Recombinase polymerase amplification assay for rapid detection of Rift Valley fever virus

Abstract

Background: Detection of nucleic acids of Rift Valley fever virus (RVFV) has been shown to be useful in field diagnostics. Objectives: To develop an isothermal ‘recombinase polymerase amplification (RPA)' assay on an ESEquant tubescanner device. Study design: RPA was adapted for RNA amplification by first developing a two-step and then a one-step-RT-RPA protocol. Several RT enzymes were tested and the best sensitivity was achieved using Transcriptor (Roche). Finally an RT-RPA pellet containing a recombinant MuLV was tested in RVFV one-step-RT-RPA. Results: The one-step-RT-RPA assay showed a sensitivity of 19 molecules detected as determined by probit analysis of eight runs using a RVFV S-segment based quantitative RNA standard and detected 20 different RVFV strains. The assays showed no cross detection of the human genome and several agents of a typical biothreat panel. It performed almost as good as the assay using glycerol buffer based Transcriptor albeit at a cost of 1-log10 step in sensitivity. The presented combination of one-step-RT-RPA and portable fluorescence reading device could be a useful tool for field or point of care diagnostics.