Patent Grant
7662562
The present invention relates, in general, to probes, methods, and kits used to determine the presence or absence of a microorganism in a sample. The probes, methods, and kits comprise at least one capture probe and/or at least one detector probe.
In the following discussion certain articles and methods will be described for background and introductory purposes. Nothing contained herein is to be construed as an “admission” of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and methods referenced herein do not constitute prior art under the applicable statutory provisions.
Bacteremia and fungemia are life-threatening infections that require timely administration of appropriate antimicrobial therapy to prevent significant mortality. The term “septicemia” is used to describe the presence of organisms within the blood in association with laboratory and/or clinical findings that are indicative of infection such as fever, chills, malaise, tachycardia, hyperventilation, shock and leucocytosis. Weinstein et al. (Rev. Infect. Dis. 5: 54-70 (1983)) determined that the overall rate of mortality was 42% among 500 episodes of bacteremia and fungemia, with approximately half of the deaths attributable directly to septicemia. It has long been recognized, however, that the majority of bacteremias and fungemias are associated with the recovery of very low numbers of organisms from the blood. Indeed, it is not uncommon for less than 1 organism/mL of blood to be present, particularly after the initiation of antimicrobial therapy. The severity of such infections and the diverse spectrum of potential pathogens, therefore, necessitate highly sensitive methods of diagnosis that are capable of identifying a broad spectrum of bacteria and fungi. Classically, diagnosis is achieved through the use of broad-based culture methods that are amenable to the growth of a wide variety of pathogens from low-level inocula. Following growth and isolation in pure culture, the organisms are identified through the application of a battery of biochemical tests. Antimicrobial susceptibility testing is then conducted to permit modification of empirical therapy to an efficacious pathogen-specific regimen that minimizes cost and toxicity. There remains, however, a need to reduce the time between collection of specimens from a patient and administration of targeted antimicrobial therapy to provide an opportunity to reduce morbidity and mortality, defray the cost of therapy and hospitalization, and minimize the spread of antimicrobial drug resistance caused by ineffective or inappropriate therapy.
The present invention relates to a method for identifying the presence of at least one microorganism in a sample, the method comprising: (a) releasing RNA or DNA from the at least one microorganism in the sample; (b) contacting the RNA or DNA with at least one capture probe capable of hybridizing to a first target sequence of the RNA or DNA, wherein the contacting is performed under conditions that permit hybridization between the first target sequence and the at least one capture probe to form a microorganism-capture probe hybrid complex, and wherein the at least one capture probe comprises at least one sequence selected from the group consisting of SEQ ID NOs:1-53, 55, 56, 61, 62, 67, 68, and 72-78; and (c) detecting the presence of the microorganism-capture probe hybrid complex by (i) contacting the RNA or DNA with at least one detector probe capable of hybridizing to a second target sequence of the RNA or DNA, wherein the detector probe comprises at least one reporter group and wherein the contacting is performed under conditions that permit hybridization between the second target sequence and the at least one detector probe to form a microorganism-capture probe-detector probe hybrid complex, and wherein the at least one detector probe also comprises at least one sequence selected from the group consisting of SEQ ID NOs:1-53, 55, 56, 61, 62, 67, 68, and 72-78; (ii) detecting the microorganism-capture probe-detector probe hybrid complex by detecting the at least one reporter group, wherein the presence of the microorganism-capture probe-detector probe hybrid complex indicates the presence of the at least one microorganism. In another embodiment, the reporter group is selected from the group consisting of a radioactive isotope, an enzyme, a fluorescent molecule and an amplification sequence. In a further embodiment, the amplification sequence initiates an amplification reaction selected from the group consisting of strand displacement amplification (SDA), polymerase chain reaction (PCR), reverse transcriptase-strand displacement amplification (RT-SDA), reverse transcriptase-polymerase chain reaction (RT-PCR), nucleic acid sequence based amplification (NASBA), transcription-mediated amplification (TMA), rolling circle amplification and Qβ replicase amplification. In an additional embodiment, detection of the microorganism-capture probe-detector probe hybrid complex is accomplished via non-specifically labeling the hybrid complex.
In an additional aspect, the first target sequence and the second target sequence comprise the same sequence. In another aspect, the capture probe is immobilized on a solid support before hybridizing to the first target sequence. In yet another aspect, the microorganism-capture probe hybrid complex is immobilized on a solid support. In a further aspect, the microorganism-capture probe-detector probe hybrid complex is immobilized on a solid support. In another aspect, the solid support is selected from the group consisting of latex beads, agarose beads, paramagnetic beads, ferric oxide, microarray chips, filter paper, nitrocellulose filters, nylon membranes, glass slides and cellular membranes. In a further aspect, the solid support is a microarray chip. In an additional aspect, two or more capture probes are immobilized on a single spot of the solid support. In a further aspect, the method described above further comprises an immobilization probe that is capable of hybridizing to the capture probe to be immobilized onto the solid support.
The methods of the present invention additionally provide a method for identifying the species of one or more microorganisms in a sample, the method comprising: (a) releasing RNA or DNA from the at least one microorganism in the sample; (b) contacting the RNA or DNA with at least one species-specific capture probe capable of hybridizing to a first target sequence of the RNA or DNA, wherein the contacting is performed under conditions that permit hybridization between the first target sequence and the at least one species-specific capture probe to form a species-specific microorganism-capture probe hybrid complex, and wherein the at least one species-specific capture probe comprises at least one sequence selected from the group consisting of SEQ ID NOs:1-53, 55, 56, 61, 62, 67, 68, and 72-78; and (c) detecting the presence of the species-specific microorganism-capture probe hybrid complex by (i) contacting the RNA or DNA with at least one detector probe capable of hybridizing to a second target sequence of the RNA or DNA, wherein the detector probe comprises at least one reporter group and wherein the contacting is performed under conditions that permit hybridization between the second target sequence and the at least one detector probe to form a species-specific microorganism-capture probe-detector probe hybrid complex, and wherein the at least one detector probe also comprises at least one sequence selected from the group consisting of SEQ ID NOs:1-53, 55, 56, 61, 62, 67, 68, and 72-78; (ii) detecting the species-specific microorganism-capture probe-detector probe hybrid complex by detecting the at least one reporter group, wherein the presence of the species-specific microorganism-capture probe-detector probe hybrid complex indicates the presence of the at least one microorganism belonging to the species. In a further embodiment, the amplification sequence initiates an amplification reaction selected from the group consisting of strand displacement amplification (SDA), polymerase chain reaction (PCR), reverse transcriptase-strand displacement amplification (RT-SDA), reverse transcriptase-polymerase chain reaction (RT-PCR), nucleic acid sequence based amplification (NASBA), transcription-mediated amplification (TMA), rolling circle amplification and Qβ replicase amplification. In another embodiment, the reporter group is selected from the group consisting of a radioactive isotope, an enzyme, a fluorescent molecule and an amplification sequence. In an additional embodiment, detection of the microorganism-capture probe-detector probe hybrid complex is accomplished via non-specifically labeling the hybrid complex.
In an additional aspect, the first target sequence and the second target sequence comprise the same sequence. In another aspect, the species-specific capture probe is immobilized on a solid support before hybridizing to the first target sequence. In yet another aspect, the species-specific microorganism-capture probe hybrid complex is immobilized on a solid support. In a further aspect, the species-specific microorganism-capture probe-detector probe hybrid complex is immobilized on a solid support. In another aspect, the solid support is selected from the group consisting of latex beads, agarose beads, paramagnetic beads, ferric oxide, microarray chips, filter paper, nitrocellulose filters, nylon membranes, glass slides and cellular membranes. In a further aspect, the solid support is a microarray chip. In an additional aspect, two or more species-specific capture probes are immobilized on a single spot of the solid support. In a further aspect, the method described above further comprises an immobilization probe that is capable of hybridizing to the capture probe to be immobilized onto the solid support.
