Abstract
African swine fever (ASF) has caused huge economic losses to the swine industry worldwide. Since there is no commercial ASF vaccine available, an early diagnosis is extremely important to prevent and control the disease. In this study, ASF virus (ASFV) capsid protein-encoding gene (p72) was selected and used to design primers for establishing a one-step visual loop-mediated isothermal amplification (LAMP) assay with neutral red, a pH-sensitive dye, as the color shift indicator. Neutral red exhibited a sharp contrast of color change from faint orange (negative) to pink (positive) during LAMP for detection of ASFV. The designed primer set targeting highly conserved region of the p72 gene was highly specific to ASFV and showed no cross-reactivity with other swine viruses. The detection limit for the one-step visual LAMP developed was 10 copies/reaction based on the recombinant plasmid containing the p72 gene of ASFV. More importantly, the developed one-step visual LAMP showed high consistency with the results of the real-time polymerase chain reaction (qPCR) method recommended by World Organization for Animal Health (OIE). Furthermore, the results demonstrate that the colorimetric detection with this LAMP assay could be directly applied for the whole blood and serum samples without requiring genome extraction. Based on our results, the developed one-step visual LAMP assay is a promising penside diagnostic tool for development of early and cost-effective ASF monitoring program that would greatly contribute to the prevention and control of ASF.
Highlights
African swine fever (ASF) is an acute and highly fatal infectious disease caused by ASF virus (ASFV)
Complimentary DNA of classical swine fever virus (CSFV), HP-PRRSV, JEV and PEDV were synthesized by reverse transcription kit (Promega, Madison, Wisconsin, United States) from total RNA extracted from infected cell culture supernatant
The results showed that the visual loop-mediated isothermal amplification (LAMP) assay gave clear positive signal at 10 copies/reaction by either to positive (Tp) analysis, direct naked-eye observation, or the agar gelbased electrophoresis
Summary
African swine fever (ASF) is an acute and highly fatal infectious disease caused by ASF virus (ASFV). ASFV is an enveloped virus with a double-stranded linear DNA. It is the only member of the Asfivirus genus, belonging to the Asfarviridae family (Alonso et al, 2018). Its genome length is between 170 and 190 kb consisting of 151 open reading frames (ORF) (Chapman et al, 2008; Villiers et al, 2010) It can encode 151 to 200 proteins among which p72 is one of the main structural proteins (García-Escudero et al, 1998). Variability of the nucleic acid sequence within the 478bp region at the C-terminal of p72 gene allows the distinction of ASFV into 24 genotypes (Galindo and Alonso, 2017)
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