A pentaplex qPCR for the simultaneous detection and differentiation of genotype I, II, I/II recombinant, and fluorescence-tagged African swine fever viruses
Year: 2026
Type: article
Abstract: African swine fever (ASF) continues to inflict severe and persistent damage to the global swine industry and food security. Since 2018, ASF viruses (ASFVs) of genotype II, genotype I, and genotype I/II recombinant with varied virulence have been reported in China. Although several ASF vaccines featuring fluorescent markers have been licensed in neighboring countries, an approved vaccine remains unavailable in China. Therefore, there is an urgent need to establish a rapid detection method to simultaneously monitor the prevalence and fluorescence-tagged ASFVs. Here, we developed a pentaplex quantitative real-time PCR (qPCR) assay targeting the B646L , X64R , MGF_360-14 L , eGFP , and mCherry genes for the simultaneous differential detection of genotype I, II, and I/II recombinant, as well as eGFP - and mCherry - tagged ASFVs. This assay demonstrated no cross-reactivity against seven non-target porcine pathogens. It achieved a limit of detection (LOD) of 50 copies/reaction for each of five plasmid targets and LODs of 0.1–1.0 50% tissue culture infective dose (TCID 50 )/reaction for genotype I, II, and I/II recombinant ASFVs. In addition, this assay demonstrated an overall concordance of 78% (39/50) with previous triplex qPCR across all 50 clinical samples. Notably, for the subset of samples with B646L cycle threshold (Ct) values <35, the concordance reached 100% (21/21), covering genotype I, II, and I/II recombinant ASFVs. This indicates that the pentaplex qPCR could effectively differentiate clinical samples with Ct values below 35. In conclusion, the pentaplex qPCR assay provides a reliable tool for the simultaneous detection and differentiation of diverse ASFVs (genotype I, II, and I/II recombinant, eGFP - and mCherry - tagged), providing supports for ASF surveillance in China.
Source: Biosafety and Health
Language: English
Subfield: Animal Science and Zoology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,500
Funders: Heilongjiang Provincial Finance Department, National Natural Science Foundation of China, Ministry of Science and Technology of the People's Republic of China, China Postdoctoral Science Foundation, Chinese Academy of Agricultural Sciences, Shanghai Municipal Commission of Agriculture and Rural Affairs, Ministry of Agriculture and Rural Affairs of the People's Republic of China
OpenAlex ID: W7170586312