agena massarray sars cov 2 variant panel v3  (agena bioscience)

Journal: Microbiology Spectrum

Article Title: A Robust, Highly Multiplexed Mass Spectrometry Assay to Identify SARS-CoV-2 Variants

doi: 10.1128/spectrum.01736-22

Figure Lengend Snippet: Detection of viral variants by the Agena MassARRAY SARS-CoV-2 Variant Panel. (A) SARS-CoV-2 genome with nucleotide positions from 5′-to-3′ direction depicted above. S gene polymorphisms targeted by the variant panel (lollipops) and corresponding amino acids are depicted below. (B) A color map depicts algorithms of target combinations that defined 16 distinct SARS-CoV-2 variants on the panel. Variant results are depicted (left) and included the WHO designation (e.g., Omicron, Delta, etc.) and corresponding PANGO lineage assignments. Note that the B.1.526.1 variant was redesignated B.1.637 to distinguish it from the Iota variant lineage ( https://cov-lineages.org/lineage_list.html , accessed April 26, 2022). The minimum number of targets required to support the corresponding variant result is indicated (right). Target results are depicted as colored cells indicating amino acid positions that did not contribute to the defined variant identity algorithm (gray). The remaining three colors reflect native amino acids (e.g., unchanged from Wuhan-Hu-1 reference) (yellow), detectable amino acid polymorphisms (red), and drop out of the given target (green), all of which contributed to the variant identity algorithm. (C) Phylogenetic composition of 391 clinical specimen viral RNA recovered for diagnostic evaluation of the variant panel. The numbers of each lineage tested are depicted in brackets.

Article Snippet: We recovered residual viral RNA from all 391 specimens from −80°C storage to undergo testing on the Agena MassARRAY SARS-CoV-2 Variant Panel v3 ( https://www.agenabio.com/products/panel/coronavirus-sars-cov-2-variant-detection-research-panel , accessed April 5, 2022).

Techniques: Variant Assay, Diagnostic Assay

Journal: Microbiology Spectrum

Article Title: A Robust, Highly Multiplexed Mass Spectrometry Assay to Identify SARS-CoV-2 Variants

doi: 10.1128/spectrum.01736-22

Figure Lengend Snippet: Diagnostic sensitivity and specificity of the Agena MassARRAY SARS-CoV-2 Variant panel. (A) Diagnostic sensitivity and (B) diagnostic specificity of 11 variant calls on the panel are depicted. The number of specimens that correspond with each variant according to WGS is annotated in brackets. Depiction of (C) diagnostic sensitivity and (D) diagnostic specificity of each of 30 distinct panel targets. The number of specimens that correspond with each amino acid polymorphism according to WGS is annotated in brackets for each target. Asterisks (*) indicate targets for which dropout results were excluded from analyses (see Materials and Methods). For target N501Y, a separate diagnostic analysis was conducted excluding BA.1 specimens (“N501Y_Excl-BA.1”). Error bars reflect 95% CI in all four panels. ND, not determined.

Article Snippet: We recovered residual viral RNA from all 391 specimens from −80°C storage to undergo testing on the Agena MassARRAY SARS-CoV-2 Variant Panel v3 ( https://www.agenabio.com/products/panel/coronavirus-sars-cov-2-variant-detection-research-panel , accessed April 5, 2022).

Techniques: Diagnostic Assay, Variant Assay

Journal: Microbiology Spectrum

Article Title: A Robust, Highly Multiplexed Mass Spectrometry Assay to Identify SARS-CoV-2 Variants

doi: 10.1128/spectrum.01736-22

Figure Lengend Snippet: Target result patterns of undefined variants on the Agena MassARRAY SARS-CoV-2 Variant Panel. (A) A color map depicts the observed target results for three undefined SARS-CoV-2 variants tested on the panel: Lambda (C.37), Mu (B.1.621), and Omicron (BA.2). Distinct target patterns were observed among each of the variant types are depicted. Cells indicate the distinct target results, including detectable native amino acid (gray), detection of target polymorphism (red), and target dropout (green). The number of specimens that yielded each of the distinct target result patterns is indicated on the right as well as the output variant ID result generated by the variant panel software. (B) A heatmap depicts the measured prevalence of each variant panel target substitution among publicly available Omicron sublineage genomes as of May 6, 2022.

