U.S. Department of the Interior

U.S. Geological Survey

Scientific Investigations Report 2009–5052

In cooperation with the Northeast Ohio Regional Sewer District

Evaluation of Real-Time Quantitative 

Polymerase Chain Reaction (qPCR) to 

Determine Escherichia coli Concentrations 

at Two Lake Erie Beaches

Euclid

Creek

Ninemile

Creek

Dugway

Brook

Doan

Brook

LAKE ERIE

Cuyahoga

River

Rocky

River

EDGEWATER

PARK

VILLA ANGELA

Cleveland

State Route 2

Study Area

OHIO

81o50' 81o

45' 81o40' 81o35'

Base map modified from U.S. Geological Survey

Cleveland North 1:24,000, 1963, photorevised

1973; Cleveland South, E. Cleveland, Lakewood

1:24,000, 1963, photorevised 1979; and North

Olmsted 1:24,000, 1963, photorevised 1985 0

0

1

1

2 3

2 3 4 MILES

4 KILOMETERS

41o30'

41o

35'

Evaluation of Real-Time Quantitative 

Polymerase Chain Reaction (qPCR) to 

Determine Escherichia coli Concentrations 

at Two Lake Erie Beaches 

By Christopher M. Kephart and Rebecca N. Bushon

In cooperation with the Northeast Ohio Regional Sewer District

Series Name 2009–5052

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

KEN SALAZAR, Secretary

U.S. Geological Survey

Suzette Kimball, Acting Director

U.S. Geological Survey, Reston, Virginia: 2009

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Although this report is in the public domain, permission must be secured from the individual copyright owners to 

reproduce any copyrighted materials contained within this report.

Suggested citation:

Kephart, C.M., Bushon, R.N., 2009, Evaluation of real-time quantitative polymerase chain reaction (qPCR) to determine 

Escherichia coli concentrations at two Lake Erie Beaches: U.S. Geological Survey Scientific Investigations Report 

2009–5052, 14 p..
iii

Contents

Abstract...........................................................................................................................................................1

Introduction.....................................................................................................................................................1

Methods of Study...........................................................................................................................................2

Sample Collection.................................................................................................................................2

Membrane Filtration and DNA Extraction/Purification ..................................................................3

qPCR Analysis........................................................................................................................................3

Quality-Assurance and Quality-Control Procedures ......................................................................3

Data Analysis.........................................................................................................................................4

Relations Between Results from qPCR and Culture-Based Methods ..................................................5

Summary and Conclusions...........................................................................................................................6

Acknowledgments.........................................................................................................................................8

References Cited............................................................................................................................................8

Appendix 1. Concentrations of Escherichia coli (E. coli) in colony-forming units per 

100 milliliters (CFU/100 mL) in composite water samples from Edgewater and

Villa Angela Beaches for the traditional culture-based method, quantitative 

polymerase chain reaction (qPCR) method, and predictions from the simple 

linear regression (SLR) model, 2006 and 2007 ...........................................................................10

Figures

1. Location of Edgewater and Villa Angela Beaches, Cleveland, Ohio....................................2

2. Example qPCR amplification plot showing the accumulation of fluorescence (Delta Rn) 

plotted against PCR cycle number.............................................................................................4

3. Compiled standard curve for E. coli...........................................................................................5

4. Escherichia coli concentrations as measured by qPCR compared to results obtained 

from the traditional culture-based method at Edgewater and Villa Angela beaches in 

2006 and 2007.................................................................................................................................6

Table

1. Method performance statistics related to the Escherichia coli bathing-water standard 

of 235 colony-forming units per 100 milliliters..........................................................................7
iv

Conversion Factors

Multiply By To obtain

Length

meter (m) 3.281 foot (ft) 

Volume

milliliter (mL) 0.03381 ounce, fluid (fl. oz)

microliter (μL) 0.00003381 ounce, fluid (fl. oz)

Mass

gram (g) 0.03527 ounce, avoirdupois (oz)

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows:

°F=(1.8×°C)+32

Concentrations of reagents are given in millimolar (mM) or micromolar (μM).

Concentrations of bacteria are given in colony-forming units per 100 milliliters (CFU/100mL). 
Abstract

During the recreational seasons of 2006 and 2007, the 

quantitative polymerase chain reaction (qPCR) method was 

used to determine Escherichia coli (E. coli) concentrations in 

samples from two Lake Erie beaches. Results from the qPCR 

method were compared to those obtained by traditional 

culturing on modified mTEC agar. Regression analysis showed 

strong, statistically significant correlations between results 

from the two methods for both years. Correlation coefficients 

at Edgewater and Villa Angela Beaches were 0.626 and 0.789 

for 2006 and 0.667 and 0.829 for 2007, respectively. Linear 

regression analyses were done to determine how well E. coli

concentrations could have been predicted from qPCR results. 

These hypothetical predictions were compared to the current 

practice of determining recreational water quality from 

E. coli concentrations determined for samples collected on the 

previous day. The qPCR method resulted in a greater percentage of correct predictions of water-quality exceedances than 

the current method for both beaches and both years. However, 

because regression equations differed somewhat between 

both sites and both years, the study did not result in any single 

relation reliable enough to use for actual real-time prediction 

of water-quality exceedances for either beach; therefore, a posterior analysis of data was done. Additional years of data may 

be needed to develop such a relation. Results from this study 

support the continued development and testing of a qPCR 

method for providing rapid and accurate estimates of E. coli

concentrations for monitoring recreational water quality.

