Abstract
African swine fever (ASF) is an acute, severe and hemorrhagic infectious disease caused by African swine fever virus (ASFV) infecting domestic pigs and wild boars. Since the outbreak of the disease in China in 2018, it has brought a great impact on China’s pig industry. Classical swine fever (CSF) is an acute contact infectious disease of pigs caused by classical swine fever virus (CSFV) infection. Clinically, acute CSF usually shows persistent high fever, anorexia, extensive congestion and bleeding of the skin and mucosa, which are similar to ASF. It is of great significance to prevent, control and accurately detect ASF and CSF in pig farms. In this study, Recombinase aided amplification (RAA) technology combined with a nucleic acid test strip (RAA-strip) was established for simple and specific detection of ASFV/CSFV. The sensitivity and preliminary clinical application results showed that the RAA test strip established in this study could detect recombinant plasmids containing ASFV/CSFV gene fragments as low as 103 copies/µL. The minimum detection limits of virus DNA/cDNA were 10 and 12 pg respectively, and there was no cross-reaction with other porcine viruses. The specificity of the method was good. We used 37–42 clinical samples to evaluate the performance of our established method, and the positive concordance rates with conventional PCR were 94.1 and 57.1%, respectively. In addition, ASFV and CSFV double RAA agarose gel electrophoresis detection methods were established. The results showed that the method had good specificity. The detection limit of this method is 106 copies for ASFV p72 gene recombinant plasmid and 105 copies for CSFV NS5B Gene recombinant plasmid. The use of this method for clinical material detection was consistent with the PCR method. In summary, the developed method of RAA-strip assay for ASFV and CSFV realized the visual detection of pathogens, and the developed method of dual RAA agarose gel electrophoresis assay for ASFV and CSFV realized the simultaneous detection of two pathogens in one reaction, with good specificity, high sensitivity and rapid reaction rate, which was expected to be clinically feasible for the differential diagnosis of ASF and CSF provided technical support.
Highlights
African swine fever (ASF) is an acute, hemorrhagic, and high contact infectious disease of pigs caused by African swine fever virus (ASFV) (Wang et al, 2021)
The Recombinase aided amplification (RAA) reaction was performed according to method 2.2 to detect primer specificity, agarose gel electrophoresis results showed (Figure 2A), three sets of primers showed specific bands corresponding to expectations at around 288, 416, 282 bp, among which the second pair of primers had the brightest band and no heterozygous bands
The second primer pair was identified as the optimal primer for ASFV RAA analysis, and probes were designed based on the targeted amplified fragment
Summary
African swine fever (ASF) is an acute, hemorrhagic, and high contact infectious disease of pigs caused by African swine fever virus (ASFV) (Wang et al, 2021). Swine usually show clinical symptoms such as high fever, respiratory failure, diarrhea after ASFV infection, and punctate hemorrhages can be observed in the skin and internal organs after necropsy of some pigs, whereas pregnant sows often experience abortion and stillbirth, etc. The ASFV genome contains 150–167 open reading frames (ORFs) and can encode more than 100 protein molecules (Xian and Xiao, 2020). The ASFV p72 protein is encoded by the B646L gene and is located on the surface of the viral capsid. Due to better stability than other structural proteins of ASFV, p72 protein is often used to establish serological and molecular biological assays (Wen et al, 2019)
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