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.
Contamination risk
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.”
No comments yet