Abstract
An upgraded nicking/polymerization strategy for ultrasensitive electrochemical detection of Watermelon mosaic virus (WMV) is proposed on the basis of the exonuclease and polymerase activity of T4 DNA polymerase and Mg2+-dependent DNAzyme-assisted and hemin/G-quadruplex DNAzyme-assisted cascade amplification strategies. Briefly, the hybridized DNA of the target WMV sequence, HP1, and P1 was recognized and nicked by nicking endonuclease Nb.BbvCI, and two DNA segments (P1-25 and P1-6) were produced. P1-25 was digested in the 3′→5′ direction, and digestion was halted at the 3′-terminal G locus with the exonuclease activity of T4 DNA polymerase. When dNTP solution mix was added to the mixture, an intact enzymatic sequence of Mg2+-dependent DNAzyme was synthesized by T4 DNA polymerase, which hybridized with its substrate sequence in the loop segment of HP2 immobilized on a gold electrode and initiated the cleavage round. The caged G-quadruplex sequence was released and formed hemin/G-quadruplex-based DNAzyme, resulting in sharply increased electrochemical signals. A correlation between the differential pulse voltammetry signal and the concentration of target WMV sequence was obtained in the range from 50 fM to 1 nM, with 50 fM detection limit. Because the nicking and polymerization reactions are irreversible and share the same buffer, the cascade amplification strategy is an ultrasensitive and high-efficiency strategy, indicating potential for viral detection.
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This work was financially supported by the National Natural Science Foundation of China (grant nos. 21675074 and 31701760) and the Shandong Provincial Major Application Technology Innovation Project.
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Wang, Y., Li, B., Liu, J. et al. T4 DNA polymerase-assisted upgrade of a nicking/polymerization amplification strategy for ultrasensitive electrochemical detection of Watermelon mosaic virus. Anal Bioanal Chem 411, 2915–2924 (2019). https://doi.org/10.1007/s00216-019-01737-x
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DOI: https://doi.org/10.1007/s00216-019-01737-x