JOO/R-10/177 | January 2011 | www.epa.gov/or,
United States
Environmental Protection
Agency
Biological Sample Preparation
Collaboration Project:
Detection of Bacillus
anthracis Spores in Soil
TNAL STUDY REPORT
Office of Research and Development
National Homeland Security Research Center
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Biological Sample Preparation
Collaboration Project:
Detection of Bacillus
anthracis Spores in Soil
FINAL STUDY REPORT
Meranda D. Bradley, Ph.D.
Laura Rose, M.S.
Judith Noble-Wang, Ph.D.
Matthew Arduino, M.S., Dr.P.H.
United States Environmental Protection Agency
Office of Research and Development Centers for Disease Control and Prevention
National Homeland Security Research Center National Center for Emerging and Zoonotic Infectious Diseases
Cincinnati, Ohio 45268 Atlanta, Georgia 30329
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Disclaimer
U.S. Environmental Protection Agency (EPA), National Homeland Security Research Center and
the Centers for Disease Control and Prevention (CDC), National Center for Emerging and Zoonotic
Infectious Diseases (Proposed), under IA #DW-75-92259701 (CDC IA# C110-001), collaborated in
the development of the analysis procedure described here.
This report has been peer and administratively reviewed and has been approved for publication
as a joint EPA and CDC document. Note that approval does not signify that the contents
necessarily reflect the views of the Agency. CDC and EPA do not endorse the purchase or sale of
any commercial products or services. The findings and conclusions in this report are those of the
author(s) and do not necessarily represent CDC or EPA.
Questions concerning this document or its application should be addressed to:
Erin Silvestri, MPH
Project Officer
U.S. Environmental Protection Agency
Office of Research and Development
National Homeland Security Research Center
26 W. Martin Luther King Drive, MS NG16
Cincinnati, OH 45268
513-569-7619
Silvestri.Erin@epa.gov
Laura Rose, MS
Centers for Disease Control and Prevention
National Center for Emerging and Zoonotic Infectious Diseases (Proposed)
Division of Healthcare Quality Promotion
Clinical and Environmental Microbiology Branch
1600 Clifton Avenue
Atlanta GA, 30329
404-639-2161
Lmr8@cdc.gov
If you have difficulty accessing these PDF documents, please contact Nickel.Kathy@epa.gov or
McCall.Amelia@epa.gov for assistance.
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Foreword
Following the terrorist events of 2001, the U.S. Environmental Protection Agency's (EPA)
mission was expanded to meet critical needs related to homeland security. Presidential Directives
identified EPA as the primary federal agency responsible for the country's water supplies and
for decontamination following a chemical, biological, and/or radiological attack. To provide
scientific and technical support for EPA to meet this expanded role, EPA's National Homeland
Security Research Center (NHSRC) was established. The NHSRC research program is focused on
conducting research and delivering products that improve the capability of the Agency to carry out
its homeland security responsibilities.
As a part of its long-term goals, NHSRC has been charged with delivery of detection techniques
that will enable the rapid characterization of threats, identification of specific contaminants to
protect workers, and development of plans for recovery operations. Substantial effort and resources
have been invested in the development of molecular assays and culture techniques for pathogens;
however, initial sample collection and preparation methodologies lag behind in development. To
bridge this critical data gap, EPA collaborated with the Centers for Disease Control and Prevention
to develop and optimize an immunomagnetic separation (IMS) method for isolation of Bacillus
anthracis (BA) spores from soil. The developed BA IMS method will support environmental
remediation and recovery activities.
This report summarizes the experimental development of the method and the corresponding study
results that move EPA one step closer to achieving our homeland security mission, and our overall
mission of protecting human health and the environment.
Jonathan Herrmann, Director
National Homeland Security Research
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Acknowledgements
The following individuals and organizations served as members of the Project Team and contributed
to the development of this document are acknowledged:
U.S. Department of Health and Human Services
Centers for Disease Control and Prevention
Laura J. Rose (Principal Investigator)
Meranda D. Bradley (Co-Principal Investigator)
Matthew Arduino (QA Coordinator)
Judith Noble-Wang (Project Manager)
Michele Howard
Alicia Shams
Heather O'Connell
Stephen Morse
Leslie Dauphin
Jason Goldstein
Betsy Weirich
U.S. Environmental Protection Agency (EPA)
Office of Research and Development
National Homeland Security Research Center
Sanjiv Shah, (Project Technical Lead)
Erin Silvestri (Project Officer)
Sarah Perkins
Frank Schaefer
Eugene Rice
Naval Medical Research Center
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Contents
Disclaimer iii
Foreword
Acknowledgements vii
List of Figures xi
List of Tables xii
List of Acronyms xiii
Executive Summary xv
1.0 Introduction 1
2.0 Materials and Methods
2.1 Spore preparation 3
2.2 Inoculum preparation 3
2.3 High specific gravity sucrose extraction (HSGS) 3
2.4 Soil inoculation 3
2.5 Immunomagnetic Separation 5
2.5.1 Conjugation of tosylactivated paramagnetic beads 5
2.5.2 Exploratory Bead Retriever methods 5
2.5.3 Exploratory Method: Miltenyi Biotec Immunomagnetic Separation (IMS) 6
2.5.4 Final AIMS method: Applied Biosystems Inc, IMS 7
2.6 Spore recovery by culture 7
2.7 Spore detection by real-time PCR 7
3.0 Results and Discussion 9
3.1 High specific gravity sucrose (HSGS) extraction 9
3.2 Immunomagnetic Separation 10
3.2.1 Exploratory methods and challenges 10
3.2.2 Final method and challenges 11
3.3 Spore extraction and rapid viability PCR detection 13
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4.0 Conclusions 15
5.0 References 17
Appendix A: Conjugation of Paramagnetic Beads 19
Appendix B: Time Resolved Fluorescence Assay Summary 21
Appendix C: Final Protocols/Method for Real-World Sample Analysis 23
Appendix D: Quality Assurance and Quality Control (QA/QC) 27
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List of Figures
Figure 1. BeadRetriver™ Automated Immunomagnetic Separation Benchtop System 6
Figure 2. Average % Recovered BA spores from sterile Arizona test dust (ATD),
Minnesota loam (ML), potting soil (PS), and sand (S) using HSGS Extraction 9
Figrure 3. Percentage recovery of BA spores from soils using two different antibody
to bead ratios 10
Figure 4. Effects of preprocessing on mean percent recovery of BA spores (103/g)
from sterile Arizona test dust 11
Figure 5. SEM image of Arizona test dust and sand processed through AIMS 12
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List of Tables
Table 1. Physical and Chemical Properties of Soils 4
Table 2. AIMS Mean %R (SD) of BA Spores Recovered From All Four Sterile and Non-sterile
Preprocessed Soil Types on TSAII and PLET Agar 13
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List of Acronyms
%R Percent Recovery
ABI Applied Biosystems by Life Technologies
AIMS Automated Immunomagnetic Separation
ATCC American Type Culture Collection
ATD Arizona Test Dust
BA Bacillus anthracis
BSA Bovine Serum Albumin
CPU Colony Forming Units
CFU/g Colony Forming Units per gram of soil
CDC Centers for Disease Control and Prevention
COA Certificate of Analysis
DHHS Department of Health and Human Services
EDTA Ethylenediaminetetraacetic Acid
EPA Environmental Protection Agency
h Hour
HSGS High Specific Gravity Sucrose
IgG Immunoglobulin G
IMS Immunomagnetic Separation
LRN Laboratory Response Network
min Minute
ML Minnesota Loam
NHSRC National Homeland Security Research Center
NMRC Naval Medical Research Center
PBS Phosphate Buffered Saline
PB ST Phosphate Buffered Saline with Tween® 20
PCR Polymerase Chain Reaction
PS Potting Soil
QA Quality Assurance
QAPP Quality Assurance Project Plan
QC Quality Control
RCF Relative Centrifugal Force
RO Reverse Osmosis
RPM Revolutions per Minute
RV-PCR Rapid Viability-Polymerase Chain Reaction
PLET Polymyxin B, Lysozyme, Ethylenediaminetetraacetic Acid (EDTA),
Thallous Acetate
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s Seconds
SD Standard Deviation
SEM Scanning Electron Microscopy
SNF Sonicating and filtering
SNFS Sonicating, filtering, and settling
SNS Sonicating and settling
TRF Time Resolved Fluorescence
TSAII BBL™ Trypticase™ Soy Agar with 5% Sheep Blood
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Executive Summary
The objective of this work was to develop and optimize
an automated immunomagnetic separation (AIMS)
method for isolation of Bacillus anthracis (BA)
spores from soil. In this study, the AIMS method for
separating BA spores from various soil matrices was
developed using the Dynabeads® technology (Invitrogen
Corporation, Carlsbad, CA). This involved optimizing
paramagnetic beads conjugation with antibodies
specific to BA spores, designing an automated program
(BeadRetriever™, Invitrogen) and developing standard
procedures for separating BA spores from four soil types
using AIMS.
