30 JUNE 2024 • WORLD AQUACULTURE • WWW.WAS.ORG To evaluate the sensitivity test of the HRPzyme PCR basedColorimetric assay (OD = 410 nm) we used a concentration range of V. parahaemolyticus spanning from 10 0 to 10 7cfu mL-1. Subsequently, we compared these results with those obtained from the gel electrophoresis-based assay. To evaluate the sensitivity of the assay, a series of V. parahaemolyticus dilutions (ranging from 0 to 107 cfu mL-1) were prepared in 1x PBS buffer. The amplification of the targeted tlh gene region was carried out using a PCR thermocycler, and the success of the PCR amplification was confirmed through agarose gel electrophoresis to eliminate the possibility of false-positive signals. As illustrated in Figure 2, the band intensities observed in the gel electrophoresis directly correlated with the concentration of V. parahaemolyticus. Additionally, the colorimetric signals displayed an inverse relationship with the concentration of the tested V. parahaemolyticus, allowing for clear differentiation. The assay FIGURE 2. (a-f). Molecular confirmation and sensitivity test for detection of Vibrio parahaemolyticus using HRPzyme-Integrated PCR assay. (a) Molecular confirmation of V. parahaemolyticus using 16s rRNA. (b) Molecular confirmation of V. parahaemolyticus using specific primer for detection of thermolabile hemolysin (tlh) gene. (c) Confirmation of V. parahaemolyticus using RT-PCR. (d) Colorimetric HRPzyme-integrated PCR results of different concentration of V. parahaemolyticus. (e) Gel electrophoresis data of different concentration of V. parahaemolyticus. (f) Absorbance data of colorimetric HRPzymeintegrated PCR assay at different concentration of bacteria. FIGURE 3. (a and b). Specificity test of Colorimetric HRPzyme-integrated PCR assay results of Vibrio parahaemolyticus. (a) Gel electrophoresis data on the specificity test of V. parahaemolyticus against tested pathogens. (b) Colorimetric HRPzyme-integrated PCR results. Note: The samples were run at 60 °C for a duration of 60 min and read at the OD = 410 nm. A 100 bp ladder was used for molecular confirmation.
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