College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058, China.
Key Laboratory of Microbiol Technology and Bioinformatics of Zhejiang Province, Zhejiang Institute of Microbiology, Hangzhou, 310012, China; NMPA Key Laboratory for Testing and Risk Warning of Pharmaceutical Microbiology, Zhejiang Institute of Microbiology, Hangzhou, 310012, China.
Talanta. 2020 Jul 1;214:120818. doi: 10.1016/j.talanta.2020.120818. Epub 2020 Feb 11.
Vibrio parahaemolyticus is a major cause of seafood-associated food poisoning. It is of great significance to develop an accurate, simple and cost-effective method to identify infected seafood, especially for on-site application. Polymerase chain reaction (PCR) remains the golden standard for nucleic acid detection. But traditional methods heavily reply on sophisticated instrument and specialized operators, which limits the application for on-site detections. Here we developed a novel, specific and visualized detection method for PCR based on CRISPR/Cas12a system. On a low-cost thermal cycler, amplification reaction can be conducted easily. The CRISPR/Cas12a system was specifically designed to evaluate amplicons, eliminating false positive results. Besides the negative samples remained colorless, the positive samples generated obvious green fluorescence, which could be easily distinguished by the naked eye using a homemade UV device. The presented detection method was verified by detecting shrimp samples. The limit of detection is 1.02 × 10 copies/μL. This presented method provided a new strategy for specific endpoint detection of PCR and advanced its application in field for food safety assurance.
副溶血性弧菌是食源性食物中毒的主要原因之一。开发一种准确、简单且经济有效的方法来识别受感染的海鲜，特别是用于现场应用，具有重要意义。聚合酶链反应（PCR）仍然是核酸检测的金标准。但是，传统方法严重依赖于复杂的仪器和专业操作人员，这限制了现场检测的应用。在这里，我们开发了一种基于 CRISPR/Cas12a 系统的新型、特异性和可视化的 PCR 检测方法。在低成本的热循环仪上，可以轻松进行扩增反应。CRISPR/Cas12a 系统专门用于评估扩增产物，从而消除假阳性结果。除了阴性样本保持无色外，阳性样本产生明显的绿色荧光，使用自制的紫外装置可以通过肉眼轻松区分。该检测方法通过检测虾样进行了验证。检测限为 1.02×10 拷贝/μL。该方法为 PCR 的特定终点检测提供了一种新策略，并推进了其在食品安全保障领域的现场应用。
