<br>使用 RAA-CRISPR/Cas12b 一锅检测法 (Rcod) 快速、灵敏地检测核酸,Talanta

当前位置： X-MOL 学术 › Talanta › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

使用 RAA-CRISPR/Cas12b 一锅检测法 (Rcod) 快速、灵敏地检测核酸
Talanta ( IF 5.6 ) Pub Date : 2024-01-10 , DOI: 10.1016/j.talanta.2023.125616
Kangfeng Lin ₁ , Kaihu Yao ₂ , Xiao Li ₁ , Qinghan Li ₁ , Xiangju Guo ₁ , Weixin You ₁ , Wenjing Ren ₁ , Ya Bian ₁ , Jianguang Guo ₁ , Zhen Sun ₁ , Rui Zhang ₃ , Xiaoqing Yang ₄ , Zhiyong Li ₅ , Boan Li ₁

Affiliation

快速、灵敏、特异的方法对于核酸检测至关重要。 CRISPR/Cas12b最近被广泛应用于核酸检测。然而，由于其嗜热特性，DNA等温重组酶辅助扩增（RAA）和随后的CRISPR/Cas12b检测需要两个单独的反应，既繁琐又不方便，并且可能造成气溶胶污染。在本研究中，我们提出了一种 RAA-CRISPR/Cas12b 一锅检测法 (Rcod)，无需额外的扩增产物转移步骤即可进行检测。采用免核酸提取方法，从样品处理到响应时间小于30 min，灵敏度达到0.2拷贝/μL。在该系统中，Cas12b蛋白（AacCas12b）在37℃恒温下表现出强且特异性的β-裂解活性，而β-裂解活性较弱。这一特性减少了AacCas12b对系统中核酸的干扰。与实时PCR相比，我们的Rcod系统采用免核酸提取方法检测了221份临床样本，灵敏度和特异性分别为97.96%和99.19%。快速、灵敏、特异的Rcod系统为建立基于CRISPR的一步式核酸检测提供了思路，并可能有助于开发可靠的即时核酸检测。百日咳是一种急性呼吸道感染，具有高度传染性并可能致命，早期诊断对于百日咳的治疗至关重要。在这项研究中，我们发现 AacCas12b 在较低温度下具有高且强的特异性切割活性。开发了一种不干扰扩增的RAA-CRISPR/Cas12b一步检测平台（Rcod）。此外，Rcod与免核酸提取法相结合，可以快速、准确地对病原菌进行定性检测，且检测结果可视化，使得病原核酸检测分析过程更加简单，为病原核酸检测分析提供了新方法。快速临床诊断。

"点击查看英文标题和摘要"

Rapid and sensitive detection of nucleic acids using an RAA-CRISPR/Cas12b one-pot detection assay (Rcod)

Rapid, sensitive and specific methods are crucial for nucleic acid detection. CRISPR/Cas12b has recently been widely used in nucleic acid detection. However, due to its thermophagic property, DNA isothermal recombinase-aided amplification (RAA) and subsequent CRISPR/Cas12b detection require two separate reactions, which is cumbersome and inconvenient and may cause aerosol pollution. In this study, we propose an RAA-CRISPR/Cas12b one-pot detection assay (Rcod) for detection without additional amplification product transfer steps. The time from sample processing to response time was less than 30 min using nucleic acid extraction-free method, and the sensitivity reached 0.2 copies/μL. In this system, Cas12b protein (AacCas12b) exhibited strong and specific -cleavage activity at a constant temperature of 37 °C, while the -cleavage activity was weak. This characteristic reduces the interference of AacCas12b with nucleic acids in the system. Compared with real-time PCR, our Rcod system detected in 221 clinical samples with a sensitivity and specificity of 97.96 % and 99.19 %, respectively, with nucleic acid extraction-free method. The rapid, sensitive and specific Rcod system provides ideas for the establishment of CRISPR-based one-step nucleic acid detection and may aid the development of reliable point-of-care nucleic acid tests. Pertussis is an acute respiratory infection caused by that is highly contagious and potentially fatal, and early diagnosis is essential for the treatment of whooping cough. In this study, we found that AacCas12b has high and strongly specific -cleavage activity at lower temperatures. A RAA-CRISPR/Cas12b one-step detection platform (Rcod) without interference with amplification was developed. In addition, the combination of Rcod and nucleic acid extraction-free method can quickly and accurately detect the qualitative detection of , and the detection results are visualized, which makes the pathogen nucleic acid detection and analysis process simpler, and provides a new method for the rapid clinical diagnosis of .

更新日期：2024-01-10

点击分享查看原文

点击收藏

阅读更多本刊新发论文本刊介绍/投稿指南