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Applied and Environmental Microbiology, June 2009, p. 4185-4193, Vol. 75, No. 12
0099-2240/09/$08.00+0     doi:10.1128/AEM.00071-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Robust Detection and Identification of Multiple Oomycetes and Fungi in Environmental Samples by Using a Novel Cleavable Padlock Probe-Based Ligation Detection Assay{triangledown} ,{dagger} ,{ddagger}

R. van Doorn,1,2 M. Slawiak,1,3 M. Szemes,1,4 A. M. Dullemans,1 P. Bonants,1 G. A. Kowalchuk,2,5 and C. D. Schoen1*

Plant Research International B.V., Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands,1 NIOO-Centre for Terrestrial Ecology, P.O. Box 40, 6666 ZG Heteren, The Netherlands,2 University of Gdansk and Medical University of Gdansk, Intercollegiate Faculty of Biotechnology, Department of Biotechnology, Kladki 24, 80-822 Gdansk, Poland,3 University of Bristol, CLIC Sargent Laboratory, CMM, University Walk, Bristol BS8 1TD, United Kingdom,4 Free University of Amsterdam, Institute of Ecological Science, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands5

Received 12 January 2009/ Accepted 20 April 2009

Simultaneous detection and identification of multiple pathogenic microorganisms in complex environmental samples are required in numerous diagnostic fields. Here, we describe the development of a novel, background-free ligation detection (LD) system using a single compound detector probe per target. The detector probes used, referred to as padlock probes (PLPs), are long oligonucleotides containing asymmetric target complementary regions at both their 5' and 3' ends which confer extremely specific target detection. Probes also incorporate a desthiobiotin moiety and an internal endonuclease IV cleavage site. DNA samples are PCR amplified, and the resulting products serve as potential targets for PLP ligation. Upon perfect target hybridization, the PLPs are circularized via enzymatic ligation, captured, and cleaved, allowing only the originally ligated PLPs to be visualized on a universal microarray. Unlike previous procedures, the probes themselves are not amplified, thereby allowing a simple PLP cleavage to yield a background-free assay. We designed and tested nine PLPs targeting several oomycetes and fungi. All of the probes specifically detected their corresponding targets and provided perfect discrimination against closely related nontarget organisms, yielding an assay sensitivity of 1 pg genomic DNA and a dynamic detection range of 104. A practical demonstration with samples collected from horticultural water circulation systems was performed to test the robustness of the newly developed multiplex assay. This novel LD system enables highly specific detection and identification of multiple pathogens over a wide range of target concentrations and should be easily adaptable to a variety of applications in environmental microbiology.

* Corresponding author. Mailing address: Plant Research International B.V., Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands. Phone: 31-630453260. Fax: 31-317418094. E-mail: cor.schoen{at}wur.nl

{triangledown} Published ahead of print on 24 April 2009.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

{ddagger} Publication 4511 of The Netherlands Institute of Ecology (NIOO-KNAW).

Applied and Environmental Microbiology, June 2009, p. 4185-4193, Vol. 75, No. 12
0099-2240/09/$08.00+0     doi:10.1128/AEM.00071-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • van Doorn, R., Klerks, M. M., van Gent-Pelzer, M. P. E., Speksnijder, A. G. C. L., Kowalchuk, G. A., Schoen, C. D. (2009). Accurate Quantification of Microorganisms in PCR-Inhibiting Environmental DNA Extracts by a Novel Internal Amplification Control Approach Using Biotrove OpenArrays. Appl. Environ. Microbiol. 75: 7253-7260 [Abstract] [Full Text]  


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