Patent Grant
9624551
This application is a national phase entry of PCT/CA2012/000205, filed Mar. 8, 2012, from U.S. Provisional patent application Ser. No. 61/450,169, filed on Mar. 8, 2011, the contents of each of these applications being incorporated herein by reference in their entirety.
A computer readable form of the Sequence Listing “3244-P41273US00_SequenceListing.txt” (8,192 bytes), submitted via EFS-WEB and created on Sep. 6, 2013, is herein incorporated by reference.
Please replace the Sequence Listing currently of record with the Sequence Listing provided herewith in text format in computer readable form only.
The application discloses a method of generating catalytic nucleic acid probes useful for detecting microorganisms such as bacterial pathogens. The application also discloses catalytic nucleic acid probes and methods of using the probes for detecting microorganisms.
Prevalence of food-borne pathogens, emergence of drug-resistant bacteria and viruses, and threat of bioterrorism are pressing concerns. Early detection of pathogens prevents outbreaks.
Traditionally, microbial detection is performed using microbiological techniques[1]. Although this approach is highly accurate, it can take days (even weeks) to arrive at a definitive conclusion. Both antibody and PCR based methods represent significant advances because such tests offer high specificity and sensitivity and require significantly reduced detection times[2]. However, these methods usually require multiple analytical steps and specialized equipment. There is a significant need for both simple methods that can achieve rapid detection of known pathogens and new platforms that can be quickly put in place to create assays for a new pathogen in an unanticipated outbreak.
DNAzymes, a special class of functional nucleic acids[3], are artificial single-stranded DNA molecules with a catalytic ability[4]. They can be isolated from a random-sequence DNA pool by the technique of “in vitro selection”[5] and have been explored as unique molecular tools for an increasing number of applications[4,6].
The present disclosure describes a novel approach for bacterial pathogen detection by isolating fluorogenic DNAzymes from a random-sequence DNA library by in vitro selection using the unpurified complex extracellular mixture left behind by a specific microbe. Since both probe isolation and subsequent assaying procedures bypass tedious and time-consuming target identification steps, this method can be easily implemented for any microorganism.
The disclosure relates to a method of generating a catalytic nucleic acid probe for detecting a microorganism, the method comprising:
In one embodiment, the method also includes contacting the plurality of candidate nucleic acid molecules with a negative control target prior to contacting the plurality of candidate nucleic acids with the microorganism target and detecting a candidate nucleic acid which (i) interacts with the microorganism target, (ii) has catalytic activity to cleave the detectable substrate upon interaction with the target, thereby generating a detectable signal and (iii) does not cleave the detectable substrate upon interaction with the negative control target.
Optionally, the method further comprises:
In one embodiment, the method comprises multiple cycles of steps (A) through (E), optionally at least 5, 15, 20, or 30 cycles of steps (A) though (E). In another embodiment, the pool of candidate nucleic acid molecules becomes increasingly enriched for candidate nucleic acid molecules which (i) interact with the microorganism target and (ii) have catalytic activity to cleave the detectable substrate upon interaction with the target, thereby generating a detectable signal, upon each successive cycle. An “enriched pool” refers to a pool of molecules that has a greater proportion of candidate nucleic acid molecules compared to non-candidate nucleic acid molecules than a comparator pool. Optionally, non-candidate nucleic acid molecules are molecules that do not (i) interact with the microorganism target and (ii) have catalytic activity to cleave the detectable substrate upon interaction with the target, thereby generating a detectable signal. Other non-candidate nucleic acid molecules include molecules that cleave the substrate non-specifically (for example, in the presence of a negative control target) or generate a detectable signal non-specifically.
In one embodiment, DNA is extracted from the enriched pool corresponding to the final, or last, cycle and a nucleic acid corresponding to catalytic nucleic acid probe is sequenced and/or cloned.
In one embodiment of the present method, the catalytic nucleic acid probe is a DNAzyme. Optionally, the DNAzyme catalyses the cleavage of a RNA-DNA linkage.
In another embodiment, the detectable substrate is a single ribonucleotide linkage embedded in a DNA sequence. In another embodiment, the ribonucleotide is flanked by a fluorophore modified nucleic acid residue on one side and a quencher modified nucleic acid residue on the other side and the fluorophore is quenched until the ribonucleotide linkage is cleaved, thereby generating a fluorogenic signal.
In one embodiment of the method, the microorganism target is found in the extracellular matrix of the microorganism. Optionally, the microorganism target is found in the intracellular matrix of the microorganism.
In another embodiment, the target is a protein, a DNA molecule, a RNA molecule, a small molecule, extracellular matrix, intracellular matrix, a cell of the microorganism, or any combination thereof. Optionally, the target is crude extracellular matrix or crude intracellular matrix.
Optionally, the microorganism is a bacteria, optionally a bacterial pathogen. In one embodiment, the microorganism is E. coli, L. monocytogenes or S. typhimurium.
In yet another embodiment, the method further comprises the use of the catalytic nucleic acid probe for detecting the microorganism.
The disclosure also relates to a catalytic nucleic acid probe for detecting a microorganism, wherein the catalytic nucleic acid probe comprises a first nucleic acid region that (i) interacts with a microorganism target and (ii) has catalytic activity to cleave a detectable substrate upon interaction with the target, thereby generating a detectable signal.
Optionally, the catalytic nucleic acid probe further comprises a second nucleic acid region comprising the detectable substrate.
In one embodiment, the detectable substrate is a single ribonucleotide linkage embedded in a DNA sequence and the ribonucleotide is flanked by a fluorophore modified nucleic acid residue on one side and a quencher modified nucleic acid residue on the other side such that the fluorophore is quenched until the ribonucleotide linkage is cleaved, thereby generating a fluorogenic signal.
In another embodiment, the catalytic nucleic acid probe comprises a sequence with at least 80%, 90%, 95% or 99% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 15. Optionally, the catalytic nucleic acid probe comprises, or consists essentially of, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or residues 1-99 of SEQ ID NO: 15.
In yet another embodiment, the catalytic nucleic acid probe comprises a sequence with at least 80%, 90%, 95% or 99% sequence identity to SEQ ID NO: 5. Optionally, the catalytic nucleic acid probe comprises, or consists essentially of, SEQ ID NO: 5.
