摘要
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抗生素的过度使用导致抗生素耐药性基因（ARGs）大幅增加，这些基因已扩散到整个食品供应链。ARGs经常从加工设施转移到食品，引起重大的公共卫生问题。目前检测ARGs的标准通常取决于定量聚合酶链式反应（qPCR）和基因测序。然而，对经验丰富的人员和复杂的读出设备的依赖极大地抑制了ARG在非实验室环境中测试的扩展。改进现场检测将有助于监测ARGs污染的传播，以确保食品安全并解决公众健康问题。在此，我们开发了一种基于CRISPR-Cas12a的检测方法，用于比色检测从食品加工厂收集的洗涤水中的ARGs。在我们的测定中，用ssDNA交联剂交联DNA功能化的金纳米粒子（AuNP）。靶诱导的Cas12a反式切割用于交联剂的降解，改变AuNP的光学性质以产生容易的视觉读出。在没有DNA扩增的情况下，我们能够检测到三种具有代表性的ARG，其检测限为5nM或更低。此外，我们的测定被扩展到更复杂的培养基中，在那里，从水果洗涤设施的洗涤水中只检测到103个基因拷贝。根据我们的结果，我们的方法具有高度的特异性和敏感性。由于我们的检测方法的可负担性和简单性，这种方法可以改善ARGs的检测，以监测和防止抗微生物耐药性在食品供应链中的巨大传播。
Abstract
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The overuse of antibiotics has led to considerable increases of antibiotic resistance genes (ARGs) that have spread throughout the entire food supply chain. ARGs often transfer from processing facilities to food, causing major public health concerns. The current standard for detecting ARGs typically depends on quantitative polymerase chain reaction (qPCR) and gene sequencing. However, the reliance on experienced personnel and complicated readout equipment substantially inhibits the expansion of ARGs testing in nonlaboratory settings. Improved on-site testing will help monitor the spread of ARGs contamination to ensure food safety and address public health concerns. Herein, we developed a CRISPR-Cas12a-based assay for the colorimetric detection of ARGs in washing water collected from a food processing plant. In our assay, DNA-functionalized gold nanoparticles (AuNPs) were cross-linked with a ssDNA cross-linker. Target-induced Cas12a trans-cleavage was utilized for degradation of the cross-linkers, shifting the optical properties of the AuNPs to produce a facile visual readout. Without DNA amplification, we were able to detect three representative ARGs with a detection limit of 5 nM or lower. In addition, our assay was extended to a more complex medium, where as few as 103 gene copies in washing water from a fruit washing facility were visually detected. Based on our results, our method is both highly specific and sensitive. Due to the affordability and simplicity of our assay, this method can improve ARGs detection to monitor and prevent the immense spread of antimicrobial resistance among the food supply chain.

https://pubs.acs.org/doi/abs/10.1021/acsfoodscitech.2c00302