Author/Authors :
Zahraei، Bentolhoda نويسنده Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran , , Hashemzadeh، Mohammad Sadegh نويسنده Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran , , Najarasl، Mohammad نويسنده Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran , , Zahiriyeganeh، Samaneh نويسنده Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran , , Tat، Mahdi نويسنده Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran , , Metanat، Maliheh نويسنده , , Sepehri Rad، Nahid نويسنده 1Infectious Diseases and Tropical Medicine Research Center, Zahedan University of Medical Sciences, Zahedan, IR Iran Sepehri Rad, Nahid , Khansarinejad، Behzad نويسنده Department of Microbiology, Arak University of Medical Sciences, Arak, IR Iran , , Zafari، Ehsan نويسنده Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran , , Sharti، Mojtaba نويسنده Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran , , Dorostkar، Ruhollah نويسنده Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran ,
Abstract :
The Crimean-Congo hemorrhagic fever (CCHF) virus causes severe disease in humans, with a high mortality rate. Since, there is no approved vaccine or specific treatment for CCHF, an early and accurate diagnosis, as well as reliable surveillance, is essential for case management and patient improvement. For this research, our aim was to evaluate the application of a novel SYBR Green based one-step real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) assay for the in-house diagnosis of the CCHF virus. In this experimental study, the highly conserved S-region sequence of the CCHF viral genome was first adapted from GenBank, and the specific primers targeting this region were designed. Then, the viral RNA was extracted from 75 serum samples from different patients in eastern Iran. The sensitivity and specificity of the primers were also evaluated in positive serum samples previously confirmed to have the CCHF virus, by this one-step rRT-PCR assay, as well as a DNA sequencing analysis. From a total of 75 suspected serum samples, 42 were confirmed to be positive for CCHF virus, with no false-positives detected by the sequencing results. After 40 amplification cycles, the melting curve analysis revealed a mean melting temperature (Tm) of 86.5 ± 0.6°C (quite different from those of the primer-dimers), and the positive samples showed only a small variation in the parameters. In all of the positive samples, the predicted length of 420 bp was confirmed by electrophoresis. Moreover, the sensitivity test showed that this assay can detect less than 20 copies of viral RNA per reaction. This study showed that this novel one-step rRT-PCR assay is a rapid, reliable, repeatable, specific, sensitive, and simple tool for the detection of the CCHF virus.