In another embodiment of the disclosure, the catalytic nucleic acid probe comprises (a) a sequence with at least 80%, 90%, 95% or 99% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or residues 1-99 of SEQ ID NO: 15 and (b) a sequence with at least 80%, 90%, 95% or 99% sequence identity to SEQ ID NO: 5.
In other embodiments, the catalytic nucleic acid probe is a DNAzyme.
The disclosure also relates to a kit for detecting a microorganism, wherein the kit comprises:
Optionally, the detectable substrate is a single ribonucleotide linkage embedded in a DNA sequence. In another embodiment, the ribonucleotide is flanked by a fluorophore modified nucleic acid residue on one side and a quencher modified nucleic acid residue on the other side such that the fluorophore is quenched until the ribonucleotide linkage is cleaved, thereby generating a fluorogenic signal.
In other embodiments, the first catalytic nucleic acid comprises a sequence with at least 80%, 90%, 95% or 99% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or residues 1-99 of SEQ ID NO: 15. Optionally, the second nucleic acid comprises a sequence with at least 80%, 90%, 95% or 99% sequence identity to SEQ ID NO: 5.
In a further embodiment, the first catalytic nucleic acid and the second nucleic acid comprise a single nucleic acid probe. Optionally, the nucleic acid probe is a DNAzyme.
The disclosure also relates to a method of detecting a microorganism in a sample comprising:
In one embodiment, the detectable substrate is a single ribonucleotide linkage embedded in a DNA sequence. In another embodiment, the ribonucleotide is flanked by a fluorophore modified nucleic acid residue on one side and a quencher modified nucleic acid residue on the other side such that the fluorophore is quenched until the ribonucleotide linkage is cleaved, thereby generating a fluorogenic signal.
In one embodiment of the method, a single colony forming unit of the microorganism is detected. Optionally, at least 2, 3, 5, 10 or 20 colony forming units of the microorganism are detected.
In yet another embodiment, the microorganism is a bacterial pathogen, optionally selected from the group consisting of E. coli, L. monocytogenes and S. typhimurium.
In another embodiment, the target is a protein, a DNA molecule, a RNA molecule, a small molecule, extracellular matrix, intracellular matrix, a cell of the microorganism, or any combination thereof. Optionally, the target is crude extracellular matrix or crude intracellular matrix.
The disclosure also relates to the use of a catalytic nucleic acid probe for detecting a microorganism in a sample wherein the catalytic nucleic acid probe (i) interacts with a microorganism target and (ii) cleaves a detectable substrate upon interaction of the catalytic nucleic acid probe with the target thereby generating a signal that indicates the presence of the microorganism in the sample.
Optionally, the detectable substrate is a single ribonucleotide linkage embedded in a DNA sequence. In another embodiment, the ribonucleotide is flanked by a fluorophore modified nucleic acid residue on one side and a quencher modified nucleic acid residue on the other side such that the fluorophore is quenched until the ribonucleotide linkage is cleaved, thereby generating a fluorogenic signal.
In one embodiment, the microorganism is a bacterial pathogen, optionally selected from the group consisting of E. coli, L. monocytogenes and S. typhimurium.
In another embodiment, the target is a protein, a DNA molecule, a RNA molecule, a small molecule, extracellular matrix, intracellular matrix, a cell of the microorganism, or any combination thereof. Optionally, the target is crude extracellular matrix or crude intracellular matrix.
Embodiments of the disclosure will be shown in relation to the drawings in which the following is shown:
RFL1 (SEQ ID NO: 10): DNA library with 70 random nucleotides (N70); FS1: fluorogenic substrate (SEQ ID NO: 5); FP1 (SEQ ID NO: 6): forward PCR primer; RP1 (SEQ ID NO: 7) and RP2 (SEQ ID NO: 8): two reverse PCR primers; RFT1 (SEQ ID NO: 9): the template for ligating FS1 to RFL1.
The present disclosure relates to methods for generating catalytic nucleic acid molecules for detecting microorganisms. The disclosure also provides catalytic nucleic acid molecules for detecting microorganisms and methods of using catalytic nucleic acid molecules for detecting microorganisms.
Accordingly, in one embodiment of the disclosure, there is included a method of generating a catalytic nucleic acid molecule for detecting a microorganism, the method comprising:
As used herein, the term “microorganism” refers to a microscopic organism that comprises either a single cell or a cluster of single cells. Microorganisms include, but are not limited, to, bacteria, fungi, archaea, protists, algae, plankton and planarian. In one embodiment of the present disclosure, the microorganism is a gram-negative bacterium, for example Escherichia coli O157:H7, Escherichia coli K12, Salmonella typhimurium, Pseudomonas peli, Brevundimonas diminuta, Hafnia alvei, Yersinia ruckeri, Ochrobactrum grignonese, Achromobacter xylosoxidans, Moraxella osloensis, Acinetobacter Iwoffi, and Serratia fonticola. In another embodiment, the microorganism is a gram-positive bacterium, for example Listeria monocytogenes, Bacillus subtilis, Actinomyces orientalis, Pediococcus acidilactici, Leuconostoc mesenteroides, and Lactobacillus planturum. In specific embodiments of the invention, the microorganism is a pathogenic bacterium (for example, a bacterium that causes bacterial infection) such as Escherichia coli O157:H7, Listeria monocytogenes, Salmonella typhimurium or Clostridium difficile. In other embodiments, the microorganism is a harmless strain of bacteria.
As used in the present disclosure, “catalytic nucleic acid probes” and “catalytic nucleic acid molecules” are nucleic acid molecules that function as both binding agents and enzymes. The enzymatic activity of the catalytic nucleic acid probes of the present disclosure is activated upon interaction with a target. Catalytic nucleic acid probes are optionally DNA molecules (DNAzymes) or RNA molecules (ribozymes). In one embodiment of the present disclosure, the catalytic nucleic acid probe is a DNAzyme that cleaves a particular substrate, for example a ribonucleotide linkage.
The term “substrate” as used herein refers to a molecule that is modified, for example cleaved, by a catalytic nucleic acid probe at a cleavage site in the substrate, typically a cleavable linkage. For an RNA-cleaving DNAzyme, the substrate comprises a RNA-DNA linkage. Other substrates of catalytic nucleic acid probes include, but are not limited to, nucleic acids, modified nucleic acids, specific DNA or RNA sequences or a synthetic polymer containing a cleavable linkage such as an ester or amide. Optionally, a catalytic nucleic acid probe includes a substrate of the catalytic nucleic acid probe.
