ASTM E3131-17

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation:E3131 −17
Standard Specification for
Nucleic Acid-Based Systems for Bacterial Pathogen
Screening of Suspicious Visible Powders
This standard is issued under the fixed designation E3131; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
Evaluation of nucleic acid-based detection systems is necessary to ensure that they can achieve
required performance metrics for the intended application.These systems should be evaluated in both
laboratory and field settings to determine performance, including potential for false positive or
negative results, probability of detection (POD), and potential impacts of other substances on system
performancesuchascommonlyencounteredsuspiciouspowders.Laboratoryevaluationsestablishthe
best-case performance for a product and also serve as a means to eliminate from consideration those
products that have deficiencies or limitations before extensive cost and effort is expended for field
testing. Testing should be conducted under conditions recommended by the manufacturer. The
statistical derivation used in this specification assumes that conditions during testing remain stable.
Independent testing of biothreat or biological agent detection systems helps to establish the
reliability of results and improves first-responder and supporting agencies’confidence in these tools.
It is important that testing requirements balance the need for proven systems with the need for a
process that is not cost or time prohibitive and allows the evaluation of new technologies and assays
as they are developed. This is particularly true for nucleic acid-based detection systems because new
technologiesandproductscontinuetoemergeonthemarketandexistingassaysmayberevisedwhich
necessitates retesting.
This specification describes a statistically based testing approach for evaluating the performance of
nucleic acid-based detection systems. The approach ties performance of the system to a specified
lower confidence bound (LCB) on the POD at a known confidence level (CL) (see Fig. 1).
Testing shall be conducted to one of two performance levels (see Figs. 2 and 3): (1) ≥95% POD
with 95% CL, or (2) ≥90% POD with 90% CL. Four testing modules shall be used to evaluate
system performance (see Table 1): (1) Test Module 1—Biological agent nucleic acid inclusivity
testing; (2) Test Module 2—Biological agent nucleic acid exclusivity testing; (3) Test Module
3—Suspiciouspowdertesting(commonlyencounteredhoaxpowdersandenvironmentalmaterialthat
could interfere with test results, controls, or cause a false positive result); and (4) Test Module
4—Wholeorganismbiologicalagentspikedsuspiciouspowdertesting(impactofothermaterialonthe
ability to detect target biological agents or cause a false negative result). See Table 2 for a listing of
suspicious powders and the Annexes for the representative biological agents that shall be tested.
Three different testing tiers are also defined to reduce testing burden by allowing testing of
biological agent strain panels with fewer panel members (see Table 1). Inclusivity and exclusivity
testing tier panels are provided in Annex A4 and Annex A5. All three testing tiers shall test the full
panel of suspicious powders (Table 2) and the whole representative biological agent (see AnnexA7)
spiked into powders.
While the greatest extensiveness of test panel inclusivity and exclusivity strains and highest POD
and CL are always desirable, time and budget constraints often do not permit this extent of testing.
While some detection systems may not be able to achieve the highest performance metrics, it is still
valuable to know the level to which they can perform.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3131−17
1. Scope
1.1 General:
1.1.1 This specification provides system designers,
manufacturers, integrators, procurement personnel, end users/
practitioners, and responsible authorities a common set of
parameters to match the capabilities of biological assessment
tools with user needs.
1.1.2 This specification is not meant to provide for all uses.
Manufacturers, purchasers, and end users will need to deter-
minespecificrequirementsincluding,butnotlimitedto,useby
hazardous material (HAZMAT) teams and Urban Search and
Rescue (US&R) teams, use in explosive or other hazardous
environments or atmospheres, use with personal protective
equipment (PPE), use by firefighters or law enforcement
officers or both, special electromagnetic compatibility needs,
extended storage periods, and extended mission time. These
specific requirements may or may not be generally applicable
to all nucleic acid-based detection systems.
1.2 Operational Concepts—Nucleic acid-based detection
systems are used to detect, identify, or quantify, or combina-
tions thereof, biological hazards to support short-term tactical
decision making to protect responders and the public. The
system should provide low false-positive and false-negative
rates. Uses of these systems include survey, surveillance, and
screening of samples, particularly during a response to a
suspected biological agent incident.Afield-deployable system
should withstand the rigors associated with uses including, but
not limited to, high- and low-temperatures and storage
conditions, shock and vibration, radio frequency interference,
andrapidchangesinoperatingtemperatureandhumidity.Note
that this specification does not address testing the potential
impact of the rigors associated with use of systems in the field.
1.3 Nucleic Acid-Based System Detection Capabilities—
Manufacturers or independent third-party testing entities shall
document and verify, through testing, the capabilities of the
system.
1.4 Units—The values stated in SI units are to be regarded
as the standard. No other units of measurement are included in
this standard. Liquid concentrations of the biohazard materials
*LCB/CL—Lower confidence bound/confidence level.
are presented in number of biological agents or genome
**The number of samples to be tested shall be determined prior to any testing
equivalents per volume for pathogens such as bacteria and
and cannot be modified (for example, if 47 samples are chosen to be analyzed
(anticipating no failures) to achieve a 0.95/95 % LCB/CL and a failure occurs, spores (biological agents/mL, genome equivalents/mL (GE/
additional samples beyond the original 47 cannot be tested. Testing shall start
mL), colony forming units/mL (CFU/mL), or spores/mL).
over).
