Development of A Super-Sensitive Diagnostic Method for African Swine Fever Using CRISPR Techniques
Development of A Super-Sensitive Diagnostic Method for African Swine Fever Using CRISPR Techniques
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利用 CRISPR 技术开发非洲猪瘟超灵敏诊断方法
DOI：
10.1007/s12250-020-00323-1
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发表时间：
2021-01-07
影响因子：
5.5
通讯作者：
Zhao, Ling
中科院分区：
文献类型：
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作者：
Ren, Meishen;Mei, Hong;Zhao, Ling
非洲猪瘟(ASF)是由非洲猪瘟病毒(ASFV)引起的一种以高热、出血和高死亡率为临床症状的传染病，对全球养猪业和粮食安全构成威胁。ASFV的检疫和控制是防止养猪业感染ASFV的关键。本研究建立了重组酶聚合酶扩增(RPA)-CRISPR核酸检测方法，用于ASF的诊断。作为一种高灵敏度的检测方法，RPA-CRISPR通过实时定量聚合酶链式反应(QPCR)检测CRISPR-lwCas13a(以前称为C2c2)侧支裂解诱导的荧光信号，可以检测到ASFV的单个拷贝的质粒和基因组DNA，具有与传统的qPCR方法相同甚至更高的灵敏度。建立了一种与RPA-CRISPR联合检测ASFV的侧向流动试纸条，其灵敏度与TaqMan qPCR相同。同样，RPA-CRISPR能够区分ASFV基因组DNA和其他猪病毒的DNA/RNA，而不存在任何交叉反应。该方法也适用于ASFV临床DNA标本的诊断，对ASFV阳性和阴性标本的诊断符合率均为100%。RPA-CRISPR在养猪业和食品安全的ASFV临床检疫中具有很大的潜力。
African swine fever (ASF) is an infectious disease caused by African swine fever virus (ASFV) with clinical symptoms of high fever, hemorrhages and high mortality rate, posing a threat to the global swine industry and food security. Quarantine and control of ASFV is crucial for preventing swine industry from ASFV infection. In this study, a recombinase polymerase amplification (RPA)-CRISPR-based nucleic acid detection method was developed for diagnosing ASF. As a highly sensitive method, RPA-CRISPR can detect even a single copy of ASFV plasmid and genomic DNA by determining fluorescence signal induced by collateral cleavage of CRISPR-lwCas13a (previously known as C2c2) through quantitative real-time PCR (qPCR) and has the same or even higher sensitivity than the traditional qPCR method. A lateral flow strip was developed and used in combination with RPA-CRISPR for ASFV detection with the same level of sensitivity of TaqMan qPCR. Likewise, RPA-CRISPR is capable of distinguishing ASFV genomic DNA from viral DNA/RNA of other porcine viruses without any cross-reactivity. This diagnostic method is also available for diagnosing ASFV clinical DNA samples with coincidence rate of 100% for both ASFV positive and negative samples. RPA-CRISPR has great potential for clinical quarantine of ASFV in swine industry and food security.