Designation:D8412−21StandardGuideforQuantificationofMicrobialContaminationinLiquidFuelsandFuel-AssociatedWaterbyQuantitativePolymeraseChainReaction(qPCR)1ThisstandardisissuedunderthefixeddesignationD8412;thenumberimmediatelyfollowingthedesignationindicatestheyearoforiginaladoptionor,inthecaseofrevision,theyearoflastrevision.Anumberinparenthesesindicatestheyearoflastreapproval.Asuperscriptepsilon(´)indicatesaneditorialchangesincethelastrevisionorreapproval.1.Scope1.1Thisguidecoversproceduresforusingquantitativepolymerasechainreaction(qPCR),agenomictool,todetect,characterizeandquantifynucleicacidsassociatedwithmicro-bialDNApresentinliquidfuelsandfuel-associatedwatersamples.1.1.1Watersamplesthatmaybeusedintestinginclude,butarenotlimitedto,waterassociatedwithcrudeoilorliquidfuelsinstoragetanks,fueltanks,orpipelines.1.1.2Whiletheintentofthisguideistofocusontheanalysisoffuel-associatedsamples,theproceduresdescribedherearealsorelevanttotheanalysisofwaterusedinhydrotestingofpipesandequipment,waterinjectedintogeologicalformationstomaintainpressureand/orfacilitatetherecoveryofhydrocarbonsinoilandgasrecovery,waterco-producedduringtheproductionofoilandgas,waterinfireprotectionsprinklersystems,potablewater,industrialprocesswater,andwastewater.1.1.3Totestafuelsample,theliveandrecentlydeadmicroorganismsmustbeseparatedfromthefuelphasewhichcanincludeanyDNAfragmentsbyusingoneofvariousmethodssuchasfiltrationoranyothermicrobialcapturingmethods.1.1.4Someoftheprotocolstepsareuniversallyrequiredandareindicatedbytheuseofthewordmust.Otherprotocolstepsaretesting-objectivedependent.Atthoseprocesssteps,optionsareofferedandthebasisforchoosingamongthemareexplained.1.2Theguidedescribestheapplicationofquantitativepolymerasechainreaction(qPCR)technologytodeterminetotalbioburdenortotalmicrobialpopulationpresentinfuel-associatedsamplesusinguniversalprimersthatallowforthequantificationof16Sand18SribosomalRNAgenesthatarepresentinallprokaryotes(thatis,bacteriaandarchaea)andeucaryotes(thatis,moldand...
