Patent Application
20180298429
The instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 11, 2017, and updated on Mar. 14, 2018, is named 00501-004193-US1_ST25.txt and is 22,480 bytes in size.
The present invention relates to low-density biochips and methods for rapid detection of bacterial organisms involved in microbially influenced corrosion (MIC).
Understanding the structure and composition of microbial communities and their responses to environmental perturbations such as toxic contamination, climate change, and agricultural and industrial practices is important for the maintenance and restoration of desirable ecosystem functions. However, due to the extremely high diversity of organisms, the detection, characterization, and quantification of microbial communities in environmental or industrial samples are formidable tasks for environmental biologists. Traditional culture-based enrichment techniques for studying microbial communities have proven difficult and ultimately, provide an extremely limited view of microbial community diversity and dynamics, because the majority of naturally occurring species cannot be cultured. Even for bacteria that can be cultured, the growth rate of the main MIC-causing bacteria (the sulfate-reducing bacteria (SRB)) typically requires from three to five weeks in culture. The development and application of nucleic acid-based techniques largely eliminated the reliance on cultivation-dependent methods and consequently, greatly advanced the detection and characterization of microorganisms in natural habitats. However, the limitations of conventional nucleic acid-based detection methods prevent them from being readily adapted as high-throughput, cost-effective assessment tools for monitoring complex microbial communities, and especially within the context of MIC.
DNA or oligonucleotide-based microarray technology is a powerful functional genomics tool that allows researchers to view the physiology of a living cell from a comprehensive and dynamic molecular perspective. Compared to traditional nucleic acid hybridization with porous membranes, glass slide-based microarrays, as well as 3-D arrays offer the additional advantages of high density, high sensitivity, rapid (“real-time”) detection, automation, and low background levels. Target functional genes in environments tend to be highly diverse, and it is difficult, sometimes even experimentally impossible, to identify conserved DNA sequence regions for designing oligonucleotide probes for hybridization or primers for polymerase chain reaction (PCR) amplification. The microarray-based approach, however, does not require such sequence conservation, because all of the diverse gene sequences from different populations of the same functional group can be fabricated on arrays and used as probes to monitor their corresponding distributions in environmental samples.
Although microarray technology has been used successfully to analyze global gene expression in pure cultures, it is not clear whether it can be successfully adapted for use in complex and extreme environmental studies with sufficient specificity, sensitivity, and quantitative power [24]. First, in environmental samples, target and probe sequences can be very diverse, and it is not clear whether the performance of microarrays used with diverse environmental samples is similar to that with pure culture samples and how sequence divergence affects microarray hybridization. Second, unlike pure cultures, environmental samples are generally contaminated with substances such as humic matter, organic contaminants, and metals, which may interfere with nucleic acids-based molecular detection. Third, in contrast to pure cultures, the retrievable biomass in environmental samples is generally low. It is not clear whether microarray hybridization is sensitive enough for detecting microorganisms in environmental samples. Finally, since microarray-based hybridization has inherently high variability, it is uncertain whether microarray-based detection can be quantitative. Environmental and ecological studies often require experimental tools that not only detect the presence or absence of particular groups of microorganisms but also provide quantitative data on their in situ biological activities.
Based on the types of probes arrayed, microarrays used in environmental studies can be divided into three major classes: functional gene arrays (FGAs), community genome arrays (CGAs), and phylogenetic oligonucleotide arrays. FGAs contain probes corresponding to genes encoding key enzymes involved in various ecological and environmental processes, such as carbon fixation, nitrification, denitrification, sulfate reduction, and contaminant degradation [1-3]. Both PCR-amplified DNA fragments and oligonucleotides derived from functional genes can be used to fabricate FGAs. Thus, there are PCR product-based FGAs, as well as oligonucleotide-based FGAs. These types of arrays are useful in studying the physiological status and functional activities of microbial communities in natural environments. CGAs are constructed using whole genomic DNA isolated from pure-culture microorganisms and can be used to describe a microbial community in terms of its cultivable component [4]. Phylogenetic oligonucleotide arrays are constructed with short synthetic oligonucleotides from rRNA genes and can be used for phylogenetic analyses of microbial-community composition and structure in environmental samples [5].
Thus, novel microarray-based methods that will allow quantitative and sensitive analysis of microbial communities, especially less abundant populations in environments such as MIC, in a relatively quick and inexpensive manner, are desirable.
In certain embodiments, the present disclosure relates to an oligonucleotide probe set suitable for detection, identification, or quantification of corrosion causing bacteria in a sample, comprising a plurality of probes from Table 1, Table 3, Table 4, Table 5, or any combination thereof. In certain embodiments, the probes can be combined into one or more combinations of cassettes exemplified in Table 2.
In certain embodiments, the probes comprise SRB1, Dv, Dbacterium1, Dbulbus, Dbacter2, Dbacter1, Dsmicrobium1, Dtomaculum1, Dtomaculum2, Dscoccus, Dv 16S_1, MCR1, Archaea1, Archaea2, ARC16SRNA, Geobac, Shew, GeoS, GeoM, ShewRNA1, 16SRNA PR, FTHFS, a/bssA, Firmicutishyd A, E. coli, nirS, nirK, narG, and napA. (SEQ ID NOs: 1; 4; 5-11; 13-18; 20-22; 24-32; 80; and 84).
In certain embodiments, the probes of the set are immobilized at identifiable locations on a biochip.
In certain embodiments, the probe set further comprises a positive control probe 16SCONS_1 (SEQ ID NO:36).
In additional embodiments, the present disclosure relates to a method for determining a bacterial community composition, said method comprising the steps of:
In certain embodiments, the target DNA is single stranded.
In additional embodiments, oligonucleotide probes are immobilized on a solid support.
In additional embodiments, hybridization of at least 21 probes is indicative of corrosion causing bacterial activity.
In additional embodiments, the present disclosure relates to a BioChip comprising oligonucleotide probes from Table 1, Table 3, Table 4, Table 5, or any combination thereof, immobilized on a solid support, for detecting bacteria associated with microbially influenced corrosion (MIC).
In certain embodiments, the bacteria are selected from the group consisting of SRB: (Desulfovibrio, Desulfobacterium, Desulfobulbus, Desolfobacter, Desulfomicrobium, Desulfotomaculum ruminis, Desulfotomaculum, Desulfococcus, Methanogenic Archaea; Archaea); MRB: (Geobacter spp.; Shewanella spp.; G. sulfurreducens; G. metallireducens; Arthrobacter spp.); FB: (Acetogenic, Hydrocarbon-degrading, Firmicutes); E. coli, and NRB.
In certain embodiments, the probes are selected from the group consisting of SRB1, Dv, Dbacterium1, Dbulbus, Dbacter2, Dbacter1, Dsmicrobium1, Dtomaculum1, Dtomaculum2, Dscoccus, Dv 16S_1, MCR1, Archaea1, Archaea2, ARC16SRNA, Geobac, Shew, GeoS, GeoM, ShewRNA1, 16SRNA PR, FTHFS, a/bssA, Firmicutishyd A, E. coli, nirS, nirK, narG, napA, and 16SCONS_1. (SEQ ID NOs: 1; 4; 5-11; 13-18; 20-22; 24-32; 80; 84; and 36).
In certain embodiments, the solid support comprises a 3D-matrix material. In certain embodiments, the 3D-matrix material is dendrimer.
In additional embodiments, the present disclosure relates to a method of preparing a DNA sample for analysis comprising the following steps:
In certain embodiments, step (a) is performed at 25° C. for four hours.
In additional embodiments, fragmenting of the purified and amplified DNA is performed by a restriction enzyme that recognizes T̂TAA site. In certain embodiments, fragmenting of the purified and amplified DNA is performed using fragmentase or SaqAI.
In certain embodiments, hybridization is performed at 25° C. for 4 hours.
In additional embodiments, the present disclosure relates to a kit suitable for performing an assay that detects, identifies and/or quantitates corrosion causing bacteria in a sample, wherein said kit comprises: a) the probes from Table 1, Table 3, Table 4, Table 5, or any combination thereof, or the cassettes of Table 2, and optionally, b) additional reagents or compositions necessary to perform the assay.
In certain embodiments, the corrosion causing bacteria are selected from the group consisting of SRB: (Desulfovibrio, Desulfobacterium, Desulfobulbus, Desolfobacter, Desulfomicrobium, Desulfotomaculum ruminis, Desulfotomaculum, Desulfococcus); Methanogenic Archaea; Archaea; MRB: (Geobacter spp.; Shewanella spp.; G. sulfurreducens; G. metallireducens; Arthrobacter spp.); FB: (Acetogenic, Hydrocarbon-degrading, Firmicutes); E. coli, and NRB.
In certain embodiments, the kit further comprises a positive control probe 16SCONS_1 (SEQ ID NO:36).
In certain embodiments, the additional reagents or compositions comprise one or more of the following: sample buffer, reaction buffer, enzyme mix, Fragmentase reaction buffer, nucleotide mix, 1M GuSCN, 5 mM EDTA, or 50 mM HEPES (pH 7.5).
In yet additional embodiments, the present disclosure relates to an oligonucleotide probe set suitable for detection of bacteria associated with corrosion, comprising at least one or more of the following:
In additional embodiments, the present disclosure relates to an oligonucleotide probe set suitable for detection of combination of bacteria associated with corrosion, comprising at least one or more of the following:
In additional embodiments, the present disclosure relates to a method of detecting corrosion in a sample, said method comprising:
In additional embodiments, the method further comprises increasing the amount of one or more nitrate reducing bacteria (NRB) that comprise nitrate or nitrite reductase genes selected from the group consisting of narG, nirS/nirK, and napA in a surface or liquid environment from which the sample was obtained.
In additional embodiments, the present disclosure relates to a method of detecting corrosion susceptibility in a sample, said method comprising:
In yet additional embodiments, the method further comprises increasing the amount of one or more nitrate reducing bacteria (NRB) that comprise nitrate or nitrite reductase genes selected from the group consisting of narG, nirS/nirK, and napA in a surface or liquid environment from which the sample was obtained.
