Determination of Intracellular Bacteria

专利摘要:
This application pertains to methods for the whole-cell analysis of intracellular bacteria. The methods are capable of making a determination of whether or not a sample (e.g. a clinical sample) comprises one or more select bacteria within host cells, for example, predatory host cells such as phagocytic cells. The method is performed on substantial intact bacteria and may be performed without the use of permeabilising or lysis reagents and using PNA probes. Furthermore, the application pertains to in situ hybridization methods for Gram-positive bacteria performed using buffered saline for hybridization.
公开号:DK201300361A1
申请号:DK201300361
申请日:2013-06-16
公开日:2015-01-12
发明作者:Henrik Stender
申请人:Stender Diagnostics V Henrik Stender；
IPC主号:

专利说明:

DESCRIPTION
Determination of Intracellular Bacteria 1. Field
This application relates to the field of determination of intact intracellular bacteria and, in some embodiments, pertains to the analysis of bacteria within phagocytic cells. 2. Introduction
Traditional methods for analysis of bacteria rely on extracellular analysis where intracellular bacteria are either released into the extracellular environment or where intracellular bacteria are separated or excluded.
The methods are further complicated when the host cells are predatory cells, such as phagocytic cells, degrading the intracellular bacteria.
Conditions where bacteria are predominantly found intracellular are thus inherently difficult to determine by traditional methods. Examples include diagnosis of sepsis or bloodstream infections extracellular bacteria are cleared by either phagocytosis or antibiotic therapy resulting in false negative results by traditional methods.
Methods for determination of intracellular bacteria include conventional staining methods, such as acridine orange, which can detect bacteria, but only provide presumptive information, such as morphology, about the bacteria and thus of limited clinical value (Int J Biol Med Res. 2: 360 -368; Pejouhesh 31: 155-158). Methods providing identification of intracellular bacteria have also been described, such as determination of intracellular bacteria in polymorphonuclear leukocytes (PMN) by in situ hybridization, which have been described by Matsuhisa et al. (Biotech Histochem. 69: 31-7; Microbiol.
Immunol. 38: 511-517; US 5,358,846; US 7,651,837). This method uses enzymes for permeabilization of host cells and lysis of bacteria, respectively, to make the bare bacterial target molecules assessable, biotinylated probes in hybridization buffer containing formamide (toxic) and required overnight incubation, and signal amplification using enzyme-labeled streptavidin.
While the literature does contain various reports related to determining intracellular bacteria, these assays are hampered by the use of complex and time-consuming permeabilization and / or lysis steps, signal amplification steps and prolonged hybridization steps using toxic chemicals and / or are unable to further characterize the bacteria beyond morphology.
Therefore, there is a need for faster, simpler and less toxic methods for determination of intracellular bacteria by in situ hybridization. Such a method would further benefit if the bacteria remained substantially intact. This way, the phagocytic cells would be used to separate and concentrate the bacteria from the extracellular environment in the form of packages of bacteria to increase sensitivity and substantial intact bacteria would provide packages of target molecule to increase specificity.
SUMMARY OF THE INVENTION
Applicants have surprisingly discovered that intracellular bacteria can be determined while keeping the intracellular bacteria substantially intact and that the various methods disclosed herein may thus be performed without performing cell permeabilization or lysis step (s).
Furthermore, keeping the bacteria substantially intact for whole cell analysis has the advantage of the target molecules being concentrated within the bacteria and thus allow for analysis without the use of signal or target amplification techniques.
Surprisingly, the applications have discovered that whole cell analysis using bacteria-directed probes can be performed where the bacteria-directed probes are penetrating first the cell containing intracellular bacteria and then the bacteria without the use of permeabilization or lysis reagents.
In addition, applications have shown that methods disclosed herein can be applied for determination of intracellular bacteria subject to phagocytosis by the host cells.
Furthermore, applications have discovered that whole cell analysis methods for bacteria, including Gram-positive bacteria, such as S. aureus, may be performed without the use of toxic denaturing chemicals, such as formamide, commonly used for hybridization buffer and may in fact be used. performed using saline buffers.
By combining whole cell analysis of bacteria with intracellular analysis the methods disclosed are using the bacteria to contain the target molecules and the cells containing the bacteria to contain the bacteria hereby providing several advantages over current methods. For example, this concept offers orders of magnitudes higher target concentration compared to the target concentration as if the bacteria were lysed and / or target released into the entire sample volume hereby providing basis for higher sensitivity. As another example, the analysis of substantially intact bacteria reduces the risk of false-positive results by not detecting bare target molecules and by not detecting degraded bacteria potentially reflecting past infection or contamination thereby providing basis for higher sensitivity.
In summary, the invention offers significant improvements over prior art in the form of being faster, simpler, safer, more sensitive and / or more specific.
This method may find great utility as a clinical assay in hospitals or other applications where intracellular bacteria are found.
LETTER DESCRIPTION OF THE FIGURES
FIG. 1. Left fluorescence microscope image (A) shows blue cocci within blue PMN. Right fluorescence microscope image (B) shows green cocci identified by PNA probes whereas PMN is non-fluorescent / weak reddish background. The blue cocci observed within the PMN have the same morphology and position as the green cocci.
FIG. 2. Left fluorescence microscope image (A) shows S. aureus as bright green cocci. Right fluorescence microscope image (B) shows S. epidermidis as none / weak-fluorescent cocci. DETAILED DESCRIPTION OF THE INVENTION Definitions
For the purposes of interpreting this specification and the appended claims, the following definitions shall apply and whenever appropriate, terms used in the singular shall also include the plural and vice versa. In the event that any definition set forth below conflicts with the use of that term in any other document, the definition set forth below shall always control for purposes of interpreting the scope and intent of this specification and its associated claims. Notwithstanding the foregoing, the scope and meaning of any document incorporated herein by reference should not be altered by the definition presented below. Rather, said incorporated document should be interpreted as it would be by the ordinary practitioner based on its content and disclosure with reference to the content of the description provided herein.
The use of "or" means "and / or" unless stated otherwise or where the use of "and / or" is clearly inappropriate. The use of "a" means "one or more" unless stated otherwise or where the use of "one or more" is clearly inappropriate.
The use of "comprise," "includes," "comprising," "include," "includes," and "including" is interchangeable and not intended to be limiting. Furthermore, where the description of one or more embodiments uses the term "comprising," those skilled in the art would understand that in some specific instances, the embodiment or embodiments may alternatively be described using language "consisting essentially of" and / or "consisting of. "
As used herein, "antibody" refers to an immunoglobulin protein or to a fragment or derivative thereof which is capable of participating in antibody / antigen binding interaction (s). A discussion of the technical features of antibodies, their fragments, methods for detection of antibodies / antibody fragments and related topics can be found in the Pierce Catalog and Handbook, 1994 (Section T). Antibodies include, for example, various classes and isotypes of immunoglobulins such as IgA, IgD, IgE, IgG1, IgG2a, IgG2b, IgG3, and IgM. Antibody fragments include molecules such as Fab, scFv, F (ab '), sub.2 and Fab' molecules. Antibody derivatives include antibodies or fragments thereof having additions or substitutions, such as chimeric antibodies. Antibodies can be derived from human or animal sources, from hybridomas, through recombinant methods, or in any other way known to the art.
As used herein, "cell permeabilizing or lysing reagents or reagents" refers to a reagent, two or more reagents, a mixture of reagents or a formulation used to treat cells to thereby modify or destroy the cell's wall / outer membrane so that other analysis reagents (eg probes, detector reagents, antibodies, etc.) can penetrate (and thereby enter) said cells. Some examples of enzymes that can be used as cell permeabilizing reagents include the enzymes: lysostaphin, lysozyme, and proteinases (e.g. proteinase-K and / or achromopeptidase). When more than one reagent is used to permeabilize cells, the permeabilizing reagents can be added sequentially, simultaneously, or a combination of some reagents being added sequentially and some being added simultaneously. In short, there is no limit to the extent to which the reagent or reagents are contacted with the bacteria as long as the process adequately permeabilizes the cells. In some embodiments, methods disclosed herein can be practiced by contacting the sample with a cell permeabilizing or lysing reagent or reagents.
As used herein, "chimera" refers to an oligomer comprising subunits of two or more different classes of subunits. For example, a chimera can comprise subunits of deoxyribonucleic acid (DNA) and locked nucleic acid (LNA), can comprise subunits of DNA and ribonucleic acid (RNA), can comprise subunits of DNA and peptide nucleic acid (PNA), can comprise subunits of DNA, LNA and PNA or can comprise subunits of RNA and LNA, etc. It is understood that what the literature refers to as LNA probes are typically chimeras (according to this definition), since said "LNA probes" usually incorporate only one or a few LNA nucleotides into an oligomer. The remaining nucleotides are typically standard DNA or RNA nucleotides.
As used herein, "bacteria-directed labeled probe or probes" refers to a probe or probes that are labeled with one or more labels (in some embodiments the probe or probes will comprise only a single label), where said probe or probes are selected to bind with a high degree of specificity to a target in the chromosomal DNA, the RNA and / or a plasmid of bacteria sought to be determined in the assay (eg the select bacteria). The chromosomal DNA, RNA and / or plasmid target is selected because it codes for (and / or is associated with) the select bacteria sought to be determined in the assay.
As used herein, "determining" refers to making a decision based on investigation, data, reasoning and / or calculation. Some examples of determining include detecting, identifying and / or locating bacteria as appropriate based on the context / usage of the term herein.
As used herein, "fixation" refers to specimen preservation and / or sterilization where cellular nucleic acid (DNA and RNA) integrity and cellular morphology are substantially maintained. Fixation can be performed either chemically using one or more solutions containing one or more fixing agent (s) and / or mechanically, such as for example by preparing a smear on a microscope slide and subsequently heating the smear either by passing the slide through a flame or placing the slide on a heat block.
As used herein, "fixative reagent or reagents" means a reagent, two or more reagents, a mixture of reagents, a formulation or even a process (with or without associated use of reagent (s) (including mixture (s) or formulation (s) s)) to treat bacterial cells to thereby preserve and / or prepare said bacterial cells for microscopic analysis. Some examples of fixative reagents include paraformaldehyde, gluteraldehyde, methanol and ethanol. When more than one reagent is used to fix, the reagents may be added. sequentially, simultaneously, or a combination of some reagents being added sequentially and some being added simultaneously.In some embodiments, methods disclosed herein can be practiced by contacting the sample with a fixative reagent or reagents.
As used herein, "in the aggregate" refers to considering relevant subject matter as a whole rather than piecemeal.
As used herein, "label" refers to a structural unit (or structural units as the case may be) of a composition (e.g., a hybridization probe) that renders the composition detectable by instrument and / or method. Non-limiting examples of labels include fluorophores, chromophores, haptens, radioisotopes and quantum dots. In some embodiments, two or more of the foregoing can be used in combination to render the composition detectable or independently (uniquely) detectable. Some words that are synonymous (i.e. interchangeable) with "label" are "detectable moiety", "tag" and "marker".
As used herein, "mixed population" refers to a mixture of two or more different strains of bacteria.
As used herein, "nucleic acid" refers to a nucleobase containing polymer formed from nucleotide subunits composed of a nucleobase, a ribose or 2'-deoxyribose sugar and a phosphate group. Some examples of nucleic acid are DNA and RNA.
As used herein, "nucleic acid analog" refers to a nucleobase containing polymer formed from subunits which comprise a nucleobase and a sugar moiety that is not ribose or 2'-deoxyribose and / or a linkage (between the sugar units) that is not a phosphate group. A non-limiting example of a nucleic acid analog is a locked nucleic acid (LNA: See, for example, U.S. Pat. Nos. 6,043,060,7,053,199,7,217,805 and 7,427,672).
See: Janson and During, "Peptide Nucleic Acids, Morpholinos and Related Antisense Biomolecules", Chapter 7, "Chemistry of Locked Nucleic Acids (LNA)", Springer Science & Business, 2006 for a summary of the chemistry of LNA.
As used herein, "nucleic acid mimic" refers to a nucleobase containing polymer formed from subunits that comprise a nucleobase and a backbone structure that is not a sugar moiety (or that comprises a sugar moiety) but that can never specifically bind to a nucleic acid. An example of a nucleic acid mimic is peptide nucleic acid (PNA: See, for example, 5,539,082, 5,527,675,5,623,049, 5,714,331,5,718,262,5,736,336, 5,773,571,5,766,855,5,786,461,5,837,459,5,891,625,5,972,610, 5,986,053, 5,986,053 W092 / 20703). Another example of a nucleic acid mimic is a morpholino oligomer. (See Janson and During, "Peptide Nucleic Acids, Morpholinos and Related Antisense Biomolecules", Chapter 6, "Morpholinos and PNAs Compared", Springer Science & Business, 2006 for a discussion of the differences between PNAs and morpholinos. A further example of a nucleic acid mimic is the pyrrolidinyl polyamide (PP). A PP is an oligomeric polymer comprising a nucleobase and polyamide backbone as described in U.S. Pat. Nos. 6,403,763,6,713,603, 6,716,961 and 7,098,321 as well as Vilaivan et al., "Hybridization of Pyrrolidinyl Peptide Nucleic Acids and DNA: Selectivity, Base-Pairing
Specificity and Direction of Binding ", Organic Letters, 8 (9): 1897-1900 (2006).
As used herein, "nucleobase" refers to those naturally occurring and those non-naturally occurring heterocyclic moieties commonly known to those that generate polymers that can sequence specifically bind to nucleic acids. Nonlimiting examples of suitable nucleobases include: adenine, cytosine, guanine, thymine, uracil, 5-propynyl-uracil, 2-thio-5-propynyl-uracil, 5-methylcytosine, pseudoisocytosine, 2-thiouracil and 2-thiothymine, 2-aminopurine , N9 - (2-amino-6-chloropurine), N9 - (2,6-diaminopurine), hypoxanthine, N9- (7-deaza-guanine), N9- (7-deaza-8-aza-guanine) and N8 - (7-deaza-8-aza-adenine). Other non-limiting examples of suitable nucleobase include those nucleobases illustrated in FIGS. 2 (A) and 2 (B) of Buchardt et al. (W092 / 20702 or W092 / 20703).
As used herein, "one or more probe / chromosomal DNA, probe / RNA and / or probe / plasmid complexes" refers to a complex or complexes formed by: 1) binding of a probe or probes to a target in a molecule or molecules of the chromosomal DNA of a bacterial cell or cells of a sample; 2) binding a probe or probes to a target in a molecule or molecules of the RNA of a bacterial cell or cells of a sample; and / or 3) binding of a probe or probes to a target in a molecule or molecules of the nucleic acid of a plasmid of a bacterial cell or cells of a sample. Typically, formation of the probe / chromosomal DNA, probe / mRNA and / or probe / plasmid complex or complexes is used herein to determine the select bacteria of a sample.
