Change search
ReferencesLink to record
Permanent link

Direct link
Color-coded bead based readout from droplet PCR for the detection of pathogen biomarkers
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.ORCID iD: 0000-0003-3618-9944
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

We present a workflow using fluorescent color-coded Luminex beads to detect the

outcome of droplet PCR assay. The assay was performed to detect three important

poultry pathogens: avian influenza, infectious laryngotracheitis virus and

campylobacter. Droplet-based TaqMan PCR has been commonly used for detection

of rare and significant biomarkers in clinical samples. However, the spectral overlap

of fluorescent TaqMan probes limits the detection to 5 different targets in a single

assay. The color codes of the Luminex detection beads allowed accurate classification

of the different bead sets used in this assay concurrently. The target-specific capture

probes coupled to distinct bead sets enabled capture and detection of target DNA in

the droplet. The capture assay detected target DNA of all three poultry pathogens with

high specificity, from samples with average target concentration of 1 template per

droplet. This workflow demonstrates that the detection panel of droplet PCR assay

can be increased to potentially detect multiple targets in a sample by utilizing the

scalability offered by the color-coded detection beads.

National Category
Diagnostic Biotechnology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-192575OAI: oai:DiVA.org:kth-192575DiVA: diva2:971045
Note

QC 20160921

Available from: 2016-09-15 Created: 2016-09-15 Last updated: 2016-09-21Bibliographically approved
In thesis
1. Droplet microfluidics for single cell and nucleic acid analysis
Open this publication in new window or tab >>Droplet microfluidics for single cell and nucleic acid analysis
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Droplet microfluidics is an emerging technology for analysis of single cells and biomolecules at high throughput. The controlled encapsulation of particles along with the surrounding microenvironment in discrete droplets, which acts as miniaturized reaction vessels, allows millions of particles to be screened in parallel. By utilizing the unit operations developed to generate, manipulate and analyze droplets, this technology platform has been used to miniaturize a wide range of complex biological assays including, but not limited to, directed evolution, rare cell detection, single cell transcriptomics, rare mutation detection and drug screening.

The aim of this thesis is to develop droplet microfluidics based methods for analysis of single cells and nucleic acids. In Paper I, a method for time-series analysis of mammalian cells, using automated fluorescence microscopy and image analysis technique is presented. The cell-containing droplets were trapped on-chip and imaged continuously to assess the viability of hundreds of isolated individual cells over time. This method can be used for studying the dynamic behavior of cells. In Paper II, the influence of droplet size on cell division and viability of mammalian cell factories during cultivation in droplets is presented. The ability to achieve continuous cell division in droplets will enable development of mammalian cell factory screening assays in droplets. In Paper III, a workflow for detecting the outcome of droplet PCR assay using fluorescently color-coded beads is presented. This workflow was used to detect the presence of DNA biomarkers associated with poultry pathogens in a sample. The use of color-coded detection beads will help to improve the scalability of the detection panel, to detect multiple targets in a sample. In Paper IV, a novel unit operation for label-free enrichment of particles in droplets using acoustophoresis is presented. This technique will be useful for developing droplet-based assays that require label-free enrichment of cells/particles and removal of droplet content. In general, droplet microfluidics has proven to be a versatile tool for biological analysis. In the years to come, droplet microfluidics could potentially be used to improve clinical diagnostics and bio-based production processes.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2016. 58 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:17
Keyword
Acoustophoresis, Biomarker detection, Cell behavior analysis, Cell factories, Droplet microfluidics, Droplet PCR, High throughput biology, Label-free enrichment, Single cell analysis
National Category
Other Engineering and Technologies not elsewhere specified
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-192668 (URN)978-91-7729-125-1 (ISBN)
Public defence
2016-10-21, Gard-aulan, Nobels väg 18, Solna, 10:00 (English)
Opponent
Supervisors
Note

QC 20160926

Available from: 2016-09-26 Created: 2016-09-19 Last updated: 2016-09-26Bibliographically approved

Open Access in DiVA

No full text

Search in DiVA

By author/editor
Periyannan Rajeswari, Prem KumarSoderberg, Lovisa MAndersson Svahn, HeleneJönsson, Håkan
By organisation
Proteomics and Nanobiotechnology
Diagnostic Biotechnology

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Total: 12 hits
ReferencesLink to record
Permanent link

Direct link