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| Image created by Dr. Michael J. Miller |
African swine fever (ASF), caused by the African swine fever virus (ASFV) represents a major threat to the global pig industry and has since brought major economic loss. ASFVs are divided into 24 genotypes based on their B646L gene, with only genotypes I and II circulating globally. A rapid ASFV genotyping method is not only a powerful tool for ASF diagnosis and epidemiological investigation but also a critical supporting technology for the future application of live attenuated vaccines
To that end, a team of researchers from China developed an isothermal 'one-pot' CRISPR-Cas12i3/Cas13d-based assay, designated OBServe.v2, to detect two amplified targets from multiplex recombinase polymerase amplification (RPA) in a single tube. They reported their results in the Journal of Integrative Agriculture.
“Using a serially diluted synthetic plasmid, OBServe.v2 exhibits sensitivity comparable to that of a commercially available Real-time PCR Kit (Lijian, Qingdao, China), with the capability to detect either genotype I or II ASFVs at concentrations as low as 8 copies μL-1,” shares corresponding author Gaiping Zhang, an academician and professor at Peking University. “For clinical samples, it achieved 100% diagnostic sensitivity and specificity, with no cross-reactivity against other common swine viruses.”
“When compared with real-time PCR, OBServe.v2 successfully identified all ASFV-positive samples with Ct values ranging from 14.6 to 36.57, achieving 100% sensitivity and specificity,” adds co-corresponding author Jianguo Zhao, a professor at Chinese Academy of Sciences.
Additionally, the assay eliminates the high contamination risk associated with conventional two-step CRISPR-Cas based diagnostics and demonstrates complete concordance with the classic PCR-sequencing genotyping method.
“OBServe.v2 provides a visual, accurate and rapid alternative to existing ASFV genotyping methods, with 100% sensitivity and specificity, while requiring minimal equipment,” adds co-corresponding author Yanfang Wang, a professor at Chinese Academy of Agricultural Sciences. “It will be particularly valuable for rapid on-site ASFV surveillance and LAV strains selection.”
Reference:
Zhe Wang, Haili Wang, Xinghui Yan, Jin Li, Yu Wang, Guosong Qin, Wenbo Sun, Yanfang Wang, Gaiping Zhang, Jianguo Zhao, A one-pot Cas12i3/Cas13d-based assay for rapid portable identification of genotypes I and II African swine fever viruses, Journal of Integrative Agriculture, Volume 24, Issue 11, 2025, Pages 4456-4459, ISSN 2095-3119, https://doi.org/10.1016/j.jia.2025.02.013.