An Integrated PMA Pretreatment Instrument for Simultaneous Quantitative Detection of Vibrio parahaemolyticus and Vibrio cholerae in Aquatic Products

Traditional hazard identification techniques for <i>Vibrio parahaemolyticus</i> 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 <i>V. parahaemolyticus</i> and <i>V. cholerae</i> 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 10¹-10² CFU/mL in shrimp farming environment water and 10²-10³ CFU/g in shrimp (<i>Litopenaeus vannamei</i>) and oyster (<i>Crassostrea gigas</i>). Notably, it effectively discriminated viable bacteria from 10⁶ 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.