ASTM D8412-21

Standard Guide for Quantification of Microbial Contamination in Liquid Fuels and Fuel-Associated Water by Quantitative Polymerase Chain Reaction (qPCR)

SIGNIFICANCE AND USE
5.1 This guide provides a protocol for detecting, characterizing, and quantifying nucleic acids (that is, DNA) of living and recently dead microorganisms in fuels and fuel-associated waters by means of a culture independent qPCR procedure. Microbial contamination is inferred when elevated DNA levels are detected in comparison to the expected background DNA level of a clean fuel and fuel system.  
5.2 A sequence of protocol steps is required for successful qPCR testing.  
5.2.1 Quantitative detection of microorganisms depends on the DNA-extraction protocol and selection of appropriate oligonucleotide primers.  
5.2.2 The preferred DNA extraction protocol depends on the type of microorganism present in the sample and potential impurities that could interfere with the subsequent qPCR reaction.  
5.2.3 Primers vary in their specificity. Some 16S and 18S RNA gene regions present in the DNA of prokaryotic and eukaryotic microorganisms appear to have been conserved throughout evolution and thus provide a reliable and repeatable target for gene amplification and detection. Amplicons targeting these conserved nucleotide sequences are useful for quantifying total population densities. Other target DNA regions are specific to a metabolic class (for example, sulfate reducing bacteria) or individual taxon (for example, the bacterial species Pseudomonas aeruginosa). Primers targeting these unique nucleotide sequences are useful for detecting and quantifying specific microbes or groups of microbes known to be associated with biodeterioration.  
5.3 Just as the quantification of microorganisms using microbial growth media employs standardized formulations of growth conditions enabling the meaningful comparison of data from different laboratories (Practice D6974), this guide seeks to provide standardization to detect, characterize, and quantify nucleic acids associated with living and recently dead microorganisms in fuel-associated samples using qPCR.
Note 3: Many primers, an...
SCOPE
1.1 This guide covers procedures for using quantitative polymerase chain reaction (qPCR), a genomic tool, to detect, characterize and quantify nucleic acids associated with microbial DNA present in liquid fuels and fuel-associated water samples.  
1.1.1 Water samples that may be used in testing include, but are not limited to, water associated with crude oil or liquid fuels in storage tanks, fuel tanks, or pipelines.  
1.1.2 While the intent of this guide is to focus on the analysis of fuel-associated samples, the procedures described here are also relevant to the analysis of water used in hydrotesting of pipes and equipment, water injected into geological formations to maintain pressure and/or facilitate the recovery of hydrocarbons in oil and gas recovery, water co-produced during the production of oil and gas, water in fire protection sprinkler systems, potable water, industrial process water, and wastewater.  
1.1.3 To test a fuel sample, the live and recently dead microorganisms must be separated from the fuel phase which can include any DNA fragments by using one of various methods such as filtration or any other microbial capturing methods.  
1.1.4 Some of the protocol steps are universally required and are indicated by the use of the word must. Other protocol steps are testing-objective dependent. At those process steps, options are offered and the basis for choosing among them are explained.  
1.2 The guide describes the application of quantitative polymerase chain reaction (qPCR) technology to determine total bioburden or total microbial population present in fuel-associated samples using universal primers that allow for the quantification of 16S and 18S ribosomal RNA genes that are present in all prokaryotes (that is, bacteria and archaea) and eucaryotes (that is, mold and yeast collectively termed fungi), respectively.  
1.3 This guide describes laboratory protocols. As described in Practice D7464, the...

General Information

• Status: Published
• Publication Date: 30-Nov-2021
• ICS: 07.100.01 - Microbiology in general
• Technical Committee: D02 - Petroleum Products, Liquid Fuels, and Lubricants
• Drafting Committee: D02.14 - Stability, Cleanliness and Compatibility of Liquid Fuels

