Detection of Circulating SARS-CoV-2 Variants of Concern (VOCs) Using a Multiallelic Spectral Genotyping Assay

Area of Science:

• Virology
• Molecular Biology
• Public Health

Background:

• Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continuously evolves, leading to variants of concern (VOCs) with increased transmissibility and immune evasion.
• Traditional methods like next-generation sequencing (NGS) are effective but too slow for rapid outbreak response.
• There is a critical need for fast, accurate diagnostic tools to monitor and screen populations for emerging SARS-CoV-2 VOCs.

Purpose of the Study:

• To develop and validate a novel molecular beacon-based real-time RT-PCR assay for the rapid detection and discrimination of SARS-CoV-2 VOCs.
• To target specific deletions and insertions within SARS-CoV-2 genomes for enhanced diagnostic accuracy.
• To provide a cost-effective and time-efficient alternative to NGS for VOC surveillance.

Main Methods:

• Designed a real-time RT-PCR assay utilizing five molecular beacons targeting specific deletions and insertions in SARS-CoV-2 VOCs.
• Optimized assay conditions to allow all molecular beacons to function simultaneously under identical real-time RT-PCR parameters.
• Validated the assay using reference strains and clinical nasopharyngeal samples previously characterized by NGS.

Main Results:

• The developed assay successfully detected and discriminated between various SARS-CoV-2 VOCs.
• All molecular beacons performed effectively under the same real-time RT-PCR conditions, enhancing efficiency.
• The assay demonstrated high accuracy and reliability in genotype confirmation for tested VOC samples.

Conclusions:

• The molecular beacon-based real-time RT-PCR assay is a valuable tool for rapid and accurate detection and discrimination of SARS-CoV-2 VOCs.
• This assay offers a time- and cost-efficient solution for population screening and monitoring of emerging variants.
• Implementation of this assay can contribute to limiting the spread of SARS-CoV-2 and protecting public health.