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Development of a One-Step SYBR Green I Real-Time RT-PCR Assay for the Detection and Quantitation of Araraquara and Rio Mamore Hantavirus

Hantaviruses are members of the family Bunyaviridae and are an emerging cause of disease worldwide with high lethality in the Americas. In Brazil, the diagnosis for hantaviruses is based on immunologic techniques associated with conventional RT-PCR. A novel one-step SYBR Green real-time RT-PCR was d...

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Autores principales: Machado, Alex Martins, de Souza, William Marciel, de Pádua, Michelly, Machado, Aline Rafaela da Silva Rodrigues, Figueiredo, Luiz Tadeu Moraes
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3798901/
https://www.ncbi.nlm.nih.gov/pubmed/24056673
http://dx.doi.org/10.3390/v5092272
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author Machado, Alex Martins
de Souza, William Marciel
de Pádua, Michelly
Machado, Aline Rafaela da Silva Rodrigues
Figueiredo, Luiz Tadeu Moraes
author_facet Machado, Alex Martins
de Souza, William Marciel
de Pádua, Michelly
Machado, Aline Rafaela da Silva Rodrigues
Figueiredo, Luiz Tadeu Moraes
author_sort Machado, Alex Martins
collection PubMed
description Hantaviruses are members of the family Bunyaviridae and are an emerging cause of disease worldwide with high lethality in the Americas. In Brazil, the diagnosis for hantaviruses is based on immunologic techniques associated with conventional RT-PCR. A novel one-step SYBR Green real-time RT-PCR was developed for the detection and quantitation of Araraquara hantavirus (ARAV) and Rio Mamore hantavirus (RIOMV). The detection limit of assay was 10copies/µL of RNA in vitro transcribed of segment S. The specificity of assay was evaluated by melting curve analysis, which showed that the Araraquara virus amplified product generated a melt peak at 80.83 ± 0.89 °C without generating primer-dimers or non-specific products. The assay was more sensitive than conventional RT-PCR and we detected two samples undetected by conventional RT-PCR. The one-step SYBR Green real-time quantitative RT-PCR is specific, sensible and reproducible, which makes it a powerful tool in both diagnostic applications and general research of ARAV and RIOMV and possibly other Brazilian hantaviruses.
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spelling pubmed-37989012013-10-21 Development of a One-Step SYBR Green I Real-Time RT-PCR Assay for the Detection and Quantitation of Araraquara and Rio Mamore Hantavirus Machado, Alex Martins de Souza, William Marciel de Pádua, Michelly Machado, Aline Rafaela da Silva Rodrigues Figueiredo, Luiz Tadeu Moraes Viruses Article Hantaviruses are members of the family Bunyaviridae and are an emerging cause of disease worldwide with high lethality in the Americas. In Brazil, the diagnosis for hantaviruses is based on immunologic techniques associated with conventional RT-PCR. A novel one-step SYBR Green real-time RT-PCR was developed for the detection and quantitation of Araraquara hantavirus (ARAV) and Rio Mamore hantavirus (RIOMV). The detection limit of assay was 10copies/µL of RNA in vitro transcribed of segment S. The specificity of assay was evaluated by melting curve analysis, which showed that the Araraquara virus amplified product generated a melt peak at 80.83 ± 0.89 °C without generating primer-dimers or non-specific products. The assay was more sensitive than conventional RT-PCR and we detected two samples undetected by conventional RT-PCR. The one-step SYBR Green real-time quantitative RT-PCR is specific, sensible and reproducible, which makes it a powerful tool in both diagnostic applications and general research of ARAV and RIOMV and possibly other Brazilian hantaviruses. MDPI 2013-09-19 /pmc/articles/PMC3798901/ /pubmed/24056673 http://dx.doi.org/10.3390/v5092272 Text en © 2013 by the authors; licensee MDPI, Basel, Switzerland. http://creativecommons.org/licenses/by/3.0/ This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
spellingShingle Article
Machado, Alex Martins
de Souza, William Marciel
de Pádua, Michelly
Machado, Aline Rafaela da Silva Rodrigues
Figueiredo, Luiz Tadeu Moraes
Development of a One-Step SYBR Green I Real-Time RT-PCR Assay for the Detection and Quantitation of Araraquara and Rio Mamore Hantavirus
title Development of a One-Step SYBR Green I Real-Time RT-PCR Assay for the Detection and Quantitation of Araraquara and Rio Mamore Hantavirus
title_full Development of a One-Step SYBR Green I Real-Time RT-PCR Assay for the Detection and Quantitation of Araraquara and Rio Mamore Hantavirus
title_fullStr Development of a One-Step SYBR Green I Real-Time RT-PCR Assay for the Detection and Quantitation of Araraquara and Rio Mamore Hantavirus
title_full_unstemmed Development of a One-Step SYBR Green I Real-Time RT-PCR Assay for the Detection and Quantitation of Araraquara and Rio Mamore Hantavirus
title_short Development of a One-Step SYBR Green I Real-Time RT-PCR Assay for the Detection and Quantitation of Araraquara and Rio Mamore Hantavirus
title_sort development of a one-step sybr green i real-time rt-pcr assay for the detection and quantitation of araraquara and rio mamore hantavirus
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3798901/
https://www.ncbi.nlm.nih.gov/pubmed/24056673
http://dx.doi.org/10.3390/v5092272
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