Baltelle
The Business of Innovation
 Environmental Technology
 Verification Program
 Advanced Monitoring
 Systems Center
 Test/QA Plan for
 Verification of
 Rapid PCR Technologies

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    TEST/QA PLAN
           for
     Verification of
Rapid PCR Technologies
         May 2004
        Prepared by
         Battelle
      505 King Avenue
  Columbus, OH 43201-2693

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                                TABLE OF CONTENTS

                                                                                   Page

1.0 Introduction	  1
       1.1  Test Objective	  1
       1.2  Test Description  	  1
       1.3  Organization and Responsibility	  2
              1.3.1Battelle  	  2
              1.3.2 Vendors	  5
              1.3.3 EPA	  5
              1.3.4 Supporting Organizations	  6

2.0 Verification Approach  	  7
       2.1  Scope of Testing	  7
       2.2  Experimental Design	  8
       2.3  Test Samples	  10
              2.3.1 PT Samples	10
              2.3.2 Drinking Water Samples	12
              2.3.3 QC Samples  	13
              2.3.4 Field Portability Samples  	14
       2.4  Reference Method	14

3.0 Materials and Equipment 	15
       3.1  Testing Supplies  	15
       3.2  Field Analysis Supplies  	15
       3.3  Special Facilities	15

4.0 Procedures	17
       4.1  Test Sample Collection, Preparation, and Storage	17
       4.2  Sample Identification 	17
       4.3  Sample Analysis  	18
              4.3.1 Drinking Water Characterization 	18
              4.3.2 Stock Solutions Confirmatory Methodologies	19
       4.4  Rapid PCR Technologies  	19
       4.5  Schedule  	19

5.0 Data Handling and Reporting	20
       5.1  Data Acquisition and Review	20
       5.2  Data Analysis  	21
              5.2.1 Accuracy	21
              5.2.2 Precision	22
              5.2.3 Specificity	22
              5.2.4 False Positive/False Negative Responses	22

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                        TABLE OF CONTENTS (CONTINUED)
             5.2.5 Matrix Interferences	23
             5.2.6 Field Portability  	23
       5.3 Reporting  	23

6.0 Quality Assurance/Quality Control  	25
       6.1 Sample Chain-of-Custody Procedures	25
       6.2 Rapid PCR Technology Calibration  	25
       6.3 Equipment Calibration	25
       6.4 QC of Stock Solutions Preparation and DW Characterization 	26
       6.5 Audits	26
             6.5.1 Technical Systems Audit  	26
             6.5.2 Data Quality Audit 	26
       6.6 QA/QC Reporting	27
       6.7 Corrective Action	27

7.0 Health and Safety  	28
       7.1 Standard/Test Sample Preparation  	28
       7.2 Handling During Verification Testing  	28
       7.3 Testing Anthrax, E. coli, F. tularensis, Brucella suis, and Plague	28

8.0 References	29
                                  LIST OF TABLES

Table 1. Infective/Lethal Dose of Target Contaminants  	9

Table 2. Summary of Test Samples for Rapid PCR Technology Verification	11

Table 3. Physio-Chemical Characterization of Drinking Water	18

Table 4. Summary of Data Recording Process  	21



                                 LIST OF FIGURES

Figure 1.  Organization Chart 	3

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 ETV ADVANCED MONITORING SYSTEMS CENTER


      Test/QA Plan for Verification of
         Rapid PCR Technologies

              Version 1.0

               May 2004

              APPROVAL:
Name	

Company

Date

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DISTRIBUTION LIST

Matt Scullion
Technical Sales Associate
Idaho Technology Inc.
390WakaraWay
Salt Lake City, UT 84108

Jonas E. Ruiz, M.B.A.
Senior Product Specialist
Molecular Microbiology Group
Applied Biosystems
850 Lincoln Centre Drive
Foster City, CA 94404

Willem Folkerts
Associate Director, Biodefense
Invitrogen
7335 Executive Way
Frederick, MD 21704

Elizabeth A. Betz
U.S. Environmental Protection Agency -
HEASD
National Exposure Research Laboratory
E205-01 EPAMailroom
Research Triangle Park, NC 27711

Robert Fuerst
U.S. Environmental Protection Agency -
HEASD
National Exposure Research Laboratory
D205-05 EPA Mailroom
Research Triangle Park, NC 27711

Karen Riggs
Amy Dindal
Stephanie Buehler
Zachary Willenberg
Battelle
505 King Ave.
Columbus, OH 43201

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                                    1.0 INTRODUCTION
                                      1.1  Test Objective

       This test/quality assurance (QA) plan provides procedures for a performance verification
test of rapid polymerase chain reaction (PCR) technologies for the analysis of biological agents
and pathogens in drinking and distilled water, under a specific set of test conditions. The
verification test will be conducted under the auspices of the U.S. Environmental Protection
Agency (EPA) through the Environmental Technology Verification (ETV) Program. The
purpose of ETV is to provide objective and quality-assured performance data on environmental
technologies so that users, developers, regulators, and consultants can make informed decisions
about purchasing and applying these technologies. ETV verification does not imply approval,
certification, or designation by EPA or Battelle, but rather a quantitative assessment of the
performance of a technology under  specified test conditions. The objective of this verification
test of rapid PCR technologies is to evaluate their ability to rapidly detect specific biological
agents and pathogens that are particularly toxic to humans and their susceptibility to interferents
in several drinking water matrices.

                                    1.2 Test Description

       The verification test will be  performed by Battelle, which is managing the ETV
Advanced Monitoring Systems (AMS) Center through a cooperative agreement with EPA.  The
scope of the AMS Center covers verification of monitoring technologies for contaminants and
natural species in air, water, and soil.  In performing the verification test, Battelle will follow the
procedures specified in this test/QA plan  and will comply with the data quality requirements in
the "Quality Management Plan for the ETV Advanced Monitoring Systems Center" (AMS
QMP)1.
       Rapid PCR technologies are based on polymerase chain reactions combined with real-
time detection capabilities. These technologies can be both lab-based (e.g., weighing over 75
Ibs. and measuring 20 in. x 21 in. x  15 in.) and field portable (e.g., weighing 50 Ibs. and
measuring 19 in. x 14 in. x 10 in.).  The PCR process involves enzyme-mediated reactions which
allow for target DNA replication and amplification through a series of temperature cycles. Each
series of temperature cycles, which  can consist of up to 30 or more cycles, constitutes a run on a
given rapid PCR technology. Depending on the design of the technology, multiple  samples can
be measured in one PCR run.  For most rapid PCR technologies, reactions are monitored in real-
time during the PCR process through the  use of hybridization probes and DNA-specific dyes in

