2008 Phytophthora Workshop in Italy

1. Nanobiotechnology
for Phytophthora
diagnosis
M. Riedel, S. Julich, L. Dressler, R. Möller, S. Wagner, A. Breitenstein, T. Henkel and
S. Werres
which
Phytophthora?
Riedel et al. : Nanobiotechnology 3rd International Phytophthora, Pythium and related genera workshop 26.08.2008

2. aim of the project
Development of a detection system
- species specific - sensitive
- robust - easy to handle
- applicable in the field - results within short time
chip-based diagnostic system
Riedel et al. : Nanobiotechnology 3rd International Phytophthora, Pythium and related genera workshop 26.08.2008

3. functional principle of the chip system
combined microchip for chip based PCR amplification
(PCR chip) and DNA detection (DNA chip) via hybri-
dization
scheme of micro-
systemic implemen- PCR chip DNA chip
tation of automa-
tized chip systems
combined with an
integrated micro-
fluidic system for
anlysis of nucleic
acids
buffer PCR complete control elements for waste pump
wash solutions mix chip-system temperature control and
enzymes electric detection
Riedel et al. : Nanobiotechnology 3rd International Phytophthora, Pythium and related genera workshop 26.08.2008

4. PCR chip module
on chip amplification und labeling of DNA by PCR
single PCR chip
bondpad
temperature sensor
detection window
circular fluid anchor
heater
results: amplification of P. software
PDA with control and analysis ramorum
ITS2 fragment identical with conventional
for chip-based PCR amplification
thermocycler and PCR-chip
Riedel et al. : Nanobiotechnology 3rd International Phytophthora, Pythium and related genera workshop 26.08.2008

5. DNA chip module
detection of Phytophthora DNA fragments on chip
Phytophthora specific DNA of closely
first results: hybridization probes
related Phytophthora taxa
spotted at elektrode gap (10 µm)
unspecific hybridization:
P. lateralis : P. ramorum
example for species specific probes ::
example for species specific probes
P.ramorum
P.ramorum GGAAACTTGT GTTGGCGGCT TCGGCTGCTG
GGAAACTTGT GTTGGCGGCT TCGGCTGCTG
P.lateralis GGAAATTTGT GTTGGCAGCT TCGGCTGTTG
P.lateralis GGAAATTTGT GTTGGCAGCT TCGGCTGTTG
specific hybridization:
detection of hybrisization results by
silver enhancement - electrical P. ramorum
P. ramorum : or optical
analysis possible
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

6. 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 !
Riedel et al. : Nanobiotechnology 3rd International Phytophthora, Pythium and related genera workshop 26.08.2008