Development and evaluation of serotype-specific recombinase polymerase amplification combined with lateral flow dipstick assays for the diagnosis of foot-and-mouth disease virus serotype A, O and Asia1

Abstract

Background: Foot-and-mouth disease (FMD) caused by foot-and-mouth disease virus (FMDV) is one of the most highly infectious diseases in livestock, and leads to huge economic losses. Early diagnosis and rapid differentiation of FMDV serotype is therefore integral to the prevention and control of FMD. In this study, a series of serotype-specific reverse transcription recombinase polymerase amplification assays combined with lateral flow dipstick (RPA-LFD) were establish to differentiate FMDV serotypes A, O or Asia 1, respectively.

Results: The serotype-specific primers and probes of RPA-LFD were designed to target conserved regions of the FMDV VP1 gene sequence, and three primer and probe sets of serotype-specific RPA-LFD were selected for amplification of FMDV serotypes A, O or Asia 1, respectively. Following incubation at 38 °C for 20 min, the RPA amplification products could be visualized by LFD. Analytical sensitivity of the RPA assay was then determined with ten-fold serial dilutions of RNA of VP1 gene and the recombinant vector respectively containing VP1 gene from FMDV serotypes A, O or Asia1, the detection limits of these assays were 3 copies of plasmid DNA or 50 copies of viral RNA per reaction. Moreover, the specificity of the assay was assessed, and there was no cross reactions with other viruses leading to bovine vesicular lesions. Furthermore, 126 clinical samples were respectively detected with RPA-LFD and real-time PCR (rPCR), there was 98.41% concordance between the two assays, and two samples were positive by RPA-LFD but negative in rPCR, these were confirmed as FMDV-positive through viral isolation in BHK-21 cells. It showed that RPA-LFD assay was more sensitive than the rPCR method in this study.

Conclusion: The development of serotype-specific RPA-LFD assay provides a rapid, sensitive, and specific method for differentiation of FMDV serotype A, O or Asia1, respectively. It is possible that the serotype-specific RPA-LFD assay may be used as a integral protocol for field detection of FMDV.

Keywords: FMDV; Lateral flow dipstick; Recombinase polymerase amplification; Serotype-specific.

Fig. 1

Screening of the primers/probes for the FMDV serotype-specific RPA-LFD assay. a Agarose gel electrophoresis and LFD detection of RT-RPA products amplified with different primer and probe sets of FMDV serotype A. Lane M was DNA Marker DL1000. A1 to A9 were different primer and probe sets. A6: the optimal primer and probe set, and the estimated size of the RPA amplified fragment were 346 bp and 334 bp. A10: negative control, (DNase-free water). A11: positive control (supplied by Twist Amp nfo kit). b Agarose gel electrophoresis and LFD detection of RT-RPA products amplified with different primers/probe sets of FMDV serotype Asia 1. As2 to As10 were different primer and probe sets. As9: the optimal primer/probe set, and the estimated size of the RPA amplified fragment were 286 bp and 182 bp. As1: negative control (DNase-free water). As10: positive control (supplied by Twist Amp nfo kit). c Agarose gel electrophoresis and LFD detection of RT-RPA products amplified with different primers/probe sets of FMDV serotype O. O1 to O9 were different primer and probe sets. O6: the optimal primer/probe set, and the estimated size of the RPA amplified fragment were 231 bp and 190 bp. O10: negative control (DNase-free water). O11: positive control (supplied by Twist Amp nfo kit)

Fig. 2

Optimization of reaction temperature and time for FMDV serotype-specific RPA-LFD assays. a The RPA-LFD performs effectively in a wide range of constant reaction temperatures. b The amplified products can be visible on the LFD at 5 min or longer

Fig. 3

The sensitivity of FMDV serotype-specific RPA-LFD assays. a Sensitivity of the standard plasmids. Molecular sensitivity of RPA-LFD was determined using 10-fold serially diluted 3 × 10⁶ to 3 × 10⁰ copies and 10⁰ copy of FMDV DNA standard plasmids per reaction as template. The minimum limits for virus detection of RPA-LFD were 3 × 10⁰ copies. b Sensitivity of the RNA standard. The cDNA of reverse transcription using 10-fold serially diluted 5 × 10⁶ to 5 × 10⁰ RNA molecular was used in molecular sensitivity of RPA-LFD. The minimum limits detection of RPA-LFD were 5 × 10⁰ RNA. A: primers/probe set of FMDV serotype A. AS: primers/probe sets of FMDV serotype Asia 1. O: primers/probe sets of FMDV serotype O. Samples were tested in triplicate with one reaction and independently repeated 3 times

Fig. 4

The specificity of the FMDV RPA-LFD assays. Other bovine viral pathogens with similar clinic and etiologies were used to assess the specificity of the assays. There was no cross-reaction with BVDV, IBRV, BEV, BEFV, BVSV and SVDV. NC: negative control. A: primers/probe set of FMDV serotype A. AS: primers/probe set of FMDV serotype Asia 1. O: primers/probe set of FMDV serotype O. Samples were tested in triplicate with one reaction and three separate assays