A Sub-Hour Point-of-Care Testing of SARS-CoV-2 Variants Identified by XNA-Based RT-qPCR

Author(s): Jonathan Li, Hong Huang, Shuo Shen, Andrew Y. Fu, Ang Cao, Qing Sun, Daniel Cataldi, Sabrina Li, Chuanyi M. Lu, Aiguo Zhang, Michael Y. Sha

Point-of-care testing (POCT) is a medical diagnostic testing at the time and place of patient care, or called bedside or near-the-patient testing. POCT has been fast-growing and showed tremendous potential in the era of precision diagnostics and personal medicine. This is exemplified by the numerous and widely-used fast antigen POCT tests (15-min only) during the COVID-19 pandemic globally. During the COVID-19 pandemic peak period while the major variants appeared, developing a fast POCT detection and identification method of SARS-CoV-2 variants attracted our interest at DiaCarta. We combined the POCT PCR system from Coyote Bioscience and our established XNA-based RT-qPCR method to detect and identify the major early virus variants as a Proof-of-Concept feasibility study. Our multiplex molecular assay uses RT-qPCR with the aid of xenonucleic acids (XNA) as molecular-clampers which are artificial genetic polymers with Watson-Crick base-pairing capability and robust DNA/RNA-binding ability, high stability, and particularly the appealing specificity --- even a single-base mismatch in XNA/DNA duplex can result in a drop of 10 – 18 °C in the melting temperature. This allows for specific and reliable detection of the SARS-CoV-2 variants, based on the unique combination of characteristic mutations on the viral spike protein domain. Our data showed that XNA-based RT-qPCR by Flash20 instrument successfully detected SARS-CoV-2 Delta and Delta Plus variants from infected or break-through patients. Furthermore, Flash20 POCT instrumentation allows for extraction-free PCR procedure. The turn-around time for Delta Variant kit on Flash20 is less than one hour --- only about 50 minutes of POCT versus regularly 4 hours for the non-POCT assay. As this pilot feasibility study, SARS-CoV-2 variants were rapidly detected with great sensitivity and specificity (PPV 100% and NPV 100%) in a randomly chosen set of clinical samples.