METHODS, COMPOSITIONS, AND KITS FOR DETECTING A CELL IN A SAMPLE
20190316169 ยท 2019-10-17
Assignee
Inventors
- Sudheendra Lakshmana (Davis, CA, US)
- Alice Ngo (West Sacramento, CA, US)
- Laura Nicole Putnam (Davis, CA, US)
- Alexander Thornton-Dunwoody (Davis, CA, US)
- Leslie Anne Jones (Davis, CA, US)
Cpc classification
C12Q1/18
CHEMISTRY; METALLURGY
C12N7/00
CHEMISTRY; METALLURGY
C12Q2563/173
CHEMISTRY; METALLURGY
C12Q1/04
CHEMISTRY; METALLURGY
C12Q2563/173
CHEMISTRY; METALLURGY
G01N27/327
PHYSICS
International classification
C12Q1/04
CHEMISTRY; METALLURGY
C12Q1/18
CHEMISTRY; METALLURGY
Abstract
Provided are methods, compositions, devices, and kits for detecting bacteria in a sample. The methods, compositions, devices, and kits are specially useful for diagnosis and prognosis of an individual suspected of having bacterial infection. The methods, compositions, devices, and kits are applicable to a wide variety of areas, for example, medical diagnostics, industries involving bacterial fermentation, dairy and wine industries, prevention of bioterrorism.
Claims
1. A method of detecting the presence of one or more types of cell in a sample comprising: a) providing one or more types of viruses, wherein each of said one or more types of viruses bind to one or more specific types of receptor of said one or more types of cell if present in said sample to form complexes of virus and cell; b) contacting said sample suspected of comprising said cell with said one or more types of viruses, wherein said one or more types of virus form complexes with said one or more types of cell if present in said sample; c) contacting said one or more types of viruses with a first set of one or more compounds to generate one or more types of viruses comprising a nucleic acid comprising said first compound; d) contacting said one or more types of cell with a second set of one or more compounds, wherein said second set of one or more compounds preferentially interacts with one or more components of said one or more types of cell in the presence of said one or more types of viruses; e) detecting said complexes by detecting said second set of one or more compounds or modifications of said second set of one or more compounds by said one or more types of cell, wherein said complexes are indicative of the presence of said one or more types of cell in said sample.
2. The method of claim 1, wherein upon contacting said one or more types of viruses with said first set of one or more compounds inhibits replication of one or more types of viruses in said one or more types of cell.
3. The method of claim 1, wherein contacting said one or more types of viruses with a first set of one or more compounds is done prior to contacting said sample suspected of comprising said cell with said one or more types of viruses.
4. The method of claim 1, wherein said one or more types of cell are one or more types of bacteria, and wherein said one or more types of viruses are one or more types of bacteriophage that bind to one or more specific types of receptor of said one or more types of bacteria.
5. The method of claim 4, wherein second set of one or more compounds preferentially binds to the nucleic acid of said one or more types of bacteria.
6. The method of claim 5, wherein second set of one or more compounds preferentially binds to the nucleic acid of said one or more types of bacteria after the formation of said complexes of bacteriophage and bacteria.
7. The method of claim 1, wherein said first set of one or more compounds comprises a first detectable label.
8. The method of claim 1, wherein said detecting further comprises detecting said first set of one or more compounds.
9. The method of claim 1, wherein said first set of one or more compounds is selected from the group consisting of propidium iodide, ethidium bromide, propidium monoazide, ethidium monoazide, a combination of, and derivatives thereof.
10. The method of claim 1, wherein said second set of one or more compounds interact with cellular nucleic acid.
11. The method of claim 1, wherein said second set of one or more compounds comprise a second detectable label.
12. The method of claim 1, wherein said second set of one or more compounds comprise a substrate for cellular enzymes.
13. The method of claim 1, wherein said second set of one or more compounds is a redox compound.
14. The method of claim 1, wherein said second set of one or more compounds is Resazurin, Resorufin, Dihydroresorufin, or a combination thereof.
15. The method of claims 14, wherein said modification of said second set of one or more compounds comprise a change in the oxidative state of Resazurin, Resorufin, Dihydroresorufin, or a combination thereof
16. The method of claim 15, wherein said detecting modifications of said second set of one or more compounds comprise measuring the change of the oxidative status of Resazurin, Resorufin, Dihydroresorufin or a combination thereof
17. The method of claim 1, further comprising measuring any modifications of said second set of one or more compounds by said one or more types of cell over a period of time and determining the kinetic profile of said modification of said second set of one or more compounds, wherein the kinetic profile is indicative of the presence of said one or more types of cell.
18. The method of claim 4, wherein said method is carried out at a temperature between about 20 C. and about 50 C.
19. (canceled)
20. The method of claim 1, wherein said second set of one or more compounds comprise Calcein derivative.
21. The method of claim 1, wherein said second set of one or more compounds comprises an antibody specific for a cellular protein.
22. (canceled)
23. The method of claim 4, further comprising incubating said complexes of said one or more types of bacteria and bacteriophage in a media comprising one or more bactericide or bacteriostatic agent prior to detecting said complexes.
24. (canceled)
25. The method of claim 23, wherein said one or more types of bacteriophages are immobilized on a solid support, and wherein complexes of said one or more types of bacteria and bacteriophage are immobilized on a solid support upon binding of the one or more types of bacteriophages to one or more specific types of receptor of said one or more types of bacteria.
26. A method of detecting one or more bactericide or bacteriostatic agent resistant bacteria in a sample comprising: a) providing a first sample comprising one or more types of bacteriophages, wherein each of said one or more types of bacteriophages bind to one or more specific types of receptor of one or more types of said bacteria if present in said sample to form complexes of bacteria and bacteriophage; and wherein said one or more types of bacteriophages are incapable of replicating inside said one or more types of bacteria; b) providing a second sample suspected of comprising one or more types of said bacteria, wherein said one or more types of said bacteria are susceptible to infection by said one or more types of bacteriophage; c) contacting said first sample with said second sample to form a sample mixture, wherein said one or more types of bacteriophages if present in said first sample forms one or more complexes with said one or more types of bacteria in said sample mixture; d) incubating said sample mixture in a media comprising one or more bactericide or bacteriostatic agent; e) contacting one or more compounds to said media; f) measuring any change of said one or more compounds in said media; wherein any change of said one or more compounds is indicative of the presence or absence of one or more types of bacteriophage infected bacteria in said sample mixture, and wherein the presence or absence of one or more types of bacteriophage infected bacteria in said sample mixture is indicative of the presence or absence of one or more types of bactericide or bacteriostatic agent resistant bacteria in said second sample.
27.-30. (canceled)
31. The method of claim 26, further comprising measuring any change of said one or more compounds in presence of said sample mixture over a period of time and determining the kinetic profile of said change of said one or more compounds, wherein the kinetic profile is indicative of the presence or absence of one or more types of bacteriophage infected bacteria in said sample mixture, and wherein the presence or absence of one or more types of bacteriophage infected bacteria in said sample mixture is indicative of the presence or absence of one or more types of bacteria in said second sample.
32. The method of claim 26, further comprising comparing said kinetic profile of the change said one or more compounds with a kinetic profile of the change said one or more compounds in the presence of one or more types of bacteria susceptible to said one or more bactericide or bacteriostatic agent, wherein a difference between kinetic profiles is indicative of the presence of one or more types of bacteria resistant to one or more bactericide or bacteriostatic agent in said second sample.
33. The method of claim 26, wherein said one or more compounds is an enzymatic substrate of said one or more types of bacteria.
34. The method of claim 26, wherein said one or more compounds is a redox compound.
35.-38. (canceled)
39. The method of claim 26, wherein said one or more types of bacteriophage is chemically treated or genetically modified.
40.-48. (canceled)
49. A kit for detecting the presence of one or more types of cell in a sample using the method of claim 26, comprising: a) a first reagent comprising one or more types of viruses, wherein each of said one or more types of viruses bind to one or more specific types of receptor of said one or more types of cell if present in said sample to form complexes of viruses and cell, wherein said one or more types of viruses are incapable of replicating inside said one or more types of cell; b) a second reagent comprising one or more compounds preferentially interacts with one or more components of said cell in the presence of said one or more types of viruses of step (a); c) buffers; d) instruction for detecting said complexes by detecting said one or more compounds or modifications of said one or more compounds by said cell, wherein detecting said complexes is indicative of the presence of said one or more types of cell in said sample.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0110] As used herein the term change or modification of a compound refers to the change in the physical, chemical, or oxidative state of the compound. In some embodiments, the change comprises covalent modification. In some embodiments, the modification is by forming a covalent bond. In some embodiments, the change is by changing the composition (chemical change such as protonation/de-protonation), changing its electronic structure (oxidation/reduction) of the detectable label. In some embodiments, a compound comprising a detectable label is modified upon association with bacteria or bacteriophage.
[0111] The term surface plasmon resonance, as used herein, refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in concentrations within a biosensor matrix, for example using the BIACORE system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).
[0112] As used herein the term at least a portion and/or grammatical equivalents thereof can refer to any fraction of a whole amount. For example, at least a portion can refer to at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9% or 100% of a whole amount.
[0113] As used herein the term about means +/10%.
[0114] Solid Support
[0115] Solid support can be two-or three-dimensional and can comprise a planar surface (e.g., a glass slide) or can be shaped. A solid support can include glass (e.g., controlled pore glass (CPG)), corrugated glass, quartz, plastic (such as polystyrene (low cross-linked and high cross-linked polystyrene), polycarbonate, polypropylene and poly(methylmethacrylate)), acrylic copolymer, polyamide, silicon, metal (e.g., alkanethiolate-derivatized gold), cellulose, nylon, latex, dextran, gel matrix (e.g., silica gel), polyacrolein, or composites.
[0116] Suitable three-dimensional solid support includes, for example, spheres, microparticles, beads, membranes, slides, plates, micromachined chips, nanoengineered chips tubes (e.g., capillary tubes), microwells, nanowells, microfluidic devices, nanofluidic devices, flow cells, channels, filters, fluidic cartridge. Solid support can include planar arrays or matrices capable of having regions that include populations of bacteriophage or bacteria.
[0117] In some embodiments, the solid support or its surface is non-planar, such as the inner or outer surface of a tube or vessel. In some embodiments, the solid support is a surface of a flow cell.
