EPA NEW ENGLAND
REGIONAL LABDRATDRY
ENVIRONMENTAL
SCIENCE FACT SHEET
r ARTNERS
RELATED
PROGRAMS
•EPA New England, Boston, MA
•EPA Office of Research &
Development, Cincinnati, OH
•USGS
•UMASS/Boston, Dept of Biology
•UMASS/Amherst, Dept. of Civil
Engineering
•MA Dept. of Environmental
Protection
•MA Water Resource Authority
•MA Dept. of Conservation &
Recreation
WONTACTS
EPA New England Regional
Laboratory
11 Technology Dr.
North Chelmsford, MA
01863
1(888) 372-7341 (in NE)
1(617)918-8300
www.epa.gov/ne/lab
PnLYMERASE CHAIN REACTION
I INTRODUCTION
A/most one quarter of the 2,840 listed polluted waters of New England do not meet
water quality criteria for bacteria. Traditional microbial test methods limit state,
federal and/or tribal regulators' ability to implement appropriate and timely control
measures and/or to assess human health risk. Traditional methods only identify and
quantify fecal pollution in water. New analytical tools are needed to identify fecal
pollution sources. The polymerase chain reaction method is just such a tool, providing
an innovative way to assess microbial water pollution.
The goal of this method is to develop rapid, routine, and cost effective regional
analytical capability to discriminate between fecal pollution sources in fresh and
marine waters. To meet this goal, the New England Regional Laboratory (NERL) is
developing state-of-the-science rapid, quantitative, library-independent Real-Time Polymerase Chain
Reaction (PCR) methods to differentiate and quantify human and non-human sources of fecal
pollution.
WHAT is PCR?
PCR is a repetitive biochemical technique that mimics the natural cellular process of Deoxyribo-
nucleic Acid (DNA) replication for synthesizing copies of genetic material from either DNA or
Ribonucleic Acid (RNA). DNA, present in all organisms, whether plant or animal, is made up of
genes comprised of unique sequences of nucleotide bases. These unique base sequences are used
to identify a specific organism, a species or group of related species, or a particular strain within a
species. In three cyclically repeating steps, Denaturing, Annealing, and Extension, PCR
amplifies or makes copies of targeted fragments of the DNA. After 30 to 40 of these repetitive
cycles, copying each of the copies produced in the previous cycle, millions of copies of the
replicated nucleic acid are produced.
The product of PCR is called amplicon and Real-Time PCR meosures the amount of amplicon
being copied by labeling the DNA strands with a fluorescent marker and measuring, in real time,
the change in sample fluorescence as the copies are produced.
Each of the three steps of the PCR process is controlled by heating and/or cooling the PCR
reaction. EPA's Regional Lab currently uses a high-speed thermocycling instrument, controlling
the repetitive process and both detecting and quantifying the targeted products of interest.
continued D
&EPA
United States
Environmental Protection
Agency New England
901-F-03-004B
October 2004
www.epa.gov/ne/lab
I printed on 100% recycled paper, with a
of 50% post consumer waste, using vegetable based inks
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ENVIRONMENTAL FDRENSICS
Like phenotypic tests where colored or sheening colonies grow on agar plates allowing identification and enumeration of bacteria,
genotypic PCR techniques amplify portions of gene sequences to enable quantification and identification of the original DNA from
a specific progenitor organism, or strain of an organism. Thus, PCR allows analysts to test whether or not samples contain DNA
from known sources, asking questions such as "Is this the suspect's DNA?" or "Does this person have a specific disease?" or "Is the
fecal pollution in a water sample from human waste?"
PROGRESS TO DATE
In 2003, NERL established and validated three
different Real-Time PCR techniques to analyze water
samples from two urban beaches (Carson and
Wb//aston) in Boston and Quincy, MA for bacterial
indicators; a hydrolysis probe technique for £ coli,
a hybridization probe technique for £ coli, and a
hybridization probe technique for Enterococcus.
These ongoing Total Maximum Daily Load-funded
projects focused on removing PCR inhibitors from
environmental samples and rapid sample processing
using NERL's DNA extraction and purification ro-
botic instrumentation. The automation enables
relatively high sample through-put and precise
handling of environmental DNA samples as compared
to manual sample processing techniques.
WHAT'S IN THE FUTURE
Future efforts will build upon previous PCR
accomplishments to develop a Real-Time PCR
PRNA Coliphage quantitation and differentiation
procedure. Applied research and development in
2004 will include field assessments conducted primarily on Charles River water samples col-
lected between Waltham and Boston, MA, in dry and wet-weather conditions. Viral PCR
results will be compared to more traditional membrane filtration and MPN techniques.
MICROBIAL SOURCE TRACKING BACKGROUND
Bacteria/ F'Coliphage viruses (BCV) are an excellent indicator of viral contamination in both
marine and fresh waters and can more effectively differentiate between human and animal
sources of fecal contamination than simple £ coli- based techniques. Application of NERL's
BCV methods will determine simultaneously the relative contribution of human versus non-
human fecal contamination from different sources and indicate the potential for human viral
pathogen contamination in water bodies.
KEY FACTOR
Regulators must exercise caution when applying new methods to environmental questions.
Until now regulators have not had the ability to discriminate between specific sources of fecal
pollution and must be careful to determine how best to interpret the PCR signal derived from
human and non-human pollution. In addition to developing these analytical techniques, the
New England Regional Laboratory will work with its PCR program partners to learn how best
to apply this new technology to solve environmental problems.
The goal of this
method is to develop
rapid, routine, and
cost effective regional
analytical capability to
discriminate between
fecal pollution sources
in fresh and marine
waters.
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