A tool for the cheap and rapid screening of SARS-CoV-2 variants of concern (VoCs) by Sanger sequencing.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging virus that, since March 2020, has been responsible for a global and ongoing pandemic. Its rapid spread over the past nearly 3 years has caused novel variants to arise. To monitor the circulation and emergence of SARS-CoV-2 variants, surveillance systems based on nucleotide mutations are required. In this regard, we searched in the spike, ORF8, and nucleocapsid genes to detect variable sites among SARS-CoV-2 variants. We describe polymorphic genetic regions that enable us to differentiate between the Alpha, Beta, Gamma, Delta, and Omicron variants of concern (VoCs). We found 21 relevant mutations, 13 of which are unique for Omicron lineages BA.1/BA.1.1, BA.2, BA.3, BA.4, and BA.5. This genetic profile enables the discrimination between VoCs using only four reverse transcription PCR fragments and Sanger sequencing, offering a cheaper and faster alternative to whole-genome sequencing for SARS-CoV-2 surveillance. IMPORTANCE Our work describes a new (Sanger sequencing-based) screening methodology for SARS-CoV-2, performing PCR amplifications of a few target regions to detect diagnostic mutations between virus variants. Using the methodology developed in this work, we were able to discriminate between the following VoCs: Alpha, Beta, Gamma, Delta, and Omicron (BA.1/BA.1.1, BA.2, BA.3, BA.4, and BA.5). This becomes important, especially in low-income countries where current methodologies like next-generation sequencing have prohibitive costs. Furthermore, rapid detection would allow sanitary authorities to take rapid measures to limit the spread of the virus and therefore reduce the probability of new virus dispersion. With this methodological approach, 13 previously unreported diagnostic mutations among several Omicron lineages were found.