A pentaplex qPCR for the simultaneous detection and differentiation of genotype I, II, I/II recombinant, and fluorescence-tagged African swine fever viruses
- 1State Key Laboratory of Animal Disease Control and Prevention, National High Containment Facilities for Animal Diseases Control and Prevention, Professional Laboratory for African Swine Fever (Harbin), Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China
- 2Veterinary Diagnostic Center, Shanghai Animal Disease Control Center, Shanghai 201103, China
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 polymerase chain reaction (qPCR) assay targeting the B646L, X64R, MGF_360-14L, 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 (TCID50)/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.
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BibTeX
@article{ding2026pentaplex,
title = {{A pentaplex qPCR for the simultaneous detection and differentiation of genotype I, II, I/II recombinant, and fluorescence-tagged African swine fever viruses}},
author = {Leilei Ding and Zhigang Wang and Tianpeng Wang and Dequan Yang and Weldu Tesfagaber and Sihan Zhang and Zhenjiang Zhang and Huimin Liu and Conglu Zhang and Yuanmao Zhu and Renqiang Liu and Fang Li and Zhigao Bu and Encheng Sun and Dongming Zhao},
journal = {Biosafety and health},
year = {2026},
month = jul,
doi = {10.1016/j.bsheal.2026.07.007},
url = {https://europepmc.org/article/PMC/PMC13729067},
note = {CC BY-NC-ND},
}
RIS
TY - JOUR
TI - A pentaplex qPCR for the simultaneous detection and differentiation of genotype I, II, I/II recombinant, and fluorescence-tagged African swine fever viruses
AU - Leilei Ding
AU - Zhigang Wang
AU - Tianpeng Wang
AU - Dequan Yang
AU - Weldu Tesfagaber
AU - Sihan Zhang
AU - Zhenjiang Zhang
AU - Huimin Liu
AU - Conglu Zhang
AU - Yuanmao Zhu
AU - Renqiang Liu
AU - Fang Li
AU - Zhigao Bu
AU - Encheng Sun
AU - Dongming Zhao
PY - 2026
DA - 2026/07/24
JO - Biosafety and health
DO - 10.1016/j.bsheal.2026.07.007
UR - https://europepmc.org/article/PMC/PMC13729067
AB - 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 polymerase chain reaction (qPCR) assay targeting the B646L, X64R, MGF_360-14L, 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 (TCID50)/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.
SN - 2590-0536
SN - 2096-6962
ER -