Affordable and time-effective high throughput screening of SARS-CoV-2 variants using Denaturing High-Performance Liquid Chromatography analysis
Abstract
Introduction
Mutations in the receptor binding domain (RBD) region of SARS-CoV-2 have been shown to impact the infectivity, pathogenicity and transmissibility of new variants of concern (VOC). Even more worrisome, those mutations have the potential of causing immune escape, undermining the population immunity induced by ongoing mass vaccination programs.
Gap statement
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. There is an urgent need for novel strategies and techniques aimed at the surveillance of the active emergence and spread of the VOC.
Aim
The aim of this study was to provide a quick, cheap and straightforward Denaturing High-Performance Liquid Chromatography (DHPLC) method for the prompt identification of the SARS-CoV-2 VOC.
Methodology
Two PCRs were designed to target the RBD region, spanning residues N417 through N501 of the Spike protein. Furthermore, a DHPLC screening analysis was set up. The screening consisted of mixing the unknown sample with a standard sample of a known variant, denaturing at high temperature, renaturing at room temperature 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.
Results
The workflow was able to readily detect new variants including the P.1, the B.1.585 and the B.1. 617.2 lineages at a very affordable cost. The DHPLC analysis was robust being able to identify variants even in case of samples with very unbalanced target concentration including those samples at the limit of detection.
Conclusions
This approach has the potential of greatly expediting surveillance of the SARS-CoV-2 variants.
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