Accurate, ultrasensitive, and point-of-care (POC) diagnosis of the African swine fever virus (ASFV) remains imperative to prevent its spread and limit the losses incurred. Herein, we propose a CRISPR-Cas12a-assisted triplex amplified colorimetric assay for ASFV DNA detection with ultrahigh sensitivity and specificity. The specific recognition of recombinase aided amplification (RAA)-amplified ASFV DNA could activate the Cas12a/crRNA/ASFV DNA complex, leading to the digestion of the linker DNA (bio-L1) on magnetic beads (MBs), thereby preventing its binding of gold nanoparticles (AuNPs) network. After magnetic separation, the release of AuNPs network comprising a substantial quantity of AuNPs could lead to a discernible alteration in color and significantly amplify the plasmonic signal, which could be read by spectrophotometers or smartphones. By combining the RAA, CRISPR/Cas12a-assisted cleavage, and AuNPs network-mediated colorimetric amplification together, the assay could detect as low as 0.1 copies/μL ASFV DNA within 1 h. The assay showed an accuracy of 100% for the detection of ASFV DNA in 16 swine tissue fluid samples, demonstrating its potential for on-site diagnosis of ASFV.

中文翻译：

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利用 CRISPR-Cas12a 辅助三重扩增检测增强非洲猪瘟病毒 (ASFV) DNA 的现场诊断

对非洲猪瘟病毒 (ASFV) 进行准确、超灵敏的即时护理 (POC) 诊断对于防止其传播并限制所造成的损失仍然至关重要。在此，我们提出了一种 CRISPR-Cas12a 辅助的三重放大比色测定法，用于具有超高灵敏度和特异性的 ASFV DNA 检测。重组酶辅助扩增 (RAA) 扩增的 ASFV DNA 的特异性识别可以激活 Cas12a/crRNA/ASFV DNA 复合物，导致磁珠 (MB) 上的接头 DNA (bio-L1) 被消化，从而阻止其与 ASFV DNA 结合。金纳米颗粒（AuNP）网络。磁分离后，包含大量 AuNP 的 AuNP 网络的释放可能会导致颜色发生明显的变化，并显着放大等离子体信号，这些信号可以通过分光光度计或智能手机读取。通过将 RAA、CRISPR/Cas12a 辅助切割和 AuNPs 网络介导的比色扩增结合在一起，该检测可以在 1 小时内检测到低至 0.1 拷贝/μL 的 ASFV DNA。该检测方法对 16 份猪组织液样本中的 ASFV DNA 检测准确度为 100%，展示了其现场诊断 ASFV 的潜力。