Semi-real time electrochemical monitoring for influenza virus RNA by reverse transcription loop-mediated isothermal amplification using a USB powered portable potentiostat.
In this paper, the semi-real time electrochemical monitoring method using a screen-printed electrode, which employs reverse transcription loop-mediated isothermal amplification (RT-LAMP) for influenza virus RNA, is presented. The amplified DNA combined with methylene blue (MB), which was used as an electroactive DNA intercalator, and the electrochemical signal was monitored using square wave voltammetry in the presence of RT-LAMP reagent components. MB molecules binding to amplified DNA caused the reduction of the peak current due to the slow diffusion of MB-amplified DNA complex to the electrode surface. We successfully monitored the amplification process of DNA on the basis of RT-LAMP by measuring and analyzing the electrochemical signal of MB with only one screen-printed electrode that connected with a USB powered portable potentiostat. The peak height of the current was related to the extent of amplification of DNA and the amount of input RNA. Since laborious probe immobilization is not required and both the amplification and the monitoring are possible in a single tube, our method does not suffer from potential cross-contamination. Furthermore, our method provides a new rote for the development of electrochemical hand held biosensors.
Department of Applied Chemistry, Graduate School of Engineering, Okayama University of Science, Ridai-cho, Kita-ku, Japan. nagatani@dac.ous.ac.jp
Naoki
N
Nagatani
Keiichiro
K
Yamanaka
Masato
M
Saito
Ritsuko
R
Koketsu
Tadahiro
T
Sasaki
Kazuyoshi
K
Ikuta
Toshiro
T
Miyahara
Eiichi
E
Tamiya
D055664:Electrochemical Techniques / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D008751:Methylene Blue / Q000737:chemistry; D021141:Nucleic Acid Amplification Techniques; D009975:Orthomyxoviridae / Q000235:genetics*; D012367:RNA, Viral / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism
D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase; D008751:Methylene Blue