Relations

• Refers: ASTM D4175-23a - Standard Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants
• Effective Date: 15-Dec-2023
• Refers: ASTM D4175-23e1 - Standard Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants
• Effective Date: 01-Jul-2023
• Refers: ASTM D6974-20 - Standard Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels—Filtration and Culture Procedures
• Effective Date: 01-May-2020
• Refers: ASTM D1129-13(2020)e2 - Standard Terminology Relating to Water
• Effective Date: 01-May-2020
• Refers: ASTM D7464-20 - Standard Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for Microbiological Testing
• Effective Date: 01-May-2020
• Refers: ASTM D7464-19 - Standard Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for Microbiological Testing
• Effective Date: 01-Nov-2019
• Refers: ASTM D7464-14 - Standard Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for Microbiological Testing
• Effective Date: 01-Oct-2014
• Refers: ASTM D7464-08(2013) - Standard Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for Microbiological Testing
• Effective Date: 01-May-2013
• Refers: ASTM D6974-09(2013)e2 - Standard Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels-Filtration and Culture Procedures
• Effective Date: 01-May-2013
• Refers: ASTM D6974-09(2013) - Standard Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels—Filtration and Culture Procedures
• Effective Date: 01-May-2013
• Refers: ASTM D6974-09(2013)e1 - Standard Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels—Filtration and Culture Procedures
• Effective Date: 01-May-2013
• Refers: ASTM D6469-12 - Standard Guide for Microbial Contamination in Fuels and Fuel Systems
• Effective Date: 01-Nov-2012
• Refers: ASTM D6469-11 - Standard Guide for Microbial Contamination in Fuels and Fuel Systems
• Effective Date: 01-Jan-2011
• Refers: ASTM D1129-10 - Standard Terminology Relating to Water
• Effective Date: 01-Mar-2010
• Refers: ASTM D6974-09 - Standard Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels—Filtration and Culture Procedures
• Effective Date: 01-Jun-2009

Overview

ASTM D8412-21, titled Standard Guide for Quantification of Microbial Contamination in Liquid Fuels and Fuel-Associated Water by Quantitative Polymerase Chain Reaction (qPCR), provides a comprehensive protocol for detecting and quantifying microbial DNA-specifically from living or recently dead microorganisms-in fuels and fuel-associated water samples. This guide emphasizes the use of culture-independent quantitative polymerase chain reaction (qPCR) technology, facilitating the identification and measurement of total bioburden in environments where traditional culturing methods may fall short. Implemented globally, this standard supports the need for standardized, accurate, and reproducible microbial assessment, enhancing fuel system maintenance, operational reliability, and compliance.

Key Topics

• qPCR Methodology: Procedures for extracting DNA from microorganisms present in liquid fuels or associated water samples, selection of nucleic acid primers, and conducting qPCR analyses.
• Microbial Targets: Utilization of universal primers for quantifying conserved 16S (bacteria, archaea) and 18S (fungi) ribosomal RNA genes, enabling broad detection across microbial communities.
• Sample Types: Applies to fuels (jet fuel, diesel, gasoline) and associated waters, including storage tank bottoms, pipeline transport, hydrotest water, injection water in oil production, and co-produced water.
• Detection of Contamination: Microbial contamination is inferred by elevated DNA levels compared to those found in clean fuel and system backgrounds.
• Standardization: Ensures consistency in microbial quantification across laboratories, supporting comparison and trend analysis using uniform terminology and methodology.
• Controls and Quality Assurance: Guidance on negative and positive controls to ensure data reliability and minimize measurement errors due to inhibitors or cross-contamination.

Applications

The protocols in ASTM D8412-21 offer substantial practical value across multiple industries and applications:

• Fuel System Monitoring: Rapid quantification of microbial contamination in storage tanks, pipelines, and distribution systems, aiding proactive maintenance and risk mitigation.
• Water Quality Assessment: Supports testing of water associated with fuels, including process water, wastewater, and fire protection systems, enabling comprehensive microbial management.
• Oil & Gas Industry: Facilitates analysis of hydrotest and injection waters, as well as water co-produced during oil/gas extraction, helping operators monitor and control microbial-induced corrosion and fouling.
• Industrial and Environmental Testing: Valid methodology for gross and specific detection of microbes in potable, industrial, and process water samples, supporting compliance and operational efficiency.
• Post-Biocidal Treatment Verification: Differentiates between DNA from living cells and recently dead microorganisms to support evaluation of biocidal efficacy.

Adoption of ASTM D8412-21 enhances the precision of microbial testing, reduces reliance on time-consuming culture methods, and supports the implementation of robust microbial control strategies to protect fuel quality and infrastructure.

