Identification of mutations in SARS-CoV-2 PCR primer regions

Mentes, Anikó; Papp, Krisztián; Visontai, Dávid; Stéger, József; Cochrane, Guy; Rahman, Nadim; Cummins, Carla; Yuan, David Yu; Selvakumar, Sandeep; Mansurova, Milena; O’Cathail, Colman; Sokolov, Alexey; Thorne, Ross; Koopmans, Marion; Nieuwenhuijse, David F.; Oude-Munnink, Bas; Worp, Nathalie; Amid, Clara; Csabai, István; Medgyes-Horváth, Anna; Pipek, Orsolya

doi:10.1038/s41598-022-21953-3

Cited by 12 publications

(13 citation statements)

References 50 publications

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“…It is critical to discriminate against different variants caught at entry and exit ports to support policy decisions. Compared with PCR technology, a full genome tiling array can avoid false negatives caused by mutation occurring at primer binding regions [ 17 ]. It can also provide genome sequences and mutations of the virus, which is of great significance for virus traceability, virulence, and vaccine evaluation.…”

Section: Discussionmentioning

confidence: 99%

A full genome tiling array enhanced the inspection and quarantine of SARS-CoV-2

Qi¹,

Wang

Wang³

et al. 2023

Virol J

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As the worldwide spreading epidemic of SARS-CoV-2, quick inspection and quarantine of passengers for SARS-CoV-2 infection are essential for controlling the spread of SARS-CoV-2, especially the cross-border transmission. This study reports a SARS-CoV-2 genome sequencing method based on a re-sequencing tiling array successfully used in border inspection and quarantine. The tiling array chip has four cores, with one core of 240,000 probes dedicated to the whole genome sequencing of the SAR-CoV-2 genome. The assay protocol has been improved to reduce the detection time to within one day and can detect 96 samples in parallel. The detection accuracy has been validated. This fast and simple procedure is also of low cost and high accuracy, and it is particularly suitable for the rapid tracking of viral genetic variants in custom inspection applications. Combining these properties means this method has significant application potential in the clinical investigation and quarantine of SARS-CoV-2. We used this SARS-CoV-2 genome re-sequencing tiling array to inspect and quarantine China's entry and exit ports in the Zhejiang Province. From November 2020 to January 2022, we observed the gradual shift of SARS-CoV-2 variants from the D614G type to the Delta Variant, and then to the dominance of the Omicron variant recently, consistently with the global emergency pattern of the new SARS-CoV-2 variant.

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Section: Discussionmentioning

confidence: 99%

A full genome tiling array enhanced the inspection and quarantine of SARS-CoV-2

Qi¹,

Wang

Wang³

et al. 2023

Virol J

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show abstract

“…The use of a 2x tiled primer set enabled more accurate detection of SARS-CoV-2 mutations than a single primer set, suggesting that this method may be a promising approach for detecting and monitoring the spread of SARS-CoV-2 and classifying its subtypes. In addition, we applied modified primer composition set similar to one previously implemented to help amplify large deletions in SARS-CoV-2 genome [23, 24]. Furthermore, we highlighted the benefits of employing ONT sequencing for this type of analysis.…”

Section: Discussionmentioning

confidence: 99%

Identification and Analysis of SARS-CoV-2 Mutation and Subtype using 2x tiled Primer Set with Oxford Nanopore Technologies Sequencing for Enhanced Variant Detection and Surveillance in Seoul, Korea

Han,

Lee,

Kwon

et al. 2023

Preprint

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a respiratory virus that contains RNA as its genetic material and has caused a global pandemic since its outbreak in 2020. This virus has many mutations, some of which can reduce the effectiveness of existing vaccines. Therefore, next-generation sequencing (NGS) is necessary to accurately identify new mutations. Current NGS analysis of SARS-CoV-2 uses the amplicon analysis method through a multiplex polymerase chain reaction. This study collected and validated RNA samples from patients who tested positive for SARS-CoV-2 from April to July 2022, and selected 613 samples for sequencing. The findings demonstrate the importance of long-read-based NGS analysis and 2x tiled primer set for identifying full SARS-CoV-2 genome sequence with new mutations and understanding the correlation between viral genotypes and patient characteristics for the effective management of SARS-CoV-2.

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“…Since many mutations are reported in the regions of SARS-CoV-2 genome used in the RT-qPCR protocols to molecular identification [5], we ask if this SNPs can impair the protocol capacity to identify the new variants of the virus. We stablished a workflow to validate the RT-qPCR primer and probes sets by the sorting of the new variant representative genomes around the world using GSAID initiative (Figure 2A).…”

Section: Identifying Mutation That Compromise Rt-qpcr Protocol In a W...mentioning

confidence: 99%

Validation of RT-qPCR primers and probes for new and old variants of SARS-CoV-2 in a world scale

Almeida,

Pereira

et al. 2024

Preprint

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Introduction: The demand for molecular diagnosis of pathogens has surged dramatically since the onset of the COVID-19 pandemic. In this context, different diagnostic tests have been developed to identify SARS-CoV-2 in patient samples. The emergence of new variants of SARS-CoV-2 raises questions about whether the molecular tests available for diagnosis continue to be effective in detecting the virus in biological samples. Objective: This study analyzed the viability of molecular targets directed to N, E and RdRp genes available against the new variants of SARS-CoV-2. Methodology: For this, we used bioinformatics tools to analyze SARS-CoV-2 genomic data of different variants deposited in GSAID and NCBI virus genomic databases to assess the accuracy of molecular tests available for the diagnosis of COVID-19. We also developed software for analyzing mutation frequencies in different molecular targets from the mutation database. Results: Mutation frequency analysis revealed a high rate of mutations in the N, E and RdRp genes and targets, although the target regions were more conserved. Only three SNPs were recurrent in the sequences of the variants identified in different continents and all in different targets. On the other hand, the registered mutations are not consistent and do not appear frequently in isolates of the same variant in all regions of the world. Conclusion: Our data suggest that the molecular targets designed for the first SARS-CoV-2 variants remain valid for the identification of new virus variants despite the large number of identified haplotypes. However, false negative test failures can be identified by using more than one molecular target for the same sample. Genomic regions that are under evolutive selective pressure should be avoided in the use of the diagnostic, once the emergence of new variants may affect the efficiency of molecular testing on a global scale.

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Identification of mutations in SARS-CoV-2 PCR primer regions

Cited by 12 publications

References 50 publications

A full genome tiling array enhanced the inspection and quarantine of SARS-CoV-2

A full genome tiling array enhanced the inspection and quarantine of SARS-CoV-2

Identification and Analysis of SARS-CoV-2 Mutation and Subtype using 2x tiled Primer Set with Oxford Nanopore Technologies Sequencing for Enhanced Variant Detection and Surveillance in Seoul, Korea

Validation of RT-qPCR primers and probes for new and old variants of SARS-CoV-2 in a world scale

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