Development of an on-site real-time dual detection method for norovirus and rotavirus using RPA-CRISPR/Cas12,13

IF 5.6 1区农林科学 Q1 FOOD SCIENCE & TECHNOLOGY

Food Control Pub Date : 2024-10-16 DOI:10.1016/j.foodcont.2024.110943

Xiangyun Le , Jing Jiang , Yi Hong , Juping Shi , Xuejie Liu , Junxin Xue , Xiang Wang

{"title":"Development of an on-site real-time dual detection method for norovirus and rotavirus using RPA-CRISPR/Cas12,13","authors":"Xiangyun Le , Jing Jiang , Yi Hong , Juping Shi , Xuejie Liu , Junxin Xue , Xiang Wang","doi":"10.1016/j.foodcont.2024.110943","DOIUrl":null,"url":null,"abstract":"<div><div>Norovirus (NoV) and rotavirus (RoV) are the two most prevalent foodborne viruses responsible for infectious gastroenteritis, with high contamination rates in fruits and vegetables. Monitoring and accurately identifying these pathogens are crucial for preventing human foodborne illnesses. Therefore, developing a rapid, highly specific, and sensitive detection method for NoV and RoV is essential. An on-site real time dual detection method for Noroviruses of genogroup I (GI-NoV) and Rotavirus of group A (A-RoV) was established by integrating Recombinase Polymerase Amplification (RPA) with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein (Cas) system. The DNA amplification products generated by multiplex RPA were simultaneously detected by Cas12a and Cas13a in a single tube. Utilizing the trans-cleavage preferences of Cas12a and Cas13a, the specific cleavage of DNA and RNA probes carrying different fluorescent groups within the reaction system was achieved. The results indicated that the established dual detection method had a detection limit of 10<sup>2</sup> copies/μL for GI-NoV and A-RoV. Specificity assay showed the established method only detected GI-NoV and A-RoV, with no cross-reactivity with other four foodborne viruses. Furthermore, the detection capability of this method was validated in strawberries and lettuce. The method enables the simultaneous detection of GI-NoV and A-RoV in fresh produce at 37 °C within 60 min. Detection results were interpreted by measuring fluorescence values or observing color changes under blue light at the end of the reaction. This technique required minimal instrumentation, provided excellent visual representation of results, and served as a technical reference for the rapid on-site detection of foodborne viruses in food matrices.</div></div>","PeriodicalId":319,"journal":{"name":"Food Control","volume":"168 ","pages":"Article 110943"},"PeriodicalIF":5.6000,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Food Control","FirstCategoryId":"97","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0956713524006601","RegionNum":1,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"FOOD SCIENCE & TECHNOLOGY","Score":null,"Total":0}

引用次数: 0

Abstract

Norovirus (NoV) and rotavirus (RoV) are the two most prevalent foodborne viruses responsible for infectious gastroenteritis, with high contamination rates in fruits and vegetables. Monitoring and accurately identifying these pathogens are crucial for preventing human foodborne illnesses. Therefore, developing a rapid, highly specific, and sensitive detection method for NoV and RoV is essential. An on-site real time dual detection method for Noroviruses of genogroup I (GI-NoV) and Rotavirus of group A (A-RoV) was established by integrating Recombinase Polymerase Amplification (RPA) with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein (Cas) system. The DNA amplification products generated by multiplex RPA were simultaneously detected by Cas12a and Cas13a in a single tube. Utilizing the trans-cleavage preferences of Cas12a and Cas13a, the specific cleavage of DNA and RNA probes carrying different fluorescent groups within the reaction system was achieved. The results indicated that the established dual detection method had a detection limit of 10² copies/μL for GI-NoV and A-RoV. Specificity assay showed the established method only detected GI-NoV and A-RoV, with no cross-reactivity with other four foodborne viruses. Furthermore, the detection capability of this method was validated in strawberries and lettuce. The method enables the simultaneous detection of GI-NoV and A-RoV in fresh produce at 37 °C within 60 min. Detection results were interpreted by measuring fluorescence values or observing color changes under blue light at the end of the reaction. This technique required minimal instrumentation, provided excellent visual representation of results, and served as a technical reference for the rapid on-site detection of foodborne viruses in food matrices.

查看原文本刊更多论文

利用 RPA-CRISPR/Cas 开发诺如病毒和轮状病毒的现场实时双重检测方法12,13

诺如病毒（NoV）和轮状病毒（RoV）是导致传染性肠胃炎的两种最常见的食源性病毒，在水果和蔬菜中的污染率很高。监测和准确识别这些病原体对于预防人类食源性疾病至关重要。因此，开发一种快速、高特异性和高灵敏度的 NoV 和 RoV 检测方法至关重要。通过将重组酶聚合酶扩增（RPA）与聚类正则间隔短联合重复序列（CRISPR）相关蛋白（Cas）系统相结合，建立了一种现场实时检测 I 基因组诺罗病毒（GI-NoV）和 A 组轮状病毒（A-RoV）的双重方法。在一个试管中，Cas12a 和 Cas13a 同时检测多重 RPA 产生的 DNA 扩增产物。利用 Cas12a 和 Cas13a 的反式裂解偏好，在反应体系中实现了对携带不同荧光基团的 DNA 和 RNA 探针的特异性裂解。结果表明，所建立的双重检测方法对 GI-NoV 和 A-RoV 的检测限为 102 拷贝/μL。特异性分析表明，所建立的方法只能检测到 GI-NoV 和 A-RoV，与其他四种食源性病毒没有交叉反应。此外，还在草莓和莴苣中验证了该方法的检测能力。该方法可在 37 °C 条件下，在 60 分钟内同时检测新鲜农产品中的 GI-NoV 和 A-RoV。检测结果可通过测量荧光值或在反应结束时观察蓝光下的颜色变化来解释。该技术所需仪器最少，结果直观，可作为现场快速检测食品基质中食源性病毒的技术参考。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Food Control 工程技术-食品科技

CiteScore

12.20

自引率

6.70%

发文量

758

审稿时长

33 days

期刊介绍： Food Control is an international journal that provides essential information for those involved in food safety and process control. Food Control covers the below areas that relate to food process control or to food safety of human foods: • Microbial food safety and antimicrobial systems • Mycotoxins • Hazard analysis, HACCP and food safety objectives • Risk assessment, including microbial and chemical hazards • Quality assurance • Good manufacturing practices • Food process systems design and control • Food Packaging technology and materials in contact with foods • Rapid methods of analysis and detection, including sensor technology • Codes of practice, legislation and international harmonization • Consumer issues • Education, training and research needs. The scope of Food Control is comprehensive and includes original research papers, authoritative reviews, short communications, comment articles that report on new developments in food control, and position papers.