A super convenient and specific CRISPR/Cas12a diagnostic platform for toxigenic Burkholderia gladioli based on virulence genes

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

Food Control Pub Date : 2024-09-21 DOI:10.1016/j.foodcont.2024.110904

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引用次数: 0

Abstract

In recent years, food poisoning cases caused by toxigenic Burkholderia gladioli pv. cocovenenans (BGC) have been frequently reported in Asian countries like China and Indonesia, which seriously threaten food safety and public health. BGC is the only subspecies within the Burkholderia gladioli species that can cause food poisoning in humans, but there is a high degree of sequence homology between the different subspecies, making detection and differentiation difficult. In this study, a specific DNA target was screened out from the bongkrekic acid biosynthentic gene cluster. A rapid, sensitive, and highly specific CRISPR/Cas12a molecular diagnostic platform was developed for the first time to differentiate between toxigenic and non-toxigenic subspecies. Genomic DNA was extracted from the samples after a simple boiling process. The sensitivity was evaluated from three different levels, the genomic DNA sensitivity was determined to be 10⁻⁴ ng/μL, the target DNA sensitivity was determined to be 14.9 copies/μL, and the bacterial suspension sensitivity was confirmed to be 1.52 CFU/mL. The detection limit of BGC in artificially contaminated coconut water samples was determined to be 1.50 CFU/mL without any microbial enrichment. Including sample pretreatment and DNA extraction process, the whole detection of BGC in samples can be completed within 60 min under the assistance of some small and portable devices. This super convenient diagnostic platform is suitable for food field testing and provides a theoretical basis for the specific identification of BGC.

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基于毒力基因的超便捷、特异性 CRISPR/Cas12a 诊断平台，用于诊断毒性革兰氏阴道伯克霍尔德氏菌

近年来，中国和印度尼西亚等亚洲国家频频发生由毒素型伯克霍尔德氏菌（Burkholderia gladioli pv. cocovenenans，BGC）引起的食物中毒事件，严重威胁食品安全和公众健康。BGC 是革兰氏阴沟伯克霍尔德氏菌中唯一可导致人类食物中毒的亚种，但不同亚种之间存在高度的序列同源性，因此难以检测和区分。本研究从棒曲霉素生物合成基因簇中筛选出了一个特异的 DNA 靶标。该研究首次开发了一种快速、灵敏和高度特异性的 CRISPR/Cas12a 分子诊断平台，用于区分毒素亚种和非毒素亚种。经过简单的煮沸过程，就能从样本中提取基因组 DNA。灵敏度从三个不同的水平进行了评估，基因组 DNA 灵敏度被确定为 10-4 纳克/微升，目标 DNA 灵敏度被确定为 14.9 拷贝/微升，细菌悬浮液灵敏度被确定为 1.52 CFU/毫升。在没有任何微生物富集的情况下，人工污染椰子汁样品中 BGC 的检测限被确定为 1.50 CFU/mL。包括样品预处理和 DNA 提取过程在内，在一些小型便携设备的辅助下，整个样品中 BGC 的检测可在 60 分钟内完成。这一超级便捷的诊断平台适用于食品现场检测，并为特异性鉴定 BGC 提供了理论依据。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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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.