利用RPA-CRISPR-Cas12a技术检测生态珍稀鱼类eDNA的方案。

IF 1.3 Q4 BIOCHEMICAL RESEARCH METHODS

STAR Protocols Pub Date : 2025-01-10 DOI:10.1016/j.xpro.2024.103544

Xing-Yi Wei, Yang Pei, Li Liu, Péter Hamar, De-Sheng Pei

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引用次数: 0

摘要

重组酶聚合酶扩增(RPA)-CRISPR-Cas12a-FQ系统能够对稀有鱼类的环境DNA （eDNA）进行敏感检测。在这里，我们提出了一种使用RPA进行eDNA扩增和Cas12a靶识别的方案。我们描述了鉴定目标位点，合成和纯化CRISPR RNA （crRNA）和RPA等温扩增的步骤。然后，我们详细介绍了构建eDNA CRISPR-Cas12a检测系统并验证其灵敏度的过程。该方案为监测水生生物多样性和保护工作提供了一种高灵敏度的方法，即使在低eDNA浓度下也是如此。有关本协议使用和执行的完整细节，请参阅Wei等人1。

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Protocol for detecting eDNA in ecological rare fish using RPA-CRISPR-Cas12a technology.

The recombinase polymerase amplification (RPA)-CRISPR-Cas12a-FQ system enables sensitive detection of environmental DNA (eDNA) in rare fish species. Here, we present a protocol for eDNA amplification and Cas12a for target recognition using RPA. We describe steps for identifying a target site, synthesis and purification of CRISPR RNA (crRNA), and RPA isothermal amplification. We then detail procedures for constructing the eDNA CRISPR-Cas12a detection system and verifying its sensitivity. This protocol offers a high-sensitivity approach for monitoring aquatic biodiversity and conservation efforts, even in low eDNA concentrations. For complete details on the use and execution of this protocol, please refer to Wei et al.¹.

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