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OWML: Current Projects

Project Title: IMS/ATP rapid method optimization; QPCR for E. coli and enteric viruses

Project chief:  Rebecca N. Bushon

Project support:  Christopher M. Kephart, Christina A. Likirdopulos, and Erin A. Stelzer

Cooperators:  USGS Instrumentation Committee; USGS Office of Water Quality

Project duration:  2006-2008


Introduction and problem:
E. coli analysis by quantitative PCR (QPCR) and IMS/ATP rapid method are new methods that have shown promise by the USGS and other agencies. E. coli by QPCR is a method that is being explored as a new capability for the OWML. E. coli by IMS/ATP rapid method is currently being used for several projects by the OWML; however, it has been determined that some optimization should be done to decrease the variability seen by the method. Funding from ICOM was received in FY 2006 and 2007 to optimize and test both of these methods.

Enteric virus analysis by QPCR is another method that needs to be explored as a new capability for the OWML. Optimization and testing of QPCR for enterovirus, norovirus, and adenovirus began in FY 2007.

Goals and objectives:
The objectives of this work are to (1) develop the capability to analyze for E. coli using the QPCR method and (2) optimize specific steps in the IMS/ATP rapid method for use in future projects, and (3) develop the capability to analyze for enteric viruses by QPCR.

Approach:
A DNA extraction kit experiment will be run to compare the results of several commercially-available kits that are cited in the literature. Plans are to obtain 6 of these kits and test each one using three different types of QPCR methods: 1 fecal-indicator method (E. coli), 1 virus method (adenovirus), and 1 source-tracking marker (Bacteroides human marker). The DNA extraction step occurs prior to running the QPCR reaction and produces significant variability in QPCR results. Finding a kit that will provide consistent recoveries with minimal variability will be very beneficial in fully optimizing all of the QPCR assays.

E. coli by QPCR: New DNA extraction kits will be tested and compared to previously-used kits, which have shown significant variability and inefficiency. Reagents for QPCR that are considerably less expensive have been obtained from another source and will be compared to reagents that have been previously used. These changes to the method will be tested in positive control samples, as well as environmental samples. An internal memo with results of testing will be submitted to the OWQ in late 2008.

E. coli by IMS/ATP: New antibodies will be tested and compared to those previously used. Combinations of antibodies will also be tested in several sewage and environmental samples. New less expensive reagents are also being prepared and will be tested for comparability to those previously used. The lower detection limit of the method will also be determined. A journal article describing the method performance in sewage sources is planned for publishing this year in combination with other project work.

Enteric viruses by QPCR: Virus positive controls for three viruses (enterovirus, adenovirus, and norovirus) will be prepared using a plasmid prep kit and will be tested for use in the method. Standard curves for each of the three viruses will be generated. The detection of viruses will be tested in a variety of environmental samples. The OWML has had contacts from WSCs in Iowa and Wisconsin regarding this method and hope to provide this as a service to them. As a final product, the method for analysis of samples for enteric viruses by QPCR will be documented on the OWML Web site.

 

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