Progress Report

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

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

Figures
Figure 1

Figure 1
Figure 2

Figure 2
Project Objectives for This Reporting Period
Objectives
  1. Design a new primer for subgroup IV that will accommodate all known coliphages within this group.
  2. 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.
  3. Compare efficiency of multiplexed standards generated from RNA and DNA template.
  4. Continue to extend the database of F-specific coliphages from additional animal sources.

Tasks to meet objectives

  1. The entire genome of coliphage Fi was sequenced and compared to existing sequences in GenBank. A new beacon was designed and tested.
  2. Multiplex experiments were performed using all four coliphage groups and primer/beacon pairs.
  3. Standard curves using RNA and DNA standards were performed.
  4. Fecal samples were collected from donkey, cat, dog, chicken, coyote, cow, geese, sea gulls, pigs, rabbits, sheep, squirrel, turtle. Samples were collected from the Narragansett Waste Water Treatment Facility for testing as well.

Progress on Tasks

  1. The new primer pair was designed and synthesized.
  2. The experiments were conducted.
  3. The experiments were conducted.
  4. The experiments were conducted.

Acomplishments

  1. The new primer pair for Subgroup IV is functional.
  2. The sensitivity of the four primer/beacon sets were tested by challenging each subgroup target with a million fold excess of targets from the other three subgroups.
  3. There is no difference between the DNA standards and the RNA standards. This allows us to routinely use the DNA standards to calibrate the RT-PCR.
  4. F+ coliphages were isolated from cat and chicken feces and typed using the molecular becons.

Difficulties

  1. No difficulties were encountered with this task.
  2. No difficulties were encountered with this task.
  3. No difficulties were encountered with this task.
  4. Most of the animal feces did not yield F+ coliphage plaques with the traditional top-agar overlay method. We are now testing whether we can detect F+ coliphages with RT-PCR samples that do not yield plaques.

Project Objectives for Next Reporting Period

Objectives

  1. Determine the survival rate of F+ coliphages based on RT-PCR quantification and compare to the rate determined by plaquing. This will allow us to determine the relative sensitivity, as a function of time, of the new method.
  2. Continue to expand the database by collecting feces from additional animals.
  3. Mr. Marek Kirs will defend his PhD dissertation.
  4. Continue to extend the database of F-specific coliphages from additional animal sources.

Tasks to Meet Objectives

  1. Seed surface seawater collected from Narragansett Bay with a mixture of all four F+ coliphage subgroups and sample with time for RT-PCR and plaque forming units.
  2. Collect animal feces from local farms and wildlife. In addition to isolating plaques, the samples will be prepared for RT-PCR directly.
  3. A manuscript describing the method will be submitted for publication (Sep 2005) and included in the dissertation.

Anticipated Success in Meeting Project Objectives

  1. Now that the RT-PCR method is working routinely there are no anticipated problems.
  2. We are experimenting with additional RNA isolation techniques in order to extract the RNA coliphage directly from fecal samples. In addition, we are inserting a molecular size-based clean up step prior to amplification to avoid PCR inhibition.

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

Preliminary Data
Figure 1. Sensitivity of the real-time RT-PCR reactions. Fifty copies of cDNA from one subgroup is added to 107 copies of cDNA from each of the three remaining subgroups. Å€- subgroup I, ­ subgroup II, • subgroup III, £G subgroup IV. Standard curves: (A) subgroup I, (B) subgroup II, (C) subgroup III, and (D) subgroup IV.

Figure 2. Multiplexed RNA standard and DNA standards. (A) subgroup I, (B) subgroup II, (C) subgroup III, and (D) subgroup IV.

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. 6 ­ 9 June 2005. Atlanta, GA.

Outreach Activities:
Water quality lecture presented to environmental reporters through the Metcalf Institute for Marine and Environmental Reporting. The reporters then collected water samples from the Narrow River and performed microbial water quality assays in the laboratory.

Contact with End Users:
We have continued our discussions with Dr. Rob Burgess at the EPA. This resulted in coordinated sampling in the Providence River and Narragansett Bay coupling more traditional microbial water quality assays with our real-time PCR method for microbial source tracking.

Expenditures
The expenditure during this reporting period were less than anticipated. A nocost extension was requested in order to cover Mr. Marek Kirs through the Fall 2005 semester when he is fully expected to defend his PhD dissertation on this topic.