Structure-Based Primer Design Minimizes the Risk of PCR Failure Caused by SARS-CoV-2 Mutations

Dong, Hongjie; Wang, Shuai; Zhang, Junmei; Zhang, Kundi; Zhang, Fengyu; Wang, Hongwei; Xie, Shiling; Hu, Wei; Gu, Lichuan

doi:10.3389/fcimb.2021.741147

Cited by 8 publications

(6 citation statements)

References 37 publications

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“…When the number of available SARS-CoV-2 genomes was limited, choosing conserved target genes was a crucial factor for the design of optimal PCR primers (Li et al 2020 ), and optimization of laboratory test protocols was important for effective detection (Won et al 2020 ). Checking for abundant splicing variants (Park et al 2020 ) and designing primers that can anneal to non-degenerate codons (encoding tryptophan) at the 3’-ends of sequences (Dong et al 2021 ) were also suggested to ensure highly sensitive viral nucleic acid detection. On the contrary, our current study does not rely on previous knowledge of the structural aspects of conserved genes, and our approach can be scaled easily as the number of SARS-CoV-2 genomes increases.…”

Section: Discussionmentioning

confidence: 99%

Identification of conserved regions from 230,163 SARS-CoV-2 genomes and their use in diagnostic PCR primer design

Jeong

Lee²,

Ko³

et al. 2022

Genes Genom

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Background As the rapidly evolving characteristic of SARS-CoV-2 could result in false negative diagnosis, the use of as much sequence data as possible is key to the identification of conserved viral sequences. However, multiple alignment of massive genome sequences is computationally intensive. Objective To extract conserved sequences from SARS-CoV-2 genomes for the design of diagnostic PCR primers using a bioinformatics approach that can handle massive genomic sequences efficiently. Methods A total of 230,163 full-length viral genomes were retrieved from the NCBI SARS-CoV-2 Resources and GISAID EpiCoV database. This number was reduced to 14.11% following removal of 5′-/3′-untranslated regions and sequence dereplication. Fast, reference-based, multiple sequence alignments identified conserved sequences and specific primer sets were designed against these regions using a conventional tool. Primer sets chosen among the candidates were evaluated by in silico PCR and RT-qPCR. Results Out of 17 conserved sequences (totaling 4.3 kb), two primer sets targeting the nsp2 and ORF3a genes were picked that exhibited > 99.9% in silico amplification coverage against the original dataset (230,163 genomes) when a 5% mismatch between the primers and target was allowed. In addition, the primer sets successfully detected nine SARS-CoV-2 variant RNA samples (Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Eta, Iota, and Kappa) in experimental RT-qPCR validations. Conclusion In addition to the RdRp, E, N, and S genes that are targeted commonly, our approach can be used to identify novel primer targets in SARS-CoV-2 and should be a priority strategy in the event of novel SARS-CoV-2 variants or other pandemic outbreaks. Supplementary Information The online version contains supplementary material available at 10.1007/s13258-022-01264-7.

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Section: Discussionmentioning

confidence: 99%

Identification of conserved regions from 230,163 SARS-CoV-2 genomes and their use in diagnostic PCR primer design

Jeong

Lee²,

Ko³

et al. 2022

Genes Genom

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“…Using this principle, they designed primer pairs that targeted the nucleocapsid (N) gene. Their design rendered similar sensitivity and specificity to the US and Chinese CDC-validated primers, but more importantly, performance was not influenced by frequently occurring mutations ( Dong et al, 2021 ).…”

Section: Diagnostic Methodsmentioning

confidence: 99%

Influenza A, Influenza B, and SARS-CoV-2 Similarities and Differences – A Focus on Diagnosis

et al. 2022

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In late December 2019, the first cases of viral pneumonia caused by an unidentified pathogen were reported in China. Two years later, SARS-CoV-2 was responsible for almost 450 million cases, claiming more than 6 million lives. The COVID-19 pandemic strained the limits of healthcare systems all across the world. Identifying viral RNA through real-time reverse transcription-polymerase chain reaction remains the gold standard in diagnosing SARS-CoV-2 infection. However, equipment cost, availability, and the need for trained personnel limited testing capacity. Through an unprecedented research effort, new diagnostic techniques such as rapid diagnostic testing, isothermal amplification techniques, and next-generation sequencing were developed, enabling accurate and accessible diagnosis. Influenza viruses are responsible for seasonal outbreaks infecting up to a quarter of the human population worldwide. Influenza and SARS-CoV-2 present with flu-like symptoms, making the differential diagnosis challenging solely on clinical presentation. Healthcare systems are likely to be faced with overlapping SARS-CoV-2 and Influenza outbreaks. This review aims to present the similarities and differences of both infections while focusing on the diagnosis. We discuss the clinical presentation of Influenza and SARS-CoV-2 and techniques available for diagnosis. Furthermore, we summarize available data regarding the multiplex diagnostic assay of both viral infections.

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“…Both methionine and isoleucine are nonpolar amino acids. Previous reports have suggested that M322I could bring about a slight decrease in N protein thermal stability (15) and might affect the host immune response to COVID-19 viral infection (16).…”

Section: Signature Mutations Of the Ba237 Lineagementioning

confidence: 99%

Emergence and Persistent Dominance of Omicron BA.2.3.7 Variant in Community Outbreaks in Taiwan

Shai

Gong

et al. 2022

Preprint

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Using high-throughput sequencing of the SARS-CoV-2 genome, we have analyzed 2405 PCR-positive swab samples from 2339 individuals and identified the Omicron BA.2.3.7 variant as a major lineage of the recent community outbreak in Taiwan. Since April 2022, a series of new waves of Omicron cases have surfaced in Taiwan and spread throughout the island. We conducted genomic surveillance with a high success rate and have submitted 1966 full-length Omicron sequences to GISAID. This has permitted identification of signature amino acid changes (ORF1a:L631F, S:K97E, N:M322I) in 1584 (80.6%) of the translated SARS-CoV-2 sequences. The newly established BA.2.3.7 lineage, which is relatively common in Asian countries, is now persistently present in Taiwan. By June 2022 this dominant strain had established a substantial existence in the sequence pool, resulting in additional mutations. The rapid spread and expansion of the Omicron BA.2.3.7 lineage in Asia has had an important socioeconomic impact on health policy.

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Structure-Based Primer Design Minimizes the Risk of PCR Failure Caused by SARS-CoV-2 Mutations

Cited by 8 publications

References 37 publications

Identification of conserved regions from 230,163 SARS-CoV-2 genomes and their use in diagnostic PCR primer design

Identification of conserved regions from 230,163 SARS-CoV-2 genomes and their use in diagnostic PCR primer design

Influenza A, Influenza B, and SARS-CoV-2 Similarities and Differences – A Focus on Diagnosis

Emergence and Persistent Dominance of Omicron BA.2.3.7 Variant in Community Outbreaks in Taiwan

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