[0118] In some embodiments, solid support may include Surface Enhanced Raman Spectroscopy (SERS) surface. In some embodiments, solid support may include a base support and a metallic nanostructure layer deposited upon the base support. The nanomaterial employed in the nanostructure layer may be any SERS-active metallic material, such as silver, gold, copper, platinum, titanium, chromium, combinations thereof or the like. In addition, the nanostructure layer may assume any form, such as nanoparticles, nanoaggregates, nanopores/nanodisks, nanorods, nanowires, or combinations thereof. In some embodiments, the nanoparticles are arranged on the base support by self-assembly. The base support may be any ceramic, polymer, metal, silicon, quartz (crystalline silica), glass, zinc oxide, alumina, Paraffin film, polycarbonate (PC), combinations thereof and the like. In some embodiments, the solid support is hollow metal or metal oxide nano- or microspheres.
[0119] In some embodiments, the solid support comprises microspheres or beads. As used herein, microspheres or beads or particles or grammatical equivalents herein is meant to include small discrete particles. Suitable bead compositions include, but are not limited to, plastics, ceramics, glass, polystyrene, methyl styrene, acrylic polymers, paramagnetic materials, thoria sol, carbon graphite, titanium dioxide, latex or cross-linked dextrans such as Sepharose, cellulose, nylon, cross-linked micelles and Teflon, as well as any other materials outlined herein for solid supports may all be used. Microsphere Detection Guide from Bangs Laboratories, Fishers Ind. is a helpful guide. In certain embodiments, the microspheres are magnetic microspheres or beads. In some embodiments, the beads can be color coded. For example, MicroPlex Microspheres from Luminex, Austin, Tex. may be used.
[0120] The beads need not be spherical; irregular particles may be used. Alternatively, or additionally, the beads may be porous. The bead sizes range from nanometers, i.e. lnm, to millimeters, i.e. 1 mm, with beads from about 0.01 micron to about 200 microns being preferred, and from about 0.5 to about 5 microns being particularly preferred, although in some embodiments smaller or larger beads may be used. In some embodiments, beads can be about 0.1, 0.2, 0.5, 1, 1.5, 2, 2.5, 2.8, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 15, or 20 m in diameter.
[0121] In some embodiments, the solid support has at least one of the components (materials) transparent to electromagnetic radiation. In some embodiments, the transparent substrate is conductive. In some embodiments, the transparent substrate is dielectric. In some embodiments, the solid support has at least one of its components opaque to electromagnetic radiation. In some embodiments, the opaque substrate is conductive. In some embodiments, the opaque substrate is dielectric. In some embodiments, the conductive component (material) is porous. In some embodiments, the dielectric component is porous. In some embodiments, the pore size is less than 10 nm. In some embodiments, one of the components is magnetic. In some embodiments, the substrate has a periodic structure. In some embodiments, the period of the substrate is less than a micron in dimension. In some embodiments, the period of the substrate is more than a micron in dimension. In some embodiments, the substrate has a combination of the above embodiments. In some embodiments, the substrates mentioned above generate an electromagnetic resonance.
[0122] In some embodiments, the solid support is a sensor. In some embodiments, the sensor is a piezoelectric sensor. In some embodiments, the piezoelectric sensor has at least a monolayer of gold on top or bottom of the solid support. In some embodiments, the sensors are round-shaped. In some embodiments, solid support comprises quartz crystals with gold coating. In some embodiments, the sensors are mounted on a flowcell.
[0123] In some embodiments, the solid support comprises a plurality of materials of defined compositions and property. In some embodiments, the solid support comprises a solid surface with a bilayer of different chemical composition. In some embodiments, one layer may be conductive of electricity. In some embodiments, one layer may be a metal. Exemplary metals include, but are not limited to, silver, gold, copper, platinum, titanium, chromium, Al, Ni, Cr, Fe, ceramic metals such as Indium-tin-oxide etc. or combinations thereof. In some embodiments, one layer comprises magnetic materials, e.g., Fe, Ni, Non-oxide, non-elemental magnetic material Nd2Fe14B, SmCo5 etc., non-elemental, oxide magnetic material Fe.sub.3O.sub.4, Fe.sub.2O.sub.3. In some embodiments, the second layer can be non-conductive or non-magnetic. In some embodiments, the second layer may be ceramic, polymer, metal, silicon, quartz (crystalline silica), glass, zinc oxide, alumina, Paraffin film, polycarbonate (PC), or combinations thereof.
[0124] In some embodiments, the solid support is a microarray. In some embodiments, the mircroarray may comprise different materials for the wells and the base. In some embodiments, the solid support is a bead. In some embodiments, the bead may comprise detectable label.
[0125] In some embodiments, the bacteria, bacteriophage, and/or the bacteria and bacteriophage complex may be immobilized on a solid support. In some embodiments, bacteria are immobilized on a solid support. In some embodiments, bacteriophage is immobilized on a solid support. In some embodiments, bacteria and bacteriophage complex is immobilized on a solid support. In some embodiments, the bacteria, bacteriophage, or the bacteria and bacteriophage complex are adsorbed passively on the substrate. In some embodiments, at least one bacterium, one bacteriophage, or one bacteria and bacteriophage complex is actively adsorbed by applying an external electric field. In some embodiments, the immobilization is performed under with buffers of varying concentrations and pH.
[0126] In some embodiments, the immobilization of the bacteria, bacteriophage, and/or the bacteria and bacteriophage complex to the solid support can be achieved through direct or indirect bonding to the solid support. In some embodiments, the bonding can be by covalent linkage. See, Joos et al. (1997) Analytical Biochemistry, 247:96-101; Oroskar et al. (1996) Clin. Chem., 42:1547-1555; and Khandjian (1986) Mol. Bio. Rep., 11:107-11. In some embodiments, the solid support comprises functional groups capable of immobilizing the bacteria, bacteriophage, or the bacteria and bacteriophage complex.
[0127] In some embodiments, the solid supports may comprise functional groups capable of covalently linking the bacteria, bacteriophage, or the bacteria and bacteriophage complex directly or indirectly through chemical linkers. Examples of functional groups include but are not limited to poly L-lysine, aminosilane, epoxysilane, aldehydes, carboxylic groups, azide, alkalyne, maleimide, amino groups, epoxy groups, cyano groups, ethylenic groups, hydroxyl groups, thiol groups.
[0128] In some embodiments, the immobilization is achieved through amine functional groups of a polymer, e.g., N-terminus and -amino groups of lysine residues, direct amine bonding of a terminal nucleotide of the template or a primer. In some embodiments, the immobilization is achieved through carboxylic acid/ carboxylate functional groups of a polymer, e.g., C-terminus of a protein or a peptide. In some embodiments, the polymer is an aptamer.
[0129] In some embodiments, the solid support comprises epoxide functional groups, N-Hydroxysuccinimide (NETS) group. The epoxide functional groups or the NETS group can be used to immobilize the bacteria, bacteriophage, or the bacteria and bacteriophage complex to the solid support.
[0130] In some embodiments, the immobilization of the bacteria, bacteriophage, and/or the bacteria and bacteriophage complex to the solid support can be achieved through non-covalent means. In some embodiments, non-covalently immobilizing the bacteria, bacteriophage, and/or the bacteria and bacteriophage complex to the solid support is via a binding pair, which refers herein to two molecules which form a complex through a specific interaction. Thus, the bacteria, bacteriophage, and/or the bacteria and bacteriophage complex can be immobilized on the solid support through an interaction between one member of the binding pair linked to the bacteria, bacteriophage, and/or the bacteria and bacteriophage complex and the other member of the binding pair coupled to the solid support. In a preferred embodiment, the binding pair is biotin and avidin, or variants of avidin such as streptavidin or NeutrAvidin In some embodiments, the solid support may comprise streptavidin or its variants and bacteria, bacteriophage, and/or the bacteria and bacteriophage complex may comprise biotin. Methods for biotinylating are known in the art (e.g. through primary amine by NHS-PEO12-Biotin, NHS-LC-LC-Biotin, NHS-SS-PEO4-Biotin from Pierce Chemical Co.; through sulfhydryl group by Maleimide-PEO11-Biotin, Biotin-BMCC Sulfhydryl, Iodacetyl-PEO2-Biotin).
[0131] In other embodiments, the binding pair consists of a ligand-receptor, a hormone-receptor, an antigen-antibody. Examples of such binding pair include but are not limited to digoxigenin and anti-digoxigenin antibody; 6-(2,4-dinitrophenyl) aminohexanoic acid and anti-dinitrophenyl antibody; 5-Bromo-dUTP (BrdUTP) and anti-BrdUTP antibody; N-acetyl 2-aminofluorene (AAF) and anti-AAF antibody.
[0132] The term compound in the context of detecting one or more types of cell, bacteria or bacteriophage refers to a molecule or a group of molecules that interacts a nucleic acid or a protein of a bacteriophage or bacteria, or bacterial cell wall or cell membrane. In some embodiments, the compound is permeable to bacterial cell wall and bacterial cell membrane. In some embodiments, the compound binds to dsDNA, ssDNA, dsRNA, or ssRNA. In some embodiments, the compound preferentially binds to dsDNA. In some embodiments, the compound is photoreactive. Non-limiting examples of compound binding to a nucleic acid include propidium monoazide, ethidium monoazide, based dye, propidium iodide, ethidium bromide, phenanthridine dye, acridine dye, indoles, or imidazole dye.
[0133] In some embodiments, compound binds covalently to a nucleic acid or a protein. In some embodiments, the compound intercalates between the bases of a nucleic acid. In some embodiments, the compound is modified upon contacting a bacteriophage or bacterial nucleic acid or protein. Non-limiting examples of methods of modification include forming a covalent bond, changing its composition, and changing its electronic structure. In some embodiments, the compound binds to a function group of a protein such as amine, carboxyl, phosphate, aldehyde, thiol, hydroxyl, and carbonyl. In some embodiments, the compound binding to a protein is an antibody. In some embodiments, the compound binding to a protein may be carbodiimide, NHS ester, imidoester, pentafluorophenyl ester, maleimide, haloacetyl (Bromo- or Iodo-), hydrazide, alkoxyamine, diazirine, aryl azide, isocyanate, epoxide, cyanide, and alkyne.
[0134] In some embodiments, the compound has a low dissociation constant. In some embodiments, the compound upon binding to a protein or a nucleic acid prevents other type of compounds binding to the same protein or nucleic acid. In some embodiments, a compound preferentially binds to a protein or nucleic acid in the presence of another protein or nucleic acid which is bound to a different compound. In some embodiments, this preferential binding of a compound to one set of nucleic acid or protein in the presence of another set of nucleic acid or protein that is bound to a different compound is at least in part because the different compound prevents binding of the compound to the another set of nucleic acid or protein.
[0135] In some embodiments, the compound comprises a detectable label.
[0136] The terms detectable label and tag have been used interchangeably throughout the application and refers to a molecule or a compound or a group of molecules or a group of compounds and being capable of being detected. In some embodiments, detectable label can associate with a molecule and assist in detecting the molecule with which it is associated with.
