Detection and evolution of SARS-CoV-2 coronavirus variants of concern with mass spectrometry.
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ABSTRACT:
SUBMITTER: Mann C
PROVIDER: S-EPMC8445501 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:Amplification of viral ribonucleic acid (RNA) by real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is the gold standard to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since the initial outbreak, strategies to detect and isolate patients have been important to avoid uncontrolled viral spread. Although testing capacities have been upscaled, there is still a need for reliable high throughput test systems, specifically those that require alternative consumables. Therefore, we tested and compared two different methods for the detection of viral PCR products: rRT-PCR and mass spectrometry (MS). METHODS:Viral RNA was isolated and amplified from oro- or nasopharyngeal swabs. A total of 22 samples that tested positive and 22 samples that tested negative for SARS-CoV-2 by rRT-PCR were analyzed by MS. Results of the rRT-PCR and the MS protocol were compared. RESULTS:Results of rRT-PCR and the MS test system were in concordance in all samples. Time-to-results was faster for rRT-PCR. Hands-on-time was comparable in both assays. CONCLUSIONS:MS is a fast, reliable and cost-effective alternative for the detection of SARS-CoV-2 from oral and nasopharyngeal swabs.
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Project description:A big challenge for the control of COVID-19 pandemic is the emergence of variants of concern (VOCs) or variants of interest (VOIs) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which may be more transmissible and/or more virulent and could escape immunity obtained through infection or vaccination. A simple and rapid test for SARS-CoV-2 variants is an unmet need and is of great public health importance. In this study, we designed and analytically validated a CRISPR-Cas12a system for direct detection of SARS-CoV-2 VOCs. We further evaluated the combination of ordinary reverse transcription-PCR (RT-PCR) and CRISPR-Cas12a to improve the detection sensitivity and developed a universal system by introducing a protospacer adjacent motif (PAM) near the target mutation sites through PCR primer design to detect mutations without PAM. Our results indicated that the CRISPR-Cas12a assay could readily detect the signature spike protein mutations (K417N/T, L452R/Q, T478K, E484K/Q, N501Y, and D614G) to distinguish alpha, beta, gamma, delta, kappa, lambda, and epsilon variants of SARS-CoV-2. In addition, the open reading frame 8 (ORF8) mutations (T/C substitution at nt28144 and the corresponding change of amino acid L/S) could differentiate L and S lineages of SARS-CoV-2. The low limit of detection could reach 10 copies/reaction. Our assay successfully distinguished 4 SARS-CoV-2 strains of wild type and alpha (B.1.1.7), beta (B.1.351), and delta (B.1.617.2) variants. By testing 32 SARS-CoV-2-positive clinical samples infected with the wild type (n = 5) and alpha (n = 11), beta (n = 8), and delta variants (n = 8), the concordance between our assay and sequencing was 100%. The CRISPR-based approach is rapid and robust and can be adapted for screening the emerging mutations and immediately implemented in laboratories already performing nucleic acid amplification tests or in resource-limited settings. IMPORTANCE We described CRISPR-Cas12-based multiplex allele-specific assay for rapid SARS-CoV-2 variant genotyping. The new system has the potential to be quickly developed, continuously updated, and easily implemented for screening of SARS-CoV-2 variants in resource-limited settings. This approach can be adapted for emerging mutations and implemented in laboratories already conducting SARS-CoV-2 nucleic acid amplification tests using existing resources and extracted nucleic acid.
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Project description: Not available
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Project description:BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to emerge, and effective tracking requires rapid return of results. Surveillance of variants is typically performed by whole genome sequencing (WGS), which can be financially prohibitive and requires specialized equipment and bioinformatic expertise. Genotyping approaches are rapid methods for monitoring SARS-CoV-2 variants but require continuous adaptation. Fragment analysis may represent an approach for improved SARS-CoV-2 variant detection.MethodsA multiplex fragment analysis approach (CoVarScan) was validated using PCR targeting variants by size and fluorescent color. Eight SARS-CoV-2 mutational hot spots in variants of concern (VOCs) were targeted. Three primer pairs (recurrently deleted region [RDR] 1, RDR2, and RDR3-4) flank RDRs in the S-gene. Three allele-specific primers target recurrent spike receptor binding domain mutants. Lastly, 2 primer pairs target recurrent deletions or insertions in ORF1A and ORF8. Fragments were resolved and analyzed by capillary electrophoresis (ABI 3730XL), and mutational signatures were compared to WGS results.ResultsWe validated CoVarScan using 3544 clinical respiratory specimens. The assay exhibited 96% sensitivity and 99% specificity compared to WGS. The limit of detection for the core targets (RDR1, RDR2, and ORF1A) was 5 copies/reaction. Variants were identified in 95% of samples with cycle threshold (CT) <30 and 75% of samples with a CT 34 to 35. Assay design was frozen April 2021, but all subsequent VOCs have been detected including Delta (n = 2820), Mu, (n = 6), Lambda (n = 6), and Omicron (n = 309). Genotyping results are available in as little as 4 h.ConclusionsMultiplex fragment analysis is adaptable and rapid and has similar accuracy to WGS to classify SARS-CoV-2 variants.
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