Development and evaluation of a broad reacting SYBR-green based quantitative real-time PCR for the detection of different hantaviruses

Background: Hantaviruses are endemic in most parts of the world and cause hundreds of thousand human cases of hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) annually throughout Eurasia and the Americas. They are zoonotic viruses, most commonly transmitted to humans by aerosolized rodent excreta. New hantaviruses are frequently discovered in previously unknown reservoir species and geographic areas. Consequently, there is a need to improve hantavirus diagnostics. Objectives: This paper describes the design and evaluation of a rapid and robust quantitative real-time PCR (QRT-PCR) assay able to detect a wide range of hantaviruses. Study design: Primers with the potential to detect different hantaviruses were designed from conserved regions of different hantavirus L segments, as identified from multiple sequence alignments. Results: By using SYBR-green-based QRT-PCR 100-1000 target molecules of in vitro produced RNA and less than 100 copies of hantavirus RNA from different hantavirus clades and regions of the world were detected. When using the assay on clinical samples from patients with acute HFRS, Puumala hantavirus (PUUV) RNA was confirmed in all previously positive samples. Notably, the broad reacting L-segment QRT-PCR also detected viral RNA in HFRS patient samples, previously negative by a QRT-PCR targeting the S segment of PUUV. Conclusions: This novel assay provides a powerful tool for diagnosis of hantaviruses from different clades and regions and may also be useful in surveys with the purpose of finding new hantaviruses in rodent or insectivore species. (c) 2012 Elsevier B.V. All rights reserved.