As used herein "one or more probe / chromosomal DNA, probe / RNA and / or probe / plasmid complexes" refers to a complex or complexes formed by binding of a second probe or second probes (ie a probe different from any previously mentioned probe or probes) to target within a molecule or molecules of a bacterial cell or cells of a sample. Typically, formation of the second probe / target complexes is used herein to determine another (second) select bacteria, subset of the select bacteria or the same select bacteria in the sample. It is understood that a third, fourth, fifth, sixth (etc.) probe directed to target (or bacteria of interest) could be used in any assay method described herein, any different probe directed to target can be selected to determine a different select bacteria that may be present in the sample. Often each of the second, third, fourth, fifth, sixth (etc.) probes is independently detectable from other probes used in practice of the method such that the method is practiced as a multiplex method. It is also understood that the third probe would form a third probe / target complex or complexes, the fourth probe would form a fourth probe / target complex or complexes, the fifth probe would form a fifth probe / target complex or complexes, the sixth probe would form a sixth probe / target complex or complexes, etc.
As used herein, "one or more washing reagents" refers to a reagent, two or more reagents, a mixture of reagents or a formulation which is used to remove various reagents and / or compositions from the sample and / or bacterial cells of the sample. In some embodiments, methods disclosed herein can be practiced including one or more steps pertaining to contacting the sample with one or more washing reagents.
As used herein, "pre-hybridization step" refers to the process of treating (eg contacting) a sample with a hybridization buffer that lacks a / the hybridization probe or probes for a period of time before treating (eg contacting) the sample with a hybridization buffer containing a / the hybridization probe or probes. In some embodiments, methods disclosed herein can be practiced with, or without, a pre-hybridization step.
As used herein "probe" or "hybridization probe" refers to a composition that binds to a select target. A "hybridization probe" is a probe that binds to its respective target by hybridization. Non-limiting examples of probes include nucleic acid oligomers (eg DNA, RNA, etc.) nucleic acid analog oligomers (eg locked nucleic acid (LNA)), nucleic acid mimic oligomers (eg peptide nucleic acid (PNA)), chimeras, antibodies and antibody fragments.
As used herein, "quantum dot" refers to an inorganic crystallite between about 1 nm and about 1000 nm in diameter or any integer or fraction of an integer therebetween, generally between about 2 nm and about 50 nm or any integer or fraction of an integers there between, more typically about 2 nm to about 20 nm (such as 2, 3,4,5,6,7, 8, 9,10,11,12,13,14,15,16, 17,18, 19, or 20 nm). A semiconductor nanocrystal is capable of emitting electromagnetic radiation upon excitation (i.e., the semiconductor nanocrystal is luminescent) and includes a "core" of one or more first semiconductor materials, and may be surrounded by a "shell" of a second semiconductor material. A semiconductor nanocrystals core surrounded by a semiconductor shell is referred to as a "core / shell" semiconductor nanocrystal. The surrounding "shell" material typically has a bandgap energy that is larger than the bandgap energy of the core material and may be chosen to have an atomic spacing close to that of the "core" substrate. The core and / or the shell may be a semiconductor material including, but not limited to, those of Group II-VI (ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe , CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, and the like) and III-V (GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, and the like) and IV (Ge, Si, and the like) materials, and an alloy or mixture thereof. In the scientific and patent literature the terms "semiconductor nanocrystal," "quantum dot", "Qdot.TM. nanocrystal" or simply "nanocrystal" are used interchangeably. For purposes of this specification, these terms are also equivalents of "quantum dot" as defined above.
As used herein, "rRNA-directed probe or probes" refers to a probe or probes that are selected to bind to a target or targets within a molecule or molecules of rRNA. The rRNA-directed probe or probes may be labeled with a detectable moiety or moieties or may be unlabeled. If unlabeled, complexes formed by binding of the rRNA probe to its target within a bacteria may, for example, be detected using a labeled antibody to the probe / rRNA complex or complexes (See, for example: US Pat. No. 5,612,458 to Nielsen).
Typically, the rRNA target is selected to differentiate between bacteria in the sample and thereby permit the determination of the select bacteria of the sample. Using probes directed to a target within the rRNA of a bacterium to differentiate between (and thereby determine) bacteria in a sample has long been used in ISH and FISH based assays (See for example: Amann, R., "Methodological Aspects of Fluorescence In Situ Hybridization ", Bioscience Microflora, 19 (2): 85-91 (2000) and Pemthaler et al.," Fluorescence in situ Hybridization (FISH) with rRNA-targeted Oligonucleotide Probes ", Methods in Microbiology, 30: 207-226 ( 2001)). With respect to the use of rRNA-directed PNA and LNA probes in FISH see: Cerqueira et al., "DNA Mimics for the Rapid Identification of Microorganisms by Fluorescence in Situ Hybridization (FISH)", Int. J. Mol. Sci., 9: 1944-1960 (2008). With respect to the use of rRNA-directed PNA probes for determination of staphylococci in blood samples, see: Forrest et al., "Impact of rapid in situ hybridization testing on coagulase-negative staphylococci positive blood cultures", Journal of Antimicrobial Chemotherapy, 58 : 154-158 (2006).
As used herein, "second rRNA-directed probe or probes" refers to a second probe or probes that selectively bind to a different (i.e., second) target or targets within a molecule or molecules of rRNA. The second rRNA target may exist in the same bacteria as did another (first) probe or probes used in the assay but more typically the second rRNA-directed probe or probes will be directed to a target in a different bacteria of interest such that formation and determination of a second probe / rRNA complex or complexes is used to determine a second (different) select bacteria in the sample.
As used herein, "select bacteria" refers to bacteria of interest that can be determined by practice of a method described herein. The select bacteria may be, for example, of a particular strain, species, subspecies, genus or kingdom. The select bacteria may, for example, also be a recognized group such as coagulase-negative staphylococci (CNS) or gram-positive cocci. In some embodiments, the select bacteria may be bacteria with certain virulence or antibiotic susceptibility characteristics, for example, methicillin resistance. In other embodiments, the select bacteria may be recognized by multiple characteristics, for example, being S. aureus and methicillin-resistant.
As used herein, "signal amplification" is discussed with respect to a label or labels associated (directly or indirectly) with a probe and refers to the use of specific detection methodologies to increase the signal by a factor of at least two for each label associated with the probe. probe. Signal amplification often (but not necessarily) involves the use of enzymes. Some non-limiting examples of signal amplification include tyramid signal amplification (TSA, also known as catalyzed reporter deposition (CARD)), Enzyme Labeled Fluorescence (ELF-97 product and information available from Invitrogen, Carlsbad, Calif.), Branched DNA ( bDNA) Signal Amplification (See: Collins et al., "A branched DNA signal amplification assay for quantification of nucleic acid targets below 100 molecules / ml", Nucl. Acids Res., 25 (15): 2979-2984 (1997) and Zheng et al., "Direct mecA Detection from Blood Culture Bottles by Branched-DNA Signal Amplification", J. Clin. Microbiol., 37 (12): 4192-4193 (1999), and rolling-circle amplification (RCA-See : Maruyama et al., "Visualization and Enumeration of Bacteria Carrying and Specific Gene Sequence by In Situ Rolling Circle Amplification", Applied and Environmental Microbiology, 71 (12): 7933-7940 (December 2005) and Smolin et al., "Detection of Low-Copy-Number Genomic DNA Sequences in Individual Bacterial Cells Using Peptide Nuc Leic Acid-Assisted Rolling-Circle Amplification and Fluorescence In Situ Hybridization ", Applied and Environmental Microbiology, 73 (7): 2324-2328 (2007)).
As used herein, "stained" means that a bacterial cell is directly or indirectly marked for detection with a label or labels. For example, the bacteria can be stained with one or more fluorescently labeled hybridization probes such that the bacterial cell or cells can, for example, be detected using a fluorescent microscope as described in U.S. Pat. Pat. No. 6,664,045 (See in particular FIGS. 3 (of U.S. Pat. No. 6,664,045) and the discussion associated therewith in Example 10 at col. 24-25). As is apparent in the various panels of FIG. 3 of U.S. Pat. No. 6,664,045, different bacteria of a sample can be stained with independently detectable labels (or combinations of independently labels) such that different types of bacteria in the sample appear, for example, as different colors (or otherwise possess different detectable properties). With specific reference to FIG. 3 of U.S. Pat. No. 6,664,045 for example, S. aureus bacteria are characterized as stained red (only), E. coli are characterized as stained green and red, P. aeruginosa are characterized as stained green (only) and S. typimurium are characterized as stained blue. Thus four different bacteria are determined in FIG. 3 of U.S. Pat. No. 6,664,045 using different rRNA directed labeled probes whereby the probe for each different bacteria is labeled with a unique label or combination of labels. FIGS. 2 and 3 of the present application also exhibit different types of bacteria which comprise unique independently detectable (fluorescent) stains.
As used herein, "target" or "select target" are interchangeable and refer to a molecule (or part of a molecule such as a select nucleic acid sequence) of a bacteria, such as an rRNA, mRNA, chromosomal DNA, plasmid or an antigen to which a probe is designed to specifically bind.
As used herein, "under conditions suitable for a [or" the "] probe to bind to a [or" the "] target" refers to conditions under which a probe binds to its respective target in a specific manner such as non-specific binding or probe to non-target moieties is minimized or eliminated. It is also understood that "the" may be replaced by "said" as appropriate (above and anywhere else in this specification) to indicate / acknowledge antecedent basis.
As used herein, the phrase "uniquely identifiable" is used with reference to a situation where two or more conditions of interest are distinguishable. For example, in a sample comprising at least two bacteria, one bacteria may comprise red fluorescent markers and another bacteria may comprise green fluorescent markers. Accordingly, two bacteria are "uniquely identifiable" (ie uniquely stained) using, for example, a properly equipped microscope (See for example: FIGS. 3 of U.S. Pat. No. 6,664,045 as discussed above) since the two bacteria can be distinguished using the microscope.
Bacteria may be uniquely identifiable for other reasons, such as morphology. For example, one type of bacteria may be rod-shaped and the other a cocci. In some embodiments, color and morphology can be used to distinguish / determine uniquely identifiable bacteria in a sample.
As used herein, "whole-cell" refers to cells (e.g., bacteria) in a morphologically recognizable form. "Whole-cell" is not intended to imply that the cell comprises all of its original components as it is well known that when cells may be permeabilized, they "leak" cellular constituents (See: Hoshino et al., Applied and Environmental Microbiology, 74 (16): 5068-5077 (2008) at page 5074, col. 1 and Maruyama et al., Applied and Environmental Microbiology, 71 (12): 7933-7940 (December 2005) at page 7937, col. 1). Such "leakage" is not intended to infer that an assay performed with cells that have leaked is not a whole-cell assay as discussed herein. Rather, "whole-cell" is intended to refer to substantially intact cells such that they retain their morphologically recognizable form. For example, cocci are spherical whereas other bacteria can be rod-like.
As used herein, "within bacteria" or "within the bacteria" refers to the inside of any structure (including multiple structures) of whole (intact) bacteria, such as the outer membrane, nuclear membrane, cell wall, cytoplasm and / or nucleus. For example, formation of one or more probe / rRNA complexes within bacteria of the sample may refer to formation of one or more probe / rRNA complexes within the outer membrane, nuclear membrane, cell wall and / or nucleus of said bacteria. Similarly, the probe / rRNA complex (es) can form in the cytoplasm and their presence can be used to determine the select bacteria (e.g., staphylococcus aureus) from other bacteria in a sample. However, as used herein, "within bacteria" is also, as appropriate, intended to encompass structures in contact with the outer surface of intact bacteria. For example, "within bacteria" is also intended to encompass, for example, antibody probes linked to the outer surface of a bacterium (for example as a consequence of binding to a surface protein), whereas antibody probes, for example, are used to determine select bacteria in a sample. "Within bacteria" is also, as appropriate, intended to encompass partially destroyed bacteria, i.e. due to phagocytosis, with sufficient remaining structure for determination of the select bacteria in a sample.
As used herein, "phagocyting cells" means macrophages, polymorphonuclear leukocytes (PMN) monocytes, neutrophils and eosinophils and other cells which can incorporate into themselves foreign objects, incl. phagocytes line like U937 Cell, HL60 Cell or the like.
As used herein, "intracellular bacteria" means bacteria inside other cells, most commonly, but not limited to, bacteria inside mammalian cells.
General
It is to be understood that the discussion set forth below in this "General" section may pertain to some, or all, of the various embodiments of the invention described herein.
Synthesis, Modification and Labeling of Nucleic Acids and Nucleic Acid Analogs
Nucleic acid oligomer (oligonucleotide and oligoribonucleotide) synthesis has become routine. For a detailed description of nucleic acid synthesis please see Gait, M. J., "Oligonucleotide Synthesis: a Practical Approach" IRL Press, Oxford England (1984). Persons of ordinary skill in the art will recognize that labeled and unlabeled oligonucleotides (DNA, RNA and synthetic analogues thereof) are readily available. They can be synthesized using commercially available instrumentation and reagents or they can be purchased from commercial vendors of custom manufactured oligonucleotides. PNA Synthesis and Labeling
Methods for the chemical assembly of PNAs are well known (See: U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049,5,714,331,5,718,262, 5,736,336, 5,773,571,5,766,855,5,786,461,5,837,459,5,891,625, 5,972,610,5,986,053 Some non-limiting methods for labeling PNAs are described in US Patent No. 6,110,676, WO99 / 22018, W099 / 21881, W099 / 49293 and WO99. / 37670 are otherwise well known in the art of PNA synthesis. Chemicals and instrumentation for the support bound automated chemical assembly of peptide nucleic acids are commercially available. Likewise, labeled and unlabeled PNA oligomers are available from commercial vendors of custom PNA oligomers (See: See the worldwide web at: panagene.com/pna-oligomers.php, See the worldwide web at: biosyn.com/pna_custom.aspx or See the worldwide web at: crbdiscovery.com/pna/). PNA synthesis and labeling can be found in Peter E. Nielsen, "Peptide Nucleic Acids", Taylor and Francis, (2004).
Because a PNA is a polyamide, it has a C-terminus (carboxyl terminus) and an N-terminus (amino terminus). For the purposes of designing a hybridization probe suitable for antiparallel binding to a target (the preferred orientation), the N-terminus of the PNA oligomer is the equivalent of the 5'-hydroxyl terminus of an equivalent DNA or RNA oligonucleotide.
Chimera Synthesis and Labeling / Modification
