Developing an Amplification Refractory Mutation System-Quantitative Reverse Transcription-PCR Assay for Rapid and Sensitive Screening of SARS-CoV-2 Variants of Concern

Abstract

With the emergence and wide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs), such as the Delta variant (B.1.617.2 lineage and AY sublineage), it is important to track VOCs for sourcing of transmission. Currently, whole-genome sequencing is commonly used for detecting VOCs, but this is limited by the high costs of reagents and sophisticated sequencers. In this study, common mutations in the genomes of SARS-CoV-2 VOCs were identified by analyzing more than 1 million SARS-CoV-2 genomes from public data. Among them, mutations C1709A (a change of C to A at position 1709) and C56G, respectively, were found in more than 99% of the genomes of Alpha and Delta variants and were specific to them. Then, a method using the amplification refractory mutation system combined with quantitative reverse transcription-PCR (ARMS-RT-qPCR) based on the two mutations was developed for identifying both VOCs. The assay can detect as little as 1 copy/μL of the VOCs, and the results for identifying Alpha and Delta variants in clinical samples by the ARMS-RT-qPCR assay showed 100% agreement with the results using sequencing-based methods. The whole assay can be completed in 2.5 h using commercial fluorescent PCR instruments. Therefore, the ARMS-RT-qPCR assay could be used for screening the two highly concerning variants Alpha and Delta by normal PCR laboratories in airports and in hospitals and other health-related organizations. Additionally, based on the unique mutations identified by the genomic analysis, similar molecular assays can be developed for rapid identification of other VOCs. IMPORTANCE The current stage of the pandemic, led by SARS-CoV-2 variants of concern (VOCs), underscores the necessity to develop a cost-effective and rapid molecular diagnosis assay to differentiate the VOCs. In this study, over 1 million SARS-CoV-2 genomic sequences of high quality from GISAID were analyzed and a network of the common mutations of the lineages was constructed. The conserved unique mutations specific for SARS-CoV-2 VOCs were found. Then, ARMS-RT-qPCR assays based on the two unique mutations of the Alpha and Delta variants were developed for the detection of the two VOCs. Application of the assay in clinical samples demonstrated that the current method is a convenient, cost-effective, and rapid way to screen the target SARS-CoV-2 VOCs.

Keywords: ARMS-RT-qPCR; SARS-CoV-2 variants of concern; conserved unique mutation; rapid; sensitive.

FIG 1

Identification of unique mutations of the SARS-CoV-2 variants of concern and variants under monitoring (VOCs and VUMs). (A) The relationship network between the common mutations of VOCs Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2), VUMs Kappa (B.1.617.1), Eta (B.1.525), and Iota (B.1.526), and other lineages. Among these common mutations, each mutation connected with only one lineage was considered a unique mutation for the corresponding lineage and the others, linked to at least two different lineages, were regarded as shared mutations. Additionally, the mutations shared in more than 5 different lineages are not shown in the network. (B) The genome-wide common-mutation landscapes of the two rapidly and widely spread variants Alpha (B.1.1.7) and Delta (B.1.617.2).

FIG 2

ARMS-RT-qPCR assay development. (A) Consistent comparison of the fragment amplified by the virus control targeting RBD in SARS-CoV-2 variants. (B) Validation of the two primer/probe sets for detecting the two VOCs (ND, not detectable). (C) Analytical sensitivity of the two primer/probe sets of the two VOCs to screen the corresponding VOCs. C_T, cycle threshold. Gray shadows indicate that nucleic acid cannot be amplified effectively.

FIG 3

Determination of SARS-CoV-2 Alpha (B.1.1.7) and Delta (B.1.617.2) in clinical samples through the ARMS-RT-qPCR assay. C_T, cycle threshold. Note that a C_T value of 45 was used to calculate ΔC_T when there was no amplification.

FIG 4

Scheme of the process of the ARMS-RT-qPCR assay for detecting SARS-CoV-2 variants.