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One-step real-time reverse transcription-PCR assays for detecting and subtyping pandemic influenza A/H1N1 2009, seasonal influenza A/H1N1, and seasonal influenza A/H3N2 viruses
Pandemic influenza A/H1N1 2009 (A/H1N1pdm) virus has caused significant outbreaks worldwide. A previous one-step real-time reverse transcription-PCR (rRT-PCR) assay for detecting A/H1N1pdm virus (H1pdm rRT-PCR assay) was improved since the former probe had a low melting temperature and low tolerance...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier B.V.
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7173154/ https://www.ncbi.nlm.nih.gov/pubmed/21029748 http://dx.doi.org/10.1016/j.jviromet.2010.10.018 |
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author | Nakauchi, Mina Yasui, Yoshihiro Miyoshi, Tatsuya Minagawa, Hiroko Tanaka, Tomoyuki Tashiro, Masato Kageyama, Tsutomu |
author_facet | Nakauchi, Mina Yasui, Yoshihiro Miyoshi, Tatsuya Minagawa, Hiroko Tanaka, Tomoyuki Tashiro, Masato Kageyama, Tsutomu |
author_sort | Nakauchi, Mina |
collection | PubMed |
description | Pandemic influenza A/H1N1 2009 (A/H1N1pdm) virus has caused significant outbreaks worldwide. A previous one-step real-time reverse transcription-PCR (rRT-PCR) assay for detecting A/H1N1pdm virus (H1pdm rRT-PCR assay) was improved since the former probe had a low melting temperature and low tolerance to viral mutation. To help with the screening of the A/H1N1pdm virus, rRT-PCR assays were also developed for detecting human seasonal A/H1N1 (H1 rRT-PCR assay) and A/H3N2 influenza viruses (H3 rRT-PCR assay). H1pdm, H1, and H3 rRT-PCR assays were evaluated using in vitro-transcribed control RNA, isolated viruses, and other respiratory pathogenic viruses, and were shown to have high sensitivity, good linearity (R(2) = 0.99), and high specificity. In addition, the improved H1pdm rRT-PCR assay could detect two viral strains of A/H1N1pdm, namely, A/Aichi/472/2009 (H1N1)pdm and A/Sakai/89/2009 (H1N1)pdm, which have mutation(s) in the probe-binding region of the hemagglutinin gene, without loss of sensitivity. Using the three rRT-PCR assays developed, 90 clinical specimens collected between May and October 2009 were then tested. Of these, 26, 20, and 2 samples were identified as positive for A/H1pdm, A/H3, and A/H1, respectively, while 42 samples were negative for influenza A viruses. The present results suggest that these highly sensitive and specific H1pdm, H1, and H3 rRT-PCR assays are useful not only for diagnosing influenza viruses, but also for the surveillance of influenza viruses. |
format | Online Article Text |
id | pubmed-7173154 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | Elsevier B.V. |
record_format | MEDLINE/PubMed |
spelling | pubmed-71731542020-04-22 One-step real-time reverse transcription-PCR assays for detecting and subtyping pandemic influenza A/H1N1 2009, seasonal influenza A/H1N1, and seasonal influenza A/H3N2 viruses Nakauchi, Mina Yasui, Yoshihiro Miyoshi, Tatsuya Minagawa, Hiroko Tanaka, Tomoyuki Tashiro, Masato Kageyama, Tsutomu J Virol Methods Article Pandemic influenza A/H1N1 2009 (A/H1N1pdm) virus has caused significant outbreaks worldwide. A previous one-step real-time reverse transcription-PCR (rRT-PCR) assay for detecting A/H1N1pdm virus (H1pdm rRT-PCR assay) was improved since the former probe had a low melting temperature and low tolerance to viral mutation. To help with the screening of the A/H1N1pdm virus, rRT-PCR assays were also developed for detecting human seasonal A/H1N1 (H1 rRT-PCR assay) and A/H3N2 influenza viruses (H3 rRT-PCR assay). H1pdm, H1, and H3 rRT-PCR assays were evaluated using in vitro-transcribed control RNA, isolated viruses, and other respiratory pathogenic viruses, and were shown to have high sensitivity, good linearity (R(2) = 0.99), and high specificity. In addition, the improved H1pdm rRT-PCR assay could detect two viral strains of A/H1N1pdm, namely, A/Aichi/472/2009 (H1N1)pdm and A/Sakai/89/2009 (H1N1)pdm, which have mutation(s) in the probe-binding region of the hemagglutinin gene, without loss of sensitivity. Using the three rRT-PCR assays developed, 90 clinical specimens collected between May and October 2009 were then tested. Of these, 26, 20, and 2 samples were identified as positive for A/H1pdm, A/H3, and A/H1, respectively, while 42 samples were negative for influenza A viruses. The present results suggest that these highly sensitive and specific H1pdm, H1, and H3 rRT-PCR assays are useful not only for diagnosing influenza viruses, but also for the surveillance of influenza viruses. Elsevier B.V. 2011-01 2010-10-26 /pmc/articles/PMC7173154/ /pubmed/21029748 http://dx.doi.org/10.1016/j.jviromet.2010.10.018 Text en Copyright © 2010 Elsevier B.V. All rights reserved. Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. |
spellingShingle | Article Nakauchi, Mina Yasui, Yoshihiro Miyoshi, Tatsuya Minagawa, Hiroko Tanaka, Tomoyuki Tashiro, Masato Kageyama, Tsutomu One-step real-time reverse transcription-PCR assays for detecting and subtyping pandemic influenza A/H1N1 2009, seasonal influenza A/H1N1, and seasonal influenza A/H3N2 viruses |
title | One-step real-time reverse transcription-PCR assays for detecting and subtyping pandemic influenza A/H1N1 2009, seasonal influenza A/H1N1, and seasonal influenza A/H3N2 viruses |
title_full | One-step real-time reverse transcription-PCR assays for detecting and subtyping pandemic influenza A/H1N1 2009, seasonal influenza A/H1N1, and seasonal influenza A/H3N2 viruses |
title_fullStr | One-step real-time reverse transcription-PCR assays for detecting and subtyping pandemic influenza A/H1N1 2009, seasonal influenza A/H1N1, and seasonal influenza A/H3N2 viruses |
title_full_unstemmed | One-step real-time reverse transcription-PCR assays for detecting and subtyping pandemic influenza A/H1N1 2009, seasonal influenza A/H1N1, and seasonal influenza A/H3N2 viruses |
title_short | One-step real-time reverse transcription-PCR assays for detecting and subtyping pandemic influenza A/H1N1 2009, seasonal influenza A/H1N1, and seasonal influenza A/H3N2 viruses |
title_sort | one-step real-time reverse transcription-pcr assays for detecting and subtyping pandemic influenza a/h1n1 2009, seasonal influenza a/h1n1, and seasonal influenza a/h3n2 viruses |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7173154/ https://www.ncbi.nlm.nih.gov/pubmed/21029748 http://dx.doi.org/10.1016/j.jviromet.2010.10.018 |
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