Introduction

Recreational water-quality impairment due to fecal 

pollution can adversely impact the health of the public. Levels 

of impairment from fecal pollution are assessed on the basis 

of concentrations of bacterial indicator organisms. Traditional 

culture-based methods to determine fecal-indicator bacteria concentrations typically require 18–24 hours to obtain 

results. Studies have demonstrated that fecal-indicator bacteria 

concentrations can fluctuate rapidly (Leecaster and Weisberg, 

2001; Boehm and others, 2002; Boehm, 2007). The delay 

in obtaining results when using the culture-based method 

combined with the temporal variability of bacterial concentrations can result in incorrect assessments of the current quality 

of public waters. At beaches, posting of water-quality hazard 

advisories when the exposure risk is low can result in loss of 

revenue and recreational use (Rabinovici and others, 2004). 

In contrast, not posting advisories when the risk is high can 

result in the public coming into contact with water that 

contains unsafe levels of pathogenic microorganisms. 

One study has estimated that, for one frequently used coastal 

beach, as many as 40 percent of the advisories based on traditional methods for water-quality assessment were erroneous 

(Kim and Grant, 2004).

One way to improve the accuracy and efficiency of recreational water-quality monitoring programs is the use of rapid 

methods to reduce the time required to determine bacteria 

concentrations. Recent advancements in molecular and 

immunological techniques have led to development of various 

methods capable of yielding results in less than 4 hours from 

the time of sample collection, a short enough time to permit 

managers to make public health decisions based on currentday results (Griffith and Weisberg, 2006). Many of these rapid 

methods are based on enumeration of cellular components, 

such as genetic material or cell-surface structures. These 

methods typically are very sensitive so they can be vulnerable 

to interference from complex water-sample matrices. Consequently, substantial testing is required to determine whether 

they are capable of replacing traditional culture-based methods. Recent studies evaluating rapid-detection methods have 

found several to be promising, yet there is a need for further 

evaluation and epidemiological studies to establish direct 

relations of method results to public health risk (Griffith and 

others, 2004; Noble and Weisberg, 2005).

One rapid method uses real-time quantitative polymerase 

chain reaction (qPCR) to quantify fecal-indicator bacteria. 

This method, which detects and enumerates unique genetic 

sequences within target bacteria, can be done in less than 

3 hours. The enumeration relies on the accumulation of a 

fluorescent signal that is produced during PCR cycling. 

Evaluation of Real-Time Quantitative Polymerase 

Chain Reaction (qPCR) To Determine Escherichia coli

Concentrations at Two Lake Erie Beaches

By Christopher M. Kephart and Rebecca N. Bushon
2 Evaluation of Real-Time Quantitative Polymerase Chain Reaction to Determine Escherichia coli Concentrations

A fluorescent dye is released during replication of the target 

DNA sequence; accumulation of the dye is proportional to the 

amount of target DNA present. Quantification of bacteria in 

a sample is done by means of a standard curve. The standard 

curve is developed from qPCR results for a series of samples 

with known concentrations of the target organism. 

The qPCR method for quantifying fecal-indicator bacteria has been tested for enterococci, a U.S. Environmental 

Protection Agency (USEPA)-recommended indicator for 

marine water and freshwater (Haugland and others, 2005; 

Frahm and Obst, 2003; He and Jiang, 2005) and to a lesser 

extent, Escherichia coli (E. coli), a USEPA-recommended 

indicator for freshwater (Foulds and others, 2002; Frahm and 

Obst, 2003). Additionally, an epidemiological study was done 

relating results from the qPCR rapid method for enterococci to 

swimming-associated health effects (Wade and others, 2006). 

Although the qPCR method for E. coli quantification has been 

evaluated in recent studies, there is a need for better understanding of method performance in environmental waters, 

especially samples collected at beaches. 

The U.S. Geological Survey (USGS), in cooperation 

with the Northeast Ohio Regional Sewer District (NEORSD), 

evaluated a qPCR method for determining E. coli concentrations at two Lake Erie beaches, Edgewater and Villa Angela, 

in 2006 and 2007 (fig. 1). Both beaches are sand beaches; Villa 

Angela Beach is 900 feet in length and Edgewater Beach is 

1,500 feet in length. In 2006, a total of 617,000 people visited 

both beaches combined and in 2007, a total of 616,649 people 

visited both beaches combined. According to an estimate by 

Cleveland Lakefront State Park (managers of Edgewater and 

Villa Angela Beaches), at least 90% of the visitors during both 

years were to Edgewater Beach (oral communication, 

March 2, 2009).

This report describes the performance of the qPCR 

method as compared to traditional culturing on a modified 

mTEC medium. Performance of the qPCR method was 

also evaluated for accuracy in predicting exceedances of 

water-quality standards. An effective rapid method for 

determining E. coli concentrations would enable local waterresource managers to make more informed decisions and 

could enhance public safety during recreational water use. 

