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

Importancia y uso:

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.

Subcomité:

D02.14

Referida por:

E1259-23

Volúmen:

05.05

Número ICS:

07.100.01 (Microbiology in general)

Palabras clave:

archaea; bacteria; DNA; fuel; fungi; genomics; metagenomics; microbiology; microorganisms; nucleic acids; qPCR; quantitative polymerase chain reaction;