Graphene oxide assisting the visual detection of Salmonella by CRISPR/Cas12a
Graphene oxide assisting the visual detection of Salmonella by CRISPR/Cas12a
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DOI：
10.1016/j.microc.2023.108870
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发表时间：
2023-05
影响因子：
4.8
通讯作者：
Liu Wang;Linlin Bai;Hongmei Wang;Kaiyu He;Rui Wang;Qiang Wang;Fang Zhang;Xiahong Xu
中科院分区：
文献类型：
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作者：
Liu Wang;Linlin Bai;Hongmei Wang;Kaiyu He;Rui Wang;Qiang Wang;Fang Zhang;Xiahong Xu
快速、准确地检测沙门氏菌对于确保食品安全极其重要。将核酸扩增与CRISPR/Cas12a相结合，可以实现食源性病原体的灵敏、特异检测。然而，目前基于CRISPR/Cas的方法主要依靠荧光团和猝灭剂双标记的ssDNA作为报告基因，这带来了高成本和空间位阻。在此，开发了一种新的信号输出格式。该方法利用氧化石墨烯吸附FAM-ssDNA并猝灭荧光发射，而CRISPR/Cas12a通过反式切割FAM-ssDNA来恢复荧光发射。对影响传感器性能的因素进行了优化，结果表明，3'端修饰荧光团的12 nt寡核苷酸具有最高的信噪比。信号输出方法与重组酶聚合酶扩增相结合，用于沙门氏菌检测，并具有与实时 PCR 一致的特异性和灵敏度（5 × 101 拷贝）。通过用 FAM-ssDNA 和 GO 取代双标记 ssDNA 报告基因，这种信号输出方法可节省约 65% 的成本。值得注意的是，该方法不需要实时热循环仪或任何其他复杂的仪器。除了病原体之外，这种简单方便的方法还可以扩展到检测其他目标。
Rapid and accurate detection ofSalmonellais extremely important to ensure food safety. Combining nucleic acid amplification with CRISPR/Cas12a can realize sensitive and specific detection of foodborne pathogens. However, present CRISPR/Cas-based methods mainly depend on fluorophore and quencher dual-labeled ssDNA as the reporter, which brings in high cost and steric hindrance. Herein, a new signal output format has been developed. The method takes advantage of graphene oxide adsorbing FAM-ssDNA and quenching the fluorescent emission while CRISPR/Cas12a recovering the fluorescent emission by trans-cleaving the FAM-ssDNA. Factors affecting the performance of the sensor have been optimized, and it is shown that the oligonucleotide of 12 nt with the fluorophore modified at the 3′ end displays the highest signal to noise ratio. The signal output method has been combined with recombinase polymerase amplification for detection ofSalmonellaand presents consistent specificity and sensitivity (5 × 101copies) as real-time PCR. By replacing the dual-labeled ssDNA reporter with FAM-ssDNA and GO, this signal output method saves ∼65% cost. Notably, this method does not require a real-time thermal cycler or any other sophisticated instrument. Besides pathogens, this easy and convenient method can also be extended to detect other targets.