ASTM D8412-21
(Guide)Standard Guide for Quantification of Microbial Contamination in Liquid Fuels and Fuel-Associated Water by Quantitative Polymerase Chain Reaction (qPCR)
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
Relations
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
1
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
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
1
This guide is under the jurisdiction of ASTM Committee D02 on Petroleum
2
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 service@astm.org. 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
1
----------------
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.