Methods of Study

Sample Collection

During the recreational seasons (May–September) 

of 2006 and 2007, the NEORSD conducted a routine monitoring program at Edgewater and Villa Angela Beaches. That 

program included collection of two water-quality samples at 

each beach, 5 days per week. The samples were individually 

analyzed for E. coli concentrations by use of the traditional 

Euclid

Creek

Ninemile

Creek

Dugway

Brook

Doan

Brook

LAKE ERIE

Cuyahoga

River

Rocky

River

EDGEWATER

PARK

VILLA ANGELA

Cleveland

State Route 2

Study Area

OHIO

81o50' 81o

45' 81o40' 81o35'

Base map modified from U.S. Geological Survey

Cleveland North 1:24,000, 1963, photorevised

1973; Cleveland South, E. Cleveland, Lakewood

1:24,000, 1963, photorevised 1979; and North

Olmsted 1:24,000, 1963, photorevised 1985 0

0

1

1

2 3

2 3 4 MILES

4 KILOMETERS

41o30'

41o

35'

Figure 1. Location of Edgewater and 

Villa Angela Beaches, Cleveland, Ohio.
Methods of Study 3

culture-based method. Additionally, the two samples from 

each beach were composited (Bertke, 2007) 3 days per week 

for 12 consecutive weeks, during both years. The composite 

samples were analyzed for E. coli concentrations by both the 

traditional and qPCR methods. The NEORSD collected all 

samples between 9:30 and 11:30 a.m where the water was 

1 m deep in an area of the beach used for swimming. All 

samples were collected into presterilized bottles approximately 

0.3 m below the water surface by means of a grab-sampling 

technique (Myers and others, 2007). Samples were placed on 

ice until analysis.

Membrane Filtration 

and DNA Extraction/Purification

Composited water samples were analyzed for concentrations of E. coli within 6 hours of collection at the NEORSD 

Laboratory by use of the modified mTEC membrane-filtration 

method 1603 of the USEPA (2006). Also at the NEORSD 

Laboratory, samples were processed for subsequent qPCR 

analysis by filtering 100 mL of the sample (or the maximum 

volume capable of passing through the filter) through a 0.4-μm 

polycarbonate filter (Whatman, Florham Park, N.J.). The filtration apparatus was rinsed copiously with phosphate-buffered 

saline (PBS). The filter was then aseptically folded and placed, 

with the perimeter of the filter first, inside a 2.0-mL screw-cap 

vial containing 0.3 g of acid-washed glass beads (Sigma, 

St. Louis, Mo.) and 550 μL of bead solution from the 

MO BIO UltraClean Fecal DNA Isolation Kit (MO BIO 

Laboratories, Carlsbad, Calif.). These vials were mixed briefly 

by vortexing and then stored at -70°C until further analysis by 

the USGS Ohio Water Microbiology Laboratory (OWML). 

Once the water-sample filters were received at the 

OWML, DNA was extracted from the filters and purified by 

use of a modified protocol for the MO BIO UltraClean Fecal 

DNA Isolation Kit. The initial steps of the DNA extraction-kit 

protocol were modified to accommodate the variation in 

starting material (filter-preserved DNA), and various wash 

steps were modified to enhance quantitative recovery. Briefly, 

the modifications included the following:

• Samples were processed in an eight-position mini bead 

beater (Biospec Corp., Bartlesville, Okla.) for 

2 minutes at maximum rate. 

• After centrifugation, entire supernatants were removed 

without disturbing the pellets, and subsequent reagent 

volumes were adjusted to the larger volumes of supernatant, proportionally. 

• Samples were bound to the spin filters by use of a vacuum manifold (Qiagen, Valencia, Calif.) amended with 

vacuum adapters (Promega Corp., Madison, Wis.).

The final extract volume for each sample was 50 μL. 

All DNA extracts were stored at 4°C until qPCR analysis, 

which was done within 24 hours of DNA extraction.

qPCR Analysis

Analysis by qPCR was accomplished by use of 

lyophilized bead-based reagents. The qPCR master mix was 

prepared by combining OmniMix HS beads (Cepheid, 

Sunnyvale, Calif.) and Scorpion E. coli primer and probe 

beads (Cepheid) in a microcentrifuge tube and reconstituting 

with molecular-grade water. Each OmniMix HS bead was adequate for two 25-μL reactions containing 1.5 units of TaKaRa 

hot start Taq polymerase, 200 μM deoxyribonucleotides, 

4 mM magnesium chloride, and 25 mM HEPES buffer. 

Each Scorpion bead was adequate for four 25-μL reactions 

containing 0.25 μM of a proprietary primer-probe complex 

targeting E. coli species. qPCR reactions were processed in 

optical 96-well reaction plates (Applied Biosystems, Foster 

City, Calif.) as 25-μL volumes consisting of 20 μL of the 

qPCR master mix and 5 μL of DNA extract template. The 

reactions took place in an Applied Biosystems 7500 Real-Time 

PCR System. Thermal cycling conditions were as follows: 

2 minutes at 95°C, followed by 45 cycles of 5 seconds at 95°C 

and 43 seconds at 62°C.

A standard curve was developed to quantify environmental samples. The standards were created from a pure culture 

of E. coli, American Type Culture Collection (ATCC) 25922, 

grown at 36°C in tryptic soy broth for 18 hours with shaking. 

The pure culture was diluted 1:10 in PBS. Initial attempts at 

extracting DNA from the undiluted culture were unsuccessful (data not shown); therefore, a 1:10 dilution was necessary 

prior to extraction. The pure culture was diluted further to 

determine concentrations of E. coli by use of USEPA method 

1603 (2006). The 1:10 dilution of pure culture was prepared 

for DNA extraction by filtering in duplicate through a polycarbonate filter and storing briefly at -70°C, in the same manner 

as water samples. Filters were then processed through DNA 

extraction and purification in the same manner as above. 