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combination with fluorescence detection. Other rapid PCR technologies can involve separate
detection devices that offer quick turnaround times over conventional techniques.  Results from
these methods can be qualitative or quantitative. Because rapid PCR technologies are
anticipated to serve mostly as screening tools in water monitoring scenarios, providing rapid
results as to whether or not a pathogen or biological agent is present in the water, only
qualitative results from rapid PCR technologies will be considered for this test. Quantitative
PCR testing often involves more extensive user participation and takes longer to conduct.
       This test will determine the accuracy, specificity, precision, and false positive and
negative rates of rapid PCR technologies in detecting selected biological agents and pathogens in
American Society of Testing and Materials (ASTM) Type II deionized (DI) water, in the
presence of possible interferents added to ASTM Type II DI water, and in drinking water
obtained from a variety of geographically dispersed U.S. water utilities that use various water
treatment processes. Qualitative characteristics of each rapid PCR technology, such as ease of
use,  sample throughput, and cost, will also be assessed and reported. While most of the testing
will  occur in a laboratory, the rapid PCR technologies that are designed for field use will be
tested outside of the laboratory by an experienced operator.

                              1.3 Organization and Responsibility

       The verification test will be performed by Battelle, with the participation of the vendors
who will be having the performance of their rapid PCR technologies verified.  The testing will
occur at Battelle's West Jefferson and Columbus,  Ohio laboratories and at a non-laboratory (i.e.,
field) location in the Columbus, Ohio, area. The organization chart in Figure 1 identifies the
responsibilities of the organizations and individuals associated with the verification test. Roles
and responsibilities are defined further below.

1.3.1 Battelle

       Dr. Stephanie Buehler is the AMS Center Verification Test Coordinator. In this role, Dr.
Buehler will have overall responsibility for ensuring that the technical, schedule, and cost  goals
established for the verification test are met. Specifically, she will
       •  Assemble a team of qualified technical staff to conduct the verification test.
       •  Direct the team performing the verification test in accordance with the test/QA plan.
       •  Ensure that all quality procedures specified in the test/QA plan and in the AMS QMP
          are followed.

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Prepare the draft and final test/QA plan, verification reports, and verification

statements.
Revise the draft test/QA plan, verification reports, and verification statements in

response to reviewers' comments.
Respond to any issues raised in assessment reports and audits, including instituting
corrective  action as necessary.
Serve as the primary point of contact for vendor representatives.
Coordinate distribution of the final test/QA plan, verification reports, and statements.
Establish a budget for the verification test and monitor staff effort to ensure that the
budget is not exceeded.
Ensure that confidentiality of vendor information is maintained.
   Zachary Willenberg
   Battelle AMS Center
    Quality Manager
                           Battelle
                         Management
                         Karen Riggs
                         Battelle AMS
                        Center Manager
                         Amy Dindal
                         Verification
                        Testing Leader
                   Dr. Stephanie Buehler
                        Verification
                      Test Coordinator
                           Battelle
                        Technical Staff
     Robert Fuerst
   EPA AMS Center
       Manager
     Elizabeth Betz
   EPA AMS Center
   Quality Manager
    Vendor
Representatives
                     Figure 1. Organization Chart

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       Ms. Amy Dindal is a Verification Testing Leader for the AMS Center. Ms. Dindal will
provide technical guidance and oversee the various stages of verification testing.  She will
       •  Support Dr. Buehler in preparing the test/QA plan and organizing the testing.
       •  Review the draft and final test/QA plan.
       •  Review the draft and final verification reports and verification statements.
       •  Support Dr. Buehler in responding to any issues raised in assessment reports and
          audits.

       Ms. Karen Riggs is Battelle's manager for the AMS  Center. Ms. Riggs will
       •  Review the draft and final test/QA plan.
       •  Review the draft and final verification reports and verification statements.
       •  Ensure  that necessary Battelle resources, including staff and facilities, are committed
          to the verification test.
       •  Ensure  that vendor confidentiality is maintained.
       •  Support Dr. Buehler in responding to any issues raised in assessment reports and
          audits.
       •  Maintain communication with EPA's technical and quality managers.
       •  Facilitate a stop work order if Battelle or EPA QA staff discovers adverse findings
          that will compromise test results.

       Battelle Technical Staff will conduct the testing of the rapid PCR technologies during the
verification test. The  responsibilities of the technical staff will be to
       •  Assist in the collection, receipt, and storage of drinking water samples.
       •  Prepare drinking water samples, as required for analysis.
       •  Prepare the stock solutions and the test samples as required.
       •  Perform plate enumeration to confirm concentrations of stock solutions.
       •  Perform the rapid PCR technology analyses by following the vendor's protocol.
       •  Make qualitative  observations about the operation of the rapid PCR technology.

       Mr. Zachary Willenberg is Battelle's Quality Manager for the AMS Center.
Mr.Willenberg will
       •  Review the draft and final test/QA plan.
       •  Conduct a  technical systems audit once during the verification test, or designate
          another QA manager to conduct the audit.
       •  Audit at least 10% of the verification data.

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          Prepare and distribute an assessment report for each audit.
          Verify implementation of any necessary corrective actions.
          Issue a stop work order if self audits indicate that data quality is being compromised;
          notify Battelle's AMS Center Manager if a stop work order is issued.
          Provide a summary of the QA/ quality control (QC) activities and results for the
          verification reports.
          Review the draft and final verification reports and verification statements.
          Assume overall responsibility for ensuring that the test/QA plan is followed.
1.3.2  Vendors

       The responsibilities of the vendor representatives are as follows:
       •   Review the draft test/QA plan.
       •   Approve the test/QA plan prior to test initiation.
       •   Provide off-the-shelf rapid PCR technologies for analysis of all verification test
          samples.
       •   Provide all other equipment and consumables needed to complete the PCR analyses
          on all water samples, including any necessary extraction or purification consumables
          and/or equipment.
       •   As desired, provide training to Battelle personnel on operating the rapid PCR
          technologies and associated equipment prior to testing.
       •   Provide written instructions for operation of the technology.
       •   Review the draft verification report and statement.

1.3.3  EPA

       EPA's responsibilities in the AMS Center are based on the requirements stated in the
"Environmental Technology Verification Program Quality Management Plan" (EPA QMP)2.
The roles of the specific EPA staff are as follows:

       Ms. Elizabeth Betz is EPA's AMS Center Quality Manager. For the verification test,
Ms.Betz will
       •   Review the draft test/QA plan.
       •   Perform at her option one external technical system audit during the verification test.

