WWW.WAS.ORG • WORLD AQUACULTURE • JUNE 2024 31 demonstrated high sensitivity and specificity, with the colorimetric signal showing a strong correlation with V. parahaemolyticus concentration (Figure 3). In this regard, the specificity test was performed against different pathogenic microorganisms, including K. pneumoniae, E. coli, P. aeruginosa, S. aureus, S. algae, Vibrio cholerae, and V. parahaemolyticus. To confirm the results of the colorimetric assay, we also utilized gel electrophoresis (2%). Evaluation of the HRPzyme-Integrated PCR-Based Colorimetric Platform for Aquaculture Pathogen Detection To evaluate the applicability and reliability of our proposed HRPzyme-integrated PCR-based platform for detecting Vibrio parahaemolyticus in Eastern oysters (Crassostrea virginica) and blue crabs (Callinectes sapidus), we collected samples from the muscle, heart, pericardial cavity, gonad area, and gills of C. virginica. The positive colorimetric signals obtained were compared with results from gel electrophoresis and RT-PCR methods for validation. As illustrated in Figure 4, a distinct colorimetric signal (OD410 = 0.43 ± 0.02) was evident in the organs tested, indicating contamination with V. parahaemolyticus in the C. virginica samples. These findings were further validated through PCR and RT-PCR. Specifically, no amplified bands were observed in the negative agarose gel electrophoresis of the V. parahaemolyticus samples, while a specific band of the correct size was consistently obtained in the positive samples. For the assessment of C. sapidus, hemolymph from both V. parahaemolyticus-positive and -negative samples was extracted and compared. As shown in Figure 5, the colorimetric signal generated by our HRPzyme-integrated PCR-based platform was distinctly observed in the V. parahaemolyticus-positive samples (OD410 = 0.5 ± 0.03). These results were consistently supported by gel electrophoresis and RT-PCR analysis, providing parallel confirmation. These findings strongly endorse the specificity of the HRPzyme-integrated PCR assay for detecting V. parahaemolyticus, making it a viable method for identifying this pathogen in seafood products. In this study, we suggest targeting a specific region on the tlh gene for detecting V. parahaemolyticus using the HRPzymeintegrated PCR assay. Compared to other available methods, such as gold nanoparticle-based enzyme-linked antibody assays, magnetic nanoparticles, CE-RAA-CRISPR assays, and Duplex droplet digital PCR, this proposed method has the potential to be faster and more cost-effective. However, it is essential to consider that the quantity of targeted DNA can vary significantly among different samples. This FIGURE 4. (a-f). Colorimetric HRPzyme-Integrated PCR Assay Results for Vibrio parahaemolyticus in Oysters. (a) Illustration of tested parts in oyster (1) adductor muscle, (2) heart, (3) pericardial cavity, (4) gonad area, and (5) gills. (b, f) Colorimetric HRPzyme-integrated PCR assay results showing different concentrations of V. parahaemolyticus. (c, e) Gel electrophoresis confirming the presence of V. parahaemolyticus at varying concentrations. (d) RT-PCR validation confirming the presence of V. parahaemolyticus. (CONTINUED ON PAGE 32)
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