Detecting Biological Contaminants in Water Using Rapid PCR Technologies

technical B RIEP
3 EPA
3
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Detecting Biological Contaminants in Water, Using Rapid
Polymerase Chain Reaction (PCR) Technologies
Three PCR technologies evaluated for determining the presence of biotoxins in
water
In the past, people in the United States have largely
taken the convenience of potable municipal water for
granted. However, the threat of intentional contamination
of our water supplies is becoming a concern because of a
rise in the number of terrorist acts around the world. As a
result, there is much interest in technologies that can be
used to detect a contamination event as well as dispel or
confirm the credibility of a threat.
One of these technologies uses PCR to determine the
presence of specific bacteria in water in less than four
hours. PCR involves enzyme-mediated reactions that
replicate the deoxyribonucleic acid (DNA) of the target
organisms. This replication process is facilitated through a series of temperature cycles and
amplifies the amount of DNA until it reaches detectable levels.
In June 2004, EPA tested three rapid PCR technologies:
• TaqMan® E. coli 0157:H7 Detection System (Applied Biosystems)
• R.A.P.I.D.® System (Idaho Technology Inc.)
• PathAlert™ Detection Kits (Invitrogen Corporation)
EPA tested each technology's ability to detect specific biological contaminants as well as its
propensity to register false positive and false negative responses as a result of interfering
compounds. Because rapid PCR technologies are expected to serve mainly as screening tools
in water monitoring scenarios, this testing produces only qualitative results (i.e., results indicate
only the presence or absence of a contaminant, not a concentration level).
Evaluating each rapid PCR technology required two steps: a DNA extraction and purification
step, followed by an amplification and detection step. Each of the three rapid PCR technologies
was evaluated for:
• Accuracy
• Precision
• Specificity
• False positive/negative responses
• Interferences
• Other performance factors
U.S. EPA's Homeland Security Research Program
(HSRP) develops products based on scientific
research and technology evaluations. Our products
and expertise are widely used in preventing, preparing
for, and recovering from public health and
environmental emergencies that arise from terrorist
attacks. Our research and products address
biological, radiological, or chemical contaminants that
could affect indoor areas, outdoor areas, or water
infrastructure. HSRP provides these products,
technical assistance, and expertise to support EPA's
roles and responsibilities under the National
Response Framework, statutory requirements, and
Homeland Security Presidential Directives.
This document does not constitute nor should be construed as an EPA endorsement of any particular product,
service, or technology.

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Test Design
Table 1 identifies the rapid PCR technologies that were tested using various water types fortified
(i.e., spiked) separately with contaminants and interfering compounds.
Table 1. Technologies, Contaminants, and Interfering Compounds
Technologies
Contaminants
Interfering
Compounds
Applied Biosystems
TaqMan® E. coli 0157:H7
Detection System
Escherichia coli

Idaho Technology, Inc.
R.A.P.I.D.™ System
Franc/se//a tu/arensis
Yersinia pestis
Bacillus anthracis
Brucella suis
Escherichia coli
Humic Acid
Fulvic Acid
Invitrogen Corporation
PathAlert™ Detection Kits
Francisella tularensis
Yersinia pestis
Bacillus anthracis

Three types of water samples were tested in these evaluations: performance test (PT), drinking
water (DW), and quality control (QC). PT samples were prepared with deionized (Dl) water and
fortified with the target contaminant, an interferent, or both the contaminant and interferent.
Contaminant-only PT samples were tested in a series of concentrations that included the
accepted lethal/infective dose and approximately 2, 5, 10, and 50 times the vendor-stated
detection limit.
Both interference PT samples and DW samples were used to determine the technologies'
susceptibility to false positive and false negative results. DW samples were collected from four
geographically diverse municipal sources that varied in source (ground water or surface water),
treatment (filtered or unfiltered), and disinfection process (chlorination or chloramination).
Because real-world application of rapid PCR screening relies on preconcentration of the water
sample, approximately 100 L of DWwas dechlorinated and then concentrated to 250 ml_, using
an ultrafiltration sample concentration method.
The concentrated DW samples were analyzed after being spiked with each contaminant (one at
a time) at a concentration approximately 10 times greater than each PCR's detection limit, as
well as with no contaminant spike.
All PT and DW samples were analyzed in quadruplicate. The results of each replicate sample
set were reported as a ratio of the number of positive results to the total number of replicates
(e.g., 0/4, 1/4). Method blank QC samples consisted of 10% of all samples. The number
of positive and negative control samples supplied by the vendors varied.
Performance and Results
The accuracy of the technology was determined by dividing the number of positive responses
by the overall number of analyses of spiked contaminant-only PT samples. The precision of the
sample set replicates was determined by calculating the number of consistent responses for all
the sample sets. Responses were considered consistent if all four replicates had the same
June 2008
E PA/600/S-08/007
This document does not constitute nor should be construed as an EPA endorsement of any particular product,
service, or technology.

