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.

 

 

WP6 Resuscitation and Enrichment Methods

WP Leader: Mieke Uyttendaele, UGENT, Belgium

This WP will determine the conditions of resuscitation and enrichment of the bacterial pathogens for detection by molecular biology based methods and conventional microbiological analyses. The enrichment protocols will be used in WP12 and WP13, and validated in WP15.

This WP will include definition of standard conditions for producing sub-lethal injury by exposure to sublethal stresses present in the food chain. Studies on critical points in the enrichment procedure (medium composition, atmosphere, incubation temperature and time, and microbial interaction) that affect kinetics of repair (lag-phase) and growth (generation time) will be conducted. The significance of strain variation will be established. Laboratory scale production and characterisation of phage ligands that may serve as a basis for specific capture of the respective bacterial pathogens will be carried out. Finally this WP will comprise application and optimisation of the VIT protocol for the detection of C. jejuni in situ after enrichment.

The WP includes the following tasks:

Task 6.1        Model for controlled injury and methods for determination of injury and repair of C. jejuni, E. coli (STEC), and L monocytogenes.

Task 6.2        Development of enrichment broths for injured C. jejuni, E. coli (STEC), L. monocytogenes and M. avium subsp. paratuberculosis and determination of the kinetics of repair and the kinetics of growth as determined by critical points like medium composition, atmosphere, incubation temperature and time, and microbial interactions.

Task 6.3        Isolation and characterisation of host-specific phage-derived proteins, immobilisation and development of a format for selective and rapid enrichment/isolation of C. jejuni, E. coli (STEC), and L. monocytogenes

Task 6.4        Application of existing VIT kits and development of VIT based protocols for Campylobacter jejuni.

 

 

Partner 10, Partner 11, Partner 14 and Partner 15 will be involved in this WP.

 

 

 

WP7 Staphylococcal enterotoxin expression

WP Leader: Peter Rådström, LU, Sweden

 

It is the objective of this WP to (i) detect enterotoxin producing Staphyloccocus aureus in the food chain (ii) to identify factors such as food composition, gas atmosphere, preservatives, temperature etc. which are responsible for down and up-regulating enterotoxin expression in food and to predict enterotoxin levels in food.

 

A PCR method for detection of S. aureus directed against genes encoding at least one common heat stable enterotoxin in food will be developed. Subsequently a qRT-PCR method for the quantification of staphylococcal enterotoxin gene expression will be optimised and determination of the specific production level of staphylococcal enterotoxin using an ELISA procedure with polyclonal antibodies will be used as reference. Finally information will be obtained on factors such as food composition, gas atmosphere, preservatives, temperature etc. which are responsible for down and up-regulating enterotoxin expression in food.

 

The WP will include the following tasks:

 

Task 7.1        Establishment of a PCR-based method for the detection of one common S. aureus enterotoxin gene.

Task 7.2        Establishment of a qRT-PCR reference method for S. aureus enterotoxin expression.

Task 7.3        Establishment of an immunological method for monitoring the production level of staphylococcal enterotoxin.

Task 7.4        To set up a strategy to identify factors such as food composition, gas atmosphere, preservatives, temperature etc. which are responsible for down and up-regulating enterotoxin expression in food.

 

Partner 43 will be involved in this WP.