[0137] In some embodiments, detectable label can generate a signal which can be detected. In some embodiments, the detectable label is converted to a different molecule or compound that can generate a signal which can be detected. In some embodiments, the detectable label is part of a binding pair. In some embodiments, the detectable label is associated with a solid support. In some embodiments, the detectable label is associated with a bead. In some embodiments, the detectable is further modified prior to detection. In some embodiments, such modification may include covalent modification. In some embodiments, the detectable label is associated with the bacteriophage, and/or the bacteria prior to the formation of the complex of the bacteriophage and the bacteria. In some embodiments, the detectable label is associated with the bacteriophage, bacteria, and/or the complex of bacteriophage and the bacteria after the formation of the complex.
[0138] In some embodiments, the detectable label is associated with a protein or a nucleic acid (e.g., genomic nucleic acid, fragments of genomic nucleic acid, a probe or primer) of bacteria, bacteriophage, or a complex of bacteria and bacteriophage and is used to detect the bacteria, bacteriophage, or a complex of bacteria and bacteriophage. In some embodiments, the detectable label is associated with a protein or a nucleic acid inside the bacteria, bacteriophage, or a complex of bacteria and bacteriophage.
[0139] In some embodiments, the bacteriophage, bacteria, and/or the complex of bacteriophage and the bacteria may comprise more than one type of detectable label. In some embodiments, the detectable labels of the bacteriophage, the bacteria, and their complexes are different from each other.
[0140] In some embodiments, the detectable label may be detected directly. In other embodiments, the detectable label may be a part of a binding pair, which can then be subsequently detected. Signals from the detectable label may be detected by various means and will depend on the nature of the detectable label. Detectable labels may be isotopes, fluorescent moieties, colored substances, and the like. Examples of means to detect detectable label include but are not limited to spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluoresence, or chemiluminescence, or any other appropriate means.
[0141] Detectable labels include but are not limited to fluorophores, isotopes (e.g., 32P, 33P, 35S, 3H, 14C, 125I, 131I), electron-dense reagents (e.g., gold, silver), nanoparticles, enzymes commonly used in an ELISA (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminiscent compound, photoluminescent compounds (e.g., quantum dots, lanthanide particles and lanthanide chelates), colorimetric labels (e.g., colloidal gold), magnetic labels (e.g. Dynabeads), biotin, digoxigenin, haptens, proteins for which antisera or monoclonal antibodies are available, ligands, hormones, oligonucleotides capable of forming a complex with the corresponding oligonucleotide complement.
[0142] In some embodiments, the detectable label comprises a fluorescent moiety. Non-limiting examples of fluorescent moiety include TOTO, YOYO, BOBO POPO SYBR, SYTOX, PicoGreen, OliGreen, RiboGreen, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4,5-dichloro-2,7-dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosin, eosin, rhodamine dyes (e.g., carboxytetramethyl-rhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine Green, rhodamine Red, tetramethylrhodamine (TMR), etc.), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin (AMCA), etc.), Oregon Green Dyes (e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514., etc.), Texas Red, Texas Red-X, SPECTRUM RED, SPECTRUM GREEN, cyanine dyes (e.g., CY-3, CY-5, CY-3.5, CY-5.5, etc.), Alexa Fluor dyes (e.g., Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 632, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680, etc.), BODIPYdyes (e.g., BODIPYTM FL, BODIPYTM R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY630/650, BODIPY650/665, etc.), IRDyes (e.g., IRD40, IRD 700, IRD 800, etc.), PMA (propidium monoazide), propidium iodide, ethidium monoazide (EMA) and the like. For more examples of suitable fluorescent dyes and methods for coupling fluorescent dyes to other chemical entities such as proteins and peptides, see, for example, The Handbook of Fluorescent Probes and Research Products, 9th Ed., Molecular Probes, Inc., Eugene, Oreg. Favorable properties of fluorescent labeling agents include high molar absorption coefficient, high fluorescence quantum yield, and photostability. In some embodiments, labeling fluorophores exhibit absorption and emission wavelengths in the visible (i.e., between 400 and 750 nm) rather than in the ultraviolet range of the spectrum (i.e., lower than 400 nm).
[0143] A detectable label may include more than one chemical entity such as in fluorescent resonance energy transfer (FRET). Resonance transfer results an overall enhancement of the emission intensity. For instance, see Ju et. al. (1995) Proc. Nat'l Acad. Sci. (USA) 92: 4347, the entire contents of which are herein incorporated by reference. To achieve resonance energy transfer, the first fluorescent molecule (the donor fluor) absorbs light and transfers it through the resonance of excited electrons to the second fluorescent molecule (the acceptor fluor). In one approach, both the donor and acceptor dyes can be linked together and attached to the oligo primer. Methods to link donor and acceptor dyes to a nucleic acid have been described previously, for example, in U.S. Pat. No. 5,945,526 to Lee et al., the entire contents of which are herein incorporated by reference. Donor/acceptor pairs of dyes that can be used include, for example, fluorescein/tetramethylrohdamine, IAEDANS/fluroescein, EDANS/DABCYL, fluorescein/fluorescein, BODIPY FL/BODIPY FL, and Fluorescein/ QSY 7 dye. See, e.g., U.S. Pat. No. 5,945,526 to Lee et al. Many of these dyes also are commercially available, for instance, from Molecular Probes Inc. (Eugene, Oreg.). Suitable donor fluorophores include 6-carboxyfluorescein (FAM), tetrachloro-6-carboxyfluorescein (TET), 2-chloro-7-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC), and the like.
[0144] A suitable detectable label can be an intercalating DNA/RNA dye that have dramatic fluorescent enhancement upon binding to double-stranded DNA/RNA. Examples of suitable dyes include, but are not limited to, SYBRTM and Pico Green (from Molecular Probes, Inc. of Eugene, Oreg.), ethidium bromide, propidium iodide, chromomycin, acridine orange, Hoechst 33258, TOTO-1, YOYO-1, and DAPI (4,6-diamidino-2-phenylindole hydrochloride). Additional discussion regarding the use of intercalation dyes is provided by Zhu et al., Anal. Chem. 66:1941-1948 (1994), which is incorporated by reference in its entirety.
[0145] As used herein, the term cell includes both prokaryotic cell and eukaryotic cell. Non-limiting examples of eukaryotic cell includes protozoa cell, yeast cell, plant cell, mammalian cell, human cell. Human cell can be from any human organ, blood, or tissue.
[0146] As used herein the term virus or viruses in the context of cell refers to viruses capable of specifically binding to a specific cell. Examples of such viruses are described in Lodish H, Berk A, Zipursky S L, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000. Section 6.3, Viruses: Structure, Function, and Uses., which is incorporated by reference in its entirety.
[0147] As used herein, the term bacteria refers to gram positive, gram negative and neither gram positive or gram-negative bacteria. In some embodiments, the bacteria are pathogenic to plants. In some embodiments, the bacteria are pathogenic to crop. Bacterial pathogens for plants are well known in the art. See https://en.wikipedia.org/wiki/Category:Bacterial_plant_pathogens_and_diseases. Non-limiting examples of plant bacterial pathogens include: Candidatus Liberibacter, Candidatus Phytoplasma solani, Clavibacter michiganensis, Pectobacterium atrosepticum, Pectobacterium betavasculorum, Pectobacterium carotovorum, Pectobacterium carotovorum subsp. betavasculorum, Pectobacterium wasabiae, Pseudomonas amygdali, Pseudomonas asplenii, Pseudomonas caricapapayae, Pseudomonas cichorii, Pseudomonas coronafaciens, Pseudomonas corrugata, Pseudomonas ficuserectae, Pseudomonas flavescens, Pseudomonas fuscovaginae, Pseudomonas helianthi, Pseudomonas marginalis, Pseudomonas oryzihabitans, Pseudomonas palleroniana, Pseudomonas salomonii, Pseudomonas savastanoi, Pseudomonas syringae, Pseudomonas tomato, Pseudomonas turbinellae, Pseudomonas viridiflava, Ralstonia solanacearum, Rhodococcus fascians, Xanthomonas campestris, Xanthomonas campestris pv. campestris, Xanthomonas oryzae, and Xylella fastidiosa.
[0148] In some embodiments, the bacteria are pathogenic to eukaryotic organisms of the kingdom metazoan. In some embodiments, the bacteria are pathogenic to vertebrates. In some embodiments, the bacteria are pathogenic to mammals. In some embodiments, the bacteria are pathogenic to humans. In some embodiments, the bacteria are pathogenic to farm animals. Non-limiting examples of farm animals include cows, pigs, chicken, sheep, goat, ducks, horse. In some embodiments, the bacteria are pathogenic to domestic animals. In some embodiments, the bacteria are pathogenic to marine animals, insects (bees, butterflies, and mosquitoes).
[0149] Non limiting examples of bacteria pathogenic to humans include Citrobacter freundii, Klebsiella pneumoniae, Acinetobacter, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella enteritidi, Escherichia coli, Staphylococcus aureus, Enterococci, Bacillus cereus, Escherichia coli, Listeria monocytogenes, Shigella spp., Staphylococcus aureus, Staphylococcal enteritis, Streptococcus, Vibrio cholerae, (including O1 and non-O1), Vibrio parahaemolyticus, Vibrio vulnificus, Yersinia enterocolitica and Yersinia pseudotuberculosis, Campylobacter jejuni, Clostridium perfringens, Salmonella spp., Salmonella typhimurium, Mycobacterium tuberculosis, Listeria monocytogenes, Shigella spp., Clostridium botulinum, Vibrio vulnificus, Clostridium perfringens, Bacillus cereus, Bacillus anthracis, Campylobacter coli, Yersinia pestis, Bacillus subtilis, Pseudomonas syringae, Dickeya solani, Clavibacter michiganensis, Erwinia carotovora, Agrobacterium tumefaciens, Erwinia amylovora, Enterobacter aerogenes, Enterobacter cloacae, Acinetobacter baumannii, and Clostridium difficile.
[0150] In some embodiments, the pathogenic bacteria are food borne. Non-limiting examples of food borne pathogenic bacteria include Bacillus cereus, Escherichia coli, Listeria monocytogenes, Shigella spp., Staphylococcus aureus, Staphylococcal enteritis, Streptococcus, Vibrio cholerae, (including O1 and non-O1), Vibrio parahaemolyticus, Vibrio vulnificus, Yersinia enterocolitica and Yersinia pseudotuberculosis, Campylobacter jejuni, Clostridium perfringens, Salmonella spp., Salmonella typhimurium.
[0151] Non-limiting examples of pathogenic bacteria and their corresponding bacteriophages are shown in Table 1 below.