Related Standards

ASTM D8412-21 references and is complemented by several other standards, including:

• ASTM D6469 – Guide for Microbial Contamination in Fuels and Fuel Systems
• ASTM D6974 – Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels-Filtration and Culture Procedures
• ASTM D7464 – Practice for Manual Sampling of Liquid Fuels and Associated Materials for Microbiological Testing
• ASTM D1129 – Terminology Relating to Water
• ASTM D4175 – Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants

Integrating ASTM D8412-21 with these related standards ensures thorough and consistent microbial monitoring for the fuel and associated water sector, supporting international best practices in environmental and operational safety.

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Keywords: ASTM D8412-21, qPCR, microbial contamination, liquid fuels, fuel-associated water, DNA quantification, standard guide, petroleum microbiology, environmental testing, water quality, microbial monitoring, fuel system maintenance.

Standards Content (Sample)

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:D8412 −21
Standard Guide for
Quantification of Microbial Contamination in Liquid Fuels
and Fuel-Associated Water by Quantitative Polymerase
Chain Reaction (qPCR)
This standard is issued under the fixed designation D8412; 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.
1. Scope present in all prokaryotes (that is, bacteria and archaea) and
eucaryotes (that is, mold and yeast collectively termed fungi),
1.1 This guide covers procedures for using quantitative
respectively.
polymerase chain reaction (qPCR), a genomic tool, to detect,
1.3 This guide describes laboratory protocols.As described
characterize and quantify nucleic acids associated with micro-
in Practice D7464, the qualitative and quantitative relationship
bial DNA present in liquid fuels and fuel-associated water
between the laboratory results and actual microbial communi-
samples.
tiesinthesystemsfromwhichsamplesarecollectedisaffected
1.1.1 Watersamplesthatmaybeusedintestinginclude,but
by the time delay and handling conditions between the time of
are not limited to, water associated with crude oil or liquid
sampling and time that testing is initiated.
fuels in storage tanks, fuel tanks, or pipelines.
1.4 The values stated in SI units are to be regarded as
1.1.2 While the intent of this guide is to focus on the
standard. No other units of measurement are included in this
analysis of fuel-associated samples, the procedures described
standard with the exception of the concept unit of gene
here are also relevant to the analysis of water used in
copies/mL(that is, 16S or 18S gene copies/mL) to indicate the
hydrotesting of pipes and equipment, water injected into
starting concentration of microbial DNA for the intended
geologicalformationstomaintainpressureand/orfacilitatethe
microbial targets (that is, bacteria, archaea, fungi).
recovery of hydrocarbons in oil and gas recovery, water
1.5 This standard does not purport to address all of the
co-produced during the production of oil and gas, water in fire
protection sprinkler systems, potable water, industrial process safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro-
water, and wastewater.
priate safety, health, and environmental practices and deter-
1.1.3 To test a fuel sample, the live and recently dead
mine the applicability of regulatory limitations prior to use.
microorganisms must be separated from the fuel phase which
1.6 This international standard was developed in accor-
can include any DNA fragments by using one of various
dance with internationally recognized principles on standard-
methods such as filtration or any other microbial capturing
ization established in the Decision on Principles for the
methods.
Development of International Standards, Guides and Recom-
1.1.4 Some of the protocol steps are universally required
mendations issued by the World Trade Organization Technical
and are indicated by the use of the word must. Other protocol
Barriers to Trade (TBT) Committee.
steps are testing-objective dependent. At those process steps,
options are offered and the basis for choosing among them are
2. Referenced Documents
explained.
2.1 ASTM Standards:
1.2 The guide describes the application of quantitative
D1129Terminology Relating to Water
polymerase chain reaction (qPCR) technology to determine
D4175Terminology Relating to Petroleum Products, Liquid
total bioburden or total microbial population present in fuel-
Fuels, and Lubricants
associated samples using universal primers that allow for the
