See Table 1. Within 4 h. 8 Guidance
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1 AOAC SMPR Method Performnce Requirements See Tble 1. Stndrd Method Performnce Requirements (SMPRs) for DNA-Bsed Methods of Detecting Brucell suis in Field-Deployble, Deprtment of Defense Aerosol Collection Devices Intended Use: Field-deployed use for nlysis of erosol collection filters nd/or liquids 1 Applicbility Detection of Brucell suis in collection buffers from erosol collection devices. Field-deployble ssys re preferred. 2 Anlyticl Technique Moleculr detection of nucleic cid. 3 Definitions Acceptble minimum detection level (AMDL). Predetermined minimum level of n nlyte, s specified by n expert committee which must be detected by the cndidte method t specified probbility of detection (POD). Exclusivity. Study involving pure nontrget strins, which re potentilly cross-rective, tht shll not be detected or enumerted by the cndidte method. Inclusivity. Study involving pure trget strins tht shll be detected or enumerted by the cndidte method. Mximum time-to-result. Mximum time to complete n nlysis strting from the collection buffer to ssy result. Probbility of detection (POD). Proportion of positive nlyticl outcomes for qulittive method for given mtrix t specified nlyte level or concentrtion with 0.95 confidence intervl. System flse-negtive rte. Proportion of test results tht re negtive contined within popultion of known positives. System flse-positive rte. Proportion of test results tht re positive contined within popultion of known negtives. 5 System Suitbility Tests nd/or Anlyticl Qulity control The controls listed in Tble 2 shll be embedded in ssys s pproprite. Mnufcturer must provide written justifiction if controls re not embedded in the ssy. 6 Vlidtion Guidnce AOAC INTERNATIONAL Methods Committee Guidelines for Vlidtion of Biologicl Thret Agent Methods nd/or Procedures (Officil Methods of Anlysis of AOAC INTERNATIONAL, 2016, 20th Ed., Appendix I). Inclusivity nd exclusivity pnel orgnisms used for evlution must be chrcterized nd documented to truly be the species nd strins they re purported to be. If specified inclusivity or exclusivity isolte is not commercilly vilble in the United Sttes t this time, use the GenBnk ccession number to test the genomic sequence with in silico nlysis. 7 Mximum Time-to-Results Within 4 h. 8 Guidnce Orgnisms my be tested s isolted DNA, or combined to form pooled isolted DNA. Isolted DNA my be combined into pools of up to 10 exclusivity pnel orgnisms, with ech pnel orgnism represented t 10 times the AMDL. If n unexpected result occurs, ech of the exclusivity orgnisms from filed pool must be individully retested t 10 times the AMDL. If the isolte is not commercilly vilble in the United Sttes t this time, use the GenBnk ccession number to test the genomic sequence with in silico nlysis. Approved by the AOAC Stkeholder Pnel on Agent Detection Assys (SPADA). Finl Version Dte: September 1, Tble 1. Method performnce requirements Prmeter AMDL Minimum performnce requirement 2000 genomic equivlents of Brucell suis (biovr 1, type strin 1330) per ml liquid in the cndidte method smple collection buffer Probbility of detection t AMDL within smple collection buffer 0.95 Probbility of detection t AMDL in environmentl mtrix mterils 0.95 System flse-negtive rte using spiked environmentl mtrix mterils 5% System flse-positive rte using environmentl mtrix mterils 5% Inclusivity Exclusivity All inclusivity strins (Tble 3) must test positive t 2 the AMDL All exclusivity strins (Tble 4 nd Annnex I, Prt 2) must test negtive t 10 the AMDL 100% correct nlyses re expected. All discrepncies re to be retested following the AOAC INTERNATIONAL Methods Committee Guidelines for Vlidtion of Biologicl Thret Agent Methods nd/or Procedures [Officil Methods of Anlysis of AOAC INTERNATIONAL (2016) 20th Ed., AOAC INTERNATIONAL, Rockville, MD, USA, Appendix I,
