Efficient SARS-CoV-2 Surveillance during the Pandemic-Endemic Transition Using PCR-Based Genotyping Assays

Abstract

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern (VOC) pose an increased risk to public health due to higher transmissibility and/or immune escape. In this study, we assessed the performance of a custom TaqMan SARS-CoV-2 mutation panel consisting of 10 selected real-time PCR (RT-PCR) genotyping assays compared to whole-genome sequencing (WGS) for identification of 5 VOC circulating in The Netherlands. SARS-CoV-2 positive samples (N = 664), collected during routine PCR screening (15 ≤ C_T ≤ 32) between May-July 2021 and December 2021-January 2022, were selected and analyzed using the RT-PCR genotyping assays. VOC lineage was determined based on the detected mutation profile. In parallel, all samples underwent WGS with the Ion AmpliSeq SARS-CoV-2 research panel. Among 664 SARS-CoV-2 positive samples, the RT-PCR genotyping assays classified 31.2% as Alpha (N = 207); 48.9% as Delta (N = 325); 19.4% as Omicron (N = 129), 0.3% as Beta (N = 2), and 1 sample as a non-VOC. Matching results were obtained using WGS in 100% of the samples. RT-PCR genotyping assays enable accurate detection of SARS-CoV-2 VOC. Furthermore, they are easily implementable, and the costs and turnaround time are significantly reduced compared to WGS. For this reason, a higher proportion of SARS-CoV-2 positive cases in the VOC surveillance testing can be included, while reserving valuable WGS resources for identification of new variants. Therefore, RT-PCR genotyping assays would be a powerful method to include in SARS-CoV-2 surveillance testing. IMPORTANCE The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genome changes constantly. It is estimated that there are thousands of variants of SARS-CoV-2 by now. Some of those variants, variants of concern (VOC), pose an increased risk to public health due to higher transmissibility and/or immune escape. Pathogen surveillance helps researchers, epidemiologists, and public health officials to monitor the evolution of infectious diseases agents, alert on the spread of pathogens, and develop counter measures like vaccines. The technique used for the pathogen surveillance is called sequence analysis which makes it possible to examine the building blocks of SARS-CoV-2. In this study, a new PCR method based on the detection of specific changes of those building blocks is presented. This method enables a fast, accurate and cheap determination of different SARS-CoV-2 VOC. Therefore, it would be a powerful method to include in SARS-CoV-2 surveillance testing.

Keywords: RT-PCR genotyping assays; SARS-CoV-2; variant of concern.

FIG 1

SARS-CoV-2 variants of concern detected in The Netherlands, 2021-2022. (A) Nine RT-PCR genotyping assays that were selected to test the randomly selected SARS-CoV-2 positive samples in the period of May-July 2021 (N = 331). (B) Between May and July 2021 (N = 331), the Delta variant (N = 121) became dominant over the Alpha variant (N = 207). (C) Three RT-PCR genotyping assays that were selected to test the randomly selected SARS-CoV-2 positive samples in the period of December 2021-January 2022 (N = 333). (D) During December 2021 and January 2022 (N = 333), the Omicron variant (N = 129) became dominant over the Delta variant (N = 204).

FIG 2

Phylogenetic tree of SARS-CoV-2 variants of concern detected in The Netherlands, 2021-2022. A total of 669 SARS-CoV-2 genomes, including 660 of the 664 sequenced genomes from this study plus 9 reference sequences from GenBank, are depicted. Four genomes were excluded due to multiple regions with inconclusive sequencing. The sequences (N = 669) show clusters matching the 9 known SARS-CoV-2 clades included in this study: 19A (original Wuhan strain), 20D (non-VOC/VOI SARS-CoV-2 lineage C.36.3.1), 20H (Beta), 20I (Alpha), 20J (Gamma), 21I (Delta), 21J (Delta), 21K (Omicron, subtype BA.1), and 21L (Omicron, subtype BA.2).