ABSTRACT
As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to circulate, multiple variants of concern (VOC) have emerged. New variants pose challenges for diagnostic platforms since sequence diversity can alter primer/probe binding sites (PBS), causing false-negative results. The Agena MassARRAY(R) SARS-CoV-2 Panel utilizes reverse-transcription polymerase chain reaction and mass-spectrometry to detect five multiplex targets across N and ORF1ab genes. Herein, we utilize a dataset of 256 SARS-CoV-2-positive specimens collected between April 11, 2021-August 28, 2021 to evaluate target performance with paired sequencing data. During this timeframe, two targets in the N gene (N2, N3) were subject to the greatest sequence diversity. In specimens with N3 dropout, 69% harbored the Alpha-specific A28095U polymorphism that introduces a 3-mismatch to the N3 forward PBS and increases risk of target dropout relative to specimens with 28095A (relative risk (RR): 20.02; p<0.0001; 95% Confidence Interval (CI): 11.36-35.72). Furthermore, among specimens with N2 dropout, 90% harbored the Delta-specific G28916U polymorphism that creates a 3-mismatch to the N2 probe PBS and increases target dropout risk (RR: 11.92; p<0.0001; 95% CI: 8.17-14.06). These findings highlight the robust capability of Agena MassARRAY(R) SARS-CoV-2 Panel target results to reveal circulating virus diversity and underscore the power of multi-target design to capture VOC.
Subject(s)
Coronavirus InfectionsABSTRACT
The COVID-19 pandemic sparked rapid development of SARS-CoV-2 diagnostics. However, emerging variants pose the risk for target dropout and false-negative results secondary to primer/probe binding site (PBS) mismatches. The Agena MassARRAY(R) SARS-CoV-2 Panel combines RT-PCR and MALDI-TOF mass-spectrometry to probe for five targets across N and ORF1ab genes, which provides a robust platform to accommodate PBS mismatches in divergent viruses. Herein, we utilize a deidentified dataset of 1,262 SARS-CoV-2-positive specimens from Mount Sinai Health System (New York City) from December 2020 through April 2021 to evaluate target results and corresponding sequencing data. Overall, the level of PBS mismatches was greater in specimens with target dropout. Of specimens with N3 target dropout, 57% harbored an A28095T substitution that is highly-specific for the alpha (B.1.1.7) variant of concern. These data highlight the benefit of redundancy in target design and the potential for target performance to illuminate the dynamics of circulating SARS-CoV-2 variants.