2 Tble 2. Controls Control Description Implementtion Positive control Negtive control Inhibition control Designed to demonstrte n pproprite test response. The positive control should be included t low but esily detectble concentrtion, nd should monitor the performnce of the entire ssy. The purpose of using low concentrtion of positive control is to demonstrte tht the ssy sensitivity is performing t previously determined level of sensitivity. It is recommended tht technique (i.e., unique distinguishble signture) is used to confirm whether the positive control is the cuse of positive signl generted by smple. Designed to demonstrte tht the ssy itself does not produce detection in the bsence of the trget orgnism. The purpose of this control is to rule out cuses of flse positives, such s contmintion in the ssy or test. Designed to specificlly ddress the impct of smple or smple mtrix on the ssy s bility to detect the trget orgnism. Single use per smple (or smple set) run Single use per smple (or smple set) run Single use per smple (or smple set) run ANNEX I Environmentl Fctors for Vlidting Biologicl Thret Agent Detection Assys [Adpted from the Environmentl Fctors Pnel pproved by SPADA on June 10, 2010.] The Environmentl Fctors Studies supplement the biologicl thret gent ner-neighbor exclusivity testing pnel. There re three prts to Environmentl Fctors Studies: Prt 1 Environmentl mtrix smples; Prt 2 Environmentl orgnisms study; nd Prt 3 Potentil interferents pplicble to Deprtment of Defense pplictions (dded in June 2015 for the Deprtment of Defense project). Prt 1: Environmentl Mtrix Smples Aerosol Environmentl Mtrices Method developers shll obtin environmentl mtrix smples tht re representtive nd consistent with the collection method tht is nticipted to ultimtely be used in the field. This includes considertions tht my be encountered when the collection system is deployed opertionlly such s collection medium, durtion of collection, diversity of geogrphicl res tht will be smpled, climtic/environmentl conditions tht my be encountered nd sesonl chnges in the regions of deployment. Justifictions for the selected conditions tht were used to generte the environmentl mtrix nd limittions of the vlidtion bsed on those criteri must be documented. Method developers shll test the environmentl mtrix smples for interference using smples inoculted with trget biologicl thret gent sufficient to chieve 95% probbility of detection. Cross-rectivity testing will include sufficient smples nd replictes to ensure ech environmentl condition is dequtely represented. Prt 2: Environmentl Pnel Orgnisms This list is comprised of identified orgnisms from the environment. Inclusion of ll environmentl pnel orgnisms is not requirement if method developer provides pproprite justifiction tht the intended use of the ssy permits the exclusion of specific pnel orgnisms. Justifiction for exclusion of ny environmentl pnel orgnism(s) must be documented nd submitted. Orgnisms nd cell lines my be tested s isolted DNA, or s pools of isolted DNA. Isolted DNA my be combined into pools of up to 10 pnel orgnisms, with ech pnel orgnism represented t 10 times the AMDL, where possible. The combined DNA pools re tested in the presence (t 2 times the AMDL) nd bsence of the trget gene or gene frgment. If n unexpected result occurs, ech of the individul environmentl orgnisms from filed pool must be individully retested t 10 times the AMDL with nd without the Tble 3. Inclusivity pnel No. Strin designtion Biovr ATCC/BEI/GB Accession No. Avilble from Comments 1 B. suis ATCC BEI NR B. suis Thomsen 2 ATCC BEI NR B. suis ATCC BEI NR B. suis 40 4 ATCC BEI NR-305 Swine, USA Hre, Denmrk Swine, USA Reindeer, Russi 5 B. suis ACBK b GenBnk Mouse, Russi 6 B. suis S2 NA ALOS b GenBnk Nturlly ttenuted vccine strin used in Chin The Brucell Working Group recognizes tht B. suis biovr 5 is difficult to distinguish from the other B. suis biovrs. The working group concluded tht B. suis biovr 5 should be included s prt of the B. suis inclusivity pnel with cution tht B. suis biovr 5 my be very difficult to differentite from other B. suis biovrs. However, the SMPR does not require cndidte ssys to differentite biovrs. b Avilble in the whole genome dtbse t GenBnk.
