Qin Yulong, Xiong Rongrong, Zhao Yong, Zhang Zhaohuan, Yin Yachang
College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China.
International Research Center for Food and Health, Shanghai Ocean University, Shanghai 201306, China.
Foods. 2025 Jun 21;14(13):2166. doi: 10.3390/foods14132166.
Traditional hazard identification techniques for often neglect the distinction between viable and nonviable bacteria in aquatic products, leading to overestimated disease risks and uncertainties in risk assessments. To address this limitation, we developed an automated PMA pretreatment instrument that integrates dark incubation and photo-crosslinking into a unified workflow, allowing customizable parameters such as incubation time, light exposure duration, and mixing speed while maintaining stable temperatures (<±1 °C fluctuation) to preserve bacterial DNA integrity. Leveraging this system, a duplex qPCR assay was optimized for simultaneous quantitative detection of and in aquatic products and environmental samples. The assay demonstrated robust performance with 90-110% amplification efficiencies across diverse matrices, achieving low limits of detection (LODs) of 10-10 CFU/mL in shrimp farming environment water and 10-10 CFU/g in shrimp () and oyster (). Notably, it effectively discriminated viable bacteria from 10 CFU/mL(g) nonviable cells and showed strong correlation with ISO-standard methods in real-world sample validation. This integrated platform offers a rapid, automated solution for accurate viable bacterial quantification, with significant implications for food safety, pathogen surveillance, and risk management in aquatic industries.
传统的水产品危害识别技术往往忽视了活菌与死菌之间的区别,导致疾病风险被高估,风险评估存在不确定性。为解决这一局限性,我们开发了一种自动化的PMA预处理仪器,该仪器将暗培养和光交联整合到一个统一的工作流程中,允许设置诸如培养时间、光照持续时间和混合速度等可定制参数,同时保持稳定的温度(波动<±1°C)以保持细菌DNA的完整性。利用该系统,优化了一种双重qPCR检测方法,用于同时定量检测水产品和环境样品中的 和 。该检测方法在不同基质中表现出强大的性能,扩增效率为90 - 110%,在对虾养殖环境水中的检测限低至10⁻¹⁰ CFU/mL,在对虾( )和牡蛎( )中的检测限低至10⁻¹⁰ CFU/g。值得注意的是,它能有效区分10 CFU/mL(g)的死菌中的活菌,并且在实际样品验证中与ISO标准方法显示出很强的相关性。这个集成平台为准确的活菌定量提供了一种快速、自动化的解决方案,对水产行业的食品安全、病原体监测和风险管理具有重要意义。