Riedel et al. : Nanobiotechnology 3rd International Phytophthora, Pythium and related genera workshop 26.08.2008

Nanobiotechnologyfor Phytophthora

diagnosisM. Riedel, S. Julich, L. Dressler, R. Möller, S. Wagner, A. Breitenstein, T. Henkel and

S. Werres

whichPhytophthora?

Riedel et al. : Nanobiotechnology 3rd International Phytophthora, Pythium and related genera workshop 26.08.2008

Development of a detection system

- species specific - sensitive- robust - easy to handle - applicable in the field - results within short time

aim of the project

chip-based diagnostic system

Riedel et al. : Nanobiotechnology 3rd International Phytophthora, Pythium and related genera workshop 26.08.2008

functional principle of the chip system

combined microchip for chip based PCR amplification (PCR chip) and DNA detection (DNA chip) via hybri-dization

bufferwash solutions

enzymes

PCRmix

complete chip-system

control elements for temperature control and

electric detection

waste pump

PCR chip DNA chipscheme of micro-systemic implemen-tation of automa-tized chip systems combined with an integrated micro-fluidic system for anlysis of nucleic acids

Riedel et al. : Nanobiotechnology 3rd International Phytophthora, Pythium and related genera workshop 26.08.2008

PCR chip module

on chip amplification und labeling of DNA by PCR

single PCR chip

bondpad

temperature sensor

detection window

circular fluid anchor

heater

bondpad

temperature sensor

detection window

circular fluid anchor

heater

PDA with control and analysis software for chip-based PCR amplificationresults: amplification of P. ramorumITS2 fragment identical with conventional thermocycler and PCR-chip

Riedel et al. : Nanobiotechnology 3rd International Phytophthora, Pythium and related genera workshop 26.08.2008

detection of hybrisization results bysilver enhancement - electrical or opticalanalysis possible

DNA chip module

detection of Phytophthora DNA fragments on chip

example for species specific probes :

P.ramorum GGAAACTTGT GTTGGCGGCT TCGGCTGCTGP.lateralis GGAAATTTGT GTTGGCAGCT TCGGCTGTTG

example for species specific probes :

P.ramorum GGAAACTTGT GTTGGCGGCT TCGGCTGCTGP.lateralis GGAAATTTGT GTTGGCAGCT TCGGCTGTTG

Phytophthora specific DNA probesspotted at elektrode gap (10 µm)first results: hybridization of closely related Phytophthora taxa

unspecific hybridization:

P. lateralis : P. ramorum

specific hybridization:

P. ramorum : P. ramorum

combined gold chip with 42 detec-tion sites and microfluidic

Riedel et al. : Nanobiotechnology 3rd International Phytophthora, Pythium and related genera workshop 26.08.2008

Acknowledgements

We would like to thank the Bundesanstalt für Ernährung

und Landwirtschaft (BLE) for funding our project

and all partners at JKI, IPHT und BECIT !