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Adenoviral detection by recombinase polymerase amplification and vertical flow paper microarray.
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.ORCID iD: 0000-0003-4727-6138
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2019 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 411, no 4, p. 813-822Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Springer, 2019. Vol. 411, no 4, p. 813-822
Keywords [en]
Adenoviral, RPA, VFM
National Category
Medical Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-244426DOI: 10.1007/s00216-018-1503-yISI: 000456132900003PubMedID: 30498984Scopus ID: 2-s2.0-8505759966OAI: oai:DiVA.org:kth-244426DiVA, id: diva2:1290466
Note

QC 20190222

Available from: 2019-02-20 Created: 2019-02-20 Last updated: 2019-02-22Bibliographically approved
In thesis
1. Novel planar and particle-based microarrays for point-of-care diagnostics
Open this publication in new window or tab >>Novel planar and particle-based microarrays for point-of-care diagnostics
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Point-of-care assays are easy-to-use, portable and inexpensive tests that can

be used to aid diagnostics by measuring levels of disease-specific molecules

in settings where access to advanced laboratory equipment and trained

personnel are limited, such as at the patient's bedside or in low resource

parts of developing countries. In order to achieve high multiplexing

capacities, such assays can be based on planar microarrays consisting of

spots immobilized on a flat surface or on particle-based microarrays based

on populations of encoded particles. The aim of the work presented in this

thesis is to develop new point-of-care amenable planar and particle-based

microarrays that allow for highly multiplexed assays while maintaining low

sample-to-result times, complexity and instrumentation requirements.

Paper I demonstrates the use graphically encoded particles for colorimetric

detection of autoantibodies using a consumer-grade flatbed scanner. Four

graphical characters on the surface of each particle allows for millions of

codes and the use of gold nanoparticles as a detection label allows both the

code and the signal intensity to be read out in a single image.

Paper II describes a signal enhancement method that increases the

sensitivity of gold nanoparticle detection on planar microarrays. Using this

method, detection of allergen-specific IgE can be carried out using a

consumer-grade flatbed scanner instead of a more expensive fluorescence

scanner without sacrificing assay performance.

Paper III demonstrates the use of an isothermal DNA amplification method

for detection of adenoviral DNA on a paper-based microarray. Using an

isothermal amplification method eliminates the need for a thermocycler,

reducing the instrumentation required for such detection.

Paper IV shows the use of solid-phase PCR to amplify bacterial DNA directly

on the surface of particles. This strategy reduces assay time by eliminating

the need for separate amplification and hybridisation steps.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2019. p. 88
Series
TRITA-CBH-FOU ; 2019:13
Keywords
Planar microarrays, Particle-based microarrays, Point-of-care diagnostics, Colorimetric detection, Signal enhancement, Isothermal DNA amplification, Solid-phase DNA amplification
National Category
Medical Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-244430 (URN)978-91-7873-101-5 (ISBN)
Public defence
2019-03-22, Science for Life Laboratory, room Air & Fire, Tomtebodavägen 23A, Solna, 10:00 (English)
Opponent
Supervisors
Note

QC 20190221

Available from: 2019-02-21 Created: 2019-02-20 Last updated: 2019-02-21Bibliographically approved

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Gantelius, JesperSvahn Andersson, Helene

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