Article Snippet: We recovered residual viral RNA from all 391 specimens from −80°C storage to undergo testing on the Agena MassARRAY SARS-CoV-2 Variant Panel v3 ( https://www.agenabio.com/products/panel/coronavirus-sars-cov-2-variant-detection-research-panel , accessed April 5, 2022).

Techniques: Variant Assay, Generated, Software

Journal: The Journal of Molecular Diagnostics : JMD

Article Title: RT-PCR/MALDI-TOF diagnostic target performance reflects circulating SARS-CoV-2 variant diversity in New York City

doi: 10.1016/j.jmoldx.2022.04.003

Figure Lengend Snippet: RT-PCR/MALDI-TOF target detection rate in SARS-CoV-2-positive specimens. (A) Heatmap depicting the proportion of SARS-CoV-2-positive specimens that have detectable RT-PCR/MALDI-TOF targets (N1, N2, N3, ORF1A, ORF1AB) by week from April 11 through August 28, 2021. The total number of SARS-CoV-2-positive specimens and the number of SARS-CoV-2-positive specimens sequenced by pathogen surveillance are depicted above each week (column). Grey boxes indicate weeks where no specimens were positive for SARS-CoV-2 on this platform. (B) Stacked area plots depicting frequencies of SARS-CoV-2 variants reported by publicly-available NYC Department of Health surveillance data within the same timeframe.

Article Snippet: Primer and probe sequences for each Agena target were obtained from published FDA EUA documentation for the Agena MassARRAY SARS-CoV-2 Panel ( ) .

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Molecular Diagnostics : JMD

Article Title: RT-PCR/MALDI-TOF diagnostic target performance reflects circulating SARS-CoV-2 variant diversity in New York City

doi: 10.1016/j.jmoldx.2022.04.003

Figure Lengend Snippet: Alignment of SARS-CoV-2 haplotypes to RT-PCR/MALDI-TOF targets. Multiple sequence alignment of haplotype sequences to forward, reverse, and probe binding sites for (A) N1, (B) N2, (C) N3, (D) ORF1A, and (E) ORF1AB targets. In each alignment, the reference sequence (NC_045512.2; https://www.ncbi.nlm.nih.gov/nuccore/ , last accessed April 15, 2022) is annotated above comprised of color-coded nucleotides. The coordinates of the reference sequence are annotated at the bottom corners of each alignment. Each row represents a haplotype sequence that aligns to the target site. Absolute counts of each haplotype (e.g., Hap_1, Hap_2, etc.) within the dataset and counts stratified by variant lineage (e.g., Alpha, Delta, Iota) are depicted to the right side of each haplotype sequence. Positions of primer/probe sequences are indicated by arrows where tail to arrowhead reflects 5’ to 3’ directionality. Grey boxes with black dotted borders outline PBSs. Dots represent conserved nucleotides at each position and mismatched nucleotides are indicated. PBS mismatches are indicated by arrows; red arrows reflect mismatches that are significantly associated with target dropout. Notably, the association of the G28881U mismatch to the N2 forward PBS (panel C, red “(U)”) with target dropout is distinguished from the G28881A mismatch. Nucleotide diversity (π) of sequences at each PBS is indicated.

Article Snippet: Primer and probe sequences for each Agena target were obtained from published FDA EUA documentation for the Agena MassARRAY SARS-CoV-2 Panel ( ) .

