title: Development of a Rift Valley fever real-time RT-PCR assay that can detect all three genome segments.
abstract: Outbreaks of Rift Valley fever in Kenya, Madagascar, Mauritania, and South Africa had devastating effects on livestock and human health. In addition, this disease is a food security issue for endemic countries. There is growing concern for the potential introduction of RVF into non-endemic countries. A number of single-gene target amplification assays have been developed for the rapid detection of RVF viral RNA. This paper describes the development of an improved amplification assay that includes two confirmatory target RNA segments (L and M) and a third target gene, NSs, which is deleted in the Clone 13 commercial vaccine and other candidate vaccines. The assay also contains an exogenous RNA control added during the PCR setup for detection of amplification inhibitors. The assay was evaluated initially with samples from experimentally infected animals, after which clinical veterinary and human samples from endemic countries were tested for further evaluation. The assay has a sensitivity range of 66.7-100% and a specificity of 92.0-100% depending on the comparison. The assay has an overall sensitivity of 92.5%, specificity of 95% and a positive predictive value of 98.7%. The single-tube assay provides confirmation of the presence of RVFV RNA for improved confidence in diagnostic results and a "differentiate infected from vaccinated animals" (DIVA)--compatible marker for RVFV NSs--deleted vaccines, which is useful for RVF endemic countries, but especially important in non-endemic countries.
authors.affiliation: Arthropod-Borne Animal Diseases Research Unit, Agricultural Research Service, United States Department of Agriculture, Manhattan, KS 66502, USA. Electronic address: William.wilson@ars.usda.gov.
authors.forename: William C
authors.initials: WC
authors.lastname: Wilson
authors.forename: Marco
authors.initials: M
authors.lastname: Romito
authors.forename: Dane C
authors.initials: DC
authors.lastname: Jasperson
authors.forename: Hana
authors.initials: H
authors.lastname: Weingartl
authors.forename: Yatinder S
authors.initials: YS
authors.lastname: Binepal
authors.forename: Moabi R
authors.initials: MR
authors.lastname: Maluleke
authors.forename: David B
authors.initials: DB
authors.lastname: Wallace
authors.forename: Petrus Jansen
authors.initials: PJ
authors.lastname: van Vuren
authors.forename: Janusz T
authors.initials: JT
authors.lastname: Paweska
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000235:genetics; D016679:Genome, Viral / Q000235:genetics; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012295:Rift Valley Fever / Q000175:diagnosis* / Q000662:veterinary* / Q000821:virology; D012296:Rift Valley fever virus / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers
keywords: Cattle; Real-time RT-PCR; Rift Valley fever virus; Sheep