Abstract
African swine fever (ASF) is a highly contagious and usually deadly porcine infectious disease listed as a notifiable disease by the World Organization for Animal Health (OIE). It has brought huge economic losses worldwide, especially since 2018, the first outbreak in China. As there are still no effective vaccines available to date, diagnosis of ASF is essential for its surveillance and control, especially in areas far from city with limited resources and poor settings. In this study, a sensitive, specific, rapid, and simple molecular point of care testing for African swine fever virus (ASFV) B646L gene in blood samples was established, including treatment of blood samples with simple dilution and boiling for 5 min, isothermal amplification with recombinase-aided amplification (RAA) at 37°C in a water bath for 10 min, and visual readout with lateral flow assay (LFA) at room temperature for 10–15 min. Without the need to extract viral DNA in blood samples, the intact workflow from sampling to final diagnostic decision can be completed with minimal equipment requirement in 30 min. The detection limit of RAA-LFA for synthesized B646L gene-containing plasmid was 10 copies/μl, which was 10-fold more sensitive than OIE-recommended PCR and quantitative PCR. In addition, no positive readout of RAA-LFA was observed in testing classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus, pseudorabies virus and porcine circovirus 2, exhibiting good specificity. Evaluation of clinical blood samples of RAA-LFA showed 100% coincident rate with OIE-recommended PCR, in testing both extracted DNAs and treated bloods. We also found that some components in blood samples greatly inhibited PCR performance, but had little effect on RAA. Inhibitory effect can be eliminated when blood was diluted at least 32–64-fold for direct PCR, while only a 2–4 fold dilution of blood was suitable for direct RAA, indicating RAA is a better choice than PCR when blood is used as detecting sample. Taken together, we established an sensitive, specific, rapid, and simple RAA-LFA for ASFV molecular detection without the need to extract viral DNA, providing a good choice for point of care testing of ASF diagnosis in the future.
Highlights
African swine fever (ASF) is an infectious disease of domestic pigs and wild boars of all breeds and ages, usually showing symptoms like high fever and hemorrhages with a high mortality rate
A total of 37 clinical blood samples were collected from a pig farm that suffered from an ASF epidemic in Henan, China
Other viral cell cultures used in this study, including classical swine fever virus (CSFV) Shimen strain, porcine reproductive and respiratory syndrome virus (PRRSV) HN07-1 strain, porcine epidemic diarrhea virus (PEDV) Hubei2016 strain, pseudorabies virus (PRV) HeNLH/2017 strain, and porcine circovirus 2 (PCV 2) HN-LB-2016 strain, are provided by Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences
Summary
African swine fever (ASF) is an infectious disease of domestic pigs and wild boars of all breeds and ages, usually showing symptoms like high fever and hemorrhages with a high mortality rate. It endangers swine industries and brings huge economic losses each year, and is listed as a reportable disease by the World Organization for Animal Health (OIE) (Penrith and Vosloo, 2009). African swine fever virus (ASFV), the causative agent of ASF, is a large enveloped double-stranded DNA virus. It is the only member of the Asfarviridae family, Asfivirus genus, with a large viral genome around 170–194 kb, encoding at least 125 viral proteins (Dixon et al, 2013). High stability of ASFV makes eradication and prevention of ASF a challenging work during live pig production
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have