Chimeras are oligomers comprising subunits of different monomer types. In general, it is possible to use labeling techniques (with or without adaptation) applicable to the monomer types used to construct the chimera. Various labeled and unlabeled chimeric molecules are reported in the scientific literature or available from commercial sources (See: US Pat. No. 6,316,230, See the worldwide web at: biosyn.com/PNA_Synthesis.aspx, W02001 / 027326 and See the worldwide web at : sigmaaldrich.com/life-science/custom-oligos/dna-probes/product-lines/- Ina-probes .html). Therefore, persons of skill in the art can either prepare labeled chimeric molecules or purchase them from readily available sources.
labels
Non-limiting examples of labels (i.e. detectable moieties or markers) suitable for labeling probes used in the practice of this invention include a chromophore, a fluorophore, a spin label, a radioisotope, an enzyme, a hapten, a chemiluminescent compound, a quantum dot or combinations of two or more of the foregoing.
Some examples of haptens include 5 (6) -carboxyfluorescein, 2,4-dinitrophenyl, digoxigenin, and biotin.
Some examples of fluorochromes (fluorophores) include 5 (6) -carboxyfluorescein (Flu), 6 - ((7-amino-4-methylcoumarin-3-acetyl) amino) hexanoic acid (Cou), 5 (and 6) -carboxylic acid X-rhodamine (Rox), Cyanine 2 (Cy2) Dye, Cyanine 3 (Cy3) Dye, Cyanine 3.5 (Cy3.5) Dye, Cyanine 5 (Cy5) Dye, Cyanine 5.5 (Cy5.5) Dye Cyanine 7 (Cy7) Dye, Cyanine 9 (Cy9) Dye (Cyanine dyes 2, 3, 3.5,5 and 5.5 are available as NHS esters from GE Healthcare, Life Sciences, Piscataway, NJ), JOE, Tamara or the Alexa dye series (Life Technologies, Carlsbad , Calif.).
Some examples of enzymes include polymerases (e.g. Taq polymerase, Klenow PNA polymerase, T7 DNA polymerase, Sequenase, DNA polymerase 1 and phi29 polymerase), alkaline phosphatase (AP), horseradish peroxidase (HRP) and soy bean peroxidase (SBP).
Some examples of radioisotopes include .sup.l4C, .sup.32P, .sup.1291 and .sup.99Tc.
In some embodiments, spin labels can be used as labels. Spin labels are organic molecules that possess an unpaired electron spin, usually on a nitrogen atom. For example, probes can be labeled with a spin label as described in U.S. Pat. No. 7494776. Said labeled probe can then, for example, be used to stain bacteria for determination.
Independently Detectable Labels / Multiplex Analysis
In some embodiments, a multiplex method (assay) is performed. In a multiplex assay, numerous conditions of interest are examined simultaneously or sequentially. Multiplex analysis relies on the ability to sort sample components or the data associated therewith, during or after the assay is completed. A multiplex assay (as used herein), commonly relies on the use of two or more uniquely identifiable probes.
In a multiplex assay, one or more distinct independently detectable labels (typically each distinct label (or a distinct combination of labels) linked to a different probe) are used to uniquely mark (i.e. stain) two or more different bacteria of interest. In some cases, two (or more) unique labels may be directed to the same bacteria thereby generating a unique stain that results from the presence of the two (or more) unique labels in the bacteria. The ability to differentiate between and / or quantify each of the uniquely stained bacteria provides the means to multiplex the assay because the data that correlates with each uniquely marked (ie stained) bacteria can be correlated with a condition or conditions sought to be determined (e.g. select bacteria).
In practicing methods described herein, it is possible to uniquely mark bacteria so that two (or more) conditions of interest can be determined for the bacteria of the sample. For example, in the practice of some embodiments, it is possible to use a unique label to mark S. aureus bacteria in a sample as well as to use a unique label to mark bacteria in the sample that are methicillin-resistant. Thus, by analysis of the sample it is possible to determine whether the sample contains: 1) S. aureus bacteria (which are not methicillin-resistant); 2) non-S. aureus methicillin-resistant bacteria (e.g. MR-CNS); and / or 3) methicillin-resistant S. aureus bacteria.
Methods can be multiplexed in many ways and multiplexing is limited only by the number of independently detectable labels (or independently detectable probes) that can be used or detected in an assay. For example, some assays may be designed to detect and identify the presence of several (e.g., two, three, four, five, six or more) different bacteria in a sample.
Whole-Cell Assays:
Methods disclosed herein involve whole-cell assays. Whole-cell assays are performed on intact or substantially intact cells. Some examples of wholecell assays are in-situ hybridization (ISH), fluorescence in-situ hybridization (FISH) and immunocytochemistry (ICC) assays. In some embodiments, a whole-cell assay is not strictly an ISH, FISH or ICC assay. For example, whole-cell assays may involve a combination of two or more of these different assay formats (See: Goldbard et al., U.S. Pat. No. 6,524,798 entitled: "High Efficiency Methods for Combined Immunocytochemistry And In-Situ Hybridization") . More specifically, some embodiments of this invention contemplate use of oligomer (hybridization) probes used in combination with, for example, antibody probes. To the extent that the assay formats and / or components used in said assays are not mutually incompatible, this invention contemplates any combination of combined whole-cell assay formats. As discussed in more detail below, combining the assays may involve some degree of harmonization of the bonding conditions where different probe types are used in the practice of a method step. Alternatively, reprobe cycling of the sample may also be used where conditions are fixed for one probe type such that the reprobing cycle (the first cycle would actually be a probing cycle) is completed with said probe type and a new reprobing cycle is performed with the second (different) probe type (See Williams et al., U.S. Pat. No. 2005/0123959 for a discussion of whole-cell analysis using sequential steps of analysis-as used herein "reprobing cycle or reprobing cycles"). Depending on the method, probe / target complexes can be determined after each reprobing cycle, after some of the reprobing cycles or after all of the reprobing cycles. ISH:
As used herein "in situ hybridization (ISH)" refers to methods practiced using a hybridization probe directed to a nucleic acid target. The probe may be a nucleic acid (eg RNA, DNA), a nucleic acid analog (eg LNA), a nucleic acid mimic such as PNA, morpholino or PP or a chimera (eg, a DNA-RNA chimera, PNA-DNA chimera , and PNA-RNA chimera, and LNA-DNA chimera, etc.). The most widely used ISH method is "fluorescence in situ hybridization" or "FISH", in which the probe comprises one or more fluorescent labels.
Briefly, conventional in situ hybridization assays generally comprise one or more of the following steps: (1) prehybridization of the cell to increase accessibility of target DNA or RNA (e.g., denaturation with heat or alkali and / or treatment with a cell permeabilization or lysis reagent or reagents); (2) steps to reduce nonspecific binding (e.g., by blocking the hybridization capacity of repetitive sequences, e.g., using human genomic DNA); (3) pre-hybridization involving contacting the sample with hybridization solution not containing the hybridization probe; (4) hybridization of one or more hybridization probes to the nucleic acid within the bacteria; (5) washes to remove probes not bound to their respective targets; and (6) detection / determination of the probe / target complexes (e.g. by determining the stained bacteria). The reagents used in each of these steps and conditions for their use vary depending on the particular application. ISH may be carried out using a variety of detectable or detectably labeled probes (e.g., .sup.35S-labeled probes, fluorescently labeled probes, enzyme labeled probes) capable of hybridizing to a cellular nucleic acid sequence. When fluorescently labeled probes are used, the technique is called FISH.
The ISH probes may be labeled directly (e.g., using a covalently linked fluorescent label) or indirectly (e.g., through a ligand-labeled antiligand system).
Immunocytochemistry (ICC):
As used herein, immunocytochemistry refers to the use of antibody or antibody fragments to stain bacteria from a sample through the interaction of an antibody probe (or antibody fragment probe) and an antigen within bacteria. The staining may occur by using only primary antibodies or it may involve the use of (labeled) secondary antibodies. Hence, the antibody (or antibody fragment) probe can be directed to an antigen target that is specific for the select bacteria. The antibody probe can be labeled (ie direct detection) or the antibody probe / antigen target complex formed by the binding of the antibody probe to its respective antigen target within the bacteria can be determined by using labeled secondary antibody that binds to said antibody probe / antigen target complex (ie indirect detection).
No matter what is being targeted, at least one antibody is labeled with at least one detectable moiety such that when said labeled antibody binds, the bacteria is stained. Furthermore, ICC can be combined with ISH or FISH procedures to determine select bacteria according to the methods disclosed herein.
samples:
Bacteria are everywhere. A sample containing intracellular bacteria can come from any source. The source of a sample is not intended to be a limitation associated with the practice of any method disclosed herein.
Samples can be environmental samples such as samples from soil or water. Samples can come from consumer staples such as food, beverages or cosmetics. Samples can come from crime scenes (e.g. forensic analysis). Samples can come from war zones or from sites of a suspected terrorist attack (For example, for testing pathogenic bacteria, including weaponized bacteria (eg B. anthracis). Samples can come from clinical sources. Samples from clinical sources can come from any source Some non-limiting examples of clinical samples (from clinical sources) include blood, pus, sputum, spinal fluid, amniotic fluid, stool, urine, nasal swabs, throat swabs and Samples (including clinical samples) may include bacterial cultures and subcultures, or portions thereof. Samples may include samples prepared, or partially prepared, and / or fractionated for a particular analysis. For example, the sample may be a specimen that has been fixed and / or stored for a period of time.
Sample Fractionation:
Samples may be fractionated to separate or concentrate the cells optionally containing intracellular bacteria. For example, white blood cells may be separated from the blood for example by collecting the 'buffy coat' or other methods to purify, separate or concentrate the host cells.
Sample fractionation may also be virtual using, for example, antibodies to selectively stained host cells optionally containing intracellular cells, such that subsequent determination is performed on selectively stained host cells.
Intracellular Bacteria:
Bacteria may be found intracellular as part of their normal life cycle and / or bacteria may be found intracellular due to, for example, host deference mechanisms, i.e. in phagocytosis. Bacteria may also be other cellular microorganisms, such as spores or elemental bodies as long as they are cells within an host cell.
Cellular microorganisms which may cause infectious diseases and be digested with phagocytes and may include, for example, bacteria, mycete, protozoon, parasite or the like. Bacteria may include, for example, Staphylococcus, Pseudomonas, Enterococcus, Colibacillus, Streptococcus, Pneumococcus, Tubercle bacillus, Helicobacter pylori, Listeria, Yersinia, Brucellar or the like. Mycete may include, for example, Candida,
Aspergillus, Actinomyces, Coccidioides, Blastomyces or the like. Protozoon may include, for example, Karyamoebina falcata, Trichomonas vaginalis, Malaria, Toxoplasma or the like. Parasite may include, for example, Trypanosoma or the like. In particular, the causative microorganisms of sepsis or bacteremia may include, for example, Gram-positive bacteria of Staphylococcus genus (Staphylococcus aureus, Staphylococcus epidermidis) and Enterococcus genus (Enterococcus faecalis, Enterococcus faecium, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pneumoniae, Gram-negative bacteria like Enterobacteriaceae, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae (Klebsiella oxytoca, Serratia marcesens, Proteus vulgaris, Citrobacter freundii), aerophilic root of Pseudomonas genus (Pseudomonas aeruginosa), anaerobic of Clostridium (Clostridium (Bacteroides fragilis) or the like. Thus, Acinetobacter calcoaceticus, Aeromonas hydrophilia, Flavobacterium meningosepticum, Bacillus cereus can rarely fall within the causative microorganisms.
Host Cell:
Host cells may be both cells containing bacteria as part of their normal life cycle, i.e. symbiosis and / or predatory host cells digesting or degrading bacteria due to, for example, host defense mechanisms, i.e. phagocytic cells, such as white blood cells, PMN or macrophages. The host cells are typically mammalian, human, or eukaryotic cells, but any cell containing another cell for any reason is a host cell.
Probes:
Unless expressly limited by specific language or discussion herein, any probe that can be used to select a desired condition of interest (eg select bacteria) based on selective binding of said probe to its respective target can be used in the practice of embodiments of this investor tion. In some embodiments, a probe can be an antibody or antibody fragment. In some embodiments, a probe can be a peptide or protein. A probe used in the practice of embodiments of this invention may be a nucleic acid (e.g. DNA or RNA), a nucleic acid analog (e.g. LNA), a nucleic acid mimic (e.g. PNA, PP or morpholino) or a chimera. In some embodiments, the probe or probes is / are 10 to 20 nucleobase subunits in length. Probes are described herein in terms of "nucleobase subunits in length" since only nucleic acids comprise nucleotides whereas all of these different oligomer types comprise one nucleobase per subunit. Probes used in embodiments of this invention can be prepared by denovo synthesis or by other methods.
It is understood that numerous probes exist in the biological arts for detecting specific bacteria. Accordingly, the nature of the probe (for purposes of this invention) is not intended to be limited except as expressly disclosed herein.
In some embodiments, probes used in the practice of this invention may be unlabeled provided that there is an available mechanism for determining the probe / target complex formed by binding the probe to its respective target. For example, an unlabeled (primary) antibody-based probe can be determined using a secondary detectably labeled antibody that binds to said unlabeled (primary) antibody-based probe (See, for example: US Pat. No. 6,524,798 at col. 3 , lines 28-40 and U.S. Pat. No. 7,455,985 at col. 12, lines 12-63). For example, said unlabeled (primary) antibody-based probe may be used to determine the select bacteria. Thus, the complex (i.e. labeled secondary antibody / primary antibody / target complex) formed upon binding of all molecules can be determined (and hence the select bacteria) by determining said label of said secondary antibody. Other types of unlabeled probes can similarly be determined by using a labeled molecule that selectively binds to said unlabeled probe or the complex formed by binding of said unlabeled probe to its respective target (See, for example: U.S. Pat. No. 5,612,458 to Nielsen which discusses the use of antibodies to PNA-DNA complexes, etc).
In some embodiments, probes can be labeled with at least one detectable moiety (i.e., at least one label). In some embodiments, each probe will comprise only one label. In some embodiments, the probe or probes used to determine the select trait (e.g., methicillin resistance) will comprise only one label. In some embodiments, mixtures of probes (e.g. mixtures of mRNA-directed probes) are used, each probe comprising one label or two labels (i.e., a mixture of single labeled and / or dual labeled probes). In some embodiments, each probe can comprise multiple labels (e.g., two labels, three labels, four labels, five labels, six labels, etc). In some embodiments, one or more probes may comprise a single label and one or more probes may comprise multiple labels. In some embodiments, one or more of the probes may be unlabeled and one or more probes may comprise one or more labels.
In some embodiments, the label or labels can be determined directly. In some embodiments, the label or labels can be determined indirectly. In some embodiments, some of the labels can be determined directly and some determined indirectly.
