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

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-log(10) 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.