Ted Schutzbank, PhD, D(ABMM); Aleksey Nakorchevsky, PhD; Andrew Bradford, PhD
April 6, 2021
This is a partial excerpt, the full article is available as a PDF.
Agena Bioscience® has developed a Research Use Only (RUO) panel for the detection and differentiation of SARS-CoV-2 variants on the MassARRAY® System. This panel provides a robust alternative to next-generation sequencing (NGS) to screen for the presence of SARS-CoV-2 variants of concern (VOCs) in human samples. This two-well panel utilizes a one-step RT-PCR reaction to reverse transcribe viral RNA into cDNA and amplify the nucleic acid material in the same reaction. The high multiplexing capabilities of the iPLEX® Pro chemistry allow for the simultaneous detection of unique variants in this first version of the panel, with the capability to include additional VOCs quickly and easily as they emerge. Currently, the panel differentiates the B.1.1.7 (UK), B.1.351 (South Africa), B.1.248 / P.1 (Brazil), Cluster 5/Mink (Denmark) and D614G variants from the A.1 (Wuhan) lineage. The high-throughput MassARRAY System enables laboratories to cost-effectively process from hundreds up to thousands of samples per day with a single instrument, without the need for extensive bioinformatics analysis or infrastructure.
To efficiently follow the spread of VOCs a platform must overcome these NGS shortcomings. For this purpose, Agena Bioscience has developed the MassARRAY SARS-CoV-2 Variant Panel, a high throughput, low-cost, and rapid assay for the detection of 20 unique SARS-CoV-2 genetic markers. The panel is available in 96- and 384-well formats, to accommodate different testing volume needs. The assay differentiates the B.1.1.7 (UK), B.1.351 (South Africa), B.1.248 / P.1 (Brazil), Cluster 5/Mink (Denmark) and D614G variants from the A.1 (Wuhan) lineage. All genetic markers in this panel are spike protein mutations of concern that also define the particular variants. Variant D614G is included as it is an important VOC, the presence of which is known to enhance transmissibility of the affected virus. The specific variants detected are shown in Figure 3.
Figure 3: MassARRAY SARS-CoV-2 Variant Panel v1 (RUO)
The panel primers were analyzed in silico for sequence specificity to SARS-CoV-2 by performing NCBI BLAST, in silico PCR at UCSC Genome Browser, and alignment of panel primers to a multiple sequence alignment of variants intended to be interrogated by the panel. The panel primers showed 100% sequence homology to the intended targets apart from any tags utilized for mass spectrum placement.
NCBI nucleotide BLAST was used to test for cross reactivity of the primers in silico using a list of FDA-recommended micro-organisms (see Appendix A). The combination of primers for each assay (two PCR primers and one extension probe) exhibit 100% sequence homology to the conserved SARS-CoV-2 regions. All individual primers (one of two PCR primers and/or the extension probes) for each of the assays exhibit lower than the 80% homology to a cross-reactive species. The likelihood of false positive results is extremely low.
Data analysis is simplified using supporting software which qualifies the RNA sample and determines the detection status of the virus. A sample passes QC based on the results of the GAPDH assay, which serves as an internal control for the nucleic acid extraction, reverse transcription (RT), PCR, dephosphorylation (SAP), and extension steps. As the SARS-CoV-2 variant panel is designed as a reflex test for samples that have already tested positive for SARS-CoV-2, the panel also tests for the presence of the SARS-CoV-2 N-gene. Variant calling is performed according to the criteria shown in Figure 4. These thresholds for detection can be modified by the laboratory.
Figure 4: Variant Calling Summary
An example of the run report is shown in Figure 5. The report includes all relevant information including the sample ID, sample type, plate location, instrument ID, QC status, relevant messages, as well as the variant status and specific genetic markers detected for each sample.
Figure 5: Run Report
A report is also generated for each individual sample. An example of the individual sample report, shown in Figure 6, contains all relevant information for each unique sample, including software version, date, sample ID, QC information, genetic markers detected, and the variant reported.
Figure 6: Individual Sample Report
Several experiments were designed to verify the panel performance as described below. Positive samples were generated using SARS-CoV-2 negative human RNA spiked with the Synthetic SARS-CoV-2 RNA Controls from Twist Bioscience (SKU 102019). Clinical samples as well as isolates previously characterized by NGS were also utilized during verification of the panel.
The performance of the assays in detecting each individual variant is shown in Figure 7. These are plots of extension rates (yield) versus synthetic SARS-CoV-2 RNA copies input into the reaction in a background of 1 ng human RNA. These results allow the panel to detect and discriminate synthetic SARS-CoV-2 RNAs representative of the UK variant (B.1.1.7) and wild type at 25 copies per reaction input.
Figure 7: Performance of Individual Assays (Yield vs Copy Number)
Clinical samples that were pre-screened using either the Abbott RealTime SARS-CoV-2 or Agena MassARRAY SARS-CoV-2 EUA assays were also tested with the MassARRAY SARS-CoV-2 Variant Panel. The results shown in Table 3 demonstrate that the variant panel had excellent agreement with both EUA assays. There was 1 invalid sample reported with the variant panel within the low-titer SARS-CoV-2 samples due to the endogenous control for the N-gene falling below the detection threshold.
Table 3: Pre-screened Clinical Sample Results
In addition to testing clinical samples previously screened with on-market EUA assays, clinical isolates previously sequenced on the Illumina MiSeq with results deposited in GISAID were also analyzed with the MassARRAY SARS-CoV-2 Variant Panel. Table 4 shows the variants and Ct values for each of the samples.
Table 4: Clinical isolates sequenced on Illumina MiSeq
Results are shown in the test report (Figure 8). All expected variants were correctly identified given the current content within the variant panel.
Figure 8: MassARRAY SARS-Cov-2 Variant Panel Run Report for Previously Sequenced Clinical Isolates
Below (Figures 9 – 10) are examples of individual mass spectra generated from some of the clinical isolates. These are used to highlight examples of the data output for the detection of specific variants.
Figure 9: Variant B.1.2 (E484K)
Figure 10: Variant P.1 (E484K and D614G)
Results of these studies demonstrate that the Agena Bioscience MassARRAY SARS-CoV-2 Variant Panel (RUO) can be used to detect SARS-CoV-2 variants of concern and differentiate key variants. The panel represents an alternative method to NGS for the rapid screening of known variants of concern with the following key advantages:
Additionally, Agena Bioscience is in the process of incorporating additional variants of concern into the panel as they continue to emerge in the US and worldwide.11,12,13 One such variant, B.1.526, was recently identified in New York City.14 This variant carries both the E484K and N501Y genetic markers found in B.1.351 from South Africa. Neutralizing activities of convalescent plasma or vaccinee sera were determined to be lower by 7.7-fold or 3.4-fold, respectively, against B.1.526. Another new variant, B.1.429, has also been identified in Southern California.14 Both the New York and California variants are spreading rapidly within their respective communities. Other spike protein mutations, unrelated to specific variants, are appearing and spreading quickly across the US. Such genetic markers include L425R and Q677P/H, which, like other alterations in the spike protein, increase the infectivity of the virus by increasing the binding affinity to the ACE2 receptor (L425R) or enhancing the efficiency of entry into the cell (Q677P/H).
The continued emergence of SARS-CoV-2 variants worldwide underscores the importance of continued surveillance and research. The MassARRAY workflow offers a sensitive, high-throughput and robust method for the detection and differentiation of SARS-CoV-2 variants of concern.
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