A fully sealed plastic chip for multiplex PCR and its application in bacteria identification

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostics

Background:

• Multiplex PCR enables simultaneous detection of multiple DNA targets.
• Traditional multiplex PCR faces challenges due to primer interference and competition.
• Dividing multiplex PCR into single reactions is a viable strategy to mitigate these issues.

Purpose of the Study:

• To develop a novel, user-friendly, and fully sealed multiplex PCR chip.
• To enable simultaneous detection of a large number of DNA targets (63).
• To overcome limitations of traditional multiplex PCR, such as primer interference.

Main Methods:

• A plastic chip with sine-shaped infusing channels and reaction chambers was constructed.
• Primer pairs were preloaded into reaction chambers; PCR master mix and DNA template were introduced via centrifugation.
• Reversible centrifugation was used for sample loading and product recovery, with air filling channels to prevent cross-contamination.

Main Results:

• The chip successfully enabled simultaneous detection of 63 DNA targets.
• Demonstrated utility in identifying 8 pathogenic bacterial genomic DNA samples and 13 drug-resistance genes.
• The chip functions for both in situ fluorescence detection and as a sample preparation unit.

Conclusions:

• The developed multiplex PCR chip offers a simple, accurate, and versatile solution for multi-target DNA detection.
• Its high-throughput capacity and dual functionality make it suitable for clinical diagnostics and point-of-care testing.
• This technology has significant potential to advance nucleic acid-based testing applications.