3 Tble 4. Exclusivity pnel,b No. Strin designtion Biovr ATCC/BEI/Accession No. Avilble from Comments 1 B. bortus S19 1 NR NVSL S19 vccine strin, smooth 2 B. bortus RB51 1 BEI NR-2552 NVSL 3 B. bortus 86/8/59 2 ATCC BEI NR B. bortus 12 3 ATCC BEI NR B. bortus Tuly 3 ATCC BEI NR B. bortus 292 (39/94) 4 ATCC BEI NR B. bortus B ATCC BEI NR B. bortus ATCC BEI NR B. bortus 63/75 7 ATCC BEI NR B. bortus C68 9 ATCC BEI NR B. bortus ATCC BEI NR B. melitensis 16M 1 ATCC BEI NR B. melitensis 63/9 2 ATCC CP CP BEI NR B. melitensis Ether 3 ATCC BEI NR-258 Not commercilly vilble 15 B. melitensis bv. 1 str. Rev. 1 1 ACEG Not commercilly vilble 16 B. cnis RM-666 NA ATCC NR B. neotome 5K33 NA ATCC BEI NR B. ovis NA ATCC BEI NR-682 ATCC ATCC ATCC 19 B. ceti B1/94 NA AZBH Not commercilly vilble 20 B. pinnipedilis B2/94 NA ACBN Not commercilly vilble 21 Brucell spp. 83/13 NA ACBQ Not commercilly vilble 22 B. inopint BO1 NA ADEZ Not commercilly vilble 23 Brucell sp. BO2 NA ADFA Not commercilly vilble 24 B. ppionis F8/08-60(T) NA ACXD Not commercilly vilble 26 B. microti CCM 4915 NA CP CP Not commercilly vilble 27 B. vulpis NA LN LN Not commercilly vilble 28 Agrobcterium tumefciens NA ATCC 4452 ATCC RB51 vccine strin, rough Humn, Ugnd Bovine, Afric Bovine, Afric Got, USA Got, Turkey Got, Itly Elberg origin, B. melitensis vccine strin Dog Desert Wood Rt Rm, Austrli Porpoise, Scotlnd Sel, Scotlnd Rt, Austrli Humn, Oregon Humn, Austrli Novel Brucell ssocited with primtes (NVSL ) Vvole, Czech Republic Red fox, Austri
4 Tble 4. (continued) No. Strin designtion Biovr ATCC/BEI/Accession No. Avilble from Comments 29 Ochrobctrum nthropi NA ATCC ATCC 30 Ochrobctrum intermedium LMG 3301 NA SAMN The Brucell Working Group is wre tht B. cnis cn infect humns, cusing pproximtely 100 cses of humn brucellosis nnully. The working group is lso wre of the close reltionship between B. suis nd B. cnis. In fct, the txonomic clssifiction of ll Brucell spp. hs undergone debte during the lst few decdes, with some scientists proposing tht ll Brucell spp. should be reclssified s B. melitensis on the bsis of results of DNA-DNA hybridiztion, nd tht the current species should be reclssified s biovrs. However, the clssic txonomic scheme for the Brucell spp. nd existing biovrs ws repproved in 2003 [Ostermn, B., & Moriyon, I. (2006) Interntionl Committee on Systemtics of Prokryotes: Subcommittee on the Txonomy of Brucell, Int. J. Syst. Evol. Microbiol. 56, ] on the bsis of host specificity, phenotypic chrcteristics, vrying virulence, nd genotyping dt. For these resons s well s directions from DoD to focus on B. suis, the working group determined to develop this SMPR for the specific detection of B. suis. b The Brucell Working Group is wre of Russin vccines using B. bortus SR82 nd B. bortus 7579, nd other strins my lso be in use. These vccine strins were not vilble t the time this SMPR ws dopted. Consequently the working group decided not to include these vccine strins in the exclusivity pnel. trget gene or gene frgment t 2 times the AMDL in the cndidte method DNA elution buffer. DNA in this list tht lredy pper in the inclusivity or exclusivity pnel do not need to be tested gin s prt of the environmentl fctors pnel. Potentil bcteril biothret gents Bcillus nthrcis Ames Yersini pestis Colordo-92 Frncisell tulrensis subsp. tulrensis Schu-S4 Burkholderi pseudomllei Burkholderi mllei Brucell melitensis Cultivtble bcteri identified s being present in ir soil or wter Acinetobcter lwoffii Agrobcterium tumefciens Bcillus myloliquefciens Bcillus cohnii Bcillus psychroscchrolyticus Bcillus benzoevorns