Jpn. J. Infect. Dis., 58 (6), 358-362, 2005
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Original Article
Rapid and Quantitative Detection of Crimean-Congo Hemorrhagic Fever Virus by One-Step Real-Time Reverse Transcriptase-PCR
Mehmet Yapar, Hakan Aydogan1, Alaaddin Pahsa2, Bulent A. Besirbellioglu2, Hurrem Bodur3, Ahmet C. Basustaoglu1, Cakir Guney, Ismail Y. Avci2, Kenan Sener, Mohammad H. Abu Setteh4 and Ayhan Kubar*
Department of Virology, 1Department of Microbiology and Clinical Microbiology, 2Department of Infection Disease and Clinical Microbiology, Gulhane Military Medical Academy, 3Infectious Disease and Clinical Microbiology Clinic, Ankara Numune Training and Research Hospital, Ankara, Turkey and 4King Hussein Medical Center, Princess Eman Research Center for Laboratory Sciences, Amman, Jordan
(Received April 6, 2005. Accepted October 4, 2005)
*Corresponding author: Mailing address: Department of Virology, Gulhane Military Medical Academy, 06018, Etlik, Ankara, Turkey. Tel: +90-312-3043414, Fax: +90-312-3043402, E-mail: akubar@hotmail.com
SUMMARY: In this article, the development of a new TaqMan-based one-step real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay for detection and quantification of Crimean-Congo hemorrhagic fever virus (CCHFV) RNA is described. Selected oligos targeting the highly conserved S region of CCHFV were designed by using our oligo design and analysis software, Oligoware 1.0. None of the primer sequences showed genomic cross-reactivity with other viruses or cells in a BLAST (NCBI) search analysis. The sensitivity and specificity of the primers and the probe were tested using 18 serum samples from patients from East Anatolian who were suspected of having CCHFV, including 2 samples that had already been confirmed to be positive for CCHFV. Among the 16 previously unconfirmed samples, 5 were positive by TaqMan-based one-step real-time RT-PCR and 1 was positive by non-nested RT-PCR, and these results were confirmed with DNA sequencing analysis. The 2 previously confirmed CCHFV RNA samples were also positive by both TaqMan-based one-step real-time RT-PCR and non-nested RT-PCR tests. To ensure the quantitative reproducibility of TaqMan-based one-step real-time RT-PCR, the procedure was repeated several times and the same results were obtained (SD = 0.84 [maximum value]). The developed assay was able to sensitively quantify the concentration of CCHFV RNA, which ranged from 102 to 107 copies/ml per reaction, using plasmid standards generated from the CCHFV RNA (correlation coefficiency = 0.989). The results of the one-step real-time RT-PCR assay were more sensitive than those of the non-nested RT-PCR assay. It can be concluded that our one-step real-time RT-PCR assay is a reliable, reproducible, specific, sensitive and simple tool for the detection and quantification of CCHFV.