Journal
JOURNAL OF CLINICAL VIROLOGY
Volume 54, Issue 4, Pages 308-312Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jcv.2012.05.006
Keywords
RPA; Recombinase polymerase amplification; Rift Valley fever virus
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Funding
- Federal Ministry of Education and Research (BMBF) [13N10114]
- Potential release-oriented biothreat emergency diagnostics (P.R.O.B.E.)
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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. (c) 2012 Elsevier B.V. All rights reserved.
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