Viruses (Sep 2013)

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

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

DOI
https://doi.org/10.3390/v5092272
Journal volume & issue
Vol. 5, no. 9
pp. 2272 – 2281

Abstract

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Hantaviruses are members of the family Bunyaviridaeandare an emergingcause of diseaseworldwidewithhighlethalityin the Americas. In Brazil, thediagnosis forhantaviruses is basedonimmunologictechniquesassociatedwithconventionalRT-PCR.Anovelone-stepSYBRGreenreal-timeRT-PCRwasdevelopedfor the detectionandquantitationofAraraquarahantavirus(ARAV)and Rio Mamore hantavirus (RIOMV). Thedetectionlimitof assay was 10copies/µLofRNA in vitro transcribed of segment S. The specificity of assay was evaluatedbymeltingcurveanalysis, which showed thattheAraraquaravirusamplifiedproductgenerate dameltpeak at80.83±0.89°C without generating primer-dimersornon-specificproducts.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 ARAVand RIOMVand possibly other Brazilian hantaviruses.

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