RT-qPCR Assays for Rapid Detection of the N501Y, 69-70del, K417N, and E484K SARS-CoV-2 Mutations: A Screening Strategy to Identify Variants With Clinical Impact

Abstract

BackgroundSeveral variants of the SARS-CoV-2 have been documented globally during the current COVID-19 pandemic. The N501Y, 69-70del, K417N, and E484K SARS-CoV-2 mutations have been documented among the most relevant due to their potential pathogenic biological effects. This study aimed to design, validate, and propose a fast real-time RT-qPCR assay to detect SARS-CoV-2 mutations with possible clinical and epidemiological relevance in the Mexican population.MethodsTargeting spike (S) gene mutations of SARS-CoV-2 (N501Y, 69-70del, K417N, and E484K), specific primers, and probes for three specific quantitative reverse transcription PCR (RT-qPCR) assays were designed, and validated using Sanger sequencing. These assays were applied in clinical samples of 1060 COVID-19 patients from Jalisco Mexico.ResultsIn silico analyzes showed high specificity of the three assays. Amplicons of samples were confirmed through sequencing. The screening of samples of COVID-19 patients allowed the identification of the E484K mutation in nine individuals and the identification of P.2 Brazilian variant in Mexico.ConclusionThis work provides low-cost RT-qPCR assays for rapid screening and molecular surveillance of mutations with potential clinical impact. This strategy allowed the detection of E484K mutation and P.2 variant for the first time in samples from the Mexican population.

Limitation of Screening of Different Variants of SARS-CoV-2 by RT-PCR

“…Recent studies have evaluated different screening kits in comparison to NGS. The authors demonstrated a high concordance between the two methods, reinforcing the interest of screening methods [ 20 , 21 , 22 , 23 , 24 ]. Here, we showed that the PerkinElmer kit performed better than the ID Solution kit, avoiding the problem of mutation detection, notably the detection of E484K mutation.…”

Rapid Automated Screening for SARS-CoV-2 B.1.617 Lineage Variants (Delta/Kappa) through a Versatile Toolset of qPCR-Based SNP Detection

“…Interestingly, interactions of NTD deletions with a popular commercial PCR assay (TaqPath SARS-CoV-2 Assay, Thermo Fischer, Waltham, MA, USA) first directed attention to the expanding B.1.1.7 lineage in Great Britain, thereby hinting at the potential utility of specific qPCR tests for variant prediction [28,29]. The recent surge in publications regarding PCR-typing demonstrates the wide spectrum of potential methods and their varying degrees of suitability for practical use in diagnostics [9][10][11][12]14].…”

“…Spike gene mutations such as N501Y, E484K/Q, L452R, and P681H/R have occurred independently in multiple VOCs and VOIs (variants of interest), enabling dynamic adaptation of existing assay designs to detect emerging SARS-CoV-2 variants [7]. RT-qPCR is the gold standard for detection of SARS-CoV-2 in clinical samples [8] and has also been used for SARS-CoV-2 spike gene mutation detection in previous studies, often focusing on the HV69/70 deletion and the N501Y SNP (single nucleotide polymorphism) using various methods such as specific primers, simple probes, and melt curve analyses [9][10][11][12][13][14][15]. Locked nucleic acid (LNA)-Probes represent a well described method for specifically detecting SNPs by RT-PCR [16][17][18].…”

RT-qPCR assays for SARS-CoV-2 variants of concern in wastewater reveals compromised vaccination-induced immunity

“…Such an assay can help assess a variant's frequency within a population and contribute to policy guidelines. Indeed a number of publications targeting variants of concern detection using RT-qPCR ( Barreto et al, 2020 ; Bivins et al, 2021 ; Gand et al, 2021 ; Vega-Magaña et al, 2021 ; Vogels et al, 2021 ; H. Wang et al, 2021 ; Yaniv et al, 2021 ) as well as available commercial kits (e.g., TaqPath ThermoScientific, GT molecular, PerkinElmer) have emerged for individual assessments.…”