As used herein, a “detectable substrate” is a substrate where the cleavage of the substrate results in a signal that can be detected. The substrate is not detectable prior to cleavage, for example, because the signal is quenched. In one embodiment, a signal is generated or emitted when a detectable substrate is cleaved to activate a signal agent. Optionally, the signal is a fluorogenic signal, a colorimetic signal, an electrochemical signal, a surface plasmon resonance (SPR) signal or a radioactive signal.
In one embodiment of the present invention, a catalytic nucleic acid probe interacts with a microorganism target. As used herein, a “microorganism target” is a molecule, compound or substance that is present in or on the microorganism or is generated, excreted, secreted or metabolized by the microorganism. In one embodiment of the invention, a microorganism target is present in the extracellular matrix of the microorganism. In another embodiment, a microorganism target is present in the intracellular matrix of an organism. In another embodiment, a microorganism target comprises a protein, a DNA molecule, a RNA molecule, a small molecule, extracellular matrix, intracellular matrix, a cell of the microorganism, or any combination thereof. Optionally, a microorganism target is a crude or purified extracellular matrix or a crude or purified intracellular matrix. In other embodiments, a microorganism target is specific to a particular species or strain of microorganism.
As used herein, the term “interacts with a microorganism target” includes direct and indirect binding of a catalytic nucleic acid molecule to a target. In some embodiments, the binding is transitory, but sufficient to activate catalytic activity. The term “interacts with a microorganism target” is not limited to binding to a target but could include any other association between the catalytic nucleic acid molecules and the target sufficient to activate catalytic activity.
“Candidate nucleic acid molecules” are nucleic acid molecules that are screened against a microorganism target, in order to identify the catalytic nucleic acid probes of the present discourse. In one embodiment, a candidate nucleic acid molecule comprises a variable region of nucleic acid sequence and a constant region of nucleic acid sequence comprising a detectable substrate, wherein the detectable substrate is detectable only upon cleavage of the detectable substrate. As used herein, a “variable region” is a nucleic acid sequence that differs among the various candidate nucleic acid molecules. Optionally, the “variable region” comprises a random sequence of nucleic acid sequence. In one embodiment, each nucleic acid residue in the variable region has a 20-30%, optionally 25%, probability of being an A, C, G or T nucleotide. In contrast, the “constant region” of the candidate nucleic acid molecules is the same in each of the candidate nucleic acid molecules. The constant region of nucleic acid sequence is optionally a detectable substrate, for example an RNA-DNA linkage. Optionally, the RNA residue is flanked on one side by a nucleotide labeled with a fluorophore and a nucleotide labeled with a quencher on the other side.
The candidate nucleic acid molecules of the present disclosure are optionally generated from a DNA library, optionally a single stranded DNA library. In one embodiment, the DNA library is DNA library RFL1 (obtained from Yale University Keck Facilities or Integrated DNA Technologies (IDT). Optionally, the DNA library comprises single stranded DNA molecules comprising a central variable domain flanked by two non-variable regions. In one embodiment, the central variable domain is 10 to 500 nucleotides in length, optionally 20 to 250, optionally 50 to 100 nucleotides in length. The constant regions may be 5 to 50 nucleotides in length, optionally 10 to 20 nucleotides in length. DNA library RFL1 contains molecules consisting of 98 nucleic acid molecules distributed into a central random-sequence domain of 70 nucleotides and two non-variable regions of 16 nucleotides and 12 nucleotides at the 5′ and 3′ ends. Each random position in the central variable domain optionally represents a 20-30%, optionally 25%, probability of being an A, C, G or T nucleotide.
To generate candidate nucleic acid molecules, the nucleic acids molecules comprising the DNA library are optionally ligated to a detectable substrate. As described above, a “detectable substrate” is a substrate which may be modified by a catalytic nucleic acid. In one embodiment of the present disclosure, the detectable substrate is a ribonucleotide-DNA linkage. Optionally, the modification is cleavage. Modification of the detectable substrate by a catalytic nucleic acid generates a signal that is detectable (a “detectable signal”). A person skilled in the art would understand that there are numerous ways to detect the presence of cleaved molecules in the sample. Optionally, the signal is a fluorogenic signal, a colorimetric signal, an electrochemical signal, a surface plasmon resonance (SPR) signal or a radioactive signal. In some embodiments, the substrate will be linked to a suitable signalling system (such as gold nanoparticles, gold surfaces, and electrochemical-sensitive molecules). The cleavage of the substrate alters the chemical or physical behaviour of the signalling system (such as the aggregate state of gold nanoparticles, the surface plasmon of gold surfaces, and the conductivity of the electrochemically active molecules), resulting in a colorimetric, SPR or electrochemical signal.
Optionally, the ribonucleotide is flanked on one side by a nucleotide labelled with a fluorophore and on the other side by a nucleotide labelled with a quencher. Optionally, the fluorophore is fluorescein and the quencher is dabcyl. In one embodiment, cleavage of the ribonucleotide leads to the release of the nucleotide labelled with the quencher, thereby generating a fluorogenic signal.
In one embodiment of the present disclosure, the catalytic nucleic acid probes are isolated using an in vitro selection process. According to the methods of the present disclosure, a plurality of candidate nucleic acid molecules, which, as described above, comprise both a variable region of nucleic acid sequence (optionally derived from a DNA library) and a constant region of nucleic acid sequence comprising a detectable substrate, are contacted with a microorganism target. In other embodiments, at least 1000, 10,000, 100,000 or 1,000,000 candidate nucleic acid molecules are contacted with a microorganism target. Optionally, at least 1014-1016 unique molecules are contacted with a microorganism target. Optionally, contacting the molecules with a microorganism target comprises incubating the molecules in medium containing the target. In other embodiments, contacting the molecules with a microorganism target comprises incubating the molecules in extracellular matrix or intracellular matrix derived from a microorganism. The extracellular or intracellular matrix is optionally crude or purified matrix. Extracellular matrix is, optionally obtained by removing cells grown in culture media, optionally Luria-Bertani media.
