Skip Links

USGS - science for a changing world

Scientific Investigations Report 2009–5097

In cooperation with the U.S. Environmental Protection Agency, National Homeland Security Research Center

Performance of Traditional and Molecular Methods for Detecting Biological Agents in Drinking Water

By Donna S. Francy, Rebecca N. Bushon, Amie M.G. Brady, Erin E. Bertke, Christopher M. Kephart, Christina A. Likirdopulos, Brian E. Mailot, Frank W. Schaefer, III, and H.D. Alan Lindquist


Thumbnail of and link to report PDF (700 kB)

To reduce the impact from a possible bioterrorist attack on drinking-water supplies, analytical methods are needed to rapidly detect the presence of biological agents in water. To this end, 13 drinking-water samples were collected at 9 water-treatment plants in Ohio to assess the performance of a molecular method in comparison to traditional analytical methods that take longer to perform. Two 100-liter samples were collected at each site during each sampling event; one was seeded in the laboratory with six biological agents—Bacillus anthracis (B. anthracis), Burkholderia cepacia (as a surrogate for Bu. pseudomallei), Francisella tularensis (F. tularensis), Salmonella Typhi (S. Typhi), Vibrio cholerae (V. cholerae), and Cryptospordium parvum (C. parvum). The seeded and unseeded samples were processed by ultrafiltration and analyzed by use of quantiative polymerase chain reaction (qPCR), a molecular method, and culture methods for bacterial agents or the immunomagnetic separation/fluorescent antibody (IMS/FA) method for C. parvum as traditional methods. Six replicate seeded samples were also processed and analyzed.

For traditional methods, recoveries were highly variable between samples and even between some replicate samples, ranging from below detection to greater than 100 percent. Recoveries were significantly related to water pH, specific conductance, and dissolved organic carbon (DOC) for all bacteria combined by culture methods, but none of the water-quality characteristics tested were related to recoveries of C. parvum by IMS/FA. Recoveries were not determined by qPCR because of problems in quantifying organisms by qPCR in the composite seed. Instead, qPCR results were reported as detected, not detected (no qPCR signal), or +/- detected (Cycle Threshold or “Ct” values were greater than 40). Several sample results by qPCR were omitted from the dataset because of possible problems with qPCR reagents, primers, and probes. For the remaining 14 qPCR results (including some replicate samples), F. tularensis and V. cholerae were detected in all samples after ultrafiltration, B. anthracis was detected in 13 and +/- detected in 1 sample, and C. parvum was detected in 9 and +/- detected in 4 samples. Bu. cepacia was detected in nine samples, +/- detected in two samples, and not detected in three samples (for two out of three samples not detected, a different strain was used). The qPCR assay for V. cholerae provided two false positive—but late—signals in one unseeded sample. Numbers found by qPCR after ultrafiltration were significantly or nearly significantly related to those found by traditional methods for B. anthracis, F. tularensis, and V. cholerae but not for Bu. cepacia and C. parvum. A qPCR assay for S. Typhi was not available.

The qPCR method can be used to rapidly detect B. anthracis, F. tularensis, and V. cholerae with some certainty in drinking-water samples, but additional work would be needed to optimize and test qPCR for Bu. cepacia and C. parvum and establish relations to traditional methods. The specificity for the V. cholerae assay needs to be further investigated. Evidence is provided that ultrafiltration and qPCR are promising methods to rapidly detect biological agents in the Nation’s drinking-water supplies and thus reduce the impact and consequences from intentional bioterrorist events. To our knowledge, this is the first study to compare the use of traditional and qPCR methods to detect biological agents in large-volume drinking-water samples.

First posted July 13, 2009

For additional information contact:
Director, Ohio Water Science Center
U.S. Geological Survey
6480 Doubletree Avenue
Columbus, OH 43229-1111

Part or all of this report is presented in Portable Document Format (PDF); the latest version of Adobe Reader or similar software is required to view it. Download the latest version of Adobe Reader, free of charge.

Suggested citation:

Francy, D.S., Bushon, R.N., Brady, A.M.G., Bertke, E.E., Kephart, C.M., Likirdopulos, C.A., Mailot, B.E., Schaefer, F.W., III, and Lindquist, H.D.A. 2009, Performance of traditional and molecular methods for detecting biological agents in drinking water: U.S. Geological Survey Scientific Investigations Report 2009–5097, 17 p.




Methods of Study

Sampling Procedures



Traditional Methods

Molecular Methods

Initial Processing and DNA Extraction

Quantitative PCR

Data Analysis and Statistics

Analytical Results and Method Performance

Water Quality

Standard Curves and Variability of Molecular Results

Microbial Recovery and Detection

Comparison of Number of Organisms Detected by Traditional and Molecular Methods

Suggestions for Future Study

Summary and Conclusions


References Cited

Accessibility FOIA Privacy Policies and Notices

Take Pride in America logo logo U.S. Department of the Interior | U.S. Geological Survey
Page Contact Information: Contact USGS
Page Last Modified: Thursday, 10-Jan-2013 19:32:48 EST