Rapid, sensitive, and user-friendly methods are essential for the detection of nucleic acid. The combination of isothermal amplification with CRISPR/Cas12a not only demonstrates significant advantages but also holds promising prospects for widespread application. However, the requirement for separate nucleic acid preamplification and Cas12a cleavage complicates operational procedures and increases the risk of aerosol pollution. Developing an easily implementable and widely applicable one-pot system remains a challenge. In this study, we introduced a novel approach to balance the reaction kinetics between enzymatic recombinase amplification (ERA) and Cas12a enzyme activity to establish a highly sensitive one-pot ERA/Cas12a (OPERA-Cas12a) system. We systematically identified key factors affecting reactions and utilized a definitive screening design (DSD) optimization strategy to streamline the optimization process. Using porcine-specific gene as the detection target, we successfully established the OPERA-Cas12a system for pork detection, which demonstrated high specificity and sensitivity. Extending this strategy to duck-specific gene, we developed the OPERA-Cas12a system with comparable performance. Both OPERA-Cas12a systems achieved a limit of detection (LOD) of 6 copies per reaction within 60 minutes. Moreover, the detection results can be conveniently read with an LED blue light illuminator, and images can be captured using a smartphone. This OPERA-Cas12a system offers a valuable reference for developing other CRISPR/Cas-base nucleic acids detection applications.

中文翻译：

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OPERA-Cas12a：一种简化的一锅系统，用于特异性、灵敏的核酸现场检测

快速、灵敏且用户友好的方法对于核酸检测至关重要。等温扩增与CRISPR/Cas12a的结合不仅表现出显着的优势，而且具有广阔的应用前景。然而，单独的核酸预扩增和Cas12a裂解的要求使操作程序变得复杂并增加了气溶胶污染的风险。开发一种易于实施且广泛适用的一锅法系统仍然是一项挑战。在本研究中，我们引入了一种平衡酶重组酶扩增（ERA）和Cas12a酶活性之间的反应动力学的新方法，以建立高度灵敏的一锅ERA/Cas12a（OPERA-Cas12a）系统。我们系统地确定了影响反应的关键因素，并利用明确的筛选设计（DSD）优化策略来简化优化过程。以猪特异性基因为检测靶点，成功建立了用于猪肉检测的OPERA-Cas12a系统，具有较高的特异性和灵敏度。将这一策略扩展到鸭子特异性基因，我们开发了性能相当的 OPERA-Cas12a 系统。两个 OPERA-Cas12a 系统在 60 分钟内每个反应的检测限 (LOD) 均为 6 个拷贝。此外，可以使用LED蓝光照明器方便地读取检测结果，并可以使用智能手机捕获图像。该OPERA-Cas12a系统为开发其他基于CRISPR/Cas的核酸检测应用提供了有价值的参考。