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

Mentes, Anikó; Papp, Krisztián; Visontai, Dávid; Stéger, József; Group, VEO Technical Working; Csabai, István; Medgyes-Horváth, Anna; Pipek, Orsolya

doi:10.21203/rs.3.rs-1838361/v1

Cited by 5 publications

(3 citation statements)

References 37 publications

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“…As inosine preferentially pairs with cytidine, A>I mutations cause A>G and U>C transitions on the positive strand of the SARS-CoV-2 genome [23,25]. The presence of SARS-CoV-2 mutations may affect the test performance of COVID-19 diagnostic tests [26][27][28]. For this reason, they must be monitored.…”

Section: Introductionmentioning

confidence: 99%

The Mutational Landscape of SARS-CoV-2

Saldivar-Espinoza,

Garcia-Segura,

Novau-Ferré

et al. 2023

IJMS

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Mutation research is crucial for detecting and treating SARS-CoV-2 and developing vaccines. Using over 5,300,000 sequences from SARS-CoV-2 genomes and custom Python programs, we analyzed the mutational landscape of SARS-CoV-2. Although almost every nucleotide in the SARS-CoV-2 genome has mutated at some time, the substantial differences in the frequency and regularity of mutations warrant further examination. C>U mutations are the most common. They are found in the largest number of variants, pangolin lineages, and countries, which indicates that they are a driving force behind the evolution of SARS-CoV-2. Not all SARS-CoV-2 genes have mutated in the same way. Fewer non-synonymous single nucleotide variations are found in genes that encode proteins with a critical role in virus replication than in genes with ancillary roles. Some genes, such as spike (S) and nucleocapsid (N), show more non-synonymous mutations than others. Although the prevalence of mutations in the target regions of COVID-19 diagnostic RT-qPCR tests is generally low, in some cases, such as for some primers that bind to the N gene, it is significant. Therefore, ongoing monitoring of SARS-CoV-2 mutations is crucial. The SARS-CoV-2 Mutation Portal provides access to a database of SARS-CoV-2 mutations.

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Section: Introductionmentioning

confidence: 99%

The Mutational Landscape of SARS-CoV-2

Saldivar-Espinoza,

Garcia-Segura,

Novau-Ferré

et al. 2023

IJMS

View full text Add to dashboard Cite

show abstract

“…Considering the primer and probe binding site changes as the virus evolve, RT-PCR with dual target design is an important strategy to improve assay robustness to mutations in primer and probe binding sites. Some of the mutations in primer and probe sites may cause binding efficiency to drop hence could yield false negative results [34]. Examples of dual target like assays developed by Tombuloglu et al targeting viral RdRP gene, viral E gene [32] or N gene [33] and human RNase P gene as internal control.…”

Section: Plos Onementioning

confidence: 99%

The clinical characteristics of pediatric patients infected by SARS-CoV-2 Omicron variant and whole viral genome sequencing analysis

Tsang

Yu²,

Yim³

et al. 2023

PLoS ONE

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Pediatric population was generally less affected clinically by SARS-CoV-2 infection. Few pediatric cases of COVID-19 have been reported compared to those reported in infected adults. However, a rapid increase in the hospitalization rate of SARS-CoV-2 infected pediatric patients was observed during Omicron variant dominated COVID-19 outbreak. In this study, we analyzed the B.1.1.529 (Omicron) genome sequences collected from pediatric patients by whole viral genome amplicon sequencing using Illumina next generation sequencing platform, followed by phylogenetic analysis. The demographic, epidemiologic and clinical data of these pediatric patients are also reported in this study. Fever, cough, running nose, sore throat and vomiting were the more commonly reported symptoms in children infected by Omicron variant. A novel frameshift mutation was found in the ORF1b region (NSP12) of the genome of Omicron variant. Seven mutations were identified in the target regions of the WHO listed SARS-CoV-2 primers and probes. On protein level, eighty-three amino acid substitutions and fifteen amino acid deletions were identified. Our results indicate that asymptomatic infection and transmission among children infected by Omicron subvariants BA.2.2 and BA.2.10.1 are not common. Omicron may have different pathogenesis in pediatric population.

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“…Consequently, mismatches between virus genes and PCR primers/probes may occur frequently. As reported in various publications, mutations in primer or probe binding sites can impact the sensitivity of RT-PCR assays, leading to the occurrence of false-negative results [19][20][21][22]. To minimize such occurrences, it is crucial to design molecular diagnostic assays with multiple gene targets and search for a novel real-time RT-PCR target located in a conserved region of the genome.…”

Section: Introductionmentioning

confidence: 99%

Development and Evaluation of an In-House Real-Time RT-PCR Targeting nsp10 Gene for SARS-CoV-2 Detection

Yip,

Poon,

Leung

et al. 2024

IJMS

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The emergence of SARS-CoV-2 mutations poses significant challenges to diagnostic tests, as these mutations can reduce the sensitivity of commonly used RT-PCR assays. Therefore, there is a need to design diagnostic assays with multiple targets to enhance sensitivity. In this study, we identified a novel diagnostic target, the nsp10 gene, using nanopore sequencing. Firstly, we determined the analytical sensitivity and specificity of our COVID-19-nsp10 assay. The COVID-19-nsp10 assay had a limit of detection of 74 copies/mL (95% confidence interval: 48–299 copies/mL) and did not show cross-reactivity with other respiratory viruses. Next, we determined the diagnostic performance of the COVID-19-nsp10 assay using 261 respiratory specimens, including 147 SARS-CoV-2-positive specimens belonging to the ancestral strain and Alpha, Beta, Gamma, Delta, Mu, Eta, Kappa, Theta and Omicron lineages. Using a LightMix E-gene RT-PCR assay as the reference method, the diagnostic sensitivity and specificity of the COVID-19-nsp10 assay were found to be 100%. The median Cp values for the LightMix E-gene RT-PCR and our COVID-19-nsp10 RT-PCR were 22.48 (range: 12.95–36.60) and 25.94 (range 16.37–36.87), respectively. The Cp values of the COVID-19-nsp10 RT-PCR assay correlated well with those of the LightMix E-gene RT-PCR assay (Spearman’s ρ = 0.968; p < 0.0001). In conclusion, nsp10 is a suitable target for a SARS-CoV-2 RT-PCR assay.

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

Cited by 5 publications

References 37 publications

The Mutational Landscape of SARS-CoV-2

The Mutational Landscape of SARS-CoV-2

The clinical characteristics of pediatric patients infected by SARS-CoV-2 Omicron variant and whole viral genome sequencing analysis

Development and Evaluation of an In-House Real-Time RT-PCR Targeting nsp10 Gene for SARS-CoV-2 Detection

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