CRISPR/Cas12a-Based Indirect Competitive Enzyme-Linked Immunosorbent Assay for Sensitive Detection of Ochratoxin A

IF 5.7 1区农林科学 Q1 AGRICULTURE, MULTIDISCIPLINARY

Journal of Agricultural and Food Chemistry Pub Date : 2024-09-20 DOI:10.1021/acs.jafc.4c06525

Xinqi Long, Tian Zhang, Lu Yang, Chenxi Guo, Qiyang Zhao, Yongliang Cui, Chengqiu Wang, Yaohai Zhang, Yue He

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Abstract

The high toxicity and widespread contamination of ochratoxin A (OTA) make it urgent to develop a sensitive method to detect trace OTA in complex food matrices. Herein, an indirect competitive enzyme-linked immunosorbent assay (icELISA)-based on the CRISPR/Cas12a system is described. DNA amplicons with multiple activation sequences of the CRISPR/Cas12a system were pre-prepared to improve detection sensitivity. In the absence of OTA, streptavidin-mediated biotinylated DNA amplicons were captured by the biotinylated secondary antibody on the microplate. The captured DNA amplicons activated the CRISPR/Cas12a system, which thereby effectively cleaved the reporter DNA, producing strong fluorescence. The presence of OTA led to a decrease in DNA amplicons on the microplate, resulting in a decrease in activated Cas12a and ultimately a drop in fluorescence intensity. OTA in food matrices at nanogram per milliliter levels can be detected. Therefore, the new method has great potential in monitoring OTA.

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基于 CRISPR/Cas12a 的间接竞争性酶联免疫吸附试验，用于灵敏检测赭曲霉毒素 A

赭曲霉毒素 A（OTA）毒性高，污染范围广，因此迫切需要开发一种灵敏的方法来检测复杂食品基质中的痕量 OTA。本文介绍了一种基于 CRISPR/Cas12a 系统的间接竞争性酶联免疫吸附测定法（icELISA）。为提高检测灵敏度，预先制备了具有多个 CRISPR/Cas12a 系统激活序列的 DNA 扩增子。在没有 OTA 的情况下，链霉亲和素介导的生物素化 DNA 扩增子被微孔板上的生物素化二抗捕获。捕获的 DNA 扩增子激活了 CRISPR/Cas12a 系统，从而有效地裂解了报告 DNA，产生了强烈的荧光。OTA 的存在导致微孔板上的 DNA 扩增子减少，从而导致激活的 Cas12a 减少，最终导致荧光强度下降。食品基质中的 OTA 可达到每毫升毫微克的水平。因此，这种新方法在监测 OTA 方面具有很大的潜力。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Journal of Agricultural and Food Chemistry 农林科学-农业综合

CiteScore

9.90

自引率

8.20%

发文量

1375

审稿时长

2.3 months

期刊介绍： The Journal of Agricultural and Food Chemistry publishes high-quality, cutting edge original research representing complete studies and research advances dealing with the chemistry and biochemistry of agriculture and food. The Journal also encourages papers with chemistry and/or biochemistry as a major component combined with biological/sensory/nutritional/toxicological evaluation related to agriculture and/or food.