Visual and equipment-free reverse transcription recombinase polymerase amplification method for rapid detection of foot-and-mouth disease virus

Libing Liu,Mi Lin,Ruoxi Zhang,Jianchang Wang,Qingan Han,Jinfeng Wang,Ruihan Shi,Wanzhe Yuan

doi:10.1186/s12917-018-1594-x

Abstract

BackgroundFoot-and-mouth disease (FMD), which is caused by foot-and-mouth disease virus (FMDV), is a highly contagious tansboundary disease of cloven-hoofed animals and causes devastating economic damages. Accurate, rapid and simple detection of FMDV is critical to containing an FMD outbreak. Recombinase polymerase amplification (RPA) has been explored for detection of diverse pathogens because of its accuracy, rapidness and simplicity. A visible and equipment-free reverse-transcription recombinase polymerase amplification assay combined with lateral flow strip (LFS RT-RPA) was developed to detect the FMDV using primers and LF probe specific for the 3D gene.ResultsThe FMDV LFS RT-RPA assay was performed successfully in a closed fist using body heat for 15 min, and the products were visible on the LFS inspected by the naked eyes within 2 min. The assay could detect FMDV serotypes O, A and Asia1, and there were no cross-reactions with vesicular stomatitis virus (VSV), encephalomyocarditis virus (EMCV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus 2 (PCV2) and pseudorabies virus (PRV). The analytical sensitivity was 1.0 × 102 copies in vitro transcribed FMDV RNA per reaction, which was the same as a real-time RT-PCR. For the 55 samples, FMDV RNA positive rate was 45.5% (25/55) by LFS RT-RPA and 52.7% (29/55) by real-time RT-PCR. For the LFS RT-RPA assay, the positive and negative predicative values were 100% and 80%, respectively.ConclusionsThe performance of the LFS RT-RPA assay was comparable to real-time RT-PCR, while the LFS RT-RPA assay was much faster and easier to be performed. The developed FMDV LFS RT-RPA assay provides an attractive and promising tool for rapid and reliable detection of FMDV in under-equipped laboratory and at point-of-need facility, which is of great significance in FMD control in low resource settings.

Highlights

Foot-and-mouth disease (FMD), which is caused by foot-and-mouth disease virus (FMDV), is a highly contagious tansboundary disease of cloven-hoofed animals and causes devastating economic damages
Clinical and spiked samples Twelve RNA extracted from the vesicular fluid and epithelium tissue collected from pigs experimentally infected with FMDV serotype O were provided by the State Key Laboratory of Veterinary Etiological Biology (Animal Ethics Committee of the Lanzhou Veterinary Research Institute, approval number: LVRIAEC 2012– 018) and used for the clinical validation of the FMDV lateral flow strip (LFS) RT-Recombinase polymerase amplification (RPA) assay
Twenty serum samples were collected from clinically healthy pigs and twenty serum samples were collected from clinically healthy cattle, which were tested to be FMDV RNA negative by a real-time reverse transcription polymerase chain reaction (RT-PCR) [7]

Summary

Introduction

Foot-and-mouth disease (FMD), which is caused by foot-and-mouth disease virus (FMDV), is a highly contagious tansboundary disease of cloven-hoofed animals and causes devastating economic damages. A visible and equipment-free reverse-transcription recombinase polymerase amplification assay combined with lateral flow strip (LFS RT-RPA) was developed to detect the FMDV using primers and LF probe specific for the 3D gene. The RT-PCR assays are designed for use in well-equipped laboratories with reliable electrical supply and highly trained technicians, and unsuitable for being used in underequipped laboratories and in field. Several real-time RT-PCR assays have been transferred onto a portable platform and trialled successfully in field settings [7, 10, 11], expensive high precision instrumentation and consistent electrical power are still needed. When compared to current RT-PCR assays, the use of isothermal technologies reduces the need for high precision instrumentation, consistent electrical power and complex sample preparation [12]. A simple, rapid, and sensitive method is still needed for the point-of-need (PON) detection of FMDV

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Conclusion