TABLE-US-00001 TABLE 1 Pathogenic bacteria, corresponding bacteriophages and the associated diseases Bacteria Bacteriophage Disease Mycobacterium Mycobacteriophage D29, TM4, phAE159, Che12 Tuberculosis Tuberculosis (and their derivatives) Bacillus -phage, W Anthrax anthracis Campylobacter NCTC 12673 Gastroenteritis, acute jejuni enterocolitis Campylobacter GP047 Gastroenteritis, acute coli enterocolitis E. coli CBA120, AR1 and bacteriophage 56 Gastroenteritis, urinary tract infections, and neonatal meningitis E. coli O157 LG1 Yersinia pestis A1122, A1122, H, P, Y, Tal, 513, L-413C Plague Pseudomonas PA5oct, NCIMB 101116, KT28, vB_PaeM_MAG1 Pneumonia, Septic aeruginosa (MAG1) and vB_PaeP_MAG4 (MAG4) shock, Urinary tract infection, Gastrointestinal infection, skin and soft tissue infections Bacillus SPO2, 105, 22 subtilis Pseudomonas phi 6, Psa17 Bacterial speck in syringae tomato Dickeya solani vB_DsoM_LIMEstone1 and vB_DsoM_LIMEstone2 Wilts and stem rots (Dickeya spp.) Clavibacter CMP1, CN77 Systemic vascular michiganensis infection Erwinia T4, ZF40, phage 59 Plant cell wall carotovora destruction Agrobacterium PB2, PB21, PS8, R4 Crown gall disease tumefaciens Erwinia phiEa2809, vB_EamM_Ea35-70 (Ea35-70), Y2 Fireblight amylovora Enterobacter F20 Sepsis aerogenes Citrobacter phi I, phi II, and phi III Nosocomial infections freundii of the respiratory tract, urinary tract, blood Enterobacter Ent, 1 Urinary tract and cloacae respiratory tract infections Shigella spp. Sh, SboM-AG3, EP23, SP18, Stix Shigellosis Klebsiella 0507-KN2-1, KP34, JD001, KI3 Nosocomial pneumoniae infections. Acinetobacter vB_AbaM-IME-AB2, ZZ1, AB1, AP22, and phiAC-1 Nosocomial baumannii infections. Staphylococcus MR-5, Ph10, Ph12, U14, H96, JS01, 88, skin infections, such aureus YMC/09/04/R1988 MRSA BP as pimples, impetigo, boils, cellulitis, folliculitis, carbuncles, scalded skin syndrome, and abscesses, pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome, bacteremia, and sepsis Clostridium phiC2, phiC5, phiC6, phiC8, PhiCD119, phiCD6356, Diarrhea, Colitis, difficile phiCD38-2 Sepsis Listeria A511, A513, A507, A502, A505, A519, A528, A020, Listeriosis B024, B012, B035, C707, A118, P100, PSA, A006, A118, A500, B025, P35, P40, 20422-1, 805405-1 Salmonella Felix 01, SPN2T, SPN3C, SPN8T, SPN9T, SPN11T, Diarrhea, fever, and SPN13B, SPN16C, SPN4S, SPN5T, SPN6T, SPN7C, abdominal cramps SPN9C, SPN14, SPN18, SPN1S, SPN2TCW, UAB_Phi20, UAB_Phi78, and UAB_Phi87 Xanthomonas Cp1, Cp2, Cp1-sensitive, XacF1, Phil7 Citrus canker axonopodis Brucella Berkeley, Tbilisi, Firenze (Fz), Weybridge (Wb), Brucellosis S708, R/C, 1066, 281, 02, 177, 110, V, 11sa, 544, 141 Helicobacter KHP30, KHP40, HP1 Infection of the pylori stomach, peptic ulcer and stomach cancer Leptospira LE1 Leptospirosis Chlamydia Chp1, Chp2, Chp3, CPG1 CPAR39 (Cpn1), Psittacosis or Chp4, AR39 respiratory tract diseases, enteritis and chronic bowel diseases, ornithosis, pneumonia Legionella Mu Legionnaires disease pneumophila
[0152] In some embodiments, the bacteria are non-pathogenic to mammals. In some embodiments, the bacteria are non-pathogenic to humans. In some embodiments, the bacteria are mesophilic. In some embodiments, the bacteria are thermophilic.
[0153] In some embodiments, the bacteria are used in the fermentation industry. In some embodiments, the bacteria are used in the dairy industry. In some embodiments, the bacteria are used for the fermentation of milk. In some embodiments, the bacteria are used for the fermentation of milk to produce cheese. Non-limiting examples of bacteria used in the dairy industry include: Lactococcus lactis ssp. cremoris; Lactococcus lactis ssp. lactis biovar diacetylactis; Lactococcus lactis ssp. lactis; Leuconostoc mesenteroides ssp. cremoris; Lactobacillus acidophilus; Lactobacillus delbrueckii ssp. bulgaricus; Lactobacillus delbrueckii ssp. lactis; Lactobacillus helveticus; Streptococcus thermophilus; Bifidobacterium bifidus; Brevibacterium linens coryneform bacteria; and Propionibacterium freudenreichii ssp. shermanii.
[0154] In some embodiments, the bacteria are used is the wine industry for the malolactic acid fermentation to convert malic acid to lactic acid. Non-limiting examples of bacteria used in the wine industry include lactic acid bacteria of the genera: Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, and Streptococcus, Bifidobacterium, Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Sporolactobacillus, Tetragenococcus, Vagococcus, and Weissella; and Oenococcus oeni.
[0155] Non-limiting examples of bacteria used for fermentation in dairy and wine industry and their corresponding bacteriophages are shown in Table 2 below.
TABLE-US-00002 TABLE 2 bacteria used for fermentation in dairy and wine industry and their corresponding bacteriophages Bacteria Phage Lactobacillus casei A2 Lactobacillus paracasei CL1, CL2 Lactobacillus paracasei (ssp. paracasei) PL-1 Lactobacillus plantarum B1, B2, PhiJL-1, fri, g1e, LP65, JL1 Lactococcus lactis P001, 949, c6A, P087, P107, BK5-T, c2, p2, sk1, ul36, bIL170, bIL67, Q54, 1706, P335, Tuc2009, r1t Lactococcus lactis (ssp. cremoris) 1358, 936, KSY1 Lactococcus lactis (ssp. diacetylactis) P008, P270, P369 Lactococcus lactis (ssp. lactis) 1483 Lactobacillus casei A2, AT3, A2, Lactobacillus paracasei CL1, CL2 Lactobacillus paracasei (ssp. paracasei) PL-1 Oenococcus oeni Lco22 Streptococcus group C a/C7 Streptococcus mitis Streptococcus mutans M102, M102AD Streptococcus pneumoniae Cp-1 (SOCP), Dp-1 Streptococcus thermophilus Ba 24, DT1, Q1, 2972, 858, ALQ13.2, Abc2, Thermus thermophilus YS40 Leuconostoc mesenteroides pro2 Leuconostoc mesenteroides (ssp. cremoris) 400 Lactobacillus delbrueckii LdlS, Ld3, Ld17, Ld25A, lb539, LL-Ku, c5, JCL1032, LL-H, mv4, Lactobacillus helveticus AQ113 Lactobacillus gasseri KC5a Lactobacillus johnsonii Lj771 Lactobacillus gasseri adh, KC5a Lactobacillus rhamnosus Lc-Nu Lactobacillus fermentum EcoSau, EcoInf
[0156] In some embodiments, the bacteria are used in meat fermentation, vegetable fermentation, food fermentation. Exemplary list of bacteria used in meat fermentation includes, but is not limited to Lactobacillus curvatus, Lactobacillus sake, Pediococcus acidilactici, Pediococcus pentosaceus, Lactobacillus plantarum, Micrococcus varians, Staphylococcus carnosus, and Staphylococcus xylosus.
[0157] In some embodiments, the bacteria are lactic acid bacteria (LAB) used in the conversion of lactose to lactic acid. In some embodiments, the LAB belongs to the genera Aerococcus, Alloiococcus, Atopobium, Bifidobacterium, Carnobacterium, Enterococcus, Lactobacillus (Lb.), Lactococcus (L.), Leuconostoc (Leuc.), Oenococcus, Pediococcus, Streptococcus (S.), Tetragenococcus, Vagococcus and Weissella (W.)
[0158] As used herein the term susceptible to the bacteriophage refers to bacteria that have receptors for the bacteriophage that are present in a sample. As a result, the bacteriophages present in a sample can specifically bind to such receptors and infect the bacteria.
[0159] As used herein the term bactericide or bactericide sometimes abbreviated Bcidal, is a substance that kills bacteria. Bactericides are disinfectants, antiseptics, lysozyme, or antibiotics.
[0160] Non-limiting examples of disinfectants include active chlorine (i.e., hypochlorites, chloramines, dichloroisocyanurate and trichloroisocyanurate, wet chlorine, chlorine dioxide, etc.), active oxygen (peroxides, such as peracetic acid, potassium persulfate, sodium perborate, sodium percarbonate, and urea perhydrate), iodine (povidone-iodine, Lugol's solution, iodine tincture, iodinated nonionic surfactants), concentrated alcohols (mainly ethanol, 1-propanol, called also n-propanol and 2-propanol, called isopropanol and mixtures thereof; further, 2-phenoxyethanol and 1- and 2-phenoxypropanols are used), phenolic substances (such as phenol (also called carbolic acid), cresols such as thymol, halogenated (chlorinated, brominated) phenols, such as hexachlorophene, triclosan, trichlorophenol, tribromophenol, pentachlorophenol, salts and isomers thereof), cationic surfactants, such as some quaternary ammonium cations (such as benzalkonium chloride, cetyl trimethylammonium bromide or chloride, didecyldimethylammonium chloride, cetylpyridinium chloride, benzethonium chloride) and others, non-quaternary compounds, such as chlorhexidine, glucoprotamine, octenidine dihydrochloride etc.), strong oxidizers, such as ozone and permanganate solutions; heavy metals and their salts, such as colloidal silver, silver nitrate, mercury chloride, phenylmercury salts, copper sulfate, copper oxide-chloride etc.; strong acids (phosphoric, nitric, sulfuric, amidosulfuric, toluenesulfonic acids), pH<1; and alkalis (sodium, potassium, calcium hydroxides), such as of pH>13, particularly under elevated temperature (above 60 C.), kills bacteria.
[0161] Non-limiting examples of antiseptics include properly diluted chlorine preparations (e.g., Dakin's solution, 0.5% sodium or potassium hypochlorite solution, pH-adjusted to pH 7-8, or 0.5-1% solution of sodium benzenesulfochloramide (chloramine B)); some iodine preparations, such as iodopovidone in various galenics (ointment, solutions, wound plasters), in the past also Lugol's solution; peroxides such as urea perhydrate solutions and pH-buffered 0.1-0.25% peracetic acid solutions; alcohols with or without antiseptic additives, used mainly for skin antisepsis; weak organic acids such as sorbic acid, benzoic acid, lactic acid and salicylic acid; some phenolic compounds, such as hexachlorophene, triclosan and Dibromol; and cationic surfactants, such as 0.05-0.5% benzalkonium, 0.5-4% chlorhexidine, 0.1-2% octenidine solutions.
