Robust and extremely multiplexed assay for identification of SARS-CoV-2 variants
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In a current examine posted to the medRxiv* preprint server, researchers in contrast the efficiency of a extremely multiplexed mass spectrometry-based business assay to establish extreme acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants.

Study: A sturdy, extremely multiplexed mass spectrometry assay to establish SARS-CoV-2 variants. Image Credit: Design_Cells/Shutterstock


The genomically numerous SARS-CoV-2 variants differ of their transmissibility, pathogenicity, and responses to anti-SARS-CoV-2 therapeutic brokers. Therefore, the variants pose a singular menace to diagnostic laboratories and healthcare methods and should be recognized for improved coronavirus illness 2019 (COVID-19) surveillance, SARS-CoV-2 prevention, and customary of take care of COVID-19 sufferers.

For variant identification, whole-genome sequencing (WGS) is the gold customary however will not be broadly accessible to scientific and microbiological laboratories as it’s approach delicate, requires infrastructure, and is dear. This warrants the necessity to develop different strong, high-resolution, and extremely multiplexed SARS-CoV-2 assays similar to molecular assays to precisely detect SARS-CoV-2 contaminated people.

About the examine

In the current examine, researchers highlighted the diagnostic capabilities of the extremely multiplexed and business Agena MassARRAY® SARS-CoV-2 Variant Panel v3 assay in figuring out SARS-CoV-2 variants which circulated between September 2, 2020, and March 2, 2022.

For the evaluation, 391 SARS-CoV-2 scientific ribonucleic acid (RNA) samples had been obtained throughout well being methods in Bogotá, Colombia, and New York City (NYC). The samples had been beforehand subjected to WGS evaluation, the info of which was used for comparative analysis of the diagnostic efficiency of the Agena MassARRAY® SARS-CoV-2 variant panel v3. The samples comprised 349 higher respiratory tract [e.g., nasopharyngeal (n=43) anterior nares] and saliva specimens.

SARS-CoV-2 RNA was extracted from the samples and subsequently subjected to reverse transcription-polymerase chain response (RT-PCR) evaluation and next-generation sequencing, adopted by genomic assembling and PANGO (phylogenetic task of named international outbreak lineages) assignments. Sequence libraries had been ready by performing long-read Oxford Nanopore MinION sequencing.

The panel comprised built-in RT-PCR and MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) for detecting focused polymorphisms within the SARS-CoV-2 spike (S) gene. The numerous set of SARS-CoV-2 variants was examined for the extent of interrater settlement and diagnostic specificity and sensitivity throughout the variants on the molecular panel and throughout the S gene targets. Furthermore, SARS-CoV-2 genomes on the Global Initiative on Sharing Avian Influenza Data (GISAID) database (which was final accessed on 6 May 2022) had been assessed to find out the prevalence of variant panel targets throughout the Omicron subvariants.


Out of 391 specimens, 381 single variant genomic sequences had been recognized, whereas the ten remaining specimens yielded combined genomic assemblies and, thus, an inconclusive consequence. RNA extracted from the samples corresponded to 12 out of 16 variant calls on the molecular panel which included Iota (n=39), Alpha (n=40), Delta (n = 3), Delta AY.x subvariant (n = 107), and Omicron BA.1 (n=79). However, solely 11 out of 12 variant calls (excluding the D614G pressure) had been used for the settlement evaluation.

Only 62 (out of 391 specimens) had been noticed for which the variant calls had been in disagreement with WGS evaluation, of which the sequenced variant of 45 specimens didn’t have an outlined variant algorithm on the molecular panel. As a consequence, solely 17 (5%) of the RNA samples yielded outcomes that differed from these of WGS evaluation. Near-perfect interrater settlement ranges had been noticed between WGS and the assay for 9 out of 11 variant calls and 25 out of 30 targets examined on the molecular panel. The Eta and Zeta variant calls confirmed the least interrater settlement. Polymorphic targets with suboptimal settlement ranges included D215G, L242_244del, N501Y, and K1191N.

The assay demonstrated excessive diagnostic sensitivity (≥93.7%) for up to date variants (e.g., Iota, Alpha, Delta variants, and Omicron BA.1 subvariant) and excessive diagnostic specificity for all of the 11 variants examined (≥96%) and all 30 targets (≥94%) examined. The targets with the bottom sensitivities included D80A, D215G, L242_244del, and N501Y. Further, after excluding the Zeta variant, the panel demonstrated excessive constructive predictive values (≥0.9) and excessive unfavourable predictive values (≥0.984) for all of the variant calls.

Of curiosity, for the N501Y goal, 79 out of 158 samples with WGS-identified polymorphisms yielded false-negative outcomes on the variant panel, all of which belonged to the Omicron BA.1 subvariant, and on excluding the BA.1 samples, near-perfect (kappa = 0.98) interrater settlement was noticed. This indicated that genomic variations past the scope of the assay’s design may present distinctive targets for brand spanking new variants.

All 9 Omicron BA.2 specimens generated a definite goal signature on the panel assay comprising N501Y, S477N, T478K, P681H, and D614G targets and N439K goal dropout. All BA.2 specimens harbored the T22882G polymorphism. Almost all BA.2.12.1 genomes harbored the S477N, K417N, T478K, P681H, D614G, and N501Y substitutions and N439K goal dropout. Over 70% of BA.3 genomes encoded the S477N, T478, N501Y, D614G, and P681H substitutions. About 88% and 99% of BA.4 and 9 BA.5 genomes encoded the L452R substitution, respectively.


Overall, the examine findings exemplified the facility of extremely multiplexed diagnostic SARS-CoV-2 variant panels for correct variant calling and highlighted distinct goal patterns that may be utilized to establish variants not but outlined on the panel, together with Omicron BA.2.

*Important discover

medRxiv publishes preliminary scientific experiences that aren’t peer-reviewed and, subsequently, shouldn’t be considered conclusive, information scientific observe/health-related conduct, or handled as established data.

Journal reference:

  • Hernandez, M. et al. (2022) “A robust, highly multiplexed mass spectrometry assay to identify SARS-CoV-2 variants”. medRxiv. doi: 10.1101/2022.05.28.22275691. material/10.1101/2022.05.28.22275691v1

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