CA2673577A1 - Detection of corrosion-inducing prokaryotes - Google Patents

Abstract

A simple method for the collection of field samples for nucleic acid extraction and subsequential detection of microorganisms from various media such as aqueous samples, sediments and biofilms (sessile bacteria). This method is a quantitative polymerase chain reaction (PCR)-based technique for detection of SRP in various media (i.e., aqueous samples, sediments, biofilms). The principle of this technique is based on the fact that all prokaryotes (Bacteria and Archaea) able to carry out sulfate reduction possess a gene that encodes dissimilatory sulfite reductase (DSR), the key enzyme in the sulfate reduction pathway. Microbial induced corrosion is due to bacterial growth and thus, tests for the detection of bacterial growth in fluids involved in oil processing are routinely performed. Thus far, the widely accepted methods for detection of bacterial growth of microorganisms involved in corrosion are cultivation dependent and, therefore, these methods underestimate the number of microorganisms present in a sample and require long incubation times.

Description

DETECTION OF CORROSION-INDUCING PROKARYOTES
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER LOUISIANA
STATE SPONSORED RESEARCH
This research was supported in parts by Louisiana Board of Regents Research and Development program contract No. LEQSF(2003-05)-RD-A-3 1.

BACKGROUND
100011 This application claims priority to U.S. Provisional Patent Application Serial Number 60/852,227, filed on October 17, 2006, entitled QUANTIFICATION
OF
SULFATE REDUCING PROKARYOTES IN ENVIRONMENTAL SAMPLES BY USE
OF REAL-TIME PCR ASSAYS, the entire content of which is hereby incorporated by reference. This application also claims priority to U.S. Provisional Patent Application Serial Number 60/925,435, filed on April 20, 2007, entitled DETECTION OF
CORROSION-INDUCING PROKARYOTES, the entire content of which is hereby incorporated by reference.

[00021 The present invention pertains the detection of sulfate reducing prokaryotes (SRPs) in industrial water usage applications. Detection of these organisms enables the treatment of water sources with biocide, which may prevent corrosion or toxicity. A preferred embodiment of the invention comprises a method for determining the presence or absence of SRPs in a water sample by detecting the presence of disimilatory sulfate reductase gene in the nucleic acids present in the sample using a polymerase chain reaction-based assay.

100031 Biofouling and microbiologically induced corrosion (MIC) are of a great concern in many industrial processes using water or water suspensions.
For example, the monitoring of growth and presence of suspended (planktonic) as well as sessile microorganisms is frequently carried out as part of control of the quality of oil pipelines and waters utilized for fracturing. Microorganisms involved in biofouling and' corrosion are widespread in both oxic and anoxic aquatic and terrestrial environn)ents and the presence of microorganisms on a metal surface, as well as their metabolic activities, can cause corrosion and dramatically affect the efficiency of industrial processes associated with water consumption or usage. Sulfate reducing prokaryotes (SRPs) are an important group of microorganisms widespread in anoxic aquatic and terrestrial environments and are responsible for most of anaerobic degradation of organic matter. The presence of SRPs on a metal surface, as well as their metabolic activities, can cause corrosion and can dramatically affect the efficiency of oil production. As has been shown by pipeline ruptures in the past, microorganisms may contribute to corrosion of metal equipment used in oil production and transportation. The monitoring of microorganisms, which mediate biofouling and corrosion, allow the industry to reduce costs by preventing corrosion and dramatically decrease chances of enviromnental contamination from corroded pipelines.
Thus, the quantification of SRP project has indirect environmental benefits as well.

Microbiologically Induced Corrosion 100041 To prevent the corrosion of the pipelines and equipment caused by the microorganisms, the microorganisms must be eliminated. This is usually accomplished by adding antimicrobial agent or agents to the well fluids. Since the amount of microorganisms of interest present in the oil field system is an unknown quantity, in order to eliminate the microorganism of interest as completely as possible, often times, an excess amount of antimicrobial agent or agents is used to insure results. The use of excess antimicrobial agent or agents is wasteful, costly, and detrimental to the environment.
There is a need for a method that can more accurately estimate the actual amount of the microorganism of interest present in the system, thus an estimate can be made as to the amount of antimicrobial agent or agents that are needed to eliminate the microorganism, and hence avoid the waste of using excess antimicrobial agent or agents.

[0005] As a part of water quality tests, the activity, presence or growth of microorganisms involved in biofouling and/or corrosion are frequently monitored.
Biofouling and corrosion are produced as result of a synergistic relationship among microbial (mostly bacterial) communities that fonn biofilms. Components of these biofilms are bacteria such as acid producing and/or sulfate reducing bacteria, which are the most common causes for biofouling and corrosion. They thrive in water trapped in stagnant areas, such as dead legs of piping, causing pitting and crevice corrosion. There are two habitats that have to be considered when quantifying/detecting microorganisms:
free-living bacteria and attached (sessile) bacteria. Free-living bacteria (planktonic) involved in biofouling/biocorrosion if they survive, will ultimately form a biofilm.

[0006] Organisms fxom industrial water comprise a variety of types of organism, including prokaryotes, such as sulfate reducing prokaryotes. These organisms include sessile bacteria, planktonic bacteria, and a number of other microorganisms, including sulfate reducing bacteria.

[0007] All of the widely accepted or standard methods for detection of bacterial growth of microorganisms have a number of pitfalls associated with them. Most probable number (MPN) techniques as per NACE standard TM0195-2004, Item No.
21224, are most commonly used techniques for quantification of corrosion-inducing bacteria. However, since most bacteria in the environment are non-culturable, these techniques dramatically underestimate the number of microorganisms present in a sample.
The techniques requires long incubation times, up to 29 days, which prevents industry managers from using quick measures to mitigate the corrosion. Each specific MPN
technique has further drawbacks such as detection of non-specific bacteria. In case of MPN of sulfate reducing bacteria (SRP), the presence of Shewanella and Geobacteriaceae species may lead to false positive results. Tests for quantification of a total bacterial activity, such as ATP photometry (Sharp and Read, 1983) does not target specific groups of bacteria involved in biofouling and corrosion (such as acid producers or sulfate reducing bacteria). In addition, ATP measurements are difficult to interpret in terms of the number of bacteria present, since the ATP concentration in each individual cell of each bacterial species may vary substantially. There are methods that target specific groups of biocorrosive bacteria such as SRBs. Radiolabeled sulfate respirometry can be used to determine SRB growth rates (Hardy and Syrett, 1983; Rosser and Hamilton, 1983). Major disadvantages of this technique is the use of 34S labeled sulfate, a radioactive compound, which entails application for a radioactivity license, maintaining a specialized facility, training and monitoring health of personnel involved, cost associated with disposal of radioactivity, etc. Other methods for SRB detection are based on measurement of adenosylphosphosulfate (APS)-reductase activity (Gawel et al., 1991; Kremer et al., 1988). Although this biochemical test is very simple and explicitly quantitative, the presence of sediment particles, proteinases and numerous enzyme inhibitors renders it virtually useless. In addition, it has recently been shown that APS-reductase is not limited to only SRP but it is also found non-biocorrosive bacteria such as sulfur bacteria (Blazejak et al., 2006). Thus, development and application of novel culture-independent and explicitly quantitative technique for detection of biocorrosive microorganisms is long overdue. DNA-based detection techniques offer us such an opportunity.

[0008] The use of cultivation-independent, gene-based techniques has revealed a larger, not previously described microbial diversity, with a vast number of bacterial groups not previously cultured, whose physiologies remain unknown (e.g., Hugenholtz et al., 1998; Gray and Head, 2001; Buckley and Schmidt, 2002). A number of DNA-based techniques employing either quantitative DNA-DNA hybridization or PCR have been proposed for detection of microorganisms and their genes (e.g., Sahm et al., 1999;
Nedwell et al., 2004; LeLoup et al., 2004; Kondo et al., 2004). All these molecular techniques require isolation of DNA. Moreover, a DNA isolation protocol must be simple, reliable and highly reproducible in order to correlate PCR or DNA-DNA
hybridization measurements with real numbers of microorganisms present in environmental samples.
This necessity led us to development of a method for collection of microbial cells in the field and their preservation for later DNA extraction in the laboratory. We searched for the optimal conditions for transportation of samples without significant degradation of DNA
and for a best method of DNA extraction for downstream applications, such as PCR.

[0009] The DNA isolation method presented here has been tested to work equally well with aqueous, sediment and biofilms samples. Moreover, this method for collection of cells and nucleic acid extraction can be utilized with any other downstream technique for quantification of nucleic acids such as DNA-DNA hybridization techniques (Sahm et al., 1999; Nedwell et al., 2004), or various formats of quantitative PCR (i.e., competitive, MPN or real-time PCR; LeLoup et al., 2004; Kondo et al., 2004).
However, it was specifically designed for use with a quantitative real-time PCR assays presented here.

[0010] Earlier culture-independent methods for quantification of nucleic acids were corinected with DNA-DNA or RNA-DNA hybridization techniques (Sahm et al., 1999; Nedwell et al., 2004) but these methods are time consuming and low throughput and low sensitivity (Bustin et al., 2000), which made them unsuitable for detection of sequences present in environmental nucleic acid preparations at low levels.
Later, competitive PCR has also been successfully used for detection of nucleic acids in environmental samples (LeLoup et al., 2004; Kondo et al., 2004). Although this method is substantially more sensitive than the former one, it has several disadvantages. First, a special competitor DNA (or RNA) molecule should be created. In many cases, such competitor is very difficult to generate directly from the sequence to be measured and an unrelated sequence is modified to be recognized by amplification primers.
Second, this method is relatively more expensive and, most importantly, has a very low throughput.

[0011] Recently, real-time PCR, a PCR-based quanritative assay first described by Hollan et al. (1991) is the method of choice for quantification of nucleic acids (Heid et al., 1996; Bustin 2000; Peters et al., 2004). It requires very small quantities of nucleic acids (i.e., very sensitive) and provides great throughput capabilities.
Recent advances in real-time PCR thermocycler design, capabilities and software and careful choice of controls allow researchers to circumvent the drawbacks associated with real-time PCR
application to environmental samples. Real-time PCR combines the sensitivity of PCR
with real-time measurement of amplification and thus allows quantification of the original target concentration. Therefore, real-time PCR is the most sensitive and flexible method for the detection of nucleic acids to date.

[0012] Real-time PCR originally developed for clinical applications has recently been applied to several microbial ecology application (Paul et al., 1999; Wawrik et al. 2002, Suzuki et al. 2000; Gruntzig et al., 2001; Stults et al., 2001).
The work described here was directed at detection of the key metabolic gene participating in sulfate reduction using real-time PCR.