In another embodiment, the target comprises a protein, a DNA molecule, a RNA molecule, a small molecule, extracellular matrix, intracellular matrix, a cell of the microorganism, or any combination thereof. The molecules are optionally contacted with the target for at least 5, 10, 20, 30, 45 or 60 minutes. In other embodiments, the molecules are contacted with the target for 1 to 60 minutes, optionally 5 to 45 minutes, optionally 20 to 40 minutes.
In one embodiment of the present methods, the plurality of candidate nucleic acid molecules are first contacted with a negative control target prior to being contacted with the microorganism target. Optionally, the negative control target is extracellular matrix or intracellular matrix from a microorganism that is not the microorganism of interest. In other embodiments, the negative control target is a protein, a DNA molecule, a RNA molecule, a small molecule, extracellular matrix, intracellular matrix, a cell of the microorganism, or any combination thereof derived from a microorganism that is not the microorganism of interest. Only nucleic acid molecules that remain uncleaved after contact with the negative control target are then contacted with the microorganism target. Optionally, uncleaved DNA molecules are purified by polyacrylamide gel electrophoresis. One of the goals of this “negative selection” step is to remove any self-cleaving or non-specific catalytic nucleic acid molecules.
The method optionally includes selecting at least one catalytic nucleic acid molecule from the plurality of nucleic acid molecules which (i) interacts with the target and (ii) cleaves the detectable substrate upon interaction with the target, thereby generating a detectable signal. Optionally, the method also includes selecting at least one catalytic nucleic acid molecule that is not cleaved upon interaction with the negative control target. Optionally, the method also includes selecting at least one catalytic nucleic acid molecule that is not self-cleaving in the absence of the target.
In one embodiment, candidate nucleic acid molecules which are cleaved during exposure to the microorganism target are purified, optionally by dPAGE, and amplified by PCR (polymerase chain reaction). In one embodiment, the variable region of random sequence is amplified. The amplified sequences are then optionally ligated to a detectable substrate and again exposed to the microorganism target. Optionally, at least 5, 10, 15, 20 or 30 cycles of selection, amplification, ligation, contacting (exposure) and selection are performed. Each cycle of election, amplification, ligation, contacting (exposure) and selection generates an increasingly enriched pool of catalytic nucleic acid probes. As used herein, “an enriched pool” refers to a pool of molecules that has a greater proportion of candidate nucleic acid molecules compared to non-candidate nucleic acid molecules than a comparator pool. Optionally, an enriched pool has at least a 2, 5, 10, 50, 100, 200 or 500% greater proportion of candidate nucleic acid molecules compared to non-candidate nucleic acid molecules than a non-enriched comparator pool. Optionally, non-candidate nucleic acid molecules are molecules that cleave the substrate non-specifically or generate a detectable signal non-specifically.
In one embodiment, DNA is extracted from the enriched pool corresponding to the final, or last, cycle and a nucleic acid corresponding to catalytic nucleic acid probe is sequenced and/or cloned to obtain a catalytic nucleic acid probe for detecting a microorganism target.
The present disclosure also relates to catalytic nucleic acid probes, for example DNAzymes, for detecting a microorganism. The catalytic nucleic acid probes are optionally generated by the methods described herein.
The catalytic nucleic acid probes comprise the following functionalities: (a) recognition of a microorganism target and (b) catalysis of a substrate, for example an RNA-DNA linkage, such that a signal is generated, in the presence of the microorganism target. Optionally, the RNA linkage is present in the catalytic nucleic acid probe itself.
Thus in some embodiments, the catalytic nucleic acid probe (i) interacts with a microorganism target and (ii) cleaves a substrate upon interaction with the target. The cleavage of the substrate optionally generates a detectable signal. In some embodiments, the substrate is part of the catalytic DNA molecule such that the catalytic molecule comprises both (a) a first catalytic nucleic acid region that interacts with the target and (b) a second nucleic acid region comprising the substrate.
The first catalytic nucleic acid region that interacts with the target is optionally 5 to 500 base pairs in length, optionally 10 to 200 base pairs in length, optionally 20-120 base pairs in length, optionally 22 to 100 base pairs in length. In some embodiments, the first nucleic acid region comprises a sequence with at least 80%, 90%, 95% or 99% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or residues 1-99 of SEQ ID NO: 15. In other embodiments, the first nucleic acid region comprises a sequence with optionally at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 base pairs that are contiguous with sequences contained in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or residues 1-99 of SEQ ID NO: 15. In other embodiments, the first nucleic acid region comprises or consists of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or residues 1-99 of SEQ ID NO: 15. Optionally, the first nucleic acid region has catalytic activity such that it modifies a substrate upon interaction with the target.
The second nucleic acid region comprising the substrate is optionally 10 to 60 base pairs in length, optionally 20-40 base pairs in length. Optionally, the substrate is a ribonucleotide and the ribonucleotide is flanked by a fluorophore modified nucleic acid residue on one side and a quencher modified nucleic acid residue on the other side. In some embodiments, the second nucleic acid region comprises a sequence with at least 80%, 90%, 95% or 99% sequence identity to SEQ ID NO: 5. In other embodiments, the second nucleic acid region comprises or consists of SEQ ID NO: 5.
In other embodiments, the catalytic nucleic acid probe comprises a sequence that hybridizes under medium or high stringency conditions to any one of SEQ ID No: 1-5 or residues 1-99 of SEQ ID NO: 15 or the complement thereof. Medium or high stringency hybridization are well known to persons skilled in the art. Examples of hybridization conditions may be found in molecular biology reference texts such as Molecular Cloning: A Laboratory Manual by Sambrook and Russell (3rd Edition, Cold Spring Harbour Press, 2001). Optionally, high stringency conditions comprise the following: hybridization at 5× sodium chloride/sodium citrate (SCC)/5×Denhardt's solution/1.0% SDS at Tm −5° C. for 15 minutes based on the equation (Tm=81.5° C.−16.6 (Log 10 [Na+])+0.41(% G+C)−600/l) (or similar equation)), followed by a wash of 0.2×SSC/0.1% SDS at 60° C. It is understood, however, that equivalent stringencies may be achieved using alternative buffers, salts and temperatures.
In other embodiments, the second nucleic acid region is contained on a separate nucleic acid molecule from the first nucleic acid region. This arrangement is optionally referred to as “in trans”. In an “in cis” arrangement, both the first and second nucleic acid regions are present in the same molecule.
