The development of a method for the detection and estimation of CCHF virus RNA in tick species
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Date
2000-05
Authors
Du Preez, Patrick Hendrik
Journal Title
Journal ISSN
Volume Title
Publisher
University of the Free State
Abstract
Crimean Congo haemorrhagic fever (CCHF), caused by a RNA virus, is a tick-borne
viral zoonosis occurring in Europe, Asia and Africa. The fatality rate is ±30%. Rapid
and accurate diagnosis is essential. The aim of this study was to develop a reverse
transcription-polymerase chain reaction (RT-PCR) with internal control for the
detection 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 PCR
product into a pGEMEX-1 vector. Sequencing of the PCR product (381 bp) revealed
two unique restriction sites, BIn I and BstE II which were used to delete a fragment of
59 bp. The shortened PCR-product was re-inserted into E. coli. T3 RNA polymerase
produced plasmid derived RNA (322 bp) was used to spike specimens. Standard
RT-PCR was then performed. The minimum concentration of target RNA the RTPCR
can detect was estimated to be 4 x 10-5 pmol RNA, giving more or less the
same sensitivity as the PCR alone. The size difference of 59 bp is enough to
distinguish between the full-length and the deletion variant inserts when visualised
and therefore provides an internal control. RT-PCR on fifty Hyalomma ticks was negative. The CCHF virus was probably not present or at concentrations below
detection level, as RT-PCR of control CCHF virus RNA confirmed the accuracy of the
method. RT-PCR allows rapid detection of CCHF virus RNA. The constructed
internal control precludes the use of Dugbe virus, an antigenically related nairovirus.
Description
Keywords
Hemorrhagic fever, Virus diseases, Communicable diseases -- South Africa, Dissertation (M.Med.Sc. (Medical Microbiology and Virology))--University of the Free State, 2000