PCR AMPLIFICATION METHODS FOR DETECTING AND QUANTIFYING SULFATE-REDUCING BACTERIA IN OILFIELD FLUIDS

METHODES D'AMPLIFICATION D'ACP SERVANT A DETECTER ET QUANTIFIER LES BACTERIES REDUCTRICES DE SULFATE DANS LES FLUIDES DE CHAMPS PETROLIERS

English Abstract

At least one nucleic acid from a sulfate-reducing bacteria (SRB) may be
extracted
from an oilfield fluid and may be amplified by a PCR amplification method in
the
presence of at least one primer to form an amplification product. The
primer(s) may
be or include a sequence including, but not necessarily limited to, SEQ ID
NO:1,
SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID
NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and mixtures thereof. The amplification
product
may be hybridized with a probe specific for a fragment of an alpha subunit of
an APS
gene, and a presence of hybridization and a degree of hybridization may be
detected.

Claims

14

CLAIMS
What is claimed is:
1. A method of decreasing sulfate-reducing bacteria (SRB) in oilfield
fluids
comprising:
altering an amount of a microbial agent within an oilfield fluid to form an
altered oilfield fluid based on an amount of at least one SRB within an
oilfield fluid;
wherein the amount of the at least one SRB is determined by:
amplifying at least one nucleic acid of the at least one SRB in the
presence of at least one primer to form an amplification product; wherein the
at least one nucleic acid is extracted from the oilfield fluid prior to
amplifying
the at least one nucleic acid; characterized in that the at least one primer
comprises a sequence selected from the group consisting of SEQ ID NO:7,
SEQ ID NO:8, and mixtures thereof;
hybridizing the amplification product with a probe specific for a fragment
of an alpha subunit of an Adenylylsulfate Reductase gene; and
detecting a presence of hybridization and a degree of hybridization,
wherein the presence of hybridization indicates the presence of the at least
one SRB, and wherein the degree of hybridization enumerates the at least one
SRB; and
decreasing the amount of SRB by killing and/or deactivating the SRB wherein
the altered oilfield fluid comprises a decreased amount of SRB as compared to
the
oilfield fluid.
2. The method of claim 1, wherein the oilfield fluid is selected from the
group
consisting of produced waters, oilfield waters, production fluids, fracturing
fluids,
drilling fluids, completion fluids, workover fluids, packer fluids, gas
fluids, crude oils,
refinery fluids, processed crude oils, refined products, process and waste
waters,
midstream fluids, downstream fluids, and mixtures thereof.
3. The method of claim 1, wherein the probe is detectably labeled.

15

4. The method of claim 1, wherein the at least one sulfate-reducing
bacteria is
selected from the group consisting of Desulfovibrio vulgaris, Desulfovibrio
desulfuricans, Desulfovibrio aespoeensis, Thermodesulfobium narugense,
Desulfotomaculum carboxydivorans, Desulfotomaculum ruminis, Desulfovibrio
africanus, Desulfovibrio hydrothermalis, Desulfovibrio piezophilus,
Desulfobacterium
corrodens, Sulfate-reducing bacterium QLNR1, Desulfobacterium catecholicum,
Desulfobacterium catecholicum, Desulfobulbus marinus, Desulfobulbus,
Desulfobulbus propionicus, Desulfocapsa thiozymogenes, Desulfocapsa
sulfexigens,
Desulforhopalus vacuolatus, Desulforhopalus, Desulfofustis glycolicus strain,
Desulforhopalus singaporensis, Desulfobacterium, Desulfobacterium zeppelinii
strain,
Desulfobacterium autotrophicum, Desulfobacula phenolica, Desulfobacula
toluolica
Tol2, Sulfate-reducing bacterium JHAl, Desulfospira joergensenii,
Desulfobacter,
Desulfobacter postgatei, Desulfotignum, Desulfotignum balticum, Desulforegula
conservatrix, Desulfocella, Desulfobotulus sapovorans, Desulfofrigus,
Desulfonema
magnum, Desulfonema limicola, Desulfobacterium indolicum, Desulfosarcina
variabilis, Desulfatibacillum, Desulfococcus multivorans, Desulfococcus,
Desulfonema ishimotonii, Desulfococcus oleovorans Hxd3, Desulfococcus niacini,

Desulfotomaculum, Desulfotomaculum nigrificans, Desulfotomaculum ruminis,
Desulfotomaculum halophilum, Desulfotomaculum acetoxidans, Desulfotomaculum
gibsoniae, Desulfotomaculum sapomandens strain, Desulfotomaculum
thermosapovorans, Desulfotomaculum geothermicum, Desulfosporosinus meridiei,
Delta proteobacterium, Thermodesulforhabdus norvegica, Desulfacinum infernum,
Desulfacinum hydrothermale, Desulforhabdus amnigena, Desulforhabdus,
Desulforhabdus, Desulfomonile tiedjei, Desulfarculus baarsii, Desulfobacterium

anilini, Delta proteobacterium, Desulfovibrio profundus strain,
Desulfomicrobium
baculatum, Desulfocaldus hobo, Desulfovibrio, Desulfovibrio piger,
Desulfovibrio
ferrophilus, Desulfonatronovibrio hydrogenovorans, Desulfovibrio,
Desulfovibrio
acrylicus, Desulfovibrio salexigens, Desulfovibrio oxyclinae, Desulfonauticus
submarinus, Desulfothermus naphthae, Thermodesulfobacterium,
Thermodesulfobacterium hveragerdense, Thermodesulfobacterium thermophilum,
Thermodesulfatator indicus, Thermodesulfovibrio yellowstonii,
Desulfosporosinus

