Development and Application of Four Foodborne Pathogens by TaqMan Multiplex Real-Time PCR.

IF 1.9 2区农林科学 Q3 FOOD SCIENCE & TECHNOLOGY

Foodborne pathogens and disease Pub Date : 2024-04-02 DOI:10.1089/fpd.2023.0134

Yinlei Xue, Shengfang He, Meng Li, Yuanhao Qiu

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Abstract

A TaqMan multiplex real-time PCR (mRT-PCR) was developed to detect simultaneously Salmonella spp., Escherichia coli O157, Staphylococcus aureus, and Listeria monocytogenes in food samples. The method involves four sets of primers and probes tailored to the unique DNA sequences found in the invA, nuc, rfbE, and hly genes of each pathogen. The generated standard curves, correlating gene copy numbers with Ct values, demonstrated high accuracy (R² > 0.99) and efficiency (92%-104%). Meanwhile, the limit of detection was 10⁰ CFU/mL for the four target bacteria in artificially contaminated food samples after 6-8 h of enrichment. The assay's effectiveness was further verified by testing 80 naturally contaminated food samples, showing results largely in agreement with traditional culture methods. Overall, this newly developed TaqMan mRT-PCR, inclusive of a pre-enrichment step, proves to be a dependable and effective tool for detecting single or multiple pathogens in diverse food items, offering significant potential for in vitro diagnostics.

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利用 TaqMan 多重实时 PCR 技术开发和应用四种食源性病原体。

本研究开发了一种 TaqMan 多重实时 PCR（mRT-PCR）方法，用于同时检测食品样本中的沙门氏菌属、大肠杆菌 O157、金黄色葡萄球菌和单核细胞增生李斯特菌。该方法包括四组引物和探针，分别针对每种病原体的 invA、nuc、rfbE 和 hly 基因中的独特 DNA 序列。生成的标准曲线将基因拷贝数与 Ct 值相关联，显示出较高的准确度（R2 > 0.99）和效率（92%-104%）。同时，在富集 6-8 小时后，人工污染食品样本中四种目标细菌的检测限为 100 CFU/mL。通过检测 80 份自然污染的食品样本进一步验证了该检测方法的有效性，结果与传统培养方法基本一致。总之，这种新开发的包含预富集步骤的 TaqMan mRT-PCR 被证明是检测各种食品中单一或多种病原体的可靠而有效的工具，为体外诊断提供了巨大的潜力。

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来源期刊

Foodborne pathogens and disease 医学-食品科技

CiteScore

5.30

自引率

3.60%

发文量

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.