Resulting DNA extracts were each serially diluted 1:10 to a 

final dilution (including the 1:10 dilution done before DNA 

extraction) of 10-9. The final five dilutions from each of the 

duplicate DNA extracts (10-5–10-9) were analyzed by qPCR in 

triplicate in the same manner as above. A compiled standard 

curve that included three standard curve runs on different dates 

was used for final data analysis.

Quality-Assurance and Quality-Control 

Procedures

General laboratory quality-assurance (QA) procedures 

followed those of Francy and others (2007). Quality-control 

samples included whole-method, DNA extraction, and qPCR 

controls. 

Two whole-method control samples were run, consisting 

of field blanks (one per recreational season) and split replicate 

samples (one per week). Field blanks were processed by 

pouring approximately 500 mL of sterile PBS into a sterile 

collection bottle in the field and then subjecting them to the 
4 Evaluation of Real-Time Quantitative Polymerase Chain Reaction to Determine Escherichia coli Concentrations

same conditions and processes as the beach samples. Split 

replicate samples were processed by filtering a separate aliquot 

of water from the same sample bottle. 

DNA extraction controls consisted of a negative and positive extraction control, which were included with each DNA 

extraction run. The negative extraction control was processed 

by filtering approximately 100 mL of PBS and preserving the 

filter, as described for beach samples. The positive extraction 

control was processed by adding 1 μL of DNA extract from 

one of the standards for the standard curve to 4 μL of negative 

control DNA extract.

Quality-control samples for qPCR included no-template 

controls (NTCs), matrix spikes, and qPCR positive controls. 

The NTC was processed once per qPCR run by using sterile 

molecular-grade water as the DNA template. The effect of the 

matrix on qPCR was assessed by processing matrix spikes. 

In 2006, matrix spikes were analyzed by qPCR for every 

sample that was processed. In 2007, matrix spikes were 

analyzed for only a portion of the sample set, approximately 

one for every eight samples. Matrix spikes were processed by 

adding 1 μL of DNA extract from one of the standards for the 

standard curve to 4 μL of sample DNA extract to serve as the 

DNA template for qPCR. The qPCR positive control was processed by adding 1 μL of DNA extract from the same standard 

as was used for the matrix spike to 4 μL of sterile, moleculargrade water.

Data Analysis

After completion of the qPCR analyses, various settings 

in the Sequence Detection Software (SDS) (Applied 

Biosystems) were optimized to analyze the data. The optimized settings were established on the basis of the standardcurve output, and these settings were applied to each run of 

environmental samples. Output from the analysis is displayed 

in an amplification plot, where the increase in reporter dye 

fluorescence (Delta Rn) is plotted against PCR cycle number, 

as illustrated in figure 2. First, the baseline (initial cycles of 

PCR in which little change in the fluorescent signal is seen) 

was defined manually. A threshold value was then set manually as a level of increasing fluorescence that was above the 

baseline and within the exponential phase of the amplification 

curve. Cycle threshold (Ct) values were calculated by the SDS 

software for each reaction. A Ct value is the cycle at which the 

sample fluorescence passes the threshold; higher concentrations of the target sequence result in a lower Ct value because 

the fluorescent signal meets the threshold earlier in the PCR 

amplification. 

Conversion of Ct values to E. coli concentrations was 

accomplished by plotting the Ct values from each standard 

against log10-transformed E. coli concentrations that were 

determined by the traditional culture-based method. A line 

Figure 2. Example qPCR amplification plot showing the accumulation of fluorescence (Delta Rn) plotted against 

PCR cycle number.

1.000e+000

1.000e-001

1.000e-002

1.000e-003

1.000e-004

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

DELTA RN

CYCLE NUMBER
Relations Between Results from qPCR and Culture-Based Methods 5

of best fit was applied to this plot (fig. 3), and the resulting 

equation used to describe this line was used to calculate 

sample E. coli concentrations based on their Ct values. 

Correlations between the qPCR and the traditional 

culture-based method results were determined by computing 

Pearson’s correlation coefficient (r). In this study, the r-values 

were considered statistically significant if the p-values were 

less than 0.05. For data analysis, all nondetects (Ct values 

greater than 40) were set to an upper limit of 40 prior to determining CFU/100 mL from the standard curve. 

Concentrations calculated from the qPCR standard curve 

were commonly higher than concentrations obtained from the 

traditional culture-based method. Consequently, to compare 

the qPCR results to the recreational bathing-water standard, 

simple linear regression models were developed for each year 

at each beach to “predict” (in retrospect) cultivable bacterial 

concentrations as a function of the estimated concentration as 

determined by qPCR. Pearson’s correlation coefficients and 

simple linear regression equations were both computed from 

log10-transformed data.

Relations Between Results from qPCR 

and Culture-Based Methods

E. coli concentrations determined by the traditional 

culture-based method generally were higher during the 2007 

recreational season than in 2006. E. coli concentrations from 

composited samples collected at Edgewater Beach ranged 

from 13 to 760 CFU/100 mL with a median value of 

75 CFU/100 mL in 2006 and ranged from 10 to 1,800 

CFU/100 mL with a median value of 120 CFU/100 mL in 

2007 (Appendix 1–1). E. coli concentrations from composited samples taken at Villa Angela Beach ranged from 4 to 

4,100 CFU/100 mL with a median value of 230 CFU/100 mL 

in 2006 and ranged from 10 to 24,000 CFU/100 mL with a 

median value of 400 CFU/100 mL in 2007 (Appendix 1–2).

