| Autor: |
Aktas GB; Interfibio, Bioengineering and Bioelectrochemistry Group, Departament d'Enginyeria Química, Universitat Rovira i Virgili, 26 Països Catalans, 43007, Tarragona, Spain., Wichers JH; BioSensing & Diagnostics, Wageningen Food & Biobased Research, Wageningen University & Research, Bornse Weilanden 9, 6708, WG, Wageningen, The Netherlands., Skouridou V; Interfibio, Bioengineering and Bioelectrochemistry Group, Departament d'Enginyeria Química, Universitat Rovira i Virgili, 26 Països Catalans, 43007, Tarragona, Spain., van Amerongen A; BioSensing & Diagnostics, Wageningen Food & Biobased Research, Wageningen University & Research, Bornse Weilanden 9, 6708, WG, Wageningen, The Netherlands. aart.vanamerongen@wur.nl., Masip L; Interfibio, Bioengineering and Bioelectrochemistry Group, Departament d'Enginyeria Química, Universitat Rovira i Virgili, 26 Països Catalans, 43007, Tarragona, Spain. lluis.masip@urv.cat. |
| Abstrakt: |
Nucleic acid lateral flow assays (NALFA) are often performed with gold nanoparticles. These are typically associated with ligand-labeled PCR amplicons via affinity interactions of adsorbed/conjugated proteins. Otherwise, they are conjugated to specific ssDNA sequences that hybridize to the target sequence. To avoid the need to generate ssDNA and to reduce the costs associated with primer labeling and antibody use, NALFA assays were developed that allow the direct detection of PCR amplicons using conjugates of a DNA binding protein with carbon nanoparticles (CNPs). The target gene encoding 16S ribosomal RNA of Escherichia coli was amplified by PCR using a single fluorophore-labeled forward primer and a reverse primer extended with the binding sequence of the bacteriophage lambda Cro repressor protein. Three different detection approaches were evaluated: (a) scCro/CNPs conjugate (black color), (b) HRP-scCro enzyme conjugate (red color), and (c) HRP-scCro/CNPs conjugate for dual color development. The limits of detection were between 6.9 and 10.4 ng of PCR product for all three approaches. These correspond to 3.0 to 4.5 × 10 3 CFU·mL -1 . The single-step scCro/CNP approach proved to be the fastest one to perform and gave no false-positive signals. It also showed a broad dynamic range even though the signal intensities were lower compared to the enzyme-amplified tests. However, the latter ones produced some background signal. In our perception, the application of scCro in lateral flow assays to bind dsDNA appears to be an excellent alternative to the use of small tags that have to be chemically linked to synthetic primers. Finally, the approach is generic because any primer sequence can be extended with the specific scCro binding sequence. Graphical abstract Schematic presentation of the lateral flow-based fluorometric detection of DNA amplicons using conjugates of scCro DNA binding protein with (A) carbon nanoparticles, (B) HRP and (C) HRP and carbon nanoparticles. |
