A Simplified Sanger Sequencing Method for Detection of Relevant SARS-CoV-2 Variants

Area of Science:

• * Virology
• * Molecular Biology
• * Public Health

Background:

• * Genomic surveillance is crucial for tracking viral evolution and the emergence of significant SARS-CoV-2 variants.
• * New sequencing technologies have enhanced molecular surveillance, but accessible methods are needed for widespread application.
• * Sanger sequencing offers a cost-effective alternative for specific variant identification without requiring whole-genome sequencing.

Purpose of the Study:

• * To develop and validate an accessible Sanger sequencing protocol for the molecular surveillance of SARS-CoV-2 variants.
• * To identify and characterize prevalent SARS-CoV-2 variants circulating between June 2020 and February 2022.
• * To provide a more accessible platform for viral surveillance using established sequencing technology.

Main Methods:

• * A two-step RT-PCR assay was employed to amplify a 1120 bp product from SARS-CoV-2 positive samples.
• * A 1000 bp region of interest was targeted for Sanger sequencing.
• * Consensus sequences were constructed, and mutations were identified to characterize viral variants. Sequence alignment was performed against reference variant sequences.

Main Results:

• * A total of 103 SARS-CoV-2 positive samples were sequenced.
• * 69 samples harbored significant variants, including 20 BA.1, 13 Delta, 22 Gamma, and 14 Zeta variants.
• * The developed Sanger sequencing protocol proved effective in identifying these key variants.

Conclusions:

• * Sanger sequencing provides a viable and accessible method for the molecular surveillance of SARS-CoV-2 variants.
• * The developed protocol can assist public health efforts in tracking viral evolution and variant circulation.
• * This accessible platform enhances the capacity for global viral surveillance, particularly in resource-limited settings.