Development of a one-step SYBR Green I real-time RT-PCR assay for the detection and quantitation of Araraquara and Rio Mamore hantavirus

Alex Martins Machado; William Marciel de Souza; Michelly de Pádua; Aline Rafaela da Silva Rodrigues Machado; Luiz Tadeu Moraes Figueiredo

doi:10.3390/v5092272

Development of a one-step SYBR Green I real-time RT-PCR assay for the detection and quantitation of Araraquara and Rio Mamore hantavirus

Alex Martins Machado, William Marciel de Souza, Michelly de Pádua, Aline Rafaela da Silva Rodrigues Machado, Luiz Tadeu Moraes Figueiredo

Research output: Contribution to journal › Article › peer-review

Abstract

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 (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.

Original language	English (US)
Pages (from-to)	2272-2281
Number of pages	10
Journal	Viruses
Volume	5
Issue number	9
DOIs	https://doi.org/10.3390/v5092272
State	Published - Sep 19 2013
Externally published	Yes

Keywords

Araraquara virus
Hantaviruses
Real-time quantitative RT-PCR
Rio Mamore virus
SYBR Green I

ASJC Scopus subject areas

Infectious Diseases
Virology

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10.3390/v5092272

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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",

abstract = "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 (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.",

keywords = "Araraquara virus, Hantaviruses, Real-time quantitative RT-PCR, Rio Mamore virus, SYBR Green I",

author = "Machado, {Alex Martins} and {de Souza}, {William Marciel} and {de P{\'a}dua}, Michelly and {Rodrigues Machado}, {Aline Rafaela da Silva} and {Moraes Figueiredo}, {Luiz Tadeu}",

year = "2013",

month = sep,

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doi = "10.3390/v5092272",

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AU - Machado, Alex Martins

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AU - de Pádua, Michelly

AU - Rodrigues Machado, Aline Rafaela da Silva

AU - Moraes Figueiredo, Luiz Tadeu

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N2 - 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 (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.

AB - 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 (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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KW - Rio Mamore virus

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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

Abstract

Keywords

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