Development of a recombinase polymerase amplification combined with lateral-flow dipstick assay for detection of bovine ephemeral fever virus

Abstract

Bovine ephemeral fever virus (BEFV), identified as the causative pathogen of bovine ephemeral fever (BEF), is responsible for increasing numbers of epidemics/outbreaks and has a significant harmful effect on the livestock industry. Therefore, a rapid detection assay is imperative for BEFV diagnosis. In this study, we described the development of lateral-flow dipstick isothermal recombinase polymerase amplification (LFD-RPA) assays for detection of BEFV. RPA primers and LF probes were designed by targeting the specific G gene, and the amplification product can be visualized on a simple lateral flow dipstick with the naked eyes. The amplification reaction was performed at 38 °C for 20 min and LFD incubation time within 5 min. The detection limit of this assay was 8 copies per reaction, and there was no cross-reactivity with other bovine infectious viruses such as bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, bovine coronavirus, bovine parainfluenza virus type 3, bovine vesicular stomatitis virus. In addition, the assay was performed with total 128 clinical specimens and the diagnostic results were compared with conventional RT-PCR, real-time quantative(q) PCR. The result showed that the coincidence rate of BEFV LFD-RPA and real-time qPCR was 96.09% (123/128), which was higher than conventional RT-PCR. The RPA combined with LFD assay probably provides a rapid and sensitive alternative for diagnosis of BEFV infections outbreak.

Keywords: Bovine ephemeral fever virus (BEFV); Lateral flow dipstick; Recombinase polymerase amplification (RPA).

Fig. 1

Screening of the designed primers/probe of BEFV LFD-RPA assay. (A) Agarose gel electrophoresis of RPA products generated using designed primers/probes. Lane M: molecular weight standard (DNA Marker 2000). Lane1 to 8 was designed primer and probe sets: F1-1/R1-1/1LF, F1-2/R1-2/1LF, F1-3/R1-3/1LF, F1-4/R1-4/1LF, 2F-1/2R-1/2LF, 2F-2/2R-2/2LF, 3F/3R/3LF and 4F/4R/4LF, respectively. Lane 9: positive control (supplied by Twist Amp nfo kit); Lane 10: negative control (DNase-free water); Specifically, Lane 7 was primers/probe set 3F/3R/3LF, and the expected size of the product was 235bp. (B) Lateral-flow strip end-point analysis of RPA products generated by using designed primers/probe set 3F/3R/3LF. Lane 1 to 8: BEFV amplicons performed with RPA primer pair F1-1/R1-1/1LF, F1-2/R1-2/1LF, F1-3/R1-3/1LF, F1-4/R1-4/1LF, 2F-1/2R-1/2LF, 2F-2/2R-2/2LF, 3F/3R/3LF and 4F/4R/4LF, respectively. Lane 9: positive control (supplied by Twist Amp nfo kit); Lane 10: negative control (DNase-free water).

Fig. 2

Determination of reaction temperature and time. (A) The LFD-RPA works effectively in a broad range of constant reaction temperatures. (B) The LFD-RPA amplification can be visible on the LFD at 15min or longer.

Fig. 3

Analytical sensitivity of the BEFV LFD-RPA assay. (A) Sensitivity of the LFD-RPA assay. Molecular sensitivity test results of RPA using 10-fold serially diluted template of standard plasmid. Lane 1: positive control (supplied by Twist Amp nfo kit); Lane 2:negative control (DNase-free water); BEFV templates of lane 3 to 11 in these reactions ranged from 8 × 10⁷ to 8 × 10⁻¹ copies per reaction, respectively. Samples were tested in triplicate with one reaction displayed in figure for each triplicate. (B) BEFV RPA reaction products were detected on a 2% agarose gel. BEFV templates of Lane 1 to Lane 9 in these reactions ranged from 8 × 10⁷ to 8 × 10⁻¹ copies per reaction, respectively.

Fig. 4

Analytical specificity of the BEFV LFD-RPA assay. (A) Specificity of the LFD-RPA assay. The specificity of the assay was assessed for other bovine viral pathogens with similar clinic and etiologies. Lane 1: positive control of BEFV; Lanes 2 to 7: BVDV, IBRV, BPIV-3, BRSV, BcoV and VSV, respectively. Samples were tested in triplicate with one reaction displayed in figure for each triplicate. (B) The results of amplification products of the LFD-RPA on 2% agarose gel. Lane 1: positive control of BEFV; Lanes 2 to 7: BVDV, IBRV, BPIV-3, BRSV, BcoV and VSV, respectively. (C) The quality detection of RNA/DNA of BVDV, IBRV, BPIV-3, BRSV, BcoV and VSV. The RNA/DNA of BEFV, BVDV, IBRV, BPIV-3, BRSV, BcoV and VSV prepared for specificity detection were undertook PCR reaction with viral specific primers (Supplementary Table 1). The positive amplification results were shown in Lane 1, Lane 3, Lane 5, Lane 7, Lane 9, Lane 11, Lane 13, respectively. Lane 2, Lane 4, Lane 6, Lane 8, Lane 10, Lane 12, Lane 14 were negative controls with DNase-free water as template.

S.Fig. 1

Schematic drawing of the nucleotide size and the amplified gene region of 3F/3R/3LF sets. The points linked to gene sequence amplification locus and positions of the forward (3F) and reversed (3R) primers and LF probe (3LF) used in this study.