IMS technology utilizes paramagnetic beads conjugated
with BA spore antibodies (polyclonal) to separate
BA spores from spiked soil samples. Specifically,
conjugated Dynabeads® MyOne™ beads were used
for immunomagnetic separation of BA spores (Sterne
34F2; Colorado Serum Company, Denver, CO) from
four soil types; Arizona test dust (Powder Technology
Inc, Burnsville, MM), Minnesota loam, potting soil,
and sand (sources described below). Both sterile and
non-sterile soils were evaluated. Spores were recovered
from one gram of each of the soil type spiked with
101-104colony forming units (CPU). Positive controls
consisted of buffer (10X phosphate buffered saline with
0.05%Tween® 20, PBST) spiked with the same inocula,
negative controls consisted of soils without added spores.
The percent of spores recovered (%R) was determined
using the ratio of colony forming units per gram (CFU/g)
recovered from the soil to the number of CPU of the
spore stock inoculated into the gram of soil. The spore
stock was cultured onto trypticase soy agar with 5%
sheep blood plates (TSAII; BD, Franklin Lakes, NJ) and
polymyxinB, lysozyme, ethylenediaminetetraacetic acid
(EDTA)-thallous acetate (PLET) agar (non-sterile soils
only).
Although the soils were spiked with spore inocula
ranging from lO'-lO4, the lowest limit of reliable
quantitation by AIMS varied. The limit of detection
for all sterile soil types, as well as non-sterile sand and
Arizona test dust was at least 102 spores/g. The limit
of reliable quantitation (CPU >25 on each plate) for
sand and Arizona test dust (sterile and non-sterile) was
also 102 spores/g. The limit of reliable quantitation
for Minnesota loam and potting soil, however, was
103spores/g (sterile) and 104spores/g (non-sterile),
respectively. The mean %R (±SD) of BA spores per
gram of soil from inoculum levels 102 -104 varied by soil
type, with 61.39% (±9.67) from sand, 38.08% (±4.38)
from Arizona test dust, 29.09% (±5.79) from potting
soil and 15.39% (±3.44) from Minnesota loam. The %R
from the positive control (PBST) was 59.50% (±7.89).
Several published articles have evaluated various
techniques for isolating B A spore DNA from soil (Ryu et
al. 2003; Herzog et al. 2009; Gulledge et al. 2010). One
frequently cited culture based method for recovering
spores from soil is the high specific gravity sucrose
(HSGS) plus non-ionic detergent method (Dragon and
Rennie 2001). When the HSGS method was compared
to the IMS method, the %R using HSGS was lower and
the limit of reliable quantitation was 1 to 2 orders of
magnitude higher, depending upon the soil type. With
the HSGS method, the %R ranged from 0.75% - 9.0 %
with soils inoculated with 104-106 spores/g. The AIMS
method was demonstrated to improve the efficiency of
separating BA spores from soil as compared to the HSGS
extraction method.
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1.0
Introduction
The U.S. Environmental Protection Agency (EPA) has
identified detection of pathogenic microorganisms in
environmental samples following a terrorist attack as a
critical component of an effective response. Detection
of such pathogens would require development and
validation of sampling techniques that could be used
by multiple laboratories following a homeland security
event. To meet this requirement, EPA's National
Homeland Security Research Center (NHRSC), along
with other EPA divisions and sister agencies, published
a compendium of standard analytical methods, the 6th
revision in 2010 (EPA 2010). The compendium contains
suggested assays for use by laboratories tasked with
performing confirmatory analysis of environmental
samples following a homeland security event.
Sample preparation, however, remains the limiting step
in the detection techniques available today whether
for non-culture methods like PCR (polymerase chain
reaction), semi-quantitative methods, or quantitative
culture-based methods. Sample preparation includes
extraction of the analyte from sample matrix for
molecular (e.g., nucleic acid extraction) or culture-based
techniques. Extracting pathogens from environmental
matrices is challenging because the matrices are
composed of non-target biological and chemical
analytes. These non-target analytes may interfere
(compete) with the extraction of the target analyte, and
if present in the extracted product, they can inhibit PCR
detection of the target
The Centers for Disease Control and Prevention (CDC),
part of the U.S. Department of Health and Human
Services (DHHS), has extensive knowledge of potential
biological hazards. In this project, CDC, in collaboration
with EPA, developed and improved methods for
extraction of Bacillus anthracis (BA) spores from
soil. Immunomagnetic separation (IMS) technology
utilized tosylactivated beads conjugated with B A spore
antibodies (polyclonal) to separate BA spores from
spiked soil samples. IMS enables both concentration
and purification of spores from soil samples. The
IMS method for separating BA spores from various
soil matrices in this study was developed using the
Dynabeads® technology (Invitrogen Corporation,
Carlsbad, CA). Due to the size of BA spores ( 1-2
um), Dynabeads® MyOne™ beads (1 um in diameter)
were conjugated with antibodies specific for BA spores
(obtained from the Naval Medical Research Center
(NMRC, Silver Spring, MD)). Conjugated Dynabeads
MyOne beads are added to a slurry of BA spores and soil
to allow binding of the spore antigens to the antibodies
coating the beads Spore separation is performed in a
BeadRetriver™ Automated Immunomagnetic Separation
Benchtop System (Invitrogen). The magnetic beads,
along with the bound target, are collected using a
magnet. The excess solution and most of the soil is
left behind, leaving only the beads with spores attached
to the magnet. The beads are then washed to remove
inhibitors and any remaining soil. The optimum bead
conjugation parameters for the IMS protocol were
determined in this investigation. The percent recovery
(%R) of IMS method was compared to that of the
published method for extraction of spores from soil, the
high specific gravity sucrose plus non-ionic detergent
method (HSGS, Dragon and Rennie 2001).