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       •   Notify the EPA AMS Center Manager of the need for a stop work order if external
          audit indicates that data quality is being compromised.
       •   Prepare and distribute an assessment report summarizing results of external audit.
       •   Review draft verification reports and statements.

       Mr. Robert Fuerst is EPA's manager for the AMS Center. Mr. Fuerst will
      •   Review the draft test/QA plan.
      •   Approve the final test/QA plan.
      •   Review the draft verification reports and statements.
      •   Oversee the EPA review process for the verification reports and statements.
      •   Coordinate the submission of verification reports and statements for final EPA
          approval.

1.3.4   Supporting Organizations
       Physio-chemical characterization including turbidity, organic carbon,  specific
conductivity, alkalinity, pH, hardness, total organic halides, trihalomethanes and haloacetic acids
(Table 3) have been performed for all drinking water samples.  Spiked concentrations of humic
and fulvic acids in ASTM Type IIDI water will also be confirmed.  Battelle has established a
subcontract with Aqua Tech Environmental Laboratories, Inc. (hereafter called  subcontract
laboratory) to perform the physio-chemical analyses and humic and fulvic acid  concentration
confirmations.
       The Metropolitan Water District of Southern California has concentrated each drinking
water sample by a factor of 400 using ultrafiltration concentration techniques.

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                              2.0  VERIFICATION APPROACH
                                     2.1 Scope of Testing
       This test/QA plan specifically addresses verification of rapid PCR technologies that
provide measurements of Bacillus anthracis (anthrax), Escherichia coli O157:H7 (E. coli),
Francisella tularensis (F. tularensis) LVS (ATCC# 29684), Brucella suis (ATCC#23444) and/or
Yersiniapestis CO92 (plague) in drinking water. The contaminants were selected based on the
capabilities of the technologies being tested and the availability of the pathogens and biothreat
agents.  The rapid PCR technologies participating in this test will be evaluated on qualitative
results, indicating only the presence or absence of the contaminants within a specified
concentration interval.  Each rapid PCR technology will be tested only for contaminants for
which they are designed to detect, as specified by the vendor. The performance of the rapid PCR
technologies will be verified by subjecting each to various concentration levels of individual
contaminants in ASTM Type IIDI water, a single concentration of contaminant in the presence
of possible interferents (i.e., fulvic and humic acids) spiked into ASTM Type II DI water, and a
single concentration of each contaminant spiked into drinking water samples obtained from four
water utilities from different geographical locations in the United States.  Each source of
drinking water will represent a unique water treatment process. Also, both the possible
interferent samples and the drinking water matrices will be analyzed without the  addition of any
contaminant to evaluate the potential for false positive results. The performance of each rapid
PCR technology will be evaluated based on the parameters outlined below, with  confirmation of
solutions spiked with known concentrations of contaminants by available reference methods
(i.e., plate enumeration).
       The rapid PCR technologies are designed to be operated by users with technical training.
Therefore, for this verification test,  experienced operators, i.e., operators that have previous
experience using PCR technologies, that have been trained by the vendor in the operation of the
PCR technologies, will analyze the  test samples on the technology. For those technologies
which are designed to be field portable, an experienced operator trained by the vendor will also
perform tests  outside of the laboratory environment.
       A variety of natural and contaminant-fortified water samples (i.e., unspiked and spiked)
will be analyzed using the rapid PCR technologies. These matrices are examples of drinking
water types that could be monitored using rapid PCR technologies; however, this is not intended
to be an exhaustive study or to  represent all possible water types that could be tested.
       The rapid PCR technologies will be evaluated for the following parameters:
       •   Accuracy

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           Specificity
        •   Precision
        •   Matrix effects
        •   Occurrence of false positive and false negative results
        •   Field portability by experienced operator
        •   Ease of use by experienced operator
           Sample throughput.
        The PCR technologies will not be evaluated for method detection limits (MDL) because
 only the qualitative aspects of each technology will be evaluated in this verification test.
 However, the lowest concentration performance test sample to produce consistently positive
 results will be reported to help end users better understand the sensitivity of the technologies.

                                   2.2 Experimental Design

       This verification test will determine the performance capabilities of rapid PCR
technologies to detect individual contaminants in three types of samples—performance test (PT),
drinking water (DW), and quality control (QC). PT samples will include samples prepared in
ASTM Type IIDI water, including contaminant PT samples and interferent PT samples. The
contaminant PT samples will be fortified with each individual contaminants at five
concentrations.  Concentrations will include the infective/lethal dose concentration given in
Table 1 for each  contaminant and approximately 2, 5, 10, and 50 times the vendor reported limit
of detection (LOD) for each technology. The infective/lethal dose of each contaminant was
determined by calculating the concentration at which 250 mL of water is likely to cause the death
of a 70-kg person based on human LD50 or ID50 data 3. The results from quadruplicate analysis
of the contaminant PT samples and comparison with the known concentrations will provide
information on the accuracy and precision of the rapid PCR technologies. The interferent PT
samples will consist of humic and fulvic acid at two concentrations, both spiked and unspiked
with each contaminant, each analyzed in quadruplicate.

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Table 1. Infective/Lethal Dose of Target Contaminants
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     Contaminant (common
             name)
 Infective/Lethal Dose
    Concentration
  Bacillus anthracis (anthrax)
  Yersiniapestis (Plague)
  Francisella tularensis (F.
  tularensis)
  Brucella suis
  Escherichia coll O157:H7 (E.
  coif)	
200,000 spores/L
280 organisms/L
4 x 108 organisms/L

40,000 organisms/L
200 bacteria/L
       DW samples have been collected from four water systems that get water from various
sources and employ different treatment processes. The DW samples disinfected by chlorination
were obtained from surface (filtered and unfiltered) and groundwater sources.  A DW sample
from a surface water source disinfected by chloramination was also collected.  These same water
samples were concentrated by a factor of 400 at the Metropolitan Water District of Southern
California using their ultrafiltration concentration system. Each of these water samples will be
analyzed in quadruplicate, both unspiked and spiked with each contaminant at a single
concentration level approximately 10 times greater than the LOD of each rapid PCR technology.
       QC samples will include method blanks consisting of unspiked ASTM Type IIDI water,
as well as vendor-provided positive and negative controls or internal controls.  Positive, negative,
and/or internal control samples will be run with each PCR run, per the vendor  instructions, and
will be used as a quality check to  ensure the rapid PCR technology's performance. If controls are
not provided by the vendor, positive control samples will be prepared in ASTM Type II DI water
with a known concentration of a toxin specified by the vendor and will be used as a quality check
to ensure the PCR technology performance.  The number and frequency of QC samples will vary
with each rapid PCR technology,  but 10%  of all samples will be method blanks and at least 1
positive and 1 negative control will be part of every batch of samples run on each PCR
technology.
       Performance parameters, such as ease of use and reliability, will be based on documented
observations of the operators and  by the Verification Test Coordinator.  Sample throughput will

