28 JUNE 2024 • WORLD AQUACULTURE • WWW.WAS.ORG of culture-based approaches in pathogen detection. In this context, virulence genes (Vir genes) have emerged as crucial molecular markers for pathogen detection. In V. parahaemolyticus, numerous Vir genes, including tlh (thermolabile hemolysin), tdh (thermostable direct hemolysin), and trh (tdh-related hemolysin), have been identified and sequenced from isolated strains (Paria et al. 2021). These genes have been targeted for molecular detection using various clinical and laboratory assays, such as polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), and enzyme-linked immunosorbent assay (ELISA) (Alarcón Elvira et al. 2020). Recently, a novel approach has been introduced for colorimetric analysis of PCR products. This method involves the addition of horseradish peroxidase — mimicking DNAzymes (HRPzyme) at the 5’ end of the primers. HRPzymes facilitate sequence-specific hybridization, forming G-quadruplex nucleotide sequences that mimic peroxidase activity upon binding to hemin (Hanyue et al. 2023). This mimicry allows for visual detection when the DNAzymes interact with molecules like luminol, 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and 3,3’,5,5’-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2) (Garrido-Maestu et al. 2022). The short HRPzyme sequence (GGGTAGGGCGGGTTGGGT) is designed to allow primer integration in such a way that the functional HRPzyme sequence forms only after the successful amplification of the target genomic region (Ahmad et al. 2021). In this study, following the extraction of DNA and eDNA samples from the targeted sites (Figure 1, Photo 1), one-step PCR In our interconnected world, food security, safety, and the outbreak of foodborne illnesses are major concerns. According to the World Health Organization (WHO), an estimated 600 million cases of foodborne illness and 420,000 fatalities occur annually due to contaminated food products. Vibrio parahaemolyticus, a Gram-negative bacterium, significantly contributes to this global health burden (WHO 2022). Found in marine and estuarine environments, this bacterium is often isolated from seafood products and is a known cause of acute gastroenteritis in humans consuming raw or undercooked seafood. Traditional culture-based methods for detecting Vibrio species are often slow and lack specificity. Modern molecular techniques, including 16S rRNA sequencing, ELISA, isotyping, and antisera, offer improved accuracy but are often costly and complex (Cao et al. 2022). The need for cost-effective and rapid detection methods is pressing, especially in resource-poor areas. Here, we introduce a novel colorimetric detection platform for Vibrio parahaemolyticus, leveraging HRPzyme-integrated primers that facilitate a sensitive, rapid, and cost-effective diagnostic process. This innovative approach combines the specificity of PCR amplification with the simplicity of colorimetric detection, offering a promising solution for addressing the challenges associated with Vibrio detection in seafood and environmental samples. HRPzyme-Integrated PCR-Based Platform Molecular techniques, such as 16S rRNA, ELISA, isotyping, and antisera are gaining popularity to overcome the limitations Advancing Aquaculture Safety: HRPzyme-based Detection of Vibrio parahaemolyticus Ali Parsaeimehr and Gulnihal Ozbay PHOTO 1. (a and b). The aquaculture sites where samples were taken. In our interconnected world, food security, safety, and the outbreak of foodborne illnesses are major concerns. According to the World Health Organization (WHO), an estimated 600 million cases of foodborne illness and 420,000 fatalities occur annually due to contaminated food products. Vibrio parahaemolyticus, a Gram-negative bacterium, significantly contributes to this global health burden.
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