The development of a method for the detection and estimation of CCHF virus RNA in tick species

[摘要] Crimean Congo haemorrhagic fever (CCHF), caused by a RNA virus, is a tick-borneviral zoonosis occurring in Europe, Asia and Africa. The fatality rate is ±30%. Rapidand accurate diagnosis is essential. The aim of this study was to develop a reversetranscription-polymerase chain reaction (RT-PCR) with internal control for thedetection of CCHF RNA. Primers were selected for a region in the nucleocapsid-gene of the S segment. The internal control was constructed by ligating this PCRproduct into a pGEMEX-1 vector. Sequencing of the PCR product (381 bp) revealedtwo unique restriction sites, BIn I and BstE II which were used to delete a fragment of59 bp. The shortened PCR-product was re-inserted into E. coli. T3 RNA polymeraseproduced plasmid derived RNA (322 bp) was used to spike specimens. StandardRT-PCR was then performed. The minimum concentration of target RNA the RTPCRcan detect was estimated to be 4 x 10-5 pmol RNA, giving more or less thesame sensitivity as the PCR alone. The size difference of 59 bp is enough todistinguish between the full-length and the deletion variant inserts when visualisedand therefore provides an internal control. RT-PCR on fifty Hyalomma ticks was negative. The CCHF virus was probably not present or at concentrations belowdetection level, as RT-PCR of control CCHF virus RNA confirmed the accuracy of themethod. RT-PCR allows rapid detection of CCHF virus RNA. The constructedinternal control precludes the use of Dugbe virus, an antigenically related nairovirus.

[发布机构] University of the Free State