[0162] Non-limiting examples of antibiotics include beta-lactam antibiotics (penicillin derivatives (penams), cephalosporins (cephems), monobactams, and carbapenems) and vancomycin, daptomycin, fluoroquinolones, metronidazole, nitrofurantoin, co-trimoxazole, telithromycin, aminoglycosidic antibiotics.
[0163] As used herein the term bacteriostatic agent refers to a biological or chemical agent that stops bacteria from reproducing, while not necessarily killing them otherwise. Depending on their application, bacteriostatic antibiotics, disinfectants, antiseptics and preservatives can be distinguished. When bacteriostatic antimicrobials are used, the duration of therapy must be sufficient to allow host defense mechanisms to eradicate the bacteria. Upon removal of the bacteriostatic agent, the bacteria usually start to grow again. This is in contrast to bactericides, which kill bacteria.
[0164] Non-limiting examples of bacteriostatic antibiotic include Chloramphenicol, Clindamycin, Ethambutol, Lincosamides, Macrolides, Nitrofurantoin, Novobiocin, Oxazolidinone, Spectinomycin, Sulfonamides, Tetracyclines, Tigecycline, Trimethoprim.
[0165] As used herein the term cell-free bacterial receptors means bacterial receptors for bacteriophages that are substantially free of intact bacterial cells. Substantially free of intact bacterial cells means bacterial receptors that are at least 90%, 95%, 96%, 97%, 98%, 99% or more free of intact bacterial cells. In some embodiments, the bacterial receptors are part of bacterial cell wall fragments or bacterial cell membrane fragments. In some embodiments, the bacterial receptors are isolated from intact bacteria. In some embodiments, the bacterial receptors are purified. In some embodiments, the bacterial receptors are recombinant proteins.
[0166] Several amino acid sequences of bacteriophage receptor of bacteria are available in the NCBI protein database. Non-limiting examples of bacteriophage receptor of bacteria include NCBI protein database accession numbers: BAA35202.1, BAA35203, CDH64094.1, CCJ43022.1, EMD14689.1, EKU06323.1, EKU00687.1, AM074452.1, APC75070.1, BAT57805.1, CBJ83363.1, EMV90632.1, ADR59974.1 (https://www.ncbi.nlm.nih.gov/protein).
[0167] As used herein, the term bacteriophage refers to a virus that infects and can replicate within a bacterium. Bacteriophages may have a lytic cycle, a lysogenic cycle, or both. Bacteriophages attach to specific receptors on the surface of bacteria, including lipopolysaccharides, polyglycans, teichoic acids, proteins, or even flagella to form a bacteria and bacteriophage complex. This specificity means a bacteriophage can infect only certain bacteria bearing receptors to which they can bind, which in turn determines the phage's host range. Host growth conditions also influence the ability of the phage to attach and invade them.
[0168] In some embodiments, the receptor binding element of a bacteriophage binds specifically to the receptors of bacteria. In some embodiments, the receptor binding elements are receptor binding proteins of a bacteriophage.
[0169] Several bacteriophage nucleic acid sequences of bacterial receptor binding protein are available in the protein database of National Center for Biotechnology Information (NCBI). Non-limiting examples include Enterobacteria phage T4 tail fiber protein gene, tail fiber protein 36 and tail fiber protein 37 genes (GenBank Accession No: J02509), Host interaction protein of bacteriophage J1 (GenBank Accession No. KC171646.1). Each of which is incorporated by reference in the entirety.
[0170] Several bacteriophage amino acid sequences of bacterial receptor binding protein are available in the protein database of National Center for Biotechnology Information (NCBI). Non-limiting examples include Chain A, Structure Of The Receptor-binding Protein Of Bacteriophage Det7: A Podoviral Tailspike In A Myovirus (Accession No. 2V5I_A), Chain C, Structure Of Lactococcal Bacteriophage P2 Receptor Binding Protein In Complex With A Llama Vhh Domain (Accession No. BSE_C), Chain B, Structure Of Lactococcal Bacteriophage P2 Receptor Binding Protein In Complex With A Llama Vhh Domain (Accession No. 2BSE_B), Chain C, Structure Of Lactococcal Bacteriophage P2 Receptor Binding Protein (Accession No. 2BSD_C), Chain B, Structure Of Lactococcal Bacteriophage P2 Receptor Binding Protein (Accession No. 2BSD_B), Chain A, Structure Of Lactococcal Bacteriophage P2 Receptor Binding Protein (Accession No. 2BSD_A), Chain A, Structure Of The Receptor-Binding Domain Of The Bacteriophage T4 Short Tail Fibre (1OCY_A), Chain C, Structure Of The Bacteriophage T4 Long Tail Fibre Needle-Shaped Receptor-binding Tip (2XGF_C), Bacteriophage T7 tail fiber protein 37 (GenBank Accession No. AAA32514.1), Bacteriophage J1 Host interaction protein (Gen Bank Accession No. AGZ17304.1). Each of which is incorporated by reference in the entirety.
[0171] Genomes of the bacteriophages can be RNA or DNA. In some embodiments, the genome of the bacteriophage can be linear dsDNA, circular dsDNA, circular ssDNA, segmented dsRNA, linear ssRNA. In some embodiments, the phage nucleic acid is nicked. The classification by the International Committee on Taxonomy of Viruses (ICTV) according to morphology and nucleic acid is shown in Table 3.
TABLE-US-00003 TABLE 3 ICTV classification of prokaryotic (bacterial and archaeal) viruses Nucleic Order Family Morphology acid Examples Caudovirales Myoviridae Nonenveloped, Linear T4 phage, Mu, PBSX, contractile tail dsDNA P1Puna-like, P2, I3, Bcep 1, Bcep 43, Bcep 78 Caudovirales Siphoviridae Nonenveloped, Linear phage, T5 phage, phi, noncontractile tail dsDNA C2, L5, HK97, N15 (long) Caudovirales Podoviridae Nonenveloped, Linear T7 phage, T3 noncontractile tail dsDNA phage, 29, P22, P37 (short) Ligamenvirales Lipothrixviridae Enveloped, rod- Linear Acidianus filamentous shaped dsDNA virus 1 Ligamenvirales Rudiviridae Nonenveloped, rod- Linear Sulfolobus islandicus shaped dsDNA rod-shaped virus 1 Unassigned Ampullaviridae Enveloped, bottle- Linear shaped dsDNA Unassigned Bicaudaviridae Nonenveloped, Circular lemon-shaped dsDNA Unassigned Clavaviridae Nonenveloped, rod- Circular shaped dsDNA Unassigned Corticoviridae Nonenveloped, Circular isometric dsDNA Unassigned Cystoviridae Enveloped, Segmented spherical dsRNA Unassigned Fuselloviridae Nonenveloped, Circular lemon-shaped dsDNA Unassigned Globuloviridae Enveloped, Linear isometric dsDNA Unassigned Guttaviridae Nonenveloped, Circular ovoid dsDNA Unassigned Inoviridae Nonenveloped, Circular M13 filamentous ssDNA Unassigned Leviviridae Nonenveloped, Linear MS2, Q isometric ssRNA Unassigned Microviridae Nonenveloped, Circular X174 isometric ssDNA Unassigned Plasmaviridae Enveloped, Circular pleomorphic dsDNA Unassigned Tectiviridae Nonenveloped, Linear isometric dsDNA
[0172] In some embodiments, the bacteriophages are lytic phages. In some embodiments, the bacteriophages belong to Siphoviridae and Podoviridae families. In some embodiments, the bacteriophages are responsible for milk fermentation failures. In some embodiments, the bacteriophages are responsible for infecting the bacteria useful for bacterial fermentation of milk. In some embodiments, the bacteriophages are responsible for infecting the bacteria useful for malolactic fermentation in wine industry.
[0173] As used herein the phrase complex of bacteriophage and bacteria or the complex refers to a state in the bacteriophage infection in which the bacteriophages attach to specific receptors on the surface of bacteria, including lipopolysaccharides, glycoprotein, polyglycans, teichoic acids, proteins, or even flagella to form a bacteria and bacteriophage complex. The complex includes stages prior to, during, or after introduction of bacteriophage genetic material into the bacteria while the bacteriophage remains adhered to the bacterial surface.
[0174] In some embodiments, the complex is separated from the incubation mixture comprising bacteriophage and bacteria prior to detection of the complex. In some embodiments, the separation is by centrifugation, filtration, application of electric field, size exclusion chromatography or a combination thereof
[0175] In some embodiments, the complex is detectably labeled after the formation of the complex. In some embodiments, the bacteriophage and/or the bacteria are detectably labeled resulting in a detectably labeled complex. In some embodiments, the detectable labels for the bacteriophage and the bacteria are different. In some embodiments, the detectable labels for the bacteriophage, the bacteria, and/or the complex are different. In some embodiments, the complex can comprise more than one type of detectable label.
[0176] Sample can be from a human or a non-human origin. A sample may include a specimen of natural or synthetic origin. In some embodiments, sample can be a biological sample such as tissues, tissue homogenate, feces, bodily fluids, inoculums, and cheeck swab. Bodily fluids may include, but are not limited to, blood, serum, plasma, saliva, cerebral spinal fluid, tears, lactal duct fluid, lymph, sputum, mucus, pleural fluid, urine, amniotic fluid, and semen. A sample may include a bodily fluid that is acellular. An acellular bodily fluid includes less than about 1% (w/w) whole cellular material. Plasma or serum are examples of acellular bodily fluids.
[0177] In some embodiments, the sample can be fermentation samples. Non-limiting examples of fermentation samples include bacterial fermentation samples, dairy fermentation samples, yeast fermentation samples, starter culture, milk starter culture, whey starter culture. In some embodiments, the sample can be water, air, aerosol, liquid collected from facility equipment, swabbed facility surfaces, fluid samples from facilities, wash water from a facility, wash water of produce, swabbed ground samples, swabbed food sample, fermented liquid, bacterial culture, bacterial broth, sewer, soil.
[0178] In some embodiments, the sample can be food. Food can be processed, unprocessed, cooked, or raw. Non-limiting examples of food sample include raw milk, pasteurized milk, skim milk, whey, cheese, fruits, vegetables, meat, shrimp, crab, clams, fish, grains, nuts. In some embodiments, the sample can be drinks and beverages. Non-limiting examples include coconut milk, soy milk, tea, coffee, lemonade, fruit juice, vegetable juice, and soda.