The present invention further provides a method of determining the efficacy of an antimicrobial patient therapy, comprising: (a) identifying the presence or absence of a microorganism in a first patient sample according to the method claim 1; (b) identifying the presence or absence of the microorganism in a second patient sample according to the method of claim 1; wherein the first patient sample and the second patient sample are taken sequentially over time, and wherein detection of the microbial nucleic acid in the first sample and subsequent failure to detect nucleic acid in the second sample indicates a successful response to therapy; and detection of the microbial nucleic acid in the second sample indicates the continued presence of viable organisms in the sample. In an additional embodiment, the reporter group is selected from the group consisting of a radioactive isotope, an enzyme, a fluorescent molecule and an amplification sequence. In another embodiment, the amplification sequence initiates an amplification reaction selected from the group consisting of strand displacement amplification (SDA), polymerase chain reaction (PCR), reverse transcriptase-strand displacement amplification (RT-SDA), reverse transcriptase-polymerase chain reaction (RT-PCR), nucleic acid sequence based amplification (NASBA), transcription-mediated amplification (TMA), rolling circle amplification, and Qβ replicase amplification. In a further embodiment, the solid support is selected from the group consisting of latex beads, agarose beads, paramagnetic beads, ferric oxide, microarray chips, filter paper, nitrocellulose filters, nylon membranes, glass slides and cellular membranes. In an additional embodiment, the solid support is a microarray chip. In another embodiment, two or more capture probes are immobilized on a single spot of the solid support. In an additional embodiment, the method further comprises an immobilization probe that is capable of hybridizing to the capture probe to be immobilized onto the solid support. In still another embodiment, detection of the microorganism-capture probe-detector probe hybrid complex is accomplished via non-specifically labeling the hybrid complex.
The present invention provides a kit for detecting the presence or absence of at least one microorganism in a sample, comprising: (a) a solid support; (b) at least one capture probe comprising at least one capture sequence capable of hybridizing to at least one target sequence of RNA and/or DNA from the microorganism to form a microorganism-capture probe hybrid complex; wherein the at least the detector probe also comprises at least one sequence selected from the group consisting of SEQ ID NOs:1-53, 55, 56, 61, 62, 67, 68, and 72-78; (c) at least one detector probe capable of hybridizing to a second sequence of the RNA or DNA, wherein the detector probe comprises at least one reporter group, and wherein the detector probe comprises at least one sequence selected from the group consisting of SEQ ID NOs:1-53, 55, 56, 61, 62, 67, 68, and 72-78; and (d) a vessel to collect, concentrate, amplify or isolate the RNA or DNA. In one aspect, the vessel is selected from the group consisting of evacuated blood collection tubes, eppendorf tubes and test tubes. In another aspect, the solid support is selected from the group consisting of latex beads, agarose beads, paramagnetic beads, ferric oxide, microarray chips, filter paper, nitrocellulose filters, nylon membranes, glass slides and cellular membranes.
The present invention further provides an oligonucleotide for use in detecting a microorganism selected from the group consisting of Staphylococcus aureus, Escherichia coli, Staphylococcus epidermidis, Klebsiella pneumoniae, Enterococcus faecalis, Pseudomonas aeruginosa, Streptococcus pneumoniae, Streptococcus mutans, Streptococcus gordonii, Clostridium perfringens, Clostridium botulinum, Haemophilus influenzae, Enterococcus durans, Streptococcus pyogenes, Streptococcus agalacticae, Clostridium difficile and Enterococcus faecium. In one embodiment, Staphylococcus aureus is selected from the group consisting of SEQ ID NOs:1, 2, 44, 47, 50, 61, 62, 73 and 76. In another embodiment, Escherichia coli is selected from the group consisting of SEQ ID NOs:3-7, 43, 46, 49, 52, 53, 55, 56, 72, 75 and 78. In a further embodiment, Staphylococcus epidermidis is selected from the group consisting of SEQ ID NOs:8-10, 45, 48, 51, 67, 68, 74 and 77. In an additional embodiment, Klebsiella pneumoniae is selected from the group consisting of SEQ ID NOs:11-13. In yet another embodiment, Enterococcus faecalis is selected from the group consisting of SEQ ID NOs:14-16. In one aspect, Pseudomonas aeruginosal is selected from the group consisting of SEQ ID NOs:17 and 18. In another aspect, Streptococcus pneumoniae is selected from the group consisting of SEQ ID NOs:19 and 20. In a further aspect, Streptococcus mutans is selected from the group consisting of SEQ ID NOs:21 and 22. In an additional aspect, Streptococcus gordonii is selected from the group consisting of SEQ ID NOs:23 and 24. In yet another aspect Clostridium perfringens is selected from the group consisting of SEQ ID NOs:27 and 28. In another embodiment, Clostridium botulinum is selected from the group consisting of SEQ ID NOs:29 and 30. In a further embodiment, Haemophilus influenzae is selected from the group consisting of SEQ ID NOs:31 and 32. In an additional embodiment, Enterococcus durans is selected from the group consisting of SEQ ID NOs:35-37. In yet another embodiment, Streptococcus pyogenes is selected from the group consisting of SEQ ID NOs:38-40. In a further aspect, Streptococcus agalacticae is selected from the group consisting of SEQ ID NOs:41 and 42. In another aspect, Clostridium difficile is selected from the group consisting of SEQ ID NOs:25 and 26. In an additional aspect, Enterococcus faecium is selected from the group consisting of SEQ ID NOs:33 and 34.
The present invention relates to probes, methods, and kits for identifying the presence or absence of at least one microorganism in a sample. The probes of the present invention comprise single-stranded nucleic acid or nucleic acid derivatives such as a peptide nucleic acid. Probes of the present invention comprise (a) nucleic acid sequences capable of hybridizing to nucleic acid sequences specific to microorganisms and/or (b) nucleic acid sequences capable of hybridizing to another probe according to the present invention.
Probes capable of binding microorganism RNA and/or DNA are referred to herein as “capture probes” and/or “detector probes.” Capture probes are often, but need not be, immobilized to a solid support. Detector probes often, but need not, comprise a means for facilitating detection of the microorganism RNA and/or DNA. For example, detector probes often, but need not, comprise a reporter group. Methods of the present invention comprise releasing RNA and/or DNA from at least one microorganism in a sample and contacting the RNA and/or DNA with at least one capture probe under conditions that permit specific hybridization between the microorganism RNA and/or DNA and at least a portion of the probe to form a hybrid complex. A hybrid complex between a capture probe and microorganism RNA and/or DNA may be referred to herein as a “microorganism-capture probe hybrid complex.” The microorganism-capture probe hybrid complex may, but need not, be detected with a detector probe that likewise forms a specific hybrid with the microorganism RNA and/or DNA. A hybrid complex between a detector probe and microorganism RNA and/or DNA that is also hybridized to a capture probe may be referred to herein as a “microorganism-capture probe-detector probe hybrid complex.” The presence or absence of a specific hybrid complex correlates with the presence or absence of the microorganism.
The probes and/or identification methods of the present invention may be used to identify the genus and/or species of one or more microorganisms. The probes and/or identification methods of the present invention may be used to determine whether one or more microorganisms of a particular genus and/or species is present in a sample. Alternatively, the probes and/or identification methods of the present invention may be used to identify whether a sample contains one or more microorganisms belonging to a general classification category such as a taxonomic family, or to even a broader category. As a non-limiting example, the probes and/or methods of the present invention can be used to determine whether a sample contains a fungus, bacterium, virus, or parasitic microorganism. The probes and/or methods of the present invention may also be used to determine susceptibility to antimicrobial agents by determining the presence, absence and/or expression of specific markers, such as the antimicrobial drug resistance genes mecA, vanA or vanB.
