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Single-Amplicon Multiplex Real-Time Reverse Transcription-PCR with Tiled Probes To Detect SARS-CoV-2 spike Mutations Associated with Variants of Concern.

ABSTRACT: To provide an accessible and inexpensive method to surveil for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutations, we developed a multiplex real-time reverse transcription-PCR (rRT-PCR) assay, the Spike single-nucleotide polymorphism (SNP) assay, to detect specific mutations in the spike receptor binding domain. A single primer pair was designed to amplify a 348-bp region of spike, and probes were initially designed to detect K417, E484K, and N501Y. The assay was evaluated using characterized variant sample pools and residual nasopharyngeal samples. Variant calls were confirmed by SARS-CoV-2 genome sequencing in a subset of samples. Subsequently, a fourth probe was designed to detect L452R. The lower limit of 95% detection was 2.46 to 2.48 log₁₀ genome equivalents (GE)/ml for the three initial targets (∼1 to 2 GE/reaction). Among 253 residual nasopharyngeal swabs with detectable SARS-CoV-2 RNA, the Spike SNP assay was positive in 238 (94.1%) samples. All 220 samples with threshold cycle (C_T) values of <30 for the SARS-CoV-2 N2 target were detected, whereas 18/33 samples with N2 C_T values of ≥30 were detected. Spike SNP results were confirmed by sequencing in 50/50 samples (100%). Addition of the 452R probe did not affect performance for the original targets. The Spike SNP assay accurately identifies SARS-CoV-2 mutations in the receptor binding domain, and it can be quickly modified to detect new mutations that emerge.

SUBMITTER: Babiker A

PROVIDER: S-EPMC8601233 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing. A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. Toward this goal, we developed a competitive multiplex PCR-based amplicon sequencing library preparation method that a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. To determine the utility of this method, we intentionally selected PCR conditions that would cause transcript amplification products (amplicons) to converge toward equimolar concentrations (normalization) during library preparation. We then tested whether this approach would enable accurate and reproducible quantification of each transcript across multiple library preparations, and at the same time reduce (through normalization) total sequencing reads required for quantification of transcript targets across a large range of expression. We demonstrate excellent reproducibility (R²?=?0.997) with 97% accuracy to detect 2-fold change using External RNA Controls Consortium (ERCC) reference materials; high inter-day, inter-site and inter-library concordance (R²?=?0.97-0.99) using FDA Sequencing Quality Control (SEQC) reference materials; and cross-platform concordance with both TaqMan qPCR (R²??=?0.96) and whole transcriptome RNA-sequencing following "traditional" library preparation using Illumina NGS kits (R²?=?0.94). Using this method, sequencing reads required to accurately quantify more than 100 targeted transcripts expressed over a 10?-fold range was reduced more than 10,000-fold, from 2.3×10? to 1.4×10? sequencing reads. These studies demonstrate that the competitive multiplex-PCR amplicon library preparation method presented here provides the quality control, reproducibility, and reduced sequencing reads necessary for development and implementation of targeted quantitative RNA-sequencing biomarkers in molecular diagnostic testing.

Dataset Information

Single-Amplicon Multiplex Real-Time Reverse Transcription-PCR with Tiled Probes To Detect SARS-CoV-2 spike Mutations Associated with Variants of Concern.

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