[0179] In some embodiments, sample can be from a patient suspected of having a bacterial infection. Patients can be human and non-human animals. In some embodiments, sample can be from non-human animals. In some embodiments, sample can be from farm animals, marine animals, domestic animals, wild animals, insects.
[0180] In some embodiments, sample can be from plants. Non-limiting examples of plant sample include leaves, roots, stem, nuts, grains, plant extract, root extract, petals, fruit, bark, seed.
[0181] As used herein the term device refers to a widget that aids in detecting or can detect bacteria, bacteriophages or bacteriophage and bacteria complex in a sample.
[0182] In some embodiments, the device comprises a planar surface. In some embodiments, the planar surface comprises a bilayer of two different compositions. In some embodiments, one layer may be conductive of electricity. In some embodiments, one layer may be a metal. Exemplary metals include, but are not limited to silver, gold, copper, platinum, titanium, chromium, Al, Ni, Cr, Fe, ceramic metals such as Indium-tin-oxide etc. or combinations thereof In some embodiments, one layer comprises magnetic materials, e.g., Fe, Ni, Non-oxide, non-elemental magnetic material Nd.sub.2Fe.sub.14B, SmCo.sub.5 etc., non-elemental, oxide magnetic material Fe.sub.3O.sub.4, Fe.sub.2O.sub.3. In some embodiments, the second layer can be non-conductive or non-magnetic. In some embodiments, the second layer may be ceramic, polymer, metal, silicon, quartz (crystalline silica), glass, zinc oxide, alumina, Paraffin film, polycarbonate (PC), or combinations thereof In some embodiments, the planar surface is in fluid communication with the sample comprising bacteriophage, bacteria, and/or a complex of bacteriophage and bacteria.
[0183] In some embodiments, the device comprises solid support. In some embodiments, the solid supports may comprise functional groups capable of covalently linking the bacteriophage, bacteria, or the complex of bacteriophage and bacteria directly or indirectly through chemical linkers. Examples of functional groups include but are not limited to poly L-lysine, aminosilane, epoxysilane, aldehydes, amino groups, epoxy groups, cyano groups, ethylenic groups, hydroxyl groups, thiol groups, epoxide N-Hydroxysuccinimide (NHS) group. In some embodiments, the solid support may comprise one member of a binding pair and can immobilize a bacteriophage, bacteria, or the complex of bacteriophage and bacteria comprising the other member of the binding pair.
[0184] In some embodiments, the device may comprise a sensor. In some embodiments, the device may comprise plurality of sensors. In some embodiments, the sensor is a photo detector. In some embodiments, the photo detector is a spectrometer. In some embodiments, the sensor is a Raman spectrometer. In some embodiments, the sensor can detect surface plasmon resonance. In some embodiments, the sensor can detect change in mass. In some embodiments, the sensor can detect change in resonance frequency of the sensor. In some embodiments, the sensor comprises a crystal and the sensor can detect dissipation of shear movement of the crystal. In some embodiments, the sensor comprises piezoelectric crystal. In some embodiments, the sensor comprises piezoelectric crystal and a monolayer of a metal such as gold, silver etc. In some embodiments, the device comprises a sensor and a flow cell. In some embodiments, sample is flown over the sensor by a continuous flow.
[0185] In some embodiments, the device is fluidic device. In some embodiments, the fluidic device may comprise a solid support. In some embodiments, fluid is introduced to the solid support. The fluid may be stationary or the fluid may have a relative flow with respect to the solid support. In some embodiments the fluid may comprise bacteriophage, bacteria, and/or complexes of bacteriophage and bacteria. In some embodiments, the fluidic device may have two solid supports opposite to each other. In some embodiments, the fluidic device is a fluidic chamber. In some embodiments, bacteriophage, bacteria, and/or complexes of bacteriophage and bacteria are in contact with the solid support.
[0186] In some embodiments, the device comprises of at least one fluidic chamber or channel comprising at least two electrodes and at least one fluidic connection between the electrodes. In some embodiments, the device measures the membrane potential in which a change in the membrane potential is indicative of phage infection of a bacterial cell. In some embodiments, the device measures the redox potential of a molecule. In some embodiments, voltage sensitive dyes are used to measure response of the bacterial cells to phage infection. In some embodiments, application of an electric field between the electrodes allows the differential migration of the bacteriophage, bacteria, or the complex of the bacteriophage and the bacteria to the positive electrode. In some embodiments, the device may comprise a sensor to detect the differential migration of the bacteriophage, bacteria, or the complex of the bacteriophage and the bacteria. In some embodiments, a direct current is applied. In some embodiments, an alternate current is applied.
[0187] In some embodiments, the fluidic device comprises at least one main channel adapted to carry the fluid. In some embodiments, the channel comprises an interior wall. In some embodiments, the fluidic device comprises an inlet module upstream of the main channel and/or an outlet module downstream of the main channel. In some embodiments, the solid support comprises a hydrophobic surface. In some embodiments, the fluidic device comprises a force transducer that controls flow of the fluid. In some embodiments, the force transducer is an electric field, a magnetic field, a mechanical force, an optically induced force or any combination thereof
[0188] In some embodiments, the device comprises a surface plasmon resonance (SPR) system. In some embodiments, the SPR system comprises a source of polarized light, a sensor comprising a transparent element such as a prism covered with a thin metal film and linking layer, a flow chamber, a system for controlling transport of fluid, and an optical unit for detecting reflected light. The SPR principle is based on the excitation of surface plasmons in a thin layer of a metal such as gold or silver, using polarized light. Polarized light from the prism is reflected from the metal surface, and at a certain angle of light incidence the excitation of resonance in the metal film results in intensity and phase changes in the reflected light beam. An evanescent field is generated which travels in a direction perpendicular to the surface, i.e. in the direction of the fluid sample. As a result of excitation of surface plasmons the incident light is adsorbed, resulting in a decrease in the intensity of the reflected light. For each wavelength and the corresponding effective refractive index ratios between both sides of the metal layer, there is a specific angle at which a minimum in reflectivity is observed, the SPR angle. This angle increases with decreasing wavelength. When interactions occur on the metal surface within the range of the penetration depth of the evanescent field while at the glass phase the refractive index remains constant, a change in refractive index of the dielectric fluid sample occurs. This change in refractive index causes a change in the angle at which SPR occurs. This change in SPR angle is recorded as a shift in the SPR angle with time, resulting in SPR sensorgrams. The change in SPR angle of reflected light is directly proportional to the binding of the bacteriophage, bacteria, and/or the complex of bacteriophage and bacteria on the metal surface of the prism.
[0189] In some embodiments, the device comprises a calorimeter. The device measures the heat of chemical reactions or physical changes, endothermic and exothermic peaks, enthalpy as well as heat capacity.
[0190] Detection
[0191] In some embodiments, the bacteriophage, bacteria, and/or the complex is excited by a specific electromagnetic radiation (excitation) with a defined energy and amplitude and the complex is detected by a specific electromagnetic spectral signature that is generated by the complex in response to the exciting electromagnetic radiation. In some embodiments, the complex is detected by spectrometer, optical microscope, Raman spectroscopy, surface enhanced Raman spectroscopy, SPR.
[0192] In some embodiments, the bacteriophages are detected by Raman spectroscopy or surface enhanced Raman spectroscopy (SERS). Samples comprising the bacteriophage, bacteria, and the complex are introduced to a solid support comprising a metal surface. In some embodiments, the metal surface comprises nanostructures. In some embodiments, the metal surface comprises nanoparticles arranged on the solid support by self-assembly. In some embodiments, the metal surface comprises functional groups for immobilizing the bacteriophage, bacteria, and/or the complex. In some embodiments, optical excitation of the samples can be carried out at 532 nm. The corresponding Raman shift from the sample can be measured by detecting the emission from the sample by a spectrometer. In some embodiments, Raman spectroscopy or SERS can be carried out in aqueous environment.
[0193] In some embodiments, the bacteria or the bacteriophage are detected by SPR. In some embodiments, the bacteriophage or the bacteria are immobilized on flow cells of a sensor surface. When a sample comprising the other partner (i.e. bacteriophage or the bacteria) which is not immobilized and free in solution, passes through the flow cell, the resulting complex is immobilized on the flow cell and the resulting signal is detected. In some embodiments, the sample flows on the flow cell at a constant rate.
[0194] In some embodiments, the bacteria or the bacteriophage are detected by electron microscopy. In some embodiments, the solid support comprises silicon nitride. In some embodiments, the solid support comprises functional groups to immobilize the bacteriophage and/or the bacteria. In some embodiments, the sample comprising bacteriophage and/or bacteria are introduced to the solid support by a flow cell. In some embodiments, a contrast reagent is added to the sample comprising bacteriophage and/or bacteria. Non-limiting examples of contrast reagent include uranyl formate, sodium phospho-tungstate, ammonium molybdate, methyl amine tungstate. In some embodiments, the transmission electron images are recorded on a CCD camera with magnification greater than 1000.
[0195] In some embodiments, the bacteria or the bacteriophage are detected by scanning probe microscopy. In some embodiments, the detection is by atomic force microscopy. In some embodiments, the sample comprising bacteriophage, bacteria and/or the complex are introduced to the solid support. In some embodiments the bacteriophage, bacteria and/or the complex are immobilized on a solid support. In some embodiments, the solid support is glass. In some embodiments, the solid support may further comprise metal.
[0196] In some embodiments, the scanning probe is operated in constant height mode, deflection mode, tapping mode, or lift mode. In some embodiments, the morphology of the sample is recorded as height, amplitude or deflection of the probe. In some embodiments, the phage infection is deduced from the morphology data by comparing it with uninfected sample. In some embodiments, the scanning probe tip is biased by applying an alternate voltage or a direct current voltage. In some embodiments, a map of the contact potential of the phage infected bacteria is compared to the uninfected bacteria to evaluate susceptibility. In some embodiments, the probe tip comprises a thermal sensor. In some embodiments, the thermal sensor is a metal wire. In some embodiments, the probe tip forms one leg of a Wheatstone bridge. In some embodiments, the bridge is used to maintain the scanning probe tip at to measure the temperature of the scanning tip probe, or to maintain the tip at a constant temperature. In some embodiments, the heat flow from the sample is measured by at least one tip. In some embodiments, the heat flow from the sample measured by scanning at least one tip over the sample in the temperature contrast mode by measuring the resistance change of the probe. In some embodiments, the heat flow from the sample is measured in the current contrast mode by supplying additional energy to the circuit. In some embodiments, the energy required to maintain the constant current within the tip is a measured as the heat flow from the sample. In some embodiments, the difference in the heat flow from the sample to that of a negative control that does not contain the infectious phages is used to determine the infectious phages in the sample.
