Point-of-care bulk testing for SARS-CoV-2 by combining hybridization capture with improved colorimetric LAMP

Abstract

Efforts to contain the spread of SARS-CoV-2 have spurred the need for reliable, rapid, and cost-effective diagnostic methods which can be applied to large numbers of people. However, current standard protocols for the detection of viral nucleic acids while sensitive, require a high level of automation and sophisticated laboratory equipment to achieve throughputs that allow whole communities to be tested on a regular basis. Here we present Cap-iLAMP (capture and improved loop-mediated isothermal amplification) which combines a hybridization capture-based RNA extraction of gargle lavage samples with an improved colorimetric RT-LAMP assay and smartphone-based color scoring. Cap-iLAMP is compatible with point-of-care testing and enables the detection of SARS-CoV-2 positive samples in less than one hour. In contrast to direct addition of the sample to improved LAMP (iLAMP), Cap-iLAMP prevents false positives and allows single positive samples to be detected in pools of 25 negative samples, reducing the reagent cost per test to ~1 Euro per individual.

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

“…Due to its simplicity, accuracy comparable with RT-qPCR to detect SARS-CoV-2 RNA, does not require PCR machine and naked eye readable colorimetric output, RT-LAMP was the focus of massive testing campaigns (14,16,28). This screening strategy is compatible with: home, primary care clinics, point of entry Upon RNA extraction from nasopharyngeal swab-derived clinical samples we found a LoD of 20 viral genomic copies/µL, confirming previous studies based on N SARS-CoV-2 target (9,30,43). It is worth noting that when using non-clinical SARS-CoV-2 extracted RNA or synthetic target, the LoD reaches less than 0.5 cps/µL.…”

“…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.…”

“…(borders), schools, universities, sport leagues, companies and can help to achieve a safe back to work and quarantine monitoring(10,14,16,28,30). Since April 14 th , 2020 the U.S.Food and Drugs Administration (FDA) issued the emergence use authorization of Color SARS-CoV-2 RT-LAMP Diagnostic Assay from Color Health, Inc. (EUA number: EUA200539).In order to provide an affordable SARS-CoV-2 detection tools, we validate a colorimetric RT-LAMP for the COVID-19 diagnosis using clinical samples collected from different parts of Brazil.…”

A rapid near-patient detection system for SARS-CoV-2 using saliva

“…A limitation of all tests which rely on visually identifying a color change—including Saliva-Dry LAMP—is that there is some subjectivity involved in calling reactions positive or negative based on their color (especially when a reaction resembles both the positive and negative colors). However, with the fluorescent dye mix used here, positive and negative reactions differed from one another in both color and emitted light intensity, making the contrast between positive and negative reactions two-fold (unlike tests which are merely colorimetric) 38 – 41 . In the future, a mobile phone app which discriminates specific colors could be used to call reactions, thus reducing subjectivity 39 .…”

Sub-Picomolar Detection of SARS-CoV-2 RBD via Computationally-Optimized Peptide Beacons

“…Recently, there has been significant effort to detect SARS-CoV-2 via fluorescence-based readouts to allow for specific signal amplification ( 9–12 ). Such methods largely rely on binding to SARS-CoV-2 RNA or DNA, which requires isolation of nucleic acids, as described above.…”