In yet additional embodiments, the present disclosure relates to a method of detecting corrosion-causing microorganisms in a sample, the method comprising:
In certain embodiments, the one or more reductases comprise a nitrite reductase, and/or a nitrate reductase. In additional embodiments, the one or more hydrogenases comprise a periplasmic NiFe-hydrogenase, a membrane-bound NiFe-hydrogenase, a Fe-hydrogenase, hydrogenase accessory protein HypB, or combinations thereof. In yet additional embodiments, the genes comprise apr; hynAB; hydA (NiFe); hydA (Fe); hypB, or combinations thereof.
In further embodiments, the genes comprise mcr, hydA (NiFe), or a combination thereof.
In further embodiments, the genes comprise mcr; hydA (NiFe); fthfs; a/bssA; hydA (Fe); hyaA; nirS; nirK; narG; napA, or combinations thereof.
In further embodiments, the genes comprise apr; hynAB; hydA (NiFe); hydA (Fe); hypB; hyaA; mcr; fthfs; a/bssA; nirS; nirK; narG; napA, 16S rRNA, or combinations thereof.
In further embodiments, the genes comprise dsr, apr, hynB (NiFe), hydA (NiFe), hydA(Fe), hypB, 16S rRNA, or combinations thereof.
In yet further embodiments, the genes comprise dsr, apr, hynB, hydA(Fe), hydA(NiFe), mcr, fthfs, assA/bssA, hyaA, nirS; nirK; narG; napA, 16S rRNA, or combinations thereof.
In further embodiments, the genes encode one or more of the following: methyl coenzyme M reductase from Archaea; a hydrogenase from metal-reducing bacteria (MRB); fthf, assA/bssA, and hydA from fermentative bacteria (FB), narG, nirS, nirK, and napA from nitrate reducing bacteria (NRB), and hyaA from E. coli.
In further embodiments, the corrosion-causing microorganisms are selected from sulfate-reducing bacteria (SRB), methanogenic archaea, nitrate-reducing bacteria (NRB), metal-reducing bacteria (MRB), fermentative bacteria (FB).
In further embodiments, the corrosion-causing microorganisms are selected from the group consisting of SRB: (Desulfovibrio, Desulfobacterium, Desulfobulbus, Desolfobacter, Desulfomicrobium, Desulfotomaculum ruminis, Desulfotomaculum, Desulfococcus, Methanogenic Archaea; Archaea); MRB: (Geobacter spp.; Shewanella spp.; G. sulfurreducens; G. metallireducens; Arthrobacter spp.); FB: (Acetogenic, Hydrocarbon-degrading, Firmicutes); E. coli, NRB, and combinations thereof.
In further embodiments, the probes comprise one or more of the following: SEQ ID NOs: 1; 4; 5-11; 13-18; 20-22; 24-32; 80; and 84.
A large group of bacteria contribute to MIC of metal products, coatings based on iron, aluminum, manganese, iron, and even stainless steel and plastic and rubber products, which typically are in contact with water in diverse industries, such as the chemical industry (tanks, pipelines), the nuclear industry (pipes and tanks for cooling water), oil and gas industry (underground and surface), the aviation industry (fuel tanks, aluminum one-piece wing tanks), and the marine and shipbuilding industry (accelerated damage of ships and barges).
The economic loss associated with metal corrosion is huge ˜$200 billion per year (loss of time, loss of production, penalty charges, cleaning). The contribution of bacterial corrosion in different areas of the industry is estimated to be 10-77%. Identifying the extremely active microbes to determine potential sites of biocorrosion development and monitoring sites for potential intervention have proved difficult.
Embodiments of the present invention relate to a BioChip, more particularly, a low-density MIC BioChip based system utilizing detection of certain functional genes, whose products are involved in metabolic pathways related to microbially influenced corrosion (MIC). In certain aspects, the low-density MIC BioChip and related methods rely on detecting a combination of reductases and hydrogenases associated with MIC. In additional embodiments, the method further includes detecting nitrate reducing bacteria (NRB) (i.e., their nitrate and/or nitrite reductase genes), which are inhibitors of corrosion. Thus, embodiments of the proposed low-density MIC BioChip will be designed to detect at least 13 functional genes (at least 21 probes, and optionally additional genes as described, as shown in Table 5) encoding hydrogenases and reductases from four groups of sulfate-reducing bacteria (SRB), as well as optional genes encoding methyl coenzyme M reductase from Archaea, genes encoding hydrogenases from metal-reducing bacteria (MRB), genes encoding fthf, assA/bssA, and hydA from fermentative bacteria (FB), genes encoding narG, nirS/nirK, and napA from nitrate reducing bacteria (NRB), as well as hydrogenase gene hyaA from E. coli (as shown in
In additional embodiments, an extended variant of the MIC BioChip has 14 genes: 13 functional genes: apr; hynAB; hydA (NiFe); hydA (Fe); hypB; hyaA; mcr; fthfs; a/bssA; nirS; nirK; narG; napA and 16SrRNA gene (30 probes) (Table 5). 16SrRNA gene and 9 probes for the different bacteria are included as additional verifying features.
Thus, in additional embodiments, the method further includes detecting structural genes based on the 16S rRNA. Examples of such probe sets can be found at least in Table 1, Table 3, Table 4 and Table 5. Probes from any of these tables can be combined, as described below to detect the bacteria of interest as described herein.
The nitrate-reducing population may work to oppose SRB corrosion activity by the following pathways: 1) nitrite (product of nitrate reductase), but not nitrate inhibits the enzyme dissimilatory sulfite reductase [Hubert C., et al. Corrosion risk associated with microbial souring control using nitrate and nitrite. Environmental Biotechnology (2005) 68:272-282]; 2) the intermediates of nitrite reduction by nitrite reductase, nitrous oxide and nitric oxide, increase the ambient redox potential. Biological sulfide production does not occur when the redox potential is above −100 mV, and the growth of SRB can be inhibited by elevation of the redox potential [Postgate J R. The Sulphate-Reducing Bacteria (1979) Cambridge University Press, Cambridge]; 3) the intermediates of nitrite reduction by nitrite reductase inhibits the enzyme hydrogenase [Haruna S., et al. The functional complexity of [NiFe] hydrogenases in sulfate reducing bacteria (genus; Desulfovibrio spp). American J Bioscience & Bioengineering (2014) 2:1-7]; 4) soil sulfate reduction does not begin before the environment becomes exhausted of nitrates [He Q., et al. Energetic consequences of nitrite stress in Desulfovibrio vulgaris Hildenborough, inferred from global transcriptional analysis. Applied and Environ Microbiology (2006) 72:4370-4381]. Therefore, the detection the nitrate reductase genes (narG, napA) and nitrite reductase genes (nirk, nirS) using the MIC Biochip described herein works to characterize the status of nitrate-reducing population (NRB). The corresponding probes from the genes narG, napA, nirk and nirS are provided in Table 5.
In certain embodiments, the present invention provides for simultaneous detection of target nucleic acids in a sample. One of the major challenges in designing DNA probes that can be used simultaneously with other DNA is the variation in the conditions necessary for proper hybridization between the DNA probe and nucleic acid target, requiring each hybridization reaction to be carried out individually. Thus, probes that can be used simultaneously require identical hybridization characteristics. In aspects of the present invention, inventors used a novel method to evaluate BioChip probes in one reaction, using DNA cassettes, comprising all of the probes in equimolar proportions for a quick determination of the probes unsuitable for inclusion in the pilot biochip [23]. This allowed for the design of probes with identical hybridization characteristics. Thus, embodiments of the pilot BioChip of the present disclosure offer the ability to achieve simultaneous detection of many DNA targets without detriment to sensitivity.
Additional embodiments of the present invention include using the low-density MIC BioChip to obtain an estimated MIC microorganism ratio based upon detecting selected gene products from bacteria which contribute to corrosion, in combination with gene products from bacteria that inhibit or resist corrosion (e.g., nitrate reducing bacteria (NRB) and their nitrate and/or nitrite reductase genes). Based on these ratios, a corrosion scale can be developed based upon the quantity of MIC sustaining, inducing, or opposing bacteria present in the sample. In certain aspects, the present invention relates to estimating MIC microorganism ratios based upon detecting the select genes from bacteria which contribute to corrosion, in combination with genes from bacteria that inhibit or resist corrosion. For example, the MIC Biochip can detect and illustrate that Group I bacteria (Group I consists of Desulfovibrio and Desulfobacterium) dominate in the SRB (mostly, the corrosion of underground steel structures is caused by the genus Desulfovibrio), and if the signals from all the other bacterial groups are less, it means that this area is at the high-risk of corrosion. If detection of the bacteria from Groups II, III, IV are high, and Group I bacteria exhibit little or no signal, it indicates that this sample location/place is in the medium-risk of corrosion. The lowest risk areas are the places where the Group IV bacteria dominate. The use of the MIC biochip will allow for detecting the linkage between the bacterial diversity and degree of corrosion. On the basis of these results index maps of corroding regions can be developed and serve as a prognostic tool.
An aspect of the present invention relates to evaluating SRB effects on corrosion by dividing the SRB into four groups and detecting one or more of the following hydrogenase genes using the MIC BioChip (as shown in
The minimum gene panel for detecting the SRBs consists from 5 genes apr; hynAB; hydA (NiFe); hydA (Fe); hypB; (10 probes) (Table 5). The microbial populations in addition to SRB, actively participating in biocorrosion processes, are Methanogenic Archaea and metal-reducing bacteria (MRB) (
Thus, embodiments of low-density MIC BioChips and related methods provide an improved approach for determining the bacterial community contributing to potentially corrosive conditions in a variety of drilling/water/storage environments. These methods and BioChip compositions/arrays provide for high specificity while utilizing fewer probes. Advantages associated with these methods include speed of detection (e.g. 2-3 days); no requirement for cultures; unique bacterial community quantification; and an alternative to expensive chips containing thousands of genes. Further advantages also include simultaneous detection of these distinct genes based at least in part upon probe and hybridization optimizations as described herein.
