CRISPR-based diagnostic platforms for rapid detection of African swine fever virus
基于CRISPR的诊断平台快速检测非洲猪瘟病毒的研究
摘要 (Abstract)
<jats:p> African swine fever virus (ASFV) is a highly transmissible pathogen affecting swine, causing a devastating disease with high mortality rates in naive populations. Given the likelihood of significant economic impacts associated with an ASF outbreak, considerable resources have been allocated in the United States to safeguard the swine industry against this threat. Ongoing outbreaks of ASF in the Dominican Republic and Haiti further threaten the U.S. swine industry, given their proximity and involvement in movement to and from North America. Although surveillance programs are ongoing, limited point-of-care (POC) tests are available during outbreaks with the sensitivity and specificity standards of laboratory testing (e.g., real-time PCR [rtPCR]). However, the recently developed CRISPR-Cas–based testing systems may offer comparable high-quality results. We sought to develop a low-cost visual detection method for ASFV by employing a recombinase polymerase amplification (RPA)-dependent CRISPR-Cas12a technique that can be utilized in the field as a POC assay. Our CRISPR-Cas12a assay had comparable sensitivity and specificity to rtPCR, both visually and when quantified using a fluorescence reader. In whole blood samples from ASFV-suspect or ASFV-negative cases, our CRISPR assay achieved a sensitivity of 98.3% (10 <jats:sup>2</jats:sup> DNA copies) and a specificity of 100%. Test results of our RPA-CRISPR assay can be visualized in as few as 7 min, with peak fluorescence at 40 min (RPA and CRISPR steps). Our results lay the groundwork for a large-scale POC assay assessment for ASFV detection and offer a robust workflow that works with commonly submitted diagnostic samples. </jats:p>
实验设计与方法 (Experimental Design & Methods)
采用分子生物学、免疫学和临床诊断相结合的方法，系统研究病原体特征和宿主应答反应。
实验结果 (Experimental Results)
开发的诊断方法特异性达95%以上，敏感性高，可实现早期快速诊断，为疫病防控赢得了宝贵时间。
数据汇总 (Data Summary)
开发的诊断方法特异性达95%以上，敏感性高，可实现早期快速诊断，为疫病防控赢得了宝贵时间。
结论 (Conclusions)
新型诊断技术为动物疫病防控提供了有力工具。
实践意义 (Practical Significance)
对保障动物健康和公共卫生安全具有重要意义。