Citation Euler M, Wang Y, Nentwich O, Piepenburg O, Hufert FT & Weidmann M (2012) Recombinase polymerase amplification assay for rapid detection of Rift Valley fever virus. Journal of Clinical Virology, 54 (4), pp. 308-312. https://doi.org/10.1016/j.jcv.2012.05.006
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.
Keywords RPA; Recombinase polymerase amplification; Rift Valley fever virus
Journal Journal of Clinical Virology: Volume 54, Issue 4