to confirm the presence of viral RNA, which reduces the rate of false positives and enables multiplex analyses. The designers underline that the assay does not have to be stored at a low temperature, which is important for at-home tests [115].…”

Clinical validation of colorimetric RT-LAMP, a fast, highly sensitive and specific COVID-19 molecular diagnostic tool that is robust to detect SARS-CoV-2 variants of concern

“…The possibility of accessing results by naked eye, made RT-LAMP an exciting alternative that facilitates the use of COVID-19 molecular testing. Simple, scalable, cost-effective RT-LAMPbased alternatives for SARS-CoV-2 detection, has emerged during pandemics including protocols for viral inactivation, quick run, RNA extraction-free and LAMP-associated CRISPR/Cas strategies (10,11,14,16,17,(26)(27)(28)(29)(30)(31)(32). On April 14 th , 2020, the RT-LAMP received the emergency use authorization from the United States Food and Drug Administration for SARS-CoV-2 detection in COVID-19 diagnostics.…”