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Multiple pathogen biomarker detection using an encoded bead array in droplet PCR
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
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2017 (English)In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 139, p. 22-28Article in journal (Refereed) Published
Abstract [en]

We present a droplet PCR workflow for detection of multiple pathogen DNA biomarkers using fluorescent color coded Luminex beads. This strategy enables encoding of multiple singleplex droplet PCRs using a commercially available bead set of several hundred distinguishable fluorescence codes. This workflow provides scalability beyond the limited number offered by fluorescent detection probes such as TaqMan probes, commonly used in current multiplex droplet PCRs. The workflow was validated for three different Luminex bead sets coupled to target specific capture oligos to detect hybridization of three microorganisms infecting poultry: avian influenza, infectious laryngotracheitis virus and Campylobacter jejuni. In this assay, the target DNA was amplified with fluorescently labeled primers by PCR in parallel in monodisperse picoliter droplets, to avoid amplification bias. The color codes of the Luminex detection beads allowed concurrent and accurate classification of the different bead sets used in this assay. The hybridization assay detected target DNA of all three microorganisms with high specificity, from samples with average target concentration of a single DNA template molecule per droplet. This workflow demonstrates the possibility of increasing the droplet PCR assay detection panel to detect large numbers of targets in parallel, utilizing the scalability offered by the color-coded Luminex detection beads.

Place, publisher, year, edition, pages
Elsevier, 2017. Vol. 139, p. 22-28
Keyword [en]
Droplet microfluidics, Droplet PCR, Fluorescent beads, Biomarker detection, DNA hybridization
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-211322DOI: 10.1016/j.mimet.2017.04.007ISI: 000405154900005PubMedID: 28434824Scopus ID: 2-s2.0-85018453446OAI: oai:DiVA.org:kth-211322DiVA, id: diva2:1129125
Funder
Swedish Research Council Formas, 221-2011-1692Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20170801

Available from: 2017-08-01 Created: 2017-08-01 Last updated: 2017-11-29Bibliographically approved
In thesis
1. Droplet Microfluidics reverse transcription and PCR towards Single Cell and Exosome Analysis
Open this publication in new window or tab >>Droplet Microfluidics reverse transcription and PCR towards Single Cell and Exosome Analysis
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Miniaturization of biological analysis is a trend in the field of biotechnology aiming to increase resolution and sensitivity in biological assays. Decreasing the reaction volumes to analyze fewer analytes in each reaction vessel enables the detection of rare analytes in a vast background of more common variants. Droplet microfluidics is a high throughput technology for the generation, manipulation and analysis of picoliter scale water droplets an in immiscible oil. The capacity for high throughput processing of discrete reaction vessels makes droplet microfluidics a valuable tool for miniaturization of biological analysis.

In the first paper, detection methods compatible with droplet microfluidics was expanded to include SiNR FET sensors. An integrated droplet microfluidics SiNR FET sensor device capable of extracting droplet contents, transferring a train of droplets to the SiNR to measure pH was implemented and tested. In paper II, a workflow was developed for scalable and target flexible multiplex droplet PCR using fluorescently color-coded beads for target detection. The workflow was verified for concurrent detection of two microorganisms infecting poultry. The detection panel was increased to multiple targets in one assay by the use of target specific capture probes on color-coded detection beads.   In paper III, droplet microfluidics has been successfully applied to single cell processing, demonstrated in paper III, where reverse transcription was performed on 65000 individually encapsulated mammalian cells. cDNA yield was approximately equivalent for reactions performed in droplets and in microliter scale. This workflow was further developed in paper IV to perform reverse transcription PCR in microfluidic droplets for detection of exosomes based on 18S RNA content. The identification of single exosomes based on RNA content can be further developed to detect specific RNA biomarkers for disease diagnostics.

Droplet microfluidics has great potential for increasing resolution in biological analysis and to become a standard tool in disease diagnostics and clinical research.

 

 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2017. p. 69
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2017:15
Keyword
Droplet microfluidics, Reverse transcription, Droplet PCR, High Throughput biology, Single cell Analysis, Exosomes
National Category
Other Engineering and Technologies not elsewhere specified
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-216669 (URN)978-91-7729-577-8 (ISBN)
Public defence
2017-11-17, Air & Fire, Tomtebodavägen 23A, Solna, 10:00 (English)
Opponent
Supervisors
Note

QC 20171024

Available from: 2017-10-24 Created: 2017-10-23 Last updated: 2017-10-26Bibliographically approved

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