A pyrosequencing protocol for rapid identification of SARS-CoV-2 variants

Area of Science:

• Virology
• Genomics
• Molecular Diagnostics

Background:

• Next-generation sequencing (NGS) is crucial for monitoring SARS-CoV-2 variants but faces accessibility challenges in low-resource settings.
• Pyrosequencing offers a potentially faster and more cost-effective alternative for targeted mutation detection.

Purpose of the Study:

• To develop and validate a reverse transcription (RT)-PCR-pyrosequencing assay for rapid identification of SARS-CoV-2 variants.
• To assess the assay's ability to differentiate common variants circulating in the United States.

Main Methods:

• Development of an RT-PCR-pyrosequencing assay targeting over 65 spike protein gene (S) mutations in SARS-CoV-2.
• Typing of multiple SARS-CoV-2 variants including Alpha, Omicron, Beta, Delta, and Gamma, with results confirmed by NGS.
• Creation of an electronic tool to assist in variant identification based on pyrosequencing data.

Main Results:

• The assay successfully differentiated commonly reported SARS-CoV-2 variants with high confidence.
• All variants identified by pyrosequencing were confirmed by NGS data.
• The assay demonstrated potential for rapid identification of variants.

Conclusions:

• The developed RT-PCR-pyrosequencing assay is a valuable tool for rapid SARS-CoV-2 variant typing.
• This method can supplement existing surveillance efforts and aid in identifying patients eligible for specific monoclonal antibody treatments.
• The assay's efficiency makes it suitable for resource-limited settings, enhancing global SARS-CoV-2 monitoring.