NOMIS Awardee Svante Pääbo and colleagues developed a COVID-19 test called 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. Their test was presented in Nature Communications.
Introduction
The recent global outbreak of coronavirus disease 2019 (COVID-19) has led governments to take drastic measures to contain the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Lock-down measures and restrictions on travel, public gatherings, and the closing of institutions such as schools, kindergartens, and universities are usually implemented broadly, affecting the lives of infected and uninfected people alike. Reliable and economical mass testing approaches for decentralized point-of-care identification of infected individuals could greatly help to achieve more directed and efficient containment of SARS-CoV-2.
Reverse transcription followed by quantitative PCR (RT-qPCR) is the most widely used method to detect RNA viruses such as SARS-CoV-2. However, its need for expensive bulky instrumentation and shortages of resources for RNA purification has spurred the search for viable alternatives even though sensitive RNA-extraction-free SARS-CoV-2 RT-qPCR-based tests are now established. Loop-mediated isothermal amplification (LAMP) can rapidly amplify target nucleic acid sequences under isothermal conditions, and has been applied for molecular diagnostics. The reaction requires four to six primers and produces concatemers of double-stranded amplification products. These can be detected directly, using intercalating dyes (e.g., SYBR green, SYTO-dyes), by triphenylmethane dye precursors and acid hydrolysis, or by cleavage with CRISPR enzymes coupled with lateral flow color detection of the cleavage product. However, these methods require an opening of the tube after the reaction, thus posing the threat of cross-contaminating future reactions with the amplified product. Amplification can also be detected indirectly by hydroxynaptholblue or phenol red-based detection of the release of protons and/or pyrophosphate generated during DNA synthesis. Recently, a number of studies explored ways to detect SARS-CoV-2 RNA using RT-LAMP but they required time-consuming RNA isolation steps before the reaction. There have also been attempts to add sample directly into the reaction without prior purification. However, the pH of nasopharyngeal swab samples often varies and can adversely affect readouts.
In this work, we describe a method to detect SARS-CoV-2 RNA of a single-infected individual within a bulk sample composed of up to 26 individual patient samples by combining a hybridization capture-based RNA-extraction approach with smartphone app-assisted colorimetric detection of RT-LAMP products, a procedure that can be performed in <1 h.
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