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be estimated based on the time required to analyze a sample set. All rapid PCR technologies will
be tested in the laboratory; while applicable field-portable rapid PCR technologies will be
analyzed for their performance and ease of use outside of the laboratory.  Because many of the
contaminants of interest cannot be safely handled outside of special facilities, the samples
analyzed in the field will include method blank samples and non-toxic positive and negative
control samples as provided by the vendor. An experienced operator will perform analyses in  the
field, which will also include evaluations of ease of use while dressed in personal protective
equipment (PPE) (i.e., Level C PPE consisting of a splash suit and a filter-type respirator).
       Given the agent facility restrictions, vendors will not be able to operate their rapid PCR
technologies during this verification test. Each rapid PCR technology will be operated by a
Battelle staff member and tested independently. All operators will have experience with and
knowledge of PCR technologies in general. The vendor will  train the operators by means of a
visit to Battelle or a conference call prior to starting the verification test and then will be asked to
sign a consent form stating the names of the Battelle staff they have trained. Each operator will
manipulate the water samples  and  reagents to generate solutions that can be analyzed by the rapid
PCR technologies. More than one operator may be used by Battelle, but operators will be
restricted to only operating rapid PCR technologies on which they have been trained. Because of
the potential time requirements for this test, more than one operator may be assigned to each
participating technology for the duration of the test.

                                    2.3 Test Samples

       Test samples to  be used in this verification test will include PT samples, DW samples,  and
QC samples.  Table 2 lists the number and type of each sample to be analyzed for each
contaminant in the verification test. Each type of test sample is described further below.

2.3.1 PT Samples

       PT sample types (listed in Table 2) will be prepared in ASTM Type IIDI water.  The first
type of PT sample will consist of ASTM Type II DI water spiked at five concentration levels of
each individual contaminant.  The contaminant PT sample concentrations will range from the
infective/lethal dose concentration to  50 times the vendor-stated LOD.  The infective/lethal dose
concentration will be analyzed to document the response of each technology at that important
concentration level. Four concentration levels in addition to the infective/lethal dose
concentration will be analyzed by  each technology. Those concentration levels will be

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approximately 2, 5, 10, and 50 times the LOD of each individual technology. The maximum and
minimum concentrations may be limited by the available standards.  The PT samples will be
prepared for each rapid PCR technology based on the vendor-defined LOD such that samples
used for a particular technology contain 2, 5, 10, or 50 times the LOD of that technology. Each
concentration level for the PT samples will be analyzed in quadruplicate using the appropriate
rapid PCR technology for that sample.
Table 2. Summary of Test Samples for Rapid PCR Technology Verification
Performance Test
(PT)
ASTM Type II DI
Water
Drinking Water
(DW)
Filtered chlorinated
surface water
Unfiltered
chlorinated surface
water
Performance Factor
Accuracy, Specificity and
Precision
Interferent
Ease of Use, Field
Portability
Performance Factor
Matrix Effect
Sample Description
Contaminant PT sample @ infective/lethal dose
Contaminant PT sample @ 2 times the LOD(a)
Contaminant PT sample @ 5 times the LOD
Contaminant PT sample @ 10 times the LOD
Contaminant PT sample @ 50 times the LOD
Fulvic and humic acids @ a total concentration of 1
mg/L
Fulvic and humic acids @ a total concentration of 1
mg/L + contaminant @ 10 times the LOD
Fulvic and humic acids @ 5 mg/L
Fulvic and humic acids @ 5 mg/L + contaminant
@ 10 times the LOD
Analysis of method blank in level C suit
Analysis of unspiked, unconcentrated DW in level
Csuit
Analysis of method blank not in suit
Sample Description
Concentrated unspiked
Concentrated and spiked with contaminant @ 10
times the LOD
Concentrated unspiked
Concentrated and spiked with contaminant @ 10
times the LOD
Reps
4
4
4
4
4
4
4
4
4
3
3
3
Reps
4
4
4
4

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Filtered chlorinated
groundwater
Filtered
chloraminated
surface water
Quality Control
(QC)
Method Blank
Positive Control
Negative Control


Performance Factor
Quality Check
Quality Check
Quality Check

Concentrated unspiked
Concentrated and spiked with contaminant @ 10
times the LOD
Concentrated unspiked
Concentrated and spiked with contaminant @ 10
times the LOD
Sample description
DI water - 10% of all samples
Provided by vendor - at least 1 on every PCR run
Provided by vendor - at least 1 on every PCR run

Approximate total number of samples per contaminant
4
4
4
4
Reps
8
Vai00
VaiO»

85
(a) LOD in all cases is limit of detection provided by the vendor for their rapid PCR technology.
(b) Number of positive and negative controls will vary based on the number of samples that can be analyzed by the PCR
technology in each run.

       The second type of PT sample will be potential interferent samples. Because it is
anticipated that humic and fulvic acids will be major interferents in real-world uses of rapid PCR
technologies for water monitoring, four replicates of each interferent PT sample will be analyzed
to determine each rapid PCR technology's susceptibility to these commonly found interferents in
DW.  The interferent PT samples will contain humic and fulvic acids obtained from the
International Humic Substances Society spiked into ASTM Type II DI water.  Each of these
interferent mixtures will be prepared at two different concentration levels. One concentration will
be near the upper limit of what would be expected in drinking water (5 mg/L) and one
concentration at a mid-low range of what would be expected (1 mg/L).  These spiked interferent
levels will be confirmed through analysis of aliquots by the subcontract laboratory.   Also, each
contaminant will be added to these samples along with the potential interferent, at a concentration
of 10 times the LOD, and analyzed in quadruplicate.

