Emergency SARS-CoV-2 Variants of Concern: Novel Multiplex Real-Time RT-PCR Assay for Rapid Detection and Surveillance

Abstract

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread worldwide. Many variants of SARS-CoV-2 have been reported, some of which have increased transmissibility and/or reduced susceptibility to vaccines. There is an urgent need for variant phenotyping for epidemiological surveillance of circulating lineages. Whole-genome sequencing is the gold standard for identifying SARS-CoV-2 variants, which constitutes a major bottleneck in developing countries. Methodological simplification could increase epidemiological surveillance feasibility and efficiency. We designed a novel multiplex real-time reverse transcriptase PCR (RT-PCR) to detect SARS-CoV-2 variants with S gene mutations. This multiplex PCR typing method was established to detect 9 mutations with specific primers and probes (ΔHV 69/70, K417T, K417N, L452R, E484K, E484Q, N501Y, P681H, and P681R) against the receptor-binding domain of the spike protein of SARS-CoV-2 variants. In silico analyses showed high specificity of the assays. Variants of concern (VOC) typing results were found to be highly specific for our intended targets, with no cross-reactivity observed with other upper respiratory viruses. The PCR-based typing methods were further validated using whole-genome sequencing and a commercial kit that was applied to clinical samples of 250 COVID-19 patients from Taiwan. The screening of these samples allowed the identification of epidemic trends by time intervals, including B.1.617.2 in the third Taiwan wave outbreak. This PCR typing strategy allowed the detection of five major variants of concern and also provided an open-source PCR assay which could rapidly be deployed in laboratories around the world to enhance surveillance for the local emergence and spread of B.1.1.7, B.1.351, P.1, and B.1.617.2 variants and of four Omicron mutations on the spike protein (ΔHV 69/70, K417N, N501Y, P681H). IMPORTANCE COVID-19 has spread globally. SARS-CoV-2 variants of concern (VOCs) are leading the next waves of the COVID-19 pandemic. Previous studies have pointed out that these VOCs may have increased infectivity, have reduced vaccine susceptibility, change treatment regimens, and increase the difficulty of epidemic prevention policy. Understanding SARS-CoV-2 variants remains an issue of concern for all local government authorities and is critical for establishing and implementing effective public health measures. A novel SARS-CoV-2 variant identification method based on a multiplex real-time RT-PCR was developed in this study. Five SARS-CoV-2 variants (Alpha, Beta, Gamma, Delta, and Omicron) were identified simultaneously using this method. PCR typing can provide rapid testing results with lower cost and higher feasibility, which is well within the capacity for any diagnostic laboratory. Characterizing these variants and their mutations is important for tracking SAR-CoV-2 evolution and is conducive to public infection control and policy formulation strategies.

Keywords: E484K; E484R; K417N; K417T; L452R; N501Y; P681H; P681R; SARS-CoV-2; VOC genotyping; epidemiological surveillance; ΔHV 69/70.

FIG 1

Five variants of interest (VOIs) or variants of concern (VOCs) of SARS-CoV-2 strains collected from GISAID. (A) VOI (K417T and P681H) and VOC (ΔHV69-79, K417N, L452R, E484K, E484Q, N501Y, and P681R). (B) VOI and VOC positions in SARS-CoV-2 spike protein structure and related biological effects. Lime, ΔHV69-70; violet, K417; olive, L452; orange, E484; dodger blue, N501; red, D614; light sea green, P681.

FIG 2

VOI/VOC sequence-specific forward and reverse primers and probes designed for TaqMan SNP genotyping assays. (A) Spike gene position in SARS-CoV-2 genome and its amino acid functional annotation. (B) Specific primer-pair (arrow bar) and probe (long bar) positions in spike gene. Red boxes show detected mutations. Yellow, ΔHV69-70; light violet, K417N and 417T; green, L452R; brown, E484K and E484Q; gray, N5011Y; and pink, P681H and P681R.

FIG 3

Flow chart of interpretation of the VOC assays.