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 (μME-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 μL(-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.
Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, PR China.
Juan
J
Luo
Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, PR China.
Xueen
X
Fang
Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, PR China.
Daixing
D
Ye
Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, PR China.
Huixiang
H
Li
Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, PR China.
Hui
H
Chen
Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, PR China.
Song
S
Zhang
Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, PR China. Electronic address: jlkong@fudan.edu.cn.
Jilie
J
Kong
D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification*; D015374:Biosensing Techniques / Q000295:instrumentation; D003199:Computer Systems; D003217:Conductometry / Q000295:instrumentation*; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial
Electrochemistry; ITO electrode; Loop mediated isothermal amplification (LAMP); Microfluidic chip; Multiple