Bcillus megterium Bcillus horikoshii Bcillus mcroides Bcteroides frgilis Burkholderi cepci Burkholderi gldoli Burkholderi stbilis Burkholderi plntrii Chryseobcterium indologenes Clostridium srdiniense Clostridium perfringens Deinococcus rdiodurns Delfti cidovorns Escherichi coli K12 Fusobcterium nucletum Lctobcillus plntrum Legionell pneumophils Listeri monocytogenes Morxell nonliquefciens Mycobcterium smegmtis Neisseri lctmic Pseudomons eruginos Rhodobcter spheroides Riemerell ntipestifer Shewnell oneidensis Stphylococcus ureus Stenotophomons mltophili Streptococcus pneumonie Streptomyces coelicolor Synechocystis Vibrio cholere Microbil eukryotes Freshwter moebe: Acnthmoeb cstellnii Negleri fowleri Fungi: Alternri lternt Aspergillus fumgtis Aureobsidium pullulns Cldosporium cldosporioides Cldosporium spherospermum Epicoccum nigrum Eurotium mstelodmi Mucor rcemosus Pecilomyces vriotii Penicillum chrysogenum Wllemi sebi DNA from higher eukryotes Plnt pollen (if pollen is unvilble, vegettive DNA is cceptble): Ze mys (corn) Pinus spp. (pine) Gossypium spp. (cotton) Arthropods: Aedes egypti (ATCC/CCL-125 mosquito cell line) Aedes lbopictus (mosquito C6/36 cell line) Dermtophgoides pteronyssinus (dust mite; commercil source) Xenopsyll cheopis fle (Rocky Mountin Lbs) Drosophili cell line Musc domestic (housefly; ARS, USDA, Frgo, ND, USA) Gypsy moth cell lines [LED652Y cell line (bculovirus); Invitrogen]
5 Cockroch (commercil source) Tick (Amblyomm nd Dermcentor tick species for F. tulrensis detection ssys; dded by SPADA on Mrch 22, 2016) Vertebrtes: Mus musculus (ATCC/HB-123) mouse Rttus norvegicus (ATCC/CRL-1896) rt Cnis fmiliris (ATCC/CCL-183) dog Felis ctus (ATCC/CRL-8727) ct Homo spiens (HeL cell line ATCC/CCL-2) humn Gllus gllus domesticus (chicken) Cpri hirc (got; dded by SPADA on September 1, 2015) Biologicl insecticides Strins of B. thuringiensis present in commercilly vilble insecticides hve been extensively used in hoxes nd re likely to be hrvested in ir collectors. For these resons, it should be used to ssess the specificity of these thret ssys. B. thuringiensis subsp. isrelensis B. thuringiensis subsp. kurstki B. thuringiensis subsp. morrisoni Serende (fungicide) B. subtilis (QST713) Virl gents hve lso been used for insect control. Two representtive products re: Gypcheck for gypsy moths (Lymnteri dispr nucler polyhedrosis virus) Cyd-X for coddling moths (coddling moth grnulosis virus) Prt 3: Potentil Interferents Study The Potentil Interferents Study supplements the Environmentl Fctors Study, nd is pplicble to ll biologicl thret gent detection ssys for Deprtment of Defense pplictions. Tble 5 provides list of potentil interferents tht re likely to be encountered in vrious Deprtment of Defense pplictions. Method developers nd evlutors shll determine the most pproprite potentil interferents for their ppliction. Interferents shll be spiked t finl test concentrtion of 1 µg/ml directly into the smple collection buffer. Smple collection buffers spiked with potentil interferents shll by inoculted t 2 times the AMDL (or cceptble minimum identifiction level; AMIL) with one of the trget biologicl thret gents. Spiked/inoculted smple collection buffers shll be tested using the procedure specified by the cndidte method. A cndidte method tht fils t the 1 µg/ml level my be reevluted t lower concentrtions until the inhibition level is determined. It is expected tht ll smples re correctly identified s positive. Tble 5 is offered for guidnce, nd there re no mndtory minimum requirements for the number of potentil interferents to be tested.
