Wisuthiphaet Nicharee, Yang Xu, Young Glenn M, Nitin Nitin
Department of Food Science and Technology, University of California, Davis, Davis, CA, United States.
Nutrition and Food Science Department, California State Polytechnic University Pomona, Pomona, CA, United States.
Front Microbiol. 2022 Mar 4;13:853048. doi: 10.3389/fmicb.2022.853048. eCollection 2022.
Rapid detection of bacteria in water and food samples is a critical need. The current molecular methods like real-time PCR can provide rapid detection after initial enrichment. However, these methods require significant preparation steps, specialized facilities to reduce contamination, and relatively expensive reagents. This study evaluates a novel approach for detecting bacteria based on imaging of bacteriophage amplification upon infection of the target host bacteria to mitigate some of these constraints and improve the specificity of discriminating live vs. dead bacteria. Thus, this research leverages the natural ability of lytic bacteriophages to rapidly amplify their genetic material and generate progeny phages upon infecting the host bacterium. This study uses a nucleic acid staining dye, a conventional fluorescence microscope, and quantitative image analysis for imaging the amplification of bacteriophages. The sensitivity and assay time for imaging-based quantification of phage amplification for detecting were compared with RT-PCR and the standard plaque-forming assay for detection phage amplification in model systems, including coconut water and spinach wash water. The results demonstrate that the imaging approach matches both the sensitivity and speed for detecting using the RT-PCR method without requiring isolation of nucleic acids, expensive reagents, and specialized facilities. The quantitative imaging results demonstrate the detection of 10 CFU/ml of in coconut water and simulated spinach wash water with a chemical oxygen demand (COD) of 3,000 ppm within 8 h, including initial enrichment of the bacteria. In summary, the results of this study illustrate a novel phage amplification-based approach for detecting target bacteria in complex food and water samples using simple sample preparation methods and low-cost reagents.
快速检测水和食品样本中的细菌是一项迫切需求。当前的分子方法，如实时光聚合酶链式反应（real-time PCR），在初始富集后可实现快速检测。然而，这些方法需要大量的准备步骤、用于减少污染的专业设施以及相对昂贵的试剂。本研究评估了一种基于噬菌体感染靶宿主细菌后扩增成像来检测细菌的新方法，以减轻其中一些限制并提高区分活细菌与死细菌的特异性。因此，本研究利用裂解性噬菌体在感染宿主细菌后迅速扩增其遗传物质并产生子代噬菌体的天然能力。本研究使用一种核酸染色染料、一台传统荧光显微镜以及定量图像分析来对噬菌体的扩增进行成像。在包括椰子水和菠菜清洗水在内的模型系统中，将基于成像的噬菌体扩增定量检测的灵敏度和检测时间与逆转录聚合酶链式反应（RT-PCR）以及用于检测噬菌体扩增的标准噬菌斑形成试验进行了比较。结果表明，该成像方法在无需核酸分离、昂贵试剂和专业设施的情况下，在检测[具体细菌名称未给出]方面与RT-PCR方法的灵敏度和速度相当。定量成像结果表明，在8小时内，包括细菌的初始富集，能够检测出椰子水和化学需氧量（COD）为3000 ppm的模拟菠菜清洗水中每毫升含10个菌落形成单位（CFU/ml）的[具体细菌名称未给出]。总之，本研究结果说明了一种基于噬菌体扩增的新方法，该方法使用简单的样品制备方法和低成本试剂来检测复杂食品和水样本中的靶细菌。