***Testing Tier 3 does not have any inclusivity strain nucleic acid testing because
1.5 This standard does not purport to address all of the
the representative inclusivity whole biological agent for this tier is tested in Test
safety concerns, if any, associated with its use. It is the
Module 4.
responsibility of the user of this standard to establish appro-
NOTE 1—Test Modules can be performed in any order.
FIG. 1Overview of Nucleic Acid-Based Biological Agent Testing
priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use.
1.6 This international standard was developed in accor-
dance with internationally recognized principles on standard-
This specification is under the jurisdiction of ASTM Committee E54 on
ization established in the Decision on Principles for the
Homeland Security Applications and is the direct responsibility of Subcommittee
Development of International Standards, Guides and Recom-
E54.01 on CBRNE Sensors and Detectors.
mendations issued by the World Trade Organization Technical
Current edition approved Oct. 15, 2017. Published November 2017. DOI:
10.1520/E3131-17. Barriers to Trade (TBT) Committee.
E3131−17
NOTE 1—If the number of failed results is 0 out of 47 tested samples, 1 out of 79 samples, or 2 out of 107 samples, then a device meets the POD of
0.95.
FIG. 2Number of Independent Tests Required to Meet the Performance Criteria of 0.95 LCB (Horizontal Red Line) with 95% Confidence
at the Chosen Test Sample Concentration
2. Referenced Documents SMPR 2016.007Standard Method Performance Require-
2 ments (SMPRs) for Detection of Francisella tularensis in
2.1 ASTM Standards:
Aerosol Collection Devices
E2677Test Method for Determining Limits of Detection in
SMPR 2016.008Standard Method Performance Require-
Explosive Trace Detectors
3 ments (SMPRs) for DNA-Based Methods of Detecting
2.2 AOAC Standards:
Yersinia pestis in Field-Deployable, Department of De-
SMPR 2010.001Standard Method Performance Require-
fense Aerosol Collection Devices
ments for Polymerase Chain Reaction (PCR) Methods for
SMPR 2016.009Standard Method Performance Require-
Detection of Francisella tularensis in Aerosol Collection
ments (SMPRs) for DNA-Based Methods for Detecting
Filters and/or Liquids
Brucella suisinField-Deployable,DepartmentofDefense
SMPR 2010.002Standard Method Performance Require-
Aerosol Collection Devices
ments for Polymerase Chain Reaction (PCR) Methods for
SMPR 2016.010Standard Method Performance Require-
Detection of Yersinia pestis in Aerosol Collection Filters
ments (SMPRs) for DNA-Based Methods of Detecting
and/or Liquid
Burkholderia pseudomallei in Field-Deployable, Depart-
SMPR 2010.003Standard Method Performance Require-
ment of Defense Aerosol Collection Devices
ments for Polymerase Chain Reaction (PCR) Methods for
SMPR 2015.011Standard Method Performance Require-
Detection of Bacillus anthracis in Aerosol Collection
ments (SMPRs) for Detection of Coxiella burnetii
Filters and/or Liquids
2.3 ISO Standard:
SMPR 2016.006Standard Method Performance Require-
ISO Guide 34General Requirements for the Competence of
ments (SMPRs) for DNA-Based Methods of Detecting
Reference Material Producers
Bacillus anthracis in Field-Deployable, Department of
2.4 Federal Standard:
Defense Aerosol Collection Devices
18 USC 178Definitions
For referenced ASTM standards, visit the ASTM website, www.astm.org, or Available from International Organization for Standardization (ISO), ISO
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Central Secretariat, BIBC II, Chemin de Blandonnet 8, CP 401, 1214 Vernier,
Standards volume information, refer to the standard’s Document Summary page on Geneva, Switzerland, http://www.iso.org.
the ASTM website. Available from U.S. Government Printing Office, Superintendent of
Available from AOAC International, 2275 Research Blvd., Suite 300, Documents, 732 N. Capitol St., NW, Washington, DC 20401-0001, http://
Rockville, MD 20850-3250, http://www.aoac.org. www.access.gpo.gov.
E3131−17
NOTE 1—If the number of failed results is 0 out of 14 tested samples, 1 out of 31 samples, or 2 out of 44 samples, then a device meets the POD of
0.90.
FIG. 3Number of Independent Tests Required to Meet the Performance Criteria of 0.90 LCB (Horizontal Red Line) with 90% Confidence
at the Chosen Test Sample Concentration
TABLE 1 Overview of Test Samples for Testing Tiers
Test Module 4: Whole
Test Module 1: Inclusivity Test Module 2: Exclusivity
C
Test Module 3: Powders Biological Agent Spiked
A B
Nucleic Acid Nucleic Acid
D
Powders
Single whole agent
Testing Tier 1 Full panel Full panel 22
representative strain
Single whole agent
Testing Tier 2 Reduced size panel Reduced size panel 22
representative strain
Single whole agent
E
Testing Tier 3 None Reduced size panel 22
representative strain
A
Inclusivity strain panels for Testing Tiers 1–3 are listed in Annex A4.
B
Exclusivity strain panels for Testing Tiers 1–3 are listed in Annex A5.
C
See Table 2 for a list of 22 powders.
D
Whole biological agent inclusivity strains are listed in Annex A7. Note that whole biological agents are intact spores (for Bacillus) or intact cells (all others biological
agents).
