Clinical Validation of a Rapid Variant-Proof RT-RPA Assay for the Detection of SARS-CoV-2

• Published in: Diagnostics (Basel) Vol. 12; no. 5; p. 1263
• Main Authors: Cherkaoui, Dounia, Heaney, Judith, Huang, Da, Byott, Matthew, Miller, Benjamin S, Nastouli, Eleni, McKendry, Rachel A
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 19.05.2022; MDPI
• Subjects: Acids; clinical samples; Coronaviruses; COVID-19 vaccines; Diagnostic tests; Disease transmission; Genes; Genomes; isothermal amplification; Mutation; Pandemics; Pathogens; recombinase polymerase amplification; RNA polymerase; SARS-CoV-2; Severe acute respiratory syndrome coronavirus 2; University colleges; Viral infections; United Kingdom > UK; United States > US; SARS-CoV-2; recombinase polymerase amplification; clinical samples; isothermal amplification
• ISSN: 2075-4418; 2075-4418
• DOI: 10.3390/diagnostics12051263

The COVID-19 pandemic has unveiled a pressing need to expand the diagnostic landscape to permit high-volume testing in peak demand. Rapid nucleic acid testing based on isothermal amplification is a viable alternative to real-time reverse transcription polymerase chain reaction (RT-PCR) and can help close this gap. With the emergence of SARS-CoV-2 variants of concern, clinical validation of rapid molecular tests needs to demonstrate their ability to detect known variants, an essential requirement for a robust pan-SARS-CoV-2 assay. To date, there has been no clinical validation of reverse transcription recombinase polymerase amplification (RT-RPA) assays for SARS-CoV-2 variants. We performed a clinical validation of a one-pot multi-gene RT-RPA assay with the E and RdRP genes of SARS-CoV-2 as targets. The assay was validated with 91 nasopharyngeal samples, with a full range of viral loads, collected at University College London Hospitals. Moreover, the assay was tested with previously sequenced clinical samples, including eleven lineages of SARS-CoV-2. The rapid (20 min) RT-RPA assay showed high sensitivity and specificity, equal to 96% and 97%, respectively, compared to gold standard real-time RT-PCR. The assay did not show cross-reactivity with the panel of respiratory pathogens tested. We also report on a semi-quantitative analysis of the RT-RPA results with correlation to viral load equivalents. Furthermore, the assay could detect all eleven SARS-CoV-2 lineages tested, including four variants of concern (Alpha, Beta, Delta, and Omicron). This variant-proof SARS-CoV-2 assay offers a significantly faster and simpler alternative to RT-PCR, delivering sensitive and specific results with clinical samples.