100131 Sulfate reducing prokaryotes (SRP) constitute a phylogenically heterogeneous group, which however share a common metabolic pathway. We developed a culture-independent method for quantification of SRP in environmental samples. The invention was created based on the knowledge that all microorganisms that carry out sulfate reduction posses a gene that encodes for the enzyme responsible in dissimilatory sulfite reduction. Two enzymes, adenosinemonophosphate sulfate (APS) reductase (Friedrich, 2002) and dissimilatory sulfite reductase (DSR; Klein et al., 2001), mediate dissimilative sulfate reduction. APS reductase reduces APS to sulfite and DSR
catalyzes the six-electron reduction of sulfite to sulfide and is required by all SRPs.
Both genes have been targeted for studies of the diversity of SRPs (e.g., Friedrich, 2002;
Klein et al., 2001).
However, the interpretation of phylogenetic data generated using genes for these two enzymes is complicated due to numerous lateral gene transfer events. Klein and colleagues (2001) have investigated and identified the likely transfers of the dsr gene among different lineages of bacteria and archaea aiid, therefore, genes for DSR are most commonly used to describe phylogenetic diversity of sulfate-reducing prokaryotes (Chang et al, 2001;
Fishbain et a., 2003; Fukuba et al., 2003; Joulian et al., 2001; Minz et al., 1999;
Nakagawa, et al., 2004; Madrid et al., 2006). Therefore, the best molecular marker for SRP study has been the dsrAB genes, which encode for dissimilatory sulfite reductase, the key enzyme in dissimilatory sulfite reduction (Wagner et al., 1998, Klein et al., 2001;
Zvelov et al., 2005).We developed a real-time PCR-based approach to quantify gene numbers dsrAB genes encoding for dissimilatory (bi)-sulfite reductase as a proxy for sulfate reduction in waters used for oil drilling.

Real-time PCR assays [0014] A number of DNA extraction techniques are available for use in the field. A preferred DNA extraction technique would a) be highly reproducible;
b) must yield intact un-sheared DNA; c) allow for polymerase chain reaction (PCR) to be carried out on extracted DNA with no detectable non-specific products; and d) reagents used for DNA extraction and environmental contaminants, in particular, co-extracted with DNA
must not inhibit the PCR reaction.

[0015} Real-time PCR assays are a robust highly sensitive and reproducible tool for gene quantification as previously suggested by Gibson (1996), Bustin (2000), Liss (2002). However, before this real-time PCR technique can successfully be applied for nucleic acids isolated from environmental samples, several issues have to be carefully addressed. One of the major problems encountered is inhibition of real-time PCR by PCR
inhibitors (most commonly humic and fulvic acids and iron ions). Stults et al.
(2001) suggested autofluorescence and signal quenching as two other probable problems. Unless extremely high, autofluoresence should pose no problems. The background autofluorescence is typically monitored during the progression of a real-time PCR
reaction. As long as the background autofluorescence is low enough to select a cut-off value for C, quantification, autofluorescence does not affect quantification.
Quenching and inhibition, although different mechanistically, lead to detection of gene copy numbers lower than is really present in a sample. Two types of controls were incorporated in reported herein assays to address the quenching and inhibition problems:
serial dilution of samples and spiking of a sample with known amounts of standards.

[0016] Several methods previously used to study gene expression, including slot- or spot-dot hybridization and competitive RT-PCR have drawbacks, such a requirement for high quantities of RNA or low sample throughput capabilities for the latter (Ferr6, 1998). Real-time PCR surpass the biases associated with end-point amplicon detection, which is used in competitive PCR. It is very sensitive, fast and produces no radioactive waste.

[0017] An additional problem associated with end-point PCR is that, in order to be quantitative, the detection has to be carried out during exponential amplification (Morrison et al., 1994). The exponential amplification phase can be achieved after a different number of cycles for different target templates and even for samples with the same target. In real-time PCR analysis, the application of the threshold cycle C, method (Heid et al., 1996) allows for calibration in a wide dynamic range in which the number of cycles can be adjusted to the signal size. The threshold cycle is defined as the PCR cycle at which a fluorescence signal, developed by a dye-DNA complex or a free dye (depending on the fon:nat) passes a preset value. This value corresponds to an amount of amplicons generated in a few cycles if a large member of templates was present initially, or after many cycles if the PCR started with few templates. Quantification is based on the number of cycles required to reach a certain concentration of amplicons rather than on the concentration reached after a fixed number of cycles. In real-time PCR, accumulation of PCR products can be monitored using a fluorescent dye, such as SYBR Green (Higuchi et al., 1992; Wittner et al., 1997), that forms fluorescent adducts with double-stranded DNA
without compromising the polymerization reaction. The reaction can be calibrated by amplification of known amounts (gene copy number) of the target sequence and by monitoring the increase in fluorescence cycle by cycle (real-time monitoring of PCR).

[0018] Dissimilatory sulfate reductase is the key enzyme for the dissimilative sulfate reduction pathway. A high diversity of prokaryotes carrying the dsrAB
genes in mobile sediment microbial communities suggests the active participation of sulfur cycle reactions during C,g remineralization in Fe(III) rich coastal deposits (Madrid et al. 2006).
Therefore, the dsr genes appear to be a useful genetic marker to study the sulfate reduction activity in many environments. Several studies on quantification of dsrAB
genes in sediments have been described in the literature and include the work by Leloup et al (2004) and Kondo et al (2004) who used competitive PCR and by Nedwell et al.
(2004) who used slot-blot hybridization. PCR and RT-PCR is a useful method for quantifying genes. Using dsr sequence data for the sulfate reducing prokaryotes (SRPs) in mobile sediments (Madrid et al., 2006) we managed to design a pair of universal primers, which can also amplify dsr sequences from representatives of major DSR families described by Zverlov et al. (2005).

[0019] Zhu et al., Appl. Environ. Microbiol. 69(9):5354-5363, 2003 describe the characterization of microbial communities in gas industry pipelines.

100201 Zhu et al., Corrosion Paper No 05493, 2005, describe the application of quantitative, real-time PCR in monitoring microbiologically-influenced corrosion (MIC) in gas pipelines.

[0021] Zverlov et al., J. Bacteriol. 187: 2203-2208, 2005, describe the lateral gene transfer of dissimilatory (bi)dulfite reductase.

[0022] Zhu et al., Conosion 62(11): 950-955, 2006, describe the rapid detection and quantification of microbes related to microbiologically influenced corrosion using quantitative polymerase reaction.

[0023] Leloup et al., Environmental Microbiology 9(4), 131-142, 2007, describe diversity and abundance of sulfate-reducing microorganisms in the sulfate and methane zones of a marine sediment, Black Sea.

[0024] However, these method use primers which do not cover all existing conserved areas of both DSR genes (i.e., dsrA and dsrB). The 465 bp amplicon, which Zhu et al. (2005, 2006) refer to, differs in size from all amplicons generated by primers described in the current application. This means that Zhu et al. (2005, 2006) primers are not capable of amplification of all existing sulfate-reducing bacteria.
Moreover, the primers used by Zhu et al. do not include the novel dsrAB sequences described by Zverlov et al. (2005), which also may belong to bacteria involved in corrosion. Since the real-time acsav bv Z.hu et al. detects only some and not all SRPs, it is not an accurate method for the detection of SRP involved in corrosion. Because of the improved primer design, the real-time PCR-based methods described in the current application detect all SRPs which would not be detected by the Zhu method.

[00251 Another aspect of the current invention is the size of the amplicon. In general, amplicons greater than 300 bp in length do not perform optimally.
This is the case with the QuantiTect SYBR PCR kit as well as other commercial qPCR kits or protocols. Larger amplicons "wear out" the enzyme (i.e., the probability that enzyme will not finish amplification of each single target molecule and disassociate prematurely from a large fraction of them becomes very high) decreasing the efficiency of the reaction, one of the key features of real-time SYBR detection. The Zhu publications described above quantified a 465 bp amplicon. With amplicons above >300 bp in length such as those described in the current application, efficiencies of PCR assays become too low (<0.9) to reliably use SYBRGreen-based assays for quantification. Although a calibration curve can be generated for low efficiencies and it may even appear that there is a linear relationship between concentration of amplified target and Ct values, in reality, there is no more a simple correlation between Ct value and concentration.

[0026] Yet another aspect of the current invention comprises a proper collection and transportation protocol. These methods are novel, and have not been presented in previous protocols such as Zhu et al. (2005, 2006). There can be many issues associated with sample collection, preservation and transportation, and certain manipulations such as centrifugation of samples and improper transport conditions (temperature, buffer, etc) can lead to underestimation of SRP numbers. Another important aspect of the invention is DNA extraction efficiency, which can be affected by the method in which biomass is collected from samples.

SUMMARY
[0027] A preferred embodiment of this invention pertains to methods for the appropriate collection of microbial cells for the efficient extraction of their nucleic acids from any water, sediments, and biofilm environmental samples. The present invention particularly relates to oil field systems, natural water sources, and sediments; the technique is applicable for all and any industrial process where biologically mediated corrosion is a concern.

[0028] The invention comprises a fast, simple, contamination-free method for the collection of microbial cells from field samples to be used for nucleic acid extraction.
Preferred embodiments utilize pre-existing filtration equipment and similar filtration techniques for sampling, which makes this method low-cost, fast and easy to carry out.

(0029] Favorable conditions (such as buffer, temperature, etc.) have been determined for the storage and transportation of samples to maximize nucleic acid concentrations. The current invention comprises a highly reproducible, high throughput and yield procedure for DNA isolation, to be used in conjunction with the above procedures for sample processing. DNA isolated using this procedure may be suitable for all any and type of molecular analysis (PCR, DNA-DNA hybridization, etc.).

[0030] In preferred embodiments of the invention, the presence of hydrocarbons in samples does not effect sample collection, preservation, storage and consequent DNA isolation efficiency. Preferred embodiments include a fast, accurate, highly sensitive real-time PCR technique using oligonucleotide sequences which is capable of amplifying DNA fragment specific for all known sulfate reducing prokaryotes.

[0031] Another preferred embodiment further comprises novel oligonucleotides for use as primers and probes for PCR assays for specific detection and enumeration of sulfate reducing prokaryotes. PCR assay methods utilizing these primers and probes are also provided as part of the invention.

[0032] A primary object of a preferred embodiment of the invention is the monitoring of SRP present in a sample.

[0033] Still another preferred embodiment of the present invention comprises extracting nucleic acids from an aliquot of material to be tested, transporting the nucleic acids, amplifying a gene of interest, quantifying the gene of interest to obtain a value, and using the value to determine the amount of a microorganism of interest in the aliquot.

