A portable reverse transcription recombinase polymerase amplification assay for rapid detection of foot-and-mouth disease virus

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.

Figure 1. FMDV RT-RPA primers and exo-probe sequences aligned with the consensus sequence of 100 FMDV 3D genes downloaded from the Genbank.

(Geneious® 6.1.5, Biomatters Limited, New Zealand). Mismatches are indicated in bold and underlined. The consensus sequence represents nt 7847–7961 of FMDV sequence JF749843. NNN are sites of the quencher and fluropohore in following order (BHQ1-dT) (Tetrahydrofuran) (FAM-dT). Y is C & T; R: A & G.

Figure 2. FMDV RT-RPA.

Fluorescence development over time using a dilution range of 10⁷-10¹ molecules/µl of the FMDV RNA standard (Graph generated by ESEquant tubescanner software). F04+R20+P2 were employed and the analytical sensitivity was 10². 10⁷ represented by black line; 10⁶, gray; 10⁵, red; 10⁴, blue; 10³, green; 10², cyan; 10¹, dark khaki; negative control, orange.

Figure 3. Performance and analytical sensitivity of the FMDV RT-RPA assay.

A: Semi-logarithmic regression of the data collected from eight FMDV RT-RPA test runs on the RNA standard using Prism Software. It yielded results between 4–10 minutes. B: Probit regression analysis using Statistica software on data of the eight runs. The limit of detection at 95% probability (1436 RNA molecules) is depicted by a triangle.

Figure 4. Comparison between real-time RT-RPA and RT-PCR for the detection of FMDV in clinical samples During Egypt 2012 FMD outbreak.

Forty-five RNA extracts of samples collected from suspected cases of FMDV were screened. Linear regression analysis of RT-RPA threshold time (Y axis) and RT-PCR cycle threshold values (X axis) were determined by Prism software. R squared value was 0.26.