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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...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2019
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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. |
format | Online Article Text |
id | pubmed-6367508 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
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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