Abstract
African swine fever (ASF), caused by African swine fever virus (ASFV), is a devastating infectious disease of domestic pigs and wild boars, and has tremendous negative socioeconomic impact on the swine industry and food security worldwide. It is characterized as a notifiable disease by World Organisation for Animal Health (OIE). No effective vaccine or treatment against ASF has so far been available. Early detection and rapid diagnosis are of potential significance to control the spread of ASF. Recombinase-based isothermal amplification assay, recombinase polymerase amplification (RPA) developed by TwistDx (Cambridge, United Kingdom) or recombinase-aided amplification (RAA) by Qitian (Wuxi, China), is becoming a molecular tool for the rapid, specific, and cost-effective identification of multiple pathogens. In this study, we aim to investigate if RPA/RAA can be a potential candidate for on-site, rapid and primary detection of ASFV. A panel of 152 clinical samples previously well-characterized by OIE-recommended qPCR was enrolled in this study, including 20 weak positive (Ct value ≥ 30) samples. This panel was consisted of different types, such as EDTA-blood, spleen, lung, lymph node, kidney, tonsil, liver, brain. We evaluated two recombinase-based isothermal amplification assays, RPA or RAA, by targeting the ASFV B646L gene (p72), and validated the clinical performance in comparison with OIE real-time PCR. Our result showed that the analytical sensitivity of RPA and RAA was as 93.4 and 53.6 copies per reaction, respectively at 95% probability in 16 min, at 39°C. They were universally specific for all 24 genotypes of ASFV and no cross reaction to other pathogens including Classical swine fever virus (CSV), Foot-and-mouth disease virus (FMDV), Pseudorabies virus, Porcine circovirus 2 (PCV2), Porcine Reproductive and respiratory syndrome virus (PPRSV). The results on detection of various kinds of clinical samples indicated an excellent diagnostic agreement between RPA, RAA and OIE real-time PCR method, with the kappa value of 0.960 and 0.973, respectively. Compared to real-time PCR, the specificity of both RPA and RAA was 100% (94.40% ∼ 100%, 95% CI), while the sensitivity was 96.59% (90.36% ∼ 99.29%, 95% CI) and 97.73% (92.03% ∼ 99.72%, 95% CI), respectively. Our data demonstrate that the developed recombinase-based amplification assay (RPA/RAA), promisingly equipped with field-deployable instruments, offers a sensitive and specific platform for the rapid and reliable detection of ASFV, especially in the resource-limited settings for the purpose of screening and surveillance of ASF.
Highlights
African swine fever (ASF), as a notifiable disease to the World Organization for Animal Health (OIE), is a highly contagious, viral pig disease caused by African swine fever virus (ASFV)
We analyzed the sensitivity of ASFV recombinase polymerase amplification (RPA), recombinase-aided amplification (RAA) by detecting ASFV p72 recombinant plasmids at concentrations of 8.3, 41.5, 83, 415, 830, 4150, 8300 copies/μL in eight replicates (Figures 2A,B)
Our result regarding probit regression analysis showed that the detection limits of RPA and RAA at 95% probability were 93.4 copies per reaction (56.9 ∼ 619.8 copies per reaction, 95% CI) and 53.6 copies per reaction (30.7 ∼ 241.0 copies per reaction, 95% CI), respectively (Figures 2C,D)
Summary
African swine fever (ASF), as a notifiable disease to the World Organization for Animal Health (OIE), is a highly contagious, viral pig disease caused by African swine fever virus (ASFV). ASFV has been currently classified to the genus Asfivirus of the family Asfarviridae. The genome of this large, complex double-stranded DNA virus (170–193 kbp) consists of 151– 167 genes (Galindo and Alonso, 2017). ASF infection of domestic pigs and wild boars causes high fever, lethargy, digestive dysfunction, respiratory discharges, nasal discharges and abortion, with the mortality close to 100%. The diverse transmission modes include a sylvatic cycle between swine and arthropod vector (Ornithodoros moubata ticks), direct or indirect contact between susceptible animal and infected pigs, contaminated secretions (blood, feces, urine, mucus) or fomites (vehicles, equipment) (Dixon et al, 2019). No effective vaccine or antiviral treatment has been developed, while the quarantine, depopulation and sanitation strategies remain the routine ways to control the spread of ASF
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