The standard curve for E. coli that was used for interpretation of qPCR data was determined from composited data 

that was collected on three separate dates (fig.3). The standard curve had an R2

 value of 0.9497, with an amplification 

efficiency of 91.1 percent and a dynamic range of 14–23,000 

CFU/100 mL. The amplification efficiency was calculated by 

use of the equation 10(-1/slope) – 1. The dynamic range of the 

standard curve is the lowest and highest concentrations of the 

standards that were used in the regression analysis. The following equation for the standard curve was used to compute 

concentrations based on Ct values:

Predicted log CFU/100 mL = (Ct value – 44.835)/ – 3.5551

Statistically significant linear correlations were found 

between results from the qPCR method and the traditional 

culture-based method for E. coli (fig. 4). At Edgewater Beach, 

35 samples in 2006 and 45 samples in 2007 were analyzed by 

the two methods. Of the 45 samples analyzed in 2007, 17 did 

not have qPCR results as a result of a quality-control failure 

(duplicate qPCR reactions did not produce similar Ct values); 

these samples were not included in further statistical analyses. 

Outside of these 17 samples, this phenomenon has not been 

observed. Precision issues were resolved in other datasets, and 

Figure 3. Compiled standard curve for Escherichia coli. R2 = 0.9497
6 Evaluation of Real-Time Quantitative Polymerase Chain Reaction to Determine Escherichia coli Concentrations

no other data were discarded. The Pearson’s correlation coefficients relating qPCR method results and culture-based method 

results were 0.626 (p<0.0001) in 2006 and 0.667 (p<0.0001) 

in 2007. At Villa Angela Beach, 36 samples were analyzed in 

2006 and 38 samples in 2007. The Pearson’s correlation coefficients were 0.789 (p<0.0001) in 2006 and 0.829 (p<0.0001) 

in 2007. 

As shown in figure 4, the relation between the qPCR 

estimations of E. coli concentrations and the culture-based 

E. coli concentrations appeared to be different for each year. 

To determine whether the two years of data could be 

combined for each beach, the slopes of the regression 

equations were compared to determine whether they were 

statistically different (University of California, Los Angeles, 

[2008]). A p-value of less than 0.05 would indicate that the 

slopes were statistically different and that the data could not be 

combined. For both beaches, using a students t-test, the slopes 

were found to be statistically different (t = -2.13, p = 0.0369 

for Edgewater; t = -2.84, p = 0.0059 for Villa Angela); therefore, a single relation could not be used to describe the relation 

for both years at a given beach. 

Figure 4. Escherichia coli concentrations as measured by qPCR compared to results obtained from the traditional culture-based 

method at Edgewater and Villa Angela beaches in 2006 and 2007.

Edgewater

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00

log CFU/100 mL qPCR

lo

g

C

F

U/100 m

L

culture-b

a

s

e

d

m

etho

d

2006

2007

Villa Angela

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00

log CFU/100 mL qPCR

lo

g

C

F

U/100 m

L

culture-b

a

s

e

d

m

etho

d

2006

2007
Summary and Conclusions 7

As seen in the data (Appendix 1) and in the literature 

(He and Jiang, 2005; Haugland and others, 2005), the estimated bacterial concentrations, as determined by qPCR, can 

be significantly higher than concentrations determined by 

the traditional method. These findings highlight the need for 

developing new standards based on epidemiological studies 

using qPCR. Overestimation by the qPCR method can occur 

because qPCR detects target DNA regardless of cell viability. 

Consequently, simple linear regression analysis was done to 

predict E. coli concentrations from qPCR results, by site, for 

each year. These predictions, as well as the current procedure 

for determining recreational water quality (using the previous 

day’s E. coli concentrations), were compared. 

The percentage of correct classifications, false positives, and false negatives of the qPCR method with respect 

to exceedances of the recreational bathing-water standard of 

235 CFU/100 mL are listed in table 1. These percentages are 

a proportion of the total number of samples collected. False 

positive results occurred when the E. coli concentration was 

below the standard as measured by the traditional culturebased method, but qPCR results predicted exceedance of the 

standard. False negative results, which are important in that 

they result in a greater risk to human health, occurred when 

the E. coli concentration exceeded the standard as measured 

by the traditional culture-based method, but qPCR results 

predicted the concentration was below the standard. 

Table 1 also shows the statistical sensitivity and specificity results for the traditional culture-based and qPCR methods. 

Sensitivity is defined as the percentage of time a method result 

indicated an exceedance of the standard when the standard 

was actually exceeded according to culture-based analysis 

of samples collected that day. Specificity is defined as the 

percentage of time a method result indicated nonexceedance of 

the standard when culture-based analysis of samples collected 

that day indicated the standard was not exceeded.

Split replicate sample results for qPCR were combined 

for both beaches and years for a total of 20 replicate pairs. 

On the basis of a paired t-test on the log10-transformed data, 

the difference between replicate sample results was not 

statistically different from zero (t = 0.1702, p = 0.8666). 

This indicates that there is no statistical difference between 

replicate sample results.

Summary and Conclusions

Traditional culture-based methods for determining fecal 

indicator bacteria concentrations in recreational waters require 

about 18–24 hours to produce results, often leading to inaccuracies in public health reporting systems such as beach waterquality advisories. Recent emergence of rapid methods such as 

qPCR for determining fecal indicator concentrations may help 

solve the latency issue. Some advantages of using qPCR are 

that the method is both sensitive and specific (Mackay, 2004). 

