Establishment and Validation of a Rapid ERA Detection Method for Vibrio parahaemolyticus in Exported Aquatic Products.

Abstract

To address the issues of operational complexity, long duration association, and reliance on specialized equipment with existing detection methods for Vibrio parahaemolyticus, this study established a rapid detection method for V. parahaemolyticus in exported aquatic products based on the domestically developed Enzymatic Recombinase Amplification (ERA) technology. To target the thermolabile hemolysin gene (tlh) and the iron-regulated virulence regulatory protein gene (irgB) of V. parahaemolyticus, highly specific ERA primers and probes were designed and screened. Two detection platforms, a colorimetric method and a fluorescent method, were developed. Method validation results showed that this detection system achieved specific amplification for all 30 tested V. parahaemolyticus strains, with no cross-reactivity observed with 30 other common foodborne pathogenic bacteria. The detection sensitivity for both the fluorescent and colorimetric methods reached 10-1 ng/μL, with a minimum detection limit of 10 CFU/25 g for artificially contaminated samples. The entire detection process, including sample preparation, requires only approximately 20 min-significantly faster than traditional culture (24-72 h) or even conventional PCR methods. Collaborative validation across five independent laboratories confirmed excellent reproducibility, with inter-laboratory agreement yielding a Kappa coefficient of 0.98. The ERA method operates at a low, constant temperature (37-39 °C), eliminating the need for thermal cyclers. When combined with portable isothermal amplification devices and visual (colorimetric) readout, it offers a distinct advantage in terms of speed, cost-effectiveness, and suitability for resource-limited or field settings compared to existing PCR-based or culture-based platforms. This method is simple to operate, rapid, sensitive, and highly suitable for on-site application, providing a reliable and practical technical solution for the rapid screening and ris