E
Testing Tier 3 does not have any inclusivity strain nucleic acid testing because the representative inclusivity strain for this Tier is tested in Test Module 4 as a whole
biological agent.
3. Terminology organismorinfectioussubstancecapableofcausing:(1)death,
disease,orotherbiologicalmalfunctioninahuman,ananimal,
3.1 Definitions:
a plant, or other living organism; (2) deterioration of food,
3.1.1 accuracy, n—closeness of agreement between a test
water, equipment, supplies, or material of any kind; and (3)or,
result and the accepted reference value.
deleterious alteration of the environment. 18 USC 178
3.1.2 assay, n—quantitative or qualitative test used to deter-
3.1.3.1 Discussion—Also termed biothreat agent.
mine the presence or absence of a biological material.
3.1.4 calibration, n—set of operations that establish, under
3.1.3 biological agent, n—any microorganism (including,
specified conditions, the relationship between the values of
but not limited to, bacteria, viruses, fungi, rickettsiae, or
protozoa); infectious substance; or any naturally occurring, quantitiesindicatedbyameasurementinstrumentormeasuring
bioengineered, or synthesized component of any such micro- system or values represented by a material measure or a
E3131−17
TABLE 2 Suspicious Powders
3.1.10 inhibition, n—undesirable effect that can result in a
Class of Powder Powder Type false negative result and is typically caused by the presence of
Brewer’s yeast powder
compounds that interfere with the assay or detection process.
Organic, biological
Dipel dust
Milk powder
3.1.11 limit of detection, LOD, n—lowestamountofanalyte
Organic, protein-containing Infant formula
in a sample that can be detected with (stated) probability.
White flour
E2677, EP17-A2
Coffee creamer (non-dairy)
Instant pectin (2)
Acetaminophen
3.1.12 lower confidence bound, LCB, n—lowest value of a
Organic, no protein Powdered sugar
Corn starch
one-sided confidence interval; or the lowest value of a half
Polyethylene glycol 3350 (for
bandcreatedusingaprocedurethatwhenrepeatedmanytimes,
example, MiraLAX, Glycolax)
on distinct datasets, generated from the same underlying
Toothpaste powder with fluoride
Baking powder (aluminum free)
stochastic process, will include the true measure of perfor-
Calcium carbonate (antacid)
mance a proportion of times equal to the stated probability.
Baking soda
Epsom salt
3.1.13 measurement process, n—process used to detect a
Inorganic Magnesium carbonate (gym chalk)
material or determine if a system or instrument performs as
Borax
intended.
Talc
Road dust (NIST)
3.1.14 multiplexed assay, n—assay that is capable of mea-
Kaolin clay
Popcorn salt suring multiple biological agents in a single test sample.
3.1.15 near neighbor, n—organism, virus, or nucleic acid
thatissimilartoadesiredtargetbiologicalagentbutshouldnot
reference material and the corresponding values realized by
result in a positive detection result.
standards. Eurachem Selection
3.1.15.1 Discussion—Exclusivity panel members include
(1)
near neighbors.
3.1.5 confidence interval, CI, n—range of values created
3.1.16 operator, n—person operating an on-site biological
using a procedure that, when repeated many times, on distinct
assessment technology.
datasets, generated from the same underlying stochastic
3.1.17 panel, n—collection of bacteria, spores, viruses,
process, will bracket the true measure of performance, such as
nucleic acids, or suspicious powders used during testing.
probability of detection, the proportion of times stated.
3.1.17.1 Discussion—Examples of panels include inclusiv-
3.1.6 confidence level, CL, n—probability value associated
ity panel, exclusivity panel, and suspicious powder panel.
with a confidence interval; the percentage of intervals that can
3.1.18 pooling, v—act of creating a single test sample (the
be expected to include the true population parameter in the
pooled sample) that contains strains from different biological
long run.
agents.
3.1.7 exclusivity panel, n—collection of near-neighbor bio-
3.1.19 probability of detection, POD, n—proportionofposi-
logical agents, viruses, or nucleic acids used during testing.
tive analytical outcomes for a qualitative method for a given
3.1.7.1 Discussion—All exclusivity panel members should
matrix at a given biological agent level or concentration.
result in negative detection results.
SMPR 2010.003
3.1.8 genome equivalents, GE, n—number of genome cop-
3.1.20 reference material, n—material, sufficiently homog-
ies present in a given mass of deoxyribonucleic acid (DNA)
enous and stable with respect to one or more specified
that can be calculated by converting the size of a genome in
propertiesthathasbeenestablishedtobefitforitsintendeduse
base pairs to micrograms of DNA.
in the measurement process; properties can be quantitative or
3.1.8.1 Discussion—Assuming the average mass of a base
qualitative. ISO Guide 34
pair (bp) is 660 Da (g/mole) and using Avogadro’s number
(6.022 × 10 molecules/mole), the genome equivalents can be
3.1.21 sensitivity, n—change in the response of a measuring
determined by first multiplying the mass of DNA(ng) × 6.022
instrument divided by the corresponding change in the
×10 ng/g.
stimulus. Eurachem Guide
(3)
molecule
massofDNAinng 3 6.022 3 10
~ !
S D
mole
3.1.22 specificity/selectivity—ability of a measurement pro-
GE 5 (1)
g ng cedure to determine accurately and specifically the analyte of
~genomelengthinbasepairs! 3 660 310
S D
mole g interest in the presence of other components in the sample
matrix under the stated conditions of the test. Eurachem
3.1.9 inclusivity panel, n—collection of closely related bio-
Guide
logical agents, viruses, or nucleic acids used during testing.
