Modern detection and identification tools can help to provide answers to urgent questions about the incidence, prevalence, and epidemiology of currently emerging diseases. We developed highly sensitive one-step TaqMan reverse transcription-PCR assays with sensitivities ranging from 10(4) to 10(1) molecules for 11 human pathogens of the orthobunyaviruses. We compared the performances of these assays on three currently available cyclers (ABI-PRISM 7700, LightCycler, and SmartCycler). The assay for Oropouche virus (OROV) was tested using sera collected from days 1 to 5 after onset of OROV disease and was found to be greatly superior to an established nested PCR system. A mean copy number of 1.31 x 10(7) OROV RNA/ml of serum was detected. Diagnostic RNA detection can be used as early as day 1 after onset of OROV disease. The use of a mobile SmartCycler and a hands-on time of less than 3 h could help to intensify outbreak surveillance and control, especially in field studies.