The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to describe a polymer of any length, e.g., greater than about 2 bases, greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than 1,000 bases, up to about 10,000 or more bases composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, which can hybridize with nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleotides, e.g., can participate in Watson-Crick base pairing interactions. Naturally-occurring nucleotides include guanine, cytosine, adenine and thymine (G, C, A and T, respectively).
The terms “hybridize” or “hybridization”, as used herein, refer to the binding or duplexing of a nucleic acid molecule to a particular nucleotide sequence under suitable conditions.
The term “complementary”, as used herein, refers to a nucleotide sequence that base-pairs by non-covalent bonds to a target nucleic acid of interest. In the canonical Watson-Crick base pairing, adenine (A) forms a base pair with thymine (T), as does guanine (G) with cytosine (C) in DNA. In RNA, thymine is replaced by uracil (U). As such, A is complementary to T and G is complementary to C. In RNA, A is complementary to U and vice versa. Typically, “complementary” refers to a nucleotide sequence that is fully complementary to a target of interest such that every nucleotide in the sequence is complementary to every nucleotide in the target nucleic acid in the corresponding positions. In certain cases, a nucleotide sequence may be partially complementary to a target, in which not all nucleotide is complementary to every nucleotide in the target nucleic acid in all the corresponding positions.
The term “probe,” as used herein, refers to an oligonucleotide that is complementary and hybridizes to a nucleotide sequence of interest, typically to facilitate its detection.
The term “solid support” as used herein refers to any solid material able to bind oligonucleotides, e.g. by hydrophobic, ionic or covalent interaction.
The term “immobilisation” as used herein refers to reversible or irreversible association of the probes to said solid support, such as 3D matrix. If reversible, the probes remain associated with the solid support for a time sufficient for methods of the invention to be carried out.
The term “environmental sample” as used herein refers to any substance comprising bacterial community. As used herein, environmental samples include water and oil samples that comprise bacterial populations of varying genus and species that may be identified by the low density MIC BioChip described here. The environmental samples may comprise a bacterial consortium unique to a geographic region or target reservoir, or, alternatively the bacterial consortium may be adaptable to other environment sites, geographies and reservoirs. The methods of the present invention are suitable for analyzing the presence of MIC microorganism(s) in a sample originating from an oilfield or an oil well or from an oil production process, or any related equipment, surface, or systems, storage tanks or pipes.
High density microarray and low density biochips. DNA biochips are divided into two major classes: high density microarray and low density biochips. Microarrays typically contain thousands and tens of thousands probes. Low density biochips usually contain tens and hundreds probes. The proposed “low-density” MIC BioChip described herein aims to detect the bacterial consortium and to feature the risk assessment using the minimum panel, consisting of from the following embodiments: 13 genes/21 probes, including 5 genes (apr; hynAB; hydA (NiFe); hydA (Fe); hypB)/10 probes required for accurate detection of SRBs; and 10 genes (mcr; hydA (NiFe); fthfs; a/bssA; hydA (Fe); hyaA; nirS; nirK; narG; napA)/11 probes for accurate detection of the next preferred members. (Table 5) Extended panel consists from 14 genes/30 probes (Table 5). In certain embodiments, additional combinations Therefore, the MIC-biochip can be considered a low density biochip.
The term “hairpin structure” as used herein refers to a polynucleotide or nucleic acid that contains a double-stranded stem segment and a single-stranded loop segment wherein the two polynucleotide or nucleic acid strands that form the double-stranded stem segment are linked and separated by the single polynucleotide or nucleic acid strand that forms the loop segment. The “hairpin structure” can further comprise 3′ and/or 5′ single-stranded region(s) extending from the double-stranded stem segment.
The term “label” as used herein refers to any atom or molecule which provides a detectable (preferably quantifiable) effect and which can be attached to a nucleic acid. The term “label” includes e.g. colored dyes; radioactive labels; binding moieties such as biotin; haptens such as digoxgenin; luminogenic, phosphorescent or fluorogenic moieties; and fluorescent dyes alone or in combination with moieties that can suppress or shift emission spectra by the energy transfer of fluorescence. Labels may provide signals, which are detectable for example by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism and enzymatic activity. A label may be a charged moiety (positive or negative charge) or may also have a neutral charge. They may include or consist of nucleic acid or protein sequence. Preferred labels are dyes and in particular preferred labels are fluorescent dyes. The amplification and the labeling can be performed either simultaneously or in subsequent steps.
The present invention also provides kits comprising the components of the combinations of the invention in kit form. A kit of the present invention includes one or more components including, but not limited to, one or more probes of the present disclosure, wherein one or more probes are used for detection of corrosion causing bacteria. In one embodiment, the kit of the present invention includes one or more components including, but not limited to, one or more probes of the present disclosure, wherein one or more probes are used for detection of target nucleic acid in a sample. In one embodiment, the kit includes a matrix comprising nucleic acid probes for detecting a labeled target nucleic acid in a sample. The nucleic acid probes are embedded in the matrix and comprise nucleotide sequences that are substantially complementary to one or more labeled nucleotide sequences in the target nucleic acid.
As a matter of convenience, one or more probes disclosed herein can be provided in a kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic or detection assay. Additives may be included into the kits, such as reagents used for DNA purification, DNA amplification, DNA fragmentation, DNA labeling, and/or DNA hybridization. In some embodiments, the kits of the present invention include sample buffer, reaction buffer, enzyme mix, Fragmentase reaction buffer, nucleotide mix, 1M GuSCN, 5 mM EDTA, or 50 mM HEPES (pH 7.5). The relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents which substantially optimize the sensitivity of the assay. Particularly, the reagents may be provided as dry powders, usually lyophilized, including excipients which on dissolution will provide a reagent solution having the appropriate concentration.
The kit's components may be pre-attached to a solid support, or may be applied to the surface of a solid support when the kit is used. In some embodiments, the signal generating means may come pre-associated with a probe of the invention or may require combination with one or more components. Optionally the kit may also comprise instructions for carrying out the methods of the invention.
The detection kits disclosed herein may also be prepared that comprise at least one of the probes disclosed herein and instructions for using the composition as a detection reagent. Containers for use in such kits may typically comprise at least one vial, test tube, flask, bottle, syringe or other suitable container, into which one or more of the detection and/or therapeutic composition(s) may be placed, and preferably suitably aliquoted. Where a second detection agent is also provided, the kit may also contain a second distinct container into which this second detection composition may be placed. Alternatively, a plurality of compounds may be prepared in a single composition, and may be packaged in a single container means, such as a vial, flask, syringe, bottle, or other suitable single container. The kits of the present invention will also typically include a means for containing the vial(s) in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vial(s) are retained. Where a radiolabel, chromogenic, fluorogenic, or other type of detectable label or detecting means is included within the kit, the labeling agent may be provided either in the same container as the detection or therapeutic composition itself, or may alternatively be placed in a second distinct container means into which this second composition may be placed and suitably aliquoted. Alternatively, the detection reagent and the label may be prepared in a single container means, and in most cases, the kit will also typically include a means for containing the vial(s) in close confinement for commercial sale and/or convenient packaging and delivery.
In accordance with the present invention, there may be numerous tools and techniques within the skill of the art, such as those commonly used in molecular immunology, cellular immunology, pharmacology, and microbiology. See, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor. N.Y.; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, N.J.; Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, N.J.; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, N.J. Production, purification, and fragmentation of polyclonal and monoclonal antibodies are described (Coligan, et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Harlow and Lane, supra). Standard techniques for characterizing ligand/receptor interactions are available (see, e.g., Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).
CGA: community genome arrays
FB: fermentative bacteria
FGA: functional gene arrays
MIC: microbially influenced corrosion
MRB: metal-reducing bacteria
NRB: nitrate-reducing bacteria
SRB: sulfate-reducing bacteria
Recent advances in microarray technology have allowed the development of nucleic acid microarrays that are customizable for a specific assay. This methodology provides for the attachment of a desired number of specific nucleic acid sequences to a miniaturized immobilized support creating a nucleic acid microarray. Subsequently, these microarrays then may be used to screen the genetic sequences of a given field sample. Selected sequences to be analyzed may be submitted to a company that has the technology to manufacture such nucleic acid microarray chips. Such companies include, but are not limited to Affymetrix, inc. or Eppendorf International. Affymetrix Inc. has developed a high density GENECHIP® Microarray and Eppendorf has created a low density SILVERQUANT® Microarray that allows for the high throughput quantification of specific mRNA. In this sense, density refers to the number of features (genetic sequences at each location), where high density refers to a greater number of features relative to low density. While higher density systems may monitor more genes in comparison to the relatively lower density systems, they may be significantly more expensive. The building of such microarrays is known, and is exemplified in U.S. Pat. Nos. 7,115,364, 7,205,104, and 7,202,026. The process generally involves photolithographic binding or spotting of target nucleotide sequences to a customized DNA microarray. Such a DNA microarray could be used to correlate fluorescent values corresponding to hybridized mRNA or DNA with quantification using target sequences from specifically identified strains to probe field samples. It is noted that in certain embodiments, a field sample mRNA may directly hybridize with target sequence or it may be converted into cDNA (complementary DNA which is more stable) to be hybridized with a target sequence.
In certain embodiments, the extended panel for a low-density MIC BioChip (30 probes) comprises a biochip based on the targeted functional gene detection and structural gene detection (16S rRNA) of 30 total probes, as shown in Table 5. In certain embodiments, an extended variant of the BioChip contains 14 genes: 13 functional genes (apr; hynAB; hydA (NiFe); hydA (Fe); hypB; hyaA; mcr; fthfs; a/bssA; nirS; nirK; narG; napA) and 1 structural gene (16SrRNA) (30 probes) (shown in Table 5). In certain embodiments, the 16SrRNA gene and 9 probes for the different bacteria are included as additional verifying features.
In additional embodiments, the low-density MIC BioChip will be in the form of an FGA.
