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
Date
2022-12-16Publisher
Πανεπιστήμιο Κύπρου, Σχολή Θετικών και Εφαρμοσμένων Επιστημών / University of Cyprus, Faculty of Pure and Applied SciencesPlace of publication
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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.