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A fluorometric lateral flow assay for visual detection of nucleic acids using a digital camera readout

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Abstract

A fluorometric lateral flow assay has been developed for the detection of nucleic acids. The fluorophores phycoerythrin (PE) and fluorescein isothiocyanate (FITC) were used as labels, while a common digital camera and a colored vinyl-sheet, acting as a cut-off optical filter, are used for fluorescence imaging. After DNA amplification by polymerase chain reaction (PCR), the biotinylated PCR product is hybridized to its complementary probe that carries a poly(dA) tail at 3΄ edge and then applied to the lateral flow strip. The hybrids are captured to the test zone of the strip by immobilized poly(dT) sequences and detected by streptavidin-fluorescein and streptavidin-phycoerythrin conjugates, through streptavidin-biotin interaction. The assay is widely applicable, simple, cost-effective, and offers a large multiplexing potential. Its performance is comparable to assays based on the use of streptavidin-gold nanoparticles conjugates. As low as 7.8 fmol of a ssDNA and 12.5 fmol of an amplified dsDNA target were detectable.

Schematic presentation of a fluorometric lateral flow assay based on fluorescein and phycoerythrin fluorescent labels for the detection of single-stranded (ssDNA) and double-stranded DNA (dsDNA) sequences and using a digital camera readout. SA: streptavidin, BSA: Bovine Serum Albumin, B: biotin, FITC: fluorescein isothiocyanate, PE: phycoerythrin, TZ: test zone, CZ: control zone.

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Acknowledgements

We acknowledge the assistance of Dr. Sotirios Tragoulias with image acquisition.

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Authors and Affiliations

  1. Department of Chemistry, University of Patras, 26504, Patras, Greece

    Maria Magiati, Areti Sevastou & Despina P. Kalogianni

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  1. Maria Magiati
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  2. Areti Sevastou
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  3. Despina P. Kalogianni
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Correspondence to Despina P. Kalogianni.

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Magiati, M., Sevastou, A. & Kalogianni, D.P. A fluorometric lateral flow assay for visual detection of nucleic acids using a digital camera readout. Microchim Acta 185, 314 (2018). https://doi.org/10.1007/s00604-018-2856-9

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  • DOI: https://doi.org/10.1007/s00604-018-2856-9

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