16

orientis, Desulfotomaculum thermobenzoicum, Desulfotomaculum solfataricum,
Desulfotomaculum luciae strain, Desulfobacca acetoxidans, Desulfovibrio
vulgaris,
Desulfovibrio desulfuricans, Desulfovibrio alaskensis, Desulfovibrio
magneticus,
Desulfosporosinus acidiphilus, Desulfotomaculum kuznetsovii, Desulfovibrio
sulfodismutans, Desulfomicrobium baculatum, Desulfonatronum lacustre,
Desulfohalobium retbaense, Desulfonauticus autotrophicus,
Thermodesulfobacterium
commune, Thermodesulfobacterium hveragerdense, Thermodesulfovibrio islandicus,

Thermodesulfovibrio, Thermodesulfobacterium, Desulfotomaculum
thermobenzoicum, Desulfotomaculum thermoacetoxidans, Desulfotomaculum
thermocisternum, Desulfotomaculum australicum, Desulfotomaculum kuznetsovii,
Desulfovibrio desulfuricans, Desulfovibrio alaskensis, Desulfovibrio vulgaris,

Desulfovibrio salexigens, Desulfosporosinus acidiphilus, Desulfosporosinus
meridiei,
Desulfosporosinus orientis, Desulfotomaculum reducens, and combinations
thereof.
5. The method of any one of claims 1-4, wherein the at least one primer is
specific for amplification of at least a fragment of an alpha subunit of an
Adenylylsulfate Reductase gene.
6. The method of any one of claims 1-4, further comprising circulating the
altered
oilfield fluid within a subterranean reservoir wellbore, wherein the altered
oilfield fluid
is selected from the group consisting of altered fracturing fluids, altered
drilling fluids,
altered completion fluids, altered workover fluids, altered packer fluids,
altered
produced waters, altered oilfield waters, altered production fluids, altered
gas fluids,
altered crude oils, altered refinery fluids, altered processed crude oils,
altered refined
products, altered process and waste waters, altered midstream fluids, altered
downstream fluids, and combinations thereof.

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7. A method of decreasing sulfate-reducing bacteria (SRB) in oilfield
fluids
comprising:
altering an amount of a microbial agent within an oilfield fluid based on an
amount of at least one SRB within the oilfield fluid to form an altered
oilfield fluid;
wherein the oilfield fluid is selected from the group consisting of oilfield
water, a
production fluid, a fracturing fluid, a drilling fluid, a completion fluid, a
workover fluid,
a packer fluid, a gas fluid, a crude oil, and mixtures thereof; wherein the
amount of
the at least one SRB is determined by:
amplifying at least one nucleic acid of the at least one SRB in the
presence of at least one primer to form an amplification product; wherein the
at least one nucleic acid is extracted from the oilfield fluid prior to
amplifying
the at least one nucleic acid; characterized in that the at least one primer
comprises a sequence selected from the group consisting of SEQ ID NO:7,
SEQ ID NO:8, and mixtures thereof;
hybridizing the amplification product with a probe specific for a fragment
of an alpha subunit of an Adenylylsulfate Reductase gene; and
detecting a presence of hybridization and a degree of hybridization;
wherein the presence of hybridization indicates the presence of the at least
one SRB; and wherein the degree of hybridization enumerates the at least one
SRB; and
decreasing the amount of SRB by killing and/or deactivating the SRB.
8. The method of claim 7, further comprising circulating the altered
oilfield fluid
within a subterranean reservoir wellbore wherein the altered oilfield fluid is
selected
from the group consisting of an altered fracturing fluid, an altered drilling
fluid, an
altered completion fluid, an altered workover fluid, an altered packer fluid,
and
combinations thereof.

18

9. A method of determining an amount of sulfate-reducing bacteria (SRB)
within
an oilfield fluid comprising:
amplifying at least one nucleic acid of at least one SRB in the presence of at

least one primer to form an amplification product; wherein the amplifying
occurs by a
PCR amplification method wherein the at least one nucleic acid is extracted
from the
oilfield fluid prior to amplifying the at least one nucleic acid; wherein the
at least one
primer comprises a selected from the group consisting of SEQ ID NO:7, SEQ ID
NO:8, and mixtures thereof;
hybridizing the amplification product with a probe specific for a fragment of
an
alpha subunit of an Adenylylsulfate Reductase gene; and
detecting a presence of hybridization and a degree of hybridization; wherein
the presence of hybridization indicates the presence of the at least one SRB;
and
wherein the degree of hybridization enumerates the at least one SRB; and
determining an amount of SRB in the oilfield fluid.
10. The method of claim 9, wherein the oilfield fluid is selected from the
group
consisting of oilfield water, a production fluid, a fracturing fluid, a
drilling fluid, a
completion fluid, a workover fluid, a packer fluid, a gas fluid, a crude oil,
and mixtures
thereof.
11. The method of claim 9, wherein the probe is detectably labeled.
12. The method of claim 9, wherein the at least one sulfate-reducing
bacteria is
selected from the group consisting of Desulfovibrio vulgaris, Desulfovibrio
desulfuricans, Desulfovibrio aespoeensis, Thermodesulfobium narugense,
Desulfotomaculum carboxydivorans, Desulfotomaculum ruminis, Desulfovibrio
africanus, Desulfovibrio hydrothermalis, Desulfovibrio piezophilus,
Desulfobacterium
corrodens, Sulfate-reducing bacterium QLNR1, Desulfobacterium catecholicum,
Desulfobacterium catecholicum, Desulfobulbus marinus, Desulfobulbus,
Desulfobulbus propionicus, Desulfocapsa thiozymogenes, Desulfocapsa
sulfexigens,
Desulforhopalus vacuolatus, Desulforhopalus, Desulfofustis glycolicus strain,
Desulforhopalus singaporensis, Desulfobacterium, Desulfobacterium zeppelinii
strain,
Desulfobacterium autotrophicum, Desulfobacula phenolica, Desulfobacula
toluolica

