WO2010109173A1 - Procédé de détection microbiologique - Google Patents

Procédé de détection microbiologique Download PDF

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Publication number
WO2010109173A1
WO2010109173A1 PCT/GB2010/000531 GB2010000531W WO2010109173A1 WO 2010109173 A1 WO2010109173 A1 WO 2010109173A1 GB 2010000531 W GB2010000531 W GB 2010000531W WO 2010109173 A1 WO2010109173 A1 WO 2010109173A1
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polynucleotide
concentration
hydrocarbon
metabolising
sample
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PCT/GB2010/000531
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Richard Hatton
Robert Sleat
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Envirogene Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/64—Geomicrobiological testing, e.g. for petroleum
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

  • the present invention relates to a method for detecting the presence of a hydrocarbon deposit and, more specifically, a method of detecting a naturally occurring hydrocarbon deposit in a geographical location.
  • WO- A-91/02086 reports on the detection of oil and gas deposits by taking soil samples from a location where there is potentially a hydrocarbon deposit. This approach is based on the theory that microbes, in particular bacteria, which are capable of metabolising hydrocarbons have a selective advantage in areas where subsurface hydrocarbon gases or vapours are present. Therefore the concentration of such microbes is higher than average in a soil sample which is located above an oil or gas deposit.
  • a first portion of a soil sample obtained from a location is exposed to a hydrocarbon gas such as ethane and the amount of a metabolite resulting from the metabolism of the hydrocarbon gas is measured over a predetermined length of time.
  • a hydrocarbon gas such as ethane
  • the amount of a metabolite resulting from the metabolism of the hydrocarbon gas is measured over a predetermined length of time.
  • a second portion of the soil sample is exposed to a substrate, such as glucose, which can generally be metabolised by all bacteria and the production of the metabolite thereof is also measured. This gives an indication of the overall microbial population in the soil sample.
  • the ratio of the microbial population which is capable of metabolising hydrocarbons to the overall microbial population is calculated to provide a normalised index of the presence of microbes which are capable of metabolising hydrocarbons.
  • the detection of an index value is indicative of the presence or absence of a hydrocarbon deposit at the location.
  • a plurality of samples can be taken at different sites across a location and the index determined at each site. The variation of index values across the location can then be mapped out to indicate the presence of a deposit within the location.
  • WO- A-91/02086 also reports on combining the index with the concentration of free hydrocarbon gas at each site in order to arrive at a multivariate index that maps seepage more reliably.
  • radioactive hydrocarbon isotopes such as carbon 14 so that the (isotopic) metabolic products can be detected and distinguished from the products of other metabolic processes.
  • radioactive isotopes require careful storage, handling and disposal.
  • supplies of hydrocarbon isotopes can easily become contaminated with compounds that disrupt the normal metabolism of microorganisms leading to inaccurate results.
  • US2002/0065609A1 reports a different approach to mineral exploration. It discloses the analysis of microbial populations in relation to sequences of their small subunit ribosomal DNA (rDNA) sequence. It hypothesises that specific polymorphisms of the 16S rDNA sequence of bacteria can be correlated to a sample parameter such as the geographical location of populations of bacteria. If the sample parameter is the presence of hydrocarbon deposits at geographical locations then the presence of bacteria containing the polymorphisms at a geographical location is indicative of the presence of a hydrocarbon deposit at the geographical location. That is to say, specific 16S rDNA polymorphisms are putative markers for bacteria suited for survival over or around hydrocarbon deposits.
  • rDNA small subunit ribosomal DNA
  • WO2005/103284 relates to multi-targeted microbial screening and monitoring methods. It involves testing for the presence/absence of microbial markers that are shared by both 'target' and 'index' microbes.
  • Index microbes are genetically distinct from target microbes but behave in a similar way under equivalent conditions. The results are used to calculate an aggregate index value. The index values are useful when the number of markers detected is not sufficient to indicate the presence of the target microbe. A threshold index value can be calculated, and if the index value is above the threshold is it indicative of the presence of target microbes.
  • WO03/012390 Another approach for the detection of hydrocarbons is provided in WO03/012390. It discloses the use of micro-arrays to analyse samples for the presence of hydrocarbons and to perform multiple tests in parallel. The probes used specifically bind to analytes of targets associated with hydrocarbons.
  • WO2009/013516 also reports a method for the detection of hydrocarbon deposits in a geographical location.
  • the method of WO09/013516 relies on comparing the concentration of a hydrocarbon metabolising marker gene with the number of bacteria at the location. The comparative results are given as a ratio.
  • This 'normalised' data termed an index value, provides information about the abundance of hydrocarbon metabolising genes at the location. It is indicative of the presence or absence of a hydrocarbon deposit.
  • index value provides information about the abundance of hydrocarbon metabolising genes at the location. It is indicative of the presence or absence of a hydrocarbon deposit.
  • there is always demand for enhanced methods of hydrocarbon deposit detection with improved accuracy and which utilise more refined processes.
  • the present invention seeks to alleviate one or more of the above problems.
  • a method for detecting the presence of a hydrocarbon deposit in a geographical location comprising the steps of:
  • ii) determining the concentration, at the same site in the location or in a sample taken from the site, of a second polynucleotide encoding a protein capable of metabolising a hydrocarbon; and iii) calculating a mathematical operation on the concentration of the first polynucleotide relative to the concentration of the second polynucleotide at the site;
  • the result of the mathematical operation is indicative of the presence or absence of a hydrocarbon deposit at the location, and wherein the first polynucleotide encodes a different protein from the second polynucleotide.
  • the mathematical operation is calculating the ratio between the first and second polynucleotide.
