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Journal of Biomedical Optics • Open Access

Application of fluorescence resonance energy transfer resolved by fluorescence lifetime imaging microscopy for the detection of enterovirus 71 infection in cells
Author(s): Vladimir Gukassyan; Yueh-Ying Hsu; Szu-Hao Kung; Fu-Jen Kao

Paper Abstract

Timely and effective virus infection detection is critical for the clinical management and prevention of the disease spread in communities during an outbreak. A range of methods have been developed for this purpose, of which classical serological and viral nucleic acids detection are the most popular. We describe an alternative, imaging-based approach that utilizes fluorescence resonance energy transfer (FRET) resolved by fluorescence lifetime imaging microscopy (FLIM) and demonstrate it on the example of enterovirus 71 (EV71) infection detection. A plasmid construct is developed with the sequence for GFP2 and DsRed2 fluorescent proteins, linked by a 12-amino-acid-long cleavage recognition site for the 2A protease (2Apro), encoded by the EV71 genome and specific for the members of Picornaviridae family. In the construct expressed in HeLa cells, the linker binds the fluorophores within the Förster distance and creates a condition for FRET to occur, thus resulting in shortening of the GFP2 fluorescence lifetime. On cells infection with EV71, viral 2Apro released to the cytoplasm cleaves the recognition site, causing disruption of FRET through separation of the fluorophores. Thus, increased GFP2 lifetime to the native values, manifested by the time-correlated single-photon counting, serves as an efficient and specific indicator of the EV71 virus infection.

Paper Details

Date Published: 1 March 2007
PDF: 8 pages
J. Biomed. Opt. 12(2) 024016 doi: 10.1117/1.2718582
Published in: Journal of Biomedical Optics Volume 12, Issue 2
Show Author Affiliations
Vladimir Gukassyan, National Yang-Ming Univ. (Taiwan)
Yueh-Ying Hsu, National Yang-Ming Univ. (Taiwan)
Szu-Hao Kung, National Yang-Ming Univ. (Taiwan)
Fu-Jen Kao, National Yang-Ming Univ. (Taiwan)

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