A chip-based universal strategy to realize multiplex PCR by using wax films for sealing and controllable release of primers

Simultaneous detection of multiple nucleic acid targets from a single sample is a common requirement in molecular diagnosis and basic research. Dividing a bulky polymerase chain reaction (PCR) into many isolated small reaction units through microfluidic technology is commonly used to realize this goal. However, previous microfluidic platforms for multiplex PCR suffer from complex structures and strict operation requirements. In this study, a chip-based universal strategy is constructed to realize multiplex PCR by using wax films for sealing and controllable release of prespotted primers. This microfluidic chip contains twenty-four reaction chambers connected in series by a single channel, and the bottom of each reaction chamber is covered by a wax film generated by solvent volatilization. These wax films can prevent the prespotted primers from being flushed away by the reaction mixture during the injection process. After thermal blocking of the connecting channel to isolate each reaction chamber, the chip is placed on a flat thermal cycler. The wax films melt during the denaturing step of PCR so that the primers are released and mixed with the reaction mixture, a process seamlessly compatible with PCR thermal cycling. After fluid flow simulation and carefully examining its basic performance, this chip was applied to genotype seven deafness-associated hotspot mutations using a competitive allele-specific PCR assay. The genotyping results of clinical samples using this chip were totally concordant with those obtained by Sanger sequencing, demonstrating the practical utility of this universal strategy for multiplex PCR detection.