  • steps i) and ii) further comprise the step of dividing the first and second polynucleotide concentrations by their respective population medians to achieve a common amplitude scale.
  • the step of determining the concentration of the first polynucleotide comprises determining the concentration of a subsequence of the first polynucleotide sequence.
  • the step of determining the concentration of the second polynucleotide comprises determining the concentration of a subsequence of the second polynucleotide sequence.
  • the subsequence comprises a consensus sequence present in a plurality of homologous genes from different micro-organisms encoding a selected protein capable of metabolising a hydrocarbon.
  • the first and/or second polynucleotide is DNA.
  • the first and/or second polynucleotide is RNA.
  • the first and/or second polynucleotide encodes a protein which metabolises a hydrocarbon selected from a Cl to C20 alkane, an optionally substituted single or multi-ring aromatic hydrocarbon, or a naphthene.
  • the first and/or second polynucleotide encodes a biphenyl dioxygenase, a toluene monooxygenase, an alkane hydroxylase, a naphthalene dioxygenase, a toluene dioxygenase, a xylene monooxygenase, a butane monooxygenase, a methane monooxygenase, a catechol 2,3, dioxygenase, a bacterial P450 oxygenase, or a eukaryotic P450 oxygenase.
  • the first or second polynucleotide is an AIkB gene.
  • the first or second polynucleotide is a XyIM gene.
  • the first or second polynucleotide is a ToID gene.
  • the first or second polynucleotide encodes a methane monooxygenase, such as particulate methane monooxygenase (pmoA).
  • methane monooxygenase such as particulate methane monooxygenase (pmoA).
  • the first or second polynucleotide encodes a catechol 2,3,dioxygenase.
  • one of the first or second polynucleotides encodes a protein capable of metabolising a short chain alkane, and wherein the other polynucleotide encodes a protein capable of metabolising a long chain alkane or an aromatic compound.
  • one of the first or second polynucleotides encodes a protein capable of metabolising an alkane, and wherein the other polynucleotide encodes a protein capable of metabolising an aromatic compound.
  • the second polynucleotide encodes a catechol 2,3,dioxygenase.
  • the concentration of the first and/or second polynucleotide is determined by quantitative PCR.
  • the sample is taken from a depth of between 0 and 100cm, preferably between 0 and 50cm, and most preferably between 10 and 40cm below the solid surface of the Earth.
  • the sample is a soil sample.
  • the sample is a marine sediment sample.
  • the sample is a freshwater sediment sample.
  • the method further comprises the step of, after taking the sample, of stabilising the nucleic acids in the sample.
  • steps (i), (ii) and (iii) are carried out with respect to a plurality of different sites at the location.
  • the method further comprises the step of correlating the results of steps (i), (ii) and (iii) at each site, thereby determining the variations in the results of the mathematical operations at different sites within the location.
  • the method further comprises the step of determining the concentration of a third polynucleotide.
  • the method further comprises the step of calculating a mathematical operation on the concentration of the first and/or second polynucleotide relative to the concentration of the third polynucleotide.
  • step of the method iii) further comprises, prior to calculating the mathematical operation on the concentration of the first polynucleotide relative to the concentration of the second polynucleotide, the step of determining the concentration of the third polynucleotide and calculating a mathematical operation on the concentration of the first and/or second polynucleotide relative to the concentration of the third polynucleotide.
  • the third polynucleotide encodes a generic bacterial protein.
  • the third polynucleotide is a EuBac gene.
  • a catechol 2,3,dioxygenase gene as a biomarker for normalising the concentration, in an environmental sample, of one or more other genes encoding a protein or proteins capable of metabolising one or more hydrocarbons, for detecting hydrocarbon deposits.
  • a method of selecting a polynucleotide encoding a protein capable of metabolising one or more hydrocarbons as a biomarker suitable for normalising the concentration, in an environmental sample, of one or more other polynucleotides encoding a protein or proteins capable of metabolising one or more hydrocarbons, in a method for detecting hydrocarbon deposits comprising;
  • the further data set comprises geochemical data.
  • the geochemical data relates to the concentration of free hydrocarbons within a solid substrate at each site.
  • the geochemical data relates to the concentration of free hydrocarbon gas or vapour above each site.
  • the presence of the first and/or second polynucleotide, wherein said polynucleotide encodes a small subunit rRNA is determined using a forward primer comprising one of SEQ ID NO:s 33 or SEQ ID NO: 39, or a sequence with at least 80% identity thereto, a reverse primer comprising SEQ ID NO:34 or SEQ ID NO: 40 respectively, or a sequence with at least 80% identity thereto, and optionally a probe comprising SEQ ID NO:35 or SEQ ID NO: 41 respectively, or a sequence with at least 80% identity thereto.
  • the presence of the first and/or second polynucleotide, wherein said polynucleotide encodes 16s rRNA is determined using a forward primer comprising SEQ ID NO:36 or SEQ ID NO: 42, or a sequence with at least 80% identity thereto, a reverse primer comprising SEQ ID NO:37 or SEQ ID NO :43 respectively, or a sequence with at least 80% identity thereto, and optionally a probe comprising SEQ ID NO:38 or SEQ ID No:44 respectively, or a sequence with at least 80% identity thereto.
  • the protein capable of metabolising a hydrocarbon is encoded by a nucleotide sequence comprising a sequence with at least 80% sequence identity to a sequence referred to in Table 2. It is preferred that the sequence has at least 90%, 95%, 99% or 100% sequence identity to a sequence referred to in Table 2.
  • Table 2 provides the GenBank accession numbers of the sequences. The preferred sequences are those present in the GenBank Database on 28 th July 2008.
