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

doi:10.3390/genes13050734

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

Genes (Basel). 2022 Apr 22;13(5):734. doi: 10.3390/genes13050734.

Authors

Chuanwen Jiang^{1

2}, Dagang Tao², Yuanchen Geng^{1

2}, Hao Yang^{1

2}, Bingrong Xu³, Yingyu Chen^{1

2

4

5}, Changmin Hu^{2

4

5}, Huanchun Chen^{1

2

4

5}, Shengsong Xie^{2

3

4}, Aizhen Guo^{1

2

4

5

6}

Affiliations

¹ The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
² College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
³ Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education & Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
⁴ Hubei Hongshan Laboratory, Wuhan 430070, China.
⁵ Hubei International Scientific and Technological Cooperation Base of Veterinary Epidemiology, International Research Center for Animal Disease, Ministry of Science and Technology of the People's Republic of China, Wuhan 430070, China.
⁶ Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of China Agriculture and Rural Affairs, Wuhan 430070, China.

Abstract

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.

Keywords: CRISPR-Cas12a; lumpy skin disease virus; nucleic acid detection; recombinase polymerase amplification.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
CRISPR-Cas Systems
Cattle
DNA, Viral / genetics
Lumpy skin disease virus* / genetics
Nucleotidyltransferases / genetics
Recombinases / genetics
Recombinases / metabolism
Sensitivity and Specificity

Substances

DNA, Viral
Nucleotidyltransferases
Recombinases