Determining a label directly involves determining a label's property without the use of another molecule / compound. For example, determining a fluorescent label may involve viewing a treated sample using a fluorescent microscope, using a slide scanner or using a flow cytometer. Because it is the fluorescence of the label itself that is being observed / measured in the microscope, scanner or cytometer, the determination is said to be direct.
By comparison, indirect determination involves the use of an ancillary molecule / compound that recognizes the label of the labeled probes whereby the ancillary molecule / compound (or a label thereon) is determined as a surrogate for determining the label of the labeled probe. For example, the label can be a hapten like digoxigenin. Several of the references listed in Section 8 below describe indirect methods for determining digoxigenin. In general, these methods involve the use of an anti-digoxigenin molecule (antibody) conjugated to a secondary label (e.g., enzymes like horseradish peroxidase, alkaline phosphatase or a fluorophore like fluorescein). Because it is the properties of the secondary label of the ancillary molecule (i.e. the anti-digoxigenin molecule) that is determined, this is characterized as an indirect detection method.
In practice, some probes used in embodiments of the present invention are chosen to determine a select bacterium in a sample. We refer to these as a [or "the"] "bacteria-directed" probe or probes. By "bacteria-directed" we refer to a probe or probes that find with specificity to a target within a bacteria, select bacteria. Furthermore, said bacteria-directed probe or probes are said to be "capable of determining a [or" the "] select bacteria in a [or" the "] sample" because said bacteria-directed probe or probes selectively bind to a target within the bacteria so that said select bacteria can be determined (for example by fluorescence microscopy or flow cytometry) based on formation of the probe / target complex. Thus, said bacteria-directed probe or probes are used to determine said select bacteria in said sample.
In some embodiments, the select bacteria is a gram-positive bacteria (e.g., S. aureus) and said bacteria-directed probe or probes are said to be "capable of determining a [or" the "select bacteria in a [or" the "] sample" or more specifically for staphylococcus aureus; "capable of determining Staphylococcus aureus bacteria in a [or" the "] sample". In some embodiments, other select bacteria (including as appropriate one or more gram-negative bacteria) may be selected for determination. In some cases, the sample is also contacted with one or more additional bacteria-directed probes for each select bacteria sought to be determined by practice of the method.
Often, the determination of multiple select bacteria in a sample is accomplished by using a multiplex assay. Each different type of bacteria is stained with a unique stain, combination of stains and / or unique combination of stain and cell morphology.
The probe or probes chosen to determine a select bacteria (i.e., the bacteria-directed probe or probes) can be an rRNA-directed probe or probes. Said rRNA-directed probe or probes bind with specificity to a target in the rRNA of the select bacteria. However, the bacteria-directed probe or probes need not be rRNA-directed. Rather, they may, for example, be mRNA-directed. By "mRNA-directed" we refer to a probe or probes that bind with specificity to a target in mRNA. The bacteria-directed probe or probes may also be directed to other regulatory RNAs (e.g., small RNA (sRNA) or antisense RNA (aRNA)) or chromosomal DNA or plasmid of a bacteria specific to said bacteria.
Moreover, the bacteria-directed probe or probes need not be hybridization probes. For example, the bacteria-directed probe or probes can be, for example, antibody-based (See, for example: US Pat. No. 6,231,857 and U.S. Pat. No. 7,455,985) since it is known that antibodies can be used to distinguish one. type of bacteria from another or others.
As noted several times before, the methods described herein can be practiced in multiplex mode whereby, for example; 1) two or more select bacteria are determined in a single sample; 2) two or more subsets of select bacteria are determined in a single sample; or 3) two or more select bacteria and two or more subsets of select bacteria are determined in a single sample. In general, such multiplex assays are performed by contacting the sample with additional probes as needed to determine the additional select bacteria and / or subsets of select bacteria. In some embodiments, said contacting can be done simultaneously so that all the different bacteria can be determined at the end of a single procedure. For this embodiment, the probe or probes directed to each different select bacteria can be independently detectable. In general, the labels of the various probes used in practice of the method are selected to produce different stained bacteria based on the type of bacteria. In some cases, however, it will be possible to have some identically stained bacteria, where one or more of the select bacteria is determined based on morphology of the bacteria (possibly in combination with a determination of the stain).
Rather than multiplex with different (independently detectable) labels (or uniquely stained bacteria), it is also possible to get multiple results using a reprobe cycling method (See: Published US Pat. Application No. 2005/0123959 to Williams et al. ). In a replicate cycling method, a result is obtained and then the sample is reanalyzed to determine a second, third, fourth, fifth, etc. result. Typically, in a replicate cycling method, the prior result is removed (erased) before the sample is treated to obtain the next result.
With respect to the methods disclosed herein, it is possible to use the same label type (e.g. fluorescein) to determine two or more select bacteria or subsets of select bacteria using a reprobe cycling method. In some embodiments, it is possible to determine a select bacterium and a subset in the same reprobing cycle. In some embodiments it is possible to determine a select bacteria and a select subset or another select bacteria in a different reprobing cycle. In general, a person of skill in the art can select which select bacteria are to be determined in a particular reprobing cycle by selecting the probe or probes applied to the sample during said reprobing cycle.
targets:
In general, a target can be any target molecule (or a portion thereof) that is present within the bacteria during the whole-cell assay that can be determined using a respective probe. Some non-limiting examples of targets include nucleic acid sequences present (eg select sequences within rRNA, mRNA, chromosomal DNA or plasmid DNA) within any nucleic acid of the bacteria, an antigen, an antibody, a protein, a peptide and / or a hormone.
In some embodiments, the methods disclosed herein are practiced with one bacteria-directed probe or probes capable of determining a select bacterium that may be present in a sample.
It is understood that the methods disclosed herein may be used to determine additional target (s) (for example, by multiplexing or reprobe cycling) that may be of interest in a sample and determined during practice of the methods disclosed herein. For example, it is possible to obtain additional information from the sample by contacting said sample with one or more additional probes directed to said additional target (s) whose presence within bacteria of the sample is indicative of another condition of interest (for example, another condition of clinical interest for proper diagnosis of a patient). Said additional condition of interest may be the presence of another bacteria in the sample. Said additional condition of interest may be the presence of yeast in the sample. Said additional condition of interest may be the presence of a plasmid in the select bacteria and / or in other bacteria of the sample. Said additional conditions may be virulence or antibiotic resistance. The method disclosed herein can be used in combination with numerous probes for numerous targets. Accordingly, it is possible by practice of methods disclosed herein to determine one or more additional conditions of interest based on a proper selection of targets (and the respective probe or probes for each target).
Persons of skill in the art will be able to design select suitable targets (and design appropriate probes to said suitable targets) using routine experimentation and commercially available materials and / or information.
For example, ISH is commonly used to determine select bacteria (See: Amann, R., "Methodological Aspects of Fluorescence In Situ Hybridization", Bioscience Microflora, 19 (2): 85-91 (2000) and Pernthaler et al., " Fluorescence in situ Hybridization (FISH) with rRNA-targeted Oligonucleotide Probes ", Methods in Microbiology, 30: 207-226 (2001)) including staphylococcus aureus bacteria (See: U.S. Pat. No. 6,664,045 at FIG. 3 and U.S. Pat. Application No. 2008/0008994; Cerqueira et al., "DNA Mimics for the Rapid Identification of Microorganisms by Fluorescence in Situ Hybridization (FISH)", Int. J. Mol. Sci., 9: 1944-1960 (2008); and Forrest et al., "Impact of rapid in situ hybridization testing on coagulase-negative staphylococci positive blood cultures", Journal of Antimicrobial Chemotherapy, 58: 154-158 (2006)). As previously noted (See: the section above entitled "Probes"), targets for such determinations can, for example, be rRNA. This is not intended to be a limitation, however, as the target for selecting a bacterium can, for example, be a surface antigen (See: U.S. Pat. No. 7,455,985).
Forming Probe / Target Complexes:
The select bacteria are determined by determining formation of the appropriate probe / target complexes within the bacteria of the sample. In brief, by contacting the sample with probes chosen for their affinity for their respective targets known to be associated with (and specific for) the select bacteria, the appropriate probe / target complexes will form within the bacteria of the sample.
The nature of the probe / target complex is determined by the nature of the probe and its respective target. Various types of probe / target complexes are contemplated. For example, hybridization probes for bacteria determination can be rRNA-directed or mRNA-directed. Thus, each complex formed upon binding of the probe to its target is a probe / rRNA complex or probe / mRNA complex, respectively.
Similarly, hybridization probes can be chromosome DNA-directed, or plasmid-directed. Hence, each complex formed upon binding of the probe to its respective target is a probe / chromosome DNA complex or probe / plasmid complex, respectively.
With respect to antibody probes, binding of the antibody to its antigen target produces an antibody / antigen complex.
Those of skill in the art will recognize that the probe / target complexes in the bacteria are formed under suitable binding conditions (or more correctly termed "suitable hybridization conditions" for hybridization probes). Suitable binding conditions for each probe / target complex will be determined based on the nature of the probe and target. Suffice it to say that suitable binding conditions are reflected in conditions where the interactions of the probe and its respective target are specific. Moreover, persons of ordinary skill in the art can determine suitable binding conditions for forming many types of probe / target complexes. Indeed, numerous hybridization buffers (See for example: a ready-to-use hybridization solution optimized for in situ hybridization procedures such as: See the worldwide web at: sigmaaldrich .com / catalog / ProductDetail .do N4 = H77821SIGM A & N5 = S EAR CH_CONCAT- _PNOIBRAND_KEY & F = SPEC) and / or binding buffers (See for Example; commercially available ready-to-use antibody binding buffers from ThermoScientific as described at: See the worldwide web at: piercenet.com/Products/Browse.cfm FlDI= 01010401 & WT .mc_id = go_AbPu r_Bind_pf- & gclid = CNyJ-4-uxgJoCFdxM5QodlVt5Fw) are commercially available for use in various assay formats. It is to be understood that binding conditions need not be fully optimized but rather that the conditions are merely suitable for specific binding of the probe to its respective target such that the assay produces accurate and reproducible results. Furthermore, where different types of probes (e.g., hybridization probes and antibody-based probes) are used in the same contacting step, binding conditions should be suitable for the binding of each type of probe to its respective target. For a more detailed discussion of this issue, see the section below entitled "Harmonizing Binding Conditions in Whole-Cell Assays".
Determining Probe / Target Complexes:
Once formed, the probe / target complexes can be determined. The probe / target complexes can be determined using a label associated with each different (or different type of) probe / target complex. In some embodiments, all labels associated with different (or different types of) probe / target complexes are the same. In some embodiments, different labels (or combinations of labels) are associated with each different (or different type of) probe / target complex. In some embodiments, there is a mixture of the same label associated with some of the different (or different types of) probe / target complexes and different labels associated with others of the different (or different types of) of probe / target complexes. A probe / target complex can be determined directly or indirectly. By "directly", we mean that the probe of the probe / target complex comprises a linked label which label is determined based on its own properties (See the discussion pertaining to determining direct and indirect determination of labels above in the section entitled: "Probes "). By "indirectly", we mean that the probe / target complex is determined using a secondary composition (eg a labeled antibody) that comprises a label and that binds to (or interacts with) the probe / target complex (or a label linked to the probe / target complexes), where said label is determined (Id.) as indicia of the probe / target complex. Regardless, determining the label correlates with determining the probe / target complex.
In whole-cell assays, determining the probe / target complexes can, in some embodiments, be performed by examining how the cells (i.e., the bacteria) are stained. In a letter, regardless of whether the labeling is direct or indirect, the cells become stained because the label (s) associated (directly or indirectly) with the probe / target complex or complexes is / are assimilated within (or at least on the surface of) ) the intact cells (ie bacteria). As noted previously, it is possible to use unique labels and / or unique combinations of labels for different bacteria and / or traits. Thus, any method capable of determining the stained bacteria in the sample can be used to determine the select bacteria.
For example, the select bacteria can be determined based on their visual appearance under a microscope. In some embodiments, the process can be automated so that the result can be determined using a computer and algorithm.
In some embodiments, the select bacteria can be determined using a slide scanner. Similarly, a slide scanner can be automated so that the result can be determined using a computer and algorithm.
In some embodiments, the select bacteria can be determined using a flow cytometer. Likewise, a flow cytometer can be automated so that the result can be determined using a computer and algorithm.
Furthermore, any other instrument or method suitable for determining stained cells can be used to determine the probe / target complexes formed using the inventive methods disclosed herein.
Cell Morphology:
It is an advantage of the present invention that various types of bacteria possess a unique morphology. In addition to the labels (e.g., stains) used to mark the bacteria, morphology of the cells can be used to either confirm the identity of bacteria or possibly introduce a second level of differentiation, for example, in a multiplex assay.
For example, bacilli tend to be rod-like whereas streptococci tend to be spherical (See the worldwide web at: en .wikipedia .org / wiki / B acterial_cell_structure # Cell_morphology and en.wikipedia.org/wiki/File:Bacterial_morphology_diagram.svg) . In some assays, for example, it may be that determining a yellow stained rod-like cell will confirm the presence, location and / or quantity of a select bacterium in the sample. In this case, the shape of the bacteria is used to distinguish signal (the select bacteria) from noise (other bacteria) in the assay.
In some (e.g., multiplex) assays for example, multiple cell types may be used with at least two bacteria of different morphology being stained with, for example, a yellow marker. In this case, the presence, location and / or quantity of the two select bacteria can be determined based, for example, on whether or not they are stained yellow and are rod-like or spherical in shape. Of course, an assay using this methodology can be further developed (further multiplexed) using bacteria of other known and distinguishable morphologies.
