A quadruple fluorescence quantitative PCR method for the identification of wild strains of african swine fever and gene-deficient strains.

Abstract

BACKGROUND: Originating in Africa, African swine fever (ASF) was introduced to China in 2018. This acute and highly virulent infectious disease affects domestic pigs. The World Organization for Animal Health has listed it as a statutory reportable disease, and China has listed it as a category A infectious disease. METHODS: Primers and probes were designed for four ASFV genes (B646L, EP402R, MGF505-3R, and A137R). The primers/probes were highly conserved compared with the gene sequences of 21 ASFV strains. RESULTS: After optimization, the calibration curve showed good linearity (R&#x2009;>&#x2009;0.99), the minimum concentration of positive plasmids that could be detected was 50 copies/&#xb5;L, and the minimum viral load detection limit was 10HAD/mL. Furthermore, quadruple quantitative polymerase chain reaction (qPCR) with nucleic acids from three porcine-derived DNA viruses and cDNAs from eight RNA viruses did not show amplification curves, indicating that the method was specific. In addition, 1&#x2009;&#xd7;&#x2009;10, 1&#x2009;&#xd7;&#x2009;10, and 1&#x2009;&#xd7;&#x2009;10copies/&#xb5;L of mixed plasmids were used for the quadruple qPCR; the coefficient of variation for triplicate determination between groups was <&#x2009;2%, indicating the method was reproducible. CONCLUSIONS: The results obtained by testing clinical samples containing detectable EP402R, MGF505-3R, and A137R strains with different combinations of gene deletions were as expected. Therefore, the established quadruple qPCR method was validated for the molecular diagnosis of ASF using gene-deleted ASFV strains.