A real-time microfluidic multiplex electrochemical loop-mediated isothermal amplification chip for differentiating bacteria

This report shows that loop-mediated isothermal amplification (LAMP) of nucleic acid can be integrated on a laser etched indium tin oxide (ITO) electrode-based multiplex microfluidic chip for real-time quantitative differentiation of bacteria; we call this technique microfluidic multiplex electrochemical LAMP (mu ME-LAMP) system. Three important acute upper respiratory tract infections (URTI) related bacteria, namely Mycobacterium tuberculosis (MTB), Haemophilus influenza (HIN), and Klebsiella pneumonia (KPN) were chosen for this study. We monitored the amplification process by measuring and analyzing the electrochemical signal of methylene blue (MB) through eight etched ITO electrochemical reactors. The results indicated that this assay with the ability of analyzing multiple genes qualitatively and quantitatively is highly specific, operationally simple, and cost/time effective. It exhibits high sensitivity with detection limits of 28, 17, and 16 copies mu L-1 for MTB, HIN, and KPN, respectively. The whole differentiation can be finished in a short time of 45 min, which has the potential to apply in clinical diagnosis. (C) 2014 Elsevier B.V. All rights reserved.