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On-site detection of Phytophthora spp.—single-stranded target DNA as the limiting factor to improve on-chip hybridization

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Abstract

We report on a lab-on-a-chip approach for on-site detection of Phytophthora species that allows visual signal readout. The results demonstrate the significance of single-stranded DNA (ssDNA) generation in terms of improving the intensity of the hybridization signal and to improve the reliability of the method. Conventional PCR with subsequent heat denaturation, sodium hydroxide-based denaturation, lambda exonuclease digestion and two asymmetric PCR methods were investigated for the species P. fragariae, P. kernoviae, and P. ramorum. The positioning of the capture probe within the amplified yeast GTP-binding protein (YPT1) target DNA was also of interest because it significantly influences the intensity of the signal. Statistical tests were used to validate the impact of the ssDNA generation methods and the capture-target probe position. The single-stranded target DNA generated by Linear-After-The-Exponential PCR (LATE-PCR) was found to produce signal intensities comparable to post-PCR exonuclease treatment. The LATE-PCR is the best method for the on-site detection of Phytophthora because the enzymatic digestion after PCR is more laborious and time-consuming.

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Acknowledgments

We thank Carsten F. Dormann for giving valuable support in “R” statistics and Everett Hansen for critical reading of the manuscript. The project is supported by funds of the Federal Ministry of Food, Agriculture and Consumer Protection (BMELV) based on a decision of the Parliament of the Federal Republic of Germany via the Federal Office for Agriculture and Food (BLE) under the innovation support program.

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Authors and Affiliations

  1. Institute of Photonic Technology Jena (IPHT), Jenaer BioChip Initiative, Albert-Einstein-Str. 9, 07745, Jena, Germany

    Lydia Schwenkbier, Sibyll Pollok, Jürgen Popp & Karina Weber

  2. Julius Kuehn Institute—Federal Research Centre for Cultivated Plants (JKI), Institute for Plant Protection in Horticulture and Forests, Messeweg 11/12, 38104, Braunschweig, Germany

    Stephan König, Stefan Wagner & Sabine Werres

  3. Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich Schiller University Jena, Helmholtzweg 4, 07745, Jena, Germany

    Lydia Schwenkbier, Sibyll Pollok, Jürgen Popp & Karina Weber

  4. Analytik Jena AG, Konrad-Zuse-Str. 1, 07745, Jena, Germany

    Jörg Weber & Martin Hentschel

Authors
  1. Lydia Schwenkbier
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  2. Stephan König
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  7. Jürgen Popp
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Corresponding authors

Correspondence to Sabine Werres or Karina Weber.

Additional information

Lydia Schwenkbier and Stephan König contribute equally to the paper.

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Table 1

List of sequences, isolate numbers and sequence gene bank accession numbers for in-silico analysis of the COI, ITS, and YPT1 regions. (DOCX 27 kb)

Supplement 1

FASTA alignment for COI (TXT 38 kb)

Supplement 2

FASTA alignment for ITS (TXT 50 kb)

Supplement 3

FASTA alignment for YPT1 (TXT 31 kb)

Supplement 4

YPT1 alignment used for species-specific probe generation (TXT 415 kb)

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Schwenkbier, L., König, S., Wagner, S. et al. On-site detection of Phytophthora spp.—single-stranded target DNA as the limiting factor to improve on-chip hybridization. Microchim Acta 181, 1669–1679 (2014). https://doi.org/10.1007/s00604-013-1107-3

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