Development of rapid PCR markers for distinguishing

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1. Project Title (Please provide a brief project title <20 words)
     Development of rapid PCR markers for distinguishing Cryptosporidium oocyst
       infectivity and genotype

2. Location (Location where the student / project will be based)
     SA Water House, AWQC laboratories, South Australia

3. Issue No. (from Section 17      e.g. 7a, 7b etc.)   4. Program        (i.e. DW = drinking water, WW = wastewater /
                                                       recycled water)
         1a                                                   DW

5. Project Start Date                                  6. Project End Date
     March 2011                                            November 2011

7. Nominate student             (Provide the student‟s name and e-mail address or other relevant details pertinent to the

8 Research / Organisation Member Details
Organisation: SA Water, AWQC
ABN:           69336525019
Street         250 Victoria Square
Postal         GPO Box 1751 SA 5001
Principal Supervisor                                              Associate Supervisor
Name:          Dr Brendon James King                              Name:     Dr Daniel Hoefel
Phone:         61 8 74242114                                      Phone:    61 8 74242142
Fax:                                                              Fax:
Mobile:        0401844236                                         Mobile:   0418857712
Email:                        Email:

9. Industry Collaborators Details (if applicable)
Organisation:     N/A
Industry Contact                                  Industry Contact
Name:                                             Name:
Phone:                                            Phone:
WQRA Honours Project Proposal Form 2011                                                            Page 4 of 8
Fax:                                                            Fax:
Mobile:                                                         Mobile:
Email:                                                          Email:

10. Supervision History             (Briefly outline the number of students supervisors currently supervise as well as recent
   past history of student supervision)
Both supervisors have university affiliations with South Australian institutions and have
been responsible for supervision of domestic and international students at undergraduate
and postgraduate level (refer to CVs). Currently, Dr King is supervising a TAFE placement
student until November 2010. Dr Hoefel is supervising a PhD student from Adelaide
University til 2012 and a French Intern from Université de Limoges until August 2010. Dr
King has yet to commit to supervision of any students in 2011, and plans to supervise a
single Honours Student on the project proposed.

11. Experience and Capability of Supervisors                           (Briefly outline the capability of the supervisors in the
     proposed research field (e.g. recent publications, grants etc.) and attach CV‟s to the e-mail.
         Dr King and Dr Hoefel are actively publishing work in the proposed research field
          and are involved in both initiating and collaborating on a number of successful
          research grant applications. Pleases refer to CVs for more information.

12. Identification of Background IP, Potential Project IP & Student Involvement
    (List existing background IP and potential new IP that the project may generate if relevant.)
         “no IP issues”

13. Industry Benefits          (identify knowledge gaps and briefly indicate the benefits that the project will deliver to the
   water industry and how the knowledge outcomes will be transferred)
         Currently a rapid assay to determine both Cryptosporidium oocyst infectivity and
          genotype does not exist. This project would build on the recent and successful
          research work undertaken in our laboratory in order to realize this goal. Once a
          successful assay is developed and validated, it would be envisaged that the assay
          would be NATA accredited and transferred to analytical services within the AWQC
          so it could be offered as a routine test for water service providers. It is also
          envisaged that the assay would become used by other analytical service providers.

14. Industry Contribution (Briefly clarify how the Industry Collaborators will contribute to the project)
     SA Water is a vigorous supporter of providing in-kind time of its research staff for
       training and guidance of students involved in projects of benefit to the water
       industry. The Micro R&D laboratory is excellently resourced and indisputably, a
       critical component of the premier water quality laboratories in the Southern
       Hemisphere, the Australian Water Quality Centre. We will be able to generously
       cover all consumable and expenditures costs for this project.

15. What are the benefits to your Organisation to have an Honours Project?
     SA Water is an active supporter of student supervision and mentoring on projects of
      benefit to the Water Industry.       Students often gain employment with the
      organisation after graduation, and Dr Hoefel is a prime example. Scientific
      publications have arisen from student work; research has lead to further and
      successful funding outcomes and the development of new assays.

16. Briefly describe your University’s Student Induction Process
     It would be expected that the student would be enrolled in one of three South
WQRA Honours Project Proposal Form 2011                                                                 Page 5 of 8
          Australian Universities and therefore need to meet the specific requirements
          determined by that University. However, SA Water has comprehensive induction
          processes relating to OH&S and training requirements which would need to be
          satisfied before the student can work within SA Water House. Students will be
          trained according to the Micro R&D training Matrix and records kept according to
          ISO requirements.

