WO2008066614A2 - Détection de procaryotes induisant une corrosion - Google Patents

Détection de procaryotes induisant une corrosion Download PDF

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WO2008066614A2
WO2008066614A2 PCT/US2007/022147 US2007022147W WO2008066614A2 WO 2008066614 A2 WO2008066614 A2 WO 2008066614A2 US 2007022147 W US2007022147 W US 2007022147W WO 2008066614 A2 WO2008066614 A2 WO 2008066614A2
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nucleic acid
sample
dna
industrial water
primer
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PCT/US2007/022147
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WO2008066614A3 (fr
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Vanessa Madrid
Andrei Chistoserdov
Michael Chapman
Brian Price
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Multi-Chem Group, Llc
University Of Louisiana At Lafayette
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Priority to CA002673577A priority Critical patent/CA2673577A1/fr
Publication of WO2008066614A2 publication Critical patent/WO2008066614A2/fr
Publication of WO2008066614A3 publication Critical patent/WO2008066614A3/fr
Priority to GB0908301A priority patent/GB2456116A/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • 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.
  • Biofouling and microbiologically induced corrosion are of a great concern in many industrial processes using water or water suspensions.
  • 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 enviroru ⁇ 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 are an important group of microorganisms widespread in anoxic aquatic and terrestrial environments and are responsible for most of anaerobic degradation of organic matter.
  • SRPs Sulfate reducing prokaryotes
  • 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 environmental contamination from corroded pipelines.
  • the quantification of SRP project has indirect environmental benefits as well.
  • 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.
  • Biofouling and corrosion are produced as result of a synergistic relationship among microbial (mostly bacterial) communities that form 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.
  • Organisms from 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.
  • 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 34 S 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).
  • 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.
  • 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.
  • Sulfate reducing prokaryotes constitute a phylogenically heterogeneous group, which however share a common metabolic pathway.
  • SRP Sulfate reducing prokaryotes
  • 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.
  • APS adenosinemonophosphate sulfate
  • DSR dissimilatory sulfite reductase
  • 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.
  • 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).
  • 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.
  • PCR polymerase chain reaction
  • 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.
  • the application of the threshold cycle C t method 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.
  • 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).
  • 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 org remineralization in Fe(III) rich coastal deposits (Madrid et al. 2006).
  • 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.
  • the real-time assav bv Zhu 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 realtime PCR-based methods described in the current application detect all SRPs which would not be detected by the Zhu method.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.).
  • 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.
  • 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.
  • a primary object of a preferred embodiment of the invention is the monitoring of SRP present in a sample.
  • 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.
  • 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.
  • 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;
  • FIGURE 3 shows the correlation between samples tested using the MPN method versus samples tested using the qPCR method.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • filtration of the sample will occur within one hour of sample collection, most preferably within 5 minutes of sample collection.
  • 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.
  • Temperature during transport may range between about O 0 C to about 22 0 C, preferably between about 4 0 C to about 37 0 C, most preferably below about 22 0 C.
  • Total transport time may be up to several weeks, preferably less than one week, most preferably less than about 2 days.
  • 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.
  • 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.
  • a marker gene will be detected in extracted DNA using a PCR method.
  • 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.
  • DsrUniv43F 4 RGNGGNGGNRTNRTYGGNMGNTA DsrUniv225R 5 TCNCCNGTNGMNCCRTGNAWRTT
  • N A or G or T or C or I
  • Y T or C
  • R A or G
  • K G or T
  • M A or C
  • W A or
  • 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.
  • the PCR product could be quantities of DSR gene, and the resulting value correlated with SRB concentration.
  • Non-processed samples and filters with microbial biomass collected by filtration within 5 minutes after sampling were transported at 4°C, 22 0 C and 37 0 C, immersed in one of Buffer 1 (20 mM Tris-HCl [pH 8.0] and 100 mM NaCl, 50 mM EDTA), Buffer 2 (100 mM Tris-HCl [pH 8.0], 100 mM sodium EDTA [pH 8.0], 100 mM sodium phosphate [pH 8.0], 1.5 M NaCl, 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 1 (20 mM Tris-HCl [pH 8.0] and 100 mM NaCl, 50 mM EDTA
  • Buffer 2 100 mM Tris-HCl [pH 8.0], 100 mM sodium EDTA [pH 8.0
  • 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.
  • PVDF polyvinylidene fluoride
  • Buffer 1 was the extraction buffer from Xu and Tabita (1996). Buffer 1 comprises 20 mM Tris-HCl [pH 8.0] and 100 mM NaCl, 50 mM EDTA, and was used in Method 1
  • Buffer 2 was the extraction buffer from Zhu et al. (1996). Buffer 2 comprises 100 mM Tris-HCl [pH 8.0], 100 mM sodium EDTA [pH 8.0], 100 mM sodium phosphate [pH 8.0], 1.5 M NaCl, 1% CTAB), and proteinase K final concentration of 1 mg/ml, and was used in Method 2.
  • Buffer 3 is a commercially available buffer, consisting of the 750 ⁇ l bead solution and 60 ⁇ l of Cl solution included in the PowerSoilTM DNA isolation kit MO BIO Laboratories, Carlbad, CA, and was used in Method 3.
  • Each buffer was also tested for its ability to protect chromosomal DNA in the presence/absence of proteinase K (0.5 mg ml '1 ).
  • 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), 1 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.
  • 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 0 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 0 C in the commercial buffer (buffer 3). After 2 days of incubation at these two temperatures, DNA incubated at 37 0 C began to show signs of degradation, whereas DNA stored at 22 0 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.
  • 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).
  • 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).
