Novel one-step real-time RT-PCR assay for rapid and specific diagnosis of Crimean-Congo hemorrhagic fever encountered in the Balkans

Early and accurate diagnosis of Crimean-Congo hemorrhagic fever (CCHF) is essential for the treatment and outcome of the disease and prevention of its further transmission. Molecular-based diagnostic assays now serve as the front-line tool in the diagnosis of CCHF. However, the development of real-time RT-PCR assay for the detection of Crimean-Congo hemorrhagic fever virus (CCHFV) has been hampered by a virus strain variation. The development of a one-step real-time RT-PCR assay for the detection of CCHFV is described herein. The technique is based on the fluorescence resonance energy transfer probe technology employing the endonuclease activity of Taq polymerase enzyme. The assay was designed to detect specifically the strains from a phylogenetic cluster of CCHFV which encompasses the known CCHFV strains circulating in the Balkan region. The detection system was tested using CCHFV strain Kosovo Hoti, clinical serum samples and ticks. The real-time assay described is rapid, specific and sensitive. Since the Balkan peninsula is also an endemic region for hemorrhagic fever with renal syndrome (HFRS), this method is suggested as convenient for early differential diagnosis of suspected viral hemorrhagic fever patients.