The catalytic nucleic acid molecules of the present disclosure may be synthesized using oligonucleotide synthesis methods known in the art. The entire catalytic nucleic acids may be optionally synthesized as one molecule or the first and second nucleic acid regions may be synthesized separately and combined together, for example by ligation.
The present disclosure also relates to a method for using the catalytic nucleic acid probes for indicating the presence of a microorganism. The catalytic nucleic acid probes are optionally created through the use of the methods described herein.
The catalytic nucleic acid probes described in the present disclosure are useful for detecting microorganisms in a food sample (eg. meat, fish, vegetables, fruit, eggs, dairy products etc.), a medical sample (blood, saliva, tissue sample etc.), a water sample, an environmental sample or any other sample where it is desired to know of the presence or absence of a microorganism. Optionally, the catalytic nucleic acid probes of the present disclosure are useful for identifying bacterial pathogens, optionally food borne bacterial pathogens.
The catalytic nucleic acid probes optionally detect a single live cell. In other embodiments, the catalytic nucleic acid probes optionally detect at least 0.2, 2 or 20 colony forming units.
In one embodiment of the present methods, at least a single microbial cell is isolated from a sample. The cell is optionally cultured in appropriate medium. In one embodiment, if one colony forming unit (a single live cell) is used to initiate cell growth, a culturing time of at least 4, 8, 12 or 16 hours may be used.
In one embodiment, a crude extracellular mixture is obtained from the cells by removing the cells (for example by centrifugation) from the media. In another embodiment, a protein, a DNA molecule, a RNA molecule or a small molecule is isolated from the cultured microbial cells. In another embodiment, crude intracellular matrix is obtained from the cultured cells.
Optionally, a catalytic nucleic acid probe corresponding to a specific microorganism target is exposed to, and contacted with, the extracellular matrix, intracellular matrix, protein, a DNA molecule, a RNA molecule or small molecule. Optionally, the catalytic nucleic acid probe is exposed to the whole microbial cell. The extracellular matrix, intracellular matrix, protein, a DNA molecule, a RNA molecule, small molecule or microbial cell is typically present in an appropriate media. The catalytic nucleic acid probe corresponding to a specific microorganism is exposed to the extracellular matrix, intracellular matrix, protein, a DNA molecule, a RNA molecule, small molecule and/or microbial cell optionally for at least: 5, 10, 30, 60 or 120 minutes. dPAGE analysis is optionally used to assay for cleavage of the probes. Cleavage of the probe will make the probe detectable, and detection indicates the presence of the specific microorganism corresponding to the target of the catalytic nucleic acid probe in the original sample.
In another aspect, the present disclosure includes a kit for determining the presence of a microorganism, said kit comprising
In another embodiment, the kits disclosed herein also include, without limitation, containers, instructions for use, reagents for DNAzyme activity and or bacterial culturing, and other agents commonly used in the processes described herein.
All publications, patent and patent applications are herein incorporated by reference in their entirety.
Embodiments of the disclosure will be illustrated in a non-limiting way by reference to the examples below.
To demonstrate the feasibility of this method, fluorogenic DNA probes were generated based on an RNAcleaving fluorescent DNAzyme (RFD) system [7]. These DNAzymes cleave a lone RNA linkage (R;
RFD Isolation Using the Random-Sequence DNA Library RFL1
The DNA library RFL1, the special fluorogenic substrate FS1, the forward PCR primer FP1, the two reverse PCR primers RP1 and RP2, RFT1, the template for ligating FS1 to RFL1 were purchased as synthetic oligonucleotides either from Yale University Keck Facilities or from Integrated DNA Technologies (IDT). Their sequences are provided in
The selection scheme is described as follows. Briefly, prior to the selection, two different CEM samples were prepared, one from E. coli, termed CEM-EC, and one from the control bacterium Bacillus subtilis (B. subtilis), termed CEM-BS; each CEM was prepared by removing cells grown overnight in Luria-Bertani media (LB; commonly used for culturing bacteria).
Specifically, E. coli K12 cells were grown overnight in 5 mL of LB with continuous shaking at 37° C. The cells were removed by centrifugation at 11,000 g for 5 min at room temperature. The crude supernatant was collected and aliquoted into microcentrifuge tubes and stored at −20° C. CEM-BS was prepared in an identical way as described for CEM-EC except that B. subtilis cells were grown. The CEM from other bacteria described herein were prepared in a similar way except that each bacterium was cultured in LB for a different period of time (because these bacteria have varying growth rates in LB) until the OD600 (optical density at 600 nm) of each cell culture reached ˜1.
In Vitro Selection Procedure
The following steps are depicted in
Ligation of FS1 to RFL1 (step 1 in
Purification of ligated FS1-RFL1 (step 2)—The DNA molecules in the mixture from step 1 were concentrated by ethanol precipitation and the ligated FS1-RFL1 molecules were purified by 10% dPAGE.
Negative selection (step 3)—The purified FS1-RFL1 was dissolved in 100 μL of 1× selection buffer (1×SB) (50 mM HEPES, pH 7.5, 150 mM NaCl, 15 mM MgCl2, and 0.01% Tween 20). This mixture was incubated at room temperature for 5 h. This was followed by the addition of 25 μL of CEM-BS as prepared above and 25 μL of 2×SB. The new mixture was further incubated at room temperature for 1 h. The reaction was quenched by the addition of 150 μL of stop solution containing 100 mM EDTA and 8 M urea.
Purification of uncleaved FS1-RFL1 (step 4)—After ethanol precipitation, the uncleaved FS1-RFL1 molecules from the above reaction mixture were purified by 10% dPAGE and stored at −20° C. until use.
Positive selection (step 5)—25 μL of CEM-EC was mixed with 25 μL of 2×SB and added to the uncleaved FS1-RFL1 obtained in step 4 above and the reaction volume was adjusted to 100 μL with 1×SB. The entire mixture was incubated at room temperature for 30 min before addition of 100 μL of stop solution.
Purification of cleaved products (step 6)—After ethanol precipitation, the large cleaved fragment (which contained RFL1 sequence) was purified by 10% dPAGE and used as the template for PCR in the next step. The percentage of cleaved RFL1 was also determined and used as a way to measure the progress of selection.