  • 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-HCl [pH 8.0] and 100 mM NaCl, 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-HCl [pH 8.0], 100 mM sodium EDTA [pH 8.0], 100 mM sodium phosphate [pH 8.0], 1.5 M NaCl, 1% CTAB), proteinase K final concentration of 1 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 0 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 PowerSoftTM kit. General molecular biological techniques (agarose gel electrophoresis, molecular biology buffer and reagent preparation) were described in Sambrook et al. (1989). .
  • ATCC medium Component I: 1249 Modified MgSO 4 2.0 g Baar's medium for Sodium citrate 5.O g sulfate reducers CaSO 4 -LO g
  • 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.
  • 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).
  • 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.
  • Methods 1 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 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.
  • 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.
  • 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.
  • the first set i.e., DsrUniv43F and DsrUniv225R
  • the second i.e., DsrUnivl577F and DsrUnivl712R
  • the third set (DsrlFM 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
  • the fifth set DsrUnivl712F and DsrUniv4RM
  • a binding probe could be used in the PCR reaction to detect the presence of PCR product or otherwise monitor the reaction.
  • 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.
  • 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.
  • 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 Sail, 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 IxTBE buffer (Molecular Probes, Eugene, OR).
  • 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).
  • 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).
  • 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 0 C for 3 min followed by 45 cycles at 95 0 C for 30s, optimal annealing temperature for each primer set (45 0 C for DsrUniv43F and DsrUniv225R, 43 0 C for DsrUnivl577F and DsrUnivl712R, 53 0 C for DsrUnivl712F and DsrUniv4RM and 54 0 C for DsrUnivlFM and DsrUniv225R and DsrUnivlFM and DsrUniv43R) for 30 s, 72 0 C for 30 s and 83 0 C for 10 s during which the fluorescence data were collected.
  • the threshold cycle (Q 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 0 C in 0.5 0 C increments with a dwell time at each temperature of 10 s during which the fluorescence data were collected.
  • DsrUnivlFM and DsrUniv225R (10 ⁇ M each), 1 ⁇ 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-HCl, pH8.3; 3 mM MgCl 2 ; 100 mM of KCl; 0.01% (w/v) gelatin; 0.4 mM of each dCTP, dTTP, dGTP and dATP, 0.3 U/ ⁇ l of Taq polymerase).
  • 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.
  • thermocycler model iQ iCycler equipped with an optical unit (Bio-Rad Laboratories, Hercules, CA) using the following conditions: initial incubation at 95 0 C for 3 min followed by 45 cycles at 95 0 C for 30s, 54 0 C for 30 s, 72 0 C for 30 s and 83°C for 10 s during which the fluorescence data were collected.
  • the threshold cycle (Q 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.
  • 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.
  • 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 t for the given standards. The number of gene copies in a sample is determined by the software by plotting Q value for the sample reaction against the calibration line.
  • 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.
  • Csampie gene copy number in a sample
  • 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.
  • a dye specific for double stranded DNA e.g., SYBR Green
  • 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.
  • 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.
  • the first set i.e., DsrUniv43F and DsrUniv225R
  • the second i.e., DsrUnivl577F and DsrUnivl712R
  • the third set (DsrlFM 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
  • the fifth set DsrUnivl712F and DsrUniv4RM
  • 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.
  • 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.
  • Genomic DNA from up 10 grams of sediment was extracted with a PowerSoilTM 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 cm 3 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
  • 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.
  • the optimal annealing temperatures were determined by gradient PCR with the temperature range between 4O 0 C and 65 0 C using a gradient thermocycler model iCycler (BioRad, Hercules, CA).
  • a gradient thermocycler model iCycler BioRad, Hercules, CA.
  • 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.
  • 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.
  • known quantities of genomic DNA of Desulfovib ⁇ o 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 Sail, 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 0 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).
  • 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).
  • 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).
  • 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 0 C for 3 min followed by 45 cycles at 95°C for 30s, optimal annealing temperature for each primer set (45 0 C for DsrUniv43F and DsrUniv225R, 43 0 C for DsrUnivl577F and DsrUnivl712R, 53 0 C for DsrUnivl712F and DsrUniv4RM and 54 0 C for DsrUnivlFM and DsrUniv225R and DsrUnivlFM and DsrUniv43R) for 30 s, 72°C for 30 s and 83 0 C for 10 s during which the fluorescence data were collected.
  • the threshold cycle (Q 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 0 C in 0.5 0 C increments with a dwell time at each temperature of 10 s during which the fluorescence data were collected.
  • a 20 ⁇ l real-time PCR mixture contained 1.5 ⁇ l of the primer pair DsrUnivlFM and DsrUniv225R (10 ⁇ M each), 1 ⁇ 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-HCl, pH8.3; 3 mM MgCl 2 ; 100 mM of KCl; 0.01% (w/v) gelatin; 0.4 mM of each dCTP, dTTP, dGTP and dATP, 0.3 U/ ⁇ l of Taq polymerase).
  • 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.
  • 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 0 C for 30s, 54 0 C for 30 s, 72 0 C for 30 s and 83°C for 10 s during which the fluorescence data were collected.
  • the threshold cycle (Q 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.
  • 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 t for the given standards. The number of gene copies in a sample is determined by the software by plotting Q value for the sample reaction against the calibration line.
  • E (efficiency) I0- 1/slope (Eq. 1)
  • 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.
  • Csampie gene copy number in a sample
  • Csta n dard gene copy number in a spiked 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.
  • 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 ⁇ 4.3% and ⁇ 5.4%, respectively.
  • 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.
  • 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.
  • the threshold cycle Q method 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.
  • 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).
  • 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 org 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.
  • Swinnex filter holders such as Fisher Catalog Number SXOO 047 00

