Large-Scale Nucleotide Sequence Alignment and Sequence Variability Assessment to Identify the Evolutionarily Highly Conserved Regions for Universal Screening PCR Assay Design: An Example of Influenza A Virus

Nagy, Alexander; Jiřinec, Tomáš; Černíková, Lenka; Jiřincová, Helena; Havlíčková, Martina

doi:10.1007/978-1-4939-2365-6_4

Cited by 11 publications

(6 citation statements)

References 19 publications

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“…Another source of bias may arise from the submission of isolates after passaging in the cell culture as well as sequencing artefacts including ambiguous data, short artificial insertions or deletions, incorrect sequence directions, incorrect nucleotide insertions, short sequence stretches and sequence longer than standard length [88]. Most data in the EpiCov database include the full-length data, and thus short sequences were not included in the study.…”

Section: Discussionmentioning

confidence: 99%

Presence of mismatches between diagnostic PCR assays and coronavirus SARS-CoV-2 genome

2020

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; initially named as 2019-nCoV) is responsible for the recent COVID-19 pandemic and polymerase chain reaction (PCR) is the current standard method for its diagnosis from patient samples. This study conducted a reassessment of published diagnostic PCR assays, including those recommended by the World Health Organization (WHO), through the evaluation of mismatches with publicly available viral sequences. An exhaustive evaluation of the sequence variability within the primer/probe target regions of the viral genome was performed using more than 17 000 viral sequences from around the world. The analysis showed the presence of mutations/mismatches in primer/probe binding regions of 7 assays out of 27 assays studied. A comprehensive bioinformatics approach for in silico inclusivity evaluation of PCR diagnostic assays of SARS-CoV-2 was validated using freely available software programs that can be applied to any diagnostic assay of choice. These findings provide potentially important information for clinicians, laboratory professionals and policy-makers.

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Section: Discussionmentioning

confidence: 99%

Presence of mismatches between diagnostic PCR assays and coronavirus SARS-CoV-2 genome

2020

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“…Although errors such as single nucleotide insertions, redundant stretches of primers or cloning vectors, sequence submissions in incorrect directions etc. can be recognized relatively easily [52,72] and correctable during alignment, others may be more difficult to identify, e.g. stock contamination or sequencing errors [73].…”

Section: Discussionmentioning

confidence: 99%

“…Alignment processing was performed with the AliView program [51]. Positional nucleotide numerical summary (PNNS) and entropy were calculated by the PNNS calculator and Entropy Calculator modules of the Alignment Explorer web application [52].…”

Section: Primers and Probe Selectionmentioning

confidence: 99%

A universal RT-qPCR assay for “One Health” detection of influenza A viruses

et al. 2021

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The mutual dependence of human and animal health is central to the One Health initiative as an integrated strategy for infectious disease control and management. A crucial element of the One Health includes preparation and response to influenza A virus (IAV) threats at the human-animal interface. The IAVs are characterized by extensive genetic variability, they circulate among different hosts and can establish host-specific lineages. The four main hosts are: avian, swine, human and equine, with occasional transmission to other mammalian species. The host diversity is mirrored in the range of the RT-qPCR assays for IAV detection. Different assays are recommended by the responsible health authorities for generic IAV detection in birds, swine or humans. In order to unify IAV monitoring in different hosts and apply the One Health approach, we developed a single RT-qPCR assay for universal detection of all IAVs of all subtypes, species origin and global distribution. The assay design was centred on a highly conserved region of the IAV matrix protein (MP)-segment identified by a comprehensive analysis of 99,353 sequences. The reaction parameters were effectively optimised with efficiency of 93–97% and LOD95% of approximately ten IAV templates per reaction. The assay showed high repeatability, reproducibility and robustness. The extensive in silico evaluation demonstrated high inclusivity, i.e. perfect sequence match in the primers and probe binding regions, established as 94.6% for swine, 98.2% for avian and 100% for human H3N2, pandemic H1N1, as well as other IAV strains, resulting in an overall predicted detection rate of 99% on the analysed dataset. The theoretical predictions were confirmed and extensively validated by collaboration between six veterinary or human diagnostic laboratories on a total of 1970 specimens, of which 1455 were clinical and included a diverse panel of IAV strains.

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“…Although errors such as single nucleotide insertions, redundant stretches of primers or cloning vectors, sequence submissions in incorrect directions etc. can be recognized relatively easily [51,72] and correctable during alignment, but others may be more difficult to identify [73]. In addition, it is important to consider the compositional bias which addresses significant under-or over-representation of certain sequence variants.…”

Section: Discussionmentioning

confidence: 99%

“…Alignment processing was performed with the AliView program [50]. Positional nucleotide numerical summary (PNNS) and entropy were calculated by the PNNS calculator and Entropy Calculator modules of the Alignment Explorer web application [51].…”

Section: Primers and Probe Selectionmentioning

confidence: 99%

A universal RT-qPCR assay for “One Health” detection of influenza A viruses

Nagy

Černíková

Kunteová

et al. 2020

Preprint

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AbstractThe mutual dependence of human and animal health is central to the One Health initiative as an integrated strategy for infectious disease control and management. A crucial element of the One Health includes preparation and response to influenza A virus (IAV) threats at the human-animal interface. The IAVs are characterized by extensive genetic variability, they circulate among different hosts and establish host-specific lineages. The four main host reservoirs are: avian, swine, human and equine, with occasional transmission to other mammalian species. The host diversity is mirrored in the range of the RT-qPCR assays for IAV detection. Different assays are recommended by the responsible health authorities for generic IAV detection in birds, swine or humans. In order to unify IAV monitoring in different hosts and apply the One Health approach, we developed a single RT-qPCR assay for universal detection of all IAVs of all subtypes, species origin and global distribution. The assay design was centred on a highly conserved region of the IAV MP-segment identified by a comprehensive analysis of 99,353 sequences. The reaction parameters were effectively optimised with efficiency of 93-97% and LOD95% of approximately ten IAV templates per reaction. The assay showed high repeatability, reproducibility and robustness. The extensive in silico evaluation demonstrated high inclusivity, i.e. perfect sequence match in the primers and probe binding regions, established as 94.6% for swine, 98.2% for avian and 100% for human H3N2, pandemic H1N1, as well as other IAV strains, resulting in an overall predicted detection rate of 99% on the analysed dataset. The theoretical predictions were confirmed and extensively validated by collaboration between six veterinary or human diagnostic laboratories on a total of 1970 specimens, of which 1455 were clinical and included a diverse panel of IAV strains.

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Large-Scale Nucleotide Sequence Alignment and Sequence Variability Assessment to Identify the Evolutionarily Highly Conserved Regions for Universal Screening PCR Assay Design: An Example of Influenza A Virus

Cited by 11 publications

References 19 publications

Presence of mismatches between diagnostic PCR assays and coronavirus SARS-CoV-2 genome

Presence of mismatches between diagnostic PCR assays and coronavirus SARS-CoV-2 genome

A universal RT-qPCR assay for “One Health” detection of influenza A viruses

A universal RT-qPCR assay for “One Health” detection of influenza A viruses

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