For the purposes of the present invention, the term “microorganism” is used to mean a prokaryotic organism, a bacterium, a fungus, a parasite, a protozoan, or a virus. These terms are not mutually exclusive; as two non-limiting examples, many protozoa are parasites, and all bacteria are prokaryotic organisms. A “sample,” as the term is used herein, can be derived from an animal and can include, for example, blood, urine or other body fluids, organs, tissues and any portions thereof, or can be obtained from the environment, such as air, water or soil, or from material intended for human or animal use or consumption, such as meat, fish or dairy produce, and even cosmetics. Furthermore, the methods of the present invention may be performed on the entire sample or only a portion or fraction thereof. As a non-limiting example, a sample may be whole blood from a subject, or the sample may be a collection of platelets isolated or concentrated from a subject's blood. As used herein, “subject” means an animal. The term “animal” includes, but is not limited to, birds, fish, and mammals, such as but not limited to, human and non-human primates, farm animals, and companion animals. As used herein, the terms “subject” and “patient” are used interchangeably. The sample can also be derived from in vitro cultures of cells. The cells of the cell culture can be eukaryotic or prokaryotic including, but not limited to, animal cells, plant cells, and bacterial cells. The cell cultures can be, for example, derived cells isolated from tissues, organs, or body fluid of an animal or plant. In some embodiments, the biological sample comprises animal cells that are derived from a subject. The term “sample” also encompasses a culture medium that has been inoculated with a sample taken from a mammal, food, the environment, cosmetics, or the like to permit any microorganisms present in the sample to replicate to detectable levels.
In certain embodiments, a sample is treated to concentrate or isolate microorganisms before releasing nucleic acid from them. Microorganisms may be concentrated in a sample prior to, or simultaneously with, the release of the nucleic acids. Alternatively, the DNA and/or RNA may be released prior to the concentration process.
Many methods for concentrating and/or isolating microorganisms are known in the art. Examples of ways to concentrate the microorganisms in the biological sample include, but are not limited to, using a Wampole Isolator™ tube (Wampole Laboratories, New Jersey, USA), a BD CPT™ tube (Becton, Dickinson and Company, New Jersey, USA), di-electrophoresis, traveling wave field migration, and electrophoresis. For example,
As the present invention contemplates, concentration of microorganisms and/or their nucleic acids can be accomplished in any number of steps including, but not limited to, one, two or three steps, where, in each step, the sample is progressively more concentrated. As a non-limiting example, microorganisms or their nucleic acids may be first concentrated using, for instance, a Wampole Isolator™ tube. To continue this example, the concentrated sample then may be concentrated further, separating intact microorganisms or their nucleic acids using, for instance, electrophoresis.
As used herein, the terms “nucleic acids” and “oligonucleotides” are used to mean DNA or RNA, as is recognized in the art. Nucleic acids may be single-stranded or double-stranded. Nucleic acids may be “released” from a microorganism using any means that will allow a capture probe access to hybridize the DNA or RNA. Hence, a “released” nucleic acid is a nucleic acid that is in a physical and chemical environment that allows nucleic acid probes to bind to it. Additionally, both DNA and RNA may be released by the same processes. Examples of ways that DNA or RNA may be released include, but are not limited to, lysing the microorganisms using, for example, heat, enzymes, detergents, buffers, acids, bases, chaotropes, physical shearing in the presence of beads or particles and the application of pressure. As is contemplated by the present invention, the act of collecting, isolating, or concentrating the sample, or the portion thereof to be tested, may sufficiently release the nucleic acids that are subject to capturing. For the purposes of the present invention, the DNA or RNA to be used may be, but need not be, purified, isolated or concentrated further after release. Methods for purification, isolation, and concentration of nucleic acids are well-known in the art. It is preferable that nucleic acids released from microorganisms be in single-stranded form and lack internal secondary structure before they are contacted with probe(s) according to the present invention. Accordingly, the methods of the present invention may also include one or more denaturing step, to denature any double-stranded nucleic acids or nucleic acids that possess internal secondary structure that are released from the microorganisms, prior to contacting the DNA or RNA with the capture probe. However, such a denaturing step is not required, particularly where the acts of releasing, purifying, isolating, and/or concentrating the nucleic acids also result in their denaturation. Methods for denaturation of nucleic acids are well-known in the art.
Microorganisms that are to be detected may be referred to as “target microorganisms.” Nucleic acids released by microorganisms that are to be detected may be referred to as “target nucleic acids” or “target oligonucleotides.” Target oligonucleotides will usually comprise at least one sequence that is capable of binding to a capture probe and/or a detector probe being used to detect microorganisms in a sample. Such a sequence may be referred to herein as a “target sequence.”
Once nucleic acids are released from the microorganisms, they may be contacted with one or more capture probes that are immobilized on a solid support. As an alternative, the released nucleic acids may be contacted with a free (non-immobilized) capture probe to form a hybrid complex, which is then contacted with a solid support that immobilizes the hybrid complex. As yet another alternative, capture probes may remain free (i.e., not immobilized). In such cases, hybrid complexes may be isolated by art-known means such as electrophoresis.
As used herein, a “capture probe” is a nucleic acid, or nucleic acid derivative such as a peptide nucleic acid, that is capable of binding to a released nucleic acid. A capture probe contains at least one single-stranded portion, or sequence, that is capable of contacting and hybridizing with released microorganism nucleic acids. A sequence that is capable of contacting and hybridizing with released microorganism nucleic acids may be referred to herein as a “capture sequence.” As used herein, capture probes may be classified by, for example, the microorganism(s) to which their capture sequence(s) is capable of binding. Thus, capture probes of the same “type” comprise capture sequence(s) capable of binding to the same microorganism(s). A capture probe comprises at least one capture sequence. A capture probe often also comprises, but is not required to comprise, sequences in addition to at least one capture sequence. Such additional sequences may, for example, facilitate immobilization of the capture probe.
Detector probes may be used to facilitate the detection of nucleic acids that have been released from microorganisms. Nucleic acids that have been released from microorganisms may be contacted with one or more detector probes. As used herein, a “detector probe” is a nucleic acid, or nucleic acid derivative such as a peptide nucleic acid, that is capable of binding to a released nucleic acid and that is capable of being detected, thereby facilitating detection of the released nucleic acid. A detector probe contains at least one single-stranded portion, or sequence, that is capable of contacting and hybridizing with a released microorganism nucleic acid. As with capture probes, a sequence of a detector probe that is capable of contacting and hybridizing with released microorganism nucleic acids may be referred to herein as a “capture sequence.” A detector probe is preferably bound to a reporter group to facilitate detection. A detector probe to can be bound to a reporter group before or after the detector probe is hybridized to a target oligonucleotide. Reporter groups are known in the art and are discussed in more detail hereinbelow.
The capture sequence of a detector probe will usually hybridize to a different target sequence of a target oligonucleotide than the target sequence to which the capture sequence of the capture probe hybridizes. Accordingly, a target oligonucleotide can be bound and detected by a detector probe while is bound to a capture probe. In such embodiments, either the capture probe or the detector probe may be hybridized to the target oligonucleotide first. In certain embodiments it may be desirable to utilize a capture probe and detector probe each having a capture sequence that binds to the same target sequence. In such embodiments, a target oligonucleotide may be captured by a capture probe, the capture probe-target complex may be isolated, the capture probe-target complex may be denatured, and the detector probe may them be hybridized to the target oligonucleotide.
As with capture probes, detector probes may be classified by, for example, the microorganism(s) to which their capture sequence(s) is capable of binding. Thus, detector probes of the same “type” comprise capture sequence(s) capable of binding to the same microorganism(s). A detector probe comprises at least one capture sequence. A detector probe often also comprises, but is not required to comprise, sequences in addition to at least one capture sequence. Such additional sequences may, for example, facilitate the binding of the detector probe to a reporter group.
In some embodiments, capture probes and/or detector probes comprise linker molecules such as, but not limited to, carbon chains or nucleic acid sequences that are not complementary to the target oligonucleotide. A linker molecule may serve, for example, to attach a probe to a solid surface, to bind a probe to another type of molecule (such as, for example, a protein), to attach a probe to a reporter group, or to bind a probe to another probe. A sequence that serves to immobilize a capture probe to a solid surface may be referred to herein as an “immobilization sequence.” A probe comprising an immobilization sequence may be referred to herein as an “immobilization probe.” Such non-complementary linkages may reduce steric hindrance and may also improve the kinetics of hybridization by increasing the accessibility of the probes, particularly the capture sequence(s), to the bulk solution. Examples of nucleic acid sequences that may be used as linker molecules include the human genes K-alpha (tubulin alpha-1), PPIA (peptidylprolyl isomerase A), and UBC (ubiquitin-conjugating enzyme E2A), and portions thereof.
