The diagnosis of dengue virus (DENV) has been challenging particularly in areas far from clinical laboratories. Early diagnosis of pathogens is a prerequisite for the timely treatment and pathogen control. An ideal diagnostic for viral infections should possess high sensitivity, specificity, and flexibility. In this study, we implemented dual amplification involving Cas13a and Cas12a, enabling sensitive and visually aided diagnostics for the dengue virus. Cas13a recognized the target RNA by crRNA and formed the assembly of the Cas13a/crRNA/RNA ternary complex, engaged in collateral cleavage of nearby crRNA of Cas12a. The Cas12a/crRNA/dsDNA activator ternary complex could not be assembled due to the absence of crRNA of Cas12a. Moreover, the probe, with 5′ and 3’ termini labeled with FAM and biotin, could not be separated. The probes labeled with FAM and biotin, combined the Anti-FAM and the Anti-Biotin Ab-coated gold nanoparticle, and conformed sandwich structure on the T-line. The red line on the paper strip caused by clumping of AuNPs on the T-line indicated the detection of dengue virus. This technique, utilizing an activated Cas13a system cleaving the crRNA of Cas12a, triggered a cascade that amplifies the virus signal, achieving a low detection limit of 190 fM with fluorescence. Moreover, even at 1 pM, the red color on the T-line was easily visible by naked eyes. The developed strategy, incorporating cascade enzymatic amplification, exhibited good sensitivity and may serve as a field-deployable diagnostic tool for dengue virus.

中文翻译：

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基于Cas13a和Cas12a的可现场部署的登革热病毒诊断工具


登革热病毒（DENV）的诊断一直具有挑战性，特别是在远离临床实验室的地区。病原体的早期诊断是及时治疗和控制病原体的前提。病毒感染的理想诊断应具有高敏感性、特异性和灵活性。在这项研究中，我们实现了 Cas13a 和 Cas12a 的双重扩增，从而能够对登革热病毒进行灵敏且视觉辅助的诊断。 Cas13a通过crRNA识别靶RNA，并形成Cas13a/crRNA/RNA三元复合物的组装，参与Cas12a附近crRNA的附带裂解。由于Cas12a的crRNA缺失，Cas12a/crRNA/dsDNA激活剂三元复合物无法组装。此外，5'和3'末端标记有FAM和生物素的探针无法分离。探针用FAM和生物素标记，将Anti-FAM和Anti-Biotin Ab包被的金纳米颗粒结合在一起，并在T线上形成三明治结构。试纸条上的红线是由 AuNPs 在 T 线上聚集而成的，表明检测到了登革热病毒。该技术利用激活的 Cas13a 系统切割 Cas12a 的 crRNA，触发级联放大病毒信号，实现 190 fM 的荧光检测低限。而且，即使在下午 1 点，T 线上的红色肉眼也很容易看到。所开发的策略结合了级联酶扩增，表现出良好的灵敏度，可作为登革热病毒的现场部署诊断工具。