2.3.2 Drinking Water Samples
       Drinking water samples were collected from four geographically distributed municipal
sources (Ohio, New York, California, and Florida) to evaluate the performance of the rapid PCR

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technologies with various sample matrices. These samples vary in their source and treatment and
disinfection process.  All samples have undergone either chlorination or chloramination
disinfection prior to receipt.  Samples were collected from water utility systems with the
following treatment and source characteristics:
   •   Chlorinated filtered surface water source
   •   Chlorinated unfiltered surface water source
   •   Chlorinated filtered groundwater source
   •   Chloraminated filtered surface water
       All  samples were collected in pre-cleaned high density polyethylyne (HDPE) containers.
After sample collection, to characterize the DW matrix, an aliquot of each DW sample was sent to
a subcontract laboratory to determine the following water quality parameters: concentration of
trihalomethanes, haloacetic acids, total organic halides, calcium, magnesium, pH, conductivity,
alkalinity, turbidity, organic carbon,  and hardness.  The DW samples were dechlorinated upon
arrival to the Metropolitan Water District of Southern California with sodium thiosulfate
pentahydrate to prevent the degradation of some of the contaminants by chlorine. Because real-
world applications of PCR technologies to screen water samples rely on pre-concentration of the
water sample to be analyzed, approximately 100 L of each of the above  sources of DW were
dechlorinated and then concentrated  through ultrafiltration techniques to a final volume of 250
mL.  As shown in Table 2, each DW sample will be analyzed without adding any contaminant, as
well as after fortification with each individual contaminant at a single concentration level (10
times the vendor-stated LOD).

2.3.3 QC Samples

       QC  samples will include method blank (MB) samples consisting of ASTM Type IIDI
water,  and positive and negative controls,  as provided by the vendor. All of the MB QC samples
will be exposed to identical sample preparation and analysis procedures as the test samples.
Positive and negative controls will be prepared and used according to protocol provided by the
vendor. The MB samples will be used to ensure that no sources of contamination are introduced
in the sample handling and analysis procedures.  At least 10% of the test samples (8 samples) will
be MB samples.  The vendor provided control samples will indicate to the operator whether the
rapid PCR technology is functioning properly and will be added to each run of each technology.
To the extent practicable, the test samples will be analyzed blindly by the operator such that at a
minimum samples used by the technician for the analysis are marked with a non-identifying
number.

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2.3.4  Field Portability Samples

       Those PCR technologies that are designed to be field portable will be tested outside of the
laboratory by an experienced operator. Because many of the contaminants being tested are highly
toxic and unsafe to be handled outside of a special facility, method blank samples and non-toxic
positive and negative control samples as provided by the vendor will be analyzed in the field
portability test. Unconcentrated drinking water samples will also be tested to simulate real-world
applications.  Because these technologies are meant to be used by first-responders, their
performance in the field will also be evaluated under simulated first response conditions by
having the operator dressed in Level C PPE. One set of MB samples will be tested without the
use of a protective suit.

                                  2.4  Reference Method

       For all contaminants, a plate enumeration technique of quantifying bacteria will be
followed to confirm the concentration of the stock solution of these contaminants. If necessary,
aliquots of the organism to be used for testing may be frozen during the plate enumeration process
to ensure that the proper solution concentration is being tested and prevent the organism from
multiplying during storage while awaiting the results of the confirmation analysis.  For anthrax
and plague plate enumeration, the Battelle standard operating  procedure (SOP) to be followed is
SOP No: MREF X-0544 "Standard Operating Procedure (SOP) for the Enumeration of BL-2 and
BL-3 Bacteria Samples Via the Spread Plate Technique." For E. coli, F. tularensis., and Brucella
suis, general laboratory practices for bacteria plate enumeration will be followed.

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                             3.0 MATERIALS AND EQUIPMENT

       In general, this verification test will rely on rapid PCR technology materials and
equipment provided by the vendors. Battelle will provide the following equipment and materials
for the collection, preparation, storage, and shipment of test samples.

                                   3.1  Testing Supplies

       The following supplies will be needed throughout testing for sample collection and
preparation of the DW and QC samples:
       •   ASTM Type IIDI water
       •   Various laboratory supplies necessary for accurate preparation of the test samples (i.e.,
          volumetric pipettes, pipette bulbs, Eppendorf micro pipettes/pipette tips, volumetric
          flasks, disposable pipettes, etc.)
       •   Various smaller sizes of pre-cleaned HDPE and glass containers for sample aliquot
          storage
       •   Standards of contaminant and interferents with a known level of purity (NIST
          traceable or equivalent)
       •   Sodium thiosulfate pentahydrate
       •   n,n-diethyl-p-phenylenediamine (DPD) tablet
       •   Personal protective equipment.

                               3.2  Field Analysis Supplies

       For the analysis of the method blank samples and unconcentrated, unspiked drinking
water samples in the field, Battelle will provide the water used for analysis and the Level C
personal protective equipment (PPE). The operators will depend on only supplies provided by the
vendor to analyze the samples.

                                   3.3  Special Facilities

       The contaminants to be evaluated in this verification test require special handling and/or
special facilities. Plague, anthrax, and Brucella suis can only be handled in laboratories that are
specially designed and certified for the use of chemical and biological agents and by operators

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who are trained in their use.  Battelle's Medical Research and Evaluation Facility (MREF), which
is a Department of Defense laboratory-scale facility conducting research with chemical and
biological agents, will provide the facilities and staff for verification testing of plague, anthrax,
and Brucella suis. The MREF is licensed to ship, receive, and handle select agents, as defined by
the Centers for Disease Control and Prevention (CDC)5.  The facility maintains state-of-the-art
equipment and professional and technical staffing trained to safely conduct testing  and evaluation
of hazardous chemical and biological materials.
       The MREF and its personnel have the demonstrated capability for storing and safely
handling plague, anthrax, and Brucella suis. Biological agent use will be according to the CDC
Select Agents Program (32 CFR 626 and 627)5 administered through the Biological Defense
Safety Program and the Battelle MREF Facility Safety Plan.  All PCR technologies used in this
building will have to go through a decontamination process involving hydrogen peroxide vapor
spray to eliminate the possibility of any anthrax or plague remaining on or in the instrument. The
decontamination step has previously been used on computers and other sensitive electronic
equipment without harm to the technology.
       E. coli and F. tularensis are not required to be used in a specially designed facility as are
anthrax, plague, and Brucella suis, but they do require special handling, thus testing of these
bacteria will take place in qualified laboratories in either the Battelle Columbus Operations or
MREF facilities.

