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Updated: Jul 21, 2025
Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species
Published on: November 8, 2016
Multiplex PCR-Lateral Flow Dipstick Method for Detection of Thermostable Direct Hemolysin (TDH) Producing V.
Jirakrit Saetang1, Phutthipong Sukkapat1, Suriya Palamae1
1International Center of Excellence in Seafood Science and Innovation, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai 90110, Songkhla, Thailand.
Abstract:
Vibrio parahaemolyticus is usually found in seafood and causes acute gastroenteritis in humans. Therefore, a detection method of pathogenic V. parahaemolyticus is necessary. Multiplex PCR combined with lateral flow dipstick (LFD) assay was developed to detect pathogenic V. parahaemolyticus. Biotin-, FAM-, and Dig-conjugated primers targeting thermolabile hemolysin (TLH) and thermostable direct hemolysin (TDH) genes were used for multiplex PCR amplification. The condition of the method was optimized and evaluated by agarose gel electrophoresis and universal lateral flow dipstick. The specificity assay was evaluated using strains belonging to seven foodborne pathogen species. The sensitivity of the method was also evaluated using DNA in the concentration range of 0.39-100 ng/reaction. The artificial spiking experiment was performed using 10 g of shrimp samples with an enrichment time of 0, 4, and 8 h with 101, 102, and 103 CFU of V. parahaemolyticus. The developed multiplex PCR-LFD assay showed no non-specific amplification with a limit of the detection of 0.78 ng DNA/reaction visualized by agarose gel electrophoresis and 0.39 ng DNA with LFD assay. The artificial spiking experiment demonstrated that this method could detect pathogenic V. parahaemolyticus at 10 CFU/10 g shrimp samples following a 4 h of enrichment. Multiplex PCR-LFD assay was therefore established for detecting pathogenic V. parahaemolyticus with high sensitivity and specificity and might be a useful tool to develop a detection kit used in the food safety sector.
Insights
A new multiplex PCR combined with lateral flow dipstick (LFD) assay effectively detects pathogenic Vibrio parahaemolyticus in seafood. This rapid method offers high sensitivity and specificity for improved food safety.
Area of Science:
- Food Microbiology
- Molecular Diagnostics
- Public Health
Background:
- Vibrio parahaemolyticus is a common cause of acute gastroenteritis from seafood consumption.
- Accurate and rapid detection methods for pathogenic V. parahaemolyticus are crucial for food safety.
- Existing methods may lack the necessary sensitivity or speed for effective screening.
Purpose of the Study:
- To develop and validate a multiplex PCR combined with lateral flow dipstick (LFD) assay.
- To detect pathogenic Vibrio parahaemolyticus strains targeting key virulence genes.
- To establish a sensitive, specific, and rapid diagnostic tool for food safety applications.
Main Methods:
- Developed a multiplex PCR assay using biotin-, FAM-, and Dig-conjugated primers targeting thermolabile hemolysin (TLH) and thermostable direct hemolysin (TDH) genes.
- Optimized reaction conditions and evaluated performance using agarose gel electrophoresis and universal lateral flow dipstick.
- Assessed specificity against seven foodborne pathogens and sensitivity using serial dilutions of target DNA; validated with spiked shrimp samples.
Main Results:
- The multiplex PCR-LFD assay demonstrated high specificity with no cross-reactivity.
- Limit of detection was 0.78 ng DNA/reaction by gel electrophoresis and 0.39 ng DNA by LFD assay.
- Successfully detected pathogenic V. parahaemolyticus at 10 CFU/10 g shrimp samples after 4 hours of enrichment.
Conclusions:
- The developed multiplex PCR-LFD assay is a highly sensitive and specific method for detecting pathogenic V. parahaemolyticus.
- This assay provides a rapid and reliable tool for pathogen detection in seafood.
- It holds potential for development into a commercial detection kit for the food safety sector.
