Development of quantitative real-time RT-PCR assay for detection and viral load determination of Crimean-Congo Hemorrhagic Fever (CCHF) virus

Abstract

Background and Aim: The CCHF (Crimean-Congo hemorrhagic fever) virus causes a severe disease in human with a case fatality rate of up to 50%. Since, there is no specific treatment or approved vaccine against CCHF viral infections, an accurate and early detection as well as a reliable surveillance and quantitative determination of viral load is necessary for patient improvement and case management. In this research, our aim was to develop a probe based one-step real-time reverse-transcription polymerase chain reaction (rRT-PCR) assay for in-house quantitative detection of CCHF virus. Methods: At first, the highly conserved S-fragment sequence of CCHF virus genome was adapted from GenBank and the specific probe and primers targeting this region were designed. Then, viral RNAs were extracted from 37 blood samples of different patients from east of Iran (Zahedan). The specificity and sensitivity of the probe and primers were also evaluated in positive blood samples, confirmed to have CCHF virus. A standard (PTG19-T vector containing S-fragment) for quantization was also constructed and the viral load was determined in some of positive samples. Results: From a total of 37 suspicious blood samples, 15 samples were confirmed to be positive for CCHF virus by this probe based one-step rRT-PCR assay and no false-positive result was detected according to sequencing data. The predicted fragment of 176 bp was also confirmed in all positive samples by gel-based electrophoresis analysis. The assay was linear between 10 to 103 copy numbers per each microliter of extracted plasmid for this technique and the viral load determined in one of patient blood samples was 55,000 viral particles per each milliliter, for example. Bioinformatics and experimental evaluations approved the specificity of this assay. The LOD of the assay was 10 (or fewer) copy numbers of viral genome per each microliter of the extracted genome. Conclusions: This research showed that the developed probe based one-step rRT-PCR assay is a specific, rapid, sensitive and the simple tool for detection and viral load determination of the CCHF virus. Keywords: CCHF Virus, Real-time RT-PCR, Quantitative, viral load, S-region.