1、Designation: E3131 17Standard Specification forNucleic Acid-Based Systems for Bacterial PathogenScreening of Suspicious Visible Powders1This standard is issued under the fixed designation E3131; the number immediately following the designation indicates the year oforiginal adoption or, in the case o
2、f revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.INTRODUCTIONEvaluation of nucleic acid-based detection systems is necessary to ensure that they can achiev
3、erequired performance metrics for the intended application. These systems should be evaluated in bothlaboratory and field settings to determine performance, including potential for false positive ornegative results, probability of detection (POD), and potential impacts of other substances on systemp
4、erformance such as commonly encountered suspicious powders. Laboratory evaluations establish thebest-case performance for a product and also serve as a means to eliminate from consideration thoseproducts that have deficiencies or limitations before extensive cost and effort is expended for fieldtest
5、ing. Testing should be conducted under conditions recommended by the manufacturer. Thestatistical derivation used in this specification assumes that conditions during testing remain stable.Independent testing of biothreat or biological agent detection systems helps to establish thereliability of res
6、ults 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 aprocess that is not cost or time prohibitive and allows the evaluation of new technologies and assaysas they are develo
7、ped. This is particularly true for nucleic acid-based detection systems because newtechnologies and products continue to emerge on the market and existing assays may be revised whichnecessitates retesting.This specification describes a statistically based testing approach for evaluating the performa
8、nce ofnucleic acid-based detection systems. The approach ties performance of the system to a specifiedlower 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 % PODwith 95 % CL, or (2
9、) 90 % POD with 90 % CL. Four testing modules shall be used to evaluatesystem performance (see Table 1): (1) Test Module 1Biological agent nucleic acid inclusivitytesting; (2) Test Module 2Biological agent nucleic acid exclusivity testing; (3) Test Module3Suspicious powder testing (commonly encounte
10、red hoax powders and environmental material thatcould interfere with test results, controls, or cause a false positive result); and (4) Test Module4Whole organism biological agent spiked suspicious powder testing (impact of other material on theability to detect target biological agents or cause a f
11、alse negative result). See Table 2 for a listing ofsuspicious 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 ofbiological agent strain panels with fewer panel members (s
12、ee Table 1). Inclusivity and exclusivitytesting tier panels are provided in Annex A4 and Annex A5. All three testing tiers shall test the fullpanel of suspicious powders (Table 2) and the whole representative biological agent (see Annex A7)spiked into powders.While the greatest extensiveness of test
13、 panel inclusivity and exclusivity strains and highest PODand 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 stillvaluable to know the level to which they c
14、an perform.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United StatesThis international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for theDevelopme
15、nt of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.11. Scope1.1 General:1.1.1 This specification provides system designers,manufacturers, integrators, procurement personnel, end users/practitioners, and respons
16、ible authorities a common set ofparameters to match the capabilities of biological assessmenttools 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-mine specific requirements including, but not limited to, use b
17、yhazardous material (HAZMAT) teams and Urban Search andRescue (US infectious substance; or any naturally occurring,bioengineered, or synthesized component of any such micro-organism or infectious substance capable of causing: (1) death,disease, or other biological malfunction in a human, an animal,a
18、 plant, or other living organism; (2) deterioration of food,water, equipment, supplies, or material of any kind; and (3)or,deleterious alteration of the environment. 18 USC 1783.1.3.1 DiscussionAlso termed biothreat agent.3.1.4 calibration, nset of operations that establish, underspecified condition
19、s, the relationship between the values ofquantities indicated by a measurement instrument or measuringsystem or values represented by a material measure or aNOTE 1If 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 o