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing, Binding Assay, Variant Assay

Journal: The Journal of Molecular Diagnostics : JMD

Article Title: RT-PCR/MALDI-TOF diagnostic target performance reflects circulating SARS-CoV-2 variant diversity in New York City

doi: 10.1016/j.jmoldx.2022.04.003

Figure Lengend Snippet: Delta-specific substitution interferes with N2 diagnostic target detection. (A) Stacked bar plots depict the composition of SARS-CoV-2 variants in 254 MSHS genomes tested by RT-PCR/MALDI-TOF. Bar plots reflect absolute number of genomes with N2 target dropout (left) and those with N2 target detection (right). Variant groups are color-coded and genomes with the G28916U polymorphism are depicted by a hatched pattern. (B) Stacked bar plot of publicly-available sequences (GISAID, see methods) from the same timeframe of this study depicting absolute numbers of variant genomes and presence or absence of the G28916U polymorphism. Color-coding and patterns are the same as in (A).

Article Snippet: Primer and probe sequences for each Agena target were obtained from published FDA EUA documentation for the Agena MassARRAY SARS-CoV-2 Panel ( ) .

Techniques: Diagnostic Assay, Reverse Transcription Polymerase Chain Reaction, Variant Assay

Journal: Viruses

Article Title: Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) including Variant Analysis by Mass Spectrometry in Placental Tissue

doi: 10.3390/v14030604

Figure Lengend Snippet: Workflow and molecular principle of SARS-CoV-2 RNA detection in FFPE tissue by MALDI-TOF mass spectrometry. RNA extraction from FFPE placental and amniotic tissue was performed using the Maxwell RSC RNA FFPE Kit (Promega) according to manufacturer’s instructions. Extracted viral RNA is then reverse transcribed into cDNA and amplified by a one-step RT-PCR reaction. The figure schematically depicts specific amplification of the SARS-CoV-2 targets N1 and N2. The forward primers are indicated in green, the reverse primers in blue. Thereafter, PCR products are treated with the shrimp alkaline phosphatase (SAP) enzyme to remove unincorporated nucleotides (dNTPs). Next, a single nucleotide extension reaction is performed, in which target-specific extension primers are elongated by a single mass-modified terminator nucleotide (A, T, C or G) complementary to the cDNA template sequence. The extension products (EP) are then analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) using the MassARRAY system (Agena Bioscience) and mass spectra of the SARS-CoV-2-specific detected cDNA fragments are generated. In the absence of viral RNA, RT-PCR as well as single nucleotide extension reactions cannot be performed and consequently, no cDNA fragments are detected by MALDI-TOF analysis. EP: extension product; UEP: unextended extension primer.

Article Snippet: For detection of the SARS-CoV-2 variants the Research Use Only (RUO) MassARRAY ® SARS-CoV-2 Variant Panel v1 (Agena Bioscience) was used.

Techniques: RNA Detection, Mass Spectrometry, RNA Extraction, Reverse Transcription, Amplification, One Step RT-PCR, Modification, Sequencing, Generated, Reverse Transcription Polymerase Chain Reaction

Journal: Viruses

Article Title: Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) including Variant Analysis by Mass Spectrometry in Placental Tissue

doi: 10.3390/v14030604

Figure Lengend Snippet: Genomic targets of the MassARRAY SARS-CoV-2 Panels . ( A ): The CE-IVD-certified MassARRAY SARS-CoV-2 Panel targets 5 genomic regions within the SARS-CoV-2 genome: ORF1, ORF1ab, N1, N2 and N3 (indicated by red arrows). The detection of at least 2 targets is required for the result “SARS-CoV-2 positive”. ORF, open reading frame; S, spike; E, envelope; M, membrane; N, nucleocapsid. ( B ): The MassARRAY SARS-CoV-2 Variant Panel v1 targets 20 specific mutations (indicated by red arrows) within the spike gene allowing the identification of 5 different SARS-CoV-2 variants. For detection of the British (B.1.1.7) variant at least 6 out of the 8 mutations H69_70 del, Y144del, N501Y, A570D, P681H, T716I, S982A, D1118H are required while for detection of the South African (B.1.351) variant at least 6 out of the 8 mutations L18F, D80A, D215G, L242_L244del, K417N, E484K, N501Y and A701V are required. The 4 mutations L18F, K417T, E484K and N501Y identify the Brazilian (P.1) variant and the 4 mutations H69_70del, Y453F, N501T and I692V allow the identification of the Danish (Mink) variant (3 out of 4 mutations required). The mutation D614G evolved very early during the pandemic spread in 2020 and is present in all variants.