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2.0
Materials and Methods
2.1 Spore preparation
Bacillus anthracis Sterne 34F2 spores (BA) (Colorado
Serum) were grown on soil extract peptone beef extract
agar (Atlas 1996) at 35°C for seven days, harvested with
sterile reverse osmosis (RO) water, and concentrated by
centrifugation at 5000 RCF (relative centrifugal force)
for 15 min. Centrifugation and washes were performed
three times. The pelleted spores were placed in 50%
ethanol (25 mL) at room temperature on a shaker table
at 100 RPM (revolutions per minute) for 1 h. The
suspension was centrifuged and washed with sterile
RO water an additional 5 times and the spores were
suspended in 5mL sterile RO water for storage at -70
°C. Spore preparations were validated by culture and
hemocytometry. The purified spore suspension density
was determined by optical density and culture.
2.2 Inoculum preparation
The inoculum was initially prepared by diluting
the frozen spore stock in sterile RO water to attain a
concentration of 10s spores/mL. This standard was
diluted in series with RO water, to make four spore
concentrations; 104, 103, 102 and 101 spores/mL. These
spore suspensions were quantified by culturing on TSAII
(BBL™ Trypticase™ Soy Agar with 5% Sheep Blood,
Becton Dickinson Microbiology Systems, Franklin
Lakes, NJ) at 35°C for 18 h. Colony forming units
(CPU) were counted and used to determine the number
of spores for inoculation of soils. A pilot experiment
comparing spore suspensions diluted in sterile RO
water and in phosphate buffered saline, pH 7.4 with
0.05% Tween® 20 surfactant (PBST) showed that the
suspension in RO water yielded fewer CPU than the
spores suspended in PBST. The PBST most likely
assisted in disaggregating spores. Thereafter, the spore
inoculum consisted of spores suspended in and diluted in
series in PBST.
2.3 High specific gravity sucrose
extraction (HSGS)
In a 15 mL conical tube, autoclaved soil (2.5 g) was
spiked with 104-106 BA spores. Sucrose extraction
solution (12 mL of 1.22 g/mL sucrose/Triton®X-100
surfactant) was added to each soil sample. The mixture
was shaken by hand for 1 min, placed on a shaker at 75
RPM for 15 min, and centrifuged at 850 RCF for 45
s. The supernatant (3mL) was transferred to a new 15
mL conical tube containing 6 mL filtered 1% bovine
serum albumin in 0.01 mol/L phosphate buffered saline
(BSA/PBS) and centrifuged in a swing bucket rotor
at 5100 RCF for 10 min. The pellet was resuspended
in 1 mL filtered 50% ethanol, gently agitated at room
temperature for 60 min, and centrifuged at 5100 RCF
for 10 min. The pelleted spores were resuspended in 1
mL 1% BSA/PBS, 0.10 mL of purified samples were
spread onto TSAII or PLET (Polymyxin B, Lysozyme,
Ethylenediaminetetraacetic Acid thallous Acetate) agar
plates, and incubated at 35°C for 24 - 48 h (Dragon and
Rennie2001).
2.4 Soil inoculation
Four soil types were obtained for this project; Arizona
test dust (Powder Technologies, Inc.), potting soil (Home
Depot, Atlanta, GA), Minnesota loam (Eagan, MM), sterile
sand (Fisher Scientific, Sewanee, GA, cat# AC61235-
5000), and non-sterile sand (Destin, FL).
The soil physical and chemical characterizations were
performed by the Plant, and Water Laboratory at the
University of Georgia, Athens, GA (Table 1).
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Table 1. Physical and Chemical Properties of Soils
Soil Type
Arizona test dust
Minnesota loam
Sand
Potting soil
Lime buffer
capacity1
NA5
371
NA
618
SoilpH2
8.08
4.51
7.87
7.46
Equivalent water
pH
8.68
5.11
8.47
8.06
Base saturation3 (Percent)
100.00
61.94
100.00
100.00
Cation exchange capacity4
29.87
4.27
0.22
26.99
Soil sample
Arizona test dust
Minnesota loam
Sand
Potting soil
Mineral Composition of Each Soil, ppm
Calcium
5423
377
28
4020
Cadmium
<0.01
0.07
0.02
<0.01
Chromium
<0.02
0.05
<0.02
0.06
Copper
0.02
0.21
0.04
0.11
Iron
0.58
34.30
7.21
3.47
Potassium
117.90
33.46
4.89
95.44
Magnesium
201.90
75.94
5.56
897.10
Soil sample
Arizona test dust
Minnesota loam
Sand
Potting soil
Mineral Composition of Each Soil, ppm
Molybdenum
0.05
<0.02
<0.02
<0.02
Sodium
176.30
9.53
4.15
24.99
Nickel
0.06
0.42
0.07
0.24
Phosphorus
6.69
14.16
0.71
30.92
Lead
<0.10
0.10
<0.10
<0.10
Zinc
0.79
2.20
0.62
3.68
Soil sample
Arizona test dust
Minnesota loam
Sand
Potting soil
Percent Composition of Each Soil
Sand
0.00
46.00
36.00
ND6
Silt
86.00
50.00
62.00
ND
Clay
14.00
4.00
2.00
ND
Total carbon
0.65
1.02
0.02
ND
1 ppm CaCO3/pH
2 Measurement of pH in dilute salt (http://www.caes.uga.edu/publications/caespubs/pubcd/C875.html)
3 Percent of soil exchange sites occupied by basic cations
4 Measures the soils ability to retain nutrients; unit of measurement millequivalent/lOOg of soil
5 Not applicable
6 Not done
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Soils were inoculated as is, or sterilized (autoclaved at
121°C, 32.5 psi for 30 min). One mL of spore suspension
was added to 1 g of soil, and 2 mL 10X buffer (positive
control). Two to 3.5 mL of 10X PBST was added to
the soils to form a slurry. An evaluation of two buffer
concentrations (IX and 10X PBST) for use in making the
slurry was conducted and the 10X was found to provide
the optimum spore recovery. The soil slurry and spiked
buffer were rotated end over end for at least 10 min,
allowed to settle for 10 min, and then filtered through a
30 um pre-separation filter (Miltenyi Biotec, Auburn,
CA), before IMS processing (see Appendix C for final
protocols/methods for real-world sample analysis).