An additional benefit of using qPCR is that the method is 

versatile and can be amended to detect different targets. 

The qPCR method can be applied for other purposes such as 

the direct quantification of pathogens (Guy and others, 2003; 

He and Jiang, 2005) and quantification of source-specific 

molecular markers (Layton and others, 2006; Noble and 

others, 2006). These applications can help answer other 

public-health research questions and provide an increased 

understanding of the microbial water quality at affected sites. 

In this study, E. coli concentrations were determined by 

use of qPCR for samples collected during the recreational 

seasons of 2006 and 2007 from two Lake Erie beaches, 

Edgewater and Villa Angela. Results were compared to 

concentrations determined by means of the traditional method 

involving membrane filtration on modified mTEC agar. 

Table 1. Method performance statistics related to the Escherichia coli bathing-water standard of 235 colony-forming units per 

100 milliliters

Year Predictive tool Sample 

size

Correct

 responsesa

False 

positivesa

False 

negativesa Sensitivityb Specificityb

Edgewater Beach

2006 qPCR 35 94% (33/35) 0% (0/35) 5.7% (2/35) 0% (0/2) 100% (33/33)

Previous day's result 24 83% (20/24) 8.3% (2/24) 8.3% (2/24) 0% (0/2) 91% (20/22)

2007 qPCR 28 75% (21/28) 7.1% (2/28) 18% (5/28) 58% (7/12) 88% (14/16)

Previous day's result 25 64% (16/25) 12% (3/25) 24% (6/25) 40% (4/10) 80% (12/15)

Villa Angela Beach

2006 qPCR 36 78% (28/36) 11% (4/36) 11% (4/36) 76% (13/17) 79% (15/19)

Previous day's result 25 52% (13/25) 24% (6/25) 24% (6/25) 50% (6/12) 54% (7/13)

2007 qPCR 38 87% (33/38) 7.9% (3/38) 5.3% (2/38) 91% (21/23) 80% (12/15)

Previous day's result 33 73% (24/33) 9.1% (3/33) 18% (6/33) 70% (14/20) 77% (10/13)

a

 In parentheses, number of responses divided by the total number of samples analyzed.

b

 In parentheses, number of responses divided by the number of exceedances (sensitivity) or nonexceedances (specificity).
8 Evaluation of Real-Time Quantitative Polymerase Chain Reaction to Determine Escherichia coli Concentrations

Statistically significant correlations between results from the 

qPCR method and results from the culture-based method were 

found for both beaches, during both years of data collection, 

with stronger correlations observed at Villa Angela Beach. 

The magnitude of correlation determined by Haugland and 

others (2005) between the qPCR method and the culture-based 

method for quantification of enterococci, another fecal-indicator microorganism, was similar to those reported in this study 

(r = 0.68). Griffith and Weisberg (2006) reported correlation 

coefficients ranging from 0.44 to 0.96 for enterococci and 

0.44 to 0.79 for E. coli in an evaluation study of multiple 

rapid-method technologies.

At each beach, data from the two sampling years were 

not combined because of a statistical difference in the slopes 

of the regression equations relating qPCR-based estimates 

of E. coli concentrations to culture-based concentrations. 

One possible reason for the different regressions for the two 

years may be from using two different lot numbers of DNA 

extraction kits. In a separate study, anecdotal data suggest that 

DNA-extraction efficiencies can vary significantly between lot 

numbers of a commercially available extraction kit. 

Estimated E. coli concentrations determined from the 

qPCR standard curve were frequently higher than concentrations determined by the traditional culture-based method. This 

may result from detection of nonculturable organisms or free 

genetic material in the water by the qPCR method. Although 

this may be viewed as a disadvantage in comparison to the 

traditional methods, these inputs still provide information 

on levels of fecal contamination. Additionally, culture-based 

methods may be underestimating contamination levels by not 

quantifying viable but nonculturable cells (Tamanai-Shacoori 

and others, 1996). 

For the purposes of this study, estimated concentrations 

from qPCR results were transformed by use of single linear 

regression models so that results could be compared with the 

current practice of predicting exceedances of the water-quality 

standard using the previous day’s E. coli concentrations. 

Comparing the methods in this way is unrealistic given that 

the transformed results were based upon a regression developed using data collected during an entire recreational season. 

At both beaches, during both years, results obtained from 

qPCR provided more accurate predictions compared to results 

obtained from the previous day’s E. coli concentrations 

(table 1); however, because of the posterior data analysis, 

results from this study could not have been provided rapidly.

The qPCR method had a lower percentage of false 

positive and false negative results than use of the previous 

day’s culture-based results (the traditional method), and it 

had greater sensitivity and specificity (with the exception of 

Edgewater Beach in 2006, where both methods were unable to 

predict two exceedances and had a sensitivity of 0 percent). In 

the cases where water-quality standards were exceeded and the 

water was considered unsafe for recreation, the results from 

the qPCR method would have resulted in fewer opportunities for public contact with levels of fecal contamination that 

exceeded the standard. For example, at Villa Angela Beach 

during the 2007 recreational season, the qPCR method would 

have resulted in posted advisories for 21 of the 23 exceedances, whereas the traditional method correctly 

predicted only 14 out of 20 exceedances.

Findings from a separate study support the use of qPCR 

at Villa Angela Beach for assessing recreational water quality (Francy and Darner, 2008). Previous studies at this beach 

(2004–7) showed that predictive modeling relying on environmental factors alone for estimating E. coli concentrations 

did not result in a more accurate forecast than the traditional 

method. Results from this study suggest that forecast improvements may be gained from other approaches, such as rapid 

methods.

