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

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, and primer and probe combinations that are not covered in this guide may be used to perform qPCR. This guide does not attempt to cover all of the possible qPCR assays and does not suggest nor imply that the qPCR assays (that is, combinations of primers and probes, and reaction conditions) discussed here are better suited for qPCR than other qPCR assays not presented here. Additional, primers, primers and probes combination, and qPCR assay conditions may be added in the future to this guide as they become available to the ASTM scientific community. Guide D6469 reviews the types of damage that uncontrolled microbial growth in fuels and fuel systems can cause.5.4 Culture-based microbiological tests depend on the ability of microbes to proliferate in liquid, solid or semisolid nutrient media, in order for microbes in a sample to be detected.5.5 There is general consensus among microbiologists that only a fraction of the microbes believed to be present in the environment have been cultured successfully.5.6 Since the mid-1990s, genetic test methods that do not rely on cultivation have been increasingly favored for the detection and quantification of microorganisms in environmental samples.5.7 qPCR is a quantitative, culture-independent method that is currently used in the medical, food, and cosmetic industries for the detection and quantification of microorganisms.5.8 Since the early 2000s, qPCR methodology has evolved and is now frequently used to quantify microorganisms in fuel-associated samples, but there is currently no standardized methodology for employing qPCR for this application (1-6).3 The purpose of this guide is to provide guidance and standardization for genetic testing of samples using qPCR to quantify total microbial populations present in fuel-associated samples.5.9 Although this guide focuses on describing recommended protocols for the quantification of total microorganisms present in fuel-associated samples using qPCR, the procedures described here can also be applied to the standardization of qPCR assays for other genetic targets and environmental matrices.5.10 Genetic techniques have great flexibility so that it is possible to design a nearly infinite number of methods to detect and quantify each and every gene. Because of this flexibility of genetic techniques, it is important to provide a standard protocol for qPCR so that data generated by different laboratories can be compared.5.11 This guide provides recommendations for primers sequences and experimental methodology for qPCR assays for the quantification of total microorganisms present in fuel-associated samples.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 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.