6 Tble 5. Potentil interferents Compound Potentil theters of opertion Group 1: Petroleum-bsed JP-8 Airfield JP-5 b Diesel/gsoline mixture Fog oil (stndrd grde fuel No. 2) Burning rubber c Nvl Nvl, ground, irfield Group 2: Exhust Gsoline exhust Jet exhust Diesel exhust Nvl, irfield Group 3: Obscurnts Terephthlic cid d Zinc chloride smoke e Solvent yellow 33 f Group 4: Environmentl Burning vegettion, irfield Rod dust Se wter (se spry) Group 5: Chemicls Brke fluid g All Brke dust h Clening solvent, MIL-L i Explosive residues: high explosives j nd rtillery propellnt k Nvl JP-8: Air Force formultion jet fuel. b JP-5: A yellow kerosene-bsed jet fuel with lower flsh point developed for use in ircrft sttioned bord ircrft crriers, where the risk from fire is prticulrly gret. JP-5 is complex mixture of hydrocrbons, contining lknes, nphthenes, nd romtic hydrocrbons. c Burning rubber (tire smoke): Gseous C1-C5 hydrocrbons: methne, ethne, isopropene, butdiene, propne. Polycyclic romtic hydrocrbons ( ng/m 3 ): prbenzo()pyrene, polychlorinted dibenzo-p-dioxins (PCDD), polychlorinted dibenzofurns (PCDF). Metls (0.7 8 mg/m 3 ): zinc, led, cdmium. d Terephthlic cid: Used in the AN/M83 hnd grende currently used by U.S. militry. All All e Zinc chloride smoke: Also known s HC smoke. Used in the M8 grende nd still used in 155 mm rtillery shells. HC smoke is composed of 45% hexchloroethne, 45% zinc oxide, nd 10% luminum. f Solvent yellow 33: IUPAC nme: 2-(2-quinolyl)-1,3-indndione. A new formultion being develop for the M18 grende. g Brke fluid: DOT 4 is the most common brke fluid, primrily composed of glycol nd borte esters. DOT 5 is silicone-bsed brke fluid. The min difference is tht DOT 4 is hydroscopic, wheres DOT 5 is hydrophobic. DOT 5 is often used in militry vehicles becuse it is more stble over time nd requires less mintennce. h Brke dust: Fe prticles cused by brsion of the cst iron brke rotor by the pd nd secondly fibers from the semi metllic elements of the brke pd. The reminder of the dust residue is crbon content within the brke pd. i MIL-L-63460: Militry Specifiction, Lubricnt, Clener nd Preservtive for Wepons nd Wepons Systems; trde nme Brek-Free CLP. Midwy USA: j High explosives: The M mm projectile is the U.S. Army/Mrine Corp s current stndrd projectile contining 10.8 kg TNT. The M795 projectile replced the M107 projectile tht contined Composition B, which is 60/40 mixture of RDX/TNT. RDX is cyclotrimethylene trinitrmine. Suggestion: test RDX/TNT together. k Artillery propellnt. Modern gun propellnts re divided into three clsses: single-bse propellnts which re minly or entirely nitrocellulose bsed, doublebse propellnts composed of combintion of nitrocellulose nd nitroglycerin, nd triple-bse composed of combintion of nitrocellulose nd nitroglycerin nd nitrogunidine. Suggestion: test totl nitrocellulose/ nitroglycerin nitrogunidine together.
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