  • the presence of the biphenyl dioxygenase protein is determined using a forward primer comprising SEQ ID NO: 13 or a sequence with at least 80% identity thereto, a reverse primer comprising SEQ ID NO: 14 or a sequence with at least 80% identity thereto, and optionally a probe comprising SEQ ID NO: 15 or a sequence with at least 80% identity thereto.
  • the presence of the catechol 2,3,dioxygenase protein is determined using a forward primer comprising SEQ ID NO: 1 or a sequence with at least 80% identity thereto, a reverse primer comprising SEQ ID NO:2 or a sequence with at least 80% identity thereto, and optionally a probe comprising SEQ ID NO:3 or a sequence with at least 80% identity thereto.
  • the presence of the naphthalene dioxygenase protein is determined using a forward primer comprising SEQ ID NO:4 or a sequence with at least 80% identity thereto, a reverse primer comprising SEQ ID NO:5 or a sequence with at least 80% identity thereto, and optionally a probe comprising SEQ ID NO:6 or a sequence with at least 80% identity thereto.
  • the presence of the toluene dioxygenase protein is determined using a forward primer comprising SEQ ID NO: 7 or a sequence with at least 80% identity thereto, a reverse primer comprising SEQ ID NO:8 or a sequence with at least 80% identity thereto, and optionally a probe comprising SEQ DD NO:9 or a sequence with at least 80% identity thereto.
  • the presence of the xylene monooxygenase protein is determined using a forward primer comprising SEQ ID NO: 10 or a sequence with at least 80% identity thereto, a reverse primer comprising SEQ ID NO:11 or a sequence with at least 80% identity thereto, and optionally a probe comprising SEQ ID NO: 12 or a sequence with at least 80% identity thereto.
  • the presence of the butane monooxygenase protein is determined using a forward primer comprising SEQ ID NO:28 or a sequence with at least 80% identity thereto, a reverse primer comprising SEQ ID NO:29 or a sequence with at least 80% identity thereto, and optionally a probe comprising SEQ ID NO:30 or a sequence with at least 80% identity thereto.
  • the presence of the alkane dehydrogenase protein is determined using a forward primer comprising one of SEQ ID NO:s 16, 19, 22 or 25, or a sequence with at least 80% identity thereto, a reverse primer comprising SEQ ID NO: 17, 20, 23 or 26 respectively, or a sequence with at least 80% identity thereto, and optionally a probe comprising SEQ ID NO: 18, 21, 24 or 27 respectively, or a sequence with at least 80% identity thereto.
  • the presence of the methane monooxygenase protein is determined using a forward primer comprising SEQ ED NO:31, or a sequence with at least 80% identity thereto, and a reverse primer comprising SEQ ID NO:32 respectively, or a sequence with at least 80% identity thereto.
  • the presence of the particulate methane monooxygenase protein is determined using a forward primer, and a reverse primer.
  • the forward primers, reverse primers and probes used for the detection of the hydrocarbon metabolising and generic polynucleotide genes have 80, 85, 90, 95, 99 or 99.5% identity with the respective SEQ ID NOs.
  • the percentage "identity" between two sequences is determined using the BLASTP algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI- BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402) using default parameters.
  • the BLAST algorithm can be accessed on the Internet using the URL http://www.ncbi.nlm.nih.gov/blast/.
  • hydrocarbon means an organic chemical compound comprising hydrogen and carbon atoms.
  • a protein is described as being "capable of metabolizing a hydrocarbon” this means that the protein has activity in facilitating or causing a chemical reaction on a hydrocarbon under in vivo conditions. This can be tested, for example, by adding a sample hydrocarbon to a reaction medium that replicates intracellular conditions and detecting the decrease in the presence of the hydrocarbon over a predetermined length of time in comparison with a control medium from which the protein is absent.
  • variable operands can be the concentrations of the first, second and/or third polynucleotides.
  • short chain alkane refers to alkanes comprising 6 carbon atoms or less.
  • long chain alkane encompasses alkanes with more than 6 carbon atoms.
  • live petroleum is used to indicate petroleum deposits with a high abundance of the biochemically labile short chain normal alkanes relative to the biochemically less labile aromatics.
  • EuBac refers to a polynucleotide which encodes a small subsection of bacterial 16S rRNA. It is highly conserved amongst most bacteria, and therefore the EuBac copy number is an example of a useful indicator of the total microbial population.
  • Figure 1 is a graph showing free or adsorbed gas, vapour or liquid hydrocarbon concentrations in surface soils versus location.
  • Figure 2 is a schematic cross-sectional view of a geographical location containing a subsurface hydrocarbon deposit.
  • Figure 3 is a block diagram demonstrating the principle of the present invention.
  • Figure 4 is a sectional display of the field site of Experimental example 1 illustrating the relationship between gene copy number values and the location of an oil seep.
  • the index value populations are each divided by their respective medians in order to reduce them to a common amplitude scale.
  • the sections are referenced in the plan view shown in Figure 5.
  • Figure 5 is a plan view of the field site of Experimental example 1 illustrating the relationship between Transform 1 index values and the location of the oil seep.
  • the index value populations are each divided by their respective medians in order to reduce them to a common amplitude scale.
  • the letter notation (A-D) refers to the section line in Figure 6.
  • Figure 6 is a sectional view of the field site of Experimental example 1 illustrating the relationship between Transform 1 index values and the location of the oil seep.
  • the sections are referenced in the plan view, Figure 5.
  • the index value populations are each divided by their respective medians in order to reduce them to a common amplitude scale.
