Affordable and time-effective high throughput screening of SARS-CoV-2 variants using Denaturing High-Performance Liquid Chromatography analysis

Mutations in the receptor binding domain region of SARS-CoV-2 have been shown to impact the infectivity, pathogenicity and transmissibility of new variants. Even more worrisome, those mutations have the potential of causing immune escape, undermining the population immunity induced by ongoing mass vaccination programs.The massive parallel sequencing techniques have taken a lead role in the detection strategies of the new variants. Nevertheless, they are still cumbersome and labour-demanding. As a matter of fact, there is an urgent need for novel strategies and techniques aimed at the surveillance of the active emergence and spread of the variants of concern.Two PCRs were designed to target the binding domain region, spanning residues N417 through N501 of the Spike protein. Furthermore, very rapid, affordable and straightforward Denaturing High-Performance Liquid Chromatography screening was set up. The screening consisted of mixing the unknown sample with a standard sample of a known sequence, denaturing at high temperature for two minutes, renaturing for 15 minutes followed by a 2-minute run using the WAVE® DHPLC system to detect the heteroduplexes which invariably originate whenever the unknown sample has a nucleotide difference with respect to the standard used. The workflow was able to readily detect new variants including the P.1 and the B.1.585 strains at a very affordable cost. This approach has the potential of greatly expediting surveillance of the SARS-CoV-2 variants.