Segmented continuous-flow multiplex polymerase chain reaction microfluidics for high-throughput and rapid foodborne...

Area of Science:

• Microfluidics
• Molecular Biology
• Food Safety

Background:

• Accurate and rapid identification of foodborne bacterial pathogens is critical for public health and the food industry.
• Existing methods often lack the high-throughput capability required for comprehensive food safety monitoring.

Purpose of the Study:

• To develop and validate a segmented continuous-flow multiplex polymerase chain reaction (SCF-MPCR) system on a microfluidic device for rapid pathogen identification.
• To achieve high-throughput, simultaneous detection of multiple foodborne pathogens with high sensitivity and minimal cross-contamination.

Main Methods:

• A spiral-channel microfluidic device made of polytetrafluoroethylene (PTFE) was designed, incorporating segmented flow regimes for sequential multiplex PCR.
• Key reaction parameters (annealing temperatures, flow rates, polymerase concentration, DNA input) were optimized for continuous-flow multiplex PCR.
• The device was tested for amplifying genomic DNA from Salmonella enterica, Listeria monocytogenes, Escherichia coli O157:H7, and Staphylococcus aureus.

Main Results:

• Optimized SCF-MPCR on the microfluidic device successfully amplified four target bacterial pathogens simultaneously in 19 minutes.
• The limit of detection was as low as 10(2) copies/μL, demonstrating high sensitivity.
• Application to real food samples (banana, milk, sausage) showed a throughput of 4x3 with no detectable cross-contamination.

Conclusions:

• The spiral-channel SCF-MPCR microfluidic device offers a rapid and high-throughput solution for multiplex pathogen detection.
• This technology has significant potential for enhancing food safety by enabling quick and accurate identification of multiple foodborne pathogens directly from food matrices.