17. List the preferred skills / interests / degree required by student                                    (Briefly indicate the
      preferred skills and knowledge as well as current undergraduate degrees which will be suitable for the project)
         Interests in the application of molecular biology and cell biology for the benefit of the
          water industry. Skill sets that would be developed would include real-time PCR,
          melt curve analysis, nucleic acid extraction, multi-parametric flow cytometry, cell
          culture, collimated beam apparatus operation and pathogen database mining.

                                        18. PROJECT OUTLINE

(Delete yes/no - what does not apply)
Yes              Is this project an extension of a Summer project?                          The summer project will look
                 more superficially at a wider array of candidate genetic markers to determine a reduced set of the most
                 promising candidates before more intensive validation and assay development is undertaken in the Honours
Yes              Is this project associated with another project? (If yes, explain clearly the association
         This project aims to build on the success of the WRF project 4133 “Identification of
          genes expressed during excystation, pre-infection and early infection as markers of
          infectivity in Cryptosporidium” where a number of candidate biomarkers were

Project Summary (approx 100 words)
    This project aims to validate a number of genetic markers which have shown
       promise as being capable of distinguishing between infectious and non-infectious
       Cryptosporidium oocysts, as well as determine genotype. The student will then
       take the most promising candidates through a more rigorous validation step before
       developing a PCR-based assay capable for detecting a single oocyst, its infectivity
       status and genotype.

Background (approx 100 words)
    Cryptosporidium spp. are currently detected in raw and treated waters using
      microscopic standard detection methodologies. However, these methodologies do
      not provide any information on the infectivity or genotype of oocysts detected.
      Recent research undertaken in our laboratory has identified critical stages essential
      for Cryptosporidium infectivity. We have also identified a number of genetic
      markers capable of distinguishing both oocyst infectivity and genotype. For the first
      time this offers us the potential for the rapid assessment of Cryptosporidium oocyst
      risk without the need to employ the costly and time inefficient techniques of cell

Project Objectives (approx 100 words)
    Identify the most promising genetic candidates capable of distinguishing between
        infectious and non-infectious oocysts as well as genotype
    Determine the most appropriate PCR assay platform and develop a highly sensitive
        PCR assay capable of detecting a single oocyst with a rapid turnaround time
    Specifically look at extraction techniques to increase sensitivity and assay
        turnaround time
WQRA Honours Project Proposal Form 2011                                    Page 6 of 8
        Validation of this assay against current cell culture infectivity assays used within the

Project Methodology (approx 100 words)
    cDNA banks have already been created from oocysts inactivated by a variety of
       stresses, a further set of cDNA banks would need to be created for more extensive
       validation of candidate genetic markers
    A RT-QPCR assay will be designed for the most promising genetic candidate/s
    Standard infectivity cell culture techniques will be used to validate the new assay,
       specifically examining the sensitivity and specificity of the assay

Project Outcomes (approx 100 words)
    Identification of genetic markers capable of distinguishing infectious from non-
       infectious oocysts, including oocyst species/genotype
    The development and validation of a new PCR based assay capable of identifying a
       single infective oocyst and simultaneously determine the oocyst species/genotype

Technology Transfer          (Identify the key forums and mechanisms to disseminate research outcomes that the project
will deliver to WQRA and its members. Include journals, publications, conferences, University exposure etc. Dot points
        Journal publications, including both industry and scientific journals
        Conference attendance and oral presentations delivered at both water industry and
         relevant scientific related forums
        University seminars

Milestones and budget items               (list funding contributors, milestone/budget items, month due and expenditure if
applicable. Add more lines as required
Funding Contributors                                                             $Amount
WQRA Stipend                                                                     $10,000
In-Kind time from Supervisors (SA Water)                                         $20,000
Expected Operating Allowance (Covered by Micro R&D)                              $15,000
Milestones                     Month/Year                                        Expenditure (cash) $
Identification    of    most June                                                N/A
promising candidate markers
Development of RT-QPCR September                                                 N/A
assay platform
Submission of thesis           November                                          N/A

***self evaluate your application against the Honours project selection criteria***

I hereby confirm that the information provided in this form to be Date
true and correct. (please insert signature)

WQRA Honours Project Proposal Form 2011                                                             Page 7 of 8

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