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Nucleic Acids Research 2005 33(8):e70; doi:10.1093/nar/gni069
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Published online 28 April 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

Methods Online

Diagnostic application of padlock probes—multiplex detection of plant pathogens using universal microarrays

Marianna Szemes, Peter Bonants, Marjanne de Weerdt, Johan Baner1, Ulf Landegren1 and Cor D. Schoen*

Plant Research International BV Droevendaalsesteeg 1, Wageningen 6708PB, The Netherlands 1Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University SE-751 85 Uppsala, Sweden

*To whom correspondence should be addressed. Tel: +31 317 476 026; Fax: +31 317 418 094; Email: cor.schoen{at}wur.nl

Received February 17, 2005. Revised March 31, 2005. Accepted March 31, 2005.

Padlock probes (PLPs) are long oligonucleotides, whose ends are complementary to adjacent target sequences. Upon hybridization to the target, the two ends are brought into contact, allowing PLP circularization by ligation. PLPs provide extremely specific target recognition, which is followed by universal amplification and microarray detection. Since target recognition is separated from downstream processing, PLPs enable the development of flexible and extendable diagnostic systems, targeting diverse organisms. To adapt padlock technology for diagnostic purposes, we optimized PLP design to ensure high specificity and eliminating ligation on non-target sequences under real-world assay conditions. We designed and tested 11 PLPs to target various plant pathogens at the genus, species and subspecies levels, and developed a prototype PLP-based plant health chip. Excellent specificity was demonstrated toward the target organisms. Assay background was determined for each hybridization using a no-target reference sample, which provided reliable and sensitive identification of positive samples. A sensitivity of 5 pg genomic DNA and a dynamic range of detection of 100 were observed. The developed multiplex diagnostic system was validated using genomic DNAs of characterized isolates and artificial mixtures thereof. The demonstrated system is adaptable to a wide variety of applications ranging from pest management to environmental microbiology.


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