Design and in silico validation of polymerase chain reaction primers to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

Davi, Maria Júlia P; Jerônimo, Selma M. B.; Lima, J. A. S.; Lanza, Daniel C.F.

doi:10.1038/s41598-021-91817-9

Cited by 13 publications

(12 citation statements)

References 25 publications

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“…The targeted RdRp, S, and N genes with specifications were shown in Table 1, while Figure 5 was used to illustrate the associated histograms. These results showed that some of the primer parameters and probes listed in the previous studies of Li et al (2020) and Davi et al (2021) should be enhanced. They also indicated that the sets used in this study did not have secondary structures.…”

Section: Primer Designmentioning

confidence: 65%

The design of Indonesian SARS‐CoV‐2 primers based on phylogenomic analysis of the SARS‐CoV‐2 clades

Nabila

Wasiati

Jati³

et al. 2022

Indones. J. Biotechnol.

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Molecular detection needs to be augmented for COVID‐19 detection in Indonesia using the PCR method with primer‐based gene analysis. This is necessary because the RNA of the SARS‐CoV‐2 virus, the causative infectious agent of the pandemic, has been mutated. Therefore, this study aimed to develop a primer design for determining SARS‐CoV‐2 clades in Indonesia using phylogenomic analysis. Data were obtained from 38 GISAID (Global Initiative on Sharing All Influenza Data) viruses and the relationships were analyzed using maximum likelihood (ML) phylogenomic analysis with a substitution model of generalized time‐reversible (GTR) to construct the tree topology. The results showed that the five types of SARS‐CoVs‐2 clades in Indonesia were L, G, GH, GR, and O. It also indicated that the GH region had the highest rate of clade at 50%, with the S clade affecting its formation. Furthermore, the genome sequences of the GH type used to design its primer were based on three genes, namely RdRp, S, and N. The RdRp and N genes were found to be conserved and hardy mutants, while the S gene occurred repeatedly. Several previous studies have stated that the designed primers produced missense mutations compared to another in silico. Therefore, three sets of primers were achieved from the GC contents and clamps, Tm range, and structural secondary indicator standards.

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Section: Primer Designmentioning

confidence: 65%

The design of Indonesian SARS‐CoV‐2 primers based on phylogenomic analysis of the SARS‐CoV‐2 clades

Nabila

Wasiati

Jati³

et al. 2022

Indones. J. Biotechnol.

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“…Considering Davi et al . ( 2021 ) reported that a supercomputer was required for the alignment of only 2,341 full viral genome sequences using Clustal Omega, ViralMSA would be the best choice for a PC-level computer. When the MAFFT (Katoh and Standley 2013 ) v7.490 program was used with the FFT-NS-2 option, it took more than 3 days to complete the alignment on the same computer.…”

Section: Resultsmentioning

confidence: 99%

“…Thus, CS ‘candidates’ would be the correct term to describe the output of post-MSA process and its manipulation. Running trimAl with the ‘-automated1’ option (a heuristic trimming method), as described previously (Davi et al 2021 ), discarded only a few sites from the original alignments and generated six subsequences covering 29,384 bp of the original 29,903 bp, suggesting that parameter optimization was required. However, as a single-threaded program, the prolonged execution time for a typical trimAl run (~ 16 h) made it unfeasible to tweak the parameters to prioritize conserved sites.…”

Section: Resultsmentioning

confidence: 99%

“…Standard MSA methods such as MUSCLE (Edgar 2004 ), MAFFT (Katoh and Standley 2013 ), and Clustal (Sievers and Higgins 2018 ) scale poorly with increasing numbers of input sequences, such that alignment of hundreds of thousands of viral genome sequences is not feasible on a Linux desktop computer. A recent study reported the extraction of CSs from Clustal Omega-generated MSAs of SARS-CoV-2 genomes (Davi et al 2021 ), but only 2,341 complete sequences were available at that time. Reference-guided MSA tools such as VIRULIGN (Libin et al 2019 ) and ViralMSA (Moshiri 2021 ) run much faster than the standard methods and can be used for a large-scale viral genome analysis.…”

Section: Introductionmentioning

confidence: 99%

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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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“…The CDC in the US also obtains the emergency use authorization (EUA) from FDA (Food and Drug Administration) for the first RT-PCR test. During the epidemic, a growing number of commercial kits for detecting SARS-CoV-2 and its variations were created, which were then employed in clinical laboratories and other organizations [ 6 – 8 ].…”

Section: Introductionmentioning

confidence: 99%

Comparison between the analytical sensitivity and clinical performance of two cobas SARS-CoV-2 tests based on high-throughput and point-of-care systems

Lin¹,

Bui²,

Chang³

et al. 2022

BioMedicine

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Objectives This study examined analytical sensitivity, specificity, and the clinical performance in detecting SARS-CoV-2 of the Cobas SARS-CoV-2 Test based on the high-throughput Cobas 6800 system and the Cobas SARS-CoV-2 & Flu A/B Test based on the point-of-care cobas Liat system. Methods The commercial reagents containing SARS-CoV-2 RNA subgenomes were diluted for assessing the sensitivity of the RT-qPCR assay. 385 nasopharyngeal swab specimens taken from contacts of COVID-19 cases were tested for the SARS-CoV-2 detection with both Cobas SARS-CoV-2 Tests. Results In analytical sensitivity assays, the Cobas SARS-CoV-2 & Flu A/B Test on the Liat system had a lower limit of detection (12.5–25 copies/mL) than the cobas SARS-CoV-2 Test on the cobas 6800 system (25–50 copies/mL). In clinical performance assays, the cobas SARS-CoV-2 Test demonstrated 89.36% (42 out of 47) PPA (positive percent agreement) and 98.82% (334 out of 338) NPA (negative percent agreement) compared to the results of the Cobas SARS-CoV-2 & Flu A/B test. Among five discordant specimens, four had the positive result of the cobas SARS-CoV-2 test, but the negative result of the cobas SARS-CoV-2 & Flu A/B Test. Moreover, these discordant specimens had the Ct values of greater than 33 for the cobas SARS-CoV-2 Test, implying a very small number of virions in the samples. Remarkably, four specimens with a presumptive positive result of the cobas SARS-CoV-2 test had been confirmed by the Cobas SARS-CoV-2 & Flu A/B Test. Next, the scatter plots of the Ct values showed a highly positive correlation between cobas SARS-CoV-2 & Flu A/B Test and the cobas SARS-CoV-2 Test (R-squared value = 0.954–0.962). Conclusions Both SARS-CoV2 tests of the cobas 6800 and Liat systems produce reliable high throughput and point-of-care assays respectively for the early virus detection and the personal care decision-making during COVID-19 pandemic.

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Design and in silico validation of polymerase chain reaction primers to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

Cited by 13 publications

References 25 publications

The design of Indonesian SARS‐CoV‐2 primers based on phylogenomic analysis of the SARS‐CoV‐2 clades

The design of Indonesian SARS‐CoV‐2 primers based on phylogenomic analysis of the SARS‐CoV‐2 clades

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

Comparison between the analytical sensitivity and clinical performance of two cobas SARS-CoV-2 tests based on high-throughput and point-of-care systems

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