Capture probes of the current invention may be “immobilized” onto a solid support. As used herein, “immobilized” means affixed to a solid support such that movement of the capture probe in a solution is limited, i.e., a capture probe that is immobilized on a solid support will not dissociate from the solid support unless it is subjected to a condition or procedure that would cause it to dissociate.
As used herein, a “solid support” is a structure or a scaffold that will not dissolve in a liquid or gas solution. Examples of solid supports include, but are not limited to, latex beads, agarose beads, sepharose beads, paramagnetic beads, ferric oxide, microarray chips, filter paper, nitrocellulose filters, nylon membranes, vessels, glass slides, and even cellular membranes. In some embodiments, the method of the present invention utilizes a three-dimensional microarray, such as, for example, the MetriGenix® Flow-Thru Chip® (MetriGenix, Inc., Maryland, USA), which facilitates increased hybridization kinetics. An example of a MetriGenix® Flow-Thru Chip® is illustrated in
Capture probes may be immobilized onto solid supports using any of the many art-known methods. Preferably, the immobilization does not adversely affect the capture probe's ability to bind to microorganism DNA and/or RNA or to other probes. A capture probe may be immobilized directly to the solid support, or it may be immobilized indirectly via attachment to another molecule that is immobilized on the solid support. For example, a capture probe may be immobilized using chemical or linker moieties such as carbon chains or polyethylene glycol (PEG). In such cases, the binding of the capture probe may be non-specific. Alternatively, methods of using chemical moieties to bind specific nucleic acid sequences are known and may be used with the present invention. As a non-limiting example, capture probes may be biotinylated, with biotin possessing the ability to bind to avidin or streptavidin. Continuing the example, the solid support may have avidin or streptavidin bound to it. Such a scheme is a non-limiting example of a method for immobilizing capture probes without adversely affecting their ability to bind microorganism DNA and/or RNA because the biotin can be located at the opposite end of the molecule from the sequence capable of binding microorganism DNA and/or RNA (which may be called a “capture sequence”), or the biotin may be located on an internal branch of the capture probe that will result in its being located at a sufficient distance from the capture sequence that the binding of the capture sequence to microorganism DNA and/or RNA is not hindered.
As another example, capture probes may also be immobilized using another single-stranded oligonucleotide probe that is itself immobilized and that is capable of hybridizing with the capture probe to be immobilized. Such oligonucleotide probes may be called “immobilization probes.” The use of immobilization probes is another non-limiting example of a method for immobilizing capture probes without adversely affecting their ability to bind microorganism DNA and/or RNA because the sequence on the capture probe that is capable of binding to the immobilization probe (which may be called an “immobilization sequence”) can be located at the opposite end of the molecule from the sequence capable of binding microorganism DNA and/or RNA (which may be called a “capture sequence”), or the immobilization sequence may be located on an internal branch of the capture probe that will result in its being located at a sufficient distance from the capture sequence that the binding of the capture sequence to microorganism DNA and/or RNA is not hindered. An example of immobilization of a capture probe via an immobilization probe is illustrated in
In some embodiments, capture probes may be immobilized directly or indirectly on a solid support in a pattern of discrete areas, or “spots.” Such a pattern, or a solid support capable of supporting such a pattern, may be referred to herein as an “array,” a “microarray,” or a “chip.” The immobilization of probes of different types to a single microarray or chip facilitates the simultaneous determination of whether different microorganisms are present in a single sample.
In certain embodiments, only a single type of capture probe is immobilized to any one spot, and different types of capture probes may be immobilized to different spots. In such embodiments, the identity of the capture probe immobilized in any given spot is known, so microorganisms hybridized to capture probes in different spots can be identified and differentiated from one another by means of their locations. Examples of immobilization of different types of capture probes in different spots of arrays are illustrated in
In other embodiments, more than one type of capture probe is immobilized in a single spot. In such embodiments, it will often be useful to use employ detector probes such that each detector probe of the same type is bound to a reporter group that is differentiable from reporter groups bound to any other type of detector probe. As a non-limiting example, one could perform an assay in which detector probes that bind to a target sequence from E. coli are labeled with fluorescein, and detector probes that bind to a target sequence from S. aureus are labeled with rhodamine. In such an assay, the presence of E. coli could be differentiated from the presence of S. aureus by the difference in the colors of the fluorescent labels. Of course, detector probes with differentiable labels may also be used in conjunction with the immobilization of different types of capture probes in different spots, thereby facilitating the performance of complex assays.
Detector probes may be attached directly or indirectly to a reporter group. As an example of an indirect attachment using a linker molecule, a detector probe may comprise a “reporter adapter sequence” linker. A reporter adapter sequence is a portion of a detector probe that is capable of binding via hybridization to a single-stranded oligonucleotide that bears a reporter group, which may be referred to herein as a “reporter probe.”
Capture probes and detector probes may be “protected” from prematurely hybridizing to random nucleic acids by having a protecting group situated on or near the capture or detector probe. As non-limiting examples, protecting groups include single-stranded nucleic acid that is partially complementary to the capture probe to be protected, or an antibody or a binding fragment thereof that binds to the single-stranded portion of the capture or detector probe to be protected.
Hybridization between a microorganism nucleic acid and a capture probe or detector probe may be referred to herein as a “hybridization event.” A hybridization event will form a “hybrid complex.” As used herein, a “hybrid complex” is a double-stranded nucleic acid comprising at least a portion of a capture probe or detector probe (usually a capture sequence) and at least a portion of a target oligonucleotide (usually a target sequence). A hybrid complex need not be double-stranded along its entire length. Furthermore, for the purposes of the present invention, a capture probe or detector probe and a target oligonucleotide need not have a complementary base pairing at every base for a hybridization event to occur. Further still, for the purposes of the present invention, a capture sequence and a target sequence need not have a complementary base pairing at every base for a hybridization event to occur. In other words, the present invention contemplates that a hybrid complex will be formed even if a target oligonucleotide hybridizes to a capture or detector probe such that a portion of the capture or detector probe or target nucleic acid is single-stranded after hybridization because the target oligonucleotide did not hybridize to the entire length of the capture or detector probe. In some embodiments of the present invention, a portion of a capture or detector probe remains single-stranded after hybridization to a target oligonucleotide. In some other embodiments, a portion of a target oligonucleotide remains single-stranded after hybridization to a capture or detector probe. In still other embodiments, portion(s) of each of a target oligonucleotide and a capture or detector probe remain(s) single-stranded after hybridization to one another. A “portion” can be one or more nucleic acids in length. Such single-stranded portions may occur within and/or outside of a capture sequence and/or a target sequence. Single-stranded portions within a capture sequence and/or target sequence may occur, as a non-limiting example, because the capture sequence and the target sequence are not 100% complementary. Single-stranded portions outside of a capture sequence and/or target sequence may occur, as a non-limiting example, because the capture and/or detector probe contains portions that are not intended to bind to the target oligonucleotide. Single-stranded portions outside of a capture sequence and/or target sequence may occur, as another non-limiting example, because the target oligonucleotide comprises sequences in addition to the target sequence. For example, a capture probe will often (but need not) comprise a sequence used to immobilize it to a solid support. As another example, a detector probe will often (but need not) comprise a sequence used to bind it to a reporter group. As yet another example, a target oligonucleotide will often (but need not) comprise sequences 3′ and/or 5′ to the target sequence(s).