  • Figure 7 is a plan view of the field site of Experimental example 1 illustrating the spatial and scalar variation in Transform 2 index values in relation to the location of the oil seep.
  • the index value populations are each divided by their respective medians in order to reduce them to a common amplitude scale.
  • the letter notation (A-D) refers to the section line in Figure 8.
  • Figure 8 is a sectional view of the field site of Experimental example 1 illustrating the relationship between Transform 2 index values and the location of the oil seep.
  • the sections are referenced in the plan view, Figure 7.
  • the index value populations are each divided by their respective medians in order to reduce them to a common amplitude scale.
  • Figure 9 is a sectional display of the field site of Experimental example 1 showing the relationship between the Transform 3 index values and the location of the Formby oil seep. Sections ABC and BD are referenced in Figure 5.
  • Figure 10 is a plan view of the field site of Experimental example 2 illustrating the spatial and scalar variation in pMoA index Transform 2 values in relation to a virgin gas field and a dry hole.
  • the index values are scaled and truncated at the low end in order to facilitate this visualisation.
  • a subsurface hydrocarbon deposit 2 is not visible from the surface 3.
  • the vertical migration of hydrocarbon gases, vapours or liquids 4 to the surface 3 from the hydrocarbon deposit results in the generation of an anomaly at the surface 3 of increased concentrations of hydrocarbons in surface soils.
  • the anomaly is directly above the hydrocarbon accumulation (an apical anomaly) but in other situations, the anomaly takes the form of a halo around the periphery of the hydrocarbon deposit (a halo anomaly).
  • a halo anomaly the concentration of migrant hydrocarbons across the geographical location 1 either as an apical or a halo anomaly is shown.
  • the present invention concerns detecting the presence of microbial and, in particular, bacterial populations which are capable of metabolising hydrocarbons. This involves determining the concentration of a first and a second polynucleotide present in a sample obtained from a site, wherein the site is to be tested for the presence of a petroleum deposit. Each of the first and second polynucleotides encodes a different hydrocarbon metabolising gene. The next step utilises a comparative mathematical operation performed on the respective concentration values of the first and second polynucleotides. The results are interpreted and are indicative of the presence or absence of a hydrocarbon deposit in the vicinity of the sample site.
  • the concentrations of the first and second polynucleotide are each divided by their respective population medians in order to bring them to a common amplitude scale before they are compared with each other.
  • the concentration of the second polynucleotide serves to act as a normalising biomarker in relation to the concentration of the first polynucleotide.
  • the method is applicable to analysis of a previously obtained sample (e.g. transported to a different location) as well as analysis in situ.
  • the concentration of a third polynucleotide is also detected.
  • This third polynucleotide encodes a generic gene, such as Eubac, which is common to the majority of bacteria and hence is useful in calculating the total microbial population.
  • the concentrations of the first and second polynucleotides can be normalised with respect to the concentration of the third polynucleotide to give an index value before they are compared with each other. This can take place before or after the optional division of the concentrations of the first and second polynucleotides with their respective population medians.
  • a plurality of polynucleotides are detected, each of which encodes a different hydrocarbon metabolizing gene, at a plurality of sample sites.
  • the concentration of each polynucleotide is compared pairwise via a mathematical operation to the concentration of each of the other polynucleotides detected at the same sample site.
  • the results are then assessed (e.g. classical statistical criteria of significance are applied) and the pairwise comparison that yields the best results in terms of accurately indicating the presence of a hydrocarbon deposit can then be selected, i.e. the optimal normalising biomarker combination is chosen. This selection is then verified by an alternative method of hydrocarbon prospecting.
  • the present invention is utilised in synergistic combination with other technology.
  • the technique of controlled source electromagnetism in which sensors are deployed over an area in order to detect hydrocarbon deposits directly, is adapted so that the sensors simultaneously retrieve a core sample at 50cm beneath the soil surface. The sample is then tested in accordance with the present invention.
  • the effectiveness of the present invention when analysing a core sample from a shallow depth, such as 50cm, is advantageous in this instance.
  • underwater vehicles such as submarines, which are currently used in various aspects of oil prospecting, are also adapted to collect shallow core samples.
  • microbial populations in the vicinity of a hydrocarbon deposit are detected by detecting the presence of genes which encode s protein capable of metabolising a hydrocarbon or a family of hydrocarbons.
  • Figure 3 shows a block diagram indicating the relationship between the presence of hydrocarbons and the expression of genes encoding hydrocarbon metabolising proteins.
  • the presence of a hydrocarbon in the environment surrounding a microbe is detected by the microbial cell via a number of mechanisms, principally via receptors on the cell surface.
  • the detection of the presence of hydrocarbon results in a raised level of a transcription of mRNA 6 which encodes a protein capable of metabolising the hydrocarbon.
  • the protein 7 is in turn, translated and expressed within the cell.
  • the protein duly oxidises the hydrocarbon 8 releasing energy and resulting in growth 9 and multiplication of the cell.
  • the multiplication of the cell leads to an increase in the number of genes 10 encoding hydrocarbon metabolising proteins in that locale, i.e. an increase in the concentration of such genes.
  • a microbe which does not contain a gene encoding a hydrocarbon metabolising protein does not metabolise hydrocarbons and does not benefit from the hydrocarbons as a source of energy in the environment.
  • the microbial population is skewed in favour of microbes containing genes encoding hydrocarbon metabolising proteins. Therefore, the concentration of DNA in a sample population is skewed in favour of DNA encoding hydrocarbon metabolising proteins, hi environments where hydrocarbons are the only source of energy, all the microbes present will contain one or more genes encoding a hydrocarbon metabolising protein in order to survive.
