We have described the successful development of a multiplex RT-qPCR testing platform for the rapid detection of SARS-CoV-2 variant strains using XNA-based molecular clamping technology. In order to develop a sensitive and specific molecular clamping assay, it is not only necessary to select a suitable set of primers and probe for a given mutant gene target, but it is also imperative to choose an XNA with appropriate sequences and desired performance characteristics. Our XNA selection process included a two-fold approach. First, during the sequence design, we excluded those improper sequences with problematic features (e.g., sequences that were too long or too short, had too high or too low Tm values, high purine content, long purine stretch, or unwanted self-complementarity within or between XNA molecules). Secondly, for each mutation assay we designed and synthesized multiple XNAs and compared their qPCR clamping robustness and clamping specificity (higher delta Ct difference between wild-type gene and mutation gene amplification). Notably, across the XNA groups, the XNA with a Tm of nearly 80°C stood out in the selection process, which is likely related to the established RT-qPCR temperature-cycling conditions42.
Using this assay, we were able to track the changes in SARS-CoV-2 variants over time. Due to its higher transmissibility compared to the original SARS-CoV-2 strain, the D614G mutant of SARS-CoV-2 became the dominant variant in the beginning of 2021 in the United States. Its sub-clade, the N501Y mutation independently emerged in the UK and South Africa and subsequently spread to North America during the first half of 2021. Our data indicated that the spread of this Alpha variant in northern California likely started in February 2021 because patient samples collected in January 2021 were all negative for the N501Y mutation, whereas the N501Y mutation was detectable in up to 5% of the samples collected in late February 2021. Although the U.S. CDC predicted that, according to an epidemiology model 43 at that time, the B.1.1.7 or Alpha variant would quickly dominate soon, in actuality the Alpha variant was soon largely replaced around the middle of 2021 by the even more potent and infectious Delta variant,. The Delta variant in turn was soon replaced by the more contagious Omicron variants in late 2021 to early 2022. Our RT-qPCR data, confirmed by Sanger or NGS sequencing, were consistent with the sequential surges of COVID-19 cases caused by the D614G mutant and the Alpha, Delta and Omicron variants in the United Stated from late 2020 to early 2022.
Availability of rapid and accurate testing platforms is critical for tackling the challenge of emerging SARS-CoV-2 variants. Amongst the many detection methods, a qPCR-based platform could serve as a rapid, cost-effective and practical testing tool for monitoring SARS-CoV-2 evolution.
A few biotechnology companies and academic institutions have been developing or have reported variant detection methods using qPCR. These methods can be categorized into one of the following: (1) mutant gene-specific or allele-specific primers and probe; (2) spike gene target failure (SGTF); (3) E gene target failure, and (4) ORF gene deletion 44–47. Some of the methods still require NGS to confirm the results, whereas other platforms require pre-testing by regular SARS-CoV-2 RT-qPCR before carrying out the variant assay. While these assays can be useful in certain circumstances 48 also emphasized the cost-effectiveness, quick and scalability, they are limited by the effectiveness and accuracy of the primers and/or probes used. This issue can limit the use of these variant testing methodologies. For example, the allele-specific PCR (AS-PCR) method quickly attracted attention for mutation detection by some test developers 49. However, AS-PCR has two inherent shortcomings: (1) due to the fixed 3′ end of the allele-specific primer, it is not always feasible to choose optimal primers for PCR amplification; and (2). high purity DNA/RNA is essential as low-quality or crude DNA/RNA samples are prone to providing inconclusive results 50.
However, XNA-based qPCR can overcome these aforementioned shortcomings because XNA sequences can be designed with more flexibility compared to restrictive primer sequence design 51, and XNA-based qPCR works better with samples of low-concentration DNA/RNA and samples with high-background mutant gene targets. Compared to AS-PCR, as demonstrated in our studies, the XNA-based RT-qPCR assay can achieve a lower LOD, about 100 copies/mL for variant detection.
The molecular clamping technology used in this study increases the sensitivity and specificity of conventional qPCR as wild-type background amplification is minimized by this method. This XNA clamping-based RT-qPCR assay can also improve multiplexing of targets, as we were able to differentiate the wild-type SARS-CoV-2 (ORF1ab gene for wild-type detection) and its variants in a single run. In addition to detecting the existing SARS-CoV-2 mutations D614G and N501Y, the assay described here also detected L452R, T478K, K417N and K417T. The presence or absence of these major mutations can be used to identify all five VOCs based on their unique mutation profiles VOCs 52 (Supplementary Table 1). As a result, this assay covers almost all SARS-CoV-2 variants (both VOC and VOI), including Delta, “Delta Plus” and Omicron, all of which have the potential to breakthrough vaccine-induced protective immunity 52. This strategy can provide an effective, rapid and easily adoptable testing platform for known and emerging SARS-CoV-2 variants in the future, and this technology can be easily adopted by any clinical laboratories that perform routine SARS-CoV-2 RT-qPCR testing. Given the past stages of the pandemic, future new variant(s) with increased transmissibility could possibly lead to a potential exponential increase in cases and/or deaths 10. Therefore, timely and convenient detection of the concerning variants is of high importance.
There are 15 mutations in the RBD of Omicron variant, including Q393R, K417N, T478K, S477N, E484A, G496S, Q498R, Y505H, G446S, N501Y, N440K, S75F, S373P, S371L and G339D 26. This posts a significant challenge for RT-qPCR based assays as this high number of mutations can render some primers dysfunctional. Interestingly, the N501Y primer, which works well for Alpha, Beta and Delta variant detection, had a lower signal for N501Y detection in Omicron variants due to the fact that the original N501Y primer was located in Omicron high mutant region. To avoid this issue in the future, we have re-designed new set of “degenerate” primers and probe to better fit the mutant area in Omicron, while still covering the original N501Y region in Alpha, Beta and Delta variants. Evaluation of this revised N501Y assay design showed that it is effective in distinguishing these variants effectively. This illustrates how this method can be rapidly adapted to test new and emerging variants while still successfully detecting older variants.
Despite this issue, our XNA-based RT-qPCR assay kit was able to identify the Omicron variant because of the presence of three mutations D614G/T478K/K417N and the absence of L452R mutation, a combination unique to the Omicron variant. Indeed, we were able to detect and differentiate Omicron and Delta variants from successfully detected the Omicron variant in patients that had breakthrough SARS-CoV-2 infections in early January 2022.
In summary, we have developed a multiplex RT-qPCR testing platform for the rapid detection of SARS-CoV-2 variants using the XNA-based molecular clamping technology. This testing platform can be easily adopted by laboratories that perform routine SARS-CoV-2 PCR testing, providing a rapid and cost-effective method in lieu of NGS-based assays, for detecting, differentiation and monitoring SARS-CoV-2 variants. In addition, this assay is easily scalable to any new variant(s) should it emerge.