On-chip quantitative PCR using integrated real-time detection by capillary electrophoresis

Abstract

Quantitative PCR (qPCR) has been widely used for the detection and monitoring of a variety of infectious diseases. PCR and CE were integrated into a microfluidic chip that was designed to achieve rapid real-time amplicon sampling, separation, and quantitation without requiring various probes. A novel chip design allows the overlapped execution of PCR and CE, minimizing the time required for CE analysis after each PCR cycle. The performance of the on-chip qPCR method was demonstrated using a 45-minutes model assay protocol for the phiX174 bacteriophage, and the multiplexing capability of the method was demonstrated by adding a second target, E. coli genomic DNA, to the model assay. The results indicate good sensitivity, reproducibility, and linearity over the tested assay range, 50 to 2 × 10(4) copies/25 μL reaction. Based on this performance, the on-chip qPCR method should be applicable to a wide variety of infectious disease detection and monitoring assays with the addition of suitable sample preparation protocols.

Keywords: CE; MicroTAS; Microfluidic chip; PCR.