Associated with morphology (although not necessarily a strict example of cell morphology), in some embodiments characteristics of the staining process can also be used to confirm or determine a result. For example, where an antibody probe interacts with a surface antigen to stain the surface of the bacteria (e.g., using an antibody based bacteria-directed probe) and a second, uniquely labeled target-directed (e.g., an mRNA-directed probe) interacts with a target inside the bacteria (eg in the cytoplasm) to thereby stain the inside of the bacteria, a unique staining pattern can result. For example, if the antibody probe is red and the target probe is green, when observed using microscope, the bacteria will appear as a red cover (or halo) surrounding a green body. Thus, bacteria of the sample are confirmed or determined based on whether or not they exhibited this particular staining pattern.
From the foregoing, it is clear that cell morphology (and staining patterns) is a feature of the present invention that can be used in determining the select gram-positive bacteria or other select bacteria sought to be determined in any methods disclosed herein. By comparison, cell morphology is not available in cell-free assays since the bacteria are destroyed.
specificity:
As noted above, probe / target complexes are formed under conditions that permit specificity of binding. Specificity of hybridization (i.e., the sequence specific binding of a hybridization probe to a nucleic acid target) is a function of various factors related to stringency and / or blocking strategy (ies). Specificity of binding also applies to antibody binding or the binding of members of any other type of ligand-ligand pair. Like hybridization specificity, specificity of binding of antibodies to antigens (or binding of one member of a ligand pair to another member) is also condition dependent. In principle, conditions are selected to optimize specificity so that non-specific binding is minimized or eliminated. However, it is to be understood that specificity of binding is a relative term which also depends on many factors, including the nature (e.g., affinity) of the compositions forming the binding complex. Below is a non-limiting discussion of various conditions / considerations. Using no more than routine experimentation in combination with the disclosure provided herein, persons of skill in the art will be able to achieve suitable conditions so that binding (or hybridization) of specific probes to their respective targets is specific (such as practice of the method produces an accurate and reproducible result). In many cases, this can be accomplished using commercially available buffers.
Blocking Probes:
In hybridization reactions, blocking probes (made of nucleic acids, nucleic analogs, nucleic acid mimics or chimeras) can be used to suppress the binding of probes to a non-target and thereby improve specificity of the formation of probe / target complexes. Especially effective blocking probes are PNA oligomers (See: Coull et al., U.S. Pat. No. 6,110,676, herein incorporated by reference and Fiandaca et al. "PNA Blocker Probes Enhance Specificity In Probe Assays", Peptide Nucleic Acids: Protocols and Applications, pp. 129-141, Horizon Scientific Press, Wymondham, UK, 1999)).
Hybridization Conditions / Stringency:
Persons of ordinary skill in the art will recognize that factors commonly used to impose or control stringency of hybridization include formamide concentration (or other chemical denaturant reagent), salt concentration (ie, ionic strength), hybridization temperature, detergent concentration, pH and presence. or absence of chaotropes. Blocking probes (See the section immediately above for a discussion of blocking probes) may also be used as a means to improve discrimination beyond the limits possible by more optimization of stringency factors. Optimal stringency for forming a probe / target complex is often found by the well-known technique of fixing several of the aforementioned stringency factors and then determining the effect of varying a single stringency factor. The same stringency factors can be modulated to thereby control the stringency of hybridization of a nucleic acid mimic, nucleic acid analog or chimera to a nucleic acid target (eg, a sequence within rRNA, mRNA or chromosomal DNA), except for some of these modified oligomers (eg PNA) hybridization may be fairly independent of ionic strength. Optimal or suitable stringency for an assay may be experimentally determined by examining each stringency factor until the desired degree of discrimination is achieved. However, optimal stringency is not required. Rather, all that is required is that the non-specific binding of probes to other than their respective targets is minimized in the assay to the extent necessary to achieve an accurate and reproducible result.
Other conditions include the use of aqueous alcohol solutions (which may also be used for fixation) (US20070128646).
The use of buffered saline, such as but not limited to 0.75 M NaCl, 5 mM EDTA, 0.10 M Tris HC1, pH 7.8 (previously used for algae (PLoS ONE 6 (10): e25527. Doi: 10.1371 / joumal.pone. 0025527), as a non-toxic hybridization buffer for bacteria and particularly Gram-positive bacteria, such as S. aureus, as exemplified in Example 2, is also within the scope of this invention.
As time to result can be an important factor particularly for clinical samples, the hybridization reactions performed in the examples provided below differ significantly from those of Matsuhisa et al., (Biotech Histochem. 69: 31-7., Inter alia, in that they were performed in less than 2 hours rather than overnight.
Suitable Antibody Binding Conditions
Suitable antibody binding conditions comprise conditions suitable for specifically binding an antibody to its antigen. Factors effecting antibody binding to its antigen (or for the binding of the ligands of a ligand-ligand complex) are substantially similar to those described above for hybridization and can be optimized in a similar manner. Suitable antibody binding conditions for various antibodies are known to persons of skill in the art. For those who are not, they can be determined. As noted above, suitable binding buffers are also commercially available.
Therefore, using the disclosure provided herein; with or without additional routine experimentation, persons of skill in the art can determine suitable antibody binding conditions. By way of additional general guidance to the practitioner, methods for preparing and using antibodies can be found in numerous references including: Molecular Probes Of The Nervous System, Volume 1, "Selected Methods For Antibody And Nucleic Acid Probes", Cold Spring Harbor Laboratory Press , 1993 by S. Hockfield et al.
Harmonizing Binding Conditions in Whole-Cell Assays:
When practicing the methods disclosed herein, persons of skill in the art may find it useful to harmonize the hybridization conditions, antibody binding conditions and other assay conditions (e.g., ligand-ligand binding conditions). For example, in some embodiments, the staining of cells with one or more hybridization probes may be performed simultaneously with, prior to, or subsequent to, an antibody binding event. Because adjustment of the same variables (pH, salt concentration, etc.) is commonly involved, aided by no more than routine experimentation, those of skill in the art will easily be able to harmonize conditions so that the assay produces a satisfactory result. A discussion of some of the problems and related solutions for harmonizing conditions for using antibody probes and hybridization probes in a single assay can be found in Goldbard et al. (U.S. Pat. No. 6,524,798) and A.beta.mus et al., "Improved In Situ Tracking of Rhizosphere Bacteria Using Dual Staining with Fluorescence-Labeled Antibodies and rRNA-Targeted Oligonucleotides", Microb. Ecol., 33: 32-40 (1997). It is also worth noting that the use of non-nucleic acid, and preferably PNA probes, can simplify the harmonization process because PNA probes bind to complementary nucleic acid (as compared to nucleic acid / nucleic acid interactions) under a wide range of conditions, thereby permitting one to tailor the conditions more closely to those suitable for the antibody antigen and / or other ligand-ligand binding.
fixing:
Whole cell assays can be performed using fixed cells. Fixing is the process of treating samples to thereby preserve and / or prepare said cells for analysis. Fixed samples can be stored for a period of time before being analyzed. A commonly used fixative reagent is paraformaldehyde. Other commonly used fixative reagents include glyoxal, glutaraldehyde, zinc salts, heat, alcohols (methanol and ethanol), acidic solutions and combinations of any two or more of these. In some embodiments, methods disclosed herein can be practiced by contacting the sample with a fixative reagent or reagents. A commonly used process for fixing cells is referred to as flame fixation or heat fixation; which process may (or may not) be accompanied by contacting the cells with a reagent or reagents. Thus, the methods disclosed herein can be practiced with a fixation step which may (or may not) include contacting the sample with a reagent or reagents.
Any fixative reagent or reagents may contain other compositions not strictly related to fixation. For example, in some embodiments one or more probes may be added to a fixation reagent or reagents. In this way, fixation and probe / target formation can be performed simultaneously. Any combination of reagents is permissible as long as the combination operates for its intended purpose much in the way that the individual reagent or reagents would if not combined.
Permeabilization or Lysis:
Permeabilization of cell lysis is the process by which the cell membrane / cell wall is modified so that reagents required to perform an assay can gain access to the target.
Some non-limiting examples of cell permeabilizing or lysis reagents include solutions / formulations comprising one or more enzymes such as lysozyme, and proteinases (e.g., proteinase-K and / or achromopeptidase). To permeabilize the cells, said enzymes can be contacted with the sample and thereby partially digest the cell membrane and / or cell wall. In some embodiments, the cell permeabilizing or lysis reagents are chemicals, mixtures of chemicals and enzymes or sequential treatment with chemical (s) and enzyme (s) in any order.
The degree of permeabilization or lysis depends on the nature of the reagents that must penetrate into the cell for practice of the particular assay. Generally, as the size of the molecule that must pass through the cell membrane / cell wall increases, a greater degree of permeabilization must be performed. Cell permeability that is too low can lead to false-negative or false-positive results (See: Pemthaler et al., "Simultaneous Fluorescence In Situ Hybridization of mRNA and rRNA in Environmental Bacteria", Applied and Environmental Microbiology, 70 (9): 5426-5433 (September 2004) at page 5429, col. 2). However, extensive treatment with the cell permeabilizing reagent or reagents can result in destruction of the bacteria cells (See:
Furukawa et al., Microbes Environ., At page 231, col. 1-2).
Permeabilization or lysis may also be performed by other mechanisms such as heating, (ultra) sound, or mechanical forces and is within the scope of the invention.
Washington:
In whole-cell assays, washing steps are commonly performed between one or more steps (or substeps) of a method to remove one or more of the components (or excess components) applied to a sample in a previous step (or substep) to thereby prepare the sample for the next method step (or substep). Washing reagents are often buffered solutions containing a salt and / or a detergent. In practice, a washing reagent is commonly referred to as a wash (ing) buffer or wash (ing) solution. Numerous washing reagents are commercially available. A washing step is often practiced after a sample is contacted with probes so that excess probe that does not selectively bind to its respective target is washed away. However, there are reports of no wash ISH-based assays (See: U.S. Pat. No. 6,905,824). Whether or not a washing step is required will depend in part on the nature of the fixative reagent or reagents as well as the probe or probes used in the assay and the means by which the determinations are made.
Signal Amplification:
In some embodiments, signal amplification of a label is used to improve upon the limits of detection of a method. In brief, signal amplification is typically used where a bacteria possesses a low copy number of a particular target and thus, a resulting small number of the respective probe / target complexes. Particularly where a determination (e.g. of the select bacteria) is based on bacteria staining, there may not be enough signal generated if the number of probe / target complexes in the bacteria is sufficiently low. However, if the signal of a single label associated with a probe / target complex can be multiplied or amplified many times, it becomes possible to make a determination even for low copy number targets in a bacterial cell.
There are several types of signal amplification techniques available. Signal amplification can be applied to both direct and indirect labeling techniques. Some non-limiting examples of signal amplification include tyramid signal amplification (TSA, also known as catalyzed reporter deposition (CARD)), Enzyme Labeled Fluorescence (ELF-97 product and information available from Invitrogen, Carlsbad, Calif.), Branched DNA ( bDNA) Signal Amplification, and rolling-circle amplification (RCA). Specific methods for using these signal amplification techniques to detect low copy number targets within bacteria are discussed in more detail in several of the references listed in Section 8, below.
Various Embodiments of the Invention
It should be understood that the order of steps or order for performing certain actions is immaterial as long as the present teachings remain operable or unless otherwise specified. Moreover, in some embodiments, two or more steps or actions can be conducted simultaneously as long as the present teachings remain operable or unless otherwise specified.
This invention pertains, inter alia, to methods for determining select bacteria of a sample, where said bacteria are located intracellular. It is understood that the methods described herein are not limited to determining one select bacteria. Rather, the methods can be used to determine multiple bacteria in a sample, incl. subsets of the same bacteria of the sample. In some embodiments, the multiple bacteria and / or multiple subsets of select bacteria will be determined using a multiplex assay. The multiplex assay can involve the use of differential staining of the bacteria whereby the different stain or stains a bacteria exhibits is used to determine the different bacteria and / or subset (s).
Therefore, in some embodiments, this invention relates to a method comprising contacting a sample with a bacteria-directed probe or probes capable of determining a select bacterium in the sample. Often the sample will be suspected of comprising one or more bacteria.
In some embodiments, the sample is treated with a permeabilizing or lysis reagent, whereas in other embodiments a permeabilization or lysis reagent is not used. It is understood that the use of permeabilization or lysis reagents may be directed towards permeabilization or lysis of the bacteria and / or the cells containing the bacteria.
In some embodiments, the hybridization buffer contains a denaturing reagent such as formamide, whereas in other embodiments a hybridization buffer does not contain a denaturing reagent. Hybridization buffer without denaturing reagents, such a formamide, may be buffered saline, such as but not limited to NaCl, Tris and EDTA, i.e. 0.75M NaCl, 5mM EDTA, 0.1M Tris, pH 7.8.
In some embodiments, signal amplification is used to increase the signal prior to determining the bacteria, whereas in other embodiments signal amplification is not used.
In preferred embodiments, the method is performed without permeabilization or lysis reagents, and / or without hybridization buffer containing denaturing chemicals and / or without the use of signal amplification. In yet another preferred embodiment, the method is performed without permeabilization or lysis reagents and without signal amplification.