BRIEF DESCRIPTION OF THE DRAWINGS
[0034] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0035] FIGURE 1 shows a typical calibration curve with a DNA standard (plasmid with a cloned copy of dsr gene) or chromosome of an SRP
(Desulfovibrio vulgaris in this case). It shows the sensitivity of approximately 5 copies of DSR gene.

[0036] FIGURE 2 shows a standard curve built using a sample with known gene copy number. Gene copy number of the sample is plotted versus the PCR
cycle at which the fluorescence value crosses the threshold line in a PCR using the sample as a template;

[0037] FIGURE 3 shows the correlation between samples tested using the MPN method versus samples tested using the qPCR method.

DETAILED DESCRIPTION OF PREFERRED EMBODIlVIENTS
[00381 The present invention relates to a procedure for the collection and processing of samples for nucleic acid extraction and consequent detection of SRPs in these samples. In a preferred embodiment, the invention includes optimal methods of collection, storage, and transporting of samples from the field to the laboratory. The technique can be adapted to oil field systems as well as natural water sources in solid or aqueous form (i.e. pits, lakes, creeks).

[00391 A preferred embodiment of the invention includes collection of a sample or aliquot in the field, separation of biomass from the sample, and transport of the sample to a testing facility. DNA of interest from the microorganism of interest present in the sample or aliquot is then extracted from the sample or aliquot at the testing facility, and amplified using specific primers in order to quantitate the concentration of sulfate reducing bacteria in the original sample.

[0040] Preferred embodiments of the invention may relate to the detection of organisms from industrial water, including prokaryotes, such as sulfate reducing prokaryotes. These organisms include sessile bacteria, planktonic bacteria, and a number of other microorganisms, including sulfate reducing bacteria.

Sample Collection [00411 In a preferred embodiment of the invention, a sample to be tested is isolated from well fluids with or without oil present. Samples may be collected in a range of volumes, preferably ranging between 250 ml and 3000 ml, although about 250 ml is suitable for most cases.

[0042] Bacteria from sample may be separated preferably by filtration or centrifugation, filtration, most preferably using a 0.1 M filter membrane. A
single sample may be divided and filtered using one or more filters, and various sizes of filters may be used, preferably from about 47 mm to about 25 mm membranes, of which 25 mm filters are best. In a preferred embodiment of the invention, filtration of the sample will occur within one hour of sample collection, most preferably within 5 minutes of sample collection.

[0043] Samples, preferably filter membranes containing biomass, may be transported in a buffer or without a buffer. The transport buffer may also include sodium molybdate, preferably at concentration of between 1 and 30 mM, to inhibit the growth of sulfate reducing bacteria.

[0044] Temperature during transport may range between about 0 C to about 22 C, preferably between about 4 C to about 37 C, most preferably below about 22 C.
Total transport time may be up to several weeks, preferably less than one week, most preferably less than about 2 days.

DNA Extraction [0045] In a preferred embodiment of the invention, DNA or other nucleic acids will be extracted from the samples. If samples are transported with filter membranes from the filtration step, the membranes may be shredded prior to DNA extraction to increase the efficiency of the extraction.

[0046] DNA may be extracted through one of several methods, preferably using the same buffer as is used for transportation. A suitable method is according to a commercially available kit, the PowerSoil Kit.

Real-time PCR

[0047] In a preferred embodiment of the invention, a marker gene will be detected in extracted DNA using a PCR method. Preferably, the marker gene will be related to the microorganism of interest, most preferably the gene will be DSR. A
preferred method of PCR would be real-time PCR, such as real-time PCR
monitored by a fluorescent dye.

Table 1. Primers and probes used for amplification of DSR genes.
Primer/probe SEQ ID NO
Primer/Probe Sequence (5'-3') name DsrUnivl577F 1 AAYATNRTNCAYACNCARGGNTR
DsrUniv1712R 2 TGNACNGCNCCRCACATRI"INRNRCARCANGC
DsrUniv 1 FM 3 ACSCAYTGGAARCAYGG
DsrUniv43F 4 RGNGGNGGNRTNRTYGGNMGNTA

DsrUniv225R 5 TCNCCNGTNGMNCCRTGNAWRTT
DsrUnivPF 6 GNGGNGGNRTNRTYGGNMGNTAYWNNGA
DsrUnivPR 7 TCNNWRTANCKNCCRAYNAYNCCNCCNC
DsrUniv43R 8 TANCKNCCRAYNAYNCCNCCNC
DsrUniv1712F 9 GCNTGYTGYNYNAAYATGTGYGGNGCNGTNCA
DsrUniv4RM 10 GTRTARCARTTNCCRCA
Where,N=AorGorTorCorI;Y=TorC;R=AorG;K=GorT;M=AorC;W=Aor T; S = C or G;

[0048] In a preferred embodiment of the invention, the PCR method will be carried out using primers directed to regions of a marker gene, most preferably primers directed to conserved regions of the DSR gene. Examples of preferred primers are listed in Table 1.

[0049] In a preferred embodiment of the invention, the PCR product could be quantities of DSR gene, and the resulting value correlated with SRB
concentration.

[0050] Well fluids with or without oil present, were collected from Comeaux, Lyons, and Caskid in Louisiana, USA. Samples of sediment and mixed water-sediment were collected from pits frequently used for fracturing (Amstrong, Falvel and Masingail pits in Texas) and natural environments such as Lake Fort Worth and Chapel Creek in Fort Worth, Texas, USA.

[0051] Non-processed samples and filters with microbial biomass collected by filtration within 5 minutes after sampling were transported at 4 C, 22 C and 37 C, immersed in one of Buffer 1 (20 mM Tris-HCI [pH 8.01 and 100 mM NaCl, 50 mM
EDTA), Buffer 2 (100 mM Tris-HCI [pH 8.0], 100 mM sodium EDTA [pH 8.0], 100 mM
sodium phosphate [pH 8.0], 1.5 M NaCI, 1% CTAB), and proteinase K final concentration of 1 mg/ml), Buffer 3 (from the Powersoil DNA Isolation Kit, MO BIO
Laboratories, Carlbad, CA) or without buffer (dry). Buffer composition is outlined in Table 2.
Transportation times of 1, 2 and 5 days were tested at each aforementioned temperature in the presence/absence of proteinase K.

[0052] Two approaches for collecting cells from samples in the laboratory were tested: (a) by centrifugation and (b) by filtration on 0.2 m polyvinylidene fluoride (PVDF) membranes. Three DNA isolation methods, one typically used for water column ("Method 1", referenced in Madrid et al., 2001; Xu and Tabita, 1996); a second used for sediment and soils ("Method 2", referenced 'in Zhu. et al., 1996) and a third with a commercially available kit ("Method 3", referenced in PowerSoilTA , Mo Bio, Carlsbad, CA) specially created for soil and sediment samples. Additional variables tested were the use of 47 mm or 25 mm membrane filters and the effect of shredding or not shredding the filters before DNA extraction.

[0053] For this series of experiments, three different buffers were tested.
Please see Table 2 for preparation of buffers.

[0054] Buffer I was the extraction buffer from Xu and Tabita (1996). Buffer 1 comprises 20 mM Tris-HCI [pH 8.0] and 100 mM NaCI, 50 mM EDTA, and was used in Method 1 [00551 Buffer 2 was the extraction buffer from Zhu et al. (1996). Buffer 2 comprises 100 mM Tris-HCI [pH 8.0], 100 mM sodium EDTA [pH 8.0], 100 mM sodium phosphate [pH 8.0], 1.5 M NaCI, 1% CTAB), and proteinase K final concentration of 1 mg/ml, and was used in Method 2.

[0056] Buffer 3 is a commercially available buffer, consisting of the 750 l bead solution and 60 l of C 1 solution included in the PowerSoilT"' DNA
isolation kit MO
BIO Laboratories, Carlbad, CA, and was used in Method 3.

[00571 Each buffer was also tested for its ability to protect chromosomal DNA
in the presence/absence of proteinase K (0.5 mg ml-1).

Table 2. Preparation of Extraction Buffer and medium ATCC 1249 Buffer 1 20 mM Tris-HCl [pH Xu and Tabita, Used in DNA
8.0] and 100 mM 1996 Extraction 1 NaCl, 50 mM EDTA
Buffer 2 100 mM Tris-HCI [pH Zhu et al., 1996 Used in DNA
8.0], 100 mM sodium Extraction 2 EDTA H 8.0], 100 mM sodium phosphate [pH 8.0], 1.5 M NaCI, 1% CTAB, proteinase K final concentration oflm ml Buffer 3 Powersoil Used in DNA
Extraction 3 Minimum Sampling Volume [0058] Sample volume was evaluated to determine the minimum volume of filtered sample required to obtain sufficient yields of DNA (Table 3). This volume is highly variable and it depends on the nature of the sample. The largest volumes can be filtered from samples that appear clear, whereas samples that contain even small traces of sediment or particles will clog the membranes more quickly. However, the later samples usually contain higher numbers of bacteria. For some natural samples (i.e., ponds, rivers, groundwater) with high numbers of particles (high to very high turbidity), I
ml of sample was enough to isolate quantities of DNA sufficient for downstream analysis (i.e., PCR).
For natural samples with traces of particles (low turbidity), 50 ml was sufficient and for non-turbid, clear samples as little as 200 ml was sufficient. However, for oil-well water there is no correlation between particle and microbial load, at least 250 ml volume is recommended. It was also concluded that collection of cells on 47 mm membranes is better, because larger volumes can be filtered through them before clogging.
If after filtration, membranes were overloaded with material, membranes were cut in two and placed the replicates into two tubes containing beads and the bead solution with the solution CI from the PowerSoil kit.

Table 3. Summary of different volume collected for various samples.

Sample Type of environmental DNA yield ( g em"3 sample) medium 30 cc 60 cc 100cc 200cc Lake Fort Worth Clear freshwater water 0.290 0.489 0.910 1.634 Chapel creek Freshwater with suspended 1.135 2.066 4.532 8.461 sediment Comeaux Water from oil pipeline 0.546 1.123 2.124 3.981 Lyons Water from oil pipeline 0.083 0.132 0.287 0.545 Catskid Water from oil pipeline 0.036 0.072 0.147 0.303 Preservation During Transportation [0059] Transportation time and storage temperatures were emulated in the laboratory. Samples shown in the Table 4a were collected and delivered to the laboratory within 5 minutes (Cypress Lake) to 2 hours on ice (Lyons and Comeaux). Results indicate that the best method of preserving DNA during transit is by immersing membrane filters with cells generated by filtration in the field in the PowerSoilTM kit commercial buffer and maintaining it at or below room temperature (not higher than 22 C) for as few days as possible. Temperature effects on DNA quality and yields are observed if samples are not properly handled. Two sets of samples were incubated at various temperatures including room temperature (22 C) and 37 C in the commercial buffer (Buffer 3). After 2 days of incubation at these two temperatures, DNA incubated at 37 C began to show signs of degradation, whereas DNA stored at 22 C remained intact. Thus, if temperature cannot be controlled during transportation, the delivery of the sample must take place within 2 days.
The presence/absence of proteinase K did not affect the quality and yield of isolated DNA
when using Buffer 3 (from the PowerSoilTM kit).

Table 4a. DNA extraction yields from samples incubated at different temperatures Total DNA extracted ( g em"3 sample) Environmental Sample medium 1 day 2 days 5 days Cypress Lake sediment 1.993 1.896 1.966 1.915 1.943 0.472 Lyons water from oil 5.178 5.171 5.174 5.167 5.173 0.904 pipeline Comeaux water from oil 1.724 1.708 1.717 1.704 1.715 0.341 pipeline D. vulgaris culture Liquid culture 5.482 5.478 5.479 5.069 5.441 2.891 D. ruminis culture Liquid culture 4.711 4.638 4.684 4.384 4.530 2.797 [0060] The delivery of dry filters from the field was also dismissed since even under other optimal conditions (i.e., time, temperature and DNA isolation methods) DNA
yield was lower than if the filters were transported immersed in a buffer (Table 4b).

Table 4b. DNA yields for membranes transported not immersed in buffer as well as immersed.

Sample DNA yield (pg cm" sample) No buffer In buffer Swamp 3.456 7.891 Comeaux 2.983 6.789 Lyons 2.314 5.912 Catskid 1.231 4.332 [0061] Results indicate that DNA is well-preserved during transit by immersing membrane filters with cells generated by filtration in the field in the PowerSoilTM kit commercial buffer and maintaining it at or below room temperature (not higher than 22 C) for as few days as possible. Temperature effects on DNA
quality and yields are observed if samples were not properly handled. Two sets of samples were incubated at various temperatures as indicated in "Methods and Materials"
including room temperature and 37 C in the commercial buffer (buffer 3). After 2 days of incubation at these two temperatures, DNA incubated at 37 C began to show signs of degradation, whereas DNA stored at 22 C remained intact. Thus, if temperature cannot be controlled during transportation, the delivery of the sample should take place within 2 days. The presence/absence of proteinase K did not affect the quality and yield of isolated DNA.

[0062] Incubation at 37 C overnight in Buffer 1 with and without proteinase K
was enough to degrade most of the DNA and, therefore, the DNA isolation method used in conjunction with Buffer 1 is not the most preferred. In addition, DNA yield obtained using Method 1 was lower than the yield obtained with Methods 2 and 3, which routinely yielded comparably high DNA quantities. For Method 2, the best way of preserving DNA
was in Buffer 2 in the presence of proteinase K. This preservation method worked well at each tested temperature. However, DNA extracted with Method 2 did not support PCR as robustly as other methods.

[0063] Filters which were delivered from the field dry yielded lower DNA than if the filters were transported in a buffer, even under other optimal conditions (i.e., time, temperature and DNA isolation methods).

Collection and handling of samples [0064] Similar volumes of samples were filtered in the field or delivered in tightly sealed glass bottles to the laboratory, transported on ice or at ambient (variable 25-35 C) temperatures. Bacterial cells from non-filtered samples were delivered to the laboratory and where either centrifuged or filtered. Centrifugation may potentially have some advantages over filtration, since a filter may become clogged preventing filtering of a large volume of a sample. Our results, however, indicate that even high speed centrifugation (10,000 x g) and long centrifugation times (30 min) are not optimal for collecting all bacterial cells. DNA could be extracted from these filters, as demonstrated in Table 5a.

Table 5a. Bacterial DNA remained in the sample following centrifugation at 10,000 x g for 30 minutes.

Sample DNA yield ( g cro sample) Supernatant Pellet Cypress Lake 1.734 2.541 Amstrong 3.113 4.523 Falvel 2.956 3.856 Masingail 3.283 4.345 D. vulgaris culture 1.345 3.941 D. ruminis culture 1.987 2.455 [00651 DNA was isolated from filters generated in the laboratory and in the field using the optimum procedure described in the above Examples. For every type of sample, filtration in the field guaranteed a higher yield of DNA than bringing and processing samples in the laboratory (see Table 5b).

Table 5b. DNA yields of liquid samples from oil pipelines filtered in the field and the laboratory at different transportation temperatures.

Sample DNA yield (Ftg cm'3 sample) Filtered in the field Filtered in laboratory Temperature at which samples 4 C ambient 4 C ambient were transported Comeaux 4.32 4.29 1.89 1.77 Lyons 3.45 3.42 1.78 1.62 [00661 DNA of filtered and pelleted samples was extracted using 3 different methods. Method 1 was applied to samples preserved in Buffer 1 (20 mM Tris-HCI
[pH
8.0] and 100 mM NaCI, 50 mM EDTA) and DNA isolation using this method was carried out according to Madrid et al. (2001). Method 2 was applied to samples preserved in Buffer 2 (100 mM Tris-HCI [pH 8.0], 100 mM sodium EDTA [pH 8.0], 100 mM sodium phosphate [pH 8.0], 1.5 M NaCI, 1% CTAB), proteinase K final concentration of mg/ml); DNA was isolated according to Zhou et al. (1996). Method 3 was applied to samples preserved in the Buffer 3 (750 l bead solution with 60 Cl solution) and DNA
isolation procedure was carried out using a PowerSoil kit according to the manufacturer instructions (Mo Bio, Carlsbad, CA). Cultures of Desulfovibrio vulgaris and Desulfotomaculum ruminis were grown in anaerobic conditions at 30 C in serum vials containing ATCC medium 1249 (for composition see Table 6.). DNA from bacterial pure cultures was isolated using either a Ultraclean microbial DNA isolation kit (Mo Bio, Carlsbad, CA) or a PowerSoilTM kit. General molecular biological techniques (agarose gel electrophoresis, molecular biology buffer and reagent preparation) were described in Sambrook et al. (1989).

Table 6. Composition of ATCC medium.
ATCC medium: Component I:
1249 Modified MgSO4 2.0 g Baar's medium for Sodium citrate 5.0 g sulfate reducers CaSO4.1.0 g NH4Cl 1.0 g Distilled water 400.0 ml Component II:
K2HPO4 0.5 g Distilled water 200.0 ml Component III:
Sodium lactate 3.5 g Yeast extract 1.0 g Distilled water 400.0 ml Adjust the pH of each component to pH 7.5 and autoclave. Mix the three components aseptically and tube under 97% N2, 3% H2 while warm to exclude as much oxygen as possible.

Component IV:
Filter-sterilize 5% Ferrous Ammonium Sulfate, Fe(NH4)2(SO4)Z, and add 0.1 ml to 5.0 ml of medium prior to inoculation.

100671 The quantity and integrity of DNA was determined using two methods.
First, the concentration was measured in a fluorometer using Picogreen dye (Molecular Probes, OR). The integrity of DNA was determined by the presence of a single non-sheared band by gel electrophoresis.

(0068] The DNA extraction efficiency was tested with pure cultures of sulfate reducing bacteria Desulfotomaculum ruminis and Desulfovibrio vulgaris.
Environmental samples were seeded with known quantities of either or both bacteria (bacterial numbers were quantified prior seeding by microscopic counts using 4-6-diamidino-phenylindole hydrochloride staining as per Kuwae and Hosokawa (1999). Then, DNA was extracted from the environmental samples and environmental samples seeded with D.
vulgaris or D.
ruminis cells and pure cultures containing the same numbers of D. vulgaris or D. ruminis cells.

[0069] To optimize DNA extraction from environmental samples, various samples were tested. Methods I and 2 had similar DNA extraction efficiencies, which somewhat increased with addition of proteinase K (see Table 8). Method 1 generated a slightly higher yield of DNA from water column samples, where as Method 2 was slightly better for sediment samples. Method 3, however, performed better with both water column and sediment samples: DNA yields were 60% higher from water samples and 2.7 times higher from sediments compared to Methods 2 and 3. With the PowerSoilTM
method, the yield of DNA during extractions was highly reproducible with deviations between independent extractions of 4.3% and did not depend on addition of proteinase K.

[0070] The quality of DNA isolated by Method 2 and 3 for downstream analysis was compared by PCR amplification using universal bacterial primers as per Madrid et a]. (2001). DNA from all samples isolated using PowerSoilTM
supported PCR, whereas only -50% of DNA samples isolated by the method 2 supported PCR.

[0071] Extraction efficiency was verified for the PowerSoilTM method using environmental samples seeded with SRBs (see methods). For comparison, another kit (UltracleanTM from Mo Bio) was tested. The yields of DNA were comparable for both kits, however, the PowerSoilm kit buffer is more preferable for sample storage and transportation. PowerSoilTM extraction method is preferred because it is designed to effectively remove PCR inhibitors which are abundant in sediment-containing samples.
Analysis of DNA yields with seeded samples and comparable quantities of pure culture cells indicated that the PowerSoilTM yields the maximum quantity of DNA
possible, i.e., DNA isolation efficiency is -100%.

100721 The extraction of DNA from soil and sediment samples is challenging due to a co-extraction of PCR inhibitors from soils. In addition, the heterogeneous nature of a soil sample as well as the different cell wall of microorganisms that inhabit soils and sediments makes the use of commercial kits very appropriate, simplifying DNA
extraction and as we have shown to be a very reliable method for extraction of DNA. This commercial kit has been previously used by other researchers in unrelated studies (Madrid et al., 2006; Hurt et al., 2001 and Dhillon et al., 2003).

[0073] As a negative control, samples were taken from the Carter #2 SWD
well, LA using membrane filters and DNA extraction Methods 2 and 3. As expected, no DNA was extracted for the negative control well because this well was under treatment with a biocide during the time of sampling, confirming that once treatment has started no cells remain alive or extractable DNA is preserved.

Table 8. Comparison of the three techniques for DNA isolation. Method 1=
Madrid et al.
(2001); Method 2 Zhou et al. (1996) and Method 3 = PowerSoilT"' kit.

Proteinase DNA yield Sample Type of media K Method g cm ,3 sample treatment Cypress Lake Water column No method 1 0.871 Cypress Lake Water with suspended sediment No method 1 1.861 Cypress Lake Water column Yes method 1 1.265 Cypress Lake Water with suspended sediment Yes method 1 2.122 Cypress Lake Water column No method 2 1.061 Cypress Lake Water with suspended sediment No method 2 2.023 Cypress Lake Water column Yes method 2 1.134 Cypress Lake Water with suspended sediment Yes method 2 2.550 Cypress Lake Water column No method 3 1.791 Cypress Lake Water with suspended sediment No method 3 6.816 Cypress Lake Clear water Yes method 3 1.876 Cypress Lake Water with suspended sediment Yes method 3 6.798 Lyons Water from oil pipeline Yes method 1 2.123 Lyons Water from oil pipeline Yes method 2 3.983 Lyons Water from oil pipeline No method 3 3.481 Cypress Lake Water with suspended sediment No UltraCleanTM 4.568 Microbial Kit Cypress Lake Water with suspended sediment No method 3 4.613 Marine sediment Mobile sediment No UltraCleanTM 2.912 (French Guiana coast) Microbial Kit Marine sediment Mobile Sediment No method 3 2.885 (French Guiana coast) D. vulgaris culture Cell suspension (10$ cells) No UltraClean'"' 3.821 Microbial Kit D. vulgaris culture Cell suspension (108 cells) No method 3 3.934 D. ruminis culture Cell suspension (108 cells) No UltraCleanTM 2.511 Microbial Kit D. ruminis culture Cell suspension (108 cells) No method 3 2.603 D. vulgaris culture and Seeded mobile sediment (108 cells) No UltraCleanTM
6.123 marine sediment Microbial Kit D. vulgaris culture and Seeded mobile sediment (108 cells) No method 3 6.097 marine sediment D. ruminis culture and Seeded mobile sediment (10a cells) No UltraCleanTM
5.467 marine sediment Microbial Kit D. ruminis culture and Seeded mobile sediment (108 cells) No method 3 5.327 marine sediment [0074] Degenerate primers for amplification of portions of dsrAB genes were designed by aligning amino acid sequences of the respective proteins previously retrieved through the analyses of metabolic gene libraries constructed for each gene of interest.
Sequences of the respective proteins from uncultured and cultured SRPs retrieved by BLAST from the GenBank database (www.ncbi.nlm.nih.gov), as well as representative sequences from each major family of a gene (as defined by Zverlov et al.
(2005) for dsr) were also included in the alignment. Primers were selected from conserved regions in alignments created by Clustal X package (Thomson et al., 1997) by both visual inspections and with the aid of the program Primer Select (Lasergene software, DNASTAR, Madison, WI). In order to minimize primer-dimer formation, Primers Select options were set for the maximum self-complementary score at 4 and the maximum 3' self-complementary score at 2. A desired amplicon length was 75 to 150 bp although longer sequences (up to 350 bp) were also deemed acceptable. Synthesis of larger amplicons during real-time PCR may lead to decreased amplification efficiencies since for longer templates, there is an increased probability of dissociation of DNA polymerase from the template. Specificity of the selected primers was screened by BLAST against available sequences in the NCBI database (http://www.ncbi.nlm.nih.gov). Potential targets amplified by each primer pair were characterized using the M-fold criterion (Santalucia et al., 1992; http://bioinfo.math.rpi.edu/-mfold/dna/forml.cgi) in order to predict formation of any secondary structure, which might form at the site of the primer binding.
Oligonucleotides were obtained from Operon Technologies (Alameda, CA). For each primer pair, the optimal annealing temperatures were determined by gradient PCR with the temperature range between 40 C and 65 C using a gradient thermocycler model iCycler (BioRad, Hercules, CA).

[00751 As a result, five sets of DSR specific primers target a conserved regions of the DSR gene, listed in Table 1, the first set (i.e., DsrUniv43F and DsrUniv225R) amplifies approximately a 180 bp region, the second (i.e., DsrUnivl577F and DsrUnivl7l2R) approximately a 150 bp region of the DSR gene,the third set (Dsr1FM
and DsrUniv225R) approximately a 225 bp region of the DSR gene, the fourth set (DsrlFM and DsrUniv43R) approximately a 70 bp region of the DSR gene and the fifth set (DsrUniv1712F and DsrUniv4RM) approximately a 330 bp region of the DSR
gene.

[0076] In a preferred embodiment, a binding probe could be used in the PCR
reaction to detect the presence of PCR product or otherwise monitor the reaction. Such a probe could contain the sequence of the gene of interest or a portion thereof.
This sequence could comprise SEQ ID NO:6 or SEQ ID NO:7, or a number of other possible target genes or regions thereof.

[0077] The plasmid pVMD (4.9 kb) was selected containing a dsrAB gene insert from a mobile sediment dsr gene library to serve as the standard for real-time PCR
assays. In several experiments, known quantities of genomic DNA of Desulfovibrio vulgaris (3.6 Mb chromosome) were used as a gene copy standard for quantification of the number of copies of dsr. Highly purified endotoxin-free plasmid DNA was extracted from 50 ml of E. coli cultures harboring the aforementioned plasmid using an UltraCleanTM
Endotoxin-free Midi Plasmid prep kit (Mo Bio, Carlsbad, CA). Plasmids were linearized with SalI, which cleaves plasmid at one single site. Linear DNA was quantified using a Picogreen DNA quantification kit (Molecular Probes, Eugene, Oregon). The endonuclease was deactivated at 65 C for 20 minutes followed by purification with Wizard SV Gel and PCR Clean-up system according to manufacturer instructions (Promega, Madison, WI). Concentrations of double-stranded standards and their transcripts were measured fluorometrically with PicoGreen reagents in 1xTBE
buffer (Molecular Probes, Eugene, OR).

100781 In a preferred embodiment of the invention, PCR product formation was monitored by determining an increase in fluorescence due to binding of SYBR green to newly synthesized double-stranded amplicons (Higuchi et al., 1992; Witter et al., 1997).
Typically a 20 pl real-time PCR mixture contained 1.5 l of each primer of a primer pair (10 M), I l of a template (0.01-2 ng Of dsDNA), 7.5 l of nuclease-free water and 10 l of iQTM SYBR Green Supennix (Bio-Rad, Hercules, CA). To prevent primer-dimer and non-specific product formation, concentration of primers in real-time PCR was optimized by 2-fold primer dilution series (Peters et al., 2004). Each assay reaction (i.e., reactions with both standard and sample) was carried out in triplicate. Real-time PCR
assays were performed in a thermocycler model iQ iCycler equipped with an optical unit (Bio-Rad Laboratories, Hercules, CA) using the following conditions: initial incubation at 95 C for 3 min followed by 45 cycles at 95 C for 30s, optimal annealing temperature for each primer set (45 C for DsrUniv43F and DsrUniv225R, 43 C for DsrUnivl577F and DsrUniv 1712R, 53 C for DsrUniv 1712F and DsrUniv4RM and 54 C for DsrUniv 1 FM
and DsrUniv225R and DsrUnivlFM and DsrUniv43R) for 30 s, 72 C for 30 s and 83 C
for 10 s during which the fluorescence data were collected. The threshold cycle (Ct value) was calculated as the cycle when the fluorescence of the sample exceeded a threshold level corresponding to 10 standard deviations from the mean of the baseline fluorescence. A
melt curve was produced by heating the samples from 75 to 95 C in 0.5 C
increments with a dwell time at each temperature of 10 s during which the fluorescence data were collected.

[00791 In the second embodiment, either of the two probes shown in the Table 1(DsrUnivPF and DsrUnvPR) labeled with FAM and Black Hole quencher was introduced in the reaction. The reaction was monitored in FAM channel of the real-time thermocyclers. Typically a 20 l real-time PCR mixture contained 1.5 l of the primer pair DsrUnivlFM and DsrUniv225R (10 M each), I l of a template (0.01-2 ng of dsDNA), 1.5 ml of a dual-labeled probe (DsrUnivPF and DsrUnvPR), 5 1 of nuclease-free water and 10 l of the master mix (30 mM Tris-HCI, pH8.3; 3 mM MgC12i 100 mM of KCI;
0.01% (w/v) gelatin; 0.4 mM of each dCTP, dTTP, dGTP and dATP, 0.3 U/ l of Taq polymerase). To prevent primer-dimer and non-specific product formation, concentration of primers and probes in real-time PCR was optimized by 2-fold primer dilution series followed by 2 fold probe dilution series (Peters et al., 2004). Each assay reaction (i.e., reactions with both standard and sample) was carried out in triplicate. Real-time PCR
assays were performed in a thermocycler model iQ iCycler equipped with an optical unit (Bio-Rad Laboratories, Hercules, CA) using the following conditions: initial incubation at 95 C for 3 min followed by 45 cycles at 95 C for 30s, 54 C for 30 s, 72 C for 30 s and 83 C for 10 s during which the fluorescence data were collected. The threshold cycle (Ct value) was calculated as the cycle when the fluorescence of the sample exceeded a threshold level corresponding to 10 standard deviations from the mean of the baseline fluorescence.

[00801 Figure 2 shows a standard curve built using a sample with known gene copy number. Gene copy number of the sample is plotted versus the PCR cycle at which the fluorescence value crosses the threshold line in a PCR using the sample as a template.

[0081] The copy number of the dsrAB genes in each standard was calculated assuming that the plasmid pVMD and DNA from reference cultures (Desulfovibrio vulgaris) contain a single copy of a target gene per genome. The molecular weight of the plasmid and bacterial genomes were calculated based on an averaged molecular weight of 660 Da base pair for DNA. The iCycler iQ Optical System Software version 3.1 (Bio-Rad Laboratories, Hercules, CA) generates a calibration line based on gene copy numbers in standard reactions and C, for the given standards. The number of gene copies in a sample is determined by the software by plotting C, value for the sample reaction against the calibration line. Real-time PCR standard curves are highly reproducible and allow the generation of highly specific and sensitive data (Bustin, 2000). Standard curves were constructed over a range from 100 to 105 dsr copies for double-stranded DNA.
The slope of each calibration curve is incorporated into the following equation by the software to determine the reaction efficiency:

E (efficiency) = 10"1/5' Pe (Eq. 1) 10082J Efficiencies between 90-105% are the best indicators of a robust, reproducible assay, and thus only assays with efficiencies of 92% or higher were considered successful for quantification purposes. The melting temperatures of the products were determined with iCycler iQ Optical System Software (version 3.1, Bio-Rad laboratories). Following each quantitative analysis, the presence of correct PCR products was verified by a melting temperature curve (a single peak representing a specific product vs. an additional nonspecific primer-dimer peak) using iCycler iQ analysis software and by detection of a single band of the expected size by 3% agarose gel electrophoresis.
Potential autofluorescence, quenching and inhibition of quantitative PCR
reactions by impurities in samples were evaluated by serial dilution of samples (10" and 10-2 must give identical copy numbers per ml of the original preparation) and by spiking samples with either a known gene copy number or known numbers of cDNA molecules of a respective standard. In the latter case, predicted and experimental gene or transcript numbers in spiked reactions were compared. If inhibition was detected for DNA reactions, samples were further purified using an SV DNA Clean up system (Promega, Madison, WI).
If the inhibition problem persisted, the gene number was calculated based on the following formula:

Csample=Cstandard/((1 + E)CtsLt(s+st) -1), (Eq. 2) where:

Csampie = gene copy number in a sample Cstandard = gene copy number in a spiked standard Cts = Ct for a sample Ct(,+,t) = Ct for a mixture of sample and standard E = efficiency of PCR reaction (determined using Eq 1 for sample with variable quantities of spiked control).

[0083] Quantification of the DSR gene was carried out by real-time PCR, which is a technique known to those of skill in the art. Without wishing to be bound by theory, real-time PCR is an existing research technique that utilizes specifically engineered DNA sequences (two amplification primers and depending on a format a probe), which are complementary and thus bind to specific sequences on DNA, in this case genes for DSR. There are two general formats of real-time PCR. In one format formation of double-stranded PCR product is monitored by binding of a dye specific for double stranded DNA (e.g., SYBR Green) to the double-stranded DNA produced as a result of PCR amplification. Thus, by following fluorescence signal of SYBR
Green-dsDNA complex one can follow PCR reaction in real-time. The signal intensity is proportional to the quantity of DNA synthesized with each cycle, which is in turn proportional to the initial concentration of specific (target) DNA in a sample. In another format, in addition to specific PCR primers, a specific probe is added to the reaction, which binds target DNA between binding sites for amplification primers. This probe is synthesized with a fluorescent dye (detector) and a fluorescence quencher attached to opposite part of the molecule. Due to physical proximity of the dye and quencher the probe emits little if any fluorescence signal. During PCR reaction this probe becomes digested by Taq DNA polymerase releasing fluorescent dye, which now is not quenched and can be detected. The quantity of the dye release is increasing with each cycle and proportional to the original quantity of the target DNA.

[0084] Five sets of DSR specific primers target a conserved regions of the DSR gene, shown in Table 7, the first set (i.e., DsrUniv43F and DsrUniv225R) amplifies approximately a 180 bp region, the second (i.e., DsrUnivl577F and DsrUnivl7l2R) approximately a 150 bp region of the DSR gene,the third set (Dsr1FM and DsrUniv225R) approximately a 225 bp region of the DSR gene, the fourth set (Dsr1FM and DsrUniv43R) approximately a 70 bp region of the DSR gene and the fifth set (DsrUnivl712F
and DsrUniv4RM) approximately a 330 bp region of the DSR gene. Cloning and sequencing of resulting PCR products corroborated primer specificity towards the DSR
gene, i.e., all and any product amplified with these two sets is a fragment of a DSR gene.
Melt-curve analysis also confirmed that only one specific product is generated as a result of assay.

Real-time PCR assays using either of the three pairs in conjunction with a double-stranding DNA binding fluorescent dye were optimized to yield consistently efficiencies of 92% or higher. We utilized absolute quantification by constructing standard curves with a plasmid containing a copy of the DSR gene. See Figure 1 as an example of such a standard curve.

[00851 The detection technique was tested with several positive and negative controls as well as samples collected at different oil production facilities.
A novel sampling and nucleic acid isolation procedure to suit this PCR-based detection technique was also developed. The novel technique allowed us to quantify DSR genes from SRP
sulfate reducing bacteria present in environmental samples such as marine sediments, freshwater sediments, marine anoxic waters and waters used for oil drilling.
Numbers of DSR genes in a sample correlated well with numbers of SRB obtained using the standard MPN method (i.e., NACE Standard TM0194-2004). See Figure 3.

[00861 A second format employs the DsrUnivlF and DsrUniv225R pair of specific DSR primers and either of the two labeled probes, DsrUniv PF or DsrUniv PR.
DsrUniv PF or DsrUniv PR are labeled with a fluorescent dye (e.g., FAM) and a quencher compatible with a dye (e.g., TAMRA or Iowa Black for FAM). Since all three primers are specific for the DSR assay no PCR product cloning and melt curve analysis is necessary.

Nucleic acid extraction [0087) Sediment samples one for genomic DNA extraction and the second for RNA extraction. Genomic DNA from up 10 grams of sediment was extracted with a PowerSoilT"' DNA Isolation Kit (MO BIO Laboratories Inc., Carlsbad, CA) according to manufacturer instructions. Concentrations of purified DNA were quantified with PicoGreen (Molecular Probes, Eugene, OR). DNA from bacterial pure cultures was isolated according Marmur (1961). DNA yield was determined fluorometrically with PicoGreen reagents in IxTBE buffer (Molecular Probes, Eugene, OR). DNA yield was expressed in g per 1 cm3 of water, sediment or sediment suspension. General molecular biological techniques (agarose gel electrophoresis, molecular biology buffer and reagent preparation) are described in Sambrook et al. (1989).

Primer design and specificity [0088] Degenerate primers for amplification of portions of dsrAB genes were designed by aligning amino acid sequences of the respective proteins previously retrieved through the analyses of metabolic gene libraries constructed for each gene of interest.
Sequences of the respective proteins from uncultured and cultured SRPs retrieved by BLAST from the GenBank database (www.ncbi.nlm.nih.gov), as well as representative sequences from each major family of a gene (as defined by Zverlov et al.
(2005) for dsr) were also included in the alignment. Primers were selected from conserved regions in alignments created by Clustal X package (Thomson et al., 1997) by both visual inspections and with the aid of the program Primer Select (Lasergene software, DNASTAR, Madison, WI). In order to minimize primer-dimer formation, Primers Select options were set for the maximum self-complementary score at 4 and the maximum 3' self-complementary score at 2. A desired amplicon length was 75 to 150 bp although longer sequences (up to 350 bp) were also deemed acceptable. Synthesis of larger amplicons during real-time PCR may lead to decreased amplification efficiencies since for longer templates, there is an increased probability of dissociation of DNA polymerase from the template. Specificity of the selected primers was screened by BLAST against available sequences in the NCBI database (http://www.ncbi.nlm.nih.gov). Potential targets amplified by each primer pair were characterized using the M-fold criterion (Santalucia et al., 1992; http://bioinfo.math.rpi.edu/-mfold/dna/forml.cgi) in order to predict formation of any secondary structure, which might form at the site of the primer binding.
Oligonucleotides were obtained from Operon Technologies (Alameda, CA). For each primer pair, the optimal annealing temperatures were determined by gradient PCR with the temperature range between 40 C and 65 C using a gradient thermocycler model iCycler (BioRad, Hercules, CA). In order to corroborate amplification of correct targets by real-time PCR primers, DNA fragments generated by amplification with PCR primers were cloned in the pGEM-T vector (as per Madrid et al, 2001 and 2006), 20 plasmids were randomly selected from each PCR clone library and inserts were sequenced.

DNA standard [0089] The plasmid pVMD (4.9 kb) was selected containing a dsrAB gene mobile sediment dsr gene library to serve as the standard for real-time PCR

assays. In several experiments, known quantities of genomic DNA of Desulfovibrio vulgaris (3.6 Mb chromosome) were used as a gene copy standard for quantification of the number of copies of dsr. Highly purified endotoxin-free plasmid DNA was extracted from 50 ml of E. coli cultures harboring the aforementioned plasmid using an U1traCleanTM
Endotoxin-free Midi Plasmid prep kit (Mo Bio, Carlsbad, CA). Plasmids were linearized with Sall, which cleaves plasmid at one single site. Linear DNA was quantified using a Picogreeri DNA quantification kit (Molecular Probes, Eugene, Oregon). The endonuclease was deactivated at 65 C for 20 minutes followed by purification with Wizard SV Gel and PCR Clean-up system according to manufacturer instructions (Promega, Madison, WI). Concentrations of double-stranded standards and their transcripts were measured fluorometrically with PicoGreen reagents in IxTBE
buffer (Molecular Probes, Eugene, OR).

Real-time PCR

[0090] In the first format, PCR product fonnation was monitored by determining an increase in fluorescence due to binding of SYBR green to newly synthesized double-stranded amplicons (Higuchi et al., 1992; Witter et al., 1997).
Typically a 20 l real-time PCR mixture contained 1.5 l of each primer of a primer pair (10 M), 1 l of a template (0.01-2 ng of dsDNA), 7.5 l of nuclease-free water and 10 l of iQTM SYBR Green Supermix (Bio-Rad, Hercules, CA). To prevent primer-dimer and non-specific product formation, concentration of primers in real-time PCR was optimized by 2-fold primer dilution series (Peters et al., 2004). Each assay reaction (i.e., reactions with both standard and sample) was carried out in triplicate. Real-time PCR
assays were performed in a thermocycler model iQ iCycler equipped with an optical unit (Bio-Rad Laboratories, Hercules, CA) using the following conditions: initial incubation at 95 C for 3 min followed by 45 cycles at 95 C for 30s, optimal annealing temperature for each primer set (45 C for DsrUniv43F and DsrUniv225R, 43 C for DsrUnivl577F and DsrUniv1712R, 53 C for DsrUnivl7l2F and DsrUniv4RM and 54 C for DsrUnivlFM and DsrUniv225R and DsrUnivlFM and DsrUniv43R) for 30 s, 72 C for 30 s and 83 C
for 10 s during which the fluorescence data were collected. The threshold cycle (C, value) was calculated as the cycle when the fluorescence of the sample exceeded a threshold level corresponding to 10 standard deviations from the mean of the baseline fluorescence. A

melt curve was produced by heating the samples from 75 to 95 C in 0.5 C
increments with a dwell time at each temperature of 10 s during which the fluorescence data were collected.

[0091] In the second fonnat, either of the two probes shown in the Table 1 (DsrUnivPF and DsrUnvPR) labeled with FAM and Black Hole quencher was introduced in the reaction. The reaction was monitored in FAM channel of the real-time thermocyclers. Typically a 20 l real-time PCR mixture contained 1.5 l of the primer pair DsrUnivlFM and DsrUniv225R (10 M each), I l of a template (0.01-2 ng of dsDNA), 1.5 ml of a dual-labeled probe (DsrUnivPF and DsrUnvPR), 5 l of nuclease-free water and 10 l of the master mix (30 mM Tris-HCI, pH8.3; 3 mM MgC12; 100 mM of KCI;
0.01 %(w/v) gelatin; 0.4 mM of each dCTP, dTTP, dGTP and dATP, 0.3 U/ l of Taq polymerase). To prevent primer-dimer and non-specific product formation, concentration of primers and probes in real-time PCR was optimized by 2-fold primer dilution series followed by 2 fold probe dilution series (Peters et al., 2004). Each assay reaction (i.e., reactions with both standard and sample) was carried out in triplicate. Real-time PCR
assays were performed in a thermocycler model iQ iCycler equipped with an optical unit (Bio-Rad Laboratories, Hercules, CA) using the following conditions: initial incubation at 95 C for 3 min followed by 45 cycles at 95 C for 30s, 54 C for 30 s, 72 C for 30 s and 83 C for 10 s during which the fluorescence data were collected. The threshold cycle (C, value) was calculated as the cycle when the fluorescence of the sample exceeded a threshold level corresponding to 10 standard deviations from the mean of the baseline fluorescence.

Calculations of gene copy number [00921 The copy number of the dsrAB genes in each standard was calculated assuming that the plasmid pVMD and DNA from reference cultures (Desulfovibrio vulgaris) contain a single copy of a target gene per genome. The molecular weight of the plasmid and bacteria] genomes were calculated based on an averaged molecular weight of 660 Da base pair for DNA. The iCycler iQ Optical System Software version 3.1 (Bio-Rad Laboratories, Hercules, CA) generates a calibration line based on gene copy numbers in standard reactions and C, for the given standards. The number of gene copies in a sample is determined by the software by plotting C, value for the sample reaction against the calibration line. Real-time PCR standard curves are highly reproducible and allow the generation of highly specific and sensitive data (Bustin, 2000). Standard curves were constructed over a range from 10 to 105 dsr copies for double-stranded DNA.
The slope of each calibration curve is incorporated into the following equation by the software to determine the reaction efficiency:

E (efficiency) = 10-11s10P (Eq. 1) (0093] Efficiencies between 90-105% are the best indicators of a robust, reproducible assay, and thus only assays with efficiencies of 92% or higher were considered successful for quantification purposes. The melting temperatures of the products were determined with iCycler iQ Optical System Software (version 3.1, Bio-Rad laboratories). Following each quantitative analysis, the presence of correct PCR products was verified by a melting temperature curve (a single peak representing a specific product vs. an additional nonspecific primer-dimer peak) using iCycler iQ analysis software and by detection of a single band of the expected size by 3% agarose gel electrophoresis.
Potential autofluorescence, quenching and inhibition of quantitative PCR
reactions by impurities in samples were evaluated by serial dilution of samples (10"1 and 10-2 must give identical copy numbers per ml of the original preparation) and by spiking samples with either a known gene copy number or known numbers of cDNA molecules of a respective standard. In the latter case, predicted and experimental gene or transcript numbers in spiked reactions were compared. If inhibition was detected for DNA
reactions, samples were further purified using an SV DNA Clean up system (Promega, Madison, WI). If inhibition problem persisted, the gene number was calculated based on the following formula:

Csample=Conda,a/((l+ E)cuia*+st) -1), (Eq= 2) where:

Csample = gene copy number in a sample Cs,aõd.d = gene copy number in a spiked standard Cts = Ct for a sample Ct(s+sc) = C, for a mixture of sample and standard E = efficiency of PCR reaction (determined using Eq 1 for sample with variable quantities of spiked control).

To determine the detection limit of the technique, we analyzed serial dilutions of standards containing known copy numbers of the target gene.

RESULTS and DISCUSSION
Nucleic acid extraction [00941 Aqueous (little quantities of suspended matter can be present) samples were filtered through DuraPore filters (Millipore), shredded, and used for DNA
isolation.
Sediment samples were used directly for DNA isolation. The yield of DNA and RNA
during extractions was highly reproducible with deviations between independent extractions of 14.3% and 5.4%, respectively. Usually, DNA preparations isolated with the Mo Bio DNA isolation kit supported subsequent PCR reactions. Samples containing large quantities of humic acids may not support PCR in a few cases and these were further purified using the SV DNA clean-up kit (Promega, Madison, WI). The losses of DNA
during this purification step were calculated to be 35-40% and this term was included in final calculations of gene copy numbers.

(0095] To further test specificity of the designed primers, PCR assays were run with positive and negative controls. The positive controls included genomic DNA of bacteria carrying the gene of interest (Desulfotomaculum ruminis and Desulfovibrio vulgaris ATCC as positive controls) and plasmids randomly chosen from French Guiana sediment gene libraries representing each of the genes of interest (Madrid et al., 2006)).
Correct amplicon size was verified by and annealing temperatures were selected based on visual observations of PCR products in 3% agarose gels and by performing melting curves analysis during real-time PCR runs. PCR conditions leading to a single band in the gel (and single peak during melting) were selected for routine analyses. The negative controls included DNA from Escherichia coli, Alcaligenes faecalis and Thiomicrospira denitrificans.

Real-time PCR assays [0096] Real-time PCR assays are a robust highly sensitive and reproducible tool for gene quantification as previously suggested by Gibson (1996), Bustin (2000), Liss (2002) and our data concur with these notions. Standard deviation among assays in this study was small (<8%) and sensitivity was high (for 1 copy of a gene per sample Ct value for dsr was 45.11). However, before this real-time PCR technique can successfully be applied for nucleic acids isolated from environmental samples, several issues have to be carefully addressed. One of the major problems encountered is inhibition of real-time PCR
by PCR inhibitors (most commonly humic and fulvic acids and iron ions). Stults et al.
(2001) suggested autofluorescence and signal quenching as two other probable problems.
Unless extremely high, autofluoresence should pose no problems. The background autofluorescence is typically monitored during the progression of a real-time PCR
reaction. As long as the background autofluorescence is low enough to select a cut-off value for Ct quantification, autofluorescence does not affect quantification.
Quenching and inhibition, although different mechanistically, lead to detection of gene copy numbers lower than is really present in a sample. Two types of controls were incorporated in reported herein assays to address the quenching and inhibition problems:
serial dilution of samples and spiking of a sample with known amounts of standards.

[0097] As mentioned above, several methods previously used to study gene expression, including slot- or spot-dot hybridization and competitive RT-PCR
have drawbacks, such a requirement of high quantities of RNA or low sample throughput capabilities for the latter (Ferr6, 1998). Real-time PCR surpass the biases associated with end-point amplicon detection, which is used in competitive PCR. It is very sensitive, fast and produces no radioactive waste. An additional problem associated with end-point PCR
is that in order to be quantitative the detection has to be carried out during exponential amplification (Morrison et al., 1994). The exponential amplification phase can be achieved after a different number of cycles for different target templates and even for samples with the same target. In real-time PCR analysis, the application of the threshold cycle C, method (Heid et al., 1996) allows for calibration in a wide dynamic range in which the number of cycles can be adjusted to the signal size. The threshold cycle is defined as the PCR cycle at which a fluorescence signal, developed by a dye-DNA complex or a free dye (depending on the format) passes a preset value. This value corresponds to an amount of amplicons generated in a few cycles if a large member of templates was present initially, or after many cycles if the PCR started with few templates. Quantification is based on the number of cycles required to reach a certain concentration of amplicons rather than on the concentration reached after a fixed number of cycles. In real-time PCR, accumulation of PCR products can be monitored using a fluorescent dye, such as SYBR Green (Higuchi et al., 1992; Wittner et al., 1997), that forms fluorescent adducts with double-stranded DNA
without compromising the polymerization reaction. The reaction can be calibrated by amplification of known amounts (gene copy number) of the target sequence and by monitoring the increase in fluorescence cycle by cycle (real-time monitoring of PCR).

[0098] Dissimilatory sulfate reductase is the key enzyme for the dissimilative sulfate reduction pathway. A high diversity of prokaryotes carrying the dsrAB
genes in mobile sediment microbial communities suggests the active participation of sulfur cycle reactions during Corg remineralization in Fe(III) rich coastal deposits (Madrid et al. 2006).
Therefore, the dsr genes appear to be a useful genetic marker to study the sulfate reduction activity in many environments. Several studies on quantification of dsrAB
genes in sediments have been described in the literature and include the work by Leloup et al (2004) and Kondo et al (2004) who used competitive PCR and by Nedwell et al.
(2004) who used slot-blot hybridization. As discussed above our method of choice is real-time PCR and RT-PCR. Using dsr sequence data for the sulfate reducing prokaryotes (SRPs) in mobile sediments (Madrid et al., 2006) we managed to design a pair of universal primers, which can also amplify dsr sequences from representatives of major DSR
families described by Zverlov et al. (2005).

Collection of aqueous samples for quantification of sulfate reducing prokaryotes Considerations [0099] Two factors need to be evaluated before collecting a sample.

A) Estimate whether or not the sample can be delivered to the laboratory for DNA
extraction as soon as possible. Depending on the time schedule there are three ways to proceed. The following instructions and recommendations are based on the assumption that the number of total bacteria present in a sample is unknown.

B) Estimate the particle load of a sample by visual examination (for details go to `General considerations' section below). The 1000m1 aliquots of samples is described for the sake of simplicity, although other sample sizes are acceptable.

A. 1) Samples delivered the same day. If a sample can be delivered the same day it is collected, the sample does not need to be filtered in the field. Go to section A.1 for detailed instructions.

A. 2) Samples 24-48 hours in transit. Samples that cannot be delivered for DNA
extraction within 8 hours need to be processed (filtered) in the field. Go to section A.2 for details.
A.3) Samples in transit for more than 2 days. Go to section A.3.

Materials needed = Brand new bottles for collection. Either glass or plastic (such as Nalgene, Fisher Catalog numbers 03-312-8,-9,10,-11) 125, 250, 500 or 1000 cc.

= Membranes PVDF 47 mm(such as Fisher Catalog Number GVWP 047 00) = Swinnex filter holders (such as Fisher Catalog Number SXOO 047 00) = Forceps (such as 09-753-50 or XX62 000 06) = Equipment for filtering (Use standard field Millipore filtering system) = One liter graduated cylinder for measuring filtered volume = Sterile gloves, nitrile = From DNA extraction kit, bead tube and solution C 1 = 1 cc syringes GENERAL INSTRUCTIONS

a) When collecting a liquid matrix sample for shipment to the lab, a volume of 1000m1 is recommended. This sample size will ensure lab personnel have sufficient amounts to work with. If you will be filtering on site, simply filter water until membranes are clogged.

b) When collecting the above mentioned glass bottle samples, introduction of oxygen must be avoided. This can be accomplished by avoiding bubbling. For this purpose when pouring, the liquid sample should run down the side of the bottle, from the inner top. Fill up to the very top, overflow if needed (and recommended), get rid of bubbles and close tight. Use leakproof bottles.

c) Bring this glass bottle to the laboratory for visual and microscopic examination.

d) If filtration is needed, by default always filter as much as possible (until membrane clogs).

e) The preferred transportation temperature of any sample irrespective of is state (on membranes, on buffer or in solution) is 4 C (about 40F).

SECTION 1: delivery to the laboratory within 8 hours: No filtering.

= Use a glass bottle and collect a sample as described in section (a). Avoid introduction of oxygen while sampling by avoiding bubbling. The liquid sample should pour down the side of the bottle. Fill up to the very top and close tight. Use leakproof bottles.

= A 1000m1 sample of water should be collected. This volume will insure an adequate volume for laboratory personnel.

= Place bottles in the dark on ice or 4 C and deliver to the lab immediately.
SECTION 2: On site filtering.

If it is possible to set up a pressure-driven filtration line go to subsection B. If no pressure (or vacuum) system is available, proceed to Subsection A.

Subsection A. Filtration using filter holders (Fisher Cat. 09-740-23D) and a hand pump = Set up a working space in a wind-free area (inside your truck). In this area set up filtration system consisting of reservoir, funnel support and hand vacuum pump = Wear gloves. With the help of sterile forceps insert a membrane in the filter holder.
The filter holder must be brand new or sterilized. Make sure the rubber o-ring is in place. Screw on tight = Add 60 gl of solution C 1 to a bead tube (use 1 cc syringes). Use a rack to hold bead tube to avoid spilling.

= FILTRATION. Collect liquid as indicated in "general instructions (b)". Pour collected liquid into the funnel of the filtration unit and apply vacuum.
Repeat with several volumes of sample until the membrane is clogged. Keep track of the volume filtered.

= Before disassembling the filtration unit, make sure the membrane is dry.
With the vacuum on, take the membrane from a clean border and fold it into a conical shape.
= Carefully place conical membrane into bead tube containing buffer Subsection B. Filtering using pressure-driven filtration = Set up filtering line same for Millipore filtration.

= Put on gloves after setting up the filtration line = Set up a working space in a wind-free area (such as inside your truck). In this area set up space for processing the Swinnex-membrane sandwich = Add 60 gl of solution C1 to a bead tube (use I cc syringes). Use a rack to hold bead tube to avoid spilling.

= With the help of sterile forceps insert a membrane into a sterile Swinnex.
The Swinnex must be brand new or sterilized. Make sure the rubber o-ring is in place.
Screw on tight = FILTRATION. Run liquid through the membrane Swinnex sandwich until the membrane is clogged. Be gentle, do not use high pressures (</= 30psi).
MEASURE THE VOLUME FILTERED.

= Disassemble the Swinnex from the line. Take the sandwich to the wind-free area.
= Dispose old gloves and put on new ones. In the outlet of the Swinnex where filtered water exits, place tubing from the hand vacuum pump. The membrane should be liquid-free before immersed into the bead tube and the hand pump will assist with this.

= Hold the vacuum and carefully open the Swinnex upright, the vacuum will hold the membrane in place. Make sure the liquid has been removed and the membrane is dry. If you open the Swinnex and the membrane still has liquid DO NOT SHAKE
the liquid away. This liquid contains bacteria and you will be discarding them, creating false negatives in the final results.

= Once the membrane is dry, take the membrane from a clean border and fold it into a conical shape.

= Carefully place conical membrane into bead tube containing buffer Subsection C. Filtration using 60 cc syringes (two people needed) = Set up a working space in a wind-free area (inside your truck). In this area prepare to receive and process the Swinnex and membrane.

= Add 60 l of solution C1 to a bead tube (use 1 cc syringes). Use a rack to hold bead tube to avoid spilling.

= With the help of sterile forceps (wiped with isopropyl alcohol) insert a membrane into a sterile Swinnex. The Swinnex must be brand new or sterilized. Make sure the rubber o-ring is in place. Screw on tight = FILTRATION. Collect liquid as indicated in "general instructions (b)". With the help of a syringe provided with 4 inches of tubing, withdraw liquid from the collection bottle. Attach syringe to the inlet of the Swinnex sandwich and press until all the liquid has passed through. Repeat with several volumes of sample until the membrane is clogged. Keep track of the volume filtered.

= Disassemble the Swinnex from the syringe. Take the sandwich to the wind-free area.

= Dispose old gloves, put on new ones. In the outlet of the Swinnex were filtered water came out connect an empty syringe with tubing. Pull plunge outwards removing liquid and air, producing vacuum. This will dry the membrane.

= Hold the vacuum and carefully open the Swinnex, the vacuum will hold the membrane in place. Make sure the liquid has been removed and the membrane is dry. If you open the Swinnex and the membrane still has liquid DO NOT SHAKE
the liquid away. This liquid contains bacteria and you will be discarding them, creating false negatives in the final results.

= Once the membrane is dry, take the membrane from a clean border and fold it into a conical shape.

= Carefully place conical membrane into bead tube containing buffer APPENDIX

= For sterilization of Swinnex units, wash thoroughly and autoclave.

= Forceps can be sterilized by thorough washing with rubbing alcohol.

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Claims (17)

1. A method of quantitating the presence of an organism in an industrial water, comprising:

adding a sample of the industrial water to a buffer to give a buffered sample;

isolating a nucleic acid from the organism, if any, in the buffered sample to give an isolated nucleic acid;

amplifying a nucleic acid sequence associated with the organism from the isolated nucleic acid to give an amplified nucleic acid; and wherein the organism in the industrial water is a sulfate reducing bacteria, wherein the nucleic acid sequence associated with the organism in the industrial water is amplified using a polymerase chain reaction;

wherein the polymerase chain reaction is carried out using a first and a second primer;
wherein the first primer and second primer are SEQ ID NO:3 and SEQ ID NO:5, SEQ ID NO:4 and SEQ ID NO:5, SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:8, or SEQ ID NO:9 and SEQ ID NO: 10, respectively;

wherein the amplification of the nucleic acid sequence associated with the organism in the industrial water is monitored using a binding probe, wherein the binding probe binds to a portion of the nucleic acid sequence associated with the organism in the industrial water;

wherein binding of the binding probe to the portion of the nucleic acid sequence is used to quantitate the amplified nucleic acid in order to indicate the amount of organism in the industrial water.

WHAT IS CLAIMED IS:

2. The method of claim 1, wherein the organism in the industrial water is a sulfate reducing bacteria.

3. The method of claim 1, wherein the nucleic acid sequence associated with the organism in the industrial water is amplified using a polymerase chain reaction.

4. The method of claim 3, wherein the polymerase chain reaction is carried out using a first and a second primer.

5. The method of claim 1, wherein the amplification of the nucleic acid sequence associated with the organism in the industrial water is monitored using a binding probe, wherein the binding probe binds to a portion of the nucleic acid sequence associated with the organism in the industrial water.

6. The method of claim 5, wherein binding of the binding probe to the portion of the nucleic acid sequence is used to quantitate the amplified nucleic acid in order to indicate the amount of organism in the industrial water.

7. The method of claim 4, wherein the first primer and second primer are DsrUniv1FM and DsrUniv225R, DsrUniv43F and DsrUniv225R, DsrUniv1577F and DsrUniv1712R, DsrUniv1FM and DsrUniv43R, or DsrUniv1712F and DsrUniv4R, respectively.

8. The method of claim 5, wherein the binding probe comprises SEQ ID NO:6 or a portion thereof still capable of directing specific PCR amplification, or SEQ ID NO:7 or a portion thereof still capable of directing specific PCR amplification.

9. The method of claim 1, further comprising the step of filtering the sample of the industrial water through a filter.

10. The method of claim 9, further comprising the step of shredding the filter before isolating nucleic acids from the sample.

11. The method of claim 9, wherein the filter comprises polyvinylidene fluoride (PVDF).

12. The method of claim 9, wherein the filter has a porosity of approximately 0.1 µm.

13. The method of claim 1, further comprising the step of transporting the sample of the industrial water to a laboratory facility.

14. The method of claim 13, wherein the sample of the industrial water is transported at a temperature of about 0°C to 22°C.

15. The method of claim 13, wherein the sample of the industrial water is transported at below about 22°C.

16. The method of claim 13 wherein the sample of the industrial water is transported to the laboratory facility within about 1 week of having been collected.

17. The method of claim 13, wherein the sample of the industrial water is transported to the laboratory facility within about 2 days of having been collected.

25. A method of detecting the presence of sulfate reducing prokaryotes in a sample which may contain said prokaryotes, comprising:

obtaining a sample susceptible of containing said prokaryote;
isolating nucleic acid from said sample;

exposing nucleic acid to a binding probe, wherein said binding probe binds to a portion of the dissimilatory sulfite reductase gene found in sulfate reducing prokaryotes;

exposing said nucleic acid to a first and second primer, amplifying said exposed nucleic acid by PCR amplification; and quantifying the PCR amplified nucleic acid by detection of said detector molecule.
26. A PCR primer composition that specifically amplifies a portion of DNA of sulfate reducing prokaryotes, the composition comprising the primers SEQ ID NO:4 or a portion thereof still capable of directing specific PCR amplification and SEQ ID NO:5 or a portion thereof still capable of directing specific PCR amplification.

27. A PCR primer composition that specifically amplifies a portion of DNA of sulfate reducing prokaryotes, the composition comprising the primers SEQ ID NO:1 or a portion thereof still capable of directing specific PCR amplification and SEQ ID NO:2 or a portion thereof still capable of directing specific PCR amplification.

28. A PCR primer composition that specifically amplifies a portion of DNA of sulfate reducing prokaryotes, the composition comprising the primers SEQ ID NO:3 or a portion thereof still capable of directing specific PCR amplification and SEQ ID NO:5 or a portion thereof still capable of directing specific PCR amplification.

29. A PCR primer composition that specifically amplifies a portion of DNA of sulfate reducing prokaryotes, the composition comprising the primers SEQ ID NO:3 or a portion thereof still capable of directing specific PCR amplification and SEQ ID NO:4 or a portion thereof still capable of directing specific PCR amplification.

30. A PCR primer composition that specifically amplifies a portion of DNA of sulfate reducing prokaryotes, the composition comprising the primers SEQ ID NO:2 or a portion thereof still capable of directing specific PCR amplification and SEQ ID NO:
10 or a portion thereof still capable of directing specific PCR amplification.

31. A PCR probe composition capable of identifying sulfate reducing prokaryotes, the composition comprising the probe SEQ ID NO:6.

32. A PCR probe composition capable of identifying sulfate reducing prokaryotes, the composition comprising the probe SEQ ID NO:7.