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EPA/600/F-10/021
science BRIEF
BUILDING A SCIENTIFIC FOUNDATION FOR SOUND ENVIRONMENTAL DECISIONS
SOIL AND WATER
WHAT IS DETECTABLE THROUGH MICROBIOLOGICAL SAMPLE PREPARATION TECHNIQUES
Issue:
The accidental or intentional
contamination of water and soil by
biological agents are real and growing
concerns with respect to human health
and the environment. These are not
unwarranted in light of the anthrax
attacks in the fall of 2001. In addition to
air and food borne biological
pathogens, the vulnerability of our
water resources to bioterrorism is a
salient problem because induced
waterborne outbreaks can involve
sizable populations. Surface water
resources can be susceptible to post
treatment contaminations, as evidenced
by the 13,000 cases of cryptosporidiosis
that occurred in a surface water supply
in Carrollton, Georgia, following
coagulation, sedimentation, filtration,
and chlorination. Another example is
the largest waterborne outbreak in
United States history where
cryptosporidiosis infected an estimated
400,000 people served by the City of
Milwaukee. Ground-water
contamination by biological agents
introduced directly or through
infiltration, also presents a formidable
challenge since ground water serves as
the source of drinking water for roughly
half of the domestic needs of the
country. To meet this challenge, the
U.S. Environmental Protection Agency
(EPA) National Homeland Security
Research Center (NHSRC), in
collaboration with experts from this
Center, which houses the Ground Water
and Ecosystems Restoration Division
(GWERD) of the National Risk
Management Research Laboratory
(NRMRL), and other federal agencies,
initiated an effort to validate, modify,
or develop sampling activities to be
used by all laboratories in the event of a
homeland security incident.
Scientific Objectives:
The preparation of environmental
samples that could potentially contain
biological pathogens is an area in need
of standardized methods. Efforts in
sample preparation development may
include, but are not limited to:
appropriate and optimal sample
collection; transport media; matrix
preparation; and validation of analyte-
specific filtration, elution,
concentration, purification, and staining
procedures. Unfortunately, most
available sampling techniques have
been developed for clinical samples. As
there are significant distinctions
between environmental samples and
clinical specimens, in terms of
concentrations of pathogens and matrix
effects, there is a need to develop
sample preparation procedures that
specifically address the isolation and
concentration of pathogens from
complex environmental matrices. To
that end, molecular and culture based-
techniques will be used to accurately
demonstrate when soil or water samples
harbor potential pathogens. It should be
noted that biomass and biodiversity
levels in subsurface samples are low
relative to a typical soil ecosystem. Soil
extraction and analytical protocols need
to be substantially modified in order to
reliably detect and identify pathogens in
subsurface samples. Therefore, the
approach requires the use of high
sensitivity methods, such as those based
on PCR. It is also important to conduct
thorough validation of these methods,
and the techniques for the collection,
preservation, and preparation of the
environmental samples. The outcome of
the present research efforts will provide
an effective interface between sample
preparation, molecular assays, and
culture techniques to provide accuracy,
precision, and reproducibility,
necessary precursors to method
standardization and adoption.
U.S. Environmental Protection Agency
Office of Research and Development
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Molecular techniques will be used to
demonstrate that environmental
samples (i.e., soil, water) harbor
potential pathogens. Initially, a subset
of analytes of concern were selected
from EPA's Standardized Analytical
Methods for Environmental Restoration
following Homeland Security Events
Revision (SAM) 3rd Edition. This
document suggests analytical methods
for use by laboratories tasked with
performing confirmatory analysis of
environmental samples following a
homeland security event. Parasites and
bacteria will be used to demonstrate
sample preparation techniques from
complex matrices that also contain
other interfering non-target biological
and chemical analytes.
The overarching goal of this project is
to develop analytical methods for
pathogens Salmonella enterica serovar
Typhi, Cryptosporidium parvum, and
Giardia lamblia in environmental water
and soil samples using real time PCR.