D6469GuideforMicrobialContaminationinFuelsandFuel
quantification of 16S and 18S ribosomal RNA genes that are
Systems
D6974Practice for Enumeration of Viable Bacteria and
This guide is under the jurisdiction of ASTM Committee D02 on Petroleum
Products, Liquid Fuels, and Lubricants and is the direct responsibility of Subcom- For referenced ASTM standards, visit the ASTM website, www.astm.org, or
mittee D02.14 on Stability, Cleanliness and Compatibility of Liquid Fuels. contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
Current edition approved Dec. 1, 2021. Published January 2022. DOI: 10.1520/ Standards volume information, refer to the standard’s Document Summary page on
D8412-21. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D8412−21
Fungi in Liquid Fuels—Filtration and Culture Procedures 3.1.10 probe-based qPCR, n—a type of qPCR reaction
D7464Practice for Manual Sampling of Liquid Fuels, As- relayingintheuseoffluorescentprobeswhichallowsdetection
sociated Materials and Fuel System Components for of single or multiple targets (that is, multiplexing) in a single
Microbiological Testing qPCR reaction.
3.1.11 quantitative PCR (qPCR), n—a PCR-based assay in
3. Terminology
which the amount of the target DNA sequence present in a
3.1 Definitions: specimen can be detected, characterized, and quantified by
measuring fluorescence from an intercalating dye or fluores-
3.1.1 For definitions of terms used in this guide, refer to
cent oligonucleotide probe.
Terminologies D1129, D4175, and the ASTM Online Termi-
3.1.11.1 Discussion—Therearemultiplespecifictechniques
nology Dictionary of Engineering Science and Technology.
bywhichqPCRcanbeaccomplishedbutingeneraltheamount
3.1.2 amplicon, n—the product of the qPCR reaction result-
of the target DNA sequence present influences the kinetics of
ing from the amplification of a genetic target using a particular
amplification and the number of amplification cycles required
pair of primers.
for the PCR reaction to produce a concentration of amplified
3.1.3 gene copies/mL, n—the unit of concentration for the
products (that is, amplicon) that exceeds a specified threshold,
qPCR assay, indicating the starting concentration of microbial
whichisdeterminedandusedtocalculatethenumberofcopies
DNA for the intended microbial targets (that is, bacteria,
of the target DNA sequence that was present in the sample.
archaea, fungi).
3.1.12 singleplex, adj—in genomic testing, indicates that a
3.1.3.1 Discussion—The 16S RNA gene is detected in
singletargetsequenceofeitherDNAorRNAistobedetected.
bacteria and archaea, and the 18S RNA gene is detected in
3.1.12.1 Discussion—Singleplex PCR is distinguished from
fungi. Both 16S and 18S RNA genes are conserved in
multiplex PCR in which multiple target sequences of either
prokaryotes and eukaryotes, respectively.
DNA or RNA are detected simultaneously.
3.1.4 intercalating dye-based qPCR, n—qPCR reaction that
3.2 Definitions of Terms Specific to This Standard:
uses a fluorescent DNA intercalating dye for detection and
3.2.1 absolute quantification, n—in PCR testing, the num-
quantification of amplicon.
berofgenecopies/mLderivedfromobservedspecimencounts
3.1.4.1 Discussion—Most common intercalating dyes are
as a function of observed reference standard counts (see
comprised of asymmetrical cyanine dyes. DNA intercalating
standards).
dyes are not used in probe-based qPCR.
3.2.2 humic acids, n—water-soluble substances found in
3.1.5 molarity (M), n—moles of solute per L of solution.
soil and other environmental matrices with the potential to
3.1.5.1 Discussion—1M=10 µM.
inhibit the polymerase chain reaction process.
3.1.6 nanomole (nmole), n—one billionth of a mole.
3.2.3 no template control (NTC), n—a negative control
3.1.6.1 Discussion—1 mole = 6.022 × 10 molecules and 1
reaction not containing DNA, which is used to detect and
-9
nanomole = 10 moles.
determine background contamination or cross contamination.
3.1.7 oligonucleotide (oligo), n—in qPCR testing,thisrefers
3.2.4 probes, n—oligonucleotides with sequence specificity
to the polynucleotide sequence of primers and probes. for the microbial target gene that are terminally labeled with a
fluorophore and quencher molecule to detect and quantify the
3.1.8 polymerase chain reaction (PCR), n—an in vitro
amplicon; probes are required in probe-based qPCR assays.
laboratory method for the enzymatic amplification of nucleic
acid sequences. 3.2.5 recently dead, adj—in microbiology, a microorganism
that is still structurally intact although it is metabolically
3.1.8.1 Discussion—Two DNA oligonucleotide primers an-
