32 JUNE 2024 • WORLD AQUACULTURE • WWW.WAS.ORG variability presents a notable challenge when attempting to establish uniform diagnostic performance criteria, particularly for specific sample types. Additionally, distinguishing between active infection and asymptomatic colonization is challenging with the HRPzymeintegrated PCR-based platform, as it does not differentiate between live and dead cells. Therefore, it is crucial to consider these limitations when using this method as a diagnostic tool and to employ complementary approaches as needed (Vuong et al. 2022; Lee et al. 2020). Comparison with existing methods and traditional culture-based methods Given the diverse impacts of foodborne pathogens on public health, ensuring the safety of food products is a primary concern during the harvesting phase. Accurate, sensitive, fast, and straightforward molecular biology-based techniques are essential for detecting these pathogens, with primer specificity playing a critical role (Lee et al. 2022; Ahmad et al. 2021). Our HRPzyme-based platform significantly reduces detection time from days to hours, allowing for timely intervention and mitigation of foodborne illness outbreaks. Furthermore, the cost of reagents and equipment for our method is substantially lower than that of advanced molecular techniques like next-generation sequencing or ELISA, making it a cost-effective solution for routine screening and surveillance. By combining the sensitivity of PCR amplification with the simplicity of colorimetric detection, our platform offers a versatile and scalable approach for detecting V. parahaemolyticus in various sample matrices, with the potential for widespread adoption in both laboratory and field settings. Future Directions While our study demonstrates the feasibility and efficacy of the HRPzyme-integrated PCR-based platform for detecting V. parahaemolyticus, several avenues for future research exist. FIGURE 5. (a-f). Colorimetric HRPzyme-integrated PCR assay results of Vibrio parahaemolyticus on Blue Crab. (a) Isolating hemolymph from the Blue Crab. (b, f) Colorimetric HRPzyme-integrated PCR results of different concentrations of V. parahaemolyticus. (c, e) Molecular confirmation of the presence of V. parahaemolyticus using gel electrophoresis data of different concentrations of V. parahaemolyticus. (d) RT-PCR confirmation of the presence of V. parahaemolyticus. While our study demonstrates the feasibility and efficacy of the HRPzyme-integrated PCR-based platform for detecting V. parahaemolyticus, several avenues for future research exist. Further validation of the assay using a larger sample size and diverse geographical locations would enhance its applicability and robustness.
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