2.5 Immunomagnetic Separation
2.5.7 Conjugation of tosylactivated
paramagnetic beads
B A anti-spore polyclonal antibodies (goat affinity
anti-anthrax antibody (IgG, lot* 031104-01 and lot*
2610006-01) were acquired from NMRC (Bethesda,
MD), purified in-house (CDC Core Facility), and initially
conjugated by Invitrogen to tosylactivated magnetic
beads. Obtaining the conjugated beads was delayed by
several months because of the merging of Invitrogen
Corporation with Applied Biosystems Inc.™ (ABI) by
Life Technologies, and subsequent reorganization of
the company. In the reorganization, ABI eliminated the
division that performs the conjugation, so arrangements
were made for the CDC Core Facility to perform the
conjugations. The conjugation method (see attached
Appendix: Protocols/methods for real-world sample
analysis) was adapted from Invitrogen's "Dynabeads
MyOne™ Tosylactivated Product Description and
Instructions for Use" (Invitrogen 2006)." Two different
temperatures and times of the reactions were evaluated
(37°C for 24 h and 20°C for 48 h), as well as two
different antibody to bead ratio (50 ug/mg and 40 ug/
mg). Optimum conditions for conjugation were found to
be 37°C for 24 h with an antibody to bead ratio of 40ug/
mg.
2.5.2 Exploratory Bead Retriever methods
Two programs were designed for the immunomagnetic
BeadRetriever system (Invitrogen Corporation, Carlsbad,
CA) to optimize recovery of spores from sand and
Arizona test dust (the soils with the largest and smallest
particle size, respectively). The alternative and the final
programs were developed using a manual magnetic
bead retriever. The parameters evaluated for optimum
recovery were (a) the volume of slurry necessary, (b)
the time needed for homogenous mixing, and (c) the
wash times. The results of these evaluations were
used to configure the KingFisher™ software (Thermo
Fisher Scientific, Waltham MA) which operates the
automated BeadRetriever system. Once the parameters
were configured, a pilot run using the automated
BeadRetriever to separate B A from spiked sand and
Arizona test dust was performed. Approximately 3 mL
total of soil slurry was divided into 3 equal portions and
placed into wells 1-3 (~1 mL per well) within a tube strip
(Figure 1). Each of these 3 tubes contained 20 uL of
antibody-conjugated MyOne beads. The automated Bead
Retriever™ was started and the beads and soil slurries
were mixed vigorously. The instrument then collected
the beads, along with the bound target B A spores, from
tubes one through three, into a fourth tube in the tube
strip where they were washed with PBST. The washed
automated immunomagnetic separation (AIMS) product,
i.e. the magnetic beads with bound target B A spores, was
then transferred to the fifth and final tube within the tube
strip and resuspended in 400 uL of PBST. The software
program is available at Life Technologies™.
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Figure 1. BeadRetreiver™Automated immunomagnetic seperation bench top system
The BeadRetriever system is capable of processing up
to 15 samples at a time. Soil and spore slurry samples
are pipetted into tube strips (A) which consists of 5 wells
linked together and held on a stationary platform. The
samples are processed by a moving platform consisting
of plastic tip comb holders (B) and magnetic rods (C)
which slide down into the plastic comb holders during
AIMS processing. Antibody conjugated paramagnetic
beads along with bound spores adhere to the plastic
enclosed magnetic rods in the first three wells and are
mixed by an up and down motion of the magnetic rods.
The platform moves the magnetic rods with captured
beads and spores up, over, and releases the collected
beads into the fourth well in the tube strip. In the fourth
well the beads with bound spores are washed in PBST
by a similar mixing action. The final washed bead with
bound spores are then collected by the magnetic rod
and placed into the fifth well of the tube strip containing
PBST (see http://tools.invitrogen.com/content/sfs/
manuals/1189_BeadRetriever_Manual.pdf).
2.5.3 Exploratory Method: Miltenyi
Biotec Immunomagnetic Separation (IMS)
Miltenyi Biotec (Auburn, CA) super-paramagnetic
particles, approximately 50nm in diameter, were also
evaluated as an alternative to the MyOne beads. It was
thought that the nano-sized beads utilized in this IMS
system would increase magnetic retrieval and spore
recovery by allowing more beads to bind to each spore.
We evaluated the manual IMS system OctoMACS™
(Miltenyi Biotec) along with their anti- mouse IgG
bead for its ability to recover 104 spores/g of soil. The
mouse IgG beads were pre-complexed with monoclonal
(mouse) BA spore antibody and used for manual
immunomagnetic separation of BA spores from sand.
Recovery using this manual system was well below the
recoveries observed using the manual Invitrogen® IMS
system and the HSGS. A preliminary study comparing
MyOne beads conjugated with polyclonal antibodies and
MyOne beads conjugated with monoclonal antibodies
revealed that the recovery was not sufficient using the
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B A monoclonal (mouse) antibody. The B A polyclonal
(goat) antibody could give better results, however, the
Miltenyi cell separation technology does not have an
appropriate microbead strategy available for goat derived
antibodies. Nonetheless, we did incorporate the Miltenyi
Biotec pre-processing step (filtering the soils with a 30
um pre-separation filter) into the final AIMS method.
2.5.4 Final AIMS method: Applied Biosystems
Inc., IMS
Three mL of the spiked soil slurry was dispensed into
three tube strips containing lOOuL of 10X PBST and
20 uL of MyOne conjugated beads. Three or more
replicates of each soil were performed. The mixture was
then gently agitated for approximately 30-35 min. The
beads, along with bound spores, were collected into one
tube, washed in 1 mL of IX PBST for approximately
10 min, and concentrated in 300 uL of IX PBST. This
magnetic bead retrieving process was accomplished with
the BeadRetriever. This AIMS system utilizes inverse
magnetic bead processing technology, which rather
than transferring liquids, transfers paramagnetic beads
through a series of tubes containing specific reagents
with the aid of internal rather than external magnetic
rods. The spores and beads captured by AIMS were
diluted in series and using the spread plate technique
cultured onto TSAll or PLET plates (non-sterile soils only)
in triplicate and incubated at 35°C for 24 h. Colonies were
counted after 24 h and the %R was determined using the
ratio of CFU/g of soil to the CPU of the inoculum (see
final protocols/methods for real-world sample analysis).
The eluent was stored at -20°C for detection by real-time
PCR.
2.6 Spore recovery by culture
The AIMS product was diluted in series and spread
plated (100 uL each) onto TSAII and PLET agar,
incubated at 35°C overnight and the colonies were then
counted. The CFU/g of soil was determined and the %R
calculated, relative to the inoculum. In order to confirm
that colony counts on both media were comparable, a
growth challenge test was conducted. TSAII and PLET
agar were inoculated with B A spores with a low level
of challenge spore concentration (10-100 CPU). The
plates were incubated at 35°C overnight and counted the
next day. Media acceptance requires that the average
number of CPU on the PLET agar must fall within ±70%
of the average number of CPU found on the TSAII plates
(adapted from USP 32, NF 27, Chapter 1227 in U.S.
Pharmacopeia 2009).
2.7 Spore detection by real-time PCR
In addition to culture, real-time PCR was evaluated for
the ability to detect the spores present in the product
of the AIMS. The DNA was extracted from the
BeadRetriever product initially using a simple boil prep
procedure (100°C for 5 min). The real-time PCR was
performed using an ABI 7500 Fast DX platform. The
cycling parameters were programmed according to the
LRN™ protocol "Detection of Bacillus anthracis DNA
by fluorogenic 5' nuclease assay using the Applied
Biosystems® 7500 Fast DX Real-Time PCR System."
