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

• Published in: Electrophoresis Vol. 37; no. 3; pp. 545 - 552
• Main Authors: Liu, Yu, Li, Chen, Li, Zhi, Chan, Samuel D, Eto, Daisuke, Wu, Warren, Zhang, Jian Ping, Chien, Ring‐Ling, Wada, Henry G, Greenstein, Michael, Satomura, Shinji
• Format: Journal Article
• Language: English
• Published: Germany Verlag Chemie 01.02.2016; Blackwell Publishing Ltd
• Subjects: Algorithms; Assaying; bacteriophages; capillary electrophoresis; Design analysis; disease detection; DNA; DNA - analysis; DNA - chemistry; DNA - genetics; Electrophoresis; Electrophoresis, Capillary - instrumentation; Electrophoresis, Capillary - methods; Equipment Design; Escherichia coli; Infectious diseases; Linearity; Microfluidic Analytical Techniques - instrumentation; Microfluidic Analytical Techniques - methods; Microfluidic chip; MicroTAS; Monitoring; PCR; Polymerase Chain Reaction - instrumentation; Polymerase Chain Reaction - methods; quantitative polymerase chain reaction; Real time; Reproduction; MicroTAS; Microfluidic chip; CE; PCR
• ISSN: 0173-0835; 1522-2683
• DOI: 10.1002/elps.201500298

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 × 10⁴ 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.