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. 2016 Jan 2;9(1):e29246.
doi: 10.5812/jjm.29246. eCollection 2016 Jan.

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

Bentolhoda Zahraei  1 , Mohammad Sadegh Hashemzadeh  1 , Mohammad Najarasl  1 , Samaneh Zahiriyeganeh  1 , Mahdi Tat  1 , Maliheh Metanat  2 , Nahid Sepehri Rad  2 , Behzad Khansari-Nejad  3 , Ehsan Zafari  1 , Mojtaba Sharti  1 , Ruhollah Dorostkar  1
Affiliations

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

Bentolhoda Zahraei et al. Jundishapur J Microbiol. .

Abstract

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.

Objectives: 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.

Patients and 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.

Keywords: Crimean-Congo; Diagnosis; Hemorrhagic Fever Virus; Real-Time Polymerase Chain Reaction.

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Figures

Figure 1.
Figure 1.. Real-Time RT-PCR Amplicons With a Length of 420 bp
Confirmed by electrophoresis (23, 25, 42, and 46 are sample numbers). Lad is a 100 bp DNA Ladder (SinaClon, Iran), NTC is not template control or negative control.
Figure 2.
Figure 2.. Melting Curve Analysis
A, Showing that the melting temperature (Tm) of the specific amplicons was 86.5°C, and different from the primer-dimers; B, serial dilutions of the positive control sample showing that the mean melting temperature (Tm) of the specific amplicons was 86.5 ± 0.6°C, and the melting temperature (Tm) of the primer-dimers was different.
Figure 3.
Figure 3.. The Melting Curve Analysis of the Positive Samples
The curve is showing only a small variation in the melting curve parameters (Tm = 86.5 ± 0.6°C). The melting temperature (Tm) of the primer-dimers was quite different.
Figure 4.
Figure 4.. Amplification Plot of Developed Rrt-Pcr Assay
Plot is showing serial dilutions of the positive control sample for the evaluation of the analytical sensitivity of the assay.
See this image and copyright information in PMC

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