ASTM D8412-21

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.

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 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 qualitative and quantitative relationship between the laboratory results and actual microbial communities in the systems from which samples are collected is affected by the time delay and handling conditions between the time of sampling and time that testing is initiated.

1.4?The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard with the exception of the concept unit of gene copies/mL (that is, 16S or 18S gene copies/mL) to indicate the starting concentration of microbial DNA for the intended microbial targets (that is, bacteria, archaea, fungi).

1.5?This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.

1.6?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.