Many groups of bacteria commonly present in oil beds directly or indirectly contribute to the process of biogenic corrosion. Sulfate-reducing bacteria (SRB) is one of the groups of bacteria most often identified as being involved in and contributing to corrosion. The SRB can be divided into six main groups based on 16S rRNA gene sequences:
8H2O→8H++8OH— (water ionization)
4Fe→4Fe2++8e− (anodic site, iron ionization)
8H++8e−→8H (the formation of a protective film at the cathode, preventing the further dissolution of the metal).
Cathodic depolarization takes place in the presence of SRB:
SO42−+8H→S2−+4H2O
In addition, the secondary reactions proceed:
Fe2++S2−→FeS
3Fe2++6OH−→3Fe(OH)2
The summary corrosion reaction is the following:
4Fe2++SO42−+4H2O−→FeS+3Fe(OH)2+2OH−
Depolarizing activity of SRB increases significantly due to the formation of hydrogen sulfide at cathodic site in the reaction:
2H++S2−→H2S
Frequent depolarization (consuming polarized hydrogen) leads to stimulation of the cathode area. In addition, the formation of the new cathodes, which are the end products of sulfate reduction to insoluble sulfides of iron and other metals, stimulates the cathode area. In view of these reactions, the following processes accelerate microbial corrosion:
The detection of SRB is an important step in MIC monitoring. As an aspect of the present invention, detection of functional genes, dissimilatory sulfite reductase (dsr) and adenosine 5′-phosphosulfate reductase (apr) (genes directly associated with the reduction of inorganic sulfate) is proposed as part of a Low Density MIC BioChip. The apr probe can be successfully used for screening of a wide spectra of sulfate-reducing bacteria: Desulfovibrio indonensis, Desulfovibrio alaskensis, Desulfovibrio vietnamensis, Desulfovibrio vulgaris, Desulfovibrio gigas, Desulfovibrio desulfuricans, Desulfomicrobium baculatus, Desulfococcus multivorans, Desulfobulbus propionicus, Desulfofrigus fragile, Desulfofrigus oceanense, Desulfotalea psychotrophila, Desulfotalea arctica, Desulfofada gelida, Desulfocinum infernum, Desulfotomaculum nigrificans, Desulfosporosinus orientis [10]. Additionally, two dsr gene probes can be used for the identification of SRB in the environmental samples [11].
As another aspect, it is proposed that detecting SRB be based in part on a division into groups I-IV on the basis of specific hydrogenase genes. The hydrogenase genes along with reductase genes of SRB are the genes encoding enzymes which largely contribute to MIC development, since biocorrosion is accelerated by hydrogenases utilizing cathodic hydrogen.
Bacterial hydrogenases can be placed into three broad categories based on the metal cofactors found at their active sites: (Fe) hydrogenases; (NiFe) hydrogenases, and (NiFeSe) hydrogenases. (Fe) hydrogenases are characterized by low affinity to hydrogen and work preferentially at its high concentration. (NiFeSe) hydrogenases are characterized by high affinity to hydrogen. (NiFe) hydrogenases can compensate for the absence of activity of two other types of hydrogenases. Hydrogenases also differ in their susceptibility to inhibitors. (Fe) hydrogenases are the most sensitive to CO and NO2−, as well as (NiFeSe) hydrogenases. (NiFe) hydrogenases are particularly resistant to inhibitors such as CO and NO2−, which are the main products of metabolism of bacterial community in the anaerobic conditions. (NiFe) hydrogenases are divided into 4 groups: 1) periplasmic proteins; 2) soluble cytoplasmic proteins; 3) bilateral membrane-bound proteins; and 4) membrane-bound outer cytoplasmic proteins.
An aspect of the present invention relates to evaluating SRB effects on corrosion by dividing the SRB into four groups and detecting one or more of the following hydrogenase genes (as shown in
Group I consists of Desulfovibrio and Desulfobacterium. Mostly, the corrosion of underground steel structures is caused by the genus Desulfovibrio. Overview of the hydrogenase genes in SRB reveals that all studied Desulfovibrio spp. possesses the genes of periplasmic (NiFe) hydrogenase (hynAB-genes), containing the highly homologous domain. The occurrence of (Fe) and (NiFeSe) hydrogenase in Desulfovibrio spp is limited [14]. Furthermore, (NiFe) hydrogenases are particularly resistant to inhibitors such as CO and NO2−, which are the main products of metabolism of bacterial community in the anaerobic conditions. The use of periplasmic (NiFe) hydrogenase gene (hynAB) strongly suggests the detection of Desulfovibrio spp. in SRB consortium. Desulfobacterium spp. possesses hynB periplasmic (NiFe) hydrogenase gene as well. The specific probe is designed on the basis of hynB gene of Desulfobacterium sp. In addition, two probes for the identification of SRB on the basis of the structural 16S rRNA genes are selected from the literature [Loy A., et al., Oligonucleotide microarray for 16S rRNA gene-based detection of all recognized lineages of sulfate-reducing prokaryotes. Appl Environ Microbiol (2002) 68:5064-5081]. These probes allow confirming the presence of Desulfovibrio spp. and Desulfobacterium spp. by the specific structural gene probes.
Group II consists of Desulfobulbus, Desulfobacter and Desulfomicrobium. They are characterized by the sequentially distinguished (NiFe) membrane-bound hydrogenase (hydA).
Group III comprises of Desulfotomaculum spp. These bacteria are gram-positive bacteria unlike other groups; this is reason why sulfate reduction occurs without periplasmic hydrogen oxidation. All hydrogenases are Fe-containing, excluding Desulfotomaculum ruminis, possessing (NiFe) hydrogenase. Corrosion is caused by bacterial genus Desulfotomaculum at high temperatures.
Group IV consists of Desulfococcus. This group can be discriminated by hydrogenase accessory protein HypB gene (hypB). Desulfococcus group is the less dangerous group of bacteria involved in steel corrosion as this group predominantly uses organic substrates as electron donors thereby not accelerating the cathodic depolarization.
In view of these correlations, detecting at least the above mentioned panel of 6 functional genes and 1 structural gene (16S rRNA) in a functional array (FGA) using the low-density MIC BioChip enables identification of the main SRB groups involved in biocorrosion (dsr, apr, hynAB (NiFe), hydA (NiFe), hydA(Fe), hypB, 16S rRNA). 12 probes were selected for identification of main SRB groups, 10 for functional genes and 2 for structural genes (Table 1, probes 1-12, e.g., SEQ ID NO:1-12). 7 probes for functional genes were designed de novo.
The selection of these microorganisms, whose metabolic products contribute to the SRB life cycle, is very beneficial for the risk assessment and monitoring of biocorrosion.
Desulfobvibrio
Desulfo-
bacterium
Desulfobulbus
Desolfobacter
Desulfo-
microbium
Desulfo-
tomaculum
Desulfococcus
Desulfobibrio
Desulfovibrio
Dv. vulgaris,
Dv. desuluricans)
Desulfo-
bacterium
Desulfobacterium
Archaea
Methanogenic
Archaea
Archaea
Archaea
Archaea
Geobacter spp
Shewanella spp
Geobacter spp
G.
sulfurreducens
G. metalli-
reducens
Shewanella spp
Shewanella spp
Firmicutes
E.coli
In addition to detecting the genes identified above relating to the SRB community, aspects of the present invention also may include detecting one or more additional genes from the following groups which contribute to accelerating biocorrosion processes:
An important factor of SRB survival is interspecies interactions with methanogens. In the absence of sulfates, SRB perform the transfer of hydrogen to other microorganisms, acting as acceptors. The genus Desulfovibrio and methanogens (functioning satellites of SRB) accomplish the transfer of hydrogen by hydrogenases, localized in the periplasmic space of the membranes.
It will be beneficial under certain conditions, to detect certain sulfate-reducing bacteria and methanogens in a monitoring program for MIC risk assessment. Monitoring programs can be expanded and improved by including the metabolic relationship of a consortium of microorganisms that significantly contribute to corrosion. Under certain conditions, the following microorganisms can be included in the monitoring of MIC risk assessment as sustaining MIC microorganisms:
A unique assortment of specific probes for low-density MIC BioChips have been identified as described herein, and in part by focusing on the cathode and anode reactions and ranking functional genes to evaluate and detect a panel of microorganisms most likely to influence MIC. Certain groups of the enzymes coded for by these functional gene probes accelerate the electrochemical processes in biocorrosion. While not wishing to be bound by theory, the microorganisms with active and developed hydrogenase systems are targeted; and in certain embodiments include detecting Desulfovibrio spp. Detection by using hydrogenase genes hynAB, hydA, and by hydrogenase accessory protein HypB gene hypB is expected to provide the ratio of sulfate-reducing microorganisms within the MIC consortium.
In certain aspects, the presently described low-density MIC biochip provides data for assessing not only the biocorrosion risk, but also estimates alternative processes such as nitrate reduction. In other aspects, the low-density MIC biochip of the present invention will provide accurate data and more comprehensive coverage of the relevant organisms involved in MIC inducing, sustaining and opposing bacteria providing a corrosion scale for the bacterial community.
In some aspects, the present invention relates to an informative arrangement of 30 oligonucleotide probes, with variations in the subsets of probes to be used for various scenarios as described herein, immobilized on an MIC biochip, such that upon hybridization with oligonucleotides in a test sample, a pattern is produced that can be interpreted with a suitable means.
In one aspect, the MIC BioChip described herein is a collection of miniaturized test sites arranged on a solid substrate that permits numerous tests to be performed simultaneously in order to achieve higher throughput and speed. MIC BioChip of the present invention comprises an arrayed series of microscopic spots of DNA oligonucleotides, each containing picomoles (10−12 moles) of a specific DNA sequence, referred to as probes. The probes are synthesized prior to deposition on the array surface and are then immobilized onto a solid support (matrix).
