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水产常见食源性病原菌三重LAMP快速检测方法的建立及应用

Establishment and application of a triple LAMP assay for rapid detection of three major foodborne pathogens in aquatic products

  • 摘要: 以副溶血弧菌、创伤弧菌和单核细胞增生李斯特氏菌3种病原的特异性基因为靶标,利用环介导等温扩增技术(loop-mediated isothermal amplification, LAMP),建立了一种三重可视化LAMP检测方法。该方法在58 ℃恒温条件下30 min即可完成检测,灵敏度达100 fg·µL−1,并能特异性区分其他常见病原菌。通过颜色变化判读结果,结合酶切反应可准确鉴定病原种类。使用该方法随机对市场上20份牡蛎和20份贻贝样品进行检测。结果表明:对40份贝类样本的副溶血弧菌、创伤弧菌和单核细胞增生李斯特氏菌3种病原检出率达100%,显著高于三重PCR法对3种病原的检出率(72.5%~90.0%)。研究结果可为水产品安全监测提供高效初筛工具。

     

    Abstract: In the report, the specific genes of Vibrio parahaemolyticus, V. vulnificus, and Listeria monocytogenes were used as the targets, a triple visual Loop-mediated isothermal amplification (LAMP) assay was developed, the method performed the rapid detection at a constant temperature of 58 ℃ within 30 min, with a sensitivity of 100 fg·µL−1, and demonstrated the high specificity by distinguishing these pathogens from other common bacteria. The results can be visually interpreted based on color changes, and the pathogen identification was further confirmed by restriction enzyme digestion. When applied to 40 randomly collected shellfish samples (20 oysters and 20 mussels), the assay achieved a 100% detection rate, significantly outperforming triple PCR (72.5%-90.0%). In conclusion, a rapid, sensitive, and specific triple visual LAMP assay for detecting V. parahaemolyticus, V. vulnificus, and L. monocytogenes was successfully established, which provide an efficient screening tool for aquatic product safety monitoring.

     

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