A microfluidic based stem-loop probes mediated isothermal amplification for multiplex detection of Vibrio...

Area of Science:

• Microfluidics
• Molecular Biology
• Food Safety

Background:

• Vibrio parahaemolyticus (VP) presents significant public health concerns.
• Existing VP detection methods lack rapid, on-site capabilities.
• There is a critical need for sensitive and specific VP detection techniques.

Purpose of the Study:

• To develop a novel, rapid, sensitive, and highly specific method for VP detection.
• To specifically detect VP and differentiate strains containing the TDH toxin gene.
• To enable simultaneous, high-throughput detection of key VP genes.

Main Methods:

• Development of a microfluidic-based stem-loop probe-mediated isothermal amplification (MSPA) method.
• Simultaneous detection of toxR, tlh, and tdh genes on a single chip within 1 hour.
• Testing MSPA with plasmid DNA, pure bacterial cultures, and artificially contaminated shrimp samples.

Main Results:

• MSPA achieved detection limits as low as 2.43 × 10^2 copies/μL for plasmid DNA and 6.5 × 10^1 CFU/μL for bacteria.
• The method demonstrated 100% specificity and excellent reproducibility (CV < 5%).
• Successful detection in artificially contaminated shrimp and 31 seafood samples, with sensitivity comparable to qPCR.

Conclusions:

• The MSPA method provides rapid, sensitive, and specific detection of Vibrio parahaemolyticus.
• MSPA can differentiate TDH-containing VP strains, crucial for risk assessment.
• This technique shows significant potential for on-site screening of pathogenic VP in seafood.