(3)
3.1.9.1 Discussion—All inclusivity panel members should
result in positive detection results.
3.1.23 spiked sample, n—sample that is created by adding a
known quantity of a biological agent to a known quantity of
6 suspicious powder.
The boldface numbers in parentheses refer to a list of references at the end of
this standard. 3.1.23.1 Discussion—Examples of spiked samples include
E3131−17
pathogens added to suspicious powders that are typically concentration (see Fig. 3). Testing shall use results from a
prepared in a liquid (buffer) suspension. single round of experiments with a fixed number of tests (that
is, if 14 samples are chosen to be tested but a failure occurs,
3.1.24 strain, n—isolates or variants of the target biological
additional samples cannot be tested. Testing shall start over).
agent(s) that the method can detect (inclusivity strains) or
4.4.1 Without a single failed result, 14 samples shall be
should not detect (exclusivity strains).
tested;
3.1.25 suspicious powder, n—any material that is used to
4.4.2 With no more than a single failed result, 31 samples
create a perceived biological threat such as a hoax powder that
shall be tested; or
may visually resemble Bacillus anthracis (Ba), ricin powder,
4.4.3 Withnomorethantwofailedresults,44samplesshall
or simply an unknown powder.
be tested.
3.1.25.1 Discussion—Suspicious powders include readily
available household items, food products, building materials,
4.4.4 The two testing plans presented in 4.3.1 – 4.3.3 and
and environmental matrices (for example, talcum powder,
4.4.1 – 4.4.3 allow demonstration of the levels of statistical
flour, drywall dust, and road dust).
performance chosen for this work: 0.95 LCB/95% CLor 0.90
LCB/90% CL. The testing plans shown are designed to be
3.1.26 testing module, n—set of samples used to establish
carriedoutbyselectingatotalnumberoftestsfortheLCB/CL
the detection technology performance for a particular type of
combination desired and then performing the number of tests
samples.
needed. The total number of failed results observed after
3.1.26.1 Discussion—Testing modules include inclusivity,
testing is completed determines if the performance level
exclusivity, suspicious powders, and whole biological agent
selected has been achieved or not.
spiked suspicious powders.
4.4.5 A more precise characterization of the statistical
4. Statistical Considerations for Testing
performance of a detection system (measured as the LCB/CL
combination) is always preferable but, as the difference in
4.1 The testing approach described herein uses a score CI
(4) to define the number of samples that need to be tested to testing resources needed between the 0.95 LCB/95% CLlevel
and the 0.90 LCB/90% level indicates, more extensive char-
achieve a desired POD estimate as indicated by the value of a
given LCB in a one-sided interval with a specified CL. In this acterizationcomesatacost.Becausethestatisticalapproachin
specification,twolevelshavebeenchosenasprovidingaccept- thisspecificationcallsformeasuringperformanceusingresults
able degrees of statistical performance: a low level and a high from a single round of experiments with a fixed number of
level. Results from selecting and executing one of the testing tests, it is advisable that the user selects the largest number of
planspresentedinthisspecificationcanbeusedtodetermineif tests that can be performed within practical limits. For
the chosen level of statistical performance has been met. example, performing 79 tests using the 0.95 LCB/95% CL
level and observing 0 failures allows the user to fulfill the
4.2 Each of the sample types listed in 4.2.1 – 4.2.4 shall be
requirements for the performance level selected, while simul-
considered as part of a separate testing module. That is, the
taneously providing a safeguard in the case a single failure is
number of samples that shall be analyzed to achieve a desired
found.
POD as demonstrated by the specified LCB and CLvalues, as
4.4.6 Similarly, deciding to test 47 samples with the goal of
described in 4.3 and 4.4, apply to test modules 4.2.1 – 4.2.4,
achieving the lower 0.90 LCB/90% CL performance level
which includes the following test modules:
allows for up to 3 failures observed during testing while still
4.2.1 Inclusivity nucleic acid testing (Test Module 1),
meeting 0.90 LCB/90% CL. Selecting the lower 0.90 LCB/
4.2.2 Exclusivity nucleic acid testing (Test Module 2),
90% performance level allows for up to 4 failures if 79
4.2.3 Suspicious powder testing (Test Module 3), and
samplesaretestedandupto7failuresif107samplesaretested
4.2.4 Whole biological agent spiked suspicious powder
while still meeting the low-level performance requirements.
testing (Test Module 4).
4.4.7 In general, only testing plans with zero, one, or two
4.3 Below is listed the minimum number of replicates
failedresultsand0.95LCB/95%CLor0.90LCB/90%CLare
required for each of the four testing modules listed in 4.2.1 –
explicitly considered in this specification. To find the LCB or
4.2.4forthecaseofLCB=0.95and95%CLatthechosentest
CL values or both corresponding to other testing results, as
concentration (see Fig. 2). Testing shall use results from a
well as determining testing requirements for other LCB/CL
single round of experiments with a fixed number of tests (that
combinations, the user is referred to Annex A8.
is, if 47 samples are chosen to be tested but a failure occurs,
4.4.8 SeeFig.1foranoverviewofhowtoplanandconduct
additional samples cannot be tested. Testing shall start over).
biological agent testing. Note that the number of samples to be
4.3.1 Without a single failed result, 47 samples shall be
tested shall be determined before any testing and cannot be
tested;
modified (for example, if you chose to test 47 samples,
4.3.2 With no more than a single failed result, 79 samples
anticipating no failures, to achieve a 0.95/95% LCB/CLand a
shall be tested; or
failureoccurs,youcannotsimplytestmoresamples—youshall
4.3.3 With no more than two failed results, 107 samples
start over).
shall be tested.
