The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
Genes (Basel). 2022 Apr 22;13(5):734. doi: 10.3390/genes13050734.
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 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 gene reached 5 copies/μL with plasmid as a template, and 10 TCID/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.
牛结节疹病（LSD）是由牛结节疹病毒（LSDV）引起的一种严重且高度传染性的痘病。为了便于早期控制 LSD，本研究旨在开发一种新的基于 基因的重组酶聚合扩增检测方法（RPA）与 CRISPR-Cas12a 荧光检测方法（RPA-Cas12a-fluorescence assay）相结合，用于快速现场 LSDV 检测。结果表明，我们的 RPA-Cas12a-fluorescence assay 检测 LSDV 基因的灵敏度以质粒为模板可达 5 拷贝/μL，以病毒基因组 DNA 为模板可达 10 TCID/mL。与其他常见的牛病毒无交叉反应。进一步对 40 份来自患有或未患有 LSD 的牛的临床样本进行现场 RPA-Cas12a-fluorescence assay 检测，其诊断灵敏度为 96.3%（95% CI：81.0-99.9%），特异性为 92.31%（95% CI：62.1-99.6%），与定量 PCR 检测方法接近。因此，我们的 RPA-Cas12a-fluorescence assay 有望用于现场快速 LSDV 检测。