According to these various methods, determination of the select bacteria involves determining the formation of probe / target complexes for the bacteria-directed probe or probes and chromosomal DNA, RNA and / or plasmid-directed labeled probe or probes, respectively. The formation of the probe / target complexes is accomplished under suitable binding conditions (or suitable hybridization conditions as appropriate). In some embodiments, formation of the respective probe / target complex or complexes will be evident based on the nature of the staining of the bacteria. Thus, for these embodiments, the select bacteria and / or subsets can be determined by analysis of the staining of individual bacteria. The staining of individual bacteria can, for example, be monitored (determined) using a microscope, slide scanner or flow cytometer.
These methods can be practiced without the use of an amplification technique (eg signal amplification of the label or labels linked to the chromosomal DNA, RNA and / or plasmid-directed labeled probe or probes or target amplification techniques such as in-situ PCR) . These methods can be practiced without contacting the sample with a cell permeabilizing reagent or reagents. In some embodiments, said single label (linked to each of the chromosomal DNA, RNA and / or plasmid-directed labeled probe or probes) comprises a fluorescent label or labels that exhibit (s) an emission maximum of less than 650 nm.
In some embodiments, these methods can be practiced using only mRNA-directed probe or probes, said probe or probes are capable of determining the select bacteria. In some embodiments, only a single mRNA-directed probe is used to determine a select bacterium or a single mRNA-directed probe is used to determine each of multiple select bacteria or subsets of bacteria of interest. In some embodiments, these methods are practiced with a mixture of mRNA-directed labeled probes.
In some embodiments, each of the mRNA-directed probe or probes comprises a single label or two labels (i.e., each probe is a single labeled or dual labeled probe). In some embodiments, said label or labels are / are a fluorescent label or labels that exhibit (s) an emission maximum of less than 650 nm.
In some embodiments, each chromosomal DNA, mRNA, and / or native plasmid-directed labeled probe comprises a single label or two labels (i.e., each probe is a single labeled or dual labeled probe). In some embodiments, the method is practiced without signal amplification of a label or labels of said chromosomal DNA, mRNA, and / or native plasmid-directed labeled probe or probes. In some embodiments, each chromosomal DNA, mRNA, and / or native plasmid-directed labeled probe comprises a single label and the method is practiced without signal amplification of said single label of said chromosomal DNA, mRNA, and / or native plasmid directed labeled probe or probes.
In some embodiments, bacteria-directed probe or probes is / are antibody-based. As such, the target for each probe is an antigen found on the surface of, or within, the select gram-positive bacteria.
In some embodiments, the bacteria-directed probe or probes is / are rRNA-directed. As such, the target for each probe is a nucleobase sequence found within rRNA of the select gram-positive bacteria.
In some embodiments, the bacteria-directed probe or probes is / are mRNA-directed. In some embodiments, the bacteria-directed probe or probes is / are directed to a regulatory RNA (e.g., sRNA or aRNA). As such, the target for each probe is a nucleobase sequence of (or within) mRNA or regulatory RNA (e.g., sRNA or aRNA), respectively.
In some embodiments, the bacteria-directed probe or probes are / are labeled with a label or labels. In some embodiments, each bacteria-directed probe is labeled with a single label or two labels (i.e., each probe is a single labeled or dual labeled probe). In some embodiments, said label or labels are fluorescent and exhibit an emission maximum of less than 650 nm. In some embodiments, one or more of said label or labels is fluorescent and exhibits an emission maximum of 650 nm or more.
In some embodiments, these methods can be practiced with or without various additional steps and / or reagents. For example, one or more washing steps maybe conducted by contacting the sample with one or more washing reagents. In some embodiments, the sample is contacted with a fixative reagent or reagents. In some embodiments, the sample is contacted with a cell permeabilizing or lysis reagent or reagents. It is understood that in some embodiments, two or more of the passing reagents may be applied to the same sample, each reagent contacting the sample one or more times. Contacting of the sample with the various reagents can be performed in any order (or simultaneously) that permits accurate determination of the select bacteria.
In some embodiments, one or more steps that are commonly performed are omitted. For example, in hybridization assays, it is common to perform a prehybridization step prior to contacting the sample with the hybridization probe or probes. In some embodiments of this invention where one or more hybridization probes are used, the method is performed with no prehybridization step. When an antibody probe or probe is used, a blocking step is often performed (or not) before the sample is contacted with said antibody probe or probes but this step may be omitted. In some embodiments, the cell permeabilization or lysis step is omitted. In some embodiments, a wash step or steps is / are omitted. Indeed any commonly performed step can be omitted where said omission does not cause the method to fail to produce an accurate result.
In some embodiments, all probes are labeled. In some embodiments, all labels are fluorescent labels. In some embodiments, these methods are conducted as an in situ hybridization (ISH) assay because all probes are hybridization probes (i.e., they hybridize to their respective targets). In some embodiments, all probes are hybridization probes and all labels are fluorescent labels. In this case the method is conducted as a fluorescence in situ hybridization (FISH) assay.
In some embodiments, more than one select bacteria can be determined. In some embodiments, this can be accomplished by multiplexing. In some embodiments, this can be accomplished by reprobe cycling the sample. In some embodiments, this can be accomplished by both multiplex and reprobe cycling the sample. Thus, in some embodiments, these methods further comprise contacting the sample with a second bacteria-directed probe or probes capable of determining a second select bacteria in the sample. It is understood that the method may also be practiced by contacting the sample with additional probes or probe sets to one or more additional select bacteria and / or subset of select bacteria.
The method is practiced on substantial intact bacteria present intracellularly by contacting the sample with one or more bacteria-directed probes comprising labels capable of determining the bacteria in said sample; and determining the one or more bacteria.
These methods can be practiced without the use of signal amplification of the label or labels linked to the bacteria-directed labeled probe or probes. However, the chromosomal DNA and / or mRNA-directed labeled probe or probes each label a single label or two labels, said label or labels can be fluorescent and have an emission maximum of less than, equal to or more than 650 nm. In some embodiments, these methods can be practiced without the use of any amplification techniques. In some embodiments, these methods can be practiced without contacting the sample with a cell permeabilizing or lysis reagent or reagents.
In some embodiments, these methods can be practiced using only mRNA-directed probe or probes said probe or probes are capable of determining mRNA associated with methicillin resistance. In some embodiments, only a single mRNA-directed probe is used to determine methicillin resistance. In some embodiments, two or more mRNA-directed probes are used to determine methicillin resistance (i.e., a mixture of mRNA-directed probes which probes can each be labeled with one or two labels).
In some embodiments, each of the mRNA-directed probe or probes comprises a single label or two labels (i.e., each probe is a single labeled or dual labeled probe). In some embodiments, said label or labels are / are fluorescent and exhibit (s) an emission maximum of less than, equal to or more than 650 nm.
In some embodiments, each chromosomal DNA and / or mRNA-directed labeled probe comprises a single label or two labels (i.e., each probe is a single labeled or dual labeled probe). In some embodiments, these methods can be practiced without signal amplification of the label or labels of said chromosomal DNA and / or mRNA-directed labeled probe or probes. In some embodiments, each chromosomal DNA and / or mRNA-directed labeled probe comprises a single label or two labels (i.e. each probe is a single labeled or dual labeled probe) and the method is practiced without signal amplification of said single label of said chromosomal and / or DNA, mRNA-directed labeled probe or probes. In some embodiments, each chromosomal DNA and / or mRNA-directed labeled probe comprises one or more labels and the method is practiced with (direct or indirect) signal amplification of said label or labels of said chromosomal and / or DNA, mRNA, directed labeled probe or probes.
In some embodiments, bacteria-directed probe or probes is / are antibody-based. As such, the target for each probe is an antigen found on the surface of, or within, the select bacteria.
In some embodiments, the bacteria-directed probe or probes is / are rRNA-directed. As such, the target for each probe is a nucleobase sequence found within the rRNA of the select bacteria. As a suitable rRNA-directed probe for determining S. aureus bacteria in clinical samples is commercially available and a study describing its use is described in: Forrest et al., "Impact of rapid in situ hybridization testing on coagulase-negative staphylococci positive blood cultures" , Journal of Antimicrobial Chemotherapy, 58: 154-158 (2006). In some embodiments, the bacteria-directed probe or probes is / are mRNA-directed or directed to other regulatory RNA (e.g., sRNA or aRNA). As such, the target for each probe is a nucleobase sequence of (or within) mRNA or regulatory RNA (e.g., sRNA or aRNA), respectively.
In some embodiments, the bacteria-directed probe or probes are / are labeled with a label or labels. In some embodiments, each bacteria-directed probe is labeled with a single label or two labels (i.e., each probe is a single labeled or dual labeled probe). In some embodiments, said label or labels are / are fluorescent and exhibit (s) an emission maximum of less than 650 nm. In some embodiments, one or more of said label or labels is / are fluorescent and exhibit (s) an emission maximum of 650 nm or more.
In some embodiments, these methods can be practiced with or without various additional steps and / or reagents. For example, one or more washing steps maybe conducted by contacting the sample with one or more washing reagents. In some embodiments, the sample is contacted with a fixative reagent or reagents. In some embodiments, the sample is contacted with a cell permeabilizing reagent or reagents.
In some embodiments, one or more steps that are commonly performed are omitted. For example, in hybridization assays, it is common to perform a prehybridization step prior to contacting the sample with the hybridization probe or probes. In some embodiments of this invention where one or more hybridization probes are used, the method is performed with no prehybridization step. When an antibody probe or probe is used, a blocking step is often performed (or not) before the sample is contacted with said antibody probe or probes but this step may be omitted. In some embodiments, the cell permeabilization step is omitted. In some embodiments, a wash step or steps is / are omitted. Indeed any step commonly performed can be omitted where said omission does not cause the method to fail to produce an accurate result.
In some embodiments, all probes are labeled. In some embodiments, all labels are fluorescent labels. In some embodiments, these methods can be conducted as an in situ hybridization (ISH) assay because all probes are hybridization probes (i.e., they hybridize to their respective targets). In some embodiments, all probes are hybridization probes and all labels are fluorescent labels. In this case, these methods can be conducted as a fluorescence in situ hybridization (FISH) assay.
In some embodiments, all labels are fluorescent labels and said method is a fluorescent in situ hybridization (FISH) assay. In some embodiments, a label or labels of said chromosomal DNA and / or mRNA-directed labeled probe or probes is / are determined directly.
In some embodiments, the chromosomal DNA and / or mRNA-directed labeled probe or probes is / are PNA. In some embodiments, the chromosomal DNA and / or mRNA-directed labeled probe or probes are / are 10 to 20 nucleobase subunits in length. In some embodiments, signal amplification is used to directly or indirectly amplify a label or labels of said chromosomal DNA and / or mRNA-directed labeled probe or probes.
In some embodiments, the method can be practiced without treating the sample with a cell permeabilizing or lysis reagent or reagents.
In some embodiments, the method can be practiced where the samples are immobilized onto a solid support such as a microscope slides, filter or bead, whereas in other embodiments, the method is practiced where the sample remains in solution.
In some embodiments, the host cells are predatory host cells, such as phagotytic cells, degrading the intracellular bacteria, whereas in other embodiments the host cells and the intracellular bacteria are living in symbiosis. Therefore, the intracellular bacteria may be viable (alive) or non-viable (dead).
In summary, there are many different ways to practice the method of the invention of which preferred embodiments can be described by: A method for the determination of one or more intracellular select bacteria, said method comprising: a. the intracellular bacteria; b. fixing the sample to maintain the intracellular bacteria substantially intact; c. contacting the sample with one or more select bacteria-directed probes comprising labels capable of determining the one or more select bacteria in said sample; and d. determining the one or more bacteria; and said method not comprising: e. contacting a cell permeabilizing or cell lysing reagent; and / or f. signal amplification of said label; and / or g. contacting a denaturing reagent.
The method above may be practiced as an in situ hybridization method where the host cells are phagocytic cells (predatory host cells), and / or the bacteria is S. aureus, and / or the bacteria-directed probes are PNA probes, and / or The labels are fluorescent, and / or the duration of the contact is less than 2 hours, and / or the hybridization buffer is non-toxic, and the sample is either attached to a solid support or in solution.
Examples
Aspects of the present teachings can be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way.
Example 1
Staphylococcus aureus were experimentally spiked into donor blood and incubated 15-45 min before ingestion of S. aureus by phagocytic cells. The PMN fraction was purified and subsequently analyzed by S. aureus PNA FISH on microscope slides in accordance with Oliveira et al., J. Clin. Microbiol 40: 247-251 (2002). Briefly, the samples were fixed onto microscope slides and hybridized with fluorescein-labeled PNA probes in hybridization buffer containing formamide for 30 minutes. Unbound PNA probes were removed by rigorous wash for 30 minutes at 55 ° C and counterstained with DAPI for visualization of both PMNs (blue) and S. aureus (blue). Examination by fluorescence microscopy showed PMNs, including S. aureus (A) using DAPI (blue) filter and only S. aureus (B) using FITC / Texas Red filter, (blue cocci are observed within the PMN with the same morphology and position as the green cocci), See Fig. First
In conclusion, intracellular bacteria in phygocytic cells can be determined by fluorescence in situ hybridization using PNA probes without the use of permeabilization or lysis reagents and without using signal amplification.
Example 2
Staphylococcus aureus and Staphylococcus epidermidis were analyzed by fluorescence in situ hybridization in accordance with PFoS ONE 6 (10): e25527 modified with PNA probe sequence and temperature (55 ° C) from J. Clin. Microbiol 40: 247-251 (2002). Briefly, the bacteria were fixed in solution using saline ethanol and washed twice (pre-hybridization) with hybridization buffer (0.75 M NaCl, 5 mM EDTA, 0.10 M Tris HCl, pH 7.8) followed by hybridization with PNA probe for 1 hour at 55 ° C. Unbound probe was removed by washing and the samples were mounted onto microscope slides. Examination by fluorescence microscopy using FITC / Texas Red filter showed strong fluorescence of S. aureus (A) and low / none fluorescence of S. epidermidis (B), see Figs. 2nd
In conclusion, S. aureus can be determined by fluorescence in situ hybridization using PNA probes in buffered saline as hybridization buffer and without immobilizing the bacteria on microscope slides during hybridization.