4.4 Below is listed the minimum number of replicates
5. Test Criteria
required for each of the four testing modules listed in 4.2.1 –
4.2.4toachieveanLCB=0.90and90%CLatthechosentest 5.1 Overview:
E3131−17
5.1.1 Select performance level (≥0.95 LCB/95% CL or sequences, a strain that contains one of the target sequences
≥0.90 LCB/90% CL) to estimate the number of samples that should not be combined with a strain that contains the other
will be required (see Fig. 1), and target sequence.
5.4.2.3 Each strain shall be present at a final concentration
5.1.2 Select testing Tier 1, 2, or 3 (see Table 2).
of 10 GE/mL in any pooled sample.
5.2 Test Sample Concentrations:
5.4.2.4 If any pooled test sample results in a failure, then
5.2.1 Test sample concentrations shall be (unless otherwise
each strain present in the pooled sample shall be tested
noted in the Annexes):
separately. If each strain present in the failed pooled sample is
5.2.1.1 The amount 10 GE/mLfor biological agent nucleic
successfully retested, then the failure of the pooled sample can
acid inclusivity panel members (Test Module 1).
be disregarded for purposes of establishing LCB/CL.
5.2.1.2 The amount 10 GE/mLfor biological agent nucleic
5.4.3 Suspicious powders shall not be pooled (Test Module
acid exclusivity panel members (Test Module 2),
3).
5.2.1.3 The amount 0.1 mg/mL for all powders listed in
5.4.4 Whole Biological Agent Pooling of Different Repre-
Table 1 (Test Module 3), and
sentative Biological Agents (Test Module 4):
5.2.1.4 The amount 2 × 10 whole biological agents/mL
5.4.4.1 Up to six representative whole biological agents
spiked into 0.1 mg/mL of powders for spiked powder testing
may be pooled into a single test sample (see Annex A7).
(Test Module 4).
NOTE 3—Burkholderia mallei and Burkholderia pseudomallei cannot
5.3 Liquids used for creating test samples shall be selected
both be present in a single test sample. However, the combined total
number of tests required for this biological agent panel (Test Module 4)
based on manufacturer guidance and are typically a buffer
includestestingofbothspecies.Forexample,toachievea0.95LBC/95%
solution.
CL with no test failures, 24 samples containing only the B. mallei
5.4 Pooling (5):
representative whole organism and 23 samples containing only the B.
pseudomallei representative whole organism could be analyzed for a total
5.4.1 Inclusivity Strain Nucleic Acid Pooling from Different
of 47 test samples.
Biological Agent Panels (Test Module 1):
5.4.4.2 Each whole biological agent shall be present at a
5.4.1.1 Up to six nucleic acid strains, one nucleic acid
final concentration of2×10 biological agents/mL in any
selected from each of the six different biological agent inclu-
pooled sample.
sivity panels listed in Annex A4 (Bacillus anthracis panel,
5.4.4.3 If any pooled test sample results in a failure, then
Brucella species panel, Burkholderia mallei/pseudomallei
each whole biological agent present in the pooled sample shall
panel, Coxiella burnetii panel, Francisella tularensis panel,
be tested separately. If each whole biological agent present in
and Yersinia pestis panel), may be pooled into a single test
the failed pooled sample is successfully retested, then the
sample. However, only a single inclusivity strain from each
failureofthepooledsamplecanbedisregardedforpurposesof
biological agent panel shall be present in a pooled sample.
establishing LCB/CL.
NOTE 1—Different biological agent strains can only be pooled if the
nucleic acid-based detection system and assay are designed to measure 5.5 Controls:
these biological agents simultaneously.
5.5.1 User-supplied controls including negative controls
NOTE 2—Different species are listed in the Brucella species inclusivity
(blanks) and positive controls (nucleic acids or whole biologi-
panel and the Burkholderia mallei/pseudomallei inclusivity panel, and
calagents)shallbeanalyzedduringthecourseoftesting.These
different subspecies are listed in the Francisella tularensis inclusivity
controls are in addition to any controls that are integral to the
panel. However, for purposes of pooling, each of these inclusivity panel
assay.
members shall be considered as the same biological agent (that is, a
pooled sample cannot contain more than one member from each different
5.5.2 Negative controls (blanks) shall be analyzed through-
biological agent inclusivity panel for: (1) Brucella species,(2) Burkhold-
outthecourseofthetestingtodemonstratethattheinstrument/
eria mallei/pseudomallei, and (3) Francisella tularensis.
assays and testing environment are free from contamination.