inactive and not susceptible to resuscitation.
neal with their complementary DNAstrands and flank (that is,
border) the segment to be amplified. The increase in amount 3.2.5.1 Discussion—Current methods available for separat-
ingwholecellsfromsamplemetricescannotseparatelivefrom
(amplification) of the DNA segments occurs during repeated
cycles consisting of three steps: heat denaturation of the dead cells.
double-stranded DNA, cooling to effect annealing of the
3.2.5.2 Discussion—Although many cells lyse immediately
primers to their complementary DNA strands, and enzymatic
upon death, a percentage of cells can remain intact for
extension of the annealed primers by DNA polymerase at its
prolonged periods after death.
optimal temperature. The repeated cycling of the three steps
3.2.5.3 Discussion—Theinabilitytoseparatelivecellsfrom
results in a near exponential increase in the amount of
ghosts makes it impossible for genomic tests to differentiate
amplicon as defined by the primers.
between analytes extracted from live and ghost cells.
Consequently, genomic data are inclusive of live and recently
3.1.9 primers, n—oligonucleotides with sequence specific-
dead cells.
ity for the microbial target gene, which is used to initiate the
polymerase chain reaction to produce amplicons.
3.2.6 standards, n—a DNA sequence spanning the full
3.1.9.1 Discussion—The primers anneal with their comple- length of the amplicon, which is serially diluted and used to
mentary DNA strands and flank the target nucleic acid se- produce a standard curve for absolute quantification of ampli-
quence. con in qPCR.
D8412−21
4. Summary of Guide to provide standardization to detect, characterize, and quantify
nucleic acids associated with living and recently dead micro-
4.1 A sample comprising a sufficient volume of fuel or
organisms in fuel-associated samples using qPCR.
fuel-associated water is collected and processed to provide the
genomic DNA required for qPCR testing.
NOTE 3—Many primers, and primer and probe combinations that are
not covered in this guide may be used to perform qPCR. This guide does
4.2 The DNA sample is tested by performing singleplex or
not attempt to cover all of the possible qPCR assays and does not suggest
multiplex qPCR. Singleplex is performed by using the primer
nor imply that the qPCR assays (that is, combinations of primers and
set for a specific microbial target with a DNA intercalating
probes,andreactionconditions)discussedherearebettersuitedforqPCR
than other qPCR assays not presented here. Additional, primers, primers
fluorescentdyeoracorrespondingfluorescenthydrolysisprobe
and probes combination, and qPCR assay conditions may be added in the
(that is, probe). Multiplex qPCR is performed by combining
future to this guide as they become available to the ASTM scientific
multiple primer and probe sets in a single reaction (that is,
community. Guide D6469 reviews the types of damage that uncontrolled
singlewell)todetectmultiplemicrobialtargetssimultaneously.
microbial growth in fuels and fuel systems can cause.
NOTE 1—The principleofthefluorescenthydrolysisprobereliesonthe 5.4 Culture-based microbiological tests depend on the abil-
degradation of the probe by the 5’ to 3’ exonuclease activity of the
ity of microbes to proliferate in liquid, solid or semisolid
polymerase, which allows the release of the fluorophore from the probe
nutrient media, in order for microbes in a sample to be
producing the fluorescence.
detected.
NOTE 2—In order to detect and accurately quantify any possible
bacteria, fungi and archaea in a sample, qPCR assays must be designed
5.5 There is general consensus among microbiologists that
with forward (FW) and reverse (RV) primers that are 100% complemen-
only a fraction of the microbes believed to be present in the
tary to the target DNA by using primers and probes with degenerate
environment have been cultured successfully.
nucleotides for polymorphic alleles.
5.6 Since the mid-1990s, genetic test methods that do not
4.3 The absolute quantification in gene copies/mL is re-
rely on cultivation have been increasingly favored for the
corded and analyzed to determine the initial microbial load in
detectionandquantificationofmicroorganismsinenvironmen-
the sample.
tal samples.
5. Significance and Use
5.7 qPCRisaquantitative,culture-independentmethodthat
5.1 This guide provides a protocol for detecting, is currently used in the medical, food, and cosmetic industries
characterizing, and quantifying nucleic acids (that is, DNA) of
for the detection and quantification of microorganisms.
living and recently dead microorganisms in fuels and fuel-