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Abstract

L'invention concerne un procédé simple pour prélever des échantillons de champ pour une extraction d'acide nucléique et une détection subséquente de micro-organismes provenant de divers milieux tels des échantillons aqueux, des sédiments et des biofilms (bactéries sessiles). Ce procédé est une technique à base de réaction en chaîne de polymérase quantitative (PCR) pour la détection de SRP dans divers milieux (c'est-à-dire des échantillons aqueux, des sédiments, des biofilms). Le principe de cette technique est fondé sur le fait que tous les procaryotes (bactérie et archéobactérie) capables d'effectuer une réduction de sulfate possèdent un gène qui encode la sulfite réductase dissimulatrice (DSR), l'enzyme clé dans le trajet de réduction de sulfate. Une corrosion induite de manière microbienne est due à une croissance bactérienne, et ainsi des tests pour la détection d'une croissance bactérienne dans des fluides impliqués dans le traitement du pétrole sont effectués de manière routinière. Jusqu'à présent, les procédés largement acceptés pour la détection d'une croissance bactérienne de micro-organismes impliquée dans la corrosion dépendent d'une culture et, par conséquent, ces procédés sous-estiment le nombre de micro-organismes présents dans un échantillon, et nécessitent de longues périodes d'incubation.
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WO2019190673A1 (fr) * 2018-03-26 2019-10-03 Buckman Laboratories International, Inc. Méthodes de quantification de la biocontamination dans des substances
CN111902546A (zh) * 2018-03-26 2020-11-06 巴克曼实验室国际公司 量化物质中的生物负荷的方法
JP2021516985A (ja) * 2018-03-26 2021-07-15 バックマン ラボラトリーズ インターナショナル，インコーポレイティド 物質におけるバイオバーデンの定量方法

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