In additional aspects, the MIC BioChip provides DNA oligonucleotide probes for key genes of metabolic pathways involved in MIC. The resulting MIC BioChip pilot comprises 30 highly specific and sensitive probes, wherein 11 probes enable identification of key SRB groups involved in the biocorrosion and allow for grading SRB groups on the corrosion scale, 18 probes are used for the identification of other MIC bacterial groups, one probe serves as positive control.
In another aspect, the present invention provides a method for detecting binding between a probe and a target sample, comprising applying the target sample including a target DNA to be bound to the oligonucleotide probe onto the MIC BioChip and detecting the target DNA specifically bound to the oligonucleotide probe. In one embodiment, fragments of DNA are labeled with a signaling substance, such as a fluorescent dye for easy detection of the target DNA. The binding between an oligonucleotide probe and a target DNA can be detected by a variety of methods, for example, a fluorescent detection method, an electrochemical detection method, a mass detection method, a charge detection method, or an optical detection method, which are currently in wide use and are classified according to the type of the signaling substance labeled to the target substance.
More specifically, the present invention provides a low-density MIC BioChip comprising 30 probes proposed for the identification of main bacterial groups involved in biocorrosion. The MIC BioChip of the invention includes 18 de novo probes based on the functional genes and 4 de novo probes based on structural genes. Structural 16S rRNA genes were used for the probe design, based on conserved regions of 16S rRNA gene which will capture as many bacterial species as possible on a biochip and also serve as positive controls, in certain instances.
In certain embodiments, the present invention provides for simultaneous detection of target nucleic acids in a sample. One of the major challenges in designing DNA probes that can be used simultaneously with other DNA is the variation in the conditions necessary for proper hybridization between the DNA probe and nucleic acid target, requiring each hybridization reaction to be carried out individually. Thus, probes that can be used simultaneously require identical hybridization characteristics. In the present invention, inventors used a novel method to evaluate BioChip probes in one reaction, using DNA cassettes, comprising all of the probes in equimolar proportions for a quick determination of the probes unsuitable for inclusion in the biochip [23]. This allowed for the design of probes with identical hybridization characteristics. Thus, the BioChip of the present disclosure offers the ability to achieve simultaneous detection of many DNA targets without detriment to sensitivity.
The inventors of the present disclosure initially designed and evaluated the activity of 38 probes (SEQ ID NO:1-38) described in Table 1, which were then subjected to experimental analysis for their hybridization capacity and sensitivity for inclusion in the pilot MIC BioChip, after the design of four test pilot BioChips.
Numerous steps are involved in the design and generation of an MIC BioChip as described herein. In general, the development of an MIC BioChip includes a combination of any of the following steps:
More specifically, designing and developing an MIC BioChip as described herein, includes any of the following: probe design, biochip matrix preparation, activation of the surface for the immobilization of oligonucleotide probes, immobilization of oligonucleotide probes onto the activated matrix, and deactivation of matrix. Additionally, several levels of analyses were completed for generation of oligonucleotide probes to be immobilized onto the matrix. Finally, preparation of target DNA and the hybridization of the target DNA is a multistep process that has also been optimized, as described herein.
General steps involved in target DNA preparation for the hybridization on a biochip are the following:
a) DNA amplification, fragmentation and labeling; and
b) Hybridization and visualization.
A wide variety of supports may be used with the invention. In one aspect, supports are rigid solids that have a surface, preferably a substantially planar surface so that single molecules to be interrogated are in the same plane. The latter feature permits efficient signal collection. Suitable solid support materials include materials such as glass, polyacrylamide-coated glass, ceramics, silica, silicon, quartz, various plastics, and the like. In one aspect, the area of a planar surface may be in the range of from 0.5 to 4 cm2. In one aspect, the solid support is glass or quartz, such as a microscope slide, having a surface that is uniformly silanized. This may be accomplished using conventional protocols, e.g. acid treatment followed by immersion in a solution of 3-glycidoxypropyl trimethoxysilane, N,N-diisopropylethylamine, and anhydrous xylene (8:1:24 v/v) at 80° C., which forms an epoxysilanized surface.
Solid support (matrix) used for the microarray/biochips can be generally divided into two main formats: two-(2D) and three-dimensional (3D). Typical 2D support constitutes glass slides (silanized and subsequently activated or modified by aldehyde, epoxy, activated carboxyl groups etc.) modified to couple with the DNA probes. DNA density on these coated substrates cannot exceed a monolayer that limits the absolute fluorescent signal intensity during biochip visualization. A 3D format considerably increases the available surface area and allows the deposition of higher probe quantities, promoting a higher sensitivity of the biochip analysis. Example of 3D format support is polyacrylamide gel-pads affixed on glass slides and activated to couple with DNA probes [17].
Another example of 3D structures is dendrimer support, which can be formed by the glass slide treatment in a special manner. Dendrimers are repetitively branched polymeric molecules chemically synthesized with well-defined shapes, size and nanoscale physicochemical properties [18]. In contrast to traditional polymers, dendrimers are unique core-shell structures possessing three basic architectural components: a core, an interior of shells (generations), and an outer shell or periphery consisting of branch cell units and terminal functional groups [19, 20]. Identical monomer units bind repeatedly around a core, sequentially building tree architecture of the polymer. Each of these layers between the core and the periphery is called generation. The generation rises after every additional interaction by a sequence of repetitive reactions. The surface of the dendrimer contains reactive terminal groups to perform a variety of functions.
Thus, according to the methods of the present invention, both 2D and 3D solid support matrices can be used to generate a BioChip of the present invention.
The inventors of the present disclosure tested two types of 3D matrices and one type of commercially available 2D matrix (Vantage aldehyde slides, Arrayit, USA) for their coupling and hybridization sensitivity (Example 1). As shown in Example 1, dendrimeric matrix was found to be the most sensitive platform. The main advantages of the dendrimeric matrix include: 1) higher sensitivity compared to that of gel matrix (approximately 2 times); and 2) following the stripping of the surface from the hybridized targets, the surface can be re-used.
A nucleic acid hybridization is a highly specific and sensitive procedure. When designing a probe, it is critical to ensure that the design meets the following well established requirements: (i) probe is specific to the target agent, (ii) predicted melting temperatures (Tm) of the probes are homogeneous, (iii) the length of oligonucleotides, preferably between 18 and 25 bases, is homogeneous, and (iv) the predicted temperature of hairpin formation is above or below temperature of hybridization.
Hybridization kinetics of nucleic acids is temperature dependent, and the specificity and efficiency depend on the hybridization temperature. One of the main difficulties with designing oligonucleotide microarrays is to achieve nearly identical melting temperatures for all probes on the array. There are several approaches for equalizing the probes' hybridization ability. The oligonucleotide probes should be designed with the same predicted melting temperature (±2° C.) by using the algorithm based on the nearest neighbor model [21].
Options and challenges associated with hybridization efficiency have been described and addressed in the field (See, for example: Yilmaz, L. S. and Noguera, D. R. (2004) “Mechanistic Approach to the Problem of Hybridization Efficiency in Fluorescent in situ Hybridization”, Applied and Environmental Microbiology, 70: 7126-7139. Yilmaz, L. S., Okten, H. E., and Noguera, D. R. (2006) “All Regions of the 16S rRNA of Escherichia coli are Accessible in situ to DNA Oligonucleotides with Sufficient Thermodynamic Affinity”, Applied and Environmental Microbiology, 72: 733-744. Yilmaz, L. S. and Noguera, D. R. (2007) “Development of Thermodynamic Models for Simulating Probe Dissociation Profiles in Fluorescence in situ Hybridization”, Biotechnology and Bioengineering, 96 (2): 349-363. Yilmaz, L. S., Bergsven, L., and Noguera, D. R. (2008) “Systematic Evaluation of Single Mismatch Stability Predictors for Fluorescence in situ Hybridization”, Environmental Microbiology, 10(10):2872-2085.) However, there remains much room for improvement of hybridization efficiency in the context of multiple probes on a biochip, as described herein below.
A critical step in biochip design is the selection of probes with identical hybridization characteristics. The theoretical profiles of probes, however, are often inconsistent with experimental data, and this can generate BioChips with irregular signal responses. In the present invention, a novel method was used to evaluate BioChip probes in one reaction, using DNA cassettes, comprising all of the probes in equimolar proportions for a quick determination of the probes unsuitable for inclusion in the biochip [23].
The evaluation of the hybridization potential of each of the 38 probes was accomplished using 16 ss (single strand) cassettes. ss cassettes present a lineal set of sequences complimentary to the studied set of probes (Example 2). The Cy3 fluorescent dye was inserted at 5′-end of ss cassette during the synthesis. The number of probes included in each cassette ranged from 3 to 8, and the size varied from 65 to 142 bases. Table 2 lists composition of ss cassettes. The variations in the fluorescent intensities on a biochip, coming from probe's size and nucleotide sequence differences were determined by the cassette method. Using this approach, the inventors identified 35 probes with similar fluorescent intensities (or binding capacity) and chose them from the initial 38 probes (Table 1) for the identification of main bacterial groups involved in the biocorrosion (Example 2, Table 3).
The application of microarrays to assessment of microbes in the environmental samples poses a number of technical challenges. One of the main challenges is the specific detection of target nucleic acids against a complex background of non-target sequences. The difficulty of the microarray approach is that all probes are hybridized simultaneously, but specific hybridization conditions often vary between probes. The extended ssDNA approach based on the hybridization of the short (100-200 bases) single stranded ssDNA (PCR fragments) with the appropriate oligonucleotide probe on the biochip was developed and used to evaluate hybridization efficiency (Example 3). This method allows for the preparation of samples in a manner that provides optimal hybridization signal intensity.
Sensitivity is another critical parameter for biochip application. The sensitivity of biochip depends on many factors, such as the quantity of the probes, on the biochip matrix (3-D array format vs. 2-D array format), and the quantity of the labeled target DNA. The pilot MIC BioChip exhibits improved sensitivity in part based on embodiments that are in the 3-D array format. Test results evaluated coupling and hybridization sensitivity of 3 matrixes: 3D gel- and dendrimeric matrixes and one type of commercially available 2D matrix (aldehyde slides, Arrayit, USA). It was shown, that the coupling capacity of the 3D-dendrimeric matrixes is 6 times higher than 3D gel-matrixes. Coupling capacities of 3D gel-matrixes and 2D matrixes are comparable. With respect to the hybridization with the single stranded compliment, the aldehyde slides show the lowest hybridization sensitivity; the hybridization characteristics of both 3D matrixes are comparable and are 3-4 times higher than 2D matrixes.
