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

IF 1.9 2区 农林科学 Q3 FOOD SCIENCE & TECHNOLOGY
Foodborne pathogens and disease Pub Date : 2024-07-01 Epub Date: 2024-03-29 DOI:10.1089/fpd.2023.0119
Xue Zhang, Yue Wang, Yixin Tang, Lihong Yang, Chenjie Zhao, Guang Yang, Pei Wang, Song Gao
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引用次数: 0

Abstract

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.

使用基于次优原位相邻位点的 crRNA 的一步式 RPA-CRISPR 检测法检测弧菌。
弧菌是一种危险的食源性病原体,约 95% 的海产品相关死亡事件都是由弧菌引起的。这突出表明,食品企业和食品安全当局迫切需要开发和使用专门的检测工具。DETECTR(DNA内切酶靶向CRISPR反式报告器)系统结合了CRISPR/Cas和重组酶聚合酶扩增(RPA)技术,已被用于开发弧菌分子检测方法。然而,由于 RPA 与 Cas12a 裂解之间的不兼容性问题尚未得到解决,因此这种检测方法需要两个步骤,既不方便又存在污染风险。在这里,我们开发了一种针对V. vulnificus的一步式RPA-CRISPR检测方法,它使用了一种特殊的crRNA,靶向具有次优原间隔邻接基序(PAM)的序列。整个检测过程在 37 摄氏度下进行,只需 40-60 分钟,结果在蓝光下可视化,并具有极高的特异性和灵敏度(每个反应可检测 4 个病原体基因组拷贝)。这项研究为检测弧菌提供了一种有价值的工具,有助于预防食源性感染,同时也是通过调整 PAM 来管理 Cas-裂解活性的一步式 RPA-CRISPR 检测的典范。
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来源期刊
Foodborne pathogens and disease
Foodborne pathogens and disease 医学-食品科技
CiteScore
5.30
自引率
3.60%
发文量
80
审稿时长
1 months
期刊介绍: Foodborne Pathogens and Disease is one of the most inclusive scientific publications on the many disciplines that contribute to food safety. Spanning an array of issues from "farm-to-fork," the Journal bridges the gap between science and policy to reduce the burden of foodborne illness worldwide. Foodborne Pathogens and Disease coverage includes: Agroterrorism Safety of organically grown and genetically modified foods Emerging pathogens Emergence of drug resistance Methods and technology for rapid and accurate detection Strategies to destroy or control foodborne pathogens Novel strategies for the prevention and control of plant and animal diseases that impact food safety Biosecurity issues and the implications of new regulatory guidelines Impact of changing lifestyles and consumer demands on food safety.
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