Development and clinical application of a novel CRISPR-Cas12a based assay for the detection of African swine fever virus
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METHODOLOGY ARTICLE
Open Access
Development and clinical application of a novel CRISPR-Cas12a based assay for the detection of African swine fever virus Xiaoying Wang1†, Sheng He1†, Na Zhao2, Xiaohong Liu1, Yongchang Cao1, Guihong Zhang3,4, Gang Wang2* and Chunhe Guo1*
Abstract Background: As 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. Results: Here 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. Conclusions: In 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. Keywords: African swine fever; CRISPR-Cas12a; nucleic acid detection; diagnosis; qPCR
Background African swine fever (ASF) is one of the most important zoonotic diseases that can lead to high mortality in domestic pigs, leading to huge economic losses [1, 2]. ASF is caused by African swine fever virus (ASFV), a large, enveloped, double-stranded DNA virus that is the sole member of the Asfarviridae family [3]. The viral genome * Correspondence: [email protected]; [email protected] † Xiaoying Wang and Sheng He contributed equally to this work. 2 Precise Genome Engineering Center, School of Life Sciences, Guangzhou University, Guangzhou, Guangdong 510006, PR China 1 State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, North Third Road, Guangzhou Higher Education Mega Center, Guangzhou, Guangdong 510006, PR China Full list of author information is available at the end of the article
comprises between 170 and 190 kilobases that encode more than 150 ORFs and approximately 165 viral proteins [4]. The viral capsid protein p72, encoded by the B646L gene, is highly conserved and well-characterized [5, 6], making it a widely used target for both nucleic acid detection and phylogenetic analysis [7, 8]. African swine fever was first documented in Kenya in 1921 and later discovered in most countries in Africa, and it was introduced into Europe in the middle of the last century [1, 9]. In 2007, an outbreak of this disease was r
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