|
Near-Real-Time Detection of Microbial Waterborne Agents to Inform Risk Analysis
Start Date 05/01/2007
End Date 04/30/2008
Primary Partner: Syracuse University
Primary Contact: Raina, Ramesh - Professor
Other Project Contacts:
Driscoll, Charles.T. - Co-Principal Investigator
Coleman, Margaret (Peg) - Co-Principal Investigator
Project Type: CARTI I
Technical Description:
Reported cases of waterborne disease (e.g., cryptosporidiosis) doubled in Upstate New York in 2005. Reducing the number of cases is clearly in the public interest, and determining the sources and conditions of the contamination is a logical first step in reducing these cases. The usefulness of water monitoring for pathogens is limited because efficient rapid methods for sampling and detecting pathogens are lacking. While water quality indicators are sometimes used as a surrogate to direct monitoring, no link exists between the indicators and many human diseases of concern, including cryptosporidiosis. Part of the problem in monitoring pathogens such as Cryptosporidium, and using the monitoring data as a human disease indicator, is that it is unclear what water concentrations of the organism are required to cause cryptosporidiosis. Low concentrations of Cryptosporidium (<50 oocysts/100L source water) in UK monitoring programs reportedly caused no disease. Local data are insufficient for microbial risk assessment and effective risk management. Optimization of local water systems requires an improved understanding of the supply, transport, and populations of pathogens.
Expected Outcomes:
The goal of this one-year pilot study was to develop Real Time-PCR (RT-PCR) based rapid and quantitative method to detect these pathogens and to generate preliminary data to inform the design of a comprehensive multi-year study that would characterize waterborne microbes in drinking waters and associated human disease.
Accomplishments:
• Results of our experiments have shown that RT-PCR is a sensitive method to detect Cryptosporidium and Giardia in lake water.
• We found that the most efficient method to isolate the DNA from oocysts and cysts is freeze-thaw followed by extraction using QIAamp DNA minikit (Qiagen).
• Use of BSA (100 μg/ml) was found to be most efficient in suppressing effects of PCR inhibitors.
• Based on the preliminary results of the reconstituted samples, the recovery of oocysts and cysts from the system blank samples was ~80% and from the lake water samples, ~50%.
• RT-PCR was found to be reproducible in plate-to-plate and well-to-well replicate experiments.
• Although our pilot experiments are quite encouraging, more replicate experiments will have to be done to develop confidence in this method to detect Cryptosporidium and Giardia in lake water.
Benefits:
Developing a reliable near real time method to identify waterborne pathogens in water sources will greatly enhance efforts to reduce human disease caused by these organisms.
For more information: http://biology.syr.edu/raina/index.html
Back To Research Projects List
|