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                                   4.0 PROCEDURES

                   4.1  Test Sample Collection, Preparation, and Storage

       Stock solutions of each contaminant and interferent will be prepared in ASTM Type IIDI
water or appropriate reagent from certified standards. The concentration of all stock solutions
will be confirmed using a plate enumeration method.  If possible, solutions will be prepared on a
daily basis for all bacteria. However, because the concentration of some organisms can change
over the course of a day, aliquots of the solution to be used for spiking the samples may be
prepared and frozen while the confirmation analyses are performed.  Samples confirmed to
contain adequate levels of the organism would then be thawed and used as needed.
       PT samples will be prepared in ASTM Type II DI water using the aforementioned stock
solutions.  Aliquots of each stock solution will be diluted to the appropriate concentration using
volumetric pipettes and glassware. The DW samples were collected as described in Section 2.3.2.
Because free chlorine will degrade many of the contaminants and interferences during storage, the
samples were immediately dechlorinated with sodium thiosulfate pentahydrate (or other
dechlorination reagents as per vendor protocol).  The dechlorination of the DW was qualitatively
confirmed by adding a diethyl-p-phenylene diamine (DPD) tablet to an aliquot of DW. If the
water did not turn pink, the dechlorination process was determined to be successful.  If the water
did turn pink, additional dechlorinating reagent was added and the dechlorination confirmation
procedure was repeated.  Once dechlorination was confirmed, 100 L of each DW sample was
concentrated as described previously, and approximately 25 L remained unconcentrated.  The
dechlorinated concentrated DW samples will be analyzed unspiked and spiked. Aliquots of each
bacteria stock solution will be diluted with DW samples to the appropriate concentration. All
spiked DW samples will  go through the appropriate vendor-specified DNA preparation/isolation
procedure on the same day as they are spiked.

                                4.2  Sample Identification

       Aliquots to be analyzed by  each rapid PCR technology will be drawn from the PT, QC or
DW samples and placed in sample containers with unique identification (ID) numbers. A master
log of the samples and sample ID numbers for each rapid PCR technology will be maintained by
Battelle.  The ID number, date, person collecting, sample location, and time of collection was
recorded on a chain-of-custody form for all field samples.

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                                   4.3 Sample Analysis

4.3.1 Drinking Water Characterization

       Table 3 lists the methods used to characterize the DW samples collected from the various
water sources by the subcontract laboratory. An aliquot of each DW sample was sent to Aqua
Tech Environmental Laboratory prior to concentration to determine the water quality parameters
listed in Table 3.  Because the water samples were collected and then transported and tested at
later dates, some of the water quality parameters may have changed slightly prior to testing with
the rapid PCR technologies. Information produced on physio-chemical parameters of drinking
water by the subcontract laboratory will be utilized for verifying performance of the rapid PCR
technologies.
Table 3. Physio-Chemical Characterization of Drinking Water"
Parameter
Turbidity
Organic carbon
Specific conductivity
Alkalinity
pH
Hardness
Total organic halides
Trihalomethane s
Haloacetic acids
Method
EPA 180. 16
SM53107
SM25107
SM 23207
EPA 150.1s
EPA 130.2s
SM 53207
EPA 524.29
EPA 552.210
Method Detection
Limits^
0.067 ntu
0.7 mg/L
2 (imho
2 mg/L
NA
5 mg/L
5 Mg/L
0.5 (ig/L/analyte
1.0 (ig/L/analyte
(a) Physio-chemical DW characterization to be performed by the subcontract laboratory
(b) Method detection limits based on standard methods.

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4.3.2  Stock Solution Confirmatory Methodologies

       The concentration of all contaminants will be confirmed by a plate enumeration method
following Battelle SOP No.: MERF X-054 and Battelle general laboratory practices for plate
enumeration. For the interferent samples, the concentration of humic and fulvic acid will be
confirmed by Standard Method 53107 for total and dissolved organic carbon by the subcontract
laboratory.
                              4.4  Rapid PCR Technologies
       Each vendor will provide rapid PCR technologies, consumables (e.g., reagents and
controls), and other necessary equipment (e.g. DNA extraction and purification reagents and/or
equipment) for the analysis of all samples.  The full set of samples listed in Table 2, unless
otherwise noted, will be analyzed by each rapid PCR technology for each applicable contaminant.
The analyses will be performed according to the vendor's recommended procedures as described
in the standard written instructions or manual provided with the rapid PCR technologies.
Calibration and maintenance of the rapid PCR technologies will be performed as specified in the
written instructions or manual.
       Rapid PCR technology results will be recorded manually by the operators on data sheets
designed specifically for this verification test. Where applicable, electronic results from the
supplied technology software will also be coded and stored. In addition to the rapid PCR
technology results, the data sheets will include records of the time required for sample analysis
and the operator observations concerning the use of the rapid PCR technology (e.g., ease of use,
maintenance, etc.).

                                      4.5  Schedule
       The verification test described here  will take place during approximately four to seven
weeks in late May - July 2004 at Battelle's  laboratories in West Jefferson and Columbus, Ohio,
and at a nearby  non-laboratory location. It  will be necessary for participating vendors to provide
their rapid PCR technologies to Battelle prior to testing so staff may become familiar with
operating the rapid PCR technologies before testing begins. Vendor staff are requested to provide
training in operating the rapid PCR technologies either in person or by teleconference. Unused
rapid PCR technologies and associated equipment will be returned to the vendors at the
completion of report writing. As appropriate, rapid PCR technologies will be decontaminated by
being exposed to a vapor of hydrogen peroxide  before being returned to the vendor.

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                        5.0 DATA HANDLING AND REPORTING
                            5.1 Data Acquisition and Review
       Various types of data will be acquired and recorded electronically or manually by Battelle
during this verification test. Table 4 summarizes the type of data to be recorded. All data and
observations will be documented by Battelle staff on data sheets or in laboratory record books.
Results from the reference methods will be compiled in written or electronic format.
       Records received by or generated by Battelle staff in the verification test will be reviewed
by a another Battelle staff member within two weeks of receipt or generation, respectively, before
the records are used to calculate, evaluate, or report verification results.  This review will be
performed by a Battelle technical staff member involved in the verification test, but not the staff
member that originally received or generated the record. The review will be documented by the
person performing the review by adding his/her initials and date to a hard copy of the record
being reviewed. This hard copy will then be returned to the Battelle staff member who will be
storing the record.  In addition, data calculations performed by Battelle will be  spot-checked by
Battelle technical staff to  ensure that calculations are performed correctly. Calculations to be
checked include any statistical calculations described in this test/QA plan. The data obtained
from this verification test will be compiled and reported independently for each rapid PCR
technology. The Verification Report prepared and provided to the vendor will  address only the
vendor's technology submitted for performance verification (not other technologies submitted by
other vendors that might also have been verified under the ETV program).

