Comparative Evaluation of Rapid Isothermal Amplification and Antigen Assays for Screening Testing of SARS-CoV-2

Abstract

Human transmission of SARS-CoV-2 and emergent variants of concern continue to occur globally, despite mass vaccination campaigns. Public health strategies to reduce virus spread should therefore rely, in part, on frequent screening with rapid, inexpensive, and sensitive tests. We evaluated two digitally integrated rapid tests and assessed their performance using stored nasal swab specimens collected from individuals with or without COVID-19. An isothermal amplification assay combined with a lateral flow test had a limit of detection of 10 RNA copies per reaction, and a positive percent agreement (PPA)/negative percent agreement (NPA) during the asymptomatic and symptomatic phases of 100%/100% and 95.83/100%, respectively. Comparatively, an antigen-based lateral flow test had a limit of detection of 30,000 copies and a PPA/NPA during the asymptomatic and symptomatic phases of 82.86%/98.68% and 91.67/100%, respectively. Both the isothermal amplification and antigen-based lateral flow tests had optimized detection of SARS-CoV-2 during the peak period of transmission; however, the antigen-based test had reduced sensitivity in clinical samples with qPCR Ct values greater than 29.8. Low-cost, high-throughput screening enabled by isothermal amplification or antigen-based techniques have value for outbreak control.

Keywords: SARS-CoV-2; antigen test; isothermal molecular test; screening; surveillance; testing.

Figure 1

Schematic of RT-RPA assay versus antigen-based test. (A) In RT-RTPA, viral RNA is coped to cDNA by reverse transcriptase, then degraded by RNase H. Using a forward and a FAM-labeled reverse pair of primers specific to a target sequence, the cDNA product is amplified by RPA, then denatured and hybridized to a biotinylated probe. FAM-labeled and biotin-labeled products are detected on a lateral flow strip using molecules specific for FAM and biotin and nanoparticles. (B) In an antigen test, protein targets are detected by a lateral flow strip using protein-specific antibodies and nanoparticles. (C) A mobile phone application was used to image capture, machine-read, and quantify test results. The average pixel intensity is quantified at the test line, control line, and background areas. The background-subtracted test line signal is then normalized to the background-subtracted control line and expressed at % of control. − (red), test signal below the limit of detection; + (orange), low test signal; ++ (blue), medium test signal; +++ (green), high test signal.

Figure 2

Analytical sensitivity of the RT-RPA assay and the antigen test using nasal swab dilution specimens. (A) Lateral flow strips for the RT-RPA reactions with dilution specimens containing RNA copies ranging from 0 to 1000. (B) Plot from the RT-RPA assay results quantified by the mobile phone application. The x-axis corresponds to dilutions’ specimens with known input copies of SARS-CoV-2 RNA. The y-axis corresponds to background subtracted test signal normalized to the control line for each lateral flow strip. Test results (purple dots) less than 10% of control are considered negative results, which is indicated by the black dashed line. (C) Lateral flow strips for the antigen tests with dilution specimens containing RNA copies ranging from 0 to 200,000. (D) Plot from the antigen tests results quantified by the mobile phone application. The x-axis corresponds to dilutions’ specimens with known input copies of SARS-CoV-2 RNA. The y-axis corresponds to background subtracted test signal normalized to the control line for each lateral flow strip. Test results (blue dots) less than 10% of control are considered negative results, which is indicated by the black dashed line.

Figure 3

Clinical performance of the RT-RPA assay and the antigen test using nasal swab specimens collected from individuals with or without COVID-19. (A) Comparative evaluation of the RT-RPA assay (purple) and the antigen test (blue) using nasal swab specimens from asymptomatic cases. Comparative performance between the tests was plotted according to qPCR positive (Ct values between <20 to <40) and negative results. (B) Comparative evaluation of the RT-RPA assay (purple) and the antigen test (blue) using nasal swab specimens from symptomatic cases. Comparative performance between the tests was plotted according to qPCR positive (Ct values between <20 to <40) and negative results.