  • the concentration of genes encoding hydrocarbon metabolising proteins varies not only due to the number of cells containing such genes in the population but also the number of copies of such genes within each cell.
  • bacterial cells containing a plasmid with such a gene will have a selective advantage in a hydrocarbon-rich environment over cells which do not contain such a plasmid.
  • a cell which contains multiple plasmids incorporating such a gene may have a selective advantage over a cell containing plasmid with only a single copy of a hydrocarbon metabolising protein encoding gene. Therefore, in such embodiments, the detection of genes encoding hydrocarbon metabolising proteins is particularly sensitive.
  • RNA and, in particular, mRNA molecules encoding hydrocarbon metabolising proteins are detected instead.
  • mRNA transcripts exist for a relatively short period of time within a cell and therefore the detection of such an mRNA transcript is indicative of the cell actively metabolising hydrocarbons at the time of sampling.
  • the DNA gene copy number is an integrative measure of the micororganisms exposure to specific hydrocarbons over a period of time.
  • the relative concentrations of DNA and mRNA encoding hydrocarbon metabolising proteins are compared to give an indication of the history of the presence of hydrocarbons in the environment of the microbial population. For example, if the concentration of DNA encoding hydrocarbon metabolising proteins in a microbial population is found to be average but the concentration of mRNA encoding hydrocarbon metabolising proteins is found to be well above average then this may be an indication that there is no underlying hydrocarbon deposit in the environment of the microbial population and that the presence of the high concentration of mRNA transcripts is due to human intervention (e.g. the vehicle of an individual taking the samples).
  • hydrocarbons which are metabolised by microbes In order to carry out embodiments of the present invention, it is necessary to identify hydrocarbons which are metabolised by microbes; proteins which effect the oxidation process; and genes which encode the proteins.
  • the number of organic compounds comprising petroleum hydrocarbons numbers in the thousands. While there are far fewer compounds found in thermogenically derived gas or vapour, they are still plentiful. Hydrocarbons which migrate from a naturally occurring subsurface reservoir may be grouped into two categories based. They are grouped not according to their structure, but on their volatility; namely volatile, semi-volatile or liquid. The volatiles are generally found as gases or vapours at standard temperature and pressure conditions while semi-volatiles are generally liquid under similar conditions but can volatilise very easily.
  • the main chemical component of gas or vapour migrating from a naturally occurring sub-surface reservoir to the surface is methane.
  • Methane can be generated thermogenically and also biogenically, and therefore there is a risk of "false positive” results if detecting the presence of methane.
  • it is still useful to determine the concentration of this gene hi preferred embodiments, a C2 to C20 alkane is detected (longer chained alkanes are found in decreasing concentrations in migrating gas or vapour) or straight chain alkanes and branch chain alkanes are detected.
  • Alkenes are also detected, as are simple and alkylated single multi-ring aromatics and saturated rings (napthenes).
  • Alkanes are types of organic hydrocarbon compounds which only have single carbon-carbon bonds.
  • Acyclic alkanes have the general formula C n H (2n+2 ).
  • the enzymology of microbial alkane oxidation is well known in the art with many reviews available (see for example Oil & Gas Science and Technology - Rev. IFP, Vol. 58 (2003) pp 427-440).
  • alkane hydroxylases Straight-chain hydrocarbons are oxidized by a group of enzymes known as alkane hydroxylases. These enzymes introduce oxygen atoms derived from molecular oxygen into the alkane substrate.
  • Alkane degrading yeast strains contain multiple alkane hydroxylases belonging to the P450 superfamily, while many bacteria contain membrane-bound alkane hydroxylase systems.
  • Short-chain alkanes are thought to be oxidized by alkane hydroxylases related to the soluble and particulate methane monooxygenases.
  • Some embodiments involve the detection and determination of the concentration of methane monooxygenases, e.g. the detection of soluble methane monooxygenase (sMMO) using primers mmoXl-mmoX2 (see Table 1), as described in Miguez et al., Microbiol Ecology (1997), 33:21-31.
  • sMMO soluble methane monooxygenase
  • primers mmoXl-mmoX2 see Table 1
  • polynucleotides encoding e.g. particulate methane monooxygenase gene (pmoA) are detected.
  • Alternative embodiments comprise assays based on the membrane bound alkane hydroxylase (alkB), which is thought to target longer chain alkanes.
  • Aromatic hydrocarbons are hydrocarbons which incorporate one or more planar sets of six carbon atoms connected by delocalized electrons, i.e. a benzene ring.
  • the enzymology of microbially mediated aromatic oxidation is also well known in the art.
  • C23DO catechol 2,3 dioxygneases
  • the individual pathways of aromatic biodegradation are usually initiated through the action of either a dioxygenase or a monooxygenase.
  • a dioxygenase or a monooxygenase.
  • biphenyl dioxygenase is involved in the oxidative biodegradation of phenol whilst toluene monooxygenase is involved in the oxidative biodegradation of toluene.
  • references of reports on detection of genes responsible for aromatic oxidation in environmental samples include the following: Applied & Environmental Microbiology, 65, 80-87 (1999); Applied & Environmental Microbiology, 56, 254-259 (1990); Applied & Environmental Microbiology, 67, 1542-1550 (2001); Applied & Environmental Microbiology, 66, 80-8678-6837 (2000); and Applied & Environmental Microbiology, 69, 3350-3358 (2003)
  • Naphthenes are cycloalkanes, i.e. they are types of alkanes which have one or more rings of carbon atoms in their chemical structure. They consist of carbon and hydrogen only and there are no double bonds between the carbon atoms.
