Rapid and sensitive pathogen detection methods are critical for disease diagnosis and treatment. RPA-CRISPR/Cas12 systems have displayed remarkable potential in pathogen detection. A self-priming digital PCR chip is a powerful and attractive tool for nucleic detection. However, the application of the RPA-CRISPR/Cas12 system to the self-priming chip still has great challenges due to the problems of protein adsorption and two-step detection mode of RPA-CRISPR/Cas12. In this study, an adsorption-free self-priming digital chip was developed and a direct digital dual-crRNAs (3D) assay was established based on the chip for ultrasensitive detection of pathogens. This 3D assay combined the advantages of rapid amplification of RPA, specific cleavage of Cas12a, accurate quantification of digital PCR, and point-of-care testing (POCT) of microfluidics, enabling accurate and reliable digital absolute quantification of Salmonella in POCT. Our method can provide a good linear relationship of Salmonella detection in the range from 2.58 × 10¹ to 2.58 × 10⁴ cells/mL with a limit of detection ∼0.2 cells/mL within 30 min in a digital chip by targeting the invA gene of Salmonella. Moreover, the assay could directly detect Salmonella in milk without nucleic acid extraction. Therefore, the 3D assay has the significant potential to provide accurate and rapid pathogen detection in POCT. This study provides a powerful nucleic detection platform and facilitates the application of CRISPR/Cas-assisted detection and microfluidic chips.

中文翻译：

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用于病原体超灵敏检测的无吸附自吸直接数字双 crRNA CRISPR/Cas12a 辅助芯片

快速灵敏的病原体检测方法对于疾病的诊断和治疗至关重要。RPA-CRISPR/Cas12 系统在病原体检测方面显示出非凡的潜力。自吸式数字 PCR 芯片是一种强大而有吸引力的核酸检测工具。然而，由于RPA-CRISPR/Cas12的蛋白吸附和两步检测模式等问题，RPA-CRISPR/Cas12系统在自吸芯片上的应用仍面临很大挑战。在这项研究中，开发了一种无吸附自吸数字芯片，并基于该芯片建立了直接数字双 crRNAs (3D) 检测方法，用于病原体的超灵敏检测。这种 3D 分析结合了 RPA 的快速扩增、Cas12a 的特异性切割、数字 PCR 的准确定量和微流体的即时检测 (POCT) 的优势，POCT 中的沙门氏菌。^{我们的方法可以在 2.58 × 10 1}到 2.58 × 10 ^{4 个}细胞/mL 的范围内提供良好的沙门氏菌检测线性关系，通过靶向 inv A基因，在数字芯片中 30 分钟内检测限为 ∼0.2 个细胞/mL沙门氏菌。_ 此外，该测定无需核酸提取即可直接检测牛奶中的沙门氏菌。因此，3D 检测具有在 POCT 中提供准确、快速的病原体检测的巨大潜力。本研究提供了一个强大的核酸检测平台，促进了CRISPR/Cas辅助检测和微流控芯片的应用。