5.4.1.2 Each strain shall be present at a final concentration
5.5.2.1 A negative control shall be tested at least once per
of 10 GE/mL in any pooled sample.
ten samples analyzed or at least once each day that assays are
5.4.1.3 If any pooled test sample results in a failure, then
performed or whichever is greater.
each strain present in the pooled sample shall be tested
5.5.2.2 Negativecontrolsdonotcounttowardthenumberof
separately. If each strain present in the failed pooled sample is
tests required and failures do not count towards the LCB/CL
successfully retested, then the failure of the pooled sample can
performance metrics. If problems with negative controls occur
be disregarded for purposes of establishing LCB/CL.
and are not resolved, then testing shall be stopped until the
5.4.2 Exclusivity Strain Nucleic Acid Pooling from the Same
source of the problem can be identified and corrected.
or Different Biological Agent/Near-Neighbor Panels (Test
5.5.3 Positive controls (a known material such as purified
Module 2):
genomic deoxyribonucleic acid (DNA) or whole organism)
5.4.2.1 Up to ten exclusivity strain nucleic acids from the
shall be analyzed throughout the course of the testing to
same or different biological agent exclusivity panels may be demonstrate that the nucleic acid-based detection system is
pooled into a single test sample.
performing as expected.
5.4.2.2 Care should be taken to ensure pooling of certain 5.5.3.1 Apositivecontrolshallbetestedatleastonceperten
strains does not result in a false positive result for the assay samples analyzed or at least once each day that assays are
being tested. For example, if an assay is detecting two target performed or whichever is greater.
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5.5.3.2 Positive controls do not count toward the number of 5.7.3 Total time from sample to answer; and
tests required and failures do not count towards the LCB/CL 5.7.4 Total cost of ownership (for example, hardware,
performance metrics. If problems with positive controls occur consumables, and training needs), understanding that the
and are not resolved, then testing shall be stopped until the reagent cost, shelf-life, instrument maintenance, and upgrades
source of the problem can be identified and corrected. are significant contributors to the total cost.
5.5.3.3 Manufacturer-supplied controls do not count toward
6. Hazards
the number of tests required and failures or successes do not
6.1 BSL-3 and BSL-4 biological agents are very dangerous
count towards the LCB/CL performance metrics. If problems
with manufacturer controls occur, the target analyte results for and shall only be handled by experienced individuals/
institutions with appropriate controls and facilities. BSL-2
that test sample are invalidated and the test shall be repeated.
biological agents also pose risks and are potentially dangerous
If problems with manufacturer-supplied controls are not
if not handled properly.
resolved, then testing shall be stopped until the source of the
problem can be identified and corrected.
7. Keywords
5.6 Suspicious Powders—These sample types may include
7.1 biodetection assay; biological agent; biological assess-
commonly encountered hoax powders, environmental material
ment; biothreat agent; confidence interval; confidence level;
such as road dust, and other matrices such as drywall dust.
deoxyribonucleic acid; DNA; field screening; first responder;
PowdersinadditiontothoseinTable1canbetestedasdeemed
homeland security; nucleic acid; pathogen; PCR; polymerase
necessary.
chain reaction; probability of detection; spore; suspicious
5.7 Additional Factors to Consider that Could Impact Use
powder
and Performance:
5.7.1 Ease of use;
See http://www.cdc.gov/biosafety/publications/bmbl5/bmbl5_sect_iv.pdf for a
5.7.2 Weight and size; more in-depth discussion of the distinction between BSL conditions.
ANNEXES
(Mandatory Information)
A1. INCLUSIVITY AND EXCLUSIVITY TEST PANEL AVAILABILITY
A1.1 The sources mentioned in the following are sugges- A1.2.6 Deutsche Sammlung von Mikroorganismen und
tions only and their inclusion in this specification does not Zellkulturen GmbH (DSMZ), Germany;
imply their availability nor does it ensure their quality.
A1.2.7 European Culture Collections’ Organisation
(ECCO);
A1.2 Thefollowingarepotentialsourcesofbiologicalagent
A1.2.8 Microbiologics, United States;
nucleic acids and, to some degree, whole biological agents:
A1.2.9 National Collection of Industrial and Marine Bacte-
A1.2.1 American Type Culture Collection (ATCC), United
ria (NCIMB), United Kingdom;
States;
A1.2.10 National Collection of Type Cultures (NCTC)/
A1.2.2 Biodefense and Emerging Infections Research Re-
Public Health England, United Kingdom;
sources Repository (BEI Resources), United States;
A1.2.11 Northern Regional Research Laboratory (NRRL),
A1.2.3 Belgian Co-Ordinated Collections of Micro-
namely, Agricultural Research Service; Culture Collection,
organisms (BCCM), Belgium;
United States;
A1.2.4 Colorado Serum Company, United States;
A1.2.12 Robert Koch Institute, Germany; and
A1.2.5 Defense Biological Product Assurance Office (for-
A1.2.13 United Kingdom National Culture Collection
merly the Critical Reagents Program CRP)), United States; (UKNCC), United Kingdom.
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A2. TEST PANEL DOCUMENTATION
A2.1 Documentationofallnucleicacidsandwholebiologi- A2.2 Documentation shall include all organizations con-
cal agents used in testing shall include, at a minimum, tacted and other relevant information for any inclusivity or
sufficient information to trace the material used to its source
exclusivity panel members that cannot be obtained. Any
(for example, organism name including genus and species;
alternate panel strains that are acquired shall be clearly
strain, biovar or serovar (if applicable); lot number; manufac-
documented as to which original panel member strain they are
turer name; catalog or product number or both; purchase date;
replacing.
and organism storage and growth conditions).
