CRISPR/Cas12a based fluorescence-enhanced lateral flow biosensor for detection of Staphylococcus aureus

清脆的 重组酶聚合酶扩增 金黄色葡萄球菌 检出限 荧光 生物 DNA 化学 细菌 色谱法 遗传学 基因 环介导等温扩增 物理 量子力学
作者
Baoqing Zhou,Qinghua Ye,Fan Li,Xinran Xiang,Yuting Shang,Chu‐Fang Wang,Yanna Shao,Liang Xue,Jumei Zhang,Juan Wang,Yu Ding,Moutong Chen,Qingping Wu
出处
期刊:Sensors and Actuators B-chemical [Elsevier BV]
卷期号:351: 130906-130906 被引量:89
标识
DOI:10.1016/j.snb.2021.130906
摘要

Rapid, accurate point-of-care testing (POCT) for pathogenic bacteria detection is the key for avoiding foodborne diseases caused by pathogens or their toxins. The lateral flow biosensor (LFB) based on clustered regularly interspaced short palindromic repeats (CRISPR/Cas) has displayed remarkable potential for pathogen diagnosis. In this study, we report a novel CRISPR/Cas12a-based fluorescence enhanced LFB in conjunction with functionalized quantum dots, combined with recombinase-assisted amplification (RAA), to establish low-cost, simple, and sensitive detection of Staphylococcus aureus, namely CRA-LFB (CRISPR/Cas-recombinase-assisted amplification based LFB). The CRA-LFB assay is characterized by Cas12a-mediated trans-cleavage activation induced by the target DNA to digest biotin-DNA probes, caused no complementarity to the capture probe immobilized on the test (T) line, and resulted in an undetectable T line fluorescence signal on LFB. The naked eye or fluorescence strip reader was used to determine the fluorescence intensity of the T and control lines. The limit of detection (at optimal conditions) was as low as 75 aM of genomic DNA, and 5.4 × 102 cfu/mL of S. aureus in pure cultures were detected. Moreover, this CRA-LFB assay can rapidly and accurately detect S. aureus in spiked and natural meat and vegetable samples. The CRA-LFB method yielded high specificity and no interference from other nontargeted bacteria. The results exhibited high-resolution, high-intensity fluorescent signals obtained within 70 min. This bacterial detection platform is simple, low cost, almost equipment-free, and fully qualified for food and clinical diagnosis onsite testing requirements.
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