{"title":"A One-Step RPA-CRISPR Assay Using crRNA Based on Suboptimal Protospacer Adjacent Motif for <i>Vibrio vulnificus</i> Detection.","authors":"Xue Zhang, Yue Wang, Yixin Tang, Lihong Yang, Chenjie Zhao, Guang Yang, Pei Wang, Song Gao","doi":"10.1089/fpd.2023.0119","DOIUrl":null,"url":null,"abstract":"<p><p><i>Vibrio vulnificus</i> 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 <i>V. vulnificus</i>. 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 <i>V. vulnificus</i> 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 <i>V. vulnificus</i>, aiding in foodborne infection prevention, and exemplifies one-step RPA-CRISPR assays managing Cas-cleavage activity through PAM adjustments.</p>","PeriodicalId":12333,"journal":{"name":"Foodborne pathogens and disease","volume":null,"pages":null},"PeriodicalIF":1.9000,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Foodborne pathogens and disease","FirstCategoryId":"97","ListUrlMain":"https://doi.org/10.1089/fpd.2023.0119","RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/3/29 0:00:00","PubModel":"Epub","JCR":"Q3","JCRName":"FOOD SCIENCE & TECHNOLOGY","Score":null,"Total":0}
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.
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.