Rapid and Accurate Identification of SARS-CoV-2 Variants Using Real Time PCR Assays.

Area of Science:

• Virology
• Genomics
• Molecular Diagnostics

Background:

• Genomic surveillance is crucial for understanding SARS-CoV-2 epidemiology and the impact of viral variants.
• Variants can affect diagnostic tests, transmissibility, disease severity, therapeutic efficacy, and immune evasion.
• Whole-genome sequencing (WGS) is resource-intensive, limiting its widespread use in laboratories.

Purpose of the Study:

• To evaluate the performance of multiplexed real-time reverse transcription-PCR (RT-PCR) assays for identifying SARS-CoV-2 variants.
• To compare RT-PCR variant detection with WGS, including the Omicron variant.

Main Methods:

• SARS-CoV-2 specimens were tested using allele-specific primers and detection technology (PlexPrime® and PlexZyme®) for spike gene mutations.
• RT-PCR detection of specific mutations was compared against WGS results for variant identification.

Main Results:

• RT-PCR detection of spike gene mutations (N501Y, E484K, S982A) showed 100% correlation with WGS for 29 lineages, including Alpha, Beta, and Gamma.
• RT-PCR effectively screened for the Delta variant (B.1.617.2 and AY sublineages) by incorporating the P681R mutation.
• High agreement was observed between RT-PCR and WGS for Delta variant detection (97.8%-100%) and 100% concordance for Omicron and Delta specimens in a blinded panel.

Conclusions:

• Multiplexed real-time RT-PCR genotyping demonstrates strong agreement with WGS for SARS-CoV-2 variant screening.
• RT-PCR offers a viable alternative to WGS when resources are limited or cost is a concern.
• RT-PCR assays can supplement sequencing efforts, providing rapid results for timely patient management.