A One-Step RPA-CRISPR Assay Using crRNA Based on Suboptimal Protospacer Adjacent Motif for Vibrio vulnificus Detection

作者：Xue Zhang, Yue Wang, Yixin Tang, Lihong Yang, Chenjie Zhao, Guang Yang, Pei Wang, Song Gao · 发表于：Foodborne Pathogens and Disease · 年份：2024 · DOI：10.1089/fpd.2023.0119 · 被引用次数：8 · 研究领域：CRISPR and Genetic Engineering、Vibrio bacteria research studies、Aquaculture disease management and microbiota

Vibrio vulnificus is a hazardous foodborne pathogen responsible for approximately 95% of seafood-related deaths. This highlights the urgent requirement for specialized detection tools to be developed and used by food enterprises and food safety authorities. The DETECTR (DNA endonuclease targeted CRISPR trans reporter) system that combines CRISPR/Cas and recombinase polymerase amplification (RPA) has been utilized to develop a molecular detection assay for V. vulnificus. However, because the incompatibility between RPA and Cas12a cleavage has not been addressed, it is a two-step assay that lacks convenience and presents contamination risk. Here, we developed a one-step RPA-CRISPR assay for V. vulnificus using a special crRNA targeting a sequence with a suboptimal protospacer adjacent motif (PAM). The entire assay, conducted at 37°C, takes only 40–60 min, yields results visualized under blue light, and exhibits exceptional specificity and sensitivity (detecting 4 pathogen genome copies per reaction). This study offers a valuable tool for detecting V. vulnificus, aiding in foodborne infection prevention, and exemplifies one-step RPA-CRISPR assays managing Cas-cleavage activity through PAM adjustments.