African swine fever (ASF) is one of the most severe diseases of pigs. In this study, a CRISPR-Cas12a (also known as Cpf1) system coupled with nucleic acid amplification was optimized for the detection of ASF virus (ASFV). Two novel single-stranded DNA-fluorophore-quencher (ssDNA-FQ) reporters were developed to increase the brightness of the fluorescent signal for the visualization of nucleic acid detection. The CRISPR-Cas12a system was used to simultaneously cleave the polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP) amplicons and the newly developed ssDNA-FQ reporter, resulting in fluorescence that could be easily detected in multiple platforms, especially on cheap and portable blue or UV light transilluminators. This specific cleavage with fluorescence reveals the presence of the amplicon and confirms its identity, thereby preventing false-positive test results from nonspecific amplicons. This method is also uninterfered by the presence of large amounts of irrelevant background DNA and displays no cross-reactivity with other porcine DNA or RNA viruses. When coupled with LAMP, the Cas12a platform can detect a plasmid containing p72 with as few as 2 copies/μL reaction. Our results indicate that the CRISPR-Cas12a enhanced fluorescence assay coupled with nucleic acid amplification is robust, convenient, specific, confirmatory, affordable, and potentially adaptable for ASF diagnosis.

中文翻译：

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应用 CRISPR-Cas12a 增强荧光检测结合核酸扩增灵敏检测非洲猪瘟病毒。

非洲猪瘟（ASF）是猪最严重的疾病之一。在本研究中，针对 ASF 病毒 (ASFV) 的检测，优化了结合核酸扩增的 CRISPR-Cas12a（也称为 Cpf1）系统。开发了两种新型单链 DNA 荧光团淬灭剂 (ssDNA-FQ) 报告基因，以增加荧光信号的亮度，用于核酸检测的可视化。CRISPR-Cas12a 系统用于同时切割聚合酶链反应 (PCR) 或环介导等温扩增 (LAMP) 扩增子和新开发的 ssDNA-FQ 报告基因，从而产生可在多个平台上轻松检测到的荧光，尤其是在便宜且便携的蓝光或紫外光透照器。这种特殊的荧光切割揭示了扩增子的存在并确认了其身份，从而防止来自非特异性扩增子的假阳性测试结果。该方法也不受存在大量无关背景 DNA 的干扰，并且不会与其他猪 DNA 或 RNA 病毒发生交叉反应。当与 LAMP 结合使用时，Cas12a 平台可以检测含有 p72 的质粒，反应量低至 2 个拷贝/μL。我们的结果表明，CRISPR-Cas12a 增强型荧光检测与核酸扩增相结合是稳健、方便、特异、确证、负担得起的，并且可能适用于 ASF 诊断。Cas12a 平台可以检测含有 p72 的质粒，反应量低至 2 个拷贝/μL。我们的结果表明，CRISPR-Cas12a 增强型荧光检测与核酸扩增相结合是稳健、方便、特异性、确证、经济实惠的，并且可能适用于 ASF 诊断。Cas12a 平台可以检测含有 p72 的质粒，反应量低至 2 个拷贝/μL。我们的结果表明，CRISPR-Cas12a 增强型荧光检测与核酸扩增相结合是稳健、方便、特异、确证、负担得起的，并且可能适用于 ASF 诊断。