The primers and probes were obtained from the LRN™
and are described in Hoffmaster et al. (2002). The only
modification made was that the PCR was performed
using IX PCR master mix [Light Cycler FastStart PLUS
DNA Master Hybprobe (Roche Molecular Biochemicals,
Indianapolis, IN)]. We evaluated two DNA extraction
kits, the MO BIO UltraClean® Soil DNA Isolation Kit
(MO BIO Laboratories, Inc., Carlsbad, CA) and the
QIAamp DNA Blood Mini Kit (Qiagen Inc., Valencia,
CA), for DNA extraction from the AIMS product. Both
kits are supported by the LRN™. The MO BIO soil
kit was used by collaborators at Lawrence Livermore
National Labs. Both kits were evaluated for their
efficiency of DNA purification from AIMS products
(from spiked potting soil and Arizona test dust with 103
spores/g of soil).
-------
-------
3.0
Results and Discussions
3.1 High specific gravity sucrose
(HSGS) extraction
This study was designed to improve existing methods
to extract BA spores from soil samples. The HSGS
method exploits the difference in specific gravities
between B A spores and spores from other organisms
and from background materials in order to isolate the
spores. The HSGS method is inexpensive relative to
immunomagnetic separation; however, we found the
technique to be highly variable. Dragon and Rennie
(2001) found that using HSGS, the %R of BA (ATCC
4229) spores (2 x!05to 8xl05) seeded into 2.5 g of
sterile field soil was 4.5%. Using this sucrose flotation
method, we recovered 0.75 - 9% BA spores on TSAII
from 1 g of spiked sterile soils (Figure 2). As seen in
Figure 2, the best average %R for each soil were 5% (106
spores/g) from Arizona test dust, 3.7% (104 spores/g)
from Minnesota loam, 9% (104 spores/g) from potting
soil, and 5.8% (105 spores/g) from sand. The standard
deviations decreased slightly as the inoculum level
increased. We also observed that this method yielded the
highest %R from potting soil, regardless of the inoculum
level tested. Dragon and Rennie (2001) also found
potting soil to yield the highest %R when compared to
spiked field soils.
% Recovered B. anthracis spores from sterile Arizona test
dust. Minnesota loam, potting soil, and sand
12.00 n
10.00
1.00
6.00
2.00
0.00
456
(Log 10 } B. anthracis spores inoculated/ g soil
n Arizona test dust a Minnesota loam n potting soil csand
Figure 2. The mean percent of spores recovered using HSGS plus non-ionic detergent extraction (Y-axis) (n>3)
is shown for each spiked BA spore inoculum level (shown on the X-axis) and for each sterile soil type: Arizona
test dust, Minnesota loam, potting soil, and sand. Error bars shown in graph represent standard deviations.
-------
3.2 Immunomagnetic Separation
3.2.7 Exploratory methods and challenges
3.2.1.1 Alternate IMS system
Soon after obtaining the BeadRetriever system from
Invitrogen, a merger between Invitrogen and Applied
Biosy stems led to reconstruction of the division
responsible for programming the BeadRetriever system
and conjugating the MyOne beads with our antibody.
This restructuring impeded the programming of the
BeadRetriever system and delayed obtaining conjugated
beads; therefore, an alternative IMS method was
explored. The Miltenyi Biotec IMS system was selected
as an alternative because it uses nanoscale paramagnetic
beads. Theoretically, smaller beads should allow for
multiple beads to bind to each B A spore. This is a
different approach than the Invitrogen system's lum
MyOne beads in which fewer beads bind per spore
simply because the spore and the bead are of equivalent
size. Unfortunately, all spore recovery attempts with
the Miltenyi system failed, most likely due to the use
of monoclonal, rather than polyclonal, antibodies.
Fortunately, we were able to get the BeadRetriever King
Fisher™ Software programmed by Applied Biosystems
technicians, and were able to continue with the
BeadRetriever instrument as originally planned.
3.2.1.2 Conjugation of tosylactivatedparamagnetic
beads
The performance of beads conjugated at 40 ug IgG/
mg beads and 50 ug IgG/mg beads were evaluated
using the three soil types with lower spore recovery
rates. Results showed that the %R of spores from soil
was greater when the beads were conjugated with an
antibody to bead ratio of 40 ug IgG/mg (Figure 3). The
one unexplained exception was potting soil spiked with
102 BA spores. The %R of spores from positive controls
(buffer) was greater when using beads conjugated with
an antibody to bead ratio of 50 ug IgG/mg. We found
that conjugation at the different times and temperatures
evaluated (20°C for 48 h and 37°C for 24 h) did not
affect the %R (data not shown). We therefore chose to
conjugate the antibody to the beads at 37°C because of
the shorter incubation time.
% Recovery of B. anthracis spores from soil using two
different antibody to bead ratios
100.00
150 ug IgG/mg
140 ng IgG/mg
Soil type and inoculum (Log10 CFU/g)
Figure 3. Comparison of the mean percent of BA spores recovered (n>3) from Arizona test dust (AID);
Minnesota loam (ML); potting soil (PS), and PBST (buffer positive control) spiked with 102-104 spores/g of soil
using conjugated bead concentrations of 50 jig IgG/mg and 40 fig IgG/mg.
During the course of the experiments, we received more
antibodies from NMRC with a different lot number
than previously used. In order to confirm comparable
performance, we evaluated the two antibodies,
conjugated to the beads in the same manner (37°C for
24 h), for their ability to recover spores from soil and
buffer. The results indicated little difference in the %R
when using beads conjugated with either of the two lot
numbers of antibody.
-------
3.2.2 Final method and challenges
Our optimization of the AIMS protocol revealed
that separation of the B A spores from soil was best
accomplished by preprocessing the soil slurry samples.
sonicating and vortexing (3 min each) the spore slurry to
disrupt clumps, filtering the slurry through a 30 um pore
size filter, allowing the slurry to settle, and removing the
liquid from the top of the sediment and placing it in the
Bead Retriever tube tray. The preprocessing procedures
improved the recovery of BA spores in all soil types and
decreased the amount of unrecovered spores in the wash.
These improvements were most noticeable with Arizona
test dust (Figure 4).
Effects of preprocessing on mean percent recovery of B,
anthracis spores (103/g)from sterile Arizona test dust
o
«
«_
o
3>
to
Q>
O
Q.
8
CO 10
o
o
• % Recovered
• % Unrecoveiecl
SNFS
SNF
No
Preprocessing methods
Figure 4. Effects of Preprocessing on the mean recovery of BA spores from sterile Arizona test dust.
(Experiment done in duplicate.) Unrecovered spores = percent of spores recovered in IMS wash tube.
Preprocessing abbreviations: SNFS - sonicating, filtering, and settling, and; SNF-sonicating and filtering;
SNS- sonicating and settling and, No- no preprocessing.
The AIMS system program was designed for the
immunomagnetic separation of spores from Arizona
test dust and sand. Each program specifies the amount
of time the beads are allowed contact with the soil, the
amount of vigor used in agitation of the slurry and beads.
and the length of time for the wash step. The entire
AIMS procedure consists of two steps: soil extraction
which includes all of the preprocessing of samples
and automated immunomagnetic separation by the
BeadRetriever system.