A3. TEST PANEL QUALITY ASSURANCE
A3.1 Nucleic acids or biological agents shall undergo nec- should be demonstrated to be free of PCR inhibitors by using
essary quality assurance assessment before use. The concen- a reference measurement technique before testing (for
trations of all nucleic acid samples obtained should be verified example, real-time PCR using published primer/probes). This
usingaDNAintercalatingdye-basedassayorsimilarassay.An testing only ensures that nucleic acids can be amplified and
ultraviolet-visible (UV-VIS) spectrophotometric quantification doesnotnecessarilyensurethecorrectspeciesorstrainorboth
method is not recommended because this method is prone to were received. If nucleic acid is extracted from organisms, a
overestimation of DNAconcentration as a result of potentially quantification step should be performed after extraction using
contaminating ribonucleic acid (RNA). For polymerase chain an intercalating dye as described earlier or other suitable
reaction (PCR) systems, any nucleic acid samples obtained method.
A4. TEST MODULE 1: TESTING TIER INCLUSIVITY PANELS
A4.1 Test Module 1: Testing Tier 1 Inclusivity Panels: sequence type is not available, a strain different than those
listed in Table A4.3, but from the same organism, shall be
Nucleic acid from the following inclusivity panel members
selected, if available.
shall be tested for the applicable biological agent(s):
A4.1.3.1 For multiplexed assays, only one panel member
A4.1.1 Bacillus anthracis—If any listed strain is not
from those listed in Table A4.3 can be present in any pooled
available, an alternate strain shall be selected from the same
sample. Burkholderia mallei and Burkholderia pseudomallei
variable number tandem repeat (VNTR) group. If an alternate
cannot both be present in a single multiplexed test sample.
strain from the same VNTR group is not available, a strain
Note that the combined total number of tests required for this
from the same genotype shall be selected. If an alternate strain
biologicalagentnucleicacidpanelincludesthetotalnumberof
from the same genotype is not available, a strain different than test samples conducted of both species. For example, to
thoselistedinTableA4.1,butfromthesameorganism,shallbe achieve a 0.95 LCB/95% CLwith no test failures, 24 samples
containing only a B. mallei strain and 23 samples containing
selected, if available.
only a B. pseudomallei strain could be analyzed for a total of
A4.1.2 Brucella species—If any listed strain is not
47testsamples.Anapproximate50:50splitof B. malleiand B.
available, an alternate strain shall be selected from: (1) the
pseudomallei panel members shall be used.
same biovar for B. abortus, B. melitensis, and B. suis species
A4.1.4 Coxiella burnetii—If any listed strain is not
and (2) a different strain for B. canis, B. ceti, B. inopinata, B.
available, an alternate strain shall be selected from the same
microti, B. neotomae, B. ovis, B. papionis, B. pinipedialis, and
genomic group. If an alternate strain from the same genomic
B. vulpis species. If an alternate strain is not available, a strain
group is not available, a strain different than those listed in
different than those listed in Table A4.2, but from the same
Table A4.4, but from the same organism, shall be selected, if
organism, shall be selected if available. For multiplexed
available.
assays, only one panel member from those listed in TableA4.2
A4.1.5 Francisella tularensis—If any listed strain is not
can be present in any pooled sample.
available, an alternate strain shall be selected from the same
A4.1.3 Burkholderia mallei/pseudomallei—If any listed
type (if applicable). If an alternate strain from the same type is
strainisnotavailable,analternatestrainshallbeselectedfrom
not available, a strain different than those listed in TableA4.5,
the same sequence type. If an alternate strain from the same but from the same organism, shall be selected, if available. For
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TABLE A4.1 Test Module 1: Tier 1 Bacillus anthracis Inclusivity TABLE A4.3 Test Module 1: Tier 1 Burkholderia mallei/
A A
Panel pseudomallei Inclusivity Panel
Species Strain Notes Brukholderia mallei
Bacillus anthracis Ames pXO1+, pXO2+; VNTR Species Strain Notes
group A3b; genotype 62 B. mallei India 86-567-2 Sequence Type 40
Bacillus anthracis BA0018 pXO1+, pXO2+; VNTR B. mallei China 5 Sequence Type 40
group A1a; genotype 7 B. mallei 37 Sequence Type 40
Bacillus anthracis BA1035 pXO1+, pXO2+; VNTR
B. mallei China 7 Sequence Type 40
group B1; genotype 82 B. mallei GB5 Sequence Type 40
Bacillus anthracis K3 pXO1+, pXO2+; VNTR
B. mallei GB7 Sequence Type 100
group A3c; genotype 67 B. mallei Strain 6 Sequence Type 40
Bacillus anthracis Ohio ACB pXO1+, pXO2+; VNTR Burkholderia pseudomallei
group A3d; genotype 68 B. pseudomallei 576 Sequence Type 501
Bacillus anthracis PAK-1 pXO1+, pXO2+; VNTR B. pseudomallei 1026b Sequence Type 102
group A2; genotype 29
B. pseudomallei 7894 Sequence Type 11
B
Bacillus anthracis Pasteur pXO1–, pXO2+; VNTR B. pseudomallei HBPUB10134a Sequence Type 228
group A1a; genotype 8
B. pseudomallei K96243 Sequence Type 10
Bacillus anthracis RA3 pXO1+, pXO2+; VNTR B. pseudomallei MSHR305 Sequence Type 36
group B2; genotype 80
B. pseudomallei MSHR668 Sequence Type 129
Bacillus anthracis SK-102 pXO1+, pXO2+; VNTR B. pseudomallei MSHR840 Sequence Type 257
group A4; genotype 69
B. pseudomallei MSHR1655 Sequence Type 131
C
Bacillus anthracis Sterne pXO1+, pXO2–; VNTR B. pseudomallei RF80 Sequence Type 507
group A3b; genotype
A
Adapted from SMPR 2016.010.
