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In silico re-assessment of a diagnostic RT-qPCR assay for universal detection of Influenza A viruses

The ongoing evolution of microbial pathogens represents a significant issue in diagnostic PCR/qPCR. Many assays are burdened with false negativity due to mispriming and/or probe-binding failures. Therefore, PCR/qPCR assays used in the laboratory should be periodically re-assessed in silico on public...

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Autores principales: Nagy, Alexander, Jiřinec, Tomáš, Jiřincová, Helena, Černíková, Lenka, Havlíčková, Martina
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6367508/
https://www.ncbi.nlm.nih.gov/pubmed/30733500
http://dx.doi.org/10.1038/s41598-018-37869-w
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author Nagy, Alexander
Jiřinec, Tomáš
Jiřincová, Helena
Černíková, Lenka
Havlíčková, Martina
author_facet Nagy, Alexander
Jiřinec, Tomáš
Jiřincová, Helena
Černíková, Lenka
Havlíčková, Martina
author_sort Nagy, Alexander
collection PubMed
description The ongoing evolution of microbial pathogens represents a significant issue in diagnostic PCR/qPCR. Many assays are burdened with false negativity due to mispriming and/or probe-binding failures. Therefore, PCR/qPCR assays used in the laboratory should be periodically re-assessed in silico on public sequences to evaluate the ability to detect actually circulating strains and to infer potentially escaping variants. In the work presented we re-assessed a RT-qPCR assay for the universal detection of influenza A (IA) viruses currently recommended by the European Union Reference Laboratory for Avian Influenza. To this end, the primers and probe sequences were challenged against more than 99,000 M-segment sequences in five data pools. To streamline this process, we developed a simple algorithm called the SequenceTracer designed for alignment stratification, compression, and personal sequence subset selection and also demonstrated its utility. The re-assessment confirmed the high inclusivity of the assay for the detection of avian, swine and human pandemic H1N1 IA viruses. On the other hand, the analysis identified human H3N2 strains with a critical probe-interfering mutation circulating since 2010, albeit with a significantly fluctuating proportion. Minor variations located in the forward and reverse primers identified in the avian and swine data were also considered.
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spelling pubmed-63675082019-02-14 In silico re-assessment of a diagnostic RT-qPCR assay for universal detection of Influenza A viruses Nagy, Alexander Jiřinec, Tomáš Jiřincová, Helena Černíková, Lenka Havlíčková, Martina Sci Rep Article The ongoing evolution of microbial pathogens represents a significant issue in diagnostic PCR/qPCR. Many assays are burdened with false negativity due to mispriming and/or probe-binding failures. Therefore, PCR/qPCR assays used in the laboratory should be periodically re-assessed in silico on public sequences to evaluate the ability to detect actually circulating strains and to infer potentially escaping variants. In the work presented we re-assessed a RT-qPCR assay for the universal detection of influenza A (IA) viruses currently recommended by the European Union Reference Laboratory for Avian Influenza. To this end, the primers and probe sequences were challenged against more than 99,000 M-segment sequences in five data pools. To streamline this process, we developed a simple algorithm called the SequenceTracer designed for alignment stratification, compression, and personal sequence subset selection and also demonstrated its utility. The re-assessment confirmed the high inclusivity of the assay for the detection of avian, swine and human pandemic H1N1 IA viruses. On the other hand, the analysis identified human H3N2 strains with a critical probe-interfering mutation circulating since 2010, albeit with a significantly fluctuating proportion. Minor variations located in the forward and reverse primers identified in the avian and swine data were also considered. Nature Publishing Group UK 2019-02-07 /pmc/articles/PMC6367508/ /pubmed/30733500 http://dx.doi.org/10.1038/s41598-018-37869-w Text en © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Nagy, Alexander
Jiřinec, Tomáš
Jiřincová, Helena
Černíková, Lenka
Havlíčková, Martina
In silico re-assessment of a diagnostic RT-qPCR assay for universal detection of Influenza A viruses
title In silico re-assessment of a diagnostic RT-qPCR assay for universal detection of Influenza A viruses
title_full In silico re-assessment of a diagnostic RT-qPCR assay for universal detection of Influenza A viruses
title_fullStr In silico re-assessment of a diagnostic RT-qPCR assay for universal detection of Influenza A viruses
title_full_unstemmed In silico re-assessment of a diagnostic RT-qPCR assay for universal detection of Influenza A viruses
title_short In silico re-assessment of a diagnostic RT-qPCR assay for universal detection of Influenza A viruses
title_sort in silico re-assessment of a diagnostic rt-qpcr assay for universal detection of influenza a viruses
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6367508/
https://www.ncbi.nlm.nih.gov/pubmed/30733500
http://dx.doi.org/10.1038/s41598-018-37869-w
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