A Study on the Electrochemical Real-Time Polymerase Chain Reaction System：Application to Detection of Mycobacterium tuberculosis IS6110 Target Sequence

This work aims at developing a real-time PCR (RT-PCR) sensor based on the electrochemical method, and the ultimate goal is to provide an alternative to the application of optical RT-PCR system. Although the optical system has been used generally, but at this moment the technique is limited to the usage of laboratory due to its large size. Since the electrochemical system is based on small components and electric analytical methods, it is more suitable for the development for portable Q-PCR than the optical system. This study focus on four parts as listed below: (1) The effect of redox mediator addition on PCR; (2) electrochemical PCR system utilizing potassium ferricyanide; (3) electrochemical PCR system utilizing methylene blue; (4) detection of Mycobacterium tuberculosis IS6110 target sequence using the proposed electrochemical PCR system. We used analytical tools both alternative current voltammetry and gel electrophoresis as the major. According to the results, we found that 1mM potassium ferricyanide and ABTS could be used in PCR to amplify the target sequence Also methylene blue, Hoechst 33258 and RuHex can be in PCR used under the concentrations of 200 uM, 50 uM and 8 uM, respectively. In this system, the detection limit has reached 6.4 ng when using IS6110 DNA fragment as PCR the template and 3000 copies when using simulated strain of Mycobacterium tuberculosis, but the development of RT-PCR system using potassium ferricyanide is unable to provide a practical application at the present stage