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results. Specificity was determined by dividing the number of negative responses by the total
number of unspiked samples.
A false positive rate was defined as the frequency of false positive results out of the total
number of unspiked samples.
A false negative rate was defined as the frequency of false negative results out of the total
number of spiked PT (contaminant and interferent) samples and spiked DW samples. Table 2
summarizes the results of the evaluation parameters for each technology.
Table 2. Summary of Results
Technology
Contaminant
Gene Target
Cone. Range
(cfu/m L)
Accuracy
Precision
Specificity
Total False
Positives
Total False
Negatives
TaqMan1
E. co/i 0157:H7
Detection System
Escherichia coli
N/A
500-106
100%
78%
96%
1 out of 24
0 out of 52

Francisella
1
2x103-4x105
100%
95%
88%
0 out of 24
0 out of 60

tularensis
2
100%
86%
96%
0 out of 24
4 out of 60

Yersinia pestis
1
2x103-5x10*
100%
100%
100%
0 out of 24
0 out of 56
R.A.P.I.D.®
System
2
100%
100%
100%
0 out of 24
2 out of 56

1

94%
76%
100%
0 out of 24
2 out of 56
Bacillus anthracis
2
2x103-5x10'
94%
86%
100%
0 out of 24
2 out of 56


3

100%
95%
100%
0 out of 24
0 out of 56

Brucella suis
N/A
2x103-5x10"
88%
90%
100%
0 out of 24
0 out of 56

Escherichia coli
N/A
2x103-5x10"
100%
100%
100%
0 out of 24
0 out of 52
Path Alert"''
Francisella
tularensis
N/A
2x10l-5x105
100%
95%
96%
0 out of 24
0 out of 60
Detection Kits
Yersinia pestis
N/A
2x102-5x103
100%
95%
96%
0 out of 24
0 out of 56

Bacillus anthracis
N/A
2x10'-5x105
100%
95%
100%
0 out of 22
0 out of 56
N/A not applicable
In general, test concentrations ranged from 1G2 to 106 colony-forming units per milliliter (cfu/mL).
The following is a summary of the testing:
•	Minimum accuracy across all of the samples was 88%, with nine instances of 100%
accuracy.
•	Precision was 76% or above, with three instances of 100% repeatability.
•	Specificity was 88% or above, with eight instances of 100% specificity.
•	There was one false positive result and six false negative results.
•	Four of six false negative results were due to matrix interferences.
•	100% accuracy, 100% precision, 100% specificity, no false positives, and no false
negatives were achieved for two contaminants, Yersinia pestis and Escherichia coli, using
the R.A.P.I.D.®System
Several bacteria were tested using different gene targets offered by Idaho Technology, Inc.
(R.A.P.I.D.® System) and were therefore evaluated more than once by that technology.
Infective/lethal doses for Yersinia pestis (0.28 cfu/mL), Bacillus anthracis (200 cfu/mL), Brucella
suis (40 cfu/mL), and Escherichia coli (0.2 cfu/mL) were below the technologies' detection limits
and were not included in the above results.
June 2008
EPA/600/S-08/007
This document does not constitute nor should he construed as an EPA endorsement of any particular product,
service, or technology.

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CONTACT INFORMATION
For more information, visit the EPA Web site at www.epa.gov/nhsrc.
Technical Contact: Eric Koglin (koglin.eric@epa.gov)
General Feedback/Questions: Kathy Nickel (nickel.kathy@epa.gov)
June 2008
E PA/600/S-08/007
This document does not constitute nor should be construed as an EPA endorsement of any particular product,
service, or technology.

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