5.8 Since the early 2000s, qPCR methodology has evolved
associated waters by means of a culture independent qPCR
and is now frequently used to quantify microorganisms in
procedure. Microbial contamination is inferred when elevated
fuel-associated samples, but there is currently no standardized
DNA levels are detected in comparison to the expected 3
methodology for employing qPCR for this application (1-6).
background DNA level of a clean fuel and fuel system.
The purpose of this guide is to provide guidance and standard-
5.2 A sequence of protocol steps is required for successful
ization for genetic testing of samples using qPCR to quantify
qPCR testing. total microbial populations present in fuel-associated samples.
5.2.1 Quantitative detection of microorganisms depends on
5.9 Although this guide focuses on describing recom-
the DNA-extraction protocol and selection of appropriate
mended protocols for the quantification of total microorgan-
oligonucleotide primers.
isms present in fuel-associated samples using qPCR, the
5.2.2 ThepreferredDNAextractionprotocoldependsonthe
procedures described here can also be applied to the standard-
type of microorganism present in the sample and potential
ization of qPCR assays for other genetic targets and environ-
impurities that could interfere with the subsequent qPCR
mental matrices.
reaction.
5.10 Genetic techniques have great flexibility so that it is
5.2.3 Primers vary in their specificity. Some 16S and 18S
possibletodesignanearlyinfinitenumberofmethodstodetect
RNA gene regions present in the DNA of prokaryotic and
andquantifyeachandeverygene.Becauseofthisflexibilityof
eukaryotic microorganisms appear to have been conserved
genetic techniques, it is important to provide a standard
throughoutevolutionandthusprovideareliableandrepeatable
protocol for qPCR so that data generated by different labora-
target for gene amplification and detection. Amplicons target-
tories can be compared.
ing these conserved nucleotide sequences are useful for quan-
tifyingtotalpopulationdensities.OthertargetDNAregionsare
5.11 This guide provides recommendations for primers
specific to a metabolic class (for example, sulfate reducing
sequences and experimental methodology for qPCR assays for
bacteria)orindividualtaxon(forexample,thebacterialspecies
the quantification of total microorganisms present in fuel-
Pseudomonas aeruginosa). Primers targeting these unique
associated samples.
nucleotide sequences are useful for detecting and quantifying
6. Interferences, Special Precautions, and Limitations
specific microbes or groups of microbes known to be associ-
ated with biodeterioration.
6.1 Introduced Contaminants—qPCR is a very sensitive
technique and the qPCR assays described in this guide will
5.3 Just as the quantification of microorganisms using
microbial growth media employs standardized formulations of
growthconditionsenablingthemeaningfulcomparisonofdata
The boldface numbers in parentheses refer to a list of references at the end of
from different laboratories (Practice D6974), this guide seeks this standard.
D8412−21
involved in recent microbial proliferation. The buffer wash step is
detect any bacteria or fungi present in the sample, including
expected to further reduce any traces of free-DNA fragments.
those that might be introduced due to improper sample
Alternatively,ifthebackgroundlevelofDNAofagivencleanfuelsystem
handling during collection, DNAfragments present in the fuel
hasbeenascertainedthroughapropermaintenancesamplingregime,then,
system, sample processing, and testing.
it is possible to accurately assess increased microbial presence in the fuel
withouttheneedtoremoveallfree-DNAfromthesamplepriortotesting.
NOTE 4—With the understanding that work in the field cannot be
performed under complete aseptic conditions, it is expected that the user
6.5 DNA Extraction Effıciency—Avariety of procedures are
ofthisguidewillbetrainedandbecomefamiliarizedwithmicrobiological
available for extracting DNA from samples in preparation for
aseptic principles including the use of sterile sample collection bottles,
qPCRanalysis.Itisoutsidethescopeofthisguidetoprescribe
sterileconsumables,disinfectionofallequipmentthatcannotbesterilized
theextractionproceduretobeused,butitshouldberecognized
before use, and use personal equipment (for example, gloves) to prevent
introduction of human and environmental microorganisms into the fuel thattheextractionprocedurewillhaveakeybearingonresults
sample that may be detected by the qPCR test.
obtained. Procedures should be used which minimize the loss
