Novel, In-House, SYBR Green Based One-Step rRT-PCR: Rapid and Accurate Diagnosis of Crimean-Congo Hemorrhagic Fever Virus in Suspected Patients From Iran.
BACKGROUND
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.


OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran.
Bentolhoda
B
Zahraei
Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran.
Mohammad Sadegh
MS
Hashemzadeh
Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran.
Mohammad
M
Najarasl
Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran.
Samaneh
S
Zahiriyeganeh
Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran.
Mahdi
M
Tat
Research Center for Infectious Diseases and Tropical Medicine, Boo-Ali Hospital, Zahedan University of Medical Sciences, Zahedan, IR Iran.
Maliheh
M
Metanat
Research Center for Infectious Diseases and Tropical Medicine, Boo-Ali Hospital, Zahedan University of Medical Sciences, Zahedan, IR Iran.
Nahid
N
Sepehri Rad
Department of Microbiology and Immunology, Arak University of Medical Sciences, Arak, IR Iran.
Behzad
B
Khansari-Nejad
Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran.
Ehsan
E
Zafari
Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran.
Mojtaba
M
Sharti
Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran.
Ruhollah
R
Dorostkar
Crimean-Congo; Diagnosis; Hemorrhagic Fever Virus; Real-Time Polymerase Chain Reaction