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Sensors 2016, 16(11), 1916; doi:10.3390/s16111916

Optimized Detection of Plasmodium falciparum Topoisomerase I Enzyme Activity in a Complex Biological Sample by the Use of Molecular Beacons

1
Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark
2
Division of Biomedical Engineering, Department of Electronic Engineering, The Chinese University of Hong Kong, Shatin, New Territories 999077, Hong Kong, China
3
Department of Pathology, Aarhus University Hospital, 8000 Aarhus C, Denmark
*
Author to whom correspondence should be addressed.
Academic Editors: Eiichi Tamiya, Yuji Murakami, Tatsuro Endo and Mun’delanji Vestergaard
Received: 19 September 2016 / Revised: 31 October 2016 / Accepted: 4 November 2016 / Published: 15 November 2016
(This article belongs to the Special Issue Point-of-Care Biosensors)
View Full-Text   |   Download PDF [4041 KB, uploaded 15 November 2016]   |  

Abstract

The so-called Rolling Circle Amplification allows for amplification of circular DNA structures in a manner that can be detected in real-time using nucleotide-based molecular beacons that unfold upon recognition of the DNA product, which is being produced during the amplification process. The unfolding of the molecular beacons results in a fluorescence increase as the Rolling Circle Amplification proceeds. This can be measured in a fluorometer. In the current study, we have investigated the possibility of using two different molecular beacons to detect two distinct Rolling Circle Amplification reactions proceeding simultaneously and in the same reaction tube by measurement of fluorescence over time. We demonstrate the application of this fluorometric readout method, for automated and specific detection of the activity of the type IB topoisomerase from the malaria parasite Plasmodium falciparum in the presence of human cell extract containing the related topoisomerase I from humans. The obtained results point towards a future use of the presented assay setup for malaria diagnostics or drug screening purposes. In longer terms the method may be applied more broadly for real-time sensing of various Rolling Circle Amplification reactions. View Full-Text
Keywords: real-time sensing; multiplexed detection; malaria; topoisomerase I; rolling circle amplification real-time sensing; multiplexed detection; malaria; topoisomerase I; rolling circle amplification
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MDPI and ACS Style

Givskov, A.; Kristoffersen, E.L.; Vandsø, K.; Ho, Y.-P.; Stougaard, M.; Knudsen, B.R. Optimized Detection of Plasmodium falciparum Topoisomerase I Enzyme Activity in a Complex Biological Sample by the Use of Molecular Beacons. Sensors 2016, 16, 1916.

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