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A Broadly Reactive One-Step SYBR Green I Real-Time RT-PCR Assay for Rapid Detection of Murine Norovirus
A one-step SYBR Green I real-time RT-PCR assay was developed for the detection and quantification of a broad range of murine noroviruses (MNVs). The primer design was based on the multiple sequence alignments of 101 sequences of the open reading frame (ORF)1−ORF2 junction of MNV. The broad reactivit...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4029972/ https://www.ncbi.nlm.nih.gov/pubmed/24847970 http://dx.doi.org/10.1371/journal.pone.0098108 |
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author | Hanaki, Ken-Ichi Ike, Fumio Kajita, Ayako Yasuno, Wataru Yanagiba, Misato Goto, Motoki Sakai, Kouji Ami, Yasushi Kyuwa, Shigeru |
author_facet | Hanaki, Ken-Ichi Ike, Fumio Kajita, Ayako Yasuno, Wataru Yanagiba, Misato Goto, Motoki Sakai, Kouji Ami, Yasushi Kyuwa, Shigeru |
author_sort | Hanaki, Ken-Ichi |
collection | PubMed |
description | A one-step SYBR Green I real-time RT-PCR assay was developed for the detection and quantification of a broad range of murine noroviruses (MNVs). The primer design was based on the multiple sequence alignments of 101 sequences of the open reading frame (ORF)1−ORF2 junction of MNV. The broad reactivity and quantitative capacity of the assay were validated using 7 MNV plasmids. The assay was completed within 1 h, and the reliable detection limit was 10 copies of MNV plasmid or 0.063 median tissue culture infective doses per milliliter of RAW264 cell culture-propagated viruses. The diagnostic performance of the assay was evaluated using 158 mouse fecal samples, 91 of which were confirmed to be positive. The melting curve analysis demonstrated the diversity of MNV in the samples. This is the first report of a broadly reactive one-step SYBR Green I real-time RT-PCR assay for detecting of MNVs. The rapid and sensitive performance of this assay makes it a powerful tool for diagnostic applications. |
format | Online Article Text |
id | pubmed-4029972 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-40299722014-05-28 A Broadly Reactive One-Step SYBR Green I Real-Time RT-PCR Assay for Rapid Detection of Murine Norovirus Hanaki, Ken-Ichi Ike, Fumio Kajita, Ayako Yasuno, Wataru Yanagiba, Misato Goto, Motoki Sakai, Kouji Ami, Yasushi Kyuwa, Shigeru PLoS One Research Article A one-step SYBR Green I real-time RT-PCR assay was developed for the detection and quantification of a broad range of murine noroviruses (MNVs). The primer design was based on the multiple sequence alignments of 101 sequences of the open reading frame (ORF)1−ORF2 junction of MNV. The broad reactivity and quantitative capacity of the assay were validated using 7 MNV plasmids. The assay was completed within 1 h, and the reliable detection limit was 10 copies of MNV plasmid or 0.063 median tissue culture infective doses per milliliter of RAW264 cell culture-propagated viruses. The diagnostic performance of the assay was evaluated using 158 mouse fecal samples, 91 of which were confirmed to be positive. The melting curve analysis demonstrated the diversity of MNV in the samples. This is the first report of a broadly reactive one-step SYBR Green I real-time RT-PCR assay for detecting of MNVs. The rapid and sensitive performance of this assay makes it a powerful tool for diagnostic applications. Public Library of Science 2014-05-21 /pmc/articles/PMC4029972/ /pubmed/24847970 http://dx.doi.org/10.1371/journal.pone.0098108 Text en © 2014 Hanaki et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Hanaki, Ken-Ichi Ike, Fumio Kajita, Ayako Yasuno, Wataru Yanagiba, Misato Goto, Motoki Sakai, Kouji Ami, Yasushi Kyuwa, Shigeru A Broadly Reactive One-Step SYBR Green I Real-Time RT-PCR Assay for Rapid Detection of Murine Norovirus |
title | A Broadly Reactive One-Step SYBR Green I Real-Time RT-PCR Assay for Rapid Detection of Murine Norovirus |
title_full | A Broadly Reactive One-Step SYBR Green I Real-Time RT-PCR Assay for Rapid Detection of Murine Norovirus |
title_fullStr | A Broadly Reactive One-Step SYBR Green I Real-Time RT-PCR Assay for Rapid Detection of Murine Norovirus |
title_full_unstemmed | A Broadly Reactive One-Step SYBR Green I Real-Time RT-PCR Assay for Rapid Detection of Murine Norovirus |
title_short | A Broadly Reactive One-Step SYBR Green I Real-Time RT-PCR Assay for Rapid Detection of Murine Norovirus |
title_sort | broadly reactive one-step sybr green i real-time rt-pcr assay for rapid detection of murine norovirus |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4029972/ https://www.ncbi.nlm.nih.gov/pubmed/24847970 http://dx.doi.org/10.1371/journal.pone.0098108 |
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