Sensitive and Specific Detection of Lumpy Skin Disease Virus in Cattle by CRISPR-Cas12a Fluorescent Assay Coupled with Recombinase Polymerase Amplification.

Chuanwen Jiang, Dagang Tao, Yuanchen Geng, Hao Yang, Bingrong Xu, Yingyu Chen, Changmin Hu, Huanchun Chen, Shengsong Xie, Aizhen Guo

Genes 2022 Apr 22

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Lumpy skin disease (LSD) is a severe and highly infectious pox disease of cattle caused by the lumpy skin disease virus (LSDV). To facilitate early control of LSD, this study aimed to develop a new rapid on-site LSDV detection method using an orf068 gene-based recombinase polymerase amplification assay (RPA) coupled with a CRISPR-Cas12a-based fluorescence assay (RPA-Cas12a-fluorescence assay). The results showed that the sensitivity of our RPA-Cas12a-fluorescence assay for detecting LSDV orf068 gene reached 5 copies/μL with plasmid as a template, and 102 TCID50/mL with viral genomic DNA as a template. No cross-reaction with other common bovine viruses was observed. Further, an on-site RPA-Cas12a-fluorescence assay of 40 clinical samples from cattle with or without LSD showed a diagnostic sensitivity of 96.3% (95% CI: 81.0-99.9%) and specificity of 92.31% (95% CI: 62.1-99.6%), which was close to those of the quantitative PCR assay. Therefore, our RPA-Cas12a-fluorescence assay has promising prospects in on-site rapid LSDV detection.

Citation

Chuanwen Jiang, Dagang Tao, Yuanchen Geng, Hao Yang, Bingrong Xu, Yingyu Chen, Changmin Hu, Huanchun Chen, Shengsong Xie, Aizhen Guo. Sensitive and Specific Detection of Lumpy Skin Disease Virus in Cattle by CRISPR-Cas12a Fluorescent Assay Coupled with Recombinase Polymerase Amplification. Genes. 2022 Apr 22;13(5)