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

https://doi.org/10.1016/j.jcv.2012.05.006Get rights and content

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.

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 13N10114Potential 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.

References (26)

  • Qiagen. ESEQuant Tube Scanner. http://www.qiagen.com/Products/ESEQuantTubeScanner.aspx?r=603;...
  • R. Muller et al.

    Characterization of clone 13, a naturally attenuated avirulent isolate of Rift Valley fever virus, which is altered in the small segment

    Am J Trop Med Hyg

    (1995)
  • S.C. Andras et al.

    Strategies for signal amplification in nucleic acid detection

    Mol Biotechnol

    (2001)
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