Target DNA amplification prior to hybridization results in stronger signals and allows detecting specific targets even if they are present in low abundance. The steps of random fragmentation of DNA and its fluorescent labeling precede the hybridization of DNA fragments with the immobilized probes onto the matrix for the biochip visualization. The inventors have developed ten protocols with different combinations of DNA amplification, fragmentation and labeling procedures and tested them by using pure bacterial cultures (Desulfovibrio indonensis and E. coli BP) and the environmental samples (Example 4). The most effective procedure was then used for analysis of environmental samples on different biochip prototypes.
1. 1st target DNA amplification
2. Fragmentation of amplified target DNA
3. 2nd amplification and labeling simultaneously.
Thus, in one embodiment, amplified target DNA is fragmented according to any standard method known to a skilled person into smaller fragments. In the following step, the fragmented fragments are subjected to second amplification step and labeling simultaneously (Example 4). Subsequently, the fragments of labeled target DNA are subjected to a hybridizing reaction in order to obtain hybridization between the target DNA and the oligonucleotide probes present in the low density MIC BioChip. These hybridizations can be carried out according to the general knowledge in the art by a skilled person. A number of different methods of DNA preparation for the hybridization on a biochip were tested and compared and they include: 1) 1st amplification and labelling simultaneously and then fragmentation, 2) 1st amplification, fragmentation, and then labelling without 2nd amplification etc. However, not one of the other nine combinations of steps showed the highly detectable fluorescent signals and clear background as the method described above (1st amplification, fragmentation, 2nd amplification and labelling simultaneously).
By designing and testing protocols with different combinations of DNA amplification, fragmentation, and labeling procedures, the inventors have developed an efficient method for amplification of target DNA, referred to as N10 protocol (Example 4,
Finally, the inventors tested a biochip prototype using Desulfobacterium autotropicum DNA (Example 5) and showed that they can obtain successful specificity (
The inventors of the present disclosure generated two different types of 3D matrices: Gel- and two generations Dendrimeric matrices. Two types of 3D matrices and one type of commercially available 2D matrix (Vantage aldehyde slides, Arrayit, USA) were tested for their coupling and hybridization sensitivity. 5′-end of the probe was chemically modified during the synthesis by C6-Amine-group for the 2D aldehyde matrix and 3D dendrimeric matrices. 3′-end was chemically modified by Cy-3 fluorophore for the coupling signal detection. 3′-end is chemically modified during the synthesis by N3-Methyl Uridine, and 5′-end is chemically modified by Cy-3 fluorophore for the coupling signal detection on 3D Gel matrices.
The following protocol was used for dendrimeric platforms preparation:
1. Silanisation of Microscopic Slides for Glass Surface Activation (Amination)
The microscopic glass slides were immersed in 10% NaOH for overnight and subsequently washed with H2O, 1% HCl, again with H2O and finally with methanol (or ethanol). After 15 min immersion in a 3% 3-aminopropyltrimethoxysilane (APTMS) solution made in 95% methanol (or ethanol) the slide was washed in pure methanol (or ethanol), then in water, dried under a stream of nitrogen (or centrifuged) and baked at 110° C. for 15 min.
2. Dendrimeric Matrix Formation (Synthesis of Linker-System)
Synthesis was performed by repeating the following reaction steps 1 and 2 twice with the respective amines until the desired linker molecules were obtained.
3. Activation of the Surface for the Immobilization of Oligonucleotide Probes
Activation was performed by N,N′-disuccinimidylcarbonate (DSC). Specifically, activation was conducted by incubation with 1 mmol DSC and 1 mmol N,N-diisopropylethyl-amine (DIEA) in 20 ml anhydrous acetonitrile for 4 hrs. Subsequently, slides were washed with DMF and 1, 2-dichloroethane and dried under a stream of nitrogen (or centrifuged).
4. Immobilization of Oligonucleotide Probes onto the Activated Dendrimeric Matrix
250 pmol/0.1 μl oligonucleotide probes solutions in 1% DIEA in water were placed onto the activated support media. The oligonucleotide probes were modified by C6 aminolinker at 3′-end.
Spotting was performed by pins (200 nl/spot). Following attachment of the oligonucleotide probes, the slides were incubated overnight in a humid chamber at 37° C. for the immobilization and afterwards washed with H2O and methanol (or ethanol).
5. Deactivation of Dendrimeric Matrix
The glass slides surface with the immobilized probes was deactivated by a 2 hrs treatment using a solution made of 6-amino-1-hexanol (50 mM) and DIEA (150 mM) in DMF. Finally, the DNA arrays were washed with DMF, acetone, water and dried. The deactivated glass slides with the immobilized probes (ready for the hybridization) could then be stored at 4° C. (shelf-life at 4° C. is at least 6 months).
Results
Coupling signal detection revealed that the coupling capacity of probes on the 3D dendrimeric matrices is much higher than on 3D gel-matrices, while coupling capacities of 3D-gel matrices and 2D matrices are comparable. The hybridization sensitivity with the ss compliment was arranged according to the following: 3D dendrimeric matrices >3D gel-matrices >2D aldehyde matrix.
In conclusion, the main advantages of the dendrimeric matrix are: 1) higher sensitivity compared to that of gel matrix (approximately 2 times); 2) following the stripping of the surface from the hybridized targets, the surface can be re-used. The shelf-life of the dendrimeric matrix is estimated to be 3 months.
Thus, the inventors determined dendrimeric matrix to be the most sensitive platform. Accordingly, all experiments conducted for the evaluation of the hybridization capacities were performed on the dendrimeric matrices manufactured by the inventors.
Hybridization kinetics of nucleic acids is temperature dependent, and the specificity and efficiency depend on the hybridization temperature. In order to determine melting temperature (Tm), the inventors used IDT OligoAnalyzer 3.1 (http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/Default.aspx) and Primer3 program (http://simgene.com/Primer3). For the Tm calculation, the program demands not only the oligonucleotide sequence, but the buffer conditions as well. Thus, Tm and consequently the hybridization temperature depend on the buffer composition. The length of each probe was established to range from 17 to 25 bases. The GC content was calculated and only probes with GC content between 40 and 60% were selected for further analysis.
The IDT OligoAnalyzer3.1 program was used for the estimation of the conditions of the hairpin formation. The formation of hairpins (intra-molecular self-structure of the probe) significantly prevents the process of hybridization and can decrease the sensitivity of the microarray. All designed probes were verified against the GenBank nucleic acid database for specificity using the basic local alignment search tool (BLAST) program (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
While specific probes for target microorganisms could be generated following well established and above mentioned requirements, this would require an experimental confirmation of their equal hybridization capacity, as it has been estimated that between 21-34% of probes do not match their intended targets [22]. The main limitation of the existed programs and approaches for the prediction of melting temperature and consequently the hybridization temperature is that all these predictions are for free oligonucleotides but not for those bound to solid surfaces due to the limited knowledge on the thermodynamics of hybridization at solid liquid interfaces. This is the reason why probes with optimal predicted melting temperature do not necessarily perform the optimal hybridization, and this can generate biochips with irregular signal responses. Besides, it is not always possible to fit the specific probe/probes into the Tm range optimal for the most chosen probes.
The evaluation of the hybridization potential of each of the 38 probes was accomplished by using 16 single strand (ss) cassettes. ss cassettes present the lineal set of sequences complimentary to the studied set of probes. The Cy3 fluorescent dye was inserted at 5′-end of ss cassette during the synthesis. The number of probes included in each cassette ranged from 3 to 8, and the size varied from 65 to 142 bases (Table 2). The hybridization buffer was SSARC buffer (4×SSC [600 mM NaCl, 60 mM Na-citrate], 7.2% (v/v) Na-sarcosyl). The samples in the hybridization buffer were pre-heated to the hybridization temperature (45° C., 5 min). Hybridization on microscope slides was carried out under cover slips after the application of sample solution. The volume of the sample solution is calculated from 2 μl solution per 1 cm2 of the covered square. Hybridization proceeded at 45° C. for 4 hrs. After hybridization the biochip was washed once for 2 min in 2×SSC+0.2% SDS, once for 2 min in 0.2×SSC+0.2% SDS, once for 2 min in 0.2×SSC at 25° C., and dried by centrifugation.
The signal from the probe SRB1 for the identification of SRB in the bacterial consortia was used as a gold standard. The similar hybridization behavior of a cassette probe was used in the content of different cassettes. Moreover, this probe was designed in the mixture of randomly selected cassettes implicitly points with the absence of cross-hybridization between all studied probes.
The variations in the fluorescent intensities on a biochip, coming from probe's size and nucleotide sequence differences, are determined by the cassette method. 35 probes with similar fluorescent intensities (or binding capacity) have been chosen from the tested 38 probes for the identification of main bacterial groups involved in the biocorrosion (Table 3).
Desulfobvibrio
Desulfo-
bacterium
Desulfobulbus
Desolfobacter
Desulfo-
microbium
Desulfo-
tomaculum
Desulfococcus
Desulfobibrio
Desulfovibrio
Dv. vulgaris,
Dv. desuluricans)
Desulfo-
bacterium
Desulfobacterium
Archaea
Methanogenic
Archaea
Archaea
Archaea
Archaea
Geobacter spp
Shewanella spp
G.
sulfurreducens
G. metalli-
reducens
Shewanella spp
Firmicutes
E.coli
Optimization of the Probes Using Extended ssDNA Approach
As previously mentioned, the difficulty of the microarray approach is that all probes are hybridized simultaneously, but specific hybridization conditions often vary between probes. The hybridization signal intensity in the real environment has been estimated for the selected probes characterized by equal hybridization capacity. The probes are embedded in the fragment of appropriate DNA and not in the set of compliments as it is in the case of cassette. For this reason, the extended ssDNA approach has been developed. The approach is based on the hybridization of the short (100-200 bases) single stranded ssDNA (PCR fragments) with the appropriate oligonucleotide probe on the biochip. The sequence complimentary to the appropriate probe is imbedded in ssDNA fragments. The SRB strains, obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) collection, the probes and PCR primers are listed in Table 4. Individual gene-specific PCR primers were designed using Primer3Plus software (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi). The sequences complementary to the biochip probes were embedded in the one of PCR amplicon strains, which was Cy3 labeled.
