Evaluation of Real-Time Quantitative
Polymerase Chain Reaction (qPCR) to Estimate Escherichia coli
Concentrations at Lake Erie Beaches.
Project chief: Christopher Kephart
Project support: Rebecca Bushon
Northeast Ohio Regional Sewer District (NEORSD), Cleveland, Ohio
Introduction and problem:
There is a distinct need for a method to rapidly determine concentrations of fecal-indicator bacteria in recreational waters because current methods require at least 18-24 hours from sample collection to availability of results. Bacteria concentrations in the water can change appreciably within hours, and therefore, recreational users may be at risk of coming into contact with water that is not considered safe for recreation. Alternatively, recreational use and revenue may be lost if the beach is erroneously posted when the risk is low. One rapid method that has gained some favor amongst researchers is using the real-time quantitative polymerase chain reaction (qPCR) to quantify specific genetic targets within fecal-indicator bacteria.
Goals and objectives:
The overall goal of the project is to determine if the qPCR rapid method for E. coli quantification can be used at two Lake Erie beaches in Cleveland, Ohio, Edgewater and Villa Angela, to improve the timeliness and accuracy of beach advisories. The specific objectives of this study are to (1) field test a protocol for applying the qPCR rapid method for E. coli to lake-water samples, (2) compare results of the rapid method to the standard membrane-filtration method, and (3) transfer the technology to a local agency that routinely monitors for E. coli concentrations and provides a beach warning system based on their results.
During the recreational seasons (May–September) of 2006, 2007, 2008, and 2009 the Northeast Ohio Regional Sewer District (NEORSD) conducted or will be conducting a routine monitoring program for E. coli concentrations at Edgewater and Villa Angela beaches. This study augments that monitoring program by analyzing a portion of those samples by means of the qPCR rapid method. During each recreational season, 12 weeks of sampling included analysis by qPCR; one sample from each of the two beaches was analyzed three days per week. From 2006 – 2008, initial processing of water samples for qPCR was done by the NEORSD and required filtering 100 mL of water through a polycarbonate filter. The filters were shipped overnight on ice to the USGS Ohio Water Microbiology Laboratory (OWML) for subsequent DNA extraction and qPCR. During 2008, the qPCR technology was transferred from the USGS to the NEORSD and a lab-to-lab comparison was done on a portion of the samples collected. Training was done on-site at the NEORSD and continued support was provided by the USGS throughout the recreational season. Additionally in 2008, the method was optimized by incorporating new DNA extraction kits and qPCR reagents. For 2009, the NEORSD will be applying the most current DNA extraction and qPCR method for analysis and USGS scientists will provide support throughout the recreational season.
A USGS Scientific Investigations Report entitled “Evaluation of Real-Time Quantitative Polymerase Chain Reaction (qPCR) To Determine Escherichia coli Concentrations at Two Lake Erie Beaches” documents results from 2006 and 2007 will be published in 2009. Depending on funding, another report may be published documenting the most current results and the technology transfer to the NEORSD.