Development of a de novo Molecular Beacon-Based Real-Time RT-PCR Assay for the Detection and Discrimination of SARS-CoV-2 Variants of Concern

Abstract

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) emerged in late 2019 and is the causative agent of the Coronavirus Disease 2019 (COVID-19). Since its emergence it has led to a global pandemic with devastating consequences on public health. As of November 2022, there are more than 646 million SARS-CoV-2 cases and more than 6 million deaths worldwide attributed to this virus. The high rate of SARS-CoV-2 transmission and replication coupled with the constant evolutionary pressures that it receives, contribute to the accumulation of mutations and the emergence of novel variants, which can have increased epidemiological and clinical impact. In fact, some variants were termed as Variants of Concern (VOC), since they confer increased transmissibility/infectivity, immune evasion, and virulence posing a massive threat on public health. Five VOC have been designated by the World Health Organization hitherto: the Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529) VOC/lineages along with their sublineages. The constant emergence and circulation of VOC, emphasize the urgent need for the development of methods, such as Next Generation Sequencing (NGS) and Real-time Reverse Transcription-Polymerase Chain Reaction (real-time RT-PCR), that allow population screening and variant identification. Even though NGS can yield a significant amount of information, it is time-consuming, and not ideal during periods of outbreaks where screening and identifying a variant are a priority. On the other hand, real-time RT-PCR, using probes like molecular beacons, is more suitable, since it takes less time, and is highly sensitive and specific. Thus, this study focused on the development of a de novo molecular beacon-based real-time RT-PCR assay, that aimed to detect and discriminate SARS-CoV-2 VOC according to the principles of spectral genotyping. Specifically, five molecular beacons were designed to target specific deletions and an insertion, that are highly prevalent within their respective VOC: ORF1a:ΔS3675/G3676/F3677, S:ΔH69/V70, S:ΔE156/F157, S:ΔN211, S:ins214EPE, and S:ΔL242/A243/L244. The selection of these targets was a crucial design aspect of the assay, since deletions/insertions confer greater discriminatory power, in contrast to single amino acid substitutions. Following their design, all molecular beacons were examined to ascertain their thermodynamic properties, and it was confirmed that they can be used under the same real-time RT-PCR conditions, consequently improving the time and cost efficiency of the assay. The specificity of the assay was tested through real-time RT-PCR using reference and clinical samples whose lineages were known beforehand. The reference samples were obtained from the European Virus Archive goes Global (EVAg, Charité, Berlin) and were derived from cultured SARS-CoV-2 2 virus, while the clinical samples were collected using nasopharyngeal swabs from infected individuals. After performing RNA extraction and real-time RT-PCR, it was confirmed that the molecular beacons correctly identified their targets, showing that this assay can be reliably used for the detection and discrimination of VOC. Therefore, this assay provides a fast, accurate and flexible method that allows screening of the population for the detection and discrimination of SARS-CoV-2 VOC, or any emerging high-risk variant, consequently contributing to the ongoing fight against the COVID-19 pandemic.