Desulfovibrio
CAC CCC
CAA AGG GTG TCT
alaskensis,
TGC ATC
GTA CG (SEQ ID
GGC TGC
AG (SEQ
CTC ATG GCA TCC
CAG AAA TC (SEQ
Desulfo-
CAC TGG
CTT GAG ACC ATT
bacterium
AAC AGG
TCG GTT
autotropicum,
CGA TCA
GA (SEQ ID NO: 73)
AG (SEQ
GGC GCC GTT
ATA GGC TGT AG
Desulfobulbus
GCG CCA
CTC TAC AAA
propionicus,
CCC TGC
CTG GGG TGC
CGT TCA
AAG (SEQ ID
AC (SEQ
GAC CTT TGC
CTT GGA AAACA
Desulfobacter
TCA CCT
TCA CCT GGT GAA
postgatei,
GGT GAA
AAT CGG ACT (SEQ
AAT
CGGACT
CCA GGT CTG
TCC ACT GTT CC
Desulfo-
CCA CAA
GTT TCG CCG
microbium
CCT GGC
AAG AAC ATG A
baculatum,
CAT CCC
GGA AAT
CCGC CCA ATC
CCT ACA ACC T
ACC TAT
GGA AAT TCC GGA
GCC GAT
CTG GTA CA (SEQ
TGT CCC
CG
GAGGGAGTCTTCT
CCAAGCA (SEQ ID
The approach included two PCR reactions: standard and primer extension reactions. Standard PCR and primer extension reactions were performed on each DNA presented in the Table 4. The 1st standard PCR mixture (50 μl) contained 1×iProof High-Fidelity Master Mix (Bio-Rad Laboratories, USA) with 1.5 mM MgCl2, 200 μM (each) deoxynucleoside thriphosphate, 500 nM each primer, 50 ng of DNA template. PCR amplification was conducted using the following conditions identical for each primer pair: an initial denaturation step (30 s, 98° C.) was followed by 35 cycles of denaturation (10 s, 98° C.), annealing (20 s, 60° C.), and extension (15 s, 72° C.) and one terminal extension step (10 min, 72° C.). The results of the PCR amplification were assessed using agarose gel analysis of PCR fragments following DNA amplification (1.5% agarose gel, 1×TAE running buffer, ethidium bromide staining) and are shown in
Amplified DNA was purified using a PureLink Quick PCR Purification Kit (Invitrogen, USA) before single strand DNA synthesis, and used as the template DNA in the primer extension reaction. Single strand DNA samples for hybridization were synthesized using the primer extension reaction with iProof DNA polymerase in the presence of subsequent reverse or forward primers labeled by Cy3 dye (Table 4). The use of the labeled reverse or forward primer in the primer extension reaction is governed by the position of the sequence complimentary to the probe on the 1st or the 2nd strain of the template (ds PCR amplicon) DNA. The reaction was performed in 50 μl containing 1× iProof High-Fidelity Master Mix (Bio-Rad Laboratories, USA) with 1.5 mM MgCl2, 200 μM (each) deoxynucleoside thriphosphate, 1000 nM Cy3 labeled primer (Table 4), 120 ng of DNA template.
Amplification protocol was the same for all primer extension reactions, and was the same as for amplification of the first standard PCR. iProof DNA polymerase (Bio-Rad Laboratories, USA) equalizes the PCR conditions for all ran reactions and speeds the reaction. The 35 cycles PCR is completed in 1 hour. Amplified ss PCR fragment was purified using a PureLink Quick PCR Purification Kit (Invitrogen, USA) and precipitated by 10 volumes of 2% LiClO4 in acetone for 30 min at −20° C. The pellet was collected by centrifugation at 15,000 rpm for 30 min, washed 1 time with acetone and air dried. The pellet represents the labeled ssDNA ready for hybridization on the biochip. Each ssDNA fragment contains the sequence complimentary to the appropriate probe on the biochip in the environment of the other real-life sequences that models the hybridization of specific target DNA on the biochip.
To assess the hybridization signal from the oligoprobes (Table 4) each gene was amplified with single PCR. The amplified PCR of each gene was then used as a template in primer extension reaction to create single strand DNA. As the result, the single strand DNA of each gene was labeled and hybridized separately on the small microarray consisting of 6 probes, forming a part of MIC-biochip. In summary, six genes of different SRB DNA were amplified and hybridized separately with the biochip (
The quantity of DNA and fluorophore Cy3 was estimated spectrophotometrically for each ssPCR fragment. The equal quantity of each labeled ssDNA was dissolved in hybridization buffer (1 M GuSCN (guanidine thyocianate), 5 mM EDTA, 50 mM HEPES (pH 7.5), 0.2 mg/ml BSA (bovine serum albumin). The mixture was denatured at 95° C. for 5 min, and chilled on ice for 2 min. The sample was placed onto oligonucleotide probes array slides at 25° C. and covered with a glass cover slip. After hybridization for 4 hours at 25° C. in Arrayit hybridization chamber (Arrayit Corporation, USA), the slides were rinsed with 4×SSC, 7.2% Sarcosyl (Sodium lauroyl sarcosinate) for 30 sec, afterwards, the slides were washed with 0.2×SSC at room temperature for 30 sec, spun-dry at 1000 rpm for 1 min before analyzing by the portable microarray reader PA5000 (Aurora Photonics, USA). The results are presented in
The following detailed protocol was used for the extended ssDNA approach:
As shown in
The developed approach provides an opportunity to prepare the samples for the fine-tuning of the hybridization signal intensity under practical conditions, such as in 3 hours by using available DNA strains.
The sensitivity of microarray depends on the quantity of the probes, the type of the biochip matrix, and the quantity of the labeled target DNA. In the present Example, all experiments for the evaluation of DNA preparation for biochip analysis, as well as analysis of environmental samples on different biochip prototypes were performed on the 3D dendrimeric matrices manufactured in the inventor's laboratory.
Target DNA amplification before hybridization leads to stronger signals and allows for detection of specific targets even if they are present in low abundance. The steps of random fragmentation of DNA and its fluorescent labeling precede the hybridization of DNA fragments with the immobilized probes onto the matrix for the biochip visualization. Ten protocols with different combinations of DNA amplification, fragmentation and labeling procedures were designed and tested by using the pure bacterial cultures (Desulfovibrio indonensis and E. coli BP) and the environmental samples. The most optimal procedure was used for analysis of environmental samples on different biochip prototypes.
While it is understood that each of the steps outlined below and in
1st amplification was performed by using Illustra™ GenomiPhi HY DNA Amplification Kit (Catalog. No.: 25-6600-22 GE Healthcare, Life Science, USA) and subsequent purification. Fragmentation of the amplified DNA was performed by using other NEBNext dsDNA Fragmentase (Catalog. No.: M0348S New England Biolabs, USA), other FastDigest SaqAI (Catalog. No.: #FD2174 Thermo Scientific, USA) and subsequent purification; and 2nd amplification and labelling was performed simultaneously by using BioPrime® Plus Array CGH Genomic Labelling System (Catalog. No.: 18095-13 Invitrogen, USA).
As outlined above, fragmentation can be performed using dsDNA Fragmentase or by FastDigest SaqAI. Additionally, steps of the target DNA preparation and hybridization are outlined in
The developed protocol is presented schematically in
Fragmentase or restrictase SaqAI revealed less losses of DNA quantity after digestion in comparison with DNaseI. The labeling procedure by using ULYSIS® Alexa Fluor® 546 Nucleic Acid Labeling kit was substituted by BioPrime® Plus Array CGH Genomic Labelling System. The latter one was more effective because the labelling procedure is accompanied by the amplification. In addition, BioPrime® Plus Array CGH Genomic Labelling System kit contains not only Alexa Fluor® 555-aha-dCTP, but also random primers labelled at 5′-end with Alexa Fluor® 555.
The labelled target DNA was precipitated by 10 volumes of 2% LiClO4 in acetone for 30 min at −20° C. The pellet was collected by centrifugation at 15,000 rpm for 30 min, washed 1 time with acetone and air dried. The pellet represents the labeled target DNA ready for hybridization on the biochip.
Various hybridization conditions (buffers, temperature, and time) were tested, and the optimized one was selected. The labeled target DNA was dissolved in hybridization buffer (1 M GuSCN, 5 mM EDTA, 50 mM HEPES (pH 7.5), 0.2 mg/ml BSA). The mixture was denatured at 95° C. for 5 min, and chilled on ice for 2 min. The sample was placed onto oligonucleotide probes array slides at 25° C. and covered with a glass cover slip. After hybridization for 4 hours at 25° C. in Arrayit hybridization chamber (Arrayit Corporation, USA), slides were rinsed with 2×SSC, 3.6% Sarcosyl (Sodium lauroyl sarcosinate) for 30 sec, afterwards, the slides were washed with 0.2×SSC at room temperature for 30 sec, spun-dry at 1000 rpm for 1 min before analyzing by the portable microarray reader PA5000 (Aurora Photonics, USA).
The most effective combination of DNA amplification, fragmentation and labeling procedures was generated and applied to environmental samples.
