Novel Lateral Flow-Based Assay for Simple and Visual Detection of SARS-CoV-2 Mutations

Abstract

Identification of the main SARS-CoV-2 variants in real time is of interest to control the virus and to rapidly devise appropriate public health responses. The RT-qPCR is currently considered to be the reference method to screen SARS-CoV-2 mutations, but it has some limitations. The multiplexing capability is limited when the number of markers to detect increases. Moreover, the performance of this allele-specific method may be impacted in the presence of new mutations. Herein, we present a proof-of-concept study of a simple molecular assay to detect key SARS-CoV-2 mutations. The innovative features of the assay are the multiplex asymmetric one-step RT-PCR amplification covering different regions of SARS-CoV-2 S gene and the visual detection of mutations on a lateral flow DNA microarray. Three kits (Kit 1: N501Y, E484K; Kit 2: L452R, E484K/Q; Kit 3: K417N, L452R, E484K/Q/A) were developed to match recommendations for surveillance of SARS-CoV-2 variants between January and December 2021. The clinical performance was assessed using RNA extracts from 113 SARS-CoV-2-positive samples with cycle thresholds <30, and results demonstrated that our assay allows specific and sensitive detection of mutations, with a performance comparable to that of RT-qPCR. The VAR-CoV assay detected four SARS-CoV-2 targets and achieved specific and sensitive screening of spike mutations associated with the main variants of concern, with a performance comparable to that of RT-qPCR. With well-defined virus sequences, this assay can be rapidly adapted to other emerging mutations; it is a promising tool for variant surveillance.

Rapid and Sensitive Genotyping of SARS-CoV-2 Key Mutation L452R with an RPA-PfAgo Method

“…The single-copy sensitivity is the highest sensitivity that can be achieved theoretically, and is also the highest sensitivity as compared to the available SARS-CoV-2 genotyping assays. 34 − 36 According to the reports, the nucleic acid amplification and probe-based assays had sensitivities in the range of tens to hundreds of copies per reaction. 37 − 39 The CRISPR-based assays had sensitivities of around 10 copies per reaction.…”

“…According to the fluorescence signals, with as low as 10 0 copies of the SARS-CoV-2 mutant (MT) RNA, the L452R mutation could be identified (Figure ). The single-copy sensitivity is the highest sensitivity that can be achieved theoretically, and is also the highest sensitivity as compared to the available SARS-CoV-2 genotyping assays. − According to the reports, the nucleic acid amplification and probe-based assays had sensitivities in the range of tens to hundreds of copies per reaction. − The CRISPR-based assays had sensitivities of around 10 copies per reaction. , By fully utilizing Pf Ago’s specific recognition and cleavage property, the RPA- Pf Ago method exhibited a solid advantage in terms of sensitivity with which a single copy could be sufficient for the mutation identification (Figure ). The single-copy sensitivity is the highest sensitivity that can be achieved theoretically and is also the highest sensitivity as compared to the available SARS-CoV-2.…”