权利要求:
Claims (8)
[1] 1. An in situ hybridization method for the determination of one or more intracellular select bacteria in phagocytic cell(s), said method comprising: a. providing a sample comprising phagocytic cells enclosing the intracellular bacteria; b. fixing the sample to maintain the intracellular bacteria substantially intact; c. contacting the sample for less than 2 hours with one or more select bacteria-directed PNA probes comprising fluorescent labels capable of determining the one or more select bacteria in said sample; and d. determining the one or more bacteria in said host cells.
[2] 2. The method of any of the preceding claims, wherein the sample is not immobilized onto a solid support during the contacting of the bacteria-directed probes.
[3] 3. The method of any of the preceding claims, where the method is practiced without the use cell permeabilizing or lysing reagent(s) and/or step(s).
[4] 4. The method of any of the preceding claims, where said labels are not signal amplified.
[5] 5. The method of any of the preceding claims, where said sample is blood or prepared from blood.
[6] 6. An in situ hybridization method for the determination of one or more select bacteria, said method comprising: a. providing a sample comprising the bacteria; b. fixing the sample to maintain the bacteria substantially intact; c. contacting the sample for less than 2 hours with one or more select bacteria-directed PNA probes comprising fluorescent labels capable of determining the one or more select Gram- positive bacteria in said sample without the use of a denaturing or alcoholic reagent; and d. determining the one or more bacteria.
[7] 7. The method of any of the preceding claims 1-6, where the contracting comprises contacting with buffered saline reagent.
[8] 8. The method of any of the preceding claims 1-7, wherein said bacteria are S. aureus