59, 61
Bacillus anthracis Turkey 32 pXO1+, pXO2+; VNTR
group A1b; genotype 23
C
Bacillus anthracis V770-NP-1R pXO1+, pXO2–; VNTR
TABLE A4.4 Test Module 1: Tier 1 Coxiella burnetii Inclusivity
group A3A; genotype A
Panel
Species Strain Notes
Bacillus anthracis Vollum 1B pXO1+, pXO2+; VNTR
Coxiella burnetii Dugway Genomic group VI
group A4; genotype 77
Coxiella burnetii G Genomic group V
A
Adapted from SMPR 2010.003 and SMPR 2016.006.
Coxiella burnetii Henzerling Genomic group II
B
This strain should not be included in the inclusivity panel if the product being
Coxiella burnetii Idaho Goat Genomic group III
tested only uses a pXO1 assay as this strain is expected to be pXO1 negative.
Coxiella burnetii K Genomic group IV
C
These two strains should not be included in the inclusivity panel if the product
Coxiella burnetii Le B Genomic group VIII
being tested only uses a pXO2 assay as these strains are expected to be pXO2
Coxiella burnetii Nine Mile RSA439 Genomic group I
negative.
Coxiella burnetii Nine Mile RSA493 Genomic group I
Coxiella burnetii Q321 Genomic group VII
A
Adapted from SMPR 2015.011.
TABLE A4.2 Test Module 1: Tier 1 Brucella species Inclusivity
A
Panel
Species Strain Notes
TABLE A4.5 Test Module 1: Tier 1 Francisella tularensis
Brucella abortus 12 Biovar 3 A
Inclusivity Panel
Brucella abortus 292 (39/94) Biovar 4
Species Strain Notes
Brucella abortus 544 Biovar 1
Francisella tularensis 425 Type B
Brucella abortus 86/8/59 Biovar 2
subsp. holarctica
Brucella abortus 870 Biovar 6
Francisella tularensis JAP Cincinnati Type B
Brucella abortus 99-9971-135 Biovar 7
subsp. holarctica
Brucella abortus B3196 Biovar 5
Francisella tularensis LVS Type B
Brucella abortus C68 Biovar 9
B
subsp. holarctica
Brucella canis RM-666 N/A
Francisella tularensis VT68 Type B
Brucella ceti B1/94 N/A
subsp. holarctica
Brucella inopinata BO1 N/A
B
Francisella tularensis FSC147 N/A
Brucella melitensis 16M Biovar 1
subsp. mediasiatica
Brucella melitensis 63/9 Biovar 2
Francisella tularensis NM99-1823 Type A2
Brucella melitensis Ether Biovar 3
subsp. tularensis
Brucella microti CCM 4915 N/A
Francisella tularensis Scherm Type A1
Brucella neotomae 5K33 N/A
subsp. tularensis
Brucella ovis 63-390 N/A
Francisella tularensis SCHU S4 Type A1
Brucella papionis F8/08-60(T) N/A
subsp. tularensis
Brucella pinipedialis B2/94 N/A
Francisella tularensis WY96-3418 Type A2
Brucella suis 40 Biovar 4
subsp. tularensis
Brucella suis 513 Biovar 5
Brucella suis 686 Biovar 3 A
Adapted from SMPR 2010.001 and SMPR 2016.007.
B
Brucella suis 1330 Biovar 1
This subspecies is not further subdivided into types.
Brucella suis S2 Biovar 1
Brucella suis Thomsen Biovar 2
Brucella vulpis F60 or F965 N/A
A4.1.6 Yersinia pestis—If any listed strain is not available,
A
Adapted from SMPR 2016.009.
B
an alternate strain shall be selected from the same biovar. If an
N/A—These Brucella species are not further subdivided into biovars.
alternate strain from the same biovar is not available, a strain
different than those listed in Table A4.6, but from the same
organism, shall be selected, if available.
multiplexed assays, only one panel member from those listed
in Table A4.5 can be present in any pooled sample.
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TABLE A4.6 Test Module 1: Tier 1 Yersinia pestis Inclusivity
B. vulpis species. If an alternate strain is not available, a strain
A
Panel
different than those listed in Table A4.8, but from the same
Species Strain Notes
organism, shall be selected, if available. For multiplexed
Yersinia pestis A1122 Biovar Orientalis
assays, only one panel member from those listed in TableA4.8
Yersinia pestis Angola Biovar Antiqua
Yersinia pestis Antiqua Biovar Antiqua
can be present in any pooled sample.
Yersinia pestis CO92 Biovar Orientalis
A4.2.3 Burkholderia mallei/pseudomallei—If any listed
Yersinia pestis Harbin35 Biovar Mediaevalis
Yersinia pestis Java9 Biovar Orientalis
strainisnotavailable,analternatestrainshallbeselectedfrom
Yersinia pestis KIM Biovar Mediaevalis
the same sequence type. If an alternate strain from the same
Yersinia pestis Nairobi Biovar Antiqua
sequence type is not available, a strain different than those
Yersinia pestis Nicholisk 41
...