Álvarez-Rodríguez Andrés, Li Zeng, Jin Bo-Kyung, Stijlemans Benoit, Geldhof Peter, Magez Stefan
Lab of Cellular and Molecular Immunology, Brussels Center for Immunology (BCIM), Vrije Universiteit Brussel, Brussels, Belgium.
Department of Biochemistry and Microbiology (WE10), Ghent University, Ghent, Belgium.
Front Mol Biosci. 2025 Feb 14;12:1512970. doi: 10.3389/fmolb.2025.1512970. eCollection 2025.
Control of () infections remains a significant challenge in managing Surra, a widespread veterinary disease affecting both wild and domestic animals. In the absence of an effective vaccine, accurate diagnosis followed by treatment is crucial for successful disease management. However, existing diagnostic methods often fail to detect active infections, particularly in field conditions. Recent advancements in CRISPR-Cas technology, combined with state-of-the-art isothermal amplification assays, offer a promising solution. This approach has led us to the development of a RPA-CRISPR assay, a highly sensitive and specific diagnostic tool suitable for both laboratory and field settings.
First, the CRISPR-Cas12b cleavage assay was developed and optimized, and its analytical sensitivity was evaluated. Next, this technology was integrated with the RPA to create the RPA-CRISPR test, with the reaction conditions being optimized and its analytical sensitivity and specificity assessed. Finally, the test's accuracy in detecting both active and cured infections was evaluated.
The optimized CRISPR-Cas12b cleavage assay demonstrated the ability to detect target DNA at picomolar concentrations. Integrating CRISPR-Cas12b with RPA in Two-Pot and One-Pot RPA-CRISPR tests achieved up to a 100-fold increase in analytical sensitivity over RPA alone, detecting attomolar concentrations of target DNA, while maintaining analytical specificity for . Both assays exhibited performance comparable to the gold standard PCR in experimental mouse infections, validating their effectiveness for detecting active infections and assessing treatment efficacy.
The RPA-CRISPR tests prove highly effective for diagnosing active infections and assessing treatment efficacy, while being adaptable for both laboratory and field use. Thus, the RPA-CRISPR assays emerge as a promising addition to current diagnostic tools, offering efficient and reliable detection of active infections.
在苏拉病的管理中，控制（）感染仍然是一项重大挑战，苏拉病是一种广泛传播的兽医疾病，影响野生动物和家畜。在缺乏有效疫苗的情况下，准确诊断并随后进行治疗对于成功的疾病管理至关重要。然而，现有的诊断方法往往无法检测到活动性感染，尤其是在野外条件下。CRISPR-Cas技术的最新进展与最先进的等温扩增检测方法相结合，提供了一个有前景的解决方案。这种方法促使我们开发了一种RPA-CRISPR检测方法，这是一种高度灵敏且特异的诊断工具，适用于实验室和野外环境。
首先，开发并优化了CRISPR-Cas12b切割检测方法，并评估了其分析灵敏度。接下来，将该技术与RPA整合以创建RPA-CRISPR检测，优化反应条件并评估其分析灵敏度和特异性。最后，评估该检测在检测活动性和已治愈的（）感染方面的准确性。
优化后的CRISPR-Cas12b切割检测方法显示出能够检测皮摩尔浓度的（）靶DNA。在双罐和单罐RPA-CRISPR检测中将CRISPR-Cas12b与RPA整合，与单独的RPA相比，分析灵敏度提高了100倍，能够检测到阿托摩尔浓度的（）靶DNA，同时保持对（）的分析特异性。在实验小鼠感染中，两种检测方法的性能均与金标准PCR相当，验证了它们在检测活动性感染和评估治疗效果方面的有效性。
RPA-CRISPR检测在诊断活动性感染和评估治疗效果方面被证明非常有效，同时适用于实验室和野外使用。因此，RPA-CRISPR检测方法成为当前诊断工具中一个有前景的补充，能够高效可靠地检测活动性（）感染。
