A reversible valve-assisted chip coupling with integrated sample treatment and CRISPR/Cas12a for visual detection of Vibrio parahaemolyticus

环介导等温扩增 放大器 清脆的 检出限 炸薯条 重组酶聚合酶扩增 裂解缓冲液 实验室晶片 色谱法 生物 微流控 副溶血性弧菌 计算机科学 聚合酶链反应 化学 纳米技术 材料科学 溶解 分子生物学 DNA 细菌 基因 电信 生物化学 遗传学
作者
Hui Wu,Yanju Chen,Qunqing Yang,Cheng Peng,Xiaofu Wang,Mengyao Zhang,Siwenjie Qian,Junfeng Xu,Jian Wu
出处
期刊:Biosensors and Bioelectronics [Elsevier BV]
卷期号:188: 113352-113352 被引量:64
标识
DOI:10.1016/j.bios.2021.113352
摘要

Vibrio parahaemolyticus ( V. parahaemolyticus ) is regarded as a major cause of seafood-associated illnesses, which has aroused widespread public concern. Here, a rapid and convenient detection method for V. parahaemolyticus detection was established by a reversible valve-assisted chip coupling with CRISPR/Cas12a. With optimized lysis buffer, loop mediated isothermal amplification (LAMP) reagents and CRISPR reagents, the whole detection process from sampling to results could be finished within 50 min. The structure of chip was simple and the cost was low. By relying on three reversible rotary valves and the rotation direction-dependent Coriolis pseudo force, the flow order of liquid and the direction of liquid flow could be precisely controlled. The LAMP amplicons were specifically and sensitively identified by CRISPR/Cas12a. Positive amplification would produce green fluorescent signal while negative amplification generated no fluorescent signal, which could be clearly distinguished by the naked eye. With 600 μL of samples processed, the limit of detection (LOD) for both pure cultured V. parahaemolyticus or spiked shrimp samples could achieve 30 copies/reaction. These illustrated the established method displayed great feasibility for real samples detection. In the future, the chip could also combine with other amplification reactions, like PCR or recombinase polymerase amplification reaction (RPA), to conduct detection by changing the corresponding lyophilized amplification reagents. Overall, the proposed detection platform displays great potential for food safety analysis and clinical diagnostics, especially in resource-limited areas. • A reversible valve-assisted chip is established for V. parahaemolyticus detection. • The structure of chip is simple and the production cost is very low. • The detection sensitivity of the proposed method can achieve 30 copies/reaction. • Amplicons are specifically and visually detected by CRISPR/Cas12a in 5 min. • The chip can be applied in different amplification reactions to conduct detection.
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