Adenoviral detection by recombinase polymerase amplification and vertical flow paper microarray.

Nybond S, Réu P, Rhedin S, Svedberg G, Alfvén T, Gantelius J, Svahn HA

Anal Bioanal Chem 411 (4) 813-822 [2019-02-00; online 2018-11-29]

Respiratory viral infections often mimic the symptoms of infections caused by bacteria; however, restricted and targeted administration of antibiotics is needed to combat growing antimicrobial resistance. This is particularly relevant in low-income settings. In this work, we describe the use of isothermal amplification of viral DNA at 37 °C coupled to a paper-based vertical flow microarray (VFM) setup that utilizes a colorimetric detection of amplicons using functionalized gold nanoparticles. Two oligonucleotide probes, one in-house designed and one known adenoviral probe were tested and validated for microarray detection down to 50 nM using a synthetic target template. Furthermore, primers were shown to function in a recombinase polymerase amplification reaction using both synthetic template and viral DNA. As a proof-of-concept, we demonstrate adenoviral detection with four different adenoviral species associated with respiratory infections using the paper-based VFM format. The presented assay was validated with selected adenoviral species using the in-house probe, enabling detection at 1 ng of starting material with intra- and inter-assay %CV of ≤ 9% and ≤ 13%. Finally, we validate our overall method using clinical samples. Based on the results, the combination of recombinase polymerase amplification, paper microarray analysis, and nanoparticle-based colorimetric detection could thus be a useful strategy towards rapid and affordable multiplexed viral diagnostics.

PubMed 30498984

DOI 10.1007/s00216-018-1503-y

Crossref 10.1007/s00216-018-1503-y

pmc: PMC6338793
pii: 10.1007/s00216-018-1503-y