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Multiplex and quantifiable detection of nucleic acid from pathogenic fungi using padlock probes, generic real-time PCR and specific suspension array readout
Dalarna University, School of Education, Health and Social Studies, Medical Science.
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2009 (English)In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 78, no 2, p. 195-202Article in journal (Refereed) Published
Abstract [en]

A new concept for multiplex detection and quantification of microbes is here demonstrated on a range of infectious fungal species. Padlock probe methodology in conjunction with qPCR and Luminex™ technology was used for simultaneous detection of ten fungal species in one single experiment. By combining the multiplexing properties of padlock probes and Luminex™ detection with the well established quantitative characteristics of qPCR, quantitative microbe detection was done in 10-plex mode. A padlock probe is an oligonucleotide that via a ligation reaction forms circular DNA when hybridizing to specific target DNA. The region of the padlock probe that does not participate in target DNA hybridization contains generic primer sequences for amplification and a tag sequence for Luminex™ detection. This was the fundament for well performing multiplexing. Circularized padlock probes were initially amplified by rolling circle amplification (RCA), followed by a SybrGreen™ real time PCR which allowed an additive quantitative assessment of target DNA in the sample. Detection and quantification of amplified padlock probes were then done on color coded Luminex™ microspheres carrying anti-tag sequences. A novel technique, using labeled oligonucleotides to prevent reannealing of amplimers by covering the flanks of the address sequence, improved the signal to noise ratio in the detection step considerably. The method correctly detected fungi in a variety of clinical samples and offered quantitative information on fungal nucleic acid.

Place, publisher, year, edition, pages
Elsevier , 2009. Vol. 78, no 2, p. 195-202
Keywords [en]
Multiplex detection, Pathogenic fungi, Padlock probe, Rolling circle amplification, Real time PCR, Nucleic acid hybridization, Suspension array
National Category
Microbiology
Research subject
Hälsa och välfärd, Multiplex detektion av patogena agens
Identifiers
URN: urn:nbn:se:du-4148DOI: 10.1016/j.mimet.2009.05.016ISI: 000268820700015OAI: oai:dalea.du.se:4148DiVA, id: diva2:520082
Available from: 2009-08-20 Created: 2009-08-20 Last updated: 2017-12-07Bibliographically approved

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CiteExportLink to record
Permanent link

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Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • chicago-author-date
  • chicago-note-bibliography
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf