In-situ monitoring of real-time loop-mediated isothermal amplification with qcm: Detecting listeria monocytogenes

Journal article

Authors/Editors

ISARATAT PHUNG-ON

Strategic Research Themes

Sustainable Bioeconomy (Strategic Research Themes)

Publication Details

Author list: Wachiralurpan S., Phung-On I., Chanlek N., Areekit S., Chansiri K., Lieberzeit P.A.

Publisher: MDPI

Publication year: 2021

Volume number: 11

Issue number: 9

ISSN: 2079-6374

eISSN: 2079-6374

URL: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85114631824&doi=10.3390%2fbios11090308&partnerID=40&md5=06bc767299416758df721a2f4890012f

Languages: English-Great Britain (EN-GB)

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Abstract

Functionalized DNA sequences are promising sensing elements to combine with transducers for bio-sensing specific target microbes. As an application example, this paper demonstrates in situ detection of loop-mediated isothermal amplification products by hybridizing them with thiolated-ssDNA covalently anchored on the electrodes of a quartz crystal microbalance (QCM). Such hybridization leads to a frequency signal, which is suitable for monitoring real-time LAMP amplification based on mass-sensing: it detects interactions between the complementary nucleobases of LAMP products in solution and the thiolated-ssDNA probe sequence on the gold surface. Target DNA LAMP products cause irreversible frequency shifts on the QCM surfaces during hybridization in the kHz range, which result from both changes in mass and charge on the electrode surface. In order to confirm the LAMP assay working in the QCM sensing system at elevated temperature, the sky blue of positive LAMP products solution was achieved by using the Hydroxy Naphthol Blue (HNB) and agarose gel electrophoresis. Since on-QCM sensing of DNA hybridization leads to irreversible sensor responses, this work shows characterization by X-ray photoelectron spectroscopy (XPS) core spectra of S2p, N1s, Mg1s, P2p and C1s. XPS results confirmed that indeed both DNA and by-products of LAMP attached to the surface. Listeria monocytogenes DNA served to study in-situ detection of amplified LAMP products on DNA-functionalized surfaces. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

Keywords

Hly gene, LAMP products analysis, Listeria monocytogenes (L. monocytogenes), Quartz crystal microbalance (QCM), Real-time loop-mediated isothermal amplification (real-time LAMP), X-ray photoelectron spectroscopy (XPS)