  • Preferred embodiments of the present invention involve the detection of genes encoding one of the following enzymes: Alkane hydroxylase (alkB related); Catechol 2,3 dioxygenase; Napthalene dioxygenase; Toluene monooxygenase; Toluene dioxygenase; Xylene monooxygenase, biphenyl dioxygenase, or particulate methane monoxygenases (pmoA).
  • Alkane hydroxylase alkB related
  • Catechol 2,3 dioxygenase Napthalene dioxygenase
  • Toluene monooxygenase Toluene dioxygenase
  • Xylene monooxygenase biphenyl dioxygenase, or particulate methane monoxygenases (pmoA).
  • Suitable genes are those which encode one of the following enzymes: Butane monooxygenases (similar to pMMO and sMMO); Bacterial P450 oxygenases (C4-C16 n-alkanes); or Eukaryotic P450 oxygenases (C 10-Cl 6 n-alkanes).
  • the number of hydrocarbon metabolizing microbes in the vicinity increases.
  • These hydrocarbon degrading microbes are capable of using the hydrocarbons as an energy source. Therefore, in the vicinity of the deposit, the number of microbes with hydrocarbon metabolizing genes is elevated. Hence, the number of hydrocarbon metabolizing genes present in the microbial population increases.
  • the first hydrocarbons to be metabolized by microbes are short chain alkanes, followed by long chain alkanes. Next, the alkenes present in the petroleum are metabolized, and lastly microbes degrade the aromatic hydrocarbons.
  • comparing the concentration of different types of hydrocarbon metabolizing genes is advantageous for the present invention, i.e. genes that are more or less active at different stages in the degradation of the petroleum hydrocarbons.
  • a mathematical operation that compares the concentration of alkane-metabolizing genes, which are relatively abundant in a fresh petroleum deposit, with the concentration of aromatic-metabolizing genes, which are relatively resistant to degradation and therefore more likely to be relatively abundant in a degraded petroleum occurrence, is indicative of the presence or absence of 'fresh petroleum'.
  • the first polynucleotide encodes a short chain alkane metabolizing gene and that the second polynucleotide encodes an aromatic metabolizing gene, or a long chain alkane metabolizing gene.
  • the first polynucletide encodes a long chain alkane metabolizing gene
  • the second polynucleotide encodes an aromatic metabolizing gene.
  • the first polynucleotide encodes a methane, toluene or xylene metabolizing gene and the second polynucleotide encodes the aromatic metabolizing gene Cat 2,3.
  • genes referred to in Table 1 are by no means exhaustive and further genes could be used instead.
  • Other suitable genes are identified by, for example, searching public databases (e.g. GenBank and Ribosomal Database Project) for genes reported to encode hydrocarbon-metabolising proteins. Having identified a plurality of genes and their sequences in this way, it is preferred that the genes are aligned and areas of homology located in order to identify potential motifs that characterize genes encoding proteins that have this functionality. Such potential motifs are then compared with gene databases and those potential motifs that are found in genes encoding proteins not associated with hydrocarbon metabolism are discarded.
  • motifs that is to say, motifs/subsequences only found in genes encoding hydrocarbon-metabolising proteins
  • the motif subsequences range from 50 nucleotides to 100 nucleotides in length.
  • the DNA sequence(s) coding for a specific catabolic gene can be searched for in the GenBank (http://www.ncbi.nlm.nih.gov ⁇ . e.g. see Table 2, and imported into software for manipulating DNA sequences (such as DS Gene, www.accelrvs.com " ).
  • the sequences are aligned and phylogenetic analysis performed. These selected downloaded sequences are examined and consensus regions identified. These consensus sequences must show a high percentage of conformity for the sequences obtained for the resulting assay to be specific for the desired gene.
  • the consensus sequence is then exported into Primer Express software (www.appliedbiosystems.com), which analyses the consensus sequence and provides suggestions of primer/probe combinations that can be used in qPCR assays.
  • the present invention involves identifying the concentration of two genes each encoding a different hydrocarbon metabolizing protein in a sample.
  • the concentrations of each hydrocarbon metabolizing gene are then compared and one gene acts as a normalizing biomarker with respect to the other gene.
  • the concentration is determined with reference to the total microbial population in the sample so as to give an indication of the relative concentration of such genes with respect to the total microbial population, i.e. a normalized value.
  • the total microbial population is calculated by measuring the concentration of generic oligonucleotide sequences which are present in all or almost all microbes, irrespective of their capacity to metabolize hydrocarbons.
  • suitable genes from a bacterial population are provided in Table 3.
  • Two exemplary EuBac genes are provided, the first is that provided and used in the assay described in Suzuki et al. 2000, AEM, 66(11) p4605-4614, and the second is used in a modified assay which optimises the results obtained in the normalisation assays.
  • the same EuBac primer sequences have been used as reported in Suzuki et al., and the probe sequence is as reported with the exception that a FAM-minor groove binder probe has been used rather than the FAM-TAMRA probe.
  • the modified EuBac assay is run the PCR thermo-cycling conditions differ from those described in Suzuki et al.
  • the modified cycle conditions comprise 10 minutes at 95 0 C followed by 40 cycles of 95 0 C for 15 seconds and 57 0 C for 1 minute.
  • the microbial population is determined by carrying out quantitative PCR using generic primers of oligonucleotide sequences that vary slightly between strains of bacteria. Because the primers are generic, amplification of the oligonucleotide sequences takes place and is indicative of the microbial population notwithstanding differences between the oligonucleotide sequences of different bacterial strains.
  • nucleotide sequences can be identified by, for example, searching public databases for nucleotide motifs which are present in a high proportion of microorganisms, e.g. at least 80% of microorganisms.