Research:
The ongoing efforts focused on method
development, modification, and
optimization with environmental
samples seeded with S. enterica serovar
Typhi, C. parvum, and
G. lamblia to address the objectives.
Compared with traditional PCR, as
proposed in the original plan, real time
PCR is more rapid, precise, and
sensitive. Therefore, real time,
TaqMan based PCR was used in place
of traditional PCR, although traditional
PCR was still used to test primers for
specificity. Considerable effort has
been taken to design and test
primers/probes and find optimal
conditions instead of using available
primers and running conditions.
Immunomagnetic separation (IMS) is
used in EPA Method 1623 to separate
oocysts of C. parvum and cysts of G.
lamblia from water samples. There are
no methods described for DNA
extraction of C. parvum and cysts of G.
lamblia in environmental soils in this
method. Disadvantages of IMS include
variation of (oo)cyst recovery rates,
high cost, and the time-consuming
nature of this process. Extraction of
high-quality DNA is a critical step in
real time PCR detection of pathogens.
Reduction or removal of PCR inhibitors
is also an essential component in the
molecular detection of microorganisms
in environmental samples. DNA yields
extracted from environmental soils are
influenced by soil properties (e.g., pH,
contents, size, and type). Therefore,
various genomic extraction techniques
were evaluated using five different soils
as well as the ground-water samples
from the Arbuckle Simpson Aquifer
seeded with S. enterica serovar Typhi,
C. parvum, and G. lamblia.
Findings:
• When tested against S. enterica
serovar Typhi, in environmental
water and C. parvum in reagent water
the Instagene Matrix (BioRad,
Herculese CA) a buffered chelex
matrix, yielded the greatest recovery.
This was measured by the lowest
threshold cycle (Ct) value, when
compared to boiling, alkaline lysis,
and QiAmp DNA mini kit
• The PowerSoil® DNA isolation kit
(MoBio Laboratories Inc. Carlsbad,
CA) was more effective in DNA
extraction for soil samples seeded
with S. enterica serovar Typhi and
C. parvum than other kits tested
(UltraClean, Mega UltraClean, and
Regular UltraClean Soil DNA
isolation kits; all from MoBio
Laboratories). Incubation of samples
at 70°C was found to help lyse the
cells. Concentrating multiple DNA
samples onto a single spin filter did
not improve DNA yield.
• Using real time PCR, S. enterica
serovar Typhi was detected at 400
cells per ml in water and and 400
cells per gram of soil. The nested
real time PCR assay for C. parvum
detected 5 oocysts seeded in 0.5 g of
a soil matrix and 3 oocysts seeded
into a 1 ml water sample. The nested
real time assay for G. lamblia was
able to detect 10 cysts seeded into 1 g
of soil and 1 ml of water
respectively. We intend to verify
these results and publish the final
protocols for use.
Current Milestones:
• 2010 Presentation - Keeley, A. and
Q. Wu. "Microbiological Detection
Systems for Molecular Analysis of
Environmental Water and Soil
Samples." Society for Industrial
Microbiology and Biotechnology
Annual Meeting. San Francisco, CA.
• 2011 Internal Report - Development,
application, and verification of
microbiological detection limit
guidelines.
• 2011 EPA Report - Completion of
the project final report.
REFERENCES:
USEPA. "Method 1623:
Cryptosporidium and Giardia in Water
by Filtration/IMS/FA" December 2005,
US EPA, Office of Water (4607),
EPA/815/R-05/002.
USEPA. "Standardized Analytical
Methods for Environmental Restoration
following Homeland Security Events"
(Revision 3.1) November 15, 2007,
National Homeland Security Research
Center, USEPA, Office of Research and
Development, Cincinnati, OH,
EPA/600/R-07/136.
CONTACTS:
Ann Keeley, Ph.D.
USEPA/ORD/NRMRL
Ground Water & Ecosystems Restoration
Division
(580) 436-8890 keeley.ann@epa.gov
Qingzhong Wu, Ph.D.
National Research Council
(580)436-8500
wu.qingzhong@epa.gov
U.S. Environmental Protection Agency
Office of Research and Development
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