A solid support may have immobilized to or on it one or various combinations of probes that are microorganism-specific, probes that are for epidemiological markers (e.g., IS6110-based probes used for Mycobacterium tuberculosis), and/or probes that are for drug resistance markers (e.g., mecA-based probes for methicillin resistance in S. aureus or rpoB-based probes for detection of rifampin resistance in M. tuberculosis). As used herein, the term “microorganism-specific probe” includes probes that are capable of hybridizing with a target sequence derived or released from a single microorganism species. Such probes may also be referred to herein as “species-specific probes.” The term “microorganism-specific probe” also includes probes that are capable of hybridizing with target sequences from more than one species of microorganism from a single genus of microorganism (e.g., IS6110 for the detection of the M. tuberculosis complex (M. tuberculosis, M. bovis, M. microti, and M. africanum); probes based on conserved regions of the 16S rRNA, 18S rRNA, RNase P or ssrA gene sequences). For example, a microorganism-specific probe may be designed to form a hybrid with nucleic acid sequences from both S. aureus and S. epidermidis, but not with E. coli. Such probes may also be referred to herein as “genus-specific probes.” In some embodiments, a genus-specific probe will hybridize to sequences derived from all or many of the microorganisms belonging to the same genus of classification. As used herein, a “multi-genus probe” will hybridize to nucleic acid from microorganisms belonging to two or more different genera. A probe may hybridize to an antimicrobial resistance marker that may be present in one or more species, for example. Such a probe may be species-specific, genus-specific, or multi-genus, depending on how widely the antimicrobial resistance marker is distributed through phylogeny. Whether a microorganism-specific probe, as contemplated by the present invention, hybridizes to a target sequence derived or released from a single microorganism species, to target sequences derived or released from more than one microorganism species within the same genus of microorganisms, or to target sequences derived or released from microorganisms from different genuses may also depend on the hybridization and wash conditions used in the assay.
As described above for capture probes, detector probes may be microorganism-specific probes, probes that are for epidemiological markers, and/or probes that are for drug resistance markers. Various combinations of capture probes and detector probes may be used to discriminate between organisms present in a sample. For instance, a genus-specific capture probe may be used to immobilize microorganisms of a selected genus, which then may be detected as a genus with one or more genus-specific detector probes, or which may be discriminated by species with one or more species-specific detector probes. More than one type of capture probe may be used concurrently in the methods of the present invention. Likewise, more than one type of detector probe may be used concurrently in the methods of the present invention.
As non-limiting examples, oligonucleotide probes comprising one or more of the sequences set forth in the following table (Table 1) are particularly useful for detecting and identifying bacteria of the indicated species. Oligonucleotide probes comprising one or more of the sequences set forth in Table 1 can be used as capture and/or detector probes to detect nucleic acids from the indicated bacterial species. Oligonucleotide probes comprising regions that are homologous to the oligonucleotide probes set forth in Table 1 are also useful for capturing and/or detecting the indicated species. In general, oligonucleotides containing sequences that are at least about 85%, at least about 90%, at least about 95%, or about 100% homologous to the oligonucleotides of Table 1 are useful.
The sequences in Table 1 can be used as species-specific capture and/or detector sequences to detect and/or differentiate between particular species of microorganisms. Sequences from Table 1 may, but need not, comprise portion(s) of longer oligonucleotides. For example, probes according to the invention may comprise one or more sequences from Table 1 and/or additional sequences.
Staphylococcus
aureus
Staphylococcus
aureus
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Staphylococcus
epidermidis
Staphylococcus
epidermidis
Staphylococcus
epidermidis
Klebsiella
pneumoniae
Klebsiella
pneumoniae
Klebsiella
pneumoniae
Enterococcus
faecalis
Enterococcus
faecalis
Enterococcus
faecalis
Pseudomonas
aeruginosa
Pseudomonas
aeruginosa
Streptococcus
pneumoniae
Streptococcus
pneumoniae
Streptococcus
mutans
Streptococcus
mutans
Streptococcus
gordonii
Streptococcus
gordonii
Clostridium
difficile
Clostridium
difficile
Clostridium
perfringens
Clostridium
perfringens
Clostridium
botulinum
Clostridium
botulinum
Haemophilus
influenzae
Haemophilus
influenzae
Enterococcus
faecium
Enterococcus
faecium
Enterococcus
durans
Enterococcus
durans
Enterococcus
durans
Streptococcus
pyogenes
Streptococcus
pyogenes
Streptococcus
pyogenes
Streptococcus
agalacticae
Streptococcus
agalacticae
Escherichia coli
Staphylococcus
aureus
Staphylococcus
epidermidis
Escherichia coli
Staphylococcus
aureus
Staphylococcus
epidermidis
Escherichia coli
Staphylococcus
aureus
Staphylococcus
epidermidis
Escherichia coli
Escherichia coli
For the purposes of present invention, a capture probe captures (by binding to) an oligonucleotide from a sample by hybridizing with it at a sequence (e.g., a capture sequence) that is at least partially complementary to a sequence (e.g., a target sequence) of the oligonucleotide being captured. Likewise, a detector probe detects (by binding to) an oligonucleotide from a sample by hybridizing with it at a sequence (e.g., a capture sequence) that is at least partially complementary to a sequence of the oligonucleotide being captured (e.g., a target sequence). As used herein, the phrase “partially complementary” means less than 100% complementary, but at least about 85% complementary. Accordingly, the phrase “at least partially complementary” indicates that the capture sequence of a capture and/or detector probe may between about 85% complementary to about 100% complementary to a target sequence to be useful according to the present invention. A capture sequence and a target sequence of an oligonucleotide to be captured may be, as non-limiting examples, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% complementary to one another. For example, if a capture sequence is 100 bases long, and the target sequence is 95% complementary to the capture sequence, the base pairs of the capture sequence and the target sequence will match in 95 of 100 bases of the capture sequence.
As a practical matter, whether any particular nucleic acid molecule is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to a target nucleic acid can be determined conventionally using known computer programs such as, for example, the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, Wisconsin, USA). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% complementary to a reference sequence according to the present invention, the parameters are set such that the percentage of identity is calculated over the full length of the reference nucleotide sequence, whether that be the capture probe or the target nucleic acid, and that gaps in similarity of up to 5% of the total number of nucleotides in the reference sequence are allowed.
Whether the capture sequence of a capture probe and/or detector probe will hybridize to the target sequence of a target oligonucleotide depends on the degree of complementarity between the target sequence and the capture sequence, as well as both the hybridization conditions and the stringency of the wash after hybridization. As used herein, the phrase “conditions that permit hybridization” refers to hybridization parameters, as well as wash parameters, that permit hybridization between two oligonucleotides, as are understood in the art. For example, conditions that permit hybridization include, but are not limited to, more stringent hybridization and wash conditions, such as incubation at 42° C. in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing with 0.1×SSC at about 65° C., 68° C. or 70° C. Of course, hybridization and wash conditions can be set to a lower stringency. Lower stringency hybridization and wash conditions include, but are not limited to, incubation at 42° C. in a solution comprising 30% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in a solution of 2×SSC or 1×SSC or 0.5×SSC at about 55° C. or 60° C. or 65° C. As is within the capacity of one of ordinary skill in the art, the conditions to permit hybridization can be easily and routinely optimized to require a lower or higher degree of complementarity between a capture probe and/or detector probe and a target nucleic acid before hybridization will occur. For example, anionic detergents such as sodium dodecyl sulfate (SDS) may be used to enhance the stringency of hybridization or washing, and exclusion molecules such as PEG may be used to increase the effective concentration of reaction components.
A capture probe or detector probe may be an oligonucleotide or a polynucleotide, as these terms are understood in the art. A capture probe or detector probe may be, for example, at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 300, 400, 500 or 750 nucleotides in length. For convenience, the term “oligonucleotide” as used herein encompasses all of these lengths. In some embodiments, capture probes and/or detector probes may be up to and including about 2000 nucleotides in length. In some embodiments, capture probes and/or detector probes are about 15 to about 60 nucleotides in length.
The length of the capture probe and/or detector probe and the capture sequence(s) thereof and the conditions of hybridization may be tailored to form a specific complex with the nucleic acid of the intended target. The stability of a hybrid complex, commonly measured by its melting temperature, is related to the concentration of the probe, the hybridization conditions, the length of the hybrid complex and the degree of sequence identity between the capture sequence and the target sequence. The stability of a hybrid complex is decreased by mismatches and is increased by the number of base pairs in the hybrid complex. Such relationships are detailed, for example, in Sambrook et al., M
After a hybrid complex is formed, the methods of the present invention then detect the hybrid complex. If a hybrid complex is detected, the presence of the hybrid complex indicates that the target sequence was released from a microorganism in the sample tested. Accordingly, the presence of a hybrid complex indicates the presence in the sample of the microorganism and/or epidemiological marker containing the target sequence. In contrast, when a hybrid complex is not detected, the absence of the hybrid complex indicates that the target sequence was not released from a microorganism in the sample tested. Accordingly, the absence of a hybrid complex indicates that the sample most likely does not contain the microorganism and/or epidemiological marker containing the target sequence.
