Cluster 2 Summary

Cluster 2 includes WP 6, 7, 8 and 9.  Cluster 2 basically concerns itself with the development of the targets identified in cluster 1 into viable tools suitable for use in the detection and characterization of pathogens in water or for source tracking. Because one of the most problematic issues in molecular detection of pathogens in water is sample preparation we have a separate WP (6) dedicated to improving sample preparation technologies.  WP7 has the role of developing a portfolio of detection methods on different platforms suitable for different test beds (surface, ground, tap, processing water), laboratories (high, low tech) and water management systems (on-site, on-line measurement, large, small distribution systems). Because we are aware that some of the highest risk drinking water systems in Europe are very small supplies whose owners do not have the skills or resources to undertake complex or costly microbiological examinations, an important part of WP7 will be to develop low cost/ easy to use technologies.  WP8 has the objective to develop bespoke systems that integrate technologies developed in WP6 and 7.  WP9 has the responsibility of taking the technologies developed in cluster 2 and developing standard operating procedures and microbial standards suitable for use in service laboratories. In addition WP9 will undertake validation of the technologies by designing and managing appropriate external quality assurance schemes and supporting internal quality assurance though the provision of standards.

WP6: Sample Preparation – Robust and rapid sample preparation technologies. Active Months: February 2014 to January 2017

Objectives

The overall aim of this WP is to develop robust and rapid sample preparations for the subsequent molecular detection of virus, bacteria and protozoa in large volumes of water. More precisely the objectives are:

  • To improve pathogen extraction from concentrates obtained by using low-cost hollow fibre 2. To develop efficient pathogen concentration and extraction from filtrates obtained using nanotechnology-based filters.
  • To optimise and assess the concentration and extraction of pathogens using ultra-low cost glass wool filteringtechnique.
  • To develop and assess the concentration of pathogens and removal of inhibitors using novel microfluidicstructures.
  • To evaluate and compare the recovery efficiency, sensitivity, quantifiability and inhibitor removal of the abovementioned techniques via the use of (RT-)PCR techniques.
  • To address the issue of the diversity of water samples via an exchange of technologies between theparticipants across Europe.

 

Del 6.1  Review: Filtering, separation and purification techniques for molecular pathogen detection

Del 6.2  High-yield on-site pathogen concentration of 20-100L of different types of water

Del 6.3  Procedure to extract high quality nucleic acids from water concentrates for pathogen detection

Del 6.4  An evaluation of microfluidic systems to perform sample separation

 

WP7: Detection – Assessment and development of a portfolio of pathogen detection methods. Active Months: February 2014 to January 2017

Objectives

  • To develop standardised multiplex pathogen detection on RPA and real time PCR platforms 
  • To develop a point-of analysis system for virus detection and quantification 
  • To develop ATP biosensors for the detection of microbial activity in water.
  • To validate using classical and molecular detection an automated system for the detection of E. coli andenterococci
  • To develop and evaluate FISH probes for the detection of bacterial pathogens
  • To evaluate the detection of Cryptosporidium using immunofluoresence and solid-phase cytometry7. To evaluate a VOCMA platform for multiplex pathogen

 

 

Del 7.1  Optimized analytical method for ATP including opportunities for immune capture measurements

Del 7.2  Report on the mbOnline system performance

Del 7.3  Protocol report for the FISH-based detection of Campylobacter spp.

Del 7.4  Report on AqµaTAS performance to detect DNA and RNA viruses

Del 7.5  Method for multiplex detection of targets identified in cluster 1 on Luminex®2000

Del 7.6  Method for on-site detection of DNA targets available

 

 

 

WP8: Integration –integrated and automated platforms for effective pathogen detection. Active Months: February 2014 to January 2017

Objectives

  • To optimise an automated imaging system for the quantification of bacterial cell using novel imaging techniques.
  • To integrate or automate of the various filtering technologies benchmarked under WP6.
  • To develop an automated sampler for the rapid detection of living bacteria in on-line and advanced warning water detection systems.
  • To evaluate the potential for integration, mass manufacture, miniaturisation and automation of the filtering, sampling and detection  technologies proposed under WP6 and WP7, and develop a multiplex detection platform.

 

Del 8.1  Report validating the integration of the optimised system. Currently Confidential.

Del 8.2  Report describing the performance of the automated elution techniques

Del 8.3  Presentation of performance of the ATP system

Del 8.4  Performance of the on-line automated sampling module

Del 8.5  Recommendation report to WP9

Del 8.6  Report describing the prototype for multiplex detection of pathogens

 

WP9: Standardisation and validation.

Objectives

Validation of the newly developed platforms is essential for the acceptance of the molecular methods and hence the basis for a practical application. The objectives of this work package are:

  • To develop appropriate required controls for extraction and detection procedures.
  • To acquire standard operation protocols.
  • To define a validation concept.
  • To implement the external quality assessment.
  • To perform and evaluate the validation process.

 

Del 9.1  Report of the workshop and selection of target organisms.

Del 9.2  Standard operation protocols.

Del 9.3  Report of validation with nucleic acids.

Del 9.4  Report of validation with micro-organisms in different matrices.

Del 9.5  Final report for the validation and recommendations for testbed operation.