title: Development and evaluation of loop-mediated isothermal amplification assay for detection of Crimean Congo hemorrhagic fever virus in Sudan.
abstract: Crimean-Congo hemorrhagic fever (CCHF) virus (CCHFV) activity has been detected in Kordufan region of the Sudan in 2008 with high case-fatality rates in villages and rural hospitals in the region. Therefore, in the present study, a reverse transcription (RT) loop-mediated isothermal amplification (RT-LAMP) assay was developed and compared to nested RT-PCR for rapid detection of CCHFV targeting the small (S) RNA segment. A set of RT-LAMP primers, designed from a highly conserved region of the S segment of the viral genome, was employed to identify all the Sudanese CCHFV strains. The sensitivity studies indicated that the RT-LAMP detected 10fg of CCHFV RNA as determined by naked eye turbidity read out, which is more likely the way it would be read in a resource-poor setting. This level of sensitivity is good enough to detect most acute cases. Using agarose gel electrophoresis, the RT-LAMP assay detected as little as 0.1fg of viral RNA (equivalent to 50 viral particle). There was 100% agreement between results of the RT-LAMP and the nested PCR when testing 10-fold serial dilution of CCHFV RNA. The specificity studies indicated that there was no cross-reactivity with other related hemorrhagic fever viruses circulating in Sudan including, Rift Valley fever virus (RVFV), Dengue fever virus, and yellow fever virus. The RT-LAMP was performed under isothermal conditions at 63°C and no special apparatus was needed, which rendered the assay more economical and practical than real-time PCR in such developing countries, like Sudan. In addition, the RT-LAMP provides a valuable tool for rapid detection and differentiation of CCHFV during an outbreak of the disease in remote areas and in rural hospitals with resource-poor settings.
authors.affiliation: Molecular Biology Laboratory-MBL, Department of Clinical Medicine, Faculty of Veterinary Medicine, University of Khartoum, P.O. Box 32, Khartoum North, Sudan.
authors.forename: Hana A M
authors.initials: HA
authors.lastname: Osman
authors.forename: Kamal H
authors.initials: KH
authors.lastname: Eltom
authors.forename: Nasreen O
authors.initials: NO
authors.lastname: Musa
authors.forename: Nasreldin M
authors.initials: NM
authors.lastname: Bilal
authors.forename: Mustafa I
authors.initials: MI
authors.lastname: Elbashir
authors.forename: Imadeldin E
authors.initials: IE
authors.lastname: Aradaib
mesh_terms: D017931:DNA Primers / Q000235:genetics; D003225:Hemorrhagic Fever Virus, Crimean-Congo / Q000235:genetics / Q000302:isolation & purification*; D006479:Hemorrhagic Fever, Crimean / Q000175:diagnosis*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D048348:Reverse Transcription; D012680:Sensitivity and Specificity; D013397:Sudan; D013696:Temperature; D014773:Virology / Q000379:methods*
chemical_list: D017931:DNA Primers; D012367:RNA, Viral