Real-time quantitative PCR is currently the most widely used method for the human pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) identification. Due to the rapid evolution of the SARS-CoV-2 genome, novel mutations on the primer binding sites will cause the failure of PCR. Therefore, in addition to a well-designed primer set, these primers need to be updated and evaluated regularly to ensure that the rapidly evolving genome primers can be amplified. In this protocol, (1) we firstly use assembled genome sequences in the SARS-CoV-2 database to identify and characterize indels and point mutations; (2) design primers skipping the sites of mutations; (3) check the coverage of the primers with the daily update SARS-CoV-2 database; (4) redesign them if novel mutations found in the primer binding sites. Although this protocol takes SARS-CoV-2 as an example, it is suitable for other species that have genomes accumulating mutations over time.
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