CICEET Progress Report for the period 9/15/04 Through 3/15/05

Project Title: Microbial source tracking using F-specific coliphage and real-time QPCR
Principal Investigator(s): David C. Smith
Project Start Date: 1 September 2003

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ii. Determine if the four beacons can be used in a multiplexing reaction that will reduce the number of individual assays necessary for source tracking by a factor of four.

iii. Continue to extend the database of F-specific coliphages from additional animal sources.

Tasks to meet objectives
i. Three additional beacons were designed based on RNA genome sequences in database

ii. Experiments were conducted using each beacon individually with all four coliphage groups to determine their specificity.

iii. Singleplexed RT-protocols were optimized along salt, primer and beacon concentration gradients.

iv. Quantitative PCR reactions with all four coliphage groups, all four sets of PCR primers and all four molecular beacons were performed. Results from these experiments were compared with those conducted individually (above) to determine whether there are negative interactions among the primers, beacons and templates.

Progress on Tasks
i. These beacons were designed and synthesized.

ii. The experiments were conducted.

iii. The experiments were conducted.

iv. The experiments were conducted.

Accomplishments
i. The beacons are functional and behave as expected.

ii. In isolation, all four molecular beacons and primer sets show no interference with template cDNA from the other three coliphage groups.

iii. The primer, beacon and salt concentrations were optimized for the singleplexed assays.

iv. Initial results of the multiplexing RT-PCR reactions demonstrated that while all four subgroups are amplified, the reaction efficiency of subgroup IV is decreased under the multiplexing conditions.

Difficulties
i. No difficulties were encountered with this task.

ii. No difficulties were encountered with this task.

iii. No difficulties were encountered with this task.

iv. Initial results of the multiplexing RT-PCR reactions demonstrated a loss in efficiency of subgroup IV amplification. Step-wise elimination of potential interference indicates that the presence of the forward primer for subgroup III interferes with the quantitative amplification of subgroup IV. Computer analysis of the templates, beacon and primers did not reveal any potential non-specific hybridization targets among all components in the multiplexing reaction. We are using the coliphage Fi as a representative of subgroup IV. Because there is no existing sequence data in the GenBank database for this particular coliphage we designed the beacons and primers based on the sequence of other coliphages in subgroup IV (Sp, NL95). We have since sequenced the entire genome of our isolate of Fi and determined a 2-base mismatch in our reverse primer with the others in the group.

Project Objectives for Next Reporting Period

Objectives
i. Design a new primer for subgroup IV that will accommodate all coliphages within this group.

ii. Determine if the four beacons can be used in a multiplexing reaction that will reduce the number of individual assays necessary for source tracking by a factor of four.

iii. Compare efficiency of multiplexed standards generated from RNA and DNA template.

iv. Continue to extend the database of F-specific coliphages from additional animal sources.

Tasks to Meet Objectives
i. The current primer will be shifted four bases and a mixed (A and G) at one base will be used.

ii. Finalize the multiplexing reaction to simultaneously amplify all four subgroups.

iii. Fresh fecal samples will be collected in Spring and F-specific coliphages will be isolated using standard enrichment protocols.

Anticipated Success in Meeting Project Objectives
i. There are no impediments in designing another primer.

ii. Based on the new sequence data for coliphage Fi, we are confident the problem will be solved. Failing that, the four subgroups can be quantified in two multiplexing reactions instead of one using the existing primers and beacons.

iii. This portion of the project is relatively straight forward and we anticipate no problems.

Overall Project Timeline Update
We anticipate no problems finishing the multiplexing PCR project by the end of the calendar year.

Preliminary Data
See FIgures.

Dissemination
Conferences:
Kirs, M and Smith, DC. 2005. Microbial Source Tracking: Multiplex Quantitative Real-Time RT-PCR Assay for F-Specific RNA Coliphages. 105th General Meeting of the American Society of Microbiology. June Atlanta, GA. (Abstract accepted)

Outreach:
Presentation on water quality issues to Pier Middle School science classes (Narragansett, RI)

Lecture “Microbial contamination of surface waters” to the Microbial Diversity class at Brown University.

Contact with End Users
We have discussed our progress with Dr. Rob Burgess at the EPA. He has expressed interest in apply our method as soon as it becomes available. We were also contacted by a local group in Washington State inquiring about costs to analyze samples.

Expenditures
All expenditures are in line with our previous estimates. During this reporting period, the student that is supported on this grant, Mr. Marek Kirs continues to make significant progress on this project and his PhD program.