A Portable Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid Detection of Foot-and-Mouth Disease Virus

Ahmed Abd El Wahed,Ayman El-Deeb,Abeer Ahmed,Bernd Hoffmann,Mohamed El-Tholoth,Sayed Hassan,Bernd Haas,Hanaa Abd El Kader,Manfred Weidmann,Frank T Hufert,Mohamed A Shalaby

doi:10.1371/journal.pone.0071642

Abstract

Foot-and-mouth disease (FMD) is a trans-boundary viral disease of livestock, which causes huge economic losses and constitutes a serious infectious threat for livestock farming worldwide. Early diagnosis of FMD helps to diminish its impact by adequate outbreak management. In this study, we describe the development of a real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of FMD virus (FMDV). The FMDV RT-RPA design targeted the 3D gene of FMDV and a 260 nt molecular RNA standard was used for assay validation. The RT-RPA assay was fast (4–10 minutes) and the analytical sensitivity was determined at 1436 RNA molecules detected by probit regression analysis. The FMDV RT-RPA assay detected RNA prepared from all seven FMDV serotypes but did not detect classical swine fever virus or swine vesicular disease virus. The FMDV RT-RPA assay was used in the field during the recent FMD outbreak in Egypt. In clinical samples, reverse transcription polymerase chain reaction (RT-PCR) and RT-RPA showed a diagnostic sensitivity of 100% and 98%, respectively. In conclusion, FMDV RT-RPA was quicker and much easier to handle in the field than real-time RT-PCR. Thus RT-RPA could be easily implemented to perform diagnostics at quarantine stations or farms for rapid spot-of-infection detection.

Highlights

Foot-and-mouth disease (FMD) is a contagious trans-boundary disease infecting clovenhoofed animals and leads to huge economic losses [1]
FMD virus (FMDV) is a non-enveloped, positive sense single stranded RNA virus belonging to the genus Aphthovirus of the Picornaviridae family [2]
Europe and North America are free of FMDV

Summary

Introduction

FMD is a contagious trans-boundary disease infecting clovenhoofed animals and leads to huge economic losses (death of young ruminants, diminishes milk, and meat production) [1]. FMDV is a non-enveloped, positive sense single stranded RNA virus belonging to the genus Aphthovirus of the Picornaviridae family [2]. It has seven serotypes (A, O, C, SAT 1-3, and Asia1) that have a distinct geographical distribution (A and O are widely distributed across the world, SAT 1-3 mainly in Africa and Asia 1 in Asia) [3]. For example serotype O was endemic in Egypt since 1960 [5], and in 2006, type A was introduced and caused a FMD outbreak [6]. It is assumed that FMDV SAT2 was introduced from sub-Saharan Africa where it is endemic [9]

Methods

Results

Conclusion