Research, Technology and Development II
RTD Leader: Jørn Dalgaard Mikkelsen, Danisco, Denmark
WP4 Culture Independent
Techniques
WP Leader: Luca Cocolin, UNIUD, Italy
This WP will develop molecular biology based culture independent techniques
to detect viability, adhesion and virulence expression of the bacterial and
fungal pathogens. The methods will be developed on the basis of
WP2, WP5 and validated in WP15. The
methods will be used in WP8, WP9 and
WP13.
Different approaches will be used to develop molecular tools, based on
culture-independent methods, to assess the microbiological quality and
safety of food, feed and water. A PCR-DGGE method will be optimised to
profile the microorganisms present in a specific sample. The results
obtained will give information regarding the microbial ecology of the food
studied, including the presence of pathogens. The PCR-DGGE method will be
performed using DNA and RNA extracted directly from the food samples. A
second approach, to be implemented is the application of VIT techniques
using different FISH probes organised in a hierarchical structure to
identify the bacterial community present within the sample.
PCR protocols for the specific detection and identification will be
optimised. Sequences that are unique to the specific pathogen considered
will be selected and the primers designed, in a fashion that will prime
amplification only when DNA or RNA, of the pathogen under investigation is
used in the amplification. DNA or RNA will be extracted and after PCR and
Reverse Transcription PCR (RT-PCR), information on their concentration and
viability of pathogens will be obtained. In case of TBEV, the existing
real-time RT-PCR will be adapted for use in raw milk samples. Similar
approaches will be used to assess the presence in the food samples of
genetic determinants of pathogenicity and toxicity. For detection of HEV in
food samples, conventional PCR, Q-PCR, Multiplex PCR and Padlock probe
system will be developed and compared regarding their respective
sensitivity, specificity and overall usefulness. Automated RNA extraction
methods will be applied in HEV detection systems in order to facilitate
high throughput of samples. For the pathogens, virulence, stress and
adhesion genes will be selected based on results generated in WP2. Their
monitoring will be performed by specific-virulence gene PCR. Expression
analysis will be carried out, by RNA-Dot Blot analysis and Q-RT-PCR. The
results obtained during the optimisation of PCR protocols for the detection
of pathogens and their virulence, stress and adhesion genes identified in
WP2, will be used to construct microarrays applicable to
the SMEs in WP5.
The WP includes the following tasks:
Task 4.1 Development of
PCR-DGGE to directly profile microbial populations
Task 4.2 Application of
in situ based technique (VIT) for community profiling
Task 4.3 Development of
RT-PCR, Q-PCR, Multiplex PCR, specific nested PCR and Padlock system for
detection, quantification and typing of pathogens
Task 4.4 Development of
Q-RT-PCR for quantification of virulence genes
Partner 8, Partner 9, Partner 10 and Partner
11 will be involved in this WP.
WP5 Development of Arrays
WP Leader: Matthias Kuhn, Congen, Germany
The main objective of this workpackage is the development and validation of
robust microarrays to be used to detect microorganisms relevant to this
project and to monitor the existence and expression of the involved genetic
factors for virulence, toxicity and adhesion.
PCR and Q-PCR methods are fast and sensitive technologies to detect
specific sequences in a given sample but are limited to identify only a few
sequences in parallel from one sample. If a more complex profile or pattern
(>10) of specific sequences has to be identified DNA-microarrays are the
only technology which can yield these data.
Microarrays composed of discretely located DNA-probes on a solid
substrate, e.g. a glass-slide will be manufactured. Either direct DNA- or
RNA-preparations or (RT-) PCR products will be labelled with a fluorescence
dye and hybridised to the array in order to identify sequences in a sample.
Virulence and toxicity of microorganisms are a complex system of existence
and gene expression. Expression of genes is often influenced by the matrix
in which the microorganism is found or by the host that is infected. Such a
complex system can only be studied by microarrays that are able to detect
the multiplex occurrence and/or expression of all factors involved.
Microarray hybridisation can also be used to assess microbial diversity.
Microarray will be developed for different applications:
· Detection of
Listeria monocytogenes, Campylobacter jejuni, E.
coli (VTEC) and S. cerevisiae (detection and expression of
virulence, stress and adhesion)
· Monitoring
functional properties of defined protective and probiotic cultures
· Detection of
hepatitis E virus (HEV) and tickborne encephalitis virus (TBEV)
· Monitoring the
ochratoxin production pathway of fungi
The WP includes the following tasks:
Task 5.1 Collection of Data /
Generation of a Database
Task 5.2 Bioinformatics /
Microarray design
Task 5.3 Q-PCR and
Hybridisation
Task 5.4 Multiplex PCR
Task 5.5 Prototype Microarray
production
Task 5.6 Small scale
Microarray production
Partner 12 will be involved in this WP.
- last update:27 April 2006