Robust clinical detection of SARS-CoV-2 variants by RT-PCR/MALDI-TOF multitarget approach

Abstract

The coronavirus disease 2019 (COVID-19) pandemic has sparked the rapid development of severe acute respiratory syndrome coronavirus 2 (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® SARS-CoV-2 Panel combines reverse-transcription polymerase chain reaction and matrix-assisted laser desorption/ionization time-of-flight 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 data set of 1262 SARS-CoV-2-positive specimens from Mount Sinai Health System (New York City) from December 2020 to 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.

Keywords: B.1.1.7; MALDI-TOF; RT-PCR; SARS-CoV-2; diagnostic; dropout; variants.

Figure 1.. Agena target detection rate in SARS-CoV-2-positive specimens by week.

Heatmap depicting the proportion of sequenced SARS-CoV-2-positive specimens that have detectable N1, N2, N3, ORF1A, or ORF1AB targets by week from December 1, 2020 through April 24, 2021. International, national, and global statistics are indicated by dates in purple font. NYC statistics are indicated by dates in red font. Data for epidemiologic events obtained from ^,,,,. The number of sequenced SARS-CoV-2-positive specimens per week is indicated above each week (column).

Figure 2.. Impact of SARS-CoV-2 primer/probe binding site mismatches on Agena target detection results.

Number of mismatches normalized to the number of nucleotides in primer/probe binding sites (PBS length) across five Agena MassARRAY® diagnostic targets: (A) N1, (B) N2, (C) N3, (D) ORF1A, (E) ORF1AB. Each point represents the calculated mismatches per specimen consensus genome for each target PBS. Violin plots represent the distribution as density of the points grouped by primer/probe sequence (forward (For), reverse (Rev), Probe) and by target detection result (detected (magenta), dropout (turquoise)). The number of genome sequences analyzed for mismatches are depicted above each violin plot. Medians are depicted as yellow lines. Bars above distributions reflect statistical comparison of underlying distributions by Mann-Whitney test. Asterisks reflect p-values (*, p < 0.05; ****, p < 0.0001).

Figure 3.. SARS-CoV-2 positional mismatches at target primer/probe binding sites.

Line graphs depict the percentage of specimen genomes with mismatches at individual basepair positions across Agena MassARRAY® target PBSs: (A) N1, (B) N2, (C) N3, (D) ORF1A. There are three plots for each target that correspond with the forward (For), reverse (Rev), and Probe binding sites. Two line plots are depicted for each binding site to depict mismatches in genomes from specimens that yielded a detected target result (magenta) or target dropout (turquoise). The percentage represents the number of genomes with mismatches at each position relative to the number of genome sequences detected or not detected by each target (annotated in each graph).

Figure 4.. Lineage-specific substitution interferes with SARS-CoV-2 diagnostic target detection.

(A) Alignment of B.1.1.7 genomes associated with N3 target dropout. View is magnified to display mismatches across the N3 forward (For) primer binding site. Sixty-four specimen genomes are indicated by laboratory identifiers (Genome ID) and mismatches to the Wuhan-Hu-1 reference sequence (NC_045512.2) and the N3 For primer (orange) are highlighted in green. Substitutions that correspond with each of the mismatches are annotated below each panel. The lineage specific A28095T substitution that is associated with N3 target dropout is highlighted in red with white typeface font. (B) Alignment of B.1.1.7 genomes associated with N3 target detection. Sixty-three individual genomes are indicated by laboratory identifiers and mismatches are highlighted and annotated as in (A). Note for substitutions that are shared across both target results (e.g., dropout and detected), annotations are in boldface font.