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.

Fig. 1. Cap-iLAMP to detect SARS-CoV-2.

A Workflow of Cap-iLAMP involves collecting and optionally pooling up to 26 gargle lavage samples, followed by combined lysis, target RNA enrichment, and improved LAMP (iLAMP). Color hue values can be obtained using any freely available “camera color picker” application on a smartphone. B Positions of primers and biotinylated capture oligonucleotides targeting the viral Orf1a and N gene on the SARS-CoV-2 genome. C Color change induced by mixing a drop of SYBR green I in the lid of the tube after iLAMP reaction with different input copy numbers of synthetic viral RNA. D Capture efficiency of three samples with different viral loads was estimated relative to automated silica-based RNA extraction based on copy number estimates for RT-qPCR assay targeting the SARS-CoV-2 E gene. Source data are provided as a Source Data file. E Equipment necessary for Cap-iLAMP: Pipettes and pipette tips, pre-mixed reagents (including iLAMP master mix and capture bead suspension), stable buffers (lysis/binding buffer, wash buffer, low salt buffer, elution buffer), a magnetic rack, and a thermoblock. A smartphone (not depicted) is recommended for hue color scoring.

Fig. 2. Detection of SARS-CoV-2 in gargle lavage samples.

Data obtained from healthy individuals are shown in blue while SARS-CoV-2-positive patient samples are depicted in green. Solid circles indicate values of the Orf1a assay while hollow circles denote values of the N gene assay. Assays with a hue >28.5° (dotted line) are considered positive. Repeated experiments are depicted in a light color and respective samples underwent an additional freeze/thaw cycle. A Hue of individual SARS-CoV-2-negative gargle lavage samples after Cap-iLAMP measured in duplicates. B Hue of individual SARS-CoV-2-positive gargle lavage samples after Cap-iLAMP measured in duplicates. Ct values of individual patient gargle lavage samples obtained via RT-qPCR assay targeting the SARS-CoV-2 E gene are stated. C Hue of gargle lavage pools after Cap-iLAMP measured in duplicates. The Ct values of spiked-in positive samples are stated. A vertical red dotted line indicates the separation between samples with high viral loads (Ct < 24) and low viral loads (Ct ≥ 24). Samples with Ct < 24 are likely highly infectious. Source data are provided as a Source Data file.

Fig. 3. Detection of SARS-CoV-2 in cell-culture supernatants provided for assay validation.

A Ct values of individual cell-culture supernatant samples obtained via RT-qPCR assay targeting the SARS-CoV-2 E gene. B Hue of individual cell-culture supernatant samples after Cap-iLAMP measured in duplicates. Solid circles indicate values of the Orf1a assay while hollow circles denote values of the N gene assay. Data obtained from healthy individuals are shown in blue while SARS-CoV-2-positive patient samples are depicted in green. Assays with a hue >28.5° (dotted line) are considered positive. Source data are provided as a Source Data file.