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Table 4. Summary of Data Recording Process
Data to Be
Recorded
Dates and times of
test events
Calibration
information and
results for physico-
chemical
parameters
(temperature,
salinity, pH,
conductivity, etc.)
Sample collection
and preparation
information,
including chain-of-
custody
Rapid PCR
technology
procedures and
sample results
Reference method
procedures and
sample results
Where Recorded
ETV data sheets
ETV data sheets
ETV data sheets
and chain-of-
custody forms
ETV data sheets
ETV data sheets,
or data acquisition
system, as
appropriate
How Often Recorded
Start/end of test, and
at each change of a
test parameter
Prior to sample
preparation
At time of sample
collection and
preparation
Throughout test
duration
Throughout sample
analysis process
Disposition of Data(a)
Used to
organize/check test
results; manually
incorporated in data
spreadsheets as
necessary
Manually incorporated
in data spreadsheets as
necessary
Used to
organize/check test
results; manually
incorporated in data
spreadsheets as
necessary
Manually incorporated
in data spreadsheets
Transferred to
spreadsheets
(a) All activities subsequent to data recording are carried out by Battelle.



                                     5.2 Data Analysis


       The technologies participating in this verification test will only be evaluated for

qualitative results (i.e., yes/no responses to samples) based on the expected application of these

technologies as rapid screening tools. All data analyses will be based on these qualitative results.


5.2.1 Accuracy


       The accuracy will be assessed by evaluating how often the rapid PCR technology result is

positive in the presence of a concentration above the LOD.  An overall percent agreement will be

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determined by dividing the number of positive responses to the overall number of analyses of
spiked samples.

5.2.2  Precision

       Precision measures the repeatability and reproducibility of PCR technology responses.
The precision of the four replicates of each sample set will be assessed. Responses will be
considered inconsistent if one or more of the four replicates differs from the response of the other
samples in the replicate set. The overall precision for each rapid PCR technology will be assessed
by calculating the overall number of consistent responses of all the sample sets.


5.2.3  Specificity

       The specificity will assess the rapid PCR technology's ability to detect the absence of the
contaminant when it is truly absent.  An overall specificity rate will be determined by dividing the
number of negative responses by the total number of unspiked samples or method blanks.

5.2.4  False Positive/False Negative Responses

       A false positive response will be defined as a detectable or positive rapid PCR technology
response when the ASTM DI water, including interferent samples, or drinking water sample is not
spiked at all.  Because most of the contaminants being tested can occur naturally in water, and
because rapid PCR technologies cannot distinguish between live and dead organisms, each DW
sample will be plate enumerated to verify the presence or absence of the contaminant of interest.
A false positive rate will be reported as the frequency of false positive results out of the total
number of unspiked samples or method blanks, as in the specificity calculation.
       A false negative response is defined as a non-detectable response or negative response
when the sample was spiked with a contaminant at a concentration greater than the LOD.
Reagent blanks, PT (contaminant and interferent) samples, and DW  samples will be included in
the analysis. A false negative rate will be evaluated as the frequency of false negative results out
of the total number of spiked samples for a particular contaminant.

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5.2.5  Matrix Interferences

       The potential effect of the DW matrix on the rapid PCR technology performance will be
evaluated qualitatively by comparing the results for the spiked and unspiked DW samples to those
for the PT samples.  The results indicating the correct or incorrect reporting of the presence of a
contaminant will be evaluated.

5.2.6  Field Portability

       The results obtained from the measurements made on samples in the laboratory and field
setting will be compiled independently for each rapid PCR technology that is designated as being
field portable and assessed for comparability of the measurements under the different
environmental conditions.  The results obtained from unspiked, unconcentrated drinking water
samples in the field will also be evaluated.  Also, qualitative observations of each technology's
performance in a non-laboratory setting will be made by the Verification Test Coordinator and
operators. Factors such as the ease of transport and set-up, demand for electrical power, and
space requirement will be documented and discussed in the report. Dexterity issues and ease of
use will also be evaluated under a first-responder scenario where the operator will test samples in
a Level C PPE.  These results will be compared to field portability tests done without the use of a
protective suit.

                                     5.3  Reporting

       The data obtained in the verification test will be compiled separately for each rapid PCR
technology, and the  data evaluation methods described in Section 5.2 will be applied to each data
set without comparison to any other rapid PCR technology. Following completion of the data
evaluation, a draft verification report will be prepared for each rapid PCR technology.  The
verification report will individually address each rapid PCR technology submitted for
performance verification, not other technologies submitted for verification under the same test.
The verification report will describe the verification test procedures and document the results.
Each draft verification report will be submitted to the corresponding vendor for review and
comment. Each draft report will be revised in response to the comments provided by the vendor.
The revised reports will be submitted for external peer review, revised again to address the peer
review comments, and submitted to EPA for final approval.

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       A verification statement will also be prepared for each rapid PCR technology. The
verification statement is a two to four page summary of the rapid PCR technology, test
procedures, and results.  The verification statement will follow the same review and revision
process as the verification reports.  Upon final approval by EPA, each verification statement will
be signed by a senior Battelle manager and an EPA laboratory director. Final verification reports
and statements are expected to be posted on the ETV website (http://www.epa.gov/etv), and
original signed verification statements will be provided to the vendor.

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                    6.0 QUALITY ASSURANCE /QUALITY CONTROL

       The QA/QC activities associated with this verification test will focus primarily on sample
preparation and handling, data recording and analysis, and  reference laboratory analysis.  An
independent audit covering each of these areas will be performed by the Battelle Quality Manager
to ensure the quality of the verification test.

                        6.1 Sample Chain-of-Custody Procedures

       Sample custody was documented throughout collection, shipping, and analysis of the
samples from the water utility to Battelle laboratories. Similar documentation will be recorded for
shipping and analysis of samples to the subcontract laboratories. Sample chain-of-custody
procedures will be in accordance with the Battelle SOP ASAT II-00711, Sample Chain-of-Custody
for Dioxin/Furan Analysis.  The chain-of-custody form summarizes the samples collected and
analyses requested. The chain-of-custody form will track sample release from the field to the
Battelle laboratory, and from the Battelle laboratory to the subcontract laboratory. Each chain-of-
custody form will be signed by the person relinquishing samples once that person has verified that
the chain-of-custody form is accurate. The original sample chain-of-custody forms accompany
the samples; the shipper will keep a copy. Upon receipt at the sample destination, chain-of-
custody forms will be signed by the person receiving the samples once that person has verified
that all samples identified on the chain-of-custody forms are present in the shipping container.
Any discrepancies will be noted on the form and the sample receiver will immediately contact the
Verification Test Coordinator to report missing, broken, or compromised samples. The recipient
will retain a copy of the form while the original chain-of-custody form will be sent back to the
Verification Test Coordinator for storage in the study file.

                         6.2 Rapid PCR Technology Calibration

       Prior to analysis, the Verification Test Coordinator will identify which rapid PCR
technologies require calibration. All such rapid PCR technologies will be calibrated according to
vendor directed procedures. Other equipment provided by the vendor will be calibrated based on
vendor requirements.

