Droplet digital PCR and real-time PCR for the sensitive and specific detection of Vibrio vulnificus based on the novel target genes

IF 3 3区农林科学 Q2 FOOD SCIENCE & TECHNOLOGY European Food Research and Technology Pub Date : 2024-04-22 DOI:10.1007/s00217-024-04465-4

Xinping Cui, Haibo Zhou, Zhaoxin Lu, Antuo Hu, Shengyu Zhang, Xiaomei Bie, Jun Yang

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Abstract

Vibrio vulnificus poses a significant risk to public health due to its high pathogenicity and mortality rates as a common seafood-borne pathogen. Therefore, rapid, accurate, and specific detection methods for V. vulnificus are crucial for ensuring human safety and minimizing economic losses. This study identified vvhA and vv08030 as promising targets for V. vulnificus detection using bioinformatics screening and PCR analysis. Based on these specific target genes, a duplex real-time PCR (qPCR) assay and a duplex droplet digital PCR (ddPCR) assay were developed and evaluated for the rapid quantitative detection of V. vulnificus. These methods showed 100% specificity to V. vulnificus and no cross-reaction with other strains. It was proved in this research that the qPCR method can detect genomic DNA as low as 31.4 fg/μL, and the quantification range of the bacterial suspension was between 8.25 × 10² and 8.25 × 10⁷ CFU/mL. On the other hand, ddPCR exhibited greater sensitivity with genomic sensitivity reaching 3.14 fg/μL and could accurately quantify bacterial suspensions between 8.25 × 10¹–8.25 × 10⁵ CFU/mL. The feasibility of the ddPCR method in detecting V. vulnificus was assessed in spiked food samples. The lowest detectable V. vulnificus in salmon was 5.74 × 10¹ CFU/g without any pre-enrichment. These methods demonstrated high sensitivity, accuracy and rapidity, with potential applications in V. vulnificus detection strategies.

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基于新型目标基因的液滴数字 PCR 和实时 PCR 用于灵敏特异地检测弧菌

弧菌作为一种常见的海产品传播病原体，具有高致病性和高死亡率，对公众健康构成重大风险。因此，快速、准确和特异性的弧菌检测方法对于确保人类安全和减少经济损失至关重要。本研究通过生物信息学筛选和聚合酶链反应分析，确定了 vvhA 和 vv08030 作为检测 V. vulnificus 的有希望的靶基因。基于这些特异性靶基因，研究人员开发并评估了一种双链实时 PCR（qPCR）检测方法和一种双链液滴数字 PCR（ddPCR）检测方法，用于快速定量检测弧菌。这些方法对弧菌的特异性达到 100%，且与其他菌株无交叉反应。研究证明，qPCR 方法可检测到低至 31.4 fg/μL 的基因组 DNA，细菌悬浮液的定量范围为 8.25 × 102 至 8.25 × 107 CFU/mL。另一方面，ddPCR 表现出更高的灵敏度，基因组灵敏度达到 3.14 fg/μL，可准确定量 8.25 × 101-8.25 × 105 CFU/mL 之间的细菌悬浮液。ddPCR 方法检测弧菌的可行性在添加了弧菌的食品样品中进行了评估。在未进行任何预富集的情况下，三文鱼中可检测到的最低弧菌数为 5.74 × 101 CFU/g。这些方法具有高灵敏度、准确性和快速性，有望应用于弧菌检测策略。

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来源期刊

European Food Research and Technology 工程技术-食品科技

CiteScore

6.60

自引率

3.00%

发文量

232

审稿时长

2.0 months

期刊介绍： The journal European Food Research and Technology publishes state-of-the-art research papers and review articles on fundamental and applied food research. The journal''s mission is the fast publication of high quality papers on front-line research, newest techniques and on developing trends in the following sections: -chemistry and biochemistry- technology and molecular biotechnology- nutritional chemistry and toxicology- analytical and sensory methodologies- food physics. Out of the scope of the journal are: - contributions which are not of international interest or do not have a substantial impact on food sciences, - submissions which comprise merely data collections, based on the use of routine analytical or bacteriological methods, - contributions reporting biological or functional effects without profound chemical and/or physical structure characterization of the compound(s) under research.