Testing of a Biochip Prototype on the Desulfobacterium autotropicum DNA
The developed protocol for DNA amplification, fragmentation and labeling was next tested on Desulfobacterium autotropicum, DSM 3382. The results are shown in
The arrangement of the probes on the biochip is shown in
The probe Dv discriminates any Desulfovibrio in bacterial consortium on the basis of hynB (periplasmic Ni,Fe hydrogenase) gene. The similar structure is also present in the hynB (periplasmic Ni,Fe hydrogenase) gene of Desulfobacterium, where the difference is only one letter (SEQ ID NO:83: CAC CCC TGC ATC GGG TGC AG) in comparison with the Dv probe (SEQ ID NO:4: CAC CCC TGC ATC GGC TGC AG). It was found that the intensity of the fluorescent signal for this probe is significantly lower than that of the specific probe Dbacterium1 for Desulfobacterium sp.
Therefore, the Desulfobacterium sp. discrimination is accomplished by the probe Dbacterium1 on the biochip. The probes 16SCONS_1, SRB1, and Dbacterium1 revealed the similar fluorescent signal, as it has been observed when the probes have been tested by the cassette method.
The relevance of the cassette and extended ss DNA approaches for the selection of the probes with identical hybridization capacity for the inclusion in a biochip was confirmed on the bacterial DNA.
In order to develop a final pilot MIC BioChip (comprising probes listed in Table 5), the inventors used three DNA samples (A2, A21, and A13) and analyzed each using four different MIC BioChip prototypes. Different biochip prototypes provided an opportunity to analyze the efficiency of the selected probes as applied to environmental samples and to estimate the sufficient DNA quantity for the hybridization signal detection. The developed N10 protocol for DNA amplification, fragmentation and labeling described in Example 4 was used for the preparation of all DNA samples for hybridization.
The biochip prototype for A2 sample analysis was constructed from 12 probes in duplicate. 11 probes were used for the detection of SRB and their 7 genera in the bacterial consortium (SRB1, DSR2, Dv, Dbacterium1, Dbulbus, Dbacter2, Dbacter1, Dsmicrobium1, Dtomaculum2, Dtomaculum1, and Dscoccus1) and one probe 16SCONS_1 served as a positive control probe. The results are presented in
The probes SRB1 and DSR2 were chosen for detection of any SRB in bacterial consortium. The probe SRB1 was highly effective by applying it to the environmental sample A2. Among the 7 genera of SRB, 6 genera Desulfovibrio sp., Desulfobulbus sp., Desulfobacterium sp., Desulfomicrobium sp., Desulfotomaculum sp., Desolfococcus sp. were detected. The highest hybridization signals were observed for Desulfovibrio sp., Desulfobulbus sp., and Desulfobacterium sp.; followed by Desulfomicrobium sp., Desulfotomaculum sp., Desolfococcus sp. These observations indicate that Desulfovibrio sp. and Desulfobulbus sp. dominate in the SRB consortium of the sample A2. Desulfobacter sp. were not detected by applying the biochip to the sample A2.
The MIC biochip prototype for A21 sample analysis was constructed using 17 probes in duplicate. 16 probes (SRB1, Dv, Dv_hynB2, Dv 1 hynA2, Dv_2_hynA2, Dv_3_hynA2, Dv16S_1, Dbacterium1, Dbacterium_16S_2, Dbulbus, Dbacter2, Dbacter1, Dsmicrobium1, Dtomaculum2, Dtomaculum1, and Dscoccus1) were used for the detection of SRB and one probe (16SCONS_1) was used as a positive control probe. The results are presented in
Among the 7 genera of SRB, 6 genus of SRB were present in the A21 sample. The highest hybridization signals were observed for Desulfovibrio sp. and Desulfobulbus sp., followed by Desulfotomaculum sp., and Desolfococcus sp., and then by Desulfobacterium sp., and Desulfomicrobium sp. Desulfobacter sp. were not detected by applying the biochip prototype to the environmental sample A21.
In conclusion, the inventors observed that profiles of SRB in sample A2 and A21 are different. Desulfovibrio sp., Desulfobulbus sp., and Desulfobacterium sp., dominate in sample A2, while Desulfovibrio sp. and Desulfobulbus sp. dominate in sample A21. 2.5 μg of total DNA was sufficient for the visualization of the biochip results.
Another MIC biochip prototype for A13 sample analysis was constructed from 16 probes in duplicate. This prototype includes the probes for the detection of two types of bacteria: SRB and NRB. NRB can weaken the biocorrosion process caused by SRB. 11 probes (SRB1, Dv, Dv16S_1, Dbacterium1, Dbacterium_16S_2, Dbulbus, Dbacter2, Dbacter1, Dsmicrobium1, Dtomaculum 2, and Dscoccus1) were used for the detection of SRB, while 4 probes (narG, napA, nirS, nirK) were used for the detection of NRB. One probe 16SCONS_1 served as a positive control probe.
The results are shown in
Yet another MIC BioChip prototype, containing 29 probes in duplicate for the identification of key bacterial groups involved in biocorrosion, was next used for the analysis of the A2 sample. The results are shown in
12 probes (SRB1, DSR1, DSR2, Dv, Dv 16S_1, Dbacterium1, Dbacterium 16S_2, Dbulbus, Dbacter2, Dsmicrobium1, Dtomaculum 2, and Dscoccus1) were used for the detection of SRB and their 7 genera in the bacterial consortium. 6 probes (Geobac, GeoM, GeoS, Shew1, ShewRNA1, 16SRNAPR) were used for detection of MRB in the bacterial consortium. Three probes (FTHFS, a/bssA, FirmicytishydA) were used for identification of FB in the bacterial consortium. Four probes (narG, napA, nirS, nirK) were used for detection of NRB in the bacterial consortium. Three probes (16SCONS_1, 16SCONS_2, 16SCONS_3) were included for the discrimination of any bacteria in bacterial consortium (PC—positive controls).
Analysis of the A2 sample using the 29 probe BioChip revealed the same profile of hybridization signals for SRB on both biochip prototypes (
Among the MRB group, Shewanella sp. were detected, while Geobacter sp. were not. Hydrocarbon degrading bacteria were identified in the FB group. NRB were detected based on the nitrate and nitrite reductase gene probes. Finally, A2 sample did not contain detectable E. coli.
The analysis of the environmental samples by the various biochip prototypes revealed that fluorescent signals are absent for some probes. This could either indicate the absence of the appropriate group of bacteria in the studied consortium (sample), or that the probe is not suitable for bacterial detection on a specific biochip.
The functional probe Dbacterium1 and structural probe Dbacterium 16S_2 have been selected for discrimination of Desulfobacterium sp. While de novo designed probe Dbacterium1 revealed the signal, the probe Dbacterium 16S_2, selected from the literature [15] did not provide the signal in any of the studied cases, including Desulfobacterium autotropicum DNA. The absence of signal thus indicates that Dbacterium 16S_2 probe is not suitable for the bacterial detection. Accordingly Dbacterium 16S_2 probe was not included in the pilot biochip.
Three probes SRB1, DSR1, and DSR2 were included in one of the prototype MIC-BioChip for the detection of any SRB in the consortium. The analysis of the environmental samples indicated that only probe SRB1 is highly effective, while the signals from the probes DSR1, and DSR2 were barely detectable. Both these probes were selected from the literature [11]. However, in all studied cases, including Desulfobacterium autotropicum DNA, probe SRB1 (based on apr gene) provided a strong signal. It is known that dsr gene is not sufficiently conservative. As the weak signal implies that these probes are not suitable for the bacteria detection, DSR1 and DSR2 were not included in the pilot MIC BioChip. While all three positive control probes (16SCONS_1, 16SCONS_2, 16SCONS_3) revealed detectable signal, the inventors included just one positive probe 16SCONS_1 in the pilot MIC BioChip, since it was the most effective in all studied cases.
Dbacter1 and Dbacter2 functional probes were selected for detection of Desulfobacter sp. Neither of the two probes revealed signal using the environmental samples, however, the probe Dbacter2 worked well on Desulfobacter strain (
The experiments described herein allowed the inventors to develop a pilot MIC BioChip comprising 30 probes listed in Table 5. Among the 30 probes, 11 probes permit identification of the main SRB groups involved in the biocorrosion and provides a corrosion scale, 18 probes allow identification of additional MIC bacterial groups (Archea, MRB, FB, NRB, E. coli), one positive control probe (16SCONS_1).
Desulfobvibrio
Desulfo-
bacterium
Desulfobulbus
Desolfobacter
Desulfo-
bacter
Desulfo-
microbium
Desulfo-
tomaculum
Desulfococcus
Desulfobibrio
Desulfovibrio
Dv. vulgaris,
Dv. desuluricans)
Methanogenic
Archaea
Archaea
Archaea
Archaea
Geobacter spp
Shewanella spp
G.
sulfurreducens
G. metalli-
reducens
Shewanella spp
Firmicutes
E.coli
CCT ACG GGA
GGC AGC AG
The invention can be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are to be considered in all respects illustrative rather than limiting on the invention described herein. In the various embodiments of the methods and systems of the present invention, where the term comprises is used with respect to the recited steps or components, it is also contemplated that the methods and systems consist essentially of, or consist of, the recited steps or components. Further, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions can be conducted simultaneously. All percentages and ratios used herein, unless otherwise indicated, are by weight.
In the specification, the singular forms also include the plural forms, unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the case of conflict, the present specification will control.
All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g. Genbank sequences or GeneID entries), patent application, or patent, was specifically and individually indicated to be incorporated by reference. This statement of incorporation by reference is intended by Applicants, pursuant to 37 C.F.R. § 1.57(b)(1), to relate to each and every individual publication, database entry (e.g. Genbank sequences or GeneID entries), patent application, or patent, each of which is clearly identified in compliance with 37 C.F.R. § 1.57(b)(2), even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The entire disclosure of each of the patent documents, including certificates of correction, patent application documents, scientific articles, governmental reports, websites, and other references referred to herein is incorporated by reference herein in its entirety for all purposes. In case of a conflict in terminology, the present specification controls.
This present application claims priority to U.S. Provisional Patent Application Ser. No. 62/484,672 filed Apr. 12, 2017, which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62484672 | Apr 2017 | US |
