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. 2013 Sep 19;5(9):2272-81.
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

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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

Alex Martins Machado et al. Viruses. .

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 10 copies/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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Figures

Figure 1
Figure 1
Melting peaks of the one-step SYBR Green I real-time RT-PCR. (A) Melting peaks of real-time RT-PCR obtained from in vitro transcribed ARAV RNA at 103–108 copies/μL; (B) Melting peaks obtained from all positive samples. All melting curves included in A and B have similar shapes.

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