类似技术:

---

公开号 | 公开日 | 专利标题

---

US20160257997A1|2016-09-08|Methods for whole-cell analysis of gram-positive bacteria

---

US8114681B2|2012-02-14|Highly multiplexed particle-based assays

---

Perry‐O’Keefe et al.2001|Filter‐based PNA in situ hybridization for rapid detection, identification and enumeration of specific micro‐organisms

---

US20110189674A1|2011-08-04|Method for quantifying or detecting dna

---

US20180171391A1|2018-06-21|Methods and Compositions For Sorting and/or Determining Organisms

---

US20090203017A1|2009-08-13|Use of Nucleic Acid Probes to Detect Nucleotide Sequences of Interest in a Sample

---

US6593093B1|2003-07-15|Detection of group a Streptococcus

---

Kurokawa et al.1999|Abundance and distribution of bacteria carrying sltII gene in natural river water

---

EP3103884B1|2021-06-09|Nucleic acid detection or quantification method using mask oligonucleotide, and device for same

---

US20030082563A1|2003-05-01|Detection of bacillus anthracis

---

US6830888B2|2004-12-14|Detection of Legionella

---

DK178306B1|2015-11-23|Determination of Intracellular Bacteria

---

US20150218624A1|2015-08-06|Compositions and methods for detection of staphylococcus aureus

---

US9816145B2|2017-11-14|Compositions for detection of Clostridium difficile

---

US10190178B2|2019-01-29|Compositions and methods for detecting mecC-containing methicillin-resistant Staphylococcus aureus

---

US9970062B2|2018-05-15|Compositions and methods for detection of mycobacterium tuberculosis

---

JP2004147550A|2004-05-27|Nucleic acid fragment, method and kit for detecting microorganism

---

Singleton2000|Probe-Based | Methods

---

JPH11332597A|1999-12-07|Measurement of nucleic acid and detection of vero toxin-producing bacterium

同族专利:

---

公开号 | 公开日

---

US20160138090A1|2016-05-19|

---

WO2014202080A1|2014-12-24|

---

DK178306B1|2015-11-23|

---

DK201500108A1|2015-04-13|

---

EP3011057A1|2016-04-27|

---

EP3011057A4|2017-03-08|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

WO2004039831A2|2002-10-28|2004-05-13|Advandx, Inc.|Peptide nucleic acid probes for analysis of enterococcus faecium|

---

US20080108064A1|2003-11-26|2008-05-08|Advandx, Inc.|Peptide nucleic acid probes for analysis of certain staphlococcus species|

---

WO2010135480A1|2009-05-20|2010-11-25|Advandx, Inc.|Methods for whole-cell analysis of gram-positive bacteria|

---

WO2012135620A2|2011-04-01|2012-10-04|Advandx. Inc.|Molecular gram stain|

---

EP1403381B1|2001-05-31|2007-07-11|Fuso Pharmaceutical Industries Ltd.|Improved method for detecting and identifying microorganism causative of infection|

---

CA2447775A1|2001-05-31|2002-12-19|Fuso Pharmaceutical Industries, Ltd.|Process for evaluating phagocytotic function and use thereof|

---

CN101974626B|2010-10-12|2012-08-29|上海交通大学|Detection method of symbiotic bacteria in sponge cell based on quantum dot fluorescence in-situ hybridization|GB201615466D0|2016-09-12|2016-10-26|Gfc Diagnostics Ltd|Method for detecting bacteria|

法律状态:
2020-01-17| PBP| Patent lapsed|Effective date: 20190616 |

优先权:

---

申请号 | 申请日 | 专利标题

---

DKPA201300361A|DK178306B1|2013-06-16|2013-06-16|Determination of Intracellular Bacteria|

---

DK201300361|2013-06-16|DKPA201300361A| DK178306B1|2013-06-16|2013-06-16|Determination of Intracellular Bacteria|

---

EP14814049.4A| EP3011057A4|2013-06-16|2014-06-12|Determination of intracellular bacteria|

---

PCT/DK2014/000032| WO2014202080A1|2013-06-16|2014-06-12|Determination of intracellular bacteria|

---

US14/898,860| US20160138090A1|2013-06-16|2014-06-12|Determination of intracellular bacteria|

---

DK201500108A| DK201500108A1|2013-06-16|2015-02-24|Determination of lntracellular Bacterial|