Recombinase polymerase amplification assay for rapid detection of Rift Valley fever virus
Section snippets
Background
Rift Valley fever (RVFV) is a zoonotic virus causing disease in ruminants and man. It is classed as a transboundary disease in Africa by the FAO and outbreaks of RVFV have been associates with high economic costs.1, 2 Several diagnostic real time PCRs for RVFV detection have been described3, 4, 5 and shown to be of good value in mobile diagnostics.6
Recombinase polymerase amplification (RPA) is an isothermal exponential nucleic acid amplification and detection method. In this reaction the phage
Objective
To develop a highly sensitive and specific fluorescent real time RT-RPA assay for the detection of Rift Valley fever virus using an easily readable colorimetric system.8, 9
Virus culture, RNA preparation
Virus strains are listed in Table 1 and were grown on VeroE6 cells and RNA extracted as described. A S-segment based RNA standard was used as described.10
Real time RT-RPA amplicon design
Primers were designed for the S-segment using sequences with the following accession numbers: RVFV: AF134530-41, AF134543, AF134545-51, NC_002045, Y53771, D0380152-81, EU574057-87. The RPA amplicon for the detection of RVFV was designed for East- and West-African strains as well as the attenuated strains MP12 and clone 13.11, 12 The RPA probe
Sensitivity of RT-RPA
In order to determine if RPA can be used for RNA targets cDNA produced from a RVFV S-segment based RNA standard using the Transcriptor enzyme was subjected to RPA. Using this approach an analytical sensitivity of 10 molecules was achieved (Fig. 1A). Then a one-step RT-RPA was performed by adding Transcriptor to the RPA mix. We tested several additives and found that the addition of DTT improved the RT-RPA leading to the same analytical sensitivity as in two-step-RT-RPA (Fig. 1A). The standard
Discussion
Several isothermal nucleic acid amplification methods have been developed in recent years. Apart from RPA, there are T7 promotor driven amplifications (transcription mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), single primer isothermal amplification (SPIA)), strand displacement methods (strand displacement amplification (SDA), loop-mediated isothermal amplification (LAMP), smart amplification (SmartAmp)), helicase dependent amplification (HDA), and rolling
Funding
This work was supported by Federal Ministry of Education and Research (BMBF) funded project 13N10114 ‘Potential release-oriented biothreat emergency diagnostics (P.R.O.B.E.)’ under the research programme for civil security of the German Federal Government as part of the high-tech strategy for Germany.
Competing interests
Oliver Nentwich and Olaf Piepenburg are employees of TwistDx, the manufacturer and distributor of the RPA technology.
Ethical approval
None required as no clinical material was handeled.
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