Development and clinical application of a novel CRISPR-Cas12a based assay for the detection of African swine fever virus

BMC Microbiology

Guihong Zhang¹  Gang Wang²  Na Zhao²  Chunhe Guo³  Xiaoying Wang³  Sheng He³  Yongchang Cao³  Xiaohong Liu³ 

[1] College of Veterinary Medicine, South China Agricultural University, 510642, Guangzhou, Guangdong, PR China;African Swine Fever Regional Laboratory of China (Guangzhou), 510642, Guangzhou, Guangdong, PR China;Precise Genome Engineering Center, School of Life Sciences, Guangzhou University, 510006, Guangzhou, Guangdong, PR China;State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, North Third Road, Guangzhou Higher Education Mega Center, 510006, Guangzhou, Guangdong, PR China;

关键词: African swine fever;    CRISPR-Cas12a;    nucleic acid detection;    diagnosis;    qPCR;   

DOI  :  10.1186/s12866-020-01966-6

来源: Springer

【 摘 要 】

BackgroundAs no treatment or effective vaccine for African swine fever virus (ASFV) is currently available, a rapid, highly sensitive diagnostic is urgently needed to curb the spread of ASFV.ResultsHere we designed a novel CRISPR-Cas12a based assay for ASFV detection. To detect different ASFV genotypes, 19 crRNAs were designed to target the conserved p72 gene in ASFV, and several crRNAs with high activity were identified that could be used as alternatives in the event of new ASFV variants. The results showed that the sensitivity of the CRISPR-Cas12a based assay is about ten times higher than either the commercial quantitative PCR (qPCR) kit or the OIE-recommended qPCR. CRISPR-Cas12a based assay could also detect ASFV specifically without cross-reactivity with other important viruses in pigs and various virus genotypes. We also found that longer incubation times increased the detection limits, which could be applied to improve assay outcomes in the detection of weakly positive samples and new ASFV variants. In addition, both the CRISPR-Cas12a based assay and commercial qPCR showed very good consistency.ConclusionsIn summary, the CRISPR-Cas12a based assay offers a feasible approach and a new diagnostic technique for the diagnosis of ASFV, particularly in resource-poor settings.

【 授权许可】

CC BY