19

Tol2, Sulfate-reducing bacterium JHA1, Desulfospira joergensenii,
Desulfobacter,
Desulfobacter postgatei, Desulfotignum, Desulfotignum balticum, Desulforegula
conservatrix, Desulfocella, Desulfobotulus sapovorans, Desulfofrigus,
Desulfonema
magnum, Desulfonema limicola, Desulfobacterium indolicum, Desulfosarcina
variabilis, Desulfatibacillum, Desulfococcus multivorans, Desulfococcus,
Desulfonema ishimotonii, Desulfococcus oleovorans Hxd3, Desulfococcus niacini,

Desulfotomaculum, Desulfotomaculum nigrificans, Desulfotomaculum ruminis,
Desulfotomaculum halophilum, Desulfotomaculum acetoxidans, Desulfotomaculum
gibsoniae, Desulfotomaculum sapomandens strain, Desulfotomaculum
thermosapovorans, Desulfotomaculum geothermicum, Desulfosporosinus meridiei,
Delta proteobacterium, Thermodesulforhabdus norvegica, Desulfacinum infernum,
Desulfacinum hydrothermale, Desulforhabdus amnigena, Desulforhabdus,
Desulforhabdus, Desulfomonile tiedjei, Desulfarculus baarsii, Desulfobacterium

anilini, Delta proteobacterium, Desulfovibrio profundus strain,
Desulfomicrobium
baculatum, Desulfocaldus hobo, Desulfovibrio, Desulfovibrio piger,
Desulfovibrio
ferrophilus, Desulfonatronovibrio hydrogenovorans, Desulfovibrio,
Desulfovibrio
acrylicus, Desulfovibrio salexigens, Desulfovibrio oxyclinae, Desulfonauticus
submarinus, Desulfothermus naphthae, Thermodesulfobacterium,
Thermodesulfobacterium hveragerdense, Thermodesulfobacterium thermophilum,
Thermodesulfatator indicus, Thermodesulfovibrio yellowstonii,
Desulfosporosinus
orientis, Desulfotomaculum thermobenzoicum, Desulfotomaculum solfataricum,
Desulfotomaculum luciae strain, Desulfobacca acetoxidans, Desulfovibrio
vulgaris,
Desulfovibrio desulfuricans, Desulfovibrio alaskensis, Desulfovibrio
magneticus,
Desulfosporosinus acidiphilus, Desulfotomaculum kuznetsovii, Desulfovibrio
sulfodismutans, Desulfomicrobium baculatum, Desulfonatronum lacustre,
Desulfohalobium retbaense, Desulfonauticus autotrophicus,
Thermodesulfobacterium
commune, Thermodesulfobacterium hveragerdense, Thermodesulfovibrio islandicus,

Thermodesulfovibrio, Thermodesulfobacterium, Desulfotomaculum
thermobenzoicum, Desulfotomaculum thermoacetoxidans, Desulfotomaculum
thermocisternum, Desulfotomaculum australicum, Desulfotomaculum kuznetsovii,
Desulfovibrio desulfuricans, Desulfovibrio alaskensis, Desulfovibrio vulgaris,

20

Desulfovibrio salexigens, Desulfosporosinus acidiphilus, Desulfosporosinus
meridiei,
Desulfosporosinus orientis, Desulfotomaculum reducens, and combinations
thereof.
13. The method of any one of claims 9-12, wherein the at least one primer
is
specific for amplification of at least a fragment of an alpha subunit of APS
reductase
gene.
14. A PCR amplification method comprising:
amplifying at least one nucleic acid of at least one sulfate-reducing bacteria

(SRB) in the presence of at least one primer to form an amplification product;

wherein the at least one SRB is extracted from an oilfield fluid prior to
amplifying the
at least one nucleic acid; characterized in that the at least one primer
comprises a
sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, and
mixtures thereof.
15. The method of claim 14, wherein the oilfield fluid is selected from the
group
consisting of oilfield water, a production fluid, a fracturing fluid, a
drilling fluid, a
completion fluid, a workover fluid, a packer fluid, a gas fluid, a crude oil,
and mixtures
thereof.
16. The method of claim 14 or claim 15, wherein the at least one sulfate-
reducing
bacteria is selected from the group consisting of Desulfovibrio vulgaris,
Desulfovibrio
desulfuricans, Desulfovibrio aespoeensis, Thermodesulfobium narugense,
Desulfotomaculum carboxydivorans, Desulfotomaculum ruminis, Desulfovibrio
africanus, Desulfovibrio hydrothermalis, Desulfovibrio piezophilus,
Desulfobacterium
corrodens, Sulfate-reducing bacterium QLNR1, Desulfobacterium catecholicum,
Desulfobacterium catecholicum, Desulfobulbus marinus, Desulfobulbus,
Desulfobulbus propionicus, Desulfocapsa thiozymogenes, Desulfocapsa
sulfexigens,
Desulforhopalus vacuolatus, Desulforhopalus, Desulfofustis glycolicus strain,
Desulforhopalus singaporensis, Desulfobacterium, Desulfobacterium zeppelinii
strain,
Desulfobacterium autotrophicum, Desulfobacula phenolica, Desulfobacula
toluolica
Tol2, Sulfate-reducing bacterium JHA1, Desulfospira joergensenii,
Desulfobacter,

21
Desulfobacter postgatei, Desulfotignum, Desulfotignum balticum, Desulforegula
conservatrix, Desulfocella, Desulfobotulus sapovorans, Desulfofrigus,
Desulfonema
magnum, Desulfonema limicola, Desulfobacterium indolicum, Desulfosarcina
variabilis, Desulfatibacillum, Desulfococcus multivorans, Desulfococcus,
Desulfonema ishimotonii, Desulfococcus oleovorans Hxd3, Desulfococcus niacini,

Desulfotomaculum, Desulfotomaculum nigrificans, Desulfotomaculum ruminis,
Desulfotomaculum halophilum, Desulfotomaculum acetoxidans, Desulfotomaculum
gibsoniae, Desulfotomaculum sapomandens strain, Desulfotomaculum
thermosapovorans, Desulfotomaculum geothermicum, Desulfosporosinus meridiei,
Delta proteobacterium, Thermodesulforhabdus norvegica, Desulfacinum infernum,
Desulfacinum hydrothermale, Desulforhabdus amnigena, Desulforhabdus,
Desulforhabdus, Desulfomonile tiedjei, Desulfarculus baarsii, Sulfate-reducing

bacterium, Sulfate-reducing bacterium, Sulfate-reducing bacterium,
Desulfobacterium
anilini, Delta proteobacterium, Desulfovibrio profundus strain,
Desulfomicrobium
baculatum, Desulfocaldus hobo, Desulfovibrio, Desulfovibrio piger,
Desulfovibrio
ferrophilus, Desulfonatronovibrio hydrogenovorans, Desulfovibrio,
Desulfovibrio
acrylicus, Desulfovibrio salexigens, Desulfovibrio oxyclinae, Desulfonauticus
submarinus, Desulfothermus naphthae, Thermodesulfobacterium,
Thermodesulfobacterium hveragerdense, Thermodesulfobacterium thermophilum,
Thermodesulfatator indicus, Thermodesulfovibrio yellowstonii,
Desulfosporosinus
orientis, Desulfotomaculum thermobenzoicum, Desulfotomaculum solfataricum,
Desulfotomaculum luciae strain, Desulfobacca acetoxidans, Desulfovibrio
vulgaris,
Desulfovibrio desulfuricans, Desulfovibrio alaskensis, Desulfovibrio
magneticus,
Desulfosporosinus acidiphilus, Desulfotomaculum kuznetsovii, Desulfovibrio
sulfodismutans, Desulfomicrobium baculatum, Desulfonatronum lacustre,
Desulfohalobium retbaense, Desulfonauticus autotrophicus,
Thermodesulfobacterium
commune, Thermodesulfobacterium hveragerdense, Thermodesulfovibrio islandicus,

Thermodesulfovibrio, Thermodesulfobacterium, Desulfotomaculum
thermobenzoicum, Desulfotomaculum thermoacetoxidans, Desulfotomaculum
thermocisternum, Desulfotomaculum australicum, Desulfotomaculum kuznetsovii,
Desulfovibrio desulfuricans, Desulfovibrio alaskensis, Desulfovibrio vulgaris,

22
Desulfovibrio salexigens, Desulfosporosinus acidiphilus, Desulfosporosinus
meridiei,
Desulfosporosinus orientis, Desulfotomaculum reducens, and combinations
thereof.

Description

I
PCR AMPLIFICATION METHODS FOR DETECTING AND QUANTIFYING
SULFATE-REDUCING BACTERIA IN OILFIELD FLUIDS
TECHNICAL FIELD
[0001] The present invention relates to amplifying, optionally detecting and
optionally quantifying sulfate-reducing bacteria (SRB), and more specifically
relates
to rapid amplification of SRB using real-time quantitative polymerase chain
reactions
(qPCR).
BACKGROUND
[0002] The presence of SRB in many environments is undesirable, particularly
in
concentrations sufficient to cause significant corrosion of metals with
aqueous
solutions, including fresh and seawaters, having the SRB therein. SRBs are
present
in a variety of environments, including oil- and gas-bearing formations,
soils, and
wastewater. SRBs are also present in the gut of ruminant animals, particularly

domestic animals (e.g. cattle) used as protein sources for human consumption.
[0003] Sulfate-reducing bacteria, such as members of the genera Desulfovibrio
and Desulfotomaculum, may reduce sulfate and/or sulfite under suitable
conditions
(e.g. anaerobic conditions) and generate hydrogen sulfide, an odiferous, and
poisonous gas. In addition, the SRB may contact metals thereby causing
corrosion
to the metal, such as metal structures and conduits. "Sulfate-reducing
bacteria" is
defined herein to be bacteria capable of reducing sulfate to sulfite and/or
bacteria
capable of reducing sulfite to sulfide, regardless of the taxonomic group of
the
bacteria.
[0004] Traditionally, the monitoring of microbial populations has employed
microbial growth tests where a sample is diluted to various levels and used to

inoculate microbial growth media designed to favor the growth of various types
of
bacteria. After days to several weeks of incubation, the growth tests are
scored
based on the presence or absence of growth in these various microbiological
media.
Unfortunately, as numerous researchers show, only about 0.1% to about 10%
bacteria from environmental samples can actually grow in an artificial medium,
and a
significant portion of bacteria growing in the media are not actually the
target
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2
bacteria. Therefore, growth tests are unable to provide the accurate
quantification of
target bacteria in the samples. In addition, obtaining results from a serial
dilution
assay may take as long as three to four weeks.
[0005] To circumvent problems associated with such growth-based methods,
many culture-independent genetic techniques have been developed in the past
decade to detect pathogens in the field of medicine, the food industries, the
oil and
gas industries, and the like. Because many ecosystems have a relatively low
abundance of microorganisms, the polymerase chain reaction (PCR) has been
widely used to amplify the genetic signals of microbes in complex
environmental
samples. However, traditional PCR-based methods are significantly biased by
amplification efficiency and the depletion of PCR reagents.
[0006] Real-time quantitative PCR (qPCR) may be used to detect and quantify a
number of microorganisms. Quantitative PCR has also been used to determine the

abundance of microorganisms in many different types of complex environmental
samples, such as sediments, water, wastewater, and marine samples. qPCR may
provide more accurate and reproducible quantification of microorganisms
because
qPCR quantifies the PCR products during the logarithmic phase of the
reactions,
which does not occur during traditional PCR methods. Moreover, qPCR offers a
dynamic detection range of six orders of magnitude or more, does not need post-

PCR manipulation, and has the capability of high throughput analysis.
[0007] Digital PCR (dPCR) may be used to directly quantify and clonally
amplify
nucleic acids including DNA, cDNA, and/or RNA. dPCR may be more precise
method than PCR and/or qPCR. Traditional PCR carries out one reaction per
single
sample. dPCR may carry out a single reaction within a sample, but the sample
may
be separated into a large number of partitions, and the reaction may be
individually
carried out within each partition. The separation may allow for a more
reliable
collection and a more sensitive measurement of nucleic acid amounts within the

sample. dPCR may be useful for studying variations in gene sequences, such as
copy number variants, point mutations, and the like, and dPCR may be routinely

used for clonal amplification of samples for "next-generation sequencing."
[0008] It would be desirable to have a method of detecting and optionally
quantifying SRB within a sample that is cost-effective and may occur in real
time.
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3
SUMMARY
[0009] There is provided, in one form, a method of decreasing SRB in oilfield
fluids by altering an amount of a microbial agent within the oilfield fluid to
form an
altered oilfield fluid based on an amount of at least one SRB within an
oilfield fluid
where the altered oilfield fluid may have a decreased amount of SRB as
compared
to the oilfield fluid. The amount of the SRB may be determined by amplifying
at least
one nucleic acid of the SRB in the presence of at least one primer to form an
amplification product. The amplification product may be hybridized with a
probe
specific for a fragment of an alpha subunit of an APS gene. The presence of
hybridization and a degree of hybridization may be detected where the presence
of
hybridization indicates the presence of the SRB, and where the degree of
hybridization enumerates the SRB. The nucleic acid(s) may be extracted from
the
oilfield fluid prior to amplifying the nucleic acid(s). The primer(s) may have
or include
a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ

ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,
SEQ ID NO:9õ and mixtures thereof.
[0010] An alternative non-limiting embodiment of the method may also include
an
oilfield fluid, such as but not limited to an oilfield water, a production
fluid, a
fracturing fluid, a drilling fluid, a completion fluid, a workover fluid, a
packer fluid, a
gas fluid, a crude oil, and mixtures thereof.
[0011] In another non-limiting embodiment, a method of determining an amount
of
SRB within an oilfield fluid may include amplifying at least one nucleic acid
of at least
one SRB in the presence of at least one primer to form an amplification
product. The
amplifying may occur by a PCR amplification method, and the nucleic acid(s)
may
be extracted from the oilfield fluid prior to amplifying the nucleic acid(s).
The
primer(s) may include a sequence selected from the group consisting of SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and mixtures thereof. The method may
further include hybridizing the amplification product with a probe specific
for a
fragment of an alpha subunit of an APS gene, and detecting a presence of
hybridization and a degree of hybridization. The presence of hybridization may

indicate the presence of the SRB. The degree of hybridization may enumerate
the
SRB to determine an amount of SRB in the oilfield fluid.
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4
[0012] In another non-limiting embodiment, a PCR amplification method is
provided. The method may include amplifying at least one nucleic acid of at
least
one SRB in the presence of at least one primer to form an amplification
product.
SRB's DNA may be extracted from an oilfield fluid prior to amplifying the
nucleic
acid(s). The primer(s) may include a sequence selected from the group
consisting of
SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and mixtures thereof.
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5
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] In order to more fully understand the drawings referred to in the
detailed
description, a brief description of each drawing is presented here:
[0014] FIGs. 1-2, 4-5, and 7-8 (SEQ ID NO:1 through SEQ ID NO:2, SEQ ID NO:
4 through SEQ ID NO:5, and SEQ ID NO:7-8) represent the nucleotide sequences
of
a primer usable to detect SRB; and
[0015] FIGs. 3, 6, and 9 (SEQ ID NO:3, SEQ ID NO:6, and SEQ ID NO: 9)
represent the nucleotide sequence of a probe usable to detect SRB.
DETAILED DESCRIPTION
[0016] It has been discovered that an amount of antimicrobial agent may be
added to an oilfield fluid to form an altered oilfield fluid based on an
amount of at
least one SRB within an oilfield fluid. Alternatively, the amount of the
antimicrobial
agent, e.g. biocide, present may be altered within the altered oilfield fluid
based on
an amount of at least one SRB within the oilfield fluid. Non-limiting examples
of
microbial agents are those additives typically used to decrease the amount of
SRB
within an oilfield fluid. 'Decreasing' the amount of SRB may occur by killing
the
bacteria and/or by inactivating the bacteria from producing sulfur compounds,
such
as but not limited to sulfates, sulfites, mercaptans, and the like.
[0017] A polymerase chain reaction (PCR) amplification method may be used to
amplify at least one nucleic acid of at least one SRB in the presence of at
least one
primer to form an amplification product. This method of amplification,
optional
detection and optional quantification of SRBs present in a particular sample
is much
quicker than previous methods of detecting SRBs. For example, the PCR
amplification methods described below may occur in an amount of time less than

about 7 calendar days, alternatively less than 2 calendar days, or less than
24 hours
in another non-limiting embodiment. In yet another non-limiting embodiment,
the
PCR amplification methods may occur in less than 8 hours.
[0018] In an alternative embodiment, the method of amplification, optional
detection and optional quantification may occur in an amount of time less than
about
a 7 calendar days, alternatively less than 2 calendar days, or less than 24
hours in
another non-limiting embodiment. In yet another non-limiting embodiment, the
PCR
CA 3032480 2019-02-01

6
amplification, optional detection and optional quantification methods may
occur in
less than 8 hours.
[0019] 'Amplification' as defined herein refers to any in vitro method for
increasing
the number of copies of a nucleotide sequence with the use of a DNA
polymerase,
such as a PCR method of amplification in a non-limiting embodiment. PCR
amplification methods may include from about 10 cycles independently to about
50
cycles of denaturization and synthesis of a DNA molecule.
[0020] Prior to amplifying the nucleic acid(s) of the SRBs, the nucleic acids
must
first be extracted from a sample. The sample may be in any form necessary to
obtain the SRB, such as a fluid sample containing the SRB, a ground-up version
of a
field sample where it would be beneficial to determine whether the SRB are
present
in the tissue, and the like. In an alternative embodiment, a surface and/or
surface
solids suspected of having SRB contamination may be swabbed, and the swab may
be placed in a fluid to obtain the SRB fluid sample. Non-limiting examples of
a
sample may be a food product, an animal tissue, a human tissue, a water
sample, a
lab surface, a metal surface, a paper mill industry surface, a wastewater
within a
wastewater treatment facility, a sample from the paint industry, and
combinations
thereof.
[0021] The nucleic acid may be or include, DNA, RNA (e.g. mRNA), and
combinations thereof. The nucleic acid(s) from the SRB within the sample may
be
extracted from the sample prior to amplifying the nucleic acid(s). Such
extraction
techniques of the nucleic acids from the sample may be carried out by standard

techniques, which are well known to persons skilled in the art.
[0022] A non-limiting example of an extraction technique may be or include
using
the QIAamp Tissue Kit (QIAGEN, Hilden, Germany), the MP Bio Soil DNA kit, and
the like. DNA from the SRBs may be extracted from a sample using the QIAamp
Tissue Kit as is well known in the art.
[0023] Once the nucleic acid(s) are extracted, the nucleic acid(s) may be
combined with at least one primer in a reaction well to start and/or improve
the
amplification of the nucleic acids using a PCR method. The primer(s) may be or

include a sequence including, but not necessarily limited to, SEQ ID NO:1
through
SEQ ID:9 (FIGs. 1-9), and mixtures thereof. The primer(s) may be specific for
amplification of at least a fragment of an alpha subunit of an APS reductase
gene.
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7
Alternatively, the primer(s) may include an oligonucleotide from the alpha
subunit of
the APS reductase gene.
[0024] APS reductase (also known as Adenylylsulfate Reductase) allows the
reduction of adenosine phosphosulfate (APS ¨ a product of the activation of
sulfate
by ATP sulfurylase). APS reductase is a cytoplasmic enzyme containing two
subunits (alpha and beta) known to be involved only in the anaerobic
respiration of
sulfate. This enzyme may not be present in non-sulfate-reducing organisms,
since it
is not involved in the assimilatory reduction that allows the incorporation of
sulfur into
various molecules necessary for life, such as amino acids and vitamins.
Therefore,
detecting fragments of the gene(s) that may code for APS reductase may allow
for
the detection of a SRB.
[0025] 'Primer' as defined herein refers to a single-stranded oligonucleotide
that is
extended by covalent bonding of nucleotide monomers during amplification or
polymerization of a nucleic acid molecule. 'Oligonucleotide' as defined herein
refers
to a synthetic or natural molecule comprising a covalently linked sequence of
nucleotides that are joined by a phosphodiester bond between the 3' position
of the
pentose of one nucleotide and the 5' position of the pentose of the adjacent
nucleotide.
[0026] The components for a PCR method of amplification must be added to a
reaction well prior to performing the PCR method of amplification. In a non-
limiting
embodiment, the components may include the forward primer (also known as a
sense primer), the reverse primer (also known as an antisense primer), PCR
buffer,
dNTP, DNA, water, and combinations thereof. The amounts of the components
within a reaction well are very well known to those skilled in the art, and
the
components within the reaction well may vary depending on the amounts of the
other components present.
[0027] dNTPs are deoxynucleotide triphosphates included in a solution for
purposes of PCR amplification. Stock dNTP solutions may have a pH of about 7,
and the stability of dNTPs during repeated cycles of PCR may leave about 50%
of
the dNTPs remaining after about 50 PCR cycles. The concentration of each of
the
four dNTPs in solution ranges from about 20 pM to about 200 pM.
[0028] PCR methods of amplification require specific conditions of temperature

and reaction time. In one non-limiting embodiment there may be present
additional
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8
agents and/or reagents which may be useful for the fragment of the gene for
the
alpha subunit of APS reductase, to which the primers (mentioned previously)
have
hybridized, to be copied identically. Such conditions are well known to those
skilled
in the art. An average PCR program runs about 30 to about 65 cycles, but more
or
less cycles may be used depending on the conditions of the DNA, desired number
of
amplification products, time constraints, etc.
[0029] Computer processing may be used to analyze the crude amplification
products. The PCR program mentioned above is strictly a non-limiting example
and
should not be deemed to limit the invention here.
[0030] In another non-restrictive version of the PCR amplification method, an
internal amplification control can be helpful to prevent an ambiguous
interpretation of
results. In a non-limiting instance, an absence of amplification by PCR may be
due
to a difficulty including, but not necessarily limited to, to problems of
inhibition of the
reaction, or to the absence of a target, that is, the absence of DNA from the
SRB.
[0031] In another non-limiting embodiment, the amplification of one or more
fragments of the APS reductase gene can permit the detection of the fragment
of
the APS reductase gene, including, but not necessarily limited to the gene for
the
alpha subunit of the APS reductase. Optionally, gene amplification products
can be
subjected to hybridization with a probe specific for a fragment of the gene
for the
alpha subunit of the APS reductase where the probe may be labeled in a
detectable
way, including but not necessarily limited to fluorescent labeling,
radioactive labeling,
chemiluminescent labeling, enzymatic labeling, and combinations thereof.
'Gene' is
defined herein to mean a DNA sequence containing information required for
expression of a polypeptide or protein.
[0032] Hybridizing the amplification product with a probe also requires
particular
conditions of temperature, reaction time, and preventing the hybridization of
the
oligonucleotide with sequences other than the gene for the alpha subunit of
APS
reductase. In a non-limiting example, the hybridization temperature may range
from
about 55 C to about 65 C. The reaction time for the hybridization may range
from
about 0 seconds independently to about 60 seconds. As used herein with respect
to
a range, "independently" means that any threshold may be used together with
another threshold to give a suitable alternative range.
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9
[0033] The probe is a fragment of DNA used to detect the presence of
nucleotide
sequences that are complementary to the sequence in the probe. The probe
hybridizes to a single-stranded nucleic acid, whose base sequence allows probe-

target base pairing due to complementarity between the probe and the target
(e.g.
single-stranded DNA from the SRB). First, the probe may be denatured (by
heating
or under alkaline conditions, such as exposure to sodium hydroxide) into
single
stranded DNA (ssDNA) and then hybridized to the target ssDNA. The
hybridization
may occur when the target ssDNA and probe are immobilized on a membrane (e.g.
a gel) or in situ. 'Target' as used herein refers to DNA of the SRB.
[0034] A presence of hybridization and a degree of hybridization may be
detected.
The presence of hybridization may indicate the presence of the SRB, and the
degree of hybridization may enumerate the SRB.
[0035] In a non-limiting embodiment, the method may be performed by
amplifying at least one nucleic acid of at least one SRB in the presence of at
least one primer to form an amplification product where the nucleic acid(s)
are extracted from a sample prior to amplifying the nucleic acid(s). The
primer(s) may include an oligonucleotide having a nucleotide sequence
including, but not necessarily limited to, SEQ ID NO:1, SEQ ID NO: 2,
SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:
7, SEQ ID NO: 8, SEQ ID NO: 9õ and mixtures thereof;
optionally hybridizing the amplification product with a probe having a
nucleotide sequence; and
optionally detecting the hybridization complex formed between the product of
amplification and the probe to indicate the presence of SRB in the sample.
[0036] The type of sulfur-species bacteria that may be detected by the methods

may be or include, but are not limited to, Desulfovibrio vulgaris,
Desulfovibrio
desulfuricans, Desulfovibrio aespoeensis, Thermodesulfobium narugense,
Desulfotomaculum carboxydivorans, Desulfotomaculum ruminis, Desulfovibrio
africanus, Desulfovibrio hydrothermalis, Desulfovibrio piezophilus,
Desulfobacterium
corrodens, Sulfate-reducing bacterium QLNR1, Desulfobacterium catecholicum,
Desulfobacterium catecholicum, Desulfobulbus marinus, Desulfobulbus,
Desulfobulbus propionicus, Desulfocapsa thiozymo genes, Desulfocapsa
sulfexigens,
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10
Desulforhopalus vacuolatus, Desulforhopalus, Desulfofustis glycolicus strain,
Desulforhopalus sin gaporensis, Desulfobacterium, Desulfobacterium zeppelinfi
strain, Desulfobacterium auto trophicum, Desulfobacula phenolica,
Desulfobacula
toluolica To12, Sulfate-reducing bacterium JHAl, Desulfospira joergensenii,
Desulfobacter, Desulfobacter postgatei, Desulfotignum, Desulfotignum balticum,

Desulforegula conservatrix, Desulfocella, Desulfobotulus sapovorans,
Desulfofrigus,
Desulfonema magnum, Desulfonema limicola, Desulfobacterium indolicum,
Desulfosarcina variabilis, Desulfatibacillum, Desulfococcus multivorans,
Desulfococcus, Desulfonema ishimotonii, Desulfococcus oleovorans Hxd3,
Desulfococcus niacini, Desulfotomaculum, Desulfotomaculum nigrificans,
Desulfotomaculum ruminis, Desulfotomaculum halophilum, Desulfotomaculum
acetoxidans, Desulfotomaculum gibsoniae, Desulfotomaculum sapomandens strain,
Desulfotomaculum thermosapovorans, Desulfotomaculum geothermicum,
Desulfosporosinus meridiei, Delta proteobacterium, Thermodesulforhabdus
norvegica, Desulfacinum infemum, Desulfacinum hydrothermale, Desulforhabdus
amnigena, Desulforhabdus, Desulforhabdus, Desulfomonile tiedjei, Desulfarculus

baarsii, Desulfobacterium anilini, Delta proteobacterium, Desulfovibrio pro
fundus
strain, Desulfomicrobium baculatum, Desulfocaldus hobo, Desulfovibrio,
Desulfovibrio piger, Desulfovibrio ferrophilus, Desulfonatronovibrio
hydrogenovorans, Desulfovibrio, Desulfovibrio acrylicus, Desulfovibrio
salexigens,
Desulfovibrio oxyclinae, Desulfonauticus submarinus, Desulfothermus naphthae,
Thermodesulfobacterium, Thermodesulfobacterium hveragerdense,
The thermophilum, Thermodesulfatator indicus,
Thermodesulfovibrio yellowstonii, Desulfosporosinus orientis, Desulfotomaculum

thermobenzoicum, Desulfotomaculum solfataricum, Desulfotomaculum luciae
strain,
Desulfobacca acetoxidans, Desulfovibrio vulgaris, Desulfovibrio desulfuricans,

Desulfovibrio alaskensis, Desulfovibrio magneticus, Desulfosporosinus
acidiphilus,
Desulfotomaculum kuznetsovii, Desulfovibrio sulfodismutans, Desulfomicrobium
baculatum, Desulfonatronum lacustre, Desulfohalobium retbaense,
Desulfonauticus
autotrophicus, Thermodesulfobacterium commune, Thermodesulfobacterium
hveragerdense, Thermodesulfovibrio islandicus, Thermodesulfovibrio,
Thermodesulfobacterium, Desulfotomaculum thermobenzoicum, Desulfotomaculum
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11
thermoacetoxidans, Desulfotomaculum thermocistemum, Desulfotomaculum
australicum, Desulfotomaculum kuznetsovii, Desulfovibrio desulfuricans,
Desulfovibrio alaskensis, Desulfovibrio vulgaris, Desulfovibrio salexigens,
Desulfosporosinus acid/phi/us, Desulfosporosinus meridiei, Desulfosporosinus
orientis, Desulfotomaculum reducens, and combinations thereof.
[0037] In the foregoing specification, the invention has been described with
reference to specific embodiments thereof, and has been described as effective
in
providing methods and compositions for PCR amplification methods, and primers
and/or probes useful therefor. However, it will be evident that various
modifications
and changes can be made thereto without departing from the broader scope of
the
invention as set forth in the appended claims. Accordingly, the specification
is to be
regarded in an illustrative rather than a restrictive sense. For example,
specific
samples, nucleic acids, forward primers, reverse primers, probes, PCR cycles,
SRB,
internal controls (plasmids), and the like falling within the claimed
parameters, but
not specifically identified or tried in a particular composition or method,
are expected
to be within the scope of this invention.
[0038] The present invention may suitably comprise, consist or consist
essentially
of the elements disclosed and may be practiced in the absence of an element
not
disclosed. For instance, the PCR amplification method may consist of or
consist
essentially of amplifying at least one nucleic acid of at least one SRB in the
presence of at least one primer to form an amplification product; wherein the
at least
one SRB is extracted from an oilfield fluid or a solid prior to amplifying the
at least
one nucleic acid; the nucleic acid(s) is extracted from a sample prior to
amplifying
the nucleic acid(s); the primer(s) may include a nucleotide sequence of SEQ ID

NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,
SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and mixtures thereof. Oilfield
fluids
are defined herein to include, but not necessarily be limited to, crude oil
and other
fluids produced from subterranean formations, including produced waters,
oilfield
waters, production fluids, fracturing fluids, drilling fluids, completion
fluids, workover
fluids, packer fluids, gas fluids, and refinery fluids including processed
crude oil,
refined products, process and waste water, midstream fluids, downstream
fluids, and
the like.
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12
[0039] The method of determining an amount of SRB within an oilfield fluid may

consist of or consist essentially of amplifying at least one nucleic acid of
at least one
SRB in the presence of at least one primer to form an amplification product;
wherein
the amplifying occurs by a PCR amplification method wherein the at least one
nucleic acid is extracted from the oilfield fluid prior to amplifying the at
least one
nucleic acid; wherein the at least one primer comprises a sequence selected
from
the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and
mixtures thereof; hybridizing the amplification product with a probe specific
for a
fragment of an alpha subunit of an APS gene; and detecting a presence of
hybridization and a degree of hybridization; wherein the presence of
hybridization
indicates the presence of the at least one SRB; and wherein the degree of
hybridization enumerates the at least one SRB; and determining an amount of
SRB
in the oilfield fluid.
[0040] The method of decreasing SRB in oilfield fluids may consist of or
consist
essentially of adding an amount of a antimicrobial agent to an oilfield fluid
having an
amount of at least one SRB within an oilfield fluid; wherein the amount of the
at least
one SRB is determined by amplifying at least one nucleic acid of the at least
one
SRB in the presence of at least one primer to form an amplification product;
hybridizing the amplification product with a probe specific for a fragment of
an alpha
subunit of an APS gene; detecting a presence of hybridization and a degree of
hybridization; and decreasing the amount of SRB by killing and/or deactivating
the
bacteria where the altered oilfield fluid comprises a decreased amount of SRB
as
compared to the oilfield fluid.
[0041] As used herein, the terms "comprising," "including," "containing,"
"characterized by," and grammatical equivalents thereof are inclusive or open-
ended
terms that do not exclude additional, unrecited elements or method acts, but
also
include the more restrictive terms "consisting of" and "consisting essentially
of" and
grammatical equivalents thereof. As used herein, the term "may" with respect
to a
material, structure, feature or method act indicates that such is contemplated
for use
in implementation of an embodiment of the disclosure and such term is used in
preference to the more restrictive term "is" so as to avoid any implication
that other,
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13
compatible materials, structures, features and methods usable in combination
therewith should or must be, excluded.
[0042] As used herein, the singular forms "a," "an," and "the" are intended to

include the plural forms as well, unless the context clearly indicates
otherwise.
[0043] As used herein, the term "and/or" includes any and all combinations of
one
or more of the associated listed items.
[0044] As used herein, relational terms, such as "first," "second," "top,"
"bottom,"
"upper," "lower," "over," "under," etc., are used for clarity and convenience
in
understanding the disclosure and accompanying drawings and do not connote or
depend on any specific preference, orientation, or order, except where the
context
clearly indicates otherwise.
[0045] As used herein, the term "substantially" in reference to a given
parameter,
property, or condition means and includes to a degree that one of ordinary
skill in the
art would understand that the given parameter, property, or condition is met
with a
degree of variance, such as within acceptable manufacturing tolerances. By way
of
example, depending on the particular parameter, property, or condition that is

substantially met, the parameter, property, or condition may be at least 90.0%
met,
at least 95.0% met, at least 99.0% met, or even at least 99.9% met.
[0046] As used herein, the term "about" in reference to a given parameter is
inclusive of the stated value and has the meaning dictated by the context
(e.g., it
includes the degree of error associated with measurement of the given
parameter).
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Representative Drawing