Article Snippet: For detection of the SARS-CoV-2 variants the Research Use Only (RUO) MassARRAY ® SARS-CoV-2 Variant Panel v1 (Agena Bioscience) was used.

Techniques: Membrane, Variant Assay, Mutagenesis

Journal: Viruses

Article Title: Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) including Variant Analysis by Mass Spectrometry in Placental Tissue

doi: 10.3390/v14030604

Figure Lengend Snippet: Representative mass spectra of a positive sample analyzed by the CE-IVD-certified MassARRAY SARS-CoV-2 Panel. The different mass spectra display intensity and mass (in daltons) of the SARS-CoV-2 -specific cDNA fragments ORF1, ORF1ab, N1, N2 and N3. Additionally, the mass spectrum of the MS2 phage control is depicted. The red box highlights the unextended extension primer peak, whereas the blue box highlights the peak corresponding to the extension product. UEP: unextended extension primer; EP: extension product.

Article Snippet: For detection of the SARS-CoV-2 variants the Research Use Only (RUO) MassARRAY ® SARS-CoV-2 Variant Panel v1 (Agena Bioscience) was used.

Techniques: Control

Journal: Viruses

Article Title: Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) including Variant Analysis by Mass Spectrometry in Placental Tissue

doi: 10.3390/v14030604

Figure Lengend Snippet: Detection of SARS-CoV-2 RNA in placental and amniotic tissue. Placental and amniotic tissue were analyzed by MALDI-TOF MS using the CE-IVD-certified MassARRAY SARS-CoV-2 Panel ( A ) and by RT-PCR using the EURORealTime SARS-CoV-2 Kit ( B ). ( A ): The plots display the intensity of the peaks corresponding to the 5 targets ORF1, ORF1ab, N1, N2 and N3 included in the CE-IVD-certified MassARRAY SARS-CoV-2 Panel. The intensity threshold considering a peak as detected is 5. All 5 SARS-CoV-2 targets were detected in both placental and amniotic tissue. ( B ). The graphs show the fluorescence intensity ( y -axis) for each cycle ( x -axis) of the real-time amplification of the SARS-CoV-2 -specific target regions (amplification curve). The amplification curve of the positive control (PC) is highlighted in green, the one of the negative control (NC) in red. The crossing point (Cp) value is indicated for both placental tissue (p.tissue) and amniotic tissue (a.tissue).

Article Snippet: For detection of the SARS-CoV-2 variants the Research Use Only (RUO) MassARRAY ® SARS-CoV-2 Variant Panel v1 (Agena Bioscience) was used.

Techniques: Reverse Transcription Polymerase Chain Reaction, Fluorescence, Amplification, Positive Control, Negative Control

Journal: Viruses

Article Title: Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) including Variant Analysis by Mass Spectrometry in Placental Tissue

doi: 10.3390/v14030604

Figure Lengend Snippet: SARS-CoV-2 variant analysis of amniotic and placental tissue by MALDI-TOF MS. Placental and amniotic tissue were analyzed by MALDI-TOF MS using the MassARRAY SARS-CoV-2 Variant Panel v1. The figure shows the specific mutations within the SARS-CoV-2 spike gene that are assigned to the different variants B 1.1.7 ( A ), B 1.351 ( B ), P.1 ( C ) and Cluster 5/Mink ( D ). Detected mutations in placental tissue (p.tissue), amniotic tissue (a.tissue), positive controls (PC) B 1.1.7 and B 1.351 and negative control (NC) are illustrated by green-colored squares.

Article Snippet: For detection of the SARS-CoV-2 variants the Research Use Only (RUO) MassARRAY ® SARS-CoV-2 Variant Panel v1 (Agena Bioscience) was used.

Techniques: Variant Assay, Negative Control