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ARS Home » Southeast Area » Stoneville, Mississippi » Warmwater Aquaculture Research Unit » Research » Publications at this Location » Publication #427801
Research Project: Umbrella Project for Food Safety

Location: Warmwater Aquaculture Research Unit

Title: Dual Detection of Pathogenic tdh and trh Genes of Vibrio parahaemolyticus in Oysters Using Multienzyme Isothermal Rapid Amplification (MIRA) Combined with Lateral-Flow Dipstick (LFD) Assay

Author
item PARK, SEONG - Mississippi State University
item CHANG, SAM - Mississippi State University
item BI, LIN - Mississippi State University
item CHA, YUNIM - Mississippi State University
item ZHANG, YAN - Mississippi State University

Submitted to: Microbiology Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/20/2025
Publication Date: 4/22/2025
Citation: Park, S.B. 2025. Dual Detection of Pathogenic tdh and trh Genes of Vibrio parahaemolyticus in Oysters Using Multienzyme Isothermal Rapid Amplification (MIRA) Combined with Lateral-Flow Dipstick (LFD) Assay. Microbiology Research. 16(5)87. https://doi.org/10.3390/microbiolres16050087.
DOI: https://doi.org/10.3390/microbiolres16050087

Interpretive Summary: Vibrio parahaemolyticus is a common foodborne bacterium linked to seafood, especially raw oysters, and can cause serious illness. Detecting whether this bacterium is harmful typically requires laboratory-based PCR methods, which are not always feasible in field settings or resource-limited environments. This study presents a new rapid detection method that combines multienzyme isothermal rapid amplification (MIRA) with a simple lateral flow dipstick (LFD) test. The new method can detect two key pathogenic genes (tdh and trh) in just 20 minutes at 40°C, using easily visible results on a test strip—similar to a home pregnancy test. It proved highly sensitive, detecting extremely small amounts of bacterial DNA, and highly specific, with no false positives from other Vibrio or food-borne bacteria. Because of its speed, accuracy, and ease of use, this test is a promising tool for monitoring seafood safety in the field, helping protect public health and supporting the seafood industry.

Technical Abstract: Vibrio parahaemolyticus is a foodborne pathogen commonly associated with the consumption of contaminated seafood, particularly oysters. While PCR and real-time PCR are widely used to detect its pathogenicity through tdh and trh gene detection, these methods may not be practical in resource-limited settings such as field environments. To address this limitation, a rapid, sensitive, and specific duplex detection method was developed using the multienzyme isothermal rapid amplification (MIRA) assay in combination with lateral flow dipstick (LFD) technology. The assay utilized specific primer sets and probes to simultaneously amplify tdh and trh fragments tagged with 3'-FAM and 5'-Digoxigenin or Biotin during MIRA amplification, enabling the detection via respective antibody capture on the LFD strip. This duplex MIRA-LFD assay demonstrated a detection limit of 100 fg of DNA, 300 CFU/reaction for bacterial culture, and 3000 CFU/reaction for seeded oyster samples at 40 'C within 20 min. Notably, the assay exhibited no cross-reactivity with nine other Vibrio species or 18 foodborne pathogens, confirming its high specificity. Due to its simplicity, rapid turnaround time, and high sensitivity, this duplex MIRA-LFD assay offers a valuable tool for the surveillance of V. parahaemolyticus pathogenicity, aiding in public health protection and supporting the local seafood industry.