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                                6.3 Equipment Calibration

       For physio-chemical characterization, analytical equipment was calibrated by the
subcontract laboratory according to the procedure specified in the standard method. Similar
calibration will be performed for interferent conformation. Pipettes will be calibrated according
to the procedure outlined in Battelle SOP No: VI-02512. Pipettes will be calibrated semiannually
and the calibration service will provide a calibration certificate.

                   6.4  QC of Stock Solutions Preparation and DW Characterization

       Battelle QA staff will provide oversight of the stock solution preparation for all
contaminants.  The concentration of the stock solutions of all contaminants will be confirmed by
the plate enumeration method.  Additionally, each unspiked DW sample will be analyzed by this
method to confirm the absence of these bacteria. Method blanks and control spikes will be
analyzed by the subcontract laboratory performing confirmation analysis on the humic and fulvic
acid samples and water quality parameters.  Method blanks and control spikes will be analyzed
with every batch of samples processed.  Method blank and control spikes of interferences will be
analyzed in accordance with standard methods and QC limits specified therein.

                                        6.5  Audits

6.5.1  Technical Systems Audit

       The Battelle Quality Manager will conduct a technical systems audit at least once during
the course of the verification test. The purpose of this audit is to ensure that the verification test
is being performed in accordance with this test/QA plan and the AMS  QMP1, and that all
procedures described in this test/QA plan are being followed. This audit will review the
standards and methods used, compare actual test procedures  to those specified in this test/QA
plan, and review data acquisition and handling procedures.  An independent technical systems
audit may also be performed by EPA Quality Management staff during the verification test at
EPA's discretion.
       Before using an outside laboratory to perform stock solution confirmation analyses, the
Battelle Quality Manager conducts an audit of the laboratory's quality documents. The
subcontract laboratory for this test has already been audited and has been deemed qualified to
conduct confirmatory analyses as specified previously.

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6.5.2  Data Quality Audit

       At least 10% percent of the data acquired during the verification test will be audited
during the verification test.  Battelle's Quality Manager will trace the data from its initial
acquisition, through reduction and statistical analysis, to final reporting, to ensure the integrity of
the reported results. All calculations performed on the data undergoing the audit will be checked.

                                   6.6 QA/QC Reporting

       Each assessment and audit will be documented in accordance with Section 3.3.4 of the
AMS QMP1.  The results of the technical systems audit will be submitted to EPA.  Assessment
reports will include the following:
       •  Identification of any adverse findings or potential problems
       •  Response to adverse findings or potential problems
       •  Recommendations for resolving problems
       •  Confirmation that solutions have been implemented and are effective
       •  Citation of any noteworthy practices that may be of use to others.

                                   6.7 Corrective Action

       During the  course of any assessment or audit, the Battelle Quality Manager will inform
the technical staff of any immediate  corrective action that should be taken. If serious quality
problems exist, the Battelle Quality Manager is authorized to stop work. Once the assessment
report has been prepared, the Verification Test Coordinator will ensure that a response is provided
for each adverse finding or potential problem, and will implement any necessary follow-up
corrective action. The Battelle Quality Manager will ensure that follow-up corrective action has
been taken.

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                               7.0 HEALTH AND SAFETY

                          7.1  Standard/Test Sample Preparation

       All handling of solid and highly concentrated aqueous solutions of contaminants and
possible interferences will be done inside of a laboratory hood with hood sash set to the lowest
height that still allows for safe manipulation of materials. The following guidelines should be
adhered to:
   •   Personal protective equipment shall include safety glasses with side shields, a laboratory
       coat, and nitrile lab gloves. Gloves shall be immediately changed if they become
       contaminated.
   •   All contaminated waste shall be handled as hazardous waste and disposed of according to
       facility regulations.

                         7.2 Handling During Verification Testing

       Laboratory and field handling of any solutions used during the verification test will be
accomplished by taking the following precautions:
    •  All containers shall be stored and transported in double containment.
    •  Safety goggles, nitrile gloves with long cuffs, and a chemical resistant disposable lab coat
       shall be worn when handling all chemicals. Gloves shall be immediately changed if they
       become contaminated.

           7.3  Testing of Anthrax, E. coli, F. tularensis, Bmcella suis, and Plague

    Use of these contaminants in the verification of rapid PCR technologies will be done
following the safety procedures required at the MREF facility as noted in Section 3.3  and Battelle
Columbus Operation facilities.

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                                    8.0 REFERENCES

1.    Quality Management Plan (QMP) for the ETV Advanced Monitoring Systems Center,
     Version 5.0.  EPA Environmental Technology Verification Program, prepared by Battelle,
     Columbus, Ohio, March, 2004.

2.    Environmental Technology Verification Program Quality Management Plan, December
     2002 (EPA/600/R-03/021).

3.    Burrows, W. D.; Renner, S. E. Biological Warfare Agents as Threats to Potable Water,
     Environmental Health Perspectives, 107, 975-984,  1999.

4.    Battelle MREF X-054. Enumeration of BL-2 and BL-3 Bacterial Samples via the Spread
     Plate Technique. July 2003.

5.    Code of Federal Regulations, Title 32, Chapter 5, Part 626, Biological Defense Safety
     Program and Part 627, Biological Defense Safety Program, Technical Safety Requirements.

6.    EPA/600/R-93-100. EPAMethod 180.1. Turbidity  (Nephelometric), Methods for the
     Determination of Inorganic Substances in Environmental Samples. 1993.

7.    American Public Health Association, et al. Standard Methods for Examination of Water and
     Wastewater.  19th Edition.  1997. Washington D.C.

8.    EPA/600/4-79-020. Methods for Chemical Analysis of Water and Wastes. March 1983.

9.    EPA/600/R-95-131. EPA Method 524.2. Purgeable Organic Compounds by Capillary
     Column GC/Mass Spectrometry. Methods for the Determination of Organic Compounds in
     Drinking Water, Supplement III. August 1995.

10.   EPA/600/R-95-131. EPA Method 552.2. Haloacetic Acids and Dalapon by Liquid-Liquid
     Extraction, Derivatization and GC with Electron Capture Detector. Methods for the
     Determination of Organic Compounds in Drinking  Water, Supplement III. August 1995.

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                                                                                May 2004
11.   Battelle SOP ASAT 11-007. Sample Chain-of-Custody for Dioxin/Furan Analysis. October

     2000.


12.   Battelle SOP VI-025. Operation, Calibration, and Maintenance of Fixed and Adjustable

     Volume Pipettes. January, 2003.

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