20、f0.90.FIG. 3 Number of Independent Tests Required to Meet the Performance Criteria of 0.90 LCB (Horizontal Red Line) with 90 % Confidenceat the Chosen Test Sample ConcentrationTABLE 1 Overview of Test Samples for Testing TiersTest Module 1: InclusivityNucleic AcidATest Module 2: ExclusivityNucleic A
21、cidBTest Module 3: PowdersCTest Module 4: WholeBiological Agent SpikedPowdersDTesting Tier 1 Full panel Full panel 22Single whole agentrepresentative strainTesting Tier 2 Reduced size panel Reduced size panel 22Single whole agentrepresentative strainTesting Tier 3 NoneEReduced size panel 22Single wh
22、ole agentrepresentative strainAInclusivity strain panels for Testing Tiers 13 are listed in Annex A4.BExclusivity strain panels for Testing Tiers 13 are listed in Annex A5.CSee Table 2 for a list of 22 powders.DWhole biological agent inclusivity strains are listed in Annex A7. Note that whole biolog
23、ical agents are intact spores (for Bacillus) or intact cells (all others biologicalagents).ETesting 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 wholebiological agent.E3131 174reference ma
24、terial and the corresponding values realized bystandards. Eurachem Selection(1)63.1.5 confidence interval, CI, nrange of values createdusing a procedure that, when repeated many times, on distinctdatasets, generated from the same underlying stochasticprocess, will bracket the true measure of perform
25、ance, such asprobability of detection, the proportion of times stated.3.1.6 confidence level, CL, nprobability value associatedwith a confidence interval; the percentage of intervals that canbe expected to include the true population parameter in thelong run.3.1.7 exclusivity panel, ncollection of n
26、ear-neighbor bio-logical agents, viruses, or nucleic acids used during testing.3.1.7.1 DiscussionAll exclusivity panel members shouldresult in negative detection results.3.1.8 genome equivalents, GE, nnumber of genome cop-ies present in a given mass of deoxyribonucleic acid (DNA)that can be calculat
27、ed by converting the size of a genome inbase pairs to micrograms of DNA.3.1.8.1 DiscussionAssuming the average mass of a basepair (bp) is 660 Da (g/mole) and using Avogadros number(6.022 1023molecules/mole), the genome equivalents can bedetermined by first multiplying the mass of DNA (ng) 6.022109ng
28、/g.GE 5mass of DNA in ng! 3S6.022 3 1023moleculemoleDgenome length in base pairs! 3S660gmoleD3109ngg(1)3.1.9 inclusivity panel, ncollection of closely related bio-logical agents, viruses, or nucleic acids used during testing.3.1.9.1 DiscussionAll inclusivity panel members shouldresult in positive de
29、tection results.3.1.10 inhibition, nundesirable effect that can result in afalse negative result and is typically caused by the presence ofcompounds that interfere with the assay or detection process.3.1.11 limit of detection, LOD, nlowest amount of analytein a sample that can be detected with (stat
30、ed) probability.E2677, EP17-A2(2)3.1.12 lower confidence bound, LCB, nlowest value of aone-sided confidence interval; or the lowest value of a halfband created using a procedure that when repeated many times,on distinct datasets, generated from the same underlyingstochastic process, will include the
31、 true measure of perfor-mance a proportion of times equal to the stated probability.3.1.13 measurement process, nprocess used to detect amaterial or determine if a system or instrument performs asintended.3.1.14 multiplexed assay, nassay that is capable of mea-suring multiple biological agents in a
32、single test sample.3.1.15 near neighbor, norganism, virus, or nucleic acidthat is similar to a desired target biological agent but should notresult in a positive detection result.3.1.15.1 DiscussionExclusivity panel members includenear neighbors.3.1.16 operator, nperson operating an on-site biologic
33、alassessment technology.3.1.17 panel, ncollection of bacteria, spores, viruses,nucleic acids, or suspicious powders used during testing.3.1.17.1 DiscussionExamples of panels include inclusiv-ity panel, exclusivity panel, and suspicious powder panel.3.1.18 pooling, vact of creating a single test samp
34、le (thepooled sample) that contains strains from different biologicalagents.3.1.19 probability of detection, POD, nproportion of posi-tive analytical outcomes for a qualitative method for a givenmatrix at a given biological agent level or concentration.SMPR 2010.0033.1.20 reference material, nmateri