Based on this study, use of qPCR to determine E. coli

concentrations at recreational beaches may be a viable option 

for obtaining rapid results. Although this study demonstrates 

the promise of this method, adoption of qPCR as the standard 

method for determining fecal indicator concentrations for 

regulatory purposes is not yet practical. Future work should 

include development of a consensus methodology, a simplification of methods—including laboratory techniques and data 

analysis—and development of standard quality-control measures. Ultimately, epidemiological studies would be needed to 

relate qPCR results to the incidence of waterborne illness so 

that new standards can be developed.

Acknowledgments

The authors thank Rachel Noble and Denene Blackwood 

from the University of North Carolina for providing the qPCR 

protocol and guidance in using the method. The authors also 

thank Benjamin Tedrick, Eva Hatvani, Mark Citriglia, and 

Lester Stumpe from Northeast Ohio Regional Sewer District 

for their assistance in providing water samples, laboratory 

processing, and project planning. Project-related support was 

provided by the Ohio Department of Health.

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10 Evaluation of Real-Time Quantitative Polymerase Chain Reaction to Determine Escherichia coli Concentrations

Appendix 1.

Appendix 1–1. Concentrations of Escherichia coli (E. coli) in colony-forming units per 100 milliliters (CFU/100 mL) in composite 

water samples from Edgewater Beach for the traditional culture-based method, quantitative polymerase chain reaction (qPCR) 

method, and predictions from the simple linear regression (SLR) model, 2006 and 2007.—Continued

[--, not determined]

Date

E. coli – culture-based 

method, CFU/100 mL

E. coli – qPCR E. coli – SLR

predicted CFU/100 mL Average Ct value Estimated CFU/100 mL

6/7/2006 13 40.00 23 23

6/8/2006 36 38.94 46 28

6/12/2006 97 36.09 290 49

6/14/2006 17 34.43 840 67

6/19/2006 61 36.71 190 43

6/20/2006 92 32.68 2,600 93

6/21/2006 62 30.03 19,000 170

6/26/2006 40 37.24 180 42

6/27/2006 42 38.13 150 40

6/28/2006 88 34.37 1,800 84

7/6/2006 88 33.92 2,400 91

7/10/2006 170 31.86 4,500 110

7/11/2006 130 34.60 760 65

7/12/2006 100 32.89 2,300 90

7/17/2006 39 38.09 79 33

7/18/2006 330 32.00 8,200 130

7/19/2006 140 34.66 730 64

7/24/2006 15 38.98 44 28

7/25/2006 92 33.66 1,400 78

7/26/2006 75 34.49 810 66

7/31/2006 54 32.36 3,200 99

8/1/2006 39 30.55 10,000 140

8/2/2006 45 30.50 11,000 140

8/7/2006 160 29.72 18,000 170

8/8/2006 130 33.93 1,200 74

8/9/2006 26 35.84 340 51

8/14/2006 97 33.03 2,100 88

8/15/2006 760 30.35 12,000 150

8/16/2006 28 34.33 900 68

8/21/2006 82 34.70 710 63

8/22/2006 34 34.88 630 61

8/23/2006 56 35.03 570 59

9/5/2006 46 37.99 84 34

9/6/2006 190 32.55 2,900 97

9/7/2006 180 31.73 4,900 110

6/12/2007 130 37.78 96 120

6/13/2007 800 35.46 430 290

6/14/2007 21 37.86 92 120

6/19/2007 32 40.00 23 52

6/20/2007 590 32.59 1,800 670
Appendix 1. 11

Appendix 1–1. Concentrations of Escherichia coli (E. coli) in colony-forming units per 100 milliliters (CFU/100 mL) in composite 

water samples from Edgewater Beach for the traditional culture-based method, quantitative polymerase chain reaction (qPCR) 

method, and predictions from the simple linear regression (SLR) model, 2006 and 2007.—Continued

[--, not determined]

Date

E. coli – culture-based 

method, CFU/100 mL

E. coli – qPCR E. coli – SLR

predicted CFU/100 mL Average Ct value Estimated CFU/100 mL

6/21/2007 470 34.23 960 460

6/26/2007 68 40.00 23 52

6/27/2007 42 40.00 23 52

6/28/2007 700 37.29 260 220

7/3/2007 10 --a -- --

7/5/2007 53 38.36 66 97

7/6/2007 410 36.01 330 250

7/9/2007 26 --a -- --

7/10/2007 33 --a -- --

7/11/2007 100 33.94 1,200 530

7/12/2007 370 --a -- --

7/13/2007 120 --a -- --

7/17/2007 65 40.00 23 52

7/18/2007 47 --a -- --

7/19/2007 1,500 --a -- --

7/24/2007 55 --a -- --

7/25/2007 110 37.54 110 130

7/26/2007 160 --a -- --

7/27/2007 250 37.12 150 160

7/31/2007 13 40.00 23 52

8/1/2007 36 40.00 23 52

8/2/2007 17 40.00 23 52

8/6/2007 500 35.88 330 250

8/8/2007 1,800 33.98 1,100 500

8/9/2007 840 36.16 280 220

8/10/2007 600 37.18 140 150

8/14/2007 54 --a -- --

8/15/2007 120 36.46 230 200

8/16/2007 82 --a -- --

8/17/2007 390 --a -- --

8/20/2007 300 --a -- --

8/21/2007 140 37.67 100 120

8/22/2007 80 --a -- --

8/23/2007 580 --a -- --

8/24/2007 510 39.74 27 57

8/27/2007 44 --a -- --

8/28/2007 51 34.78 670 370

8/29/2007 120 --a -- --

8/30/2007 550 33.25 1,800 670

8/31/2007 110 36.92 170 170

a

 Ct value was not obtained because of a quality-control failure.
12 Evaluation of Real-Time Quantitative Polymerase Chain Reaction to Determine Escherichia coli Concentrations