Any method of detecting the hybrid complex can be used in the present invention, provided that one of skill in the art can rely on the detection methods to identify the presence of a hybrid complex. In some embodiments, at least one detector probe, in addition to the capture probe, is used to detect the hybrid complex. The detector probe hybridizes to a single-stranded portion (or “target sequence”) of the target nucleic acids that are captured.
A detector probe may comprise a reporter group to facilitate detection. As used herein, a “reporter group” means an entity that can generate a detectable signal. A reporter group may be incorporated into a detector probe, a reporter group may be directly linked or bound to a detector probe, or a reporter group may be indirectly linked or bound to a detector probe. As explained in more detail hereinabove, as an example of an indirect attachment using a linker molecule, a detector probe may comprise a reporter adapter sequence linker, which is capable of binding via hybridization to a reporter probe, which is bound to a reporter group. Detector probes that “comprise” reporter groups include detector probes that have a reporter group, reporter adapter sequence linkers, and the like incorporated therein, as well as detector probes that are directly or indirectly linked or bound to reporter groups, reporter adapter sequence linkers, and the like.
Many different reporter groups are known in the art. For example, radioactive isotopes may be used as reporter groups. Radioactive isotopes may be, for example, incorporated into or attached to a detector probe, thereby generating a radioactive probe. Examples of a radioactive isotopes that can be used as reporter groups include, but are not limited to, 32P, 33P 131I, 90Y, 188Re, 186Re, 67Cu, 198Au, 103Pd and 212Pb/212Bi.
Enzymes and/or enzyme systems that can be used to generate a detectable signal may also be used as reporter groups. Enzymes and/or other components of enzyme systems can be attached directly to a detector probe or can attach indirectly to a detector probe. For example, a detector probe may be biotinylated. As a specific, non-limiting example, a detector probe may be bound to BioTEG, which is biotin with a 15 atom tetra-ethyleneglycol spacer. Biotin possesses the ability to bind to avidin or streptavidin. Continuing the example, an enzyme such as horseradish peroxidase would be conjugated to avidin or streptavidin, thereby allowing the horseradish peroxidase to localize to the hybrid complex via binding of the biotin of the biotinylated detector probe and the avidin of the avidin-enzyme conjugate. Upon addition of a substrate, horseradish peroxidase would then generate a detectable colorimetric signal as is readily understood in the art. Additional examples of enzymes that may be used as reporter groups include, but are not limited to alkaline phosphatase, glucose oxidase, β-galactosidase, soybean peroxidase and luciferase.
Fluorescent or other detectable molecules may also be used as reporter groups and may be attached directly or indirectly to the detector probe. Non-limiting examples of detectable molecules that may be used as reporter groups include, but are not limited to, fluorescein, fluorescein isothiocyanate (FITC), rhodamine red, ROX™ (Invitrogen, California, USA), Cy™ dyes (Amersham, N.J., USA), Bodipy™ dyes (Molecular Probes, Oregon, USA), TAMRA™ dyes (Molecular Probes, Oregon, USA), TET™ (Molecular Probes, Oregon, USA), Texas Red® (Molecular Probes, Oregon, USA), europium dyes, chromogenic moieties and green fluorescent protein (GFP).
Reporter groups may also comprise an “amplification sequence,” i.e., a nucleotide sequence that can initiate nucleic acid amplification. For example, an amplification sequence may be appended to a detector probe, or an amplification sequence may be an integral part of a detector probe. An amplification sequence can initiate nucleic acid replication, or amplification, using any form of amplification including, but not limited to, strand displacement amplification (SDA), polymerase chain reaction (PCR), reverse transcriptase-strand displacement amplification (RT-SDA), reverse transcriptase-polymerase chain reaction (RT-PCR), nucleic acid sequence based amplification (NASBA), MessageAmp™ amplification (Ambion, Inc., Texas, USA), transcription-mediated amplification (TMA), rolling circle amplification, and Qβ replicase amplification. The nucleic acids may be amplified prior to contacting them with a capture probe or after they are contacted with the capture probe.
Instead of or in addition to the use of a detector probe, a hybrid complex may also be detected using such standard methods as non-specifically labeling the hybrid complex. For example, intercalating dyes, such as ethidium bromide, may be used to label the hybrid complex for detection. Other examples of non-specific labeling of the hybrid complex include, but are not limited to, acridine orange, SYBR™ Green I/II (Molecular Probes, Oregon, USA), SYBR Gold, propidium iodide and cyanine monomers or dimers.
In some embodiments, the methods and/or probes of the present invention are used to monitor the efficacy of antimicrobial patient therapy by sequential sampling of specimens over time. The detection of mRNA has been correlated with microbial viability. Hellyer et al., J Clin. Microbiol. 37: 290-295 (1999). Accordingly, the detection of RNA in a first sample, followed by administration of antimicrobial therapy and subsequent failure to detect RNA in a second sample, most likely indicates a successful response to therapy. In contrast, detection of RNA in the second sample would most likely indicate the continued presence of viable organisms in the specimen, and the need for continued therapy or a change in therapeutic regimen.
In some other embodiments, the methods and/or probes of the present invention are used to quantify the number of organisms in a sample through the use of an internal standard and by comparison of signal intensities with controls. The internal standard may be RNA or DNA that is free in solution or encapsulated, such as in an Armored RNA™ (Ambion Diagnostics, Texas, USA) particle or recombinant bacterium, to protect against degradation. The internal standard may be seeded into the sample at any point prior to detection. In some embodiments, the internal standard is seeded in the sample prior to concentration and lysis of the microorganism(s). The internal standard is detected using specific capture probes that permit distinction of the standard from target nucleic acid and from other nucleic acids that may be present. The signal generated by the internal standard in the test sample is compared to that from controls that comprise different levels of the standard nucleic acid conjugated directly to the solid phase. By plotting a curve of signal intensities for the controls, the proportion of the internal standard recovered from the specimen may be calculated. In similar fashion, controls comprising different levels of the target sequence conjugated directly to the solid phase may be used to quantify the amount of target present in the processed sample. By correcting for recovery of nucleic acid using the internal standard, the quantity of target nucleic acid in the original sample may then be calculated.
It is important to note that the methods of the present invention also have application outside the fields of human and animal infectious disease and are particularly suited to applications requiring rapid, sensitive and specific detection and/or identification of multiple analytes. Accordingly, the methods of the present invention can be used to detect microorganisms in samples in many fields including, as non-limiting examples, therapeutic monitoring, food and environmental testing and monitoring deployment of weapons of bioterrorism.
The present invention also relates to kits for detecting the presence or absence of at least one microorganism in a sample. The kits comprise a solid support, as defined hereinabove, comprising at least one capture probe, also defined hereinabove. The at least one capture probe may be a microorganism-specific probe, a probe that is for an epidemiological marker, and/or a probe that is for a drug resistance marker. The kits of the present invention also comprise at least one reporter group, as previously described hereinabove. The kits may also comprise a vessel. The vessel can be used to collect or concentrate the sample and/or to isolate the nucleic acids released from the microorganisms. The vessels may also be used when amplifying the nucleic acids released from the microorganism, if desired or necessary. Examples of vessels include, but are not limited to, evacuated blood collection tubes, eppendorf tubes, test tubes, etc. The kits may further comprise enzymes or other chemicals, such as media, detergents, buffers, acids, bases, and chaotropes used to lyse the microorganisms present in the sample. The reporter group(s) of the kits further comprises at least one detector probe. In such embodiments, the reporter group may be incorporated into or onto the detector probe, as previously described herein. As with the capture probe, the detector probe may be a universal probe or a species-specific probe. Kits of the present invention may also comprise positive, negative, and/or internal controls. In certain embodiments, kits of the present invention may comprise a sufficient number of probes and/or amounts of other components to permit the performance of only a single assay or group of related assays using probes according to the present invention. In other embodiments, kits of the present invention may comprise a sufficient number of probes and/or amounts of other components to permit the performance of multiple assays using probes according to the present invention.