  • the concentration of a first and second polynucleotide, each encoding a different hydrocarbon metabolizing protein, and optionally a third polynucleotide encoding a generic protein are determined.
  • the preferred technique for determining these concentrations is quantitative polymerase chain reaction (qPCR) which is also known as "real time PCR” (see, for example, Ding C et al "Quantitative Analysis of Nucleic Acids - The Last Few Years of Progress" J. Biochem MoI. Biol. 2004 Jan 31; 37 (l):l-10).
  • the principle underlying qPCR is that during the course of a PCR assay, the number of amplicons generated is monitored PCR cycle by PCR cycle. This is usually achieved by introducing a fluorophor into the assay system. The amount of fluorescence generated is directly proportional to the number of amplicons generated at each PCR cycle whilst the number of amplicons is a function of starting copy number and the number of PCR cycles. Therefore, by measuring the intensity of the signal (e.g. fluorescence) as the PCR cycles progress, the starting concentration of a target sequence may be determined. In particular, the PCR is monitored during the exponential phase where the first significant increase in the amount of PCR product correlates to the initial amount of target template. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed. A significant increase in fluorescence above the baseline value indicates the detection of accumulated PCR product (measured by the Ct value).
  • Absolute quantitation in PCR requires a standard curve of known copy numbers, which can be constructed using a synthesized oligonucleotide or amplicon. This amplicon is of a known concentration and by serial dilution can give a wide range of known standards. These standards then undergo PCR using exactly the same conditions as the target DNA sequence. The copy number of the target DNA sequence is then extrapolated in the sample from the calibration graph or standard curve, which is constructed plotting the log of copy number against the Ct value.
  • Exemplary apparatus includes the ABI 7300 sequence detector which performs 96 parallel wells of qPCR analyses, determines a standard curve and calculates the amount of target DNA in each of the sample wells. Thus the output is a direct measure of the abundance of the target sequence in the sample.
  • a Taqman 5' nuclease assay or a SyBr Green system are particularly preferred.
  • the Taqman 5' nuclease assay is more sensitive to the presence of a target sequence and is more specific thereto but it requires three closely linked conserved regions in the target gene.
  • the SyBr Green assay system is less sensitive and specific but only requires the presence of two conserved regions.
  • the concentrations of the first, second and third polynucleotides, or a subsequence thereof, can be determined simultaneously in a multiplex qPCR reaction.
  • forward and reverse primers and a probe are provided for both genes but the signaling system is different for each probe (e.g. the label fluoresces at a different wavelength) so that the relative quantities of each gene can be determined independently during PCR.
  • the core samples suitable for testing according to the present invention are obtained from a variety of locations.
  • the sample is a soil sample extracted from over or around a prospective oil field.
  • the sample is sediment or sand taken from a fresh or salt-water environment.
  • the microbial population of a core sample will change over time, once the sample has been removed from its original geographical location. More specifically, if a core sample is taken from a location where a subsurface hydrocarbon deposit is present then, upon removal of the sample, the microbes within the sample are deprived of their hydrocarbon source. This may affect the concentration of the first and/or second polynucleotide detected. In practice, it may be necessary for samples to be taken from a locality and analyzed by the methods of the present invention under laboratory conditions. It is therefore important that the gene copy numbers within the sample remain unchanged between removal of the sample from a location and analysis in the laboratory.
  • RNALaterTM from AmbionTM
  • RNAprotectTM from Qiagen(TM)
  • RNALaterTM from AmbionTM
  • RNAprotectTM from Qiagen(TM)
  • the present invention involves obtaining a core sample from a site.
  • the sample is dried and milled, and DNA or RNA is extracted from a representative subsample and the concentration of the DNA/RNA of interest is quantified.
  • RNA and/or DNA is extracted from soil samples.
  • Soil samples may contain contaminating substances which interfere with the PCR reaction and thus the quantitation process.
  • the comparison of the concentration of first polynucleotide to a second hydrocarbon polynucleotide or a generic microbial gene environmentally normalizes the result so that the data obtained is not detrimentally affected by contamination.
  • Different soil types may affect the efficiency of the extraction of nucleic acids, but this can also be normalized by quantification of a second hydrocarbon metabolizing hydrocarbon gene or a generic microbial gene sequence in soil samples.
  • Kits for extracting an isolated nucleic acid from soil samples are sold commercially by MoBio Laboratories, Inc. These kits require a soil sample to be added to a bead beating tube for rapid homogenization. Cell lysis occurs by both chemical and mechanical means (vortex adapter). Total genomic DNA is captured on a silicone membrane in a conventional spin column format. DNA is washed and then eluted from the spin column.
  • Epicentre Limited produces the SoilMaster DNA extraction kit which utilizes a hot detergent lysis process combined with a chromatographic step, which removes enzymatic inhibitors known to co- extract with DNA from soil and sediment samples.
  • Qbiogene Inc also produces a range of kits for extraction of DNA and RNA from soil samples.
  • the kits are based on their FastPrep TM system.
  • Soil samples were collected within and around the confirmed seepage location in order to test the survey tool.
  • the sampling sites were at approximately 100m horizontal intervals in undisturbed soil sections located between cultivated land and drainage ditches in the spatial pattern illustrated in Figure 5.
  • the soil samples were collected from the 20 cm to 30 cm subsurface interval using a spade and placed into polyethylene bags that were then stored in an ice chest until frozen at -18°C in the laboratory, pending analysis.
  • live petroleum is characterised geochemically by high abundances of biochemically labile normal alkanes relative to less labile aromatics.