PCR1 (step 7)—The PCR mixture (50 μL) contained the template prepared above, 0.5 μM each of FP1 and RP1, 200 μM each of dNTPs (dATP, dCTP, dGTP and dTTP), 1×PCR buffer (75 mM Tris-HCl, pH 9.0, 2 mM MgCl2, 50 mM KCl, 20 mM (NH4)2SO4) and 2.5 units of Thermus thermophilus DNA polymerase. The DNA was amplified using the following thermocycling steps: 94° C. for 1 min; 13 cycles of 94° C. for 30 s, 50° C. for 45 s and 72° C. for 45 s; 72° C. for 1 min.
PCR2 (step 8)—1 μL of the above PCR1 product was diluted with ddH2O to 20 μL, 1 μL of which was used as the template for this additional PCR step using primers FP1 and RP2 while following the same protocol above for PCR1. Note that the numbers of amplification cycles between different selection rounds were adjusted, typically between 13 and 15 cycles, to achieve full amplification (revealed by 1% agarose gel electrophoresis).
Purification of DNAzyme-coding strand (step 9)—As stated above, the hexaethyleneglycol spacer in RP2 prevents the amplification of the A20 fragment, making the non-DNAzyme-coding strand 20 nucleotides longer than the coding strand. The coding strand RFL1 was purified by 10% PAGE in this step.
Ligation of RFL1 to FS1 (step 10) and repetition of steps 2-10—The coding DNA strand prepared above (approximately 100 pmol, stored as dried pellet) was ligated to FS1 as follows:
100 pmol of FS1 was phosphorylated in 50 μL reaction volume with 10 μCi [γ-32P]ATP and 10 units of PNK for 10 min at 37° C. followed by the addition of non-radioactive ATP (1 μL from 50 mM stock, 1 mM final concentration) and incubation for another 20 min at 37° C. Note: for the remaining selection rounds, the reaction volume of phosphorylation of FS1 was constantly maintained at 50 μL. After quenching the reaction by heating at 90° C. for 5 min, the reaction mixture was transferred to the dried purified PCR products obtained from the step 9.
Approximately 110 pmol of RFT1 was added to this solution. The mixture was heated at 90° C. for 40 s and cooled to room temperature for 10 min. Then, 10 μL of T4 DNA ligase buffer was added and the volume of the reaction was adjusted to 100 μL with ddH2O. The ligation reaction was initiated by adding 10 units of T4 DNA ligase and continued for 2 h. After purification by 10% dPAGE, the ligated DNA product was used for the second round of selection using the same procedure as described for the first round.
A total of 20 cycles of selection were conducted; the percentage of cleaved RFL1 in step 6 in each cycle is depicted in
The DNA library was incubated in the selection buffer (SB; 50 mM HEPES, pH 7.5, 150 mM NaCl, 15 mM MgCl2, and 0.01% Tween 20) for 5 h, followed by 1-h incubation with CEM-BS diluted with 2×SB at 1:1 ratio—this combined procedure served as the “negative selection” step to remove any self-cleaving and non-specific DNAzymes. The uncleaved DNA molecules were purified by 10% denaturing polyacrylamide gel electrophoresis (dPAGE) and then incubated with CEM-EC in 1×SB for 30 min. This was the positive selection step designed to isolate fluorescent DNAzymes that can specifically respond to CEM-EC. The cleaved DNA sequences were purified by dPAGE, amplified by polymerase chain reaction (PCR) and used for the next cycle of selective amplification. After 20 iterations (the selection progress is shown in
The DNA pool from the 20th round was cloned and sequenced. Three classes of DNAzymes were discovered; the sequence of the most dominant signaling DNAzyme, named RFD-EC1, is provided in
For fluorescence based assay measured in a fluorimeter, CEM-EC and 2×SB were mixed at 1:1 ratio in a quartz crystal cuvette; such a solution maintained a steady level of fluorescence (see the constant level of fluorescence in the initial 5 min of the fluorescence plot in
To verify that the fluorescence increase of RFD-EC1 was indeed due to the cleavage of the RNA linkage, the cleavage reaction was also analyzed by 10% dPAGE. The cleavage of RFD-EC1 was expected to generate two DNA fragments; the 5′-cleavage fragment retains the fluorophore and can be detected by fluorescence imaging while the 3′-fragment does not fluoresce (see the marker lane in
Next, the response of RFD-EC1 to CEM produced by other microbes was investigated. Nine gram negative bacteria (in addition to E. coli) and 5 gram-positive bacteria (including B. subtilis, whose CEM was used during the negative selection step) were chosen for this experiment; the full names of all these bacteria can be found in the legend of
For this experiment, each bacterium was cultured in LB for a different period of time (because these bacteria have varying growth rates in LB) until the OD600 (optical density at 600 nm) of each cell culture reached ˜1. The CEM was then obtained and used to induce the cleavage of RFD-EC1 in a 1-h reaction.
None of the CEMs from these arbitrarily chosen bacteria (
To investigate the nature of the targets (proteins or small molecules) in the CEM-EC that activate RDF-EC1, the CEM-EC was treated with two proteases, trypsin (TS) and proteinase K. It was found that the CEM-EC treated with either protease was no longer able to activate RFD-EC1 (
Without being bound by theory, these observations suggest that the likely target for the DNAzyme activation is a protein.
To examine whether the observed cleavage activity of RFD-EC1 was simply caused by possible RNases that may exist in the CEM-EC, two tests were carried out. In the first test, Ribolock (RI RNase inhibitor, was added into the CEM-EC. As shown in
These results, along with the previous observation that RFSS1 (mutated RFD-EC1) failed to cleave upon contacting CEM-EC (
Next, an experiment to probe into the possible molecular weight of the target using molecular sizing column (NANOSEP OMEGA, Pall Incorporation) was carried out. CEM-EC was passed through several centrifugal columns with a cut-off molecule weight of 3K (3,000 Daltons), 10K, 30K, 50K and 100K, respectively. It was found that while the 3K, 10K and 30K filtrates of CEM-EC did not induce the cleavage of RFD-EC1, the filtrates from both 50K and 100K spin columns contained the target (
Without being bound by theory, these results suggest that the potential protein target may have a molecular weight between 30,000 and 50,000 Daltons.
Detection of Bacterial Cells
The ability to detect a single live cell is a hallmark of a bacterial detection method for many practical applications such as detection of food-borne pathogens[1]. For this reason, bacterial detection methods in such practices have an essential cell-culturing step. This method has an integrated cell-culturing step and is expected to offer the capability for single live cell detection. To verify this, the experiment described next was carried out.