ofDNAduringtheextractionprocess.Ideally,theefficiencyof
6.2 Sample Preservation—The accuracy and value of the
DNA extraction will be validated, for example, by simultane-
qPCRtestishighlydependentonthequalityofthesample.The
ous analysis of the same sample matrix spiked with DNA
time that passes between the collection of the fuel sample or
standards and the use of appropriate negative and positive
water, processing (for example, separation of the microorgan-
controls.Extractionefficiencyshouldbedemonstratedtobeno
isms from the fuel and DNA extraction) and testing (that is,
lessthan1%andideallybetterthan5%.ASTMhasaprogram
performing the qPCR) should be minimized.
todevelopandupdatestandardsrelevanttodrawingofsamples
NOTE 5—The microbial composition of a fuel sample can change upon
and preparation of samples, including DNA extraction for
retrieval from the fuel system. Thus, it is recommended that samples are
qPCR analysis.
processed within a few hours from retrieval or the fuel sample to be
refrigerated to prevent changes in microbial composition.
NOTE 6—DNA samples can degrade and factors such as nucleases,
7. Apparatus
temperature, and time may affect the speed and severity of the degrada-
tion. DNA preservation techniques including stabilization solutions, 7.1 Quantitative Real-time PCR Thermocycler, with the
refrigeration, freezing, dehydration, lyophilization may be used to miti-
following approximate specifications: ramp rate (≥3°C⁄s av-
gate or prevent DNA degradation.
erage); temperature (0°C to 100°C range, 60.2°C accuracy,
6.3 Inhibitors of qPCR—Consideration should be given to
60.4°C well-to-well uniformity); lid heats up to at least
the potential presence of polymerase chain reaction inhibitors
105°C; optical detection (able to excite and detect ≥3 multi-
and other factors in a sample that may affect the qPCR test
plexed fluorophores including emission wavelength 517nm,
results. Common inhibitors of qPCR include humic acids and
556nm, 615nm); Scan time for one channel of approximately
heavy metals.Approaches that mitigate the effect and interfer-
3s; reaction volume from 10µL to 50µL; sensitivity of one
ence of inhibitors should implemented.
gene copy of target sequence; dynamic range of ten orders
magnitude; provides singleplex and multiplex analysis soft-
NOTE 7—Multiple commercial DNA extraction kits incorporate strate-
ware for quantification.
gies to remove potential inhibitors of qPCR from the DNA sample. The
use of a DNA extraction protocols that stabilize the DNA sample is
7.2 Micropipetters, of volumes 1µL to 10µL, 10µL to
recommended.
100µL, and 100µL to 1000µL.
6.4 Limitations of qPCR—qPCRwilldetectallnucleicacids
7.3 Sterile Laminar Flow Hood, Clean Bench, qPCR
associated with microorganism whether or not the DNA was
Enclosure, or Biosafety Cabinet.
from living microbial cells or freely circulating in the fuel
sample. For the detection of biocontamination in fuel, it is
8. Reagents and Materials
DNA found inside living microbial cells which is of most
importancewhenassessingthepotentialforbiodeteriorationof
8.1 Buffer, Tris-EDTA (TE)—10mM Tris-HCl, 1mM diso-
the fuel. Cells which have recently died (for example due to
dium EDTA, pH 8.0.
application of a biocide treatment) can retain DNA within an
8.2 Master Mix—Solution containing the required reagents
intact cell structure. However, DNA derived from cells which
to perform qPCR including a hot-start DNA polymerase,
are long-dead, or which were not involved in recent active
, intercalating fluorescent dye, primers, probes,
dNTPs, MgCl
microbialproliferationinthesystemsampled,willgenerallybe
and water.
present as free-DNA fragments un-associated with cellular
material. Thus, it is recommended that strategies that reduce
NOTE 9—To produce the qPCR reaction, commercially available
thecontributionoffree-DNAtoqPCRresultsbeimplemented.
high-quality pre-made master mixes that contain all the components
required to perform the qPCR reaction, with the exception of primers,
NOTE 8—Although not yet validated for qPCR analysis of fuels, a
probes, DNA, and water may be used.
filtration process that incorporates a buffer wash step, as described in
Practice D6974 for collection of microbial cells from fuels for culture 8.3 Micropipette Tips, Plastic, (filtered micropipette tips
analysis, may be used to collect microbial cells from fuel samples for
are recommended but not required).
qPCR analysis. Because free-DNAfragments are too small to be retained
by the 0.45µm membrane filter specified in this guide, only intact cells 8.4 Primers—Forward primer and reverse prime
...