When the conjugated beads are mixed with the soil, the
critical step is the binding of the bead to the target BA
spore. This binding depends upon the specificity of the
conjugated antibody to the spore and the contact time
between the beads and the soil slurry. The antibody
specificity was tested previously, by CDC's Division of
Bioterrorism and Preparedness Response, using time
resolve fluorescence (TRF). The results showed that the
antibody can differentiate between closely related and
nonrelated bacterial strains (see appendix B). In the
TRF study, only B A spores, not vegetative cells, were
screened.
Our data show that the AIMS method recovered other
organisms in addition to B A. It is possible that the soil
aggregates contain metallic or magnetic particles that are
attracted to the magnetic rod during AIMS processing.
These soil aggregates may have other bacterial species
adhering to them. In order to determine if this were
true, we looked at Arizona test dust, which contains iron
oxide, and sand using scanning electron microscopy
(SEM). Observation by SEM of non-sterile Arizona
test dust processed AIMS without any B A spores
revealed possible magnetic particle aggregating with
MyOnebeads and other particles (Figure 5).
-------
A B
Figure 5. Scanning electron microscopy image of AIMS (automated immunomagnetic separation) processed
non-sterile Arizona test dust (A) and sand (B) (Both without BA spores and at 10,000x and 3,500x respectively).
(A) In Arizona test dust, clumps or aggregates containing beads and other particles are seen. (B). In sand, only
beads are seen.
Very few other organisms were cultured when recovering
spores from sand and Arizona test dust, but the numbers
were significant when recovering from potting soil and
Minnesota loam. For this reason, the use of the selective
agar (PLET) for recovery of B A from unknown soil
samples is recommended. A comparison of recovery on
both media (PLET and TSAII) was conducted, and the
results demonstrated that recovery of BA on PLET agar
was comparable to growth on TSAII agar. Specifically
the PLET CPUs were within 72-77% of the number of
CPUs found on TSAII agar after overnight growth at
35°C for 48 h.
We also noted that the concentration of PBST buffer
used in making the soil slurry influenced the %R. Using
10X PBST in culturing methods reduced clumping and
increased the %R when compared to IX PBST. Results
from a preliminary study showed the %R from Arizona
test dust spiked with 10 3/g BA spores were 9.23% and
0.52% when 10X PBST and IX PBST, respectively,
were used to make the slurries.
3.2.2.1 Soil and Inoculum level evaluations
The average %R for each inoculum level and
preprocessed soil type is shown in Table 2. The number
of spores recovered from all soils inoculated with 101
spores and some soil types inoculated with 102 spores
were below the limit of reliable quantitation (<25 CPUs
recovered by plate count). Therefore, the average %R is
only discussed for inoculum levels 102-104 in this report.
The %R was found to be optimal within the 102-104
range of inoculum.
The number of spores recovered does not necessarily
increase with increasing inoculum (Table 2). According
to the MyOne Tosylactivated product description and
instructions for use (Invitrogen 2006), the amount
of ligand per mg of beads is optimal at 40 ug ligand
per mg beads. Thus it seems at higher spore (ligand)
concentrations, the target sites on the beads become
saturated and can no longer bind additional spores.
-------
Table 2. AIMS Mean %R (SD) of BA Spores Recovered From All Four Sterile and Non-Sterile Preprocessed
Soil Types and PBST. Percent recovery determined from culture on TSAII and PLET Agar.
Soil Types
Inoculum level
10"
103
102
101
Overall**
10"
103
102
101
Overall**
10"
103
102
101
Overall**
Arizona test dust
Minnesota loam
Potting soil
Sand
Phosphate Buffered
Saline with Tween8
20
Avg. % Recovered BA spores/g (Std. Deviation)
Sterile TSAII
39.44 (4.57)
35.32(1.64)
25.05 (5.29)
*25.25 (5.05)
38.08 (4.38)
14.46(2.18)
16.89(3.33)
* 14.81 (4.92)
*24.23 (24.24)
15.39 (3.44)
31.28(2.75)
23.64(5.53)
*31.24(6.45)
*50.51 (0.00)
29.09 (5.79)
61.95(10.02)
55.67(3.96)
68.01 (11.25)
{25.67(2.14)
61.39(9.67)
56.12(8.98)
57.22(18.42)
67.82(11.70)
* 173. 86 (117.27)
59.50(14.88)
Nonsterile TSAII
51.07(7.39)
28.84(5.03)
48.82(9.55)
*131. 3 (113.83)
41.22(12.74)
10.09(1.76)
* 10.86 (1.94)
*10.1 (3.64)
* 116. 16 (7. 14)
10.35 (2.06)
19.52(4.80)
18.52(2.20)
*16.84(3.55)
*50.51 (0.00)
18.71 (3.64)
54.99 (9.02)
56.23 (5.87)
53.53(11.25)
*202 (0.00)
55.32 (7.74)
61.54(19.03)
62.96 (3.56)
70.38 (7.59)
* 148.2 (123.8)
64.48(16.36)
Nonsterile PLET
37.60 (6.62)
24.35 (12.25)
20.2(9.55)
*45.45 (28.56)
29.43 (11.75)
13.31 (3.40)
15.49 (5.79)
*29.63 (10.22)
*121. 21 (14.28)
17.07(10.48)
15.57(6.42)
*20.09 (16.96)
* 17.51 (5.74)
16.84(0.00)
17.78(11.81)
53.87(14.42)
48.60(18.90)
51.18(4.98)
*117.85 (119.04)
51.23(15.32)
70.99(19.53)
53.94(19.40)
60.00 (8.64)
*173.9 (133.01)
62.64(19.13)
J no preprocessing
below limit of reliable quantitation (<25 CPU)
"overall % recovered excluding 10A1 data
Sand 10A1 sterile is from experiment 10-14-2008 no preprocessing was done
3.3 Spore extraction and rapid viability
PCR detection
In evaluating the DNA extraction kits, we found that
neither kit was able to detect the chromosomal target,
most likely because the inoculum was at or below the
limit of detection. The extraction efficiencies of the
two kits were affected by the soil type. The Qiagen
Blood kit extracted DNA from Arizona test dust more
efficiently than the MO BIO soil kit. The MO BIO soil
kit extracted DNA from potting soil more efficiently than
the Qiagen Blood kit.
Both DNA extraction kits were able to extract DNA
from the AIMS products, although the results were not
consistent between replicates, and the level of detection
was still >103 spores/g of soil. Additionally, because
of the chemistry of the MO BIO soil kit (personal
communication with the MO BIO technical support
department), a consistent DNA extraction of spores from
a suspension of spores in PBS, necessary for a positive
control, was not possible.
Considering all of these challenges with the DNA
extraction kits, we decided that the best way to reduce
the level of detection and to get consistent DNA
extraction was by using the rapid viability PCR (RV-
PCR) method combined with an enzymatic lysis
protocol developed by scientists at Lawrence Livermore
Laboratories. The RV-PCR method includes an
incubation and replication step to increase the DNA copy
number. The enzymatic lysis uses Bacillus cereus Zebra
Killer lysin, currently in use within another laboratory at
CDC, and has been extensively evaluated and found to
consistently lyse vegetative BA cells.
Because of the challenges with the DNA extraction
from spores, the timeline for the RV-PCR portion of the
project was extended to December 2010. The protocol
has been finalized and work is ongoing. A separate report
will be submitted in 2011 that will include the RV-PCR
results and recommendations.