First, using culture plating method as detailed in SI, 4 E. coli stocks were prepared containing 20, 2, 0.2 and 0.02 colony-forming units (CFUs; 1 CFU=a single live cell) per 100 μL; these stocks were labeled as Stocks A-D, respectively. From each stock, 15×100 μL were taken to inoculate 15 parallel solutions of LB. Theoretically the individual tube inoculated with Stocks A, B, C and D would contain an average of 20, 2, 0.2 and 0.02 CFUs, respectively. Thus bacterial growth in all the tubes inoculated with Stocks A and B, 3 of the 15 tubes inoculated with Stock C (0.2×15=3), and none of the 15 tubes inoculated with Stock D (0.02×15=0.3≦1) was expected. All inoculated solutions were incubated at 37° C. for 24 h, CEM-EC was then prepared from each cell culture and used to activate the cleavage of RFD-EC1 in a 1-h reaction using dPAGE analysis.
The results from the experiment, shown in
The time required to generate a detectable signal if 1 CFU of E. coli was used to initiate cell growth was also investigated. From the data shown in
Finally, the number of seeding cells required to produce CEM-EC, after 6-h cell culturing, that was concentrated enough to induce a detectable signal of RFD-EC1 was examined; the data is provided in
As described above, an RNA-cleaving fluorescent DNAzyme (RFD) that reports the presence of a specific bacterium by interacting with the crude extracellular mixture (CEM) generated by this organism as cells proliferate has been created. This DNAzyme cleaves its substrate at a lone RNA linkage (R) embedded in a DNA chain and flanked by nucleotides labeled with a fluorophore (F) and a quencher (Q). In this study, the performance of the assay was improved by optimizing target sample preparation procedures, bacterial growth and DNAzyme reaction conditions.
(A) Cleavage Performance of the DNAzyme in Trans
Cleavage performances of the DNAzyme in cis (where the substrate is directly linked to the enzyme) and in trans (where the DNAzyme and the substrate exist separately) were tested. Cleavage performance of constructs EC1 (a full length DNAzyme including primer binding sites previously obtained from the in vitro selection; SEQ ID NO: 1), EC1T (a truncated version of EC1 after the primer binding sites), and EC1TM were tested in trans-acting form (where the DNAzyme and the substrate exist separately) (
Methods
Preparation of cis-Acting EC1-FS1:
cis-acting EC1-FS1 construct was generated by template mediated enzymatic ligation of FS1 to EC1. In brief, 200 pmol of FS1 was phosphorylated in 50 uL reaction volume 1 mM ATP concentration and 20 U of PNK for 30 min at 37° C. in 1×PNK buffer A. The reaction was quenched by heating at 90° C. for 5 min and cooled down to room temperature for 10 min. Equimolar EC1 and EC1 LT (ligation template) were then added to this solution, heated at 90° C. for 30 s and cooled to room temperature for 10 min. Afterwards, 10 μL of 10×T4 DNA ligase buffer (buffer info as for PNK) was added and the volume was adjusted to 100 μL with ddH2O. This was followed by the addition of 10 units of T4 DNA ligase and incubation at room temperature for 2 h. The reaction was then quenched by adding 10 μL of NaOAc (3 M, pH 7.0) and 250 μL of cold 100% ethanol. After mixing the mixture was placed at −20° C. for an hour. After ethanol precipitation, the DNAzyme was purified by 10% dPAGE.
DNAzyme Activity Assays in Trans:
Cleavage performance of constructs EC1, EC1T, and EC1TM were tested in trans-acting form. Two reaction mixtures for each construct were prepared; one as control where the reaction was conducted with the TSB and RB only excluding CEM-EC, the second one was test. The first, for the control experiment 23 μL of TSB was taken in a microcentrifuge tube followed by 25 μL of 2×RB, 1 μL of 2.5 μM FS1 and 1 μL of 25 μM of each construct in individual tube. For the test, reaction mixture was prepared similarly except adding 23 μL of CEM-EC instead of TSB. Each mixture was then incubated at room temperature for 60 min and then quenched by adding 5 μL of NaOAc (3 M) and 135 μL of ethanol (100%). After ethanol precipitation, the DNA molecules were analyzed by 10% dPAGE. Fluorescent DNA bands were visualized by fluorimager (Typhoon 9200, GE Healthcare).
(B) Target Accumulation in Intracellular Mixture
The target protein accumulated in the CEM of E. coli can also be found in its crude intracellular mixture (CIM) and could be rescued with a quick centrifugation of cells and mild heat treatment to induce the release of CIM (
Methods
Preparation of Bacterial Crude Mixtures:
E. coli was plated onto a tryptic soy agar (1.5%) and grown for 14 h at 37° C. A single colony was picked and inoculated in 2 mL of TSB and grown for 14 h (overnight) at 37° C. with shaking at 250 rpm. Afterwards, a 1% re-inoculation was prepared by taking 20 μL of the overnight culture and transferring to a fresh 2 mL of TSB. The re-inoculation was grown at 37° C. with shaking at 250 rpm until the culture reached on OD600 (optical density at 600 nm) of ˜1. Crude extracellular mixture (CEM) was prepared by separating cells from 1 mL of the culture with centrifugation at 11,000 g for 5 min at room temperature. The crude supernatant was then collected fresh in microcentrifuge tubes and stored at −20° C. Crude intracellular mixtures (CIMs) were extracted by re-suspending cell pellets in 200 μL of 1× reaction buffer (RB; 50 mM HEPES, 150 mM NaCl, 15 mM MgCl2 at pH 7.5) and heating at 50° C. for 15 min. The heat treated cell suspension was then centrifuged at 11,000 g for 5 min at room temperature. The clear supernatant was transferred to a fresh microcentifuge tube and stored at −20° C.
CIMs collected from bacteria other than E. coli listed above were prepared in a similar manner except each species was cultured in SOB for a different length of time (because each bacterium possess a different growth rate) until each culture reached an OD600 of ˜1.
