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.