Rapid detection of infectious bovine Rhinotracheitis virus using recombinase polymerase amplification assays

Abstract

Background: Infectious bovine rhinotracheitis virus (IBRV) is a major pathogen in cattle and has led to significant economic losses to the dairy industry worldwide, and therefore a more optimal method for the rapid diagnosis of IBRV infection is highly needed. In this study, we described the development of a lateral flow dipstrip (LFD) of isothermal recombinase polymerase amplification (RPA) method for rapid detection of IBRV.

Methods: Distinct regions were selected as a candidate target for designing the LFD-RPA primers and probes. The analytical sensitivity of the RPA assay was determined using ten-fold serially diluted IBRV DNA. The specificity of the assay was assessed with other viral pathogens of cattle with similar clinic and other herpesviruses. The clinical performance was evaluated by testing 106 acute-phase high fever clinical specimens.

Results: RPA primers and probe were designed to target the specific conserved UL52 region fragment of IBRV. The detection could be completed at a constant temperature of 38 °C for 25 min, and the amplification products were easily visualized on a simple LFD. The detection limit of this assay was 5 copies per reaction of IBRV DNA and there was no cross-reactivity with other viruses causing bovine gastrointestinal and respiratory infections or other herpesviruses. The assay performance on acute-phase high fever clinical samples collected from cattle with no vaccine against IBRV, which were suspected to be infected with IBRV, was validated by detecting 24 fecal, 36 blood, 38 nasal swab and 8 tissue specimens, and compared with SYBR Green I based real-time PCR. The coincidence between IBRV LFD-RPA and real-time PCR was 100%.

Conclusion: IBRV LFD-RPA was fast and much easier to serve as an alternative to the common measures used for IBRV diagnosis, as there is reduction in the use of instruments for identification of the infected animals. In addition, this assay may be the potential candidate to be used as point-of-care diagnostics in the field.

Keywords: Infectious bovine rhinotracheitis virus (IBRV); Lateral-flow dipstrip (LFD); Rapid detection; Recombinase polymerase amplification (RPA).

Fig. 1

Validating the designed primers/probe of IBRV LFD-RPA assay. a Agarose gel electrophoresis of RPA products generated using designed primers/probes. Lane M: molecular weight standard (DNA Marker1000). Lane 1 to 9: designed primer and probe sets. Especially, Lane 5 was primers/probe set 4-2F/4-2R/4-2LF, and the expected size of the product was 247 bp. b Lateral-flow strip end-point analysis of RPA products generated using optimal designed primers/probes set 4F/4R/4LF. Lane 1: positive control (supplied by Twist Ampnfo kit); Lane 2: negative control (DNase-free water); Lane 3: IBRV amplicons performed with RPA primer pair 4F/4R/4LF. Samples were tested in triplicate with one reaction displayed in figure for each triplicate

Fig. 2

Sensitivity of the LFD-RPA assay. a Molecular sensitivity of RPA using total DNA extracted from 10-fold serially diluted IBRV-infected cell as template. IBRV templates of lane 1 to 10 in these reactions extracted from ten-fold serially diluted virus range from 5 × 10⁸ to 5 × 10⁻¹ DNA copies per reaction. Samples were tested in triplicate with one reaction displayed in figure for each triplicate. b IBRV RPA reaction products (247 bp) could be detected on a 2% agarose gel. Lane 1 to 9 were represented IBRV RPA reactions extracted from ten-fold serially diluted IBRV templates range from 5 × 10⁶ to 5 × 10⁻¹ DNA copies per reaction. c Repeatability of limits detection. The sensitivity of the assay using IBRV DNA extracted from 5 copies per reaction of IBRV DNA and negative control (DNase-free water) were also evaluated in 3 replicates, respectively. d Sensitivity of conventional PCR. Molecular sensitivity of conventional PCR uses the same template amount as the IBRV LFD-RPA assay. Templates of lane 1 to 10 in these reactions extracted from ten-fold serially diluted virus genome DNA range from 5 × 10⁸ to 5 × 10⁻¹copies per reaction. Samples were tested in triplicate with one reaction displayed in figure for each triplicate

Fig. 3

Specificity of the LFD-RPA assay. a The specificity of the IBRV RPA-LFD assay was assessed for other viral pathogens genome cDNA of cattle that present similarly in the clinic other herpesviruses such as bovine herpesvirus5 (BoHV-5). Lanes 1: positive control. Lanes 2 to 8: BVDV, BPIV-3, BRSV, BEFV, BEV, BcoV and BoHV-5, respectively. Samples were tested in triplicate with one reaction displayed in figure for each triplicate. b The specificity of the IBRV RPA-LFD products could be displayed on a 2% agarose gel. Lanes 1 to 7: BVDV, BPIV-3, BRSV, BEFV, BEV, BcoV and BoHV-5, respectively, Lane 8: positive control. Lane M: molecular weight standard (DNA Marker 2000)