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 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.

Co-circulation in a single biome of the Juquitiba and Araraquara hantavirus detected in human sera in a sub-tropical region of Brazil

“…This test shows sensitivity in the detection of hantavirus in the serum of patients. Researchers obtained similar results using the SYBR Green system, thus confirming the obtained serological results when analyzing samples from patients and rodents infected with Dobrava hantavirus (DOBV) [Jakab et al, 2007], Hantaan virus [Wei et al, 2013] and ARAV [Machado et al, 2013].…”

Molecular method for the detection of Andes hantavirus infection: validation for clinical diagnostics

“…Others have previously detected hantavirus by RT-qPCR but to our knowledge, this is the first effort to validate this technique as a diagnostic tool in a large number of patients (Evander et al 2007; Ibrahim et al 2011; Machado and Souza 2013). …”

Experimental infection of Rio Mamore hantavirus in Sigmodontinae rodents

“…The RNA of the viral stock as well as that of the mouse samples was extracted using the QIAamp viral RNA extraction kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Then, for detection and quantification of RIOMV in the viral stock and in the mouse samples, we used a one-step SYBR Green I real-time RT-PCR assay ( Machado et al 2013 ). RT-PCR was performed in triplicate using the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), the SuperScript III Platinum SYBR Green One-Step Kit (Invitrogen, Carlsbad, CA, USA), and hantavirus primers that amplify 264 base pairs (bp) of the N gene in the small RNA segment of RIOMV, as previously reported ( Moreli et al 2004 ).…”

“…After RT-PCR measurements, the concentration of transcribed RNA extract from the RIOMV stock, in copies/μL, was converted to copy number using the following formula: RNA copy number (copies/μL) = (RNA concentration (g/μL)/number of nucleotides of transcript x 340) x 6.022x10 23 . ΔRn amplification results were obtained from ten-fold serial dilutions of the transcribed RNA by real-time RT-PCR and were used to create a standard curve with a known number of RIOMV RNA copies of per mL, as previously described ( Machado et al 2013 ).…”