[0197] In some embodiments, the bacteria are detected by electrochemical device. In some embodiments, the device comprises of at least one fluidic chamber or channel comprising at least two electrodes and at least one fluidic connection between the electrodes. In some embodiments, the device comprises one or more sensors. In some embodiments, the bacteriophage, bacteria, and/or the complex are immobilized on a solid support of the device. In some embodiments, sample comprising the bacteriophage, bacteria, and/or the complex are flowed at a constant rate over the sensor of the device.
[0198] In some embodiments, the electrical current is measured as a function of the applied voltage to the electrodes. In some embodiments, the device measures the membrane potential in which a change in the membrane potential is indicative of phage infection of a bacterial cell. In some embodiments, the device measures the redox potential of a molecule. In some embodiments, the pH change is measured. In some embodiments, the enzyme activity is measured (NAD-NADH, ADP-ATP). In some embodiments, voltage sensitive dyes are used to measure response of the bacterial cells to phage infection. In some embodiments, the fluorescence spectrum of a dye is monitored as a function of membrane potential change.
[0199] In some embodiments, application of an electric field between the electrodes allows the differential migration of the bacteriophage, bacteria, or the complex of the bacteriophage and the bacteria to the positive electrode. In some embodiments, the device may comprise a sensor to detect the differential migration of the bacteriophage, bacteria, or the complex of the bacteriophage and the bacteria. In some embodiments, a direct current is applied. In some embodiments, an alternate current is applied.
[0200] In some embodiments, the bacteria or the bacteriophages are detected by change in mass. In some embodiments, the bacteriophage and/or the bacteria is immobilized on a solid support. In some embodiments, the immobilization is by a covalent bond between the functional groups and the bacteriophage, bacteria, and/or the complex. In some embodiments, the immobilization is by a non-covalent means, e.g., binding of a binding pair, e.g. biotin/streptavidin.
[0201] In some embodiments, the solid support comprises a sensor. In some embodiments, the sample comprising the bacteriophage, bacteria, and/or the complex is flowed over the sensor. In some embodiments, the bacteria or the bacteriophages are measured as a change in the mass upon phage complex formation. In some embodiments, the change in the mass on the sensor is detected by measuring the change is the resonance frequency of the sensor. In some embodiments, the sensor comprises a crystal. In some embodiments, the change is the mass of the sensor is derived by measuring the dissipation of shear movement of the crystal.
[0202] In some embodiments, the sensor is a piezoelectric sensor. In some embodiments, the piezoelectric sensor has at least a monolayer of metal, e.g., gold, silver, platinum etc. on top or bottom of the sensor surface. In some embodiments, the sensors are round-shaped. In some embodiments, the sensor is a quartz crystal with gold coating and mounted on a flow cell.
[0203] In some embodiments, the bacteria are detected by detecting the bacterial nucleic acid. In some embodiments, the complexes are separated from the incubation mixture of the bacteriophage and the bacteria. The nucleic acid of the complex is isolated. The isolated nucleic acid will comprise bacterial nucleic acid and nucleic acid from the bacteriophages that specifically infect these bacteria. Bacterial DNA is large and supercoiled and plasmid DNA is supercoiled. In contrast, DNA from bacteriophage family Caudovirales is linear dsDNA. Thus, bacteriophage DNA can be separated from bacterial DNA due to the difference in size and morphology.
[0204] In some embodiments, the detection of bacterial nucleic acids is by mass spectrometry. Nucleic acid is extracted from a sample comprising bacteria and/or complex. The extracted nucleic acid is concentrated and applied on a solid support. The nucleic acid is dried. The mass spectra from the purified nucleic acid sample are recorded on a mass spectrometer device. The mass spectra of a standard healthy cells and phage infected cells are compared. A difference is indicative of phage infected bacteria.
[0205] In some embodiments, the bacteria are detected by using a calorimetric device. The endothermic and exothermic peaks of the negative control (uninfected bacteria) and the sample suspected of comprising a complex of bacteriophage and bacteria are obtained from thermograms of the calorimetry output. The peaks are compared to infer phage infected bacteria. In some embodiments, the onset temperatures of the endothermic or exothermic peaks are studied. In some embodiments, the total enthalpy of the sample is used to infer the presence of phage infected bacteria.
[0206] In some embodiments, the bacteria are detected by fluorescence. In some embodiments, the bacteria and/or the bacteriophage comprise a detectable label. In some embodiments, the bacteria and/or the bacteriophage comprise more than one detectable label, e.g., at least two, at least three detectable labels. In some embodiments, the detectable labels are different. In some embodiments, the detectable label is a fluorescent moiety. In some embodiments, a fluorescent moiety can be stimulated by a laser with the emitted light captured by a detector. The detector can be a charge-coupled device (CCD) or a confocal microscope, which records its intensity.
[0207] In some embodiments, the bacteria are detected by the change in the physiological and chemical changes due to the complexation by enzymatic reaction. In some embodiments, the bacteria are detected by the color change due to enzymatic reaction, e.g., beta galactopyranoside assay, glucuronidase, N-acetyl-b-galactosidase, esterase, glucosidase, and lactate dehydrogenase (LDH).
EXAMPLES
Example 1
Immobilization of a Complex of Bacteriophage and Bacteria on Beads
[0208] Preparation of S1 Host Bacteria
[0209] Bacterial host cell, S1 was washed twice with 1 PBS pH 7.4 buffer to remove MRS media. SYBR green (Invitrogen) stain was added to a final concentration of 2.
[0210] Preparation of PMA Stained J1 Phage
[0211] One mL of J1 phages (10.sup.8 PFU/ml) were concentrated using 50 kDa Amicon ultrafiltration filter (EMDmillipore, Billerica, Mass.). Spins were conducted at 7000 rpm for 5 minutes on a tabletop centrifuge. J1 phage was recovered in 400 L. To the concentrated J1 phage, 2.5 L of 20 mM PMA stain was added.
[0212] Preparation of Beads
[0213] 1) 200 L of BioMag (51 mg/ml) amine beads (Bangs Laboratory, Fishers, Ind.) were washed with 1 PBS pH 8.0 buffer. Beads were resuspended in final volume of 200 L 1 PBS pH 8.0 and served as the stock beads for the following experiment.
[0214] 2) To prepare BS3 (bis(sulfosuccinimidyl)suberate), (Thermo Fisher Scientific) linked magnetic beads, 5 l of stock beads was dispensed into 200 L of 1 PBS pH 8.0. Then, 20 L of 70 mM solution of BS3 was added to the beads and allowed to incubate for 45 minutes at room temperature on a rotating wheel. The BS3 linked beads were equally split into two 100 L portions and then placed on a magnetic rack for 3 minutes. The solution containing excess BS3 linker was removed from the beads. In order to attach J1 phage to the linked beads, 200 L of 10.sup.9 PFU/ml J1 stained with PMA was added to one sample of beads and incubated at room temperature for 50 minutes on a rotating wheel.
[0215] 3) The control set were non BS3-linked magnetic beads and these were prepared by dispensing 5 uL of stock magnetic beads into 200 uL 1 PBS pH 8.0. The sample was split into two 100 uL portions and placed on the magnetic rack to remove the buffer. J1 (200 uL of 10{circumflex over ()}9 PFU/ml) stained with PMA was added to one of the beads.
[0216] 4) Excess and unreacted linkers were quenched by adding 10 L of 1M Tris HCl, pH 7.5 to all four samples and incubated for 15 minutes at room temperature. The samples were placed on a magnetic rack to remove the solution.
[0217] 5) 500 L MRS was added to all 4 samples to block remaining open sites on the bead for 1 hour.
[0218] 6) Beads were washed twice with 1 PBS and ready to use.
[0219] Addition of S1 to Prepared Magnetic Beads
[0220] The SYBR green stained S1 bacteria (200 L of 10.sup.8 cells) was added to each of the four bead samples and incubated with for 5 minutes at room temperature. The beads were then washed four times with 500 L 1 PBS, pH 7.4 to remove unbound bacteria and resuspended in a final volume of 90 L 1 PBS.
[0221] Results:
[0222] Non-BS3 linked magnetic beads with passive adsorption of J1 (PMA) onto the magnetic beads: Did not show signs of bacteria binding to the beads (
[0223] Non-BS3 linked magnetic beads without exposure to bacteriophage J1 (PMA stained) had low levels of Si bacteria binding to the beads. Most of the beads were without bacteria (
[0224] BS3-linked magnetic beads comprising the linkers but without immobilizing bacteriophage J1 (PMA stained) had very low levels of Si bacteria bound to the beads. Most of the beads were without bacteria (
[0225] Bacteriophage J1 (stained with PMA) immobilized to magnetic beads with the BS3-linker showed the most bacteria bound compared to the above three samples. Even though most individual beads were not coated in bacteria, there were clumps of magnetic beads that had bound bacteria (
Example 2
Preferential Labeling of DNA with Nucleic Acid Binding Dyes
[0226] The preferential labeling of nucleic acid was tested with propidium monoazide (PMA), propidium iodide (PI) and SYTOX Orange dyes. Permeable bacterial cells were initially stained with one of the three dyes and then stained with the other two dyes individually to test the blocking of the nucleic acid or displacement of the dyes from the nucleic acid.
[0227] 0.6 ml of permeable S1 bacterial cells (10.sup.8 cfu/ml) were centrifuged at 13000 rpm. The supernatant was removed and the pellet was resuspended in 1 mL PBS. This wash step was employed to remove the media and cell debris.
[0228] In one set, 1-15 L PMA (10 mM) was added to the washed cells and incubated in the dark or under light for 5-15 min. In some cases, the sample was exposed to light for 15-30 minutes. The bacterial cell solution was equally divided into two parts. To each bacterial cell solution, SYTOX Orange (10 mM) and PI (10 mM) were added, respectively. The fluorescence was measured at different wavelength of excitation and emission corresponding to SYTOX Orange, PI and PMA dyes to observe the effect of the second dye on the presence of PMA bound to the nucleic acid.
[0229] In another set, SYTOX Orange (10 mM) was added to the washed cells and incubated in the dark or under light for 5 min. The bacterial cell solution was equally divided into two parts. To each bacterial cell solution, of PMA (10 mM) and PI (10 mM) were added, respectively.
[0230] In another set, PI (10 mM) was added to the washed cells and incubated in the dark or under light for 5-15 min. The bacterial cell solution was equally divided into two parts. To each bacterial cell solution, PMA (10 mM) and SYTOX Orange (10 mM) were added, respectively.
