College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China.
College of Food Science and Technology, Yunnan Agricultural University, Kunming 650201, China.
Biosensors (Basel). 2023 Jan 7;13(1):111. doi: 10.3390/bios13010111.
A rapid and intuitive method for detecting Vibrio parahaemolyticus (VP) was established by a designed reaction vessel which coupled CRISPR/Cas12a with loop-mediated isothermal nucleic acid amplification (LAMP). There were two spaces in the vessel-holding LAMP reaction solution and CRISPR reaction solution, respectively, which were separated with a polyvinyl alcohol (PVA) membrane. The PVA membrane could be dissolved with a water solution. The thermolabile hemolysin (TLH) gene of VP was employed as the detection target. After the target sequence of the TLH gene was amplified with LAMP, the PVA membrane would be dissolved and the CRISPR reaction solution mixed with the LAMP reaction solution. In this way, amplicons could be detected with CRISPR/Cas12a in the reaction vessel. The fluorescent signals produced by the positive samples were clearly identified by the naked eye under a UV light, while the negative samples were dark. The whole detection procedure could be finished within 35 min with a detection limit of 100 copies/µL. The designed reaction vessel is easy to produce and can effectively prevent contamination due to the opening of the reaction vessel after the LAMP reaction. Thus, it will have the potential to provide a new solution for rapid detection in the field.
建立了一种快速直观的检测副溶血性弧菌（VP）的方法，该方法采用设计的反应容器，将 CRISPR/Cas12a 与环介导等温核酸扩增（LAMP）相结合。容器中有两个空间，分别容纳 LAMP 反应溶液和 CRISPR 反应溶液，它们被聚乙烯醇（PVA）膜隔开。PVA 膜可以被水溶液溶解。VP 的热敏溶血素（TLH）基因被用作检测目标。在 TLH 基因的目标序列通过 LAMP 扩增后，PVA 膜会溶解，CRISPR 反应溶液会与 LAMP 反应溶液混合。这样，在反应容器中就可以用 CRISPR/Cas12a 检测到扩增产物。在紫外光下，阳性样本产生的荧光信号用肉眼就能清晰识别，而阴性样本则较暗。整个检测过程在 35 分钟内完成，检测限为 100 拷贝/µL。设计的反应容器易于生产，并且由于 LAMP 反应后反应容器的打开可以有效防止污染。因此，它有可能为现场快速检测提供新的解决方案。
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