The Figures provide non-limiting exemplary illustrations of embodiments of the present invention and devices and non-limiting exemplary sequences useful in practicing the present invention. One exemplary embodiment of the probes and methods of the present invention is illustrated in
Two exemplary embodiments of the use of the probes and methods of the present invention are illustrated in
In contrast, the capture probes of the embodiment illustrated in
In each of the embodiment of
Immobilization probe-capture probe combinations may be varied and customized, however. As just one non-limiting example, using
A biotinylated detector probe is bound to each target oligonucleotide via hybridization between a capture sequence of the detector probe and a second target sequence of the target oligonucleotide. Streptavidin conjugated to horseradish peroxidase enzyme is bound to the biotin molecules of the biotinylated detector probes. A horseradish peroxidase enzyme substrate is added, and a detectable signal is generated.
The following experimental examples are provided to illustrate certain embodiments of the invention, but are not intended to limit the invention. The examples and embodiments described herein are illustrative, but not limiting, of the probes, methods and kits of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in typical laboratory and which are obvious to those skilled in the art are within the spirit and scope of the invention described herein.
To prepare a density gradient, 60% iodixanol (1.32 g/ml) was diluted with Dulbecco's phosphate buffered saline to densities of 1.080, 1.101, 1.121 and 1.143 g/ml. A 2-ml volume of each of the density solutions was sequentially added to a conical centrifuge tube in order of density, starting with the highest.
A 1.25-ml volume of anticoagulated whole human blood was diluted 4-fold with Dulbecco's phosphate-buffered saline. The diluted blood was inoculated with E. coli, S. aureus or C. albicans at a final concentration of 2500 organisms/ml. The organism-spiked blood was overlayered on the density gradient. The gradient was centrifuged at 3000×g for 20 minutes at ambient temperature. Following centrifugation, 2 ml fractions were removed from the density gradient. A 0.1 -ml volume of each fraction was pipetted onto a blood agar plate and streaked for organism isolation. Each plate was incubated for approximately 18 hours at 35° C. under ambient air.
E. coli and S. aureus were isolated from the fractions with densities of between 1.101 and 1.121 and 1.121 and 1.143 g/m, respectively. C. albicans was isolated from the fraction with a density of 1.143 g/ml. Blood cells were observed in the fraction with a density between 1.080 and 1.101 g/ml. The latter data are consistent with reports in the literature, in which the density of blood is estimated to be 1.090-1.101 g/ml. These data demonstrate that organisms can be differentially separated from blood in a density solution provided that their specific gravity is higher than that of blood.
A 5-ml volume of anticoagulated whole human blood was inoculated with S. aureus or C. albicans at a final concentration of 2500 organisms/ml. After inoculation, a 2-ml volume was overlayered on a 3-ml volume of density solution at 1.090 g/ml, prepared by dilution of 60% (1.32 g/ml) iodixanol with Dulbecco's phosphate-buffered saline. The density solution was centrifuged at 5445×g for 40 minutes at ambient temperature. After centrifugation, 1-ml fractions were collected from the density solution. The organisms were isolated from the density fractions as described in Example 1. In contrast, the blood cells were observed to remain at the top of the density solution.
These data demonstrate that both S. aureus and C. albicans can be separated from whole blood by centrifugation through a density matrix. These organisms can be further processed to purify protein, DNA or RNA and be used in downstream molecular or microbiological applications.
A 5-ml volume of whole human blood was lysed with Triton X-100 at a final concentration of 1%. The detergent-treated blood was inoculated with E. coli, S. aureus or C. albicans at a final concentration 2500 organisms/ml. Sixty percent iodixanol (1.32 g/ml) was diluted to 1.090 g/ml with Dulbecco's phosphate-buffered saline. A 3-ml volume of density solution at 1.090 g/ml was overlayered with a 2-ml volume of lysed blood containing the three organisms. The density solution was centrifuged and fractions were collected as described in Examples 1 and 2. The organisms were isolated from the fractions as described in Example 1. All three organisms were recovered from the bottom of the density solution. Meanwhile, the debris from the lysed blood cells was observed at the top of the density solution.
These data show that E. coli, S. aureus and C. albicans can be separated efficiently from blood that has been lysed in Triton X-100. As described in Example 2, recovered organisms can be used in subsequent downstream molecular or microbiological applications.
The following example demonstrates the detection of E. coli-, S. aureus- and S. epidermidis-specific synthetic oligonucleotides using the MetriGenixo® 4D™ DNA chip.
MetriGenix® chips (MetriGenix, Inc., Maryland, USA) were spotted with immobilization probes of approximately 60 nucleotides in length comprising sequences from the human genes K-alpha (tubulin alpha-1) (SEQ ID NO:84), PPIA (peptidylprolyl isomerase A) (SEQ ID NO:85), and UBC (ubiquitin-conjugating enzyme E2A) (SEQ ID NO:86). The sequences of the immobilization probes (SEQ ID NOs:84-86) are set forth in Table 4b. The locations of the immobilization probes immobilized on the chips are shown in
Each of the immobilization probes was hybridized to an oligonucleotide capture probe comprising (1) an immobilization sequence of approximately 30 bases complementary to the immobilization probe and (2) and an organism-specific ssrA gene capture sequence of about 20-nucleotides. The sequences of the capture probes (SEQ ID NOs:55, 61, 67) are set forth in Table 2 and in
The capture sequences of the capture probes were designed to hybridize to sequences that are specific for E. coli, S. aureus, or S. epidermidis. The capture sequences (SEQ ID NOs:1, 6, 10) of the capture probes are underlined in
E. coli
S.
aureus
S.
epider-
midis
E. coli
S. aureus
S.
epider-
midis
E. coli ssrA
S. aureus ssrA
S. epidermidis ssrA
E. coli ssrA
S. aureus ssrA
S. epidermidis ssrA
To mimic the presence of microorganism-derived target oligonucleotides in a sample, synthetic target oligonucleotides comprising first target sequences complementary to the capture sequences of the capture probes specific for each organism were used to flood the MetriGenix® chip. The sequences of the synthetic target oligonucleotides (SEQ ID NOs:54, 60, 66) are set forth in Table 5 and in
E. coli
S. aureus
S.
epider-
midis
E. coli
S. aureus
S.
epider-
midis
The synthetic target oligonucleotides also comprised second target sequences (SEQ ID NOs:58, 64, 70; boxed in
E. coli
S. aureus
S.
epider-
midis
E. coli
S.
aureus
S.
epider-
midis
E. coli
S. aureus
S.
epider-
midis
The sample mix loaded onto to each of the chips used in this example (Chips A-E) contained 10 nM organism-specific capture probe, 100 nM biotinylated organism-specific detector probe, and 50 nM target oligonucleotide. The sample mix containing the target oligonucleotides and probes was heated on a 95° C. heat block for five minutes and immediately cooled on ice for two minutes. Reagents were flowed sequentially through MetriGenix® chips as follows using an MGX® 2000 hybridization station (MetriGenix, Inc., Maryland, USA):
All steps were performed at ambient temperature with the exception of the 2 hour hybridization incubation that was conducted at 37° C. Images of the arrays were captured on an MGX® 1200CL Detection Station using a CCD camera.
Results are depicted in
Sample processing: A 10-ml volume of anticoagulated whole human blood is seeded with 10,000 organisms each of S. aureus, S. epidermidis and E. coli and is treated with 1% (v/v) Triton X-100 for 10 minutes at ambient temperature. A density matrix is formed by diluting iodixanol (1.32 g/ml) to a density of 1.090 g/ml with Dulbecco's phosphate-buffered saline. The entire volume of lysed blood containing the aforementioned organisms is overlayered onto 15 ml of the density matrix. The organisms are separated from blood debris by centrifugation at 5440×g for 40 minutes at ambient temperature. At the end of the centrifugation step, the density matrix is decanted, and the resulting organism pellet is re-suspended in 100 μL of RNase-free water prior to the isolation of total RNA. The recovery of three organisms at this stage may be verified by growth on differential media, followed by biochemical identification.