CEM-EC and CIM-EC Activity Assay:
100 μL of a prepared 50000 CFU/mL glycerol stock was inoculated in 2 mL of TSB and grown at 37° C. for 7 h with shaking at 250 rpm. CEM and CIM were prepared similarly as described above, except the CIM was extracted in 100 μL of 1×RB. The CEM reaction consisted of 20 μL of CEM-EC, 25 μL of 2×RB, 1 μL of 2.5 μM FS1, 1 μL of 25 μM of EC1T and 3 μL ddH2O. The CIM reaction was prepared with 20 μL of CIM-EC, 28 μL of 1×RB, 1 μL of 2.5 μM FS1 and 1 μL of 25 μM of EC1T. Each reaction was then incubated at room temperature for 60 min, ethanol precipitated and analyzed by 10% dPAGE as before (
Cleavage Assay with CIMs Collected from E. Coli Grown in Various Growth Media:
100 μL of a prepared 2500 CFU/mL glycerol stock was inoculated in 2 mL of LB, LBM, SOB, SOC, TB, TH or TSB. Each culture was allowed to grow for 7 h and 1 mL was then centrifuged and subjected to CIM extraction in 50 μL 1×RB (as described previously). Reaction mixtures were then set-up with 25 μL of CIM-EC, 23 μL of 1×RB, 1 μL of 2.5 μM FS1 and 1 μL of 25 μM of EC1T as described above. After 60 min incubation at room temperature, mixtures were ethanol precipitated and analyzed by 10% dPAGE (
(C) Co-Factor, Temperature and pH
It was found that several metal ions (Ba, Mg, Mn and Ca) can support the DNAzyme activity (
Methods
Activity of EC1T with Different Divalent Metals:
Reaction mixtures were prepared to a final volume of 50 μL containing 25 μL 2×RB without MgCl2 (100 mM HEPES, 300 mM NaCl at pH 7.5), 5 μL of 150 mM M2+ (BaCl2, CdCl2, CoCl2, MgCl2, MnCl2, NiCl2, CuCl2, ZnCl2, and CaCl2), 1 μL of 2.5 μM FS1, 1 μL of 25 μM EC1T and 15.5 μL of ddH2O. Finally, 2.5 μL CIM-EC was added and the reaction was incubated at room temperature for 60 min. Reactions were then quenched, ethanol precipitated and analyzed by 10% dPAGE as before (
BaCl2 was used as the M2+ of choice and was subsequently titrated from 0 to 50 mM (final concentration) in the reaction mixture with EC1T, FS1 and CIM-EC as prepared before. Briefly, 1, 5, 7.5, 10, 15, 20 and 25 mM reactions were prepared by adding 0.5, 2.5, 3.75, 5, 7.5, 10, and 12.5 μL of 100 mM BaCl2, respectively, and a 50 mM reaction was prepared by adding 2.5 μL of 1 M BaCl2 to reaction mixtures containing 25 μL 2×RB without any M2+, 1 μL of 2.5 μM FS1, 1 μL of 25 μM EC1T. Volumes were then adjusted by adding the appropriate difference in ddH2O. Each reaction was mixed by and pipetting. Finally, 2.5 μL CIM-EC was added and the reaction was incubated at room temperature for 60 min. Reactions were then quenched, ethanol precipitated and analyzed by 10% dPAGE as before (
Reaction Temperature Dependence:
In order to find out the optimal reaction temperature, reaction cleavage reactions carried out at different temperature such as 4, 15, 23, 37 and 50° C. A 50 μL reaction volume containing 25 μL 2×RB with BaCl2 (30 mM), 1 μL of 2.5 μM FS1, 1 μL of 25 μM EC1T and 20.5 μL of ddH2O was set up. Two sets of reactions were prepared for each temperature, a control sample to which 2.5 μL of ddH2O was added instead of CIM and a test sample to which 2.5 μL of CIM-EC was added. Reactions were incubated for 60 min at their respective temperatures and then ethanol precipitated and analyzed by 10% dPAGE (
pH Dependence:
A DNAzyme pH profile was tested by preparing 2×RB solutions using different chemical compounds for each respective pH. RB's for pH 5.0, 5.5 and 6.0 were prepared with 100 mM MES, 300 mM NaCl and 30 mM BaCl2, RB's for pH 6.5, 7.0, 7.5 and 8.0 were prepared with 100 mM HEPES, 300 mM NaCl and 30 mM BaCl2 and RB's for pH 8.5 and 9.0 were prepared with 100 mM Tris-HCl, 300 mM NaCl and 30 mM BaCl2. Cleavage reactions procedures, dPAGE analysis were same as described for temperature dependency (
Optimizing the Substrate and DNAzyme Ratio (FS1:EC1T):
Substrate DNAzyme (FS1:EC1T) ratios 1:0, 1:1, 1:2, 1:5, 1:10, 1:25, 1:50, and 1:100 were tested for greatest cleavage activity. Firstly, A 50 μL reaction volume containing 25 μL of 2×RB (100 mM HEPES, 300 mM NaCl and 30 mM BaCl2, pH 7.5) and 1 μL of 2.5 μM FS1 was prepared. Ratios 1:1, 1:2 and 1:5 were prepared by adding 1, 2 and 5 μL of 2.5 μM EC1T, respectively. Ratios 1:10, 1:25 and 1:50 were prepared by adding 1, 2 and 5 μL of 25 μM EC1T, respectively. Finally, ratio 1:100 was prepared by adding 2.5 μL of 100 μM EC1T. Final volumes were adjusted as required with ddH2O. All reactions included a control and test sample for ratio and were incubated for 60 min at room temperature. Mixtures were then ethanol precipitated and analyzed by 10% dPAGE.
DNAzyme probes were generated for the bacterial pathogens Listeria monocytogenes and Salmonella typhimurium using the methods described herein.
LM-RFD (SEQ ID NO: 3) is a DNAzyme isolated for L. monocytogenes and ST-RFD (SEQ ID NO: 4) is a DNAzyme isolated for S. typhimurium.
The signalling properties of the L. monocytogenes and S. typhimurium-specific probes are shown in
The L. monocytogenes and S. typhimurium-specific probes are highly sensitive as shown in
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CA2012/000205 | 3/8/2012 | WO | 00 | 9/6/2013 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2012/119231 | 9/13/2012 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20100015608 | Kolpashchikov | Jan 2010 | A1 |
| 20110143338 | Todd | Jun 2011 | A1 |
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| 20140005072 A1 | Jan 2014 | US |
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| 61450169 | Mar 2011 | US |