  • the abundance of alkanes may be simulated using the abundance of normal alkane consumers which is in turn estimated using the AIkB-Pl gene.
  • the abundance of labile aromatics may be simulated using the generic index of aromatic consumers, the 2,3Cat gene.
  • high abundances of the AIkB-Pl gene relative to the 2,3Cat gene are deemed to signal occurrences of 'live' (fresh or only slightly altered) petroleum.
  • High relative abundances of the pMoA gene have the same meaning in respect of methane.
  • the AIkB-Pl to 2,3Cat gene copy number ratio also serves the purpose of an environmental normaliser because the subject genes do not discriminate between metabolic substrates according to provenance (soil or plant-derived hydrocarbons versus migrant petroleum).
  • Transform 1 for every soil sample, the copy number of the hydrocarbon metabolising gene was divided by the EuBac copy number and expressed as a percentage. This gives index data normalised on the total microbial abundance (the ratio of the microbial population capable of metabolizing hydrocarbons to the overall microbial population). The normalised index data was rescaled by dividing each population by its median value in order to render all of the variable data to a common amplitude scale for visualisation purposes. The result is called Transform 1.
  • Figures 5 and 6 show that Transform 1 data identify the visible seep location poorly, also identifying a number of additional anomalies presumed to represent invisible (micro) seepage.
  • the outwardly weak performance of Transform 1 at Formby is potentially attributable to the fact the survey coverage does not extend to truly background areas outside the extent of the former oilfield.
  • Transform 2 involved a singular ratio followed by rescaling.
  • the copy number of each of the hydrocarbon metabolising genes was divided by the corresponding 2,3 Cat copy number and each resulting ratio value divided by its respective population median in order to render it to a common amplitude scale.
  • the transform values thus derived exhibit improved signal/noise ratio ( Figures 7 and 8).
  • Transform 3 involved three steps, effectively combining Transforms 1 and 2.
  • the higher alkane index, AIkB-Pl that is most directly indicative of 'live' petroleum/oil, is used to illustrate the transform.
  • the 2,3Cat and AIkB-Pl index values were firstly normalised on the total microbial population by dividing them by the homologous EuBac index values. Each resulting index population was then rescaled by dividing each constituent value by the population median, enabling direct graphical comparison and also arithmetic recombination with minimal scalar artifact. Finally the transformed 2,3Cat index values thus derived were subtracted from the homologous AIkB-Pl index values to arrive at the live petroleum index illustrated in Figure 9. The Formby oil seep is identified by positive transform values.
  • the second field trial was performed in a sub-sea location, at a virgin gas field under the Barents Sea, offshore Norway. At this site some exploratory wells drilled in c. 300m of water have encountered commercially viable gas accumulations (gas discoveries) at depths exceeding 2 km sub-bottom. However, other wells drilled in the same region were unsuccessful (dry). A gas field comprising the discovery well and also a dry well were surveyed in order to test the tool in a comparative and commercially realistic context.
  • the trial survey comprised collection of seabed sediment cores for subsampling and later laboratory analysis.
  • the cores were collected using standard gravity coring equipment in c. 300m water depths at c. 1,000m horizontal intervals in a polygonal traverse across the gas field and the dry hole, as shown in Figure 10.
  • Subsamples for DNA analysis of the acquired sediment cores were collected from within the 40 - 50 cm sub-bottom interval, placed in polyethylene bags and then stored 4°C pending laboratory analysis.
  • Transform 2 was applied as explained in example 1, with the exception that the detected polynucleotide copy numbers were not divided by the population median to render them to a common amplitude scale. Each hydrocarbon metabolising gene copy number was expressed as a percentage of the EuBac copy number. Separately the copy number of the hydrocarbon metabolising gene was expressed as a percentage of the 2,3 Cat copy number. The index values thus transformed have the same meaning attributed to them in example 1.
  • the resulting pMoA Transform 2 index values are displayed in plan view Figure 10 in relation to the field circumstances.
  • the symbol size is proportional to the index value.
  • the larger symbols (high index value scores) represent methane seepage anomalies whereas the small symbols indicate the background condition where methane seepage is subdued.
  • the anomaly amplitude increases from the margin of the gas field towards the crest of the closure where the discovery well penetrates the maximum thickness of reservoired gas.
  • the index value anomalies cluster and reach their maximum in the immediate vicinity of the discovery well, thereby identifying active methane seepage emanating from the underlying reservoir.
  • the dry hole locality exhibits few, lower amplitude anomalies.

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Abstract

La présente invention concerne un procédé de détection de la présence d'un dépôt d'hydrocarbures à un emplacement géographique. Le procédé comprend les étapes de détermination de la concentration d'un premier polynucléotide et d'un deuxième polynucléotide présents à l'emplacement, les deux polynucléotides codant pour des protéines capables de métaboliser des hydrocarbures. Le premier polynucléotide code pour une protéine différente du second polynucléotide. On effectue une opération mathématique en utilisant la concentration du premier et du deuxième polynucléotide pour indiquer la présence ou l'absence d'un dépôt d'hydrocarbures à l'emplacement. La concentration d'un troisième polynucléotide, qui code pour un gène générique, est éventuellement calculée et est utilisée pour normaliser les données.
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CN107130030B (zh) * 2017-05-17 2020-12-18 山西大学 用于检测煤地质微生物古菌物种的DNA Marker及制备方法和应用
US11649478B2 (en) 2018-05-21 2023-05-16 ExxonMobil Technology and Engineering Company Identification of hot environments using biomarkers from cold-shock proteins of thermophilic and hyperthermophilic microorganisms

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