Target Preparation: Total RNA from 100 μL of the bacterial suspension is prepared using a QIAGEN® RNeasy® kit (QIAGEN, GmbH). Total bacterial RNA is recovered and re-suspended in a final volume of 60 μL of RNase-free water. Optionally, the RNA is fragmented in a fragmentation buffer (40 mM Tris, 100 mM potassium acetate, 30 mM magnesium acetate, pH 8.0) at 95° C. for 30 minutes to reduce secondary structure of the RNA.
Analysis On MetriGenix® Chips: Custom DNA chips comprising specific DNA capture probes for ssrA or RNaseP RNAs from each of the target organisms, as well as other species of potential interest, are manufactured by MetriGenix (MGX®, MetriGenix, Inc., Maryland, USA). The hybridization and detection process may take place on an MGX® 2000 hybridization station. Each capture probe comprises a target-specific region (capture sequence) of about 20 bases in length, which is immobilized on the surface of the chip via a 5′ 9-base linker that has the sequence TTT TAA AAT (SEQ ID NO:87). Capture probes for each target organism are focused in discrete areas of the chip (“spots”) permitting specific detection and identification of each species present within a sample. Negative controls for non-specific hybridization are included in which only a phosphate-buffered saline solution is spotted on the chip. Biotin-labeled DNA oligonucleotides are also spotted directly on the surface to act as positive staining controls for the chemiluminescent detection reaction. As depicted in
Detection: Specific capture of the target RNA is visualized by chemiluminescence using an MGX® 1200CL Detection Station. In the presence of hydrogen peroxide, horseradish peroxidase catalyzes the oxidation of the substrate Luminol, resulting in the emission of light that is captured by a CCD camera. The signal is collected and analyzed by MetriSof™ software that corrects the signal intensity against the local background.
Predicted Results: No signals are detected in spots corresponding to negative controls, while staining controls yield strong positive signals. Positive signals are also detected at positions corresponding to the specific organism(s) present in the original sample. There is no signal above background from spots corresponding to organisms not seeded into the original blood sample, i.e., spots corresponding to organisms other than S. aureus, S. epidermidis or E. coli.
The following example demonstrates the specific detection of E. coli and S. aureus RNA using the MetriGenix MGX™ 4D™ DNA chip (MetriGenix, Inc., Maryland, USA).
Preparation Of Chips: MetriGenix® chips were spotted with capture probes of 27-33 nucleotides in length that were directed towards (1) transcript sequences of the E. coli, S. aureus, or S. epidermidis ssrA gene (which encodes transfer-messenger RNA (“tmRNA”)); (2) transcript sequences of the E. coli, S. aureus, or S. epidermidis Ribonuclease P (“RNase P” or “mp”) gene; (3) E. coli 16S rRNA, (4) Trichomonas vaginalis 18S rRNA, or (5) Candida albicans 18S rRNA. In addition to an organism-specific capture sequence, each capture probe comprised a 9-mer immobilization sequence of 5′-TTT TAA AAT (SEQ ID NO:87), through which the capture probe was attached to the chip surface. The sequences of the capture probes, including the 5′ immobilization sequences, are set forth in Table 11. The capture sequences of the capture probes are set forth in Table 12.
The locations of the capture probes immobilized on the chips are shown in
E. coli
S. aureus
S. epider-
midis
E. coli
S. aureus
S. epider-
midis
E. coli
Trichomonas
vaginalis
Candida
albicans
E. coli
S. aureus
S. epidermidis
E. coli
S. aureus
S. epidermidis
E. coli
Trichomonas
vaginalis
Candida
albicans
Application Of Target RNA: Total RNA was isolated from E. coli, S. aureus, and S. epidermidis using a QIAGEN®-based extraction protocol. RNA (1.5 μg) from each organism was added either alone or in combination to buffer containing 1×SSPE, 0.15M NaCl, 0.01M NaH2PO4, 0.001M EDTA), 2.5% Triton X-100 and used to flood replicate MetriGenix® chips essentially as described in Example 4. Hybridization took place over 10 hours at room temperature at a flow rate of 10 μL/min. The combinations in which the RNA was used are set forth below and in Table 13. Chips were washed twice with MES buffer containing 0.88M NaCl, 0.02M EDTA, 0.5% sarcosine, 33% formamide prior to staining.
Detection Of Captured RNA: Organism-specific 50-mer detector probes that were labeled at the 3′ end with BioTEG (Biotin with a 15 atom tetra-ethyleneglycol spacer). The detector probes were washed over the chips in combinations set forth below and in Table 13. The sequences of the detector probes are set forth in Table 14. The 3′ BioTEG labels attached to the detector probes are also shown in Table 14. Streptavidin-horseradish peroxidase solution (1.25 pg/μL) was then used to flood the chip. The streptavidin molecules bound to the biotin labels on the detection probes that were, in turn, bound to the captured RNA target sequences. Unbound materials were removed by washing with 1× MES. Bound detector probes were visualized by chemiluminescence. Specifically, a chemiluminescent substrate (Luminol) was used to flood the chips. In the presence of hydrogen peroxide, horseradish peroxidase catalyzed the oxidation of the substrate Luminol, resulting in the emission of light that was captured by a CCD camera. The assays were performed on an MGX® 2000 hybridization station (MetriGenix, Inc., Maryland, USA). Images of the array were captured over a 10 second period using an MGX® 1200CL Detection Station (MetriGenix, Inc., Maryland, USA) equipped with a CCD camera. Results are depicted in
E. coli
E. coli ssrA)
E. coli rnp)
E. coli 16S rRNA)
S. aureus
S. epidermidis
E. coli ssrA)
E. coli rnp)
E. coli 16S rRNA)
S. aureus
S. aureus ssrA)
S. aureus rnp)
E. coli
S. epidermidis
S. aureus ssrA)
S. aureus rnp)
E. coli
S. aureus
E. coli
S. aureus
E. coli
‡BioTEG = 3′ Biotin with a 15 atom tetra-ethyleneglycol spacer
For all combinations studied, no signals were detected in spots corresponding to negative controls, while staining controls yielded strong positive signals. Strong positive signals were also detected at spots bearing immobilized RNA derived from a given specific organism when detector probes specific for that organism were contacted to the immobilized RNA on the chip. No signals were detected at spots bearing RNA derived from a given specific organism when no detector probes specific for that organism were contacted to the immobilized RNA on the chip. Contacting RNA derived from a given specific organism with detector probe(s) specific for different organism(s) produced no signals. These data demonstrate that both the capture probes and the detector probes used were able to selectively bind to specific target RNA. There was no cross-reaction between capture and detection systems for any of the three organisms, thereby demonstrating the ability to discriminate these species using the probes and methods of the invention.
There is no signal above background from spots corresponding to organisms not seeded into the original sample, i.e., spots corresponding to organisms other than S. aureus, S. epidermidis or E. coli.
While this invention is satisfied by embodiments in many different forms, as described in detail in connection with preferred embodiments of the invention, it is understood that the present disclosure is to be considered as exemplary of the principles of the invention and is not intended to limit the invention to the specific embodiments illustrated and described herein. Numerous variations may be made by persons skilled in the art without departure from the spirit of the invention. The scope of the invention will be measured by the appended claims and their equivalents.
This application claims priority to U.S. Provisional Application Ser. No. 60/599,858, filed Aug. 10, 2004, which is incorporated herein by reference in its entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 4563419 | Ranki et al. | Jan 1986 | A |
| 20020081588 | De Lumley-Woodyear et al. | Jun 2002 | A1 |
| Number | Date | Country |
|---|---|---|
| WO 0070086 | Nov 2000 | WO |
| WO 0210444 | Feb 2002 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20060127924 A1 | Jun 2006 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60599858 | Aug 2004 | US |
