VariantDirect: An extraction-free screening approach to detect circulating SARS-CoV-2 virus strains from pooled specimens

Abstract

Coronavirus disease (COVID-19) caused by the SARS-CoV-2 virus has exposed clinical laboratories to unprecedented challenges. With surging case numbers, clinical laboratories were forced to acquiesce and integrate multiple testing platforms with varying workflows and analytical sensitivities in order to meet testing volumes. Now a new challenge has emerged with the evolution of viral variants, both globally and locally, raising concerns for uncontrolled spread, increased disease severity, and weakened responses to vaccinations. Preliminary data suggests that these variants may be associated with higher viral titers and prolonged infections. While primarily leveraged for epidemiologic surveillance, the clinical utility of variant detection may quickly become paramount. Furthermore, laboratories must remain vigilant and nimble enough to pivot should variant identification play a role in the patient care. To prepare for the validation of clinical assays that identify important viral variants, we designed a novel method, termed VariantDirect, to screen SARS-CoV-2 positive samples for the presence of variants, focusing initially on the increasingly prevalent UK and South African (SA) variants. The detection strategy is based on primers designed to specifically target the viral receptor-binding domain mutation, N501Y, shared by the UK and SA strains. Screening for variants will be limited to nasopharyngeal swab samples of high viral titers (Ct values <25 by RT-qPCR assay, Roche Diagnostics). Pools of 9 different samples, 50 µl each, are mixed and stored at -80°C along with aliquots of the 9 original samples. These pools will then be tested, and if positive for the N501Y variant, the pooled 9 samples will be thawed and tested separately to identify the affected specimen. Most of these specimens are also being independently sequenced via a comprehensive but more resource-intensive NGS approach. Advantages of our pooled workflow are primarily in time and cost, with the capacity of screening up to 837 specimens on a single run. In addition, our collection strategy establishes a “time capsule” to document the evolution of viral strains within our geographical region. Finally, these studies serve to optimize technical parameters for the development of clinical assays. A validated nucleic acid (NA) extraction-free RT-qPCR method will be utilized for this assay. Our internal validation data showed comparable analytical sensitivities to NA extraction-based methods. Pooled samples in transport medium are diluted in normal saline at a ratio of 1:1, and then heat-inactivated in the presence of proteinase-K and ultimately analyzed on the Applied Biosystems™ 7500 Fast Dx instrument. As new variants of interest emerge, primers and probes can be quickly redesigned and validated on clinical samples within our NGS-confirmed “time capsule”. This study will provide important information needed for current or future genomic and epidemiologic studies.