35、al, sufficiently homog-enous and stable with respect to one or more specifiedproperties that has been established to be fit for its intended usein the measurement process; properties can be quantitative orqualitative. ISO Guide 343.1.21 sensitivity, nchange in the response of a measuringinstrument d
36、ivided by the corresponding change in thestimulus. Eurachem Guide(3)3.1.22 specificity/selectivityability of a measurement pro-cedure to determine accurately and specifically the analyte ofinterest in the presence of other components in the samplematrix under the stated conditions of the test. Eurac
37、hemGuide(3)3.1.23 spiked sample, nsample that is created by adding aknown quantity of a biological agent to a known quantity ofsuspicious powder.3.1.23.1 DiscussionExamples of spiked samples include6The boldface numbers in parentheses refer to a list of references at the end ofthis standard.TABLE 2
38、Suspicious PowdersClass of Powder Powder TypeOrganic, biologicalBrewers yeast powderDipel dustOrganic, protein-containingMilk powderInfant formulaWhite flourOrganic, no proteinCoffee creamer (non-dairy)Instant pectinAcetaminophenPowdered sugarCorn starchPolyethylene glycol 3350 (forexample, MiraLAX,
39、 Glycolax)InorganicToothpaste powder with fluorideBaking powder (aluminum free)Calcium carbonate (antacid)Baking sodaEpsom saltMagnesium carbonate (gym chalk)BoraxTalcRoad dust (NIST)Kaolin clayPopcorn saltE3131 175pathogens added to suspicious powders that are typicallyprepared in a liquid (buffer)
40、 suspension.3.1.24 strain, nisolates or variants of the target biologicalagent(s) that the method can detect (inclusivity strains) orshould not detect (exclusivity strains).3.1.25 suspicious powder, nany material that is used tocreate a perceived biological threat such as a hoax powder thatmay visua
41、lly resemble Bacillus anthracis (Ba), ricin powder,or simply an unknown powder.3.1.25.1 DiscussionSuspicious powders include readilyavailable household items, food products, building materials,and environmental matrices (for example, talcum powder,flour, drywall dust, and road dust).3.1.26 testing m
42、odule, nset of samples used to establishthe detection technology performance for a particular type ofsamples.3.1.26.1 DiscussionTesting modules include inclusivity,exclusivity, suspicious powders, and whole biological agentspiked suspicious powders.4. Statistical Considerations for Testing4.1 The te
43、sting approach described herein uses a score CI(4) to define the number of samples that need to be tested toachieve a desired POD estimate as indicated by the value of agiven LCB in a one-sided interval with a specified CL. In thisspecification, two levels have been chosen as providing accept-able d
44、egrees of statistical performance: a low level and a highlevel. Results from selecting and executing one of the testingplans presented in this specification can be used to determine ifthe chosen level of statistical performance has been met.4.2 Each of the sample types listed in 4.2.1 4.2.4 shall be
45、considered as part of a separate testing module. That is, thenumber of samples that shall be analyzed to achieve a desiredPOD as demonstrated by the specified LCB and CL values, asdescribed in 4.3 and 4.4, apply to test modules 4.2.1 4.2.4,which includes the following test modules:4.2.1 Inclusivity
46、nucleic acid testing (Test Module 1),4.2.2 Exclusivity nucleic acid testing (Test Module 2),4.2.3 Suspicious powder testing (Test Module 3), and4.2.4 Whole biological agent spiked suspicious powdertesting (Test Module 4).4.3 Below is listed the minimum number of replicatesrequired for each of the fo
47、ur testing modules listed in 4.2.1 4.2.4 for the case of LCB = 0.95 and 95 % CLat the chosen testconcentration (see Fig. 2). Testing shall use results from asingle round of experiments with a fixed number of tests (thatis, if 47 samples are chosen to be tested but a failure occurs,additional samples
48、 cannot be tested. Testing shall start over).4.3.1 Without a single failed result, 47 samples shall betested;4.3.2 With no more than a single failed result, 79 samplesshall be tested; or4.3.3 With no more than two failed results, 107 samplesshall be tested.4.4 Below is listed the minimum number of r
49、eplicatesrequired for each of the four testing modules listed in 4.2.1 4.2.4 to achieve an LCB = 0.90 and 90 % CL at the chosen testconcentration (see Fig. 3). Testing shall use results from asingle round of experiments with a fixed number of tests (thatis, if 14 samples are chosen to be tested but a failure occurs,additional samples cannot be tested. Testing shall start over).4.4.1 Without a single failed result, 14 samples shall betested;4.4.2 With no more than a single failed result, 31 samplesshall be tested; or4.4.3 With no