-------
-------
An optimized AIMS method was successfully
developed. The optimized method improved the %R of
BA spores from four soil types and lowered the limit
of quantitation, as compared to the extraction method,
HSGS. Percent recovery of spores from soils inoculated
with 102 to 103 spores/g with the AIMS method ranged
from 15% to 68% as compared to <9% with the HSGS
(range 104 - 106 spores/g) method. When the number of
spores in the sample was greater than 103, the amount
of target sites on the beads could have become saturated
resulting in an inability to bind additional spores. The
addition of pre-processing steps improved spore recovery
from soil. The pre-processing steps were vortexing,
sonication, filtration and allowing for soil to settle before
AIMS processing. The %R of spores from soils with
more organic matter and background organisms (i.e.,
Minnesota loam and potting soil) was lower than soil
types with less organic matter and fewer background
organisms (i.e., Arizona test dust and sand). In cases
where soil types contain high levels of background
organisms, PLET agar was helpful as a selective agar,
but an additional day of incubation was required before
countable colonies were visible. The limit of detection
of the AIMS may be enhanced with the addition of the
RV-PCR method.
4.0
Conclusion
-------
-------
5.0
References
Atlas R M. 1996. Handbook of Microbiological Media.
2nd edition. New York, NY:CRC Press.
Dragon DC, Rennie RP. 2001. Evaluation of spore
extraction and purification methods for selective
recovery of viable Bacillus anthracis spores. LettAppl
Microbiol. 33(2):lOO-5.
Gulledge J S, V A Luna, Luna, AJ, Zartman R, Cannons
AC. 2010. Detection of low numbers of Bacillus
anthracis spores in three soils using five commercial
DNA extraction methods with and without an enrichment
step. J Appl Microbiol 109(5): 1509-20.
Herzog A B, McLennan SD, Pandey, Gerba C P, Haas
CN, Rose JB, Hashsham SA. 2009. Implications of
limits of detection of various methods for Bacillus
anthracis in computing risks to human health. Appl
Environ Microbiol. 75(19): 6331-9.
Hoffmaster AR, Meyer RF, Bowen MD, Marston CK,
Weyant RS, Thurman K, Messenger SL, Minor EE,
Winchell JM, Rassmussen MV, Newton BR, Parker
JT, Morrill WE, McKinney N, Barnett GA, Sejvar JJ,
Jernigan JA, Perkins BA, Popovic T. 2002. Evaluation
and validation of a real-time polymerase chain reaction
assay for rapid identification of Bacillus anthracis.
Emerg Infect Dis. 8:1178-82
Invitrogen. 2006. Dyabeads® MyOne™ Tosylactivated.
[product description and instructions for use] Oslo,
Norway: Invitrogen Dynal AS. http://www.siercheng.
com/UploadFile/200961193927447.pdf
Ryu C, Lee K, Yoo C, Seong W K, Oh H-B. 2003.
Sensitive and rapid quantitative detection of anthrax
spores isolated from soil samples by real-time PCR.
MicrobiolImmunol 47(10): 693-9.
U.S. Department of Health and Human Services.
Public Health Service, Centers for Disease Control,
and National Institutes for Health. 2009. Biosafety in
Microbiological and Biomedical Laboratories. 5th ed.
HHS Publication No (CDC) 21-1112. Atlanta, GA:
Centers for Disease Control and Prevention, http://
www.cdc.gov/biosafetv/publications/bmbl5/index.htm
Research and Development. National Homeland
Security Research Center. 2010. Standardized analytical
methods for environmental restoration following
homeland security events. Revision 6.0. EPA/600/R-
10/122. October, 2010. http://www.epa.gov/nhsrc/
pubs/600rlO 122.pdf
U.S. Pharmacopeia 32. National Formulary 27. 2009.
Validation ofMicrobial Recovery. Vol 1 of The Official
Compendia of Standards. Baltimore, Maryland: United
Book Press.
U.S. Environmental Protection Agency. Office of
-------
-------
Appendix A
Conjugation of Paramagnetic Beads
http://tools.invitrogen.com/content/sfs/manuals/655_01_02_rev003 .pdf
-------
-------
Appendix B
Time Resolved Fluorescence
Assay Summary
TRF assay Bacillus anthracis (Spore)
Antibody Summary Information:
Agent: B. anthracis (spore assay)
Strains Tested: B. anthracis Pasteur vaccine strain,
Sterne vaccine strain, B. anthracis strains AO34, AO39,
AO62, AO102, AO149, AO158, AO174, AO188,
AO193, AO248, AO256, AO264, AO267, AO297,
AO328, AO367, AO379, AO419, AO442, AO462,
AO463, AO465, AO488, AO489, M36 (vollum), ASC-1,
ASC-3, ASC-32, ASC-38, ASC-45, ASC-58, ASC-68,
ASC-69, ASC-78, Z-l, Z-6, PB-292, PB-293
Near-neighbor Screen: Bacillus thuringiensis ssp.
kurstaki, B. thuringiensis ssp. israelensis
Unrelated Screen: Paenibacillus macerans, Bacillus
mycoides, Bacillus cereus (Laboratory Response
Network (LRN) control strain), Brevibacillus
laterosporus, Bacillus licheniformis, B. cereus, (Fri-
42), Bacillus epiphytus, Bacillus alvei, Bacillus
firmus, Bacillus amyloliquefaciens, Bacillus badius,
Brevibacillus brevis, Bacillus circulans, Bacillus
subtilis, Bacillus subtilis (globigii), Paenibacillus
polymyxa, Geobacillus stearothermophilus, Bacillus
sphaericus, Bacillus lentus, Thuricide (pesticide 0.8% B.
thuringiensis ssp. kurstaki (A.G Organics, Prosper, TX),
Dipel dust.
Negative Control Antigen: B. cereus (LRN control
strain)
Markers Targeted (if known>: B. anthracis spore coat
Ag
Sensitivity: Assay was optimized to detect 100 B.
anthracis spores total.
Optimization: Antibodies were optimized using a
range of 0.1 ug/mL to 10.0 ug/mL for both capture and
detection. The assay utilizes 0.75 ug/mL for both goat
anti-5. anthracis capture and anti-5. anthracis mouse
monoclonal detector antibodies. Assay requires 90-min
antigen incubation.
Limitations: Assay also reacts with B. anthracis
vegetative cells.
-------
-------
Appendix C
Final Protocols/Methods for
Real World Sample Analysis
Procedure Title: Automated Immunomagnetic Separation of Bacillus anthracis
spores from soil
Purpose: Immunomagnetic Separation (AIMS) of Bacillus anthracis spores from soil.
