Abstract
Background: The SARS-CoV-2 virus has assumed considerable importance during the COVID-19 pandemic. Its mutation rate is high, involving the spike (S) gene and thus there has been a rapid spread of new variants. Herein, we describe a rapid, easy, adaptable, and affordable workflow to uniquely identify all currently known variants through as few analyses. Our method only requires two conventional PCRs of the S gene and two Sanger sequencing reactions, and possibly another PCR/sequencing assay on a N gene portion to identify the B.1.160 lineage. Methods: We selected an S gene 1312 bp portion containing a set of SNPs useful for discriminating all variants. Mathematical, statistical, and bioinformatic analyses demonstrated that our choice allowed us to identify all variants even without looking for all related mutations, as some of them are shared by different variants (e.g., N501Y is found in the Alpha, Beta, and Gamma variants) whereas others, that are more informative, are unique (e.g., A57 distinctive to the Alpha variant). Results: A “weight” could be assigned to each mutation that may be present in the selected portion of the S gene. The method’s robustness was confirmed by analyzing 80 SARS-CoV-2-positive samples. Conclusions: Our workflow identified the variants without the need for whole-genome sequencing and with greater reliability than with commercial kits.
Highlights
Introduction iationsIn the continuous struggle among organisms, variability is the key to success
We describe a rapid, easy, adaptable, and affordable workflow to detect all mutations related to the SARS-CoV-2 variants described by the WHO [11], including theDelta variant, based on two conventional PCRs and two Sanger sequencing reactions
Recent genotyping analysis demonstrated that genes that encode the spike protein, other nucleoprotein, and RNA polymerase undergo very frequent mutations
Summary
Introduction iationsIn the continuous struggle among organisms, variability is the key to success. Mutations are random events that occur during the virus replication which can be selected or not, by the action of drugs or the immune system. SARS-CoV-2, identified in Wuhan in 2019, is not an exception, and mutations in the spike (S) gene, responsible for the attachment and penetration of the virus into the cell, lead to new and more transmissible lineages that, in turn, are more likely to replicate and acquire other mutations. Its mutation rate is high, involving the spike (S) gene and there has been a rapid spread of new variants. We describe a rapid, easy, adaptable, and affordable workflow to uniquely identify all currently known variants through as few analyses. PCR/sequencing assay on a N gene portion to identify the B.1.160 lineage. Conclusions: Our workflow identified the variants without the need for whole-genome sequencing and with greater reliability than with commercial kits
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