College of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, China.
State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products; Key Laboratory of Information Traceability for Agricultural Products, Ministry of Agriculture and Rural Affairs; Institute of Agro-product Safety and Nutrition, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China.
Anal Chem. 2021 Oct 26;93(42):14300-14306. doi: 10.1021/acs.analchem.1c03468. Epub 2021 Oct 13.
(), which may cause gastrointestinal disorders in humans, is a pathogen commonly found in seafood. There are many methods for detecting , yet they have some shortcomings, such as high cost, labor-intensiveness, and complicated operation, which are impractical for resource-limited settings. Herein, we present a sequence-specific, label-free, and colorimetric method for visual detection of . This method utilizes CRISPR/Cas12a to specifically recognize the loop-mediated isothermal amplification (LAMP) products for further trans-cleaving the G-quadruplex DNAzyme and depriving its peroxidase-mimicking activity. In this way, the results can be directly observed with the naked eyes the color development of 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), which displays colorless for positive samples while green for target-free samples. We term such Cas12a-crRNA preventing ABTS from developing color by trimming the G-quadruplex DNAzyme as Cascade. The proposed method can detect 9.8 CFU (per reaction) of pure cultured , and the sensitivity is comparable to real-time LAMP. It has been applied for practical use and showed the capability to detect 6.1 × 10 CFU/mL in shrimp samples. Based on this, the newly established Cascade method can be employed as a universal biosensing strategy for pathogenic bacterial testing in the field.
（）是一种常见的海鲜病原体，可能会导致人类肠胃失调。目前有许多方法可用于检测，但这些方法存在一些缺点，如成本高、劳动强度大、操作复杂，对于资源有限的环境来说并不实用。在此，我们提出了一种用于可视化检测的序列特异性、无标记和比色法。该方法利用 CRISPR/Cas12a 特异性识别环介导等温扩增（LAMP）产物，进一步切割 G-四链体 DNA 酶并剥夺其过氧化物酶模拟活性。通过这种方式，可以直接用肉眼观察结果——2,2'-联氮-双（3-乙基苯并噻唑啉-6-磺酸）（ABTS）的显色，阳性样本无色，而无靶标样本呈绿色。我们将 Cas12a-crRNA 通过修剪 G-四链体 DNA 酶来防止 ABTS 显色的这种方式称为级联反应。该方法可检测到 9.8 CFU（每个反应）的纯培养物，灵敏度与实时 LAMP 相当。它已被应用于实际用途，并显示出在虾样本中检测 6.1×10 CFU/mL 的能力。基于此，新建立的级联方法可以作为一种用于现场致病菌检测的通用生物传感策略。