Reagents: Custom coated Dynabeads® (Invitrogen Dynal MyOne™ beads, Carlsbad, CA)
Sample buffer (10X Phosphate buffered Saline pH7.4 + 0.05% Tween 20)
Equipment: Dynal® BeadRetrieversystem (Invitrogen Dynal cat# 159-50)
End over end rotator (such as VWR cat # 13916-822)
Supplies:
• 50 mL conical tubes
• 1.5 mL Eppendorf tubes
• 30 urn Pre-Separation filter (Miltenyi Biotec cat. No. 130-041-407)
• BeadRetrievertubes and strips (Invitrogen Cat. No. 159-51)
• Polymyxin lysozyme EDTA thallous acetate select agar (PLET)
• Tryptic Soy Agar II with 5% sheep blood (TSAII, BD Diagnostic Systems)
• Cell spreaders
• Pipettors (100 mL, and 1000 mL)
• Pipette tips (100 mL, and 1000 mL)
Specimen: 1 gram of soil sample, potentially contaminated with B. anthracis spores
Special safety precautions: Follow all safety precautions as outlined mBiosafety in Microbiological and Biomedical
Laboratories, 5th edition (U.S. Department of Health and Human Services 2009)
Quality control: Check sterility of PLET agar and TSA II by incubating 10% of media at 3 5°C for 7 days and check for
growth.
Procedure:
1.
2.
3.
4.
5.
Place 1 g of soil sample in a 15 mL conical tube.
Add enough 10X PBST to enable a 3 mL volume to be retrieved for AIMS. This can be estimated by subtracting the volume occupied by the soil
from the total volume as observed on the side of the tube.
Place the 15 mL conical tube (s) on the rotator and allow the tube(s) to rotate end over end for 10 min.
Remove tube from rotator, place in a tube rack and allow the soil to settle for 10 min.
Withdraw as much of the liquid from the top of the settled soil and filter it through a 30 urn pore size nylon filter (Miltenyi) that is set on top
of a second 1 5 mL conical tube.
Prepare the BeadRetriever tube strips for AIMS by placing the tube trays which consists of 5 wells into BeadRetriever rack.
Add lOOuL of 10X PBST to well 1, 2 and 3 of a BeadRetrievertube strip.
Add 20uL of beads to well 1, 2 and 3.
Add 1 mL of IX PBST to well 4.
Add 300uL of IX PBST to well 5.
7.
Mix filtered sample eluent by pipetting up and down a few times and place 1 mL of filtered sample to each of tubes 1, 2, and 3.
8.
Insert BeadRetriever tips into slot.
9.
Choose BA sand program and press start. The sample will be ready in ~ 50 min.
10.
After the cycle is finished, remove the cleaned eluent (in the final tube containing 300 uL of buffer) and place in an eppendorf tube.
11.
Mix sample by pipetting up and down a few times. If large numbers of spores are expected, serial dilutions of the AIMS product may be
necessary.
12.
Spread 0. ImLfrom well 5 onto TSAll and/or PLET plates in duplicate. Incubate plates at35°Cfor 24 h
13.
Count and record colonies on plates.
14.
Calculate the number of CFU/g soil:
mean number of colonies per plate x total dilution factor
g of soil
-------
Flowchart of AIMS method
Immunomagnetic Separation
of Bacillus anthracis spores from soil
Soil extraction
\
Ig soil into 15 ml
conical tube
\
Add 3.5 mLlOX PBST
.
Rotate tube end-over end
for 10 min
Allow soil to settle 10 min
\
f
Pipette off all liquid from top of
the soil (3mL), Filter through
the 30|j nylon mesh filter into
another 15 ml conical tube
1 1
Mix eluent by pipetting up and
down 3 times
Freeze 100 |jL eluent,
-20°Cfor PCR
Count colonies, calculate recovery
per gram soil
Bead Retriever™ preparation
Insert Bead Retriever™ rack
Place tube strips in rack slots
Place 100 ML 10X PBSTto wells 1,2 &3
Add 20 ML conjugated MyOne™ beads
towellsl,2&3
Place 1 ml of IX PBST to well 4
Place 300 pL of 1X PBST to well 5
1
Insert Bead Retriever™ tips
Place 1 ml into each of wells 1, 2 &3
Select program and press start
Wait 50 min
Mix eluent
by pipetting up and down 3 times,
Remove the eluent from tube 5 (300 |.iL '
Place in eppendorf tube
Spread plate 100 |jl_ onto PLETagar
in duplicate
Incubate @35°C overnight
-------
Limitations of AIMS
(1) False negative results may occur due to high
background contamination and may prevent
identification of growth of B A on TS All plates.
(2) The limit of detection as determined by culture is
dependent upon the soil type. Based on the four soil
types evaluated, the limit of detection for soils with
more organic material and background microorganisms
was approximately 103 spores/g. The limit of detection
for soils with less organic material and background
organisms was approximately 102 spores/g.
(3) Growth on PLET agar requires an additional 24 h
of incubation (as compared to growth on TS All) and
the resulting BA colonies are very small with slight
differences in colony size and morphology.
(4) During the pre-processing step, filtration of some soil
types may reduce recovery of BA spores, due to clogging
of the 30um mesh filter.
(5) Metallic or magnetic particles in soil can bind beads
and cause aggregation of particles and other organisms
to be recovered.
Interpretation/examination
Examine the serial dilution plates for suspect B. anthracis
colonies.
To calculate spore recovery per gram of soil from serial
dilution plates
containing between 25 and 250 B. anthracis colonies:
CFU/g =
mean number of colonies per plate x total dilution factor
g of soil
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-------
Appendix D
Quality Assurance and Quality Control
(QA/QC)
QA/QC Categories
Equipment, media and supplies
Bacillus spp. and soil sources
Immunomagnetic bead separation
DNA extraction
High density spore flotation
Real time PCR and RV-PCR
Training
Data management
Data reporting
QA/QC implemented and QAPP* deviations
As described, the certificate of analysis (COA) for all commercially available media were obtained and kept;
sterility was also assessed with no deviations. Quality control of each lot of selective PLET agar media was
performed as described.
The storage and propagation conditions for the Bacillus anthracis Sterne 34F2 veterinary vaccine strain
were as outlined in the QAPP with no deviations. Cross reactivity of the obtained antibodies was assessed
by CDC's Division of Bioterrorism and Preparedness Response and the findings are included in the report.
Physical and chemical properties of each soil type were determined and the results are found in the report.
AIMS parameters such as buffer types, buffer strength and pH were optimized for the final method as
described. For experimental trials positive and negative controls were always included. The negative control
consisted of soil slurries made with soil and buffer only. We found this to be a more relevant negative
control considering the nature of the beads and soil types. Appropriate interpretation of results was based on
the use of these controls.
Experimental trials of the DNA extraction method included BA spiked sterile soil slurries as a positive
control and buffer only as a negative control. Appropriate interpretation of the performance of the DNA
isolation kit included considered findings from the controls.
Experimental trials assessed spore recovery from spiked sterile soils and positive and negative controls as
described. Appropriate interpretation of results was based on the use of these controls.
All trials included the use of the 16S Ribosomal Ribonucleic acid (rRNA), and a plasmid containing all
select agent primer and probe sets in the detection assays as positive controls and a no template control
(water only). No deviations were observed. Findings will be covered in a another report
Standard laboratory procedures were followed and all personnel were trained on using real time PCR and
other critical equipment. No deviations were observed.
All documentation and records of testing and results were kept in a secured laboratory notebook and in
project-specific electronic files as described in the QAPP. No deviations were observed.
Monthly and quarterly progress reports were prepared and submitted to EPA's project assistant and project
manager.
*QAPP, Quality Assurance Project Plan
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United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
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