Appendix 1–2. Concentrations of Escherichia coli (E. coli) in colony-forming units per 100 milliliters (CFU/100 mL) in composite 

water samples from Villa Angela Beach for the traditional culture-based method, quantitative polymerase chain reaction (qPCR) 

method, and predictions from the simple linear regression (SLR) model, 2006 and 2007.—Continued

Date

E. coli – culture-based 

method, CFU/100 mL

E. coli – qPCR E. coli – SLR

predicted CFU/100 mL Average Ct value Estimated CFU/100 mL

6/7/2006 370 35.36 460 45

6/8/2006 230 33.46 1,600 92

6/12/2006 27 32.13 3,700 150

6/14/2006 84 37.26 140 22

6/19/2006 220 32.00 4,100 160

6/20/2006 920 28.79 33,000 540

6/21/2006 270 31.04 18,000 380

6/26/2006 280 31.40 6,000 200

6/27/2006 130 33.03 2,100 110

6/28/2006 520 29.28 24,000 450

7/6/2006 160 31.15 14,000 330

7/10/2006 960 29.04 28,000 490

7/11/2006 370 30.36 12,000 300

7/12/2006 4,100 26.89 110,000 1,100

7/17/2006 300 31.67 5,100 180

7/18/2006 980 29.35 23,000 440

7/19/2006 110 35.37 460 45

7/24/2006 1,000 28.11 51,000 690

7/25/2006 430 29.32 23,000 440

7/26/2006 510 31.64 5,100 180

7/31/2006 44 33.35 1,700 96

8/1/2006 260 30.02 15,000 340

8/2/2006 230 27.08 99,000 1,000

8/3/2006 250 30.27 12,000 300

8/7/2006 460 28.56 38,000 580

8/8/2006 140 29.54 20,000 400

8/9/2006 4 36.41 230 30

8/14/2006 70 33.07 2,000 100

8/15/2006 2,000 27.45 78,000 890

8/16/2006 40 29.85 16,000 350

8/21/2006 41 34.26 950 68

8/22/2006 140 34.42 850 64

8/23/2006 11 34.89 630 54

9/5/2006 5 40.00 23 8

9/6/2006 230 32.50 3,000 130

9/7/2006 8 40.00 23 8
Appendix 1. 13

Appendix 1–2. Concentrations of Escherichia coli (E. coli) in colony-forming units per 100 milliliters (CFU/100 mL) in composite 

water samples from Villa Angela Beach for the traditional culture-based method, quantitative polymerase chain reaction (qPCR) 

method, and predictions from the simple linear regression (SLR) model, 2006 and 2007.—Continued

Date

E. coli – culture-based 

method, CFU/100 mL

E. coli – qPCR E. coli – SLR

predicted CFU/100 mL Average Ct value Estimated CFU/100 mL

6/12/2007 59 36.72 230 47

6/13/2007 1,300 32.39 3,200 620

6/14/2007 3,100 30.43 11,000 2,100

6/19/2007 1,400 31.80 4,600 890

6/20/2007 7,600 30.34 12,000 2,300

6/21/2007 1,800 31.85 4,500 870

6/22/2007 380 32.72 2,600 510

6/26/2007 130 35.50 420 84

6/27/2007 790 29.46 21,000 3,900

6/28/2007 3,000 31.01 7,700 1,500

7/3/2007 4,600 32.18 3,600 700

7/5/2007 100 35.77 360 73

7/6/2007 620 33.26 1,800 350

7/9/2007 1,600 30.02 15,000 2,800

7/10/2007 510 31.08 7,400 1,400

7/17/2007 24,000 28.32 44,000 8,200

7/18/2007 540 33.56 1,500 300

7/19/2007 8,600 30.75 18,000 3,400

7/24/2007 80 34.77 680 140

7/25/2007 93 35.89 330 67

7/26/2007 230 35.53 410 83

7/27/2007 1,600 31.80 4,600 890

8/1/2007 18 36.60 210 43

8/2/2007 170 34.28 930 180

8/6/2007 1,300 33.38 1,700 330

8/10/2007 280 34.81 660 130

8/14/2007 130 35.83 340 69

8/15/2007 220 31.84 4,500 870

8/17/2007 410 30.66 9,100 1,700

8/20/2007 240 33.90 1,200 240

8/21/2007 270 35.72 370 75

8/22/2007 60 33.20 1,900 370

8/23/2007 7,500 28.63 36,000 6,700

8/24/2007 680 30.10 14,000 2,600

8/27/2007 10 35.78 350 71

8/28/2007 32 35.28 490 98

8/30/2007 100 33.27 1,800 350

8/31/2007 74 34.00 1,100 220
14 Evaluation of Real-Time Quantitative Polymerase Chain Reaction to Determine Escherichia coli Concentrations

Kephart and Bushon—Evaluation of Real-Time Quantitative Polymerase Chain Reaction to Determine Escherichia coli Concentrations—SIR 2009–5052

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