[0231] The fluorescence was measured at different wavelength of excitation and emission corresponding to SYTOX Orange (530/577 nm), PI (492/600 nm, 20 nm emission bandwidth) and PMA (492/600 nm, 80 nm emission bandwidth) dyes to observe the effect of the second dye on the presence of PMA bound to the nucleic acid. Exemplary excitation and emission spectra of SYTOX orange and propidium iodide dyes are shown in
TABLE-US-00004 TABLE 4 Blocking and Displacement of the Dyes Excitation/Emission Wavelengths Observation S1 without stain 16 33 46 S1 with SYTOX 1514 288 816 leak of orange (SO) SYTOX signal into PI and PMA Add PMA to the 21 58 137 PMA added SYTOX stained after staining S1 with SYTOX PMA displaces SYTOX decreasing the fluorescence of SO and increasing PMA fluorescence. Low signal in PMA channel is because efficiency of PMA is low Add PI to the 143 136 300 PI displaces SYTOX stained SYTOX dye S1 with PI 50 109 240 Add SYTOX 158 139 317 some Orange to PI displacement stained S1 but not significant Add PMA to PI 22 58 150 PMA stained S1 displaces PI. Fluorescence of PI is lowered with PMA addition S1 with PMA 20 57 129 Add PI to PMA 32 62 135 No significant Stained S1 change to Add SYTOX 31 48 126 PMA Orange to PMA fluorescence. Stained S1 PMA is not displaced by PI or SYTOX Orange. The PMA is a strong binder/blocker
Example 3
Testing of Beads for Use with SYROX Orange Dye
[0232] Magnetic beads and polystyrene beads of 1.5 m in diameter were incubated with SYROX Orange Dye. Excess dye was washed off and fluorescent readings were measured in photons per second. [0233] 1. 5M SYTOX Orange to .5% beadsMagnetic with NH2+ functionality and polystyrene with NH2+ functionality. The magnetic beads were washed by rinsing 1 PBS. A magnetic rack was used to separate the magnetic beads from solution, and remove all the liquid from the tube. Washing with 1 buffer was repeated three times. [0234] To wash polystyrene beads: [0235] a. Beads were loaded in to a 100K amicon centrifuge filter, [0236] b. Centrifuged for 5 min at 10,000 rpm. [0237] c. Beads were rinsed with 1 buffer. Centrifuged for 5 min at 10,000 rpm. Washing with 1 buffer was repeated three times. 3. The beads were resuspended in original volume. Signal was read with detector.
[0238] The results were shown in Table 5 below.
TABLE-US-00005 TABLE Non-specific binding of SYTOX orange dye to beads MB 1.5 um w PS 1.5 um w Trial MB 1.5 um SYTOX PS 1.5 um SYTOX 1 703.25 771 1303.5 59917 2 737.41 887.76 1499.29 67001.18 3 724.24 919.06 2229.65 56816 Average 722 859 1,677 61,245 Stdev 17 78 488 5,221 SYTOX orange dye binds more non-specifically to polystyrene beads.
Example 4
Detection of Bacteriophages by Preferential Labeling of DNA with Nucleic Acid Binding Dyes
[0239] A bacterial based assay was developed to capture host-specific bacteriophages. The captured bacteriophages are stained with SYTOX Orange (Molecular Probes) and SYTOX Orange has fluorescence enhancement of >500-fold upon binding DNA. SYTOX orange is a cell impermeant nucleic acid dye that stains compromised cells. In every batch of healthy cells, there exist a percentage of dead cells due to the natural life and death cell cycle. This causes unpredictable background fluorescence due to dead cells.
[0240] In order to address the dye entering normal dead cells found in all bacterial samples, a protocol was developed using commercially available nucleic acid stains to preferentially block the signal from compromised bacteria and allow us to only detect bacteriophage.
[0241] Propidium monoazide (PMA, Biotium, Fremont, California) (AB/Em, 510/610) is a cell impermeant, fluorescent dye that forms covalent bonds to nucleic acids of compromised cells. By strategically staining the bacteria-phage complex, bacteriophages can be detected specifically.
[0242] Bacterial cells were washed with PBS to remove media and cell debris before staining with PMA. The PMA dye preferentially stains compromised bacterial cells and is classified as cell impermeant. The bacterial cells are used as the capturing vehicle to attract host specific bacteriophages. Non-specific bacteriophages do not bind to bacteria and are washed away after pelleting the bacteria and a wash step.
[0243] SYTOX orange or another similar cell impermeant dye is used to stain the nucleic acids of the bacteriophages. PMA continues to block the SYTOX orange from intercalating with DNA in compromised bacterial cells. Therefore the total signal output is from captured phages.
[0244] Briefly, 0.5 ml of bacterial cells, (OD 0.1-0.9) centrifuged in 1.5 ml tube for 13 k rpm for 1 min. The supernatant was removed and the cell pellet was resuspended in 1 mL buffer to wash the cells. The wash step was repeated to remove the media and cell debris. 5-10 uL PMA (1-10 mM) was added to the washed cells and incubated in the dark for 5 minutes. The sample was exposed to light for 15 minutes to allow the covalent linking of PMA to the nucleic acid. The PMA labeled bacteria were ready to be used as the capturing vehicle of host-specific phages. Following the capture of the specific bacteriophages, 1-10 uM of SYTOX orange or another cell impermeant dye was used to stain the captured phages. The fluorescent signal was detected to detect the captured bacteriophages. The results are shown in
Example 5
Raman Spectroscopy and Surface Enhanced Raman Spectroscopy(SERS) for Bacterial Detection
[0245] For SERS internal mode, harvested bacterial cells are resuspended in AgNO3 solution (1 M) and incubated for 5 min. The cells are then collected and are washed twice in water by centrifugation at 4,500 g for 10 min at 4 C. Afterwards, the cells are resuspended in NaBH4 solution (0.5 M) for further analysis. For SERS external mode, harvested bacterial cells are first resuspended in NaBH4 solution and incubated for 5 min. The cells are then collected by centrifugation and resuspended in AgNO3 solution for further analysis. In a typical experiment, the bacterial cells are treated with 0.1% Triton X-100 (v/v) for 5 min, prior to the assay. Then, 10 L of such treated bacterial suspension (110.sup.6 CFU/mL) are mixed with 10 L of NaBH4 (0.5 M). The mixture is then incubated for 3-5 min. Subsequently, 800 L of AgNO3 (1 M) are pipetted into the mixture followed by vortexing.
[0246] Raman analysis is conducted by addition of 5 L of the bacterial suspensions obtained from above modes onto the surface of glass cover slips (0.13 mm thickness, 15 mm diameter, Ted Pella Inc., Redding, Calif., USA). SERS spectra are recorded on a benchtop Raman spectrometer (Sierra Snowy Range 785 series, Laramie, Wy., USA) under excitation wavelength of 785 nm. The exposure time is is and the number of accumulations for each measurement is 10. The spectral data are acquired over a Stokes Raman shift of 400-2,000 cm-1. For each study, three biological replicates are analyzed. To validate the results of the strain level discrimination, additional replicate is also analyzed.
Example 6
Electrochemical Detection of Bacteria
[0247] Protocol
[0248] Functionalization of Electrochemical Sensors
[0249] Thiolated capture probe is prepared at a concentration of 0.05 M in 300 M 1,6-hexanedithiol (HDT), 10 mM Tris-HCl, pH 8.0, 0.3 M NaCl, 1 mM EDTA and is incubated in the dark at room temperature for 10 min. Incubation of the thiolated capture probe with HDT ensures that the thiol group on the capture probe is reduced, resulting in more consistent results.
[0250] A stream of nitrogen is applied to bare gold 16 sensor array chip(s) for 5 sec to remove moisture and/or particulates. A 6 l of the HDT-thiolated capture probe mix is applied to the working electrode of all 16 sensors of the sensor array and store the sensor chip(s) in a covered Petri dish at 4 C. overnight. Thiolated capture probes bind directly to the bare gold electrode and the HDT acts to prevent overpacking of the capture probes and keep them in an extended conformation that promotes hybridization with the target.
[0251] The following day, the sensor chip is with deionized H2O for 2-3 sec and dry under a stream of nitrogen for 5 sec. A 6l of 10 mM Tris-HCl, pH 8.0, 0.3 M NaCl, 1 mM EDTA, 1 mM 6-mercapto-1-hexanol (MCH) is applied to the working electrode of all 16 sensors and incubate for 50 min. This and all subsequent sensor chip incubations are performed in a covered Petri dish at room temperature. MCH acts as a blocking agent, filling in any gaps where the thiolated capture probe or HDT is not present on the electrode surface.
[0252] Sample Preparation
[0253] One ml of bacterial culture is transferred in the log phase of growth (OD6000.1) to a microcentrifuge tube and centrifuge at 16,000 g for 5 min. The culture supernatant is removed. The bacterial pellet can be processed immediately or can be stored at 80 C. for later use. The bacterial pellet is thoroughly resuspended in 10 l of 1 M NaOH by applying the pipette tip to the bottom of the microcentrifuge tube and pipetting up and down several times. Incubate the suspension at room temperature for 5 min. The bacterial lysate is neutralized by adding 50 l of 1 M Phosphate Buffer, pH 7.2, containing 2.5% bovine serum albumin (BSA) and 0.25 mM of a fluorescein-modified detector probe. The neutralized lysate is incubated for 10 min at room temperature. Fluorescein-modified detector probes hybridize with bacterial rRNA target molecules.
[0254] Electrochemical Sensor Assay
[0255] The MCH is washed from the sensor chip with deionized H2O for 2-3 sec and is dried under a stream of nitrogen for 5 sec. A 4 l of neutralized bacterial lysate is applied to the working electrode of each of 14 sensors and incubate for 15 min. Target-detector probe complexes hybridize to immobilized thiolated capture probes. A 4 l of 1 nM bridging oligonucleotide in 1 M Phosphate Buffer, pH 7.2, containing 2.5% BSA and 0.25 M fluorescein-modified detector probe is applied to 2 positive control sensors (used for signal normalization) and is incubated for 15 min. The sensor chip is washed with deionized H2O for 2-3 sec and is dried under a stream of nitrogen for 5 sec. A 4 l of 0.5 U/ml anti-Fluorescein-HRP in 1 M Phosphate Buffer, pH 7.2, containing 0.5% casein is applied to the working electrode of all 16 sensors and is incubated for 15 min. The anti-Fluorescein-HRP binds to the immobilized fluorescein-modified detector probes. The sensor chip is washed with deionized H2O for 2-3 sec and is dried under a stream of nitrogen for 5 sec. A film well sticker is applied to the surface of the sensor chip and load into the sensor chip mount. A 50 l of TMB substrate is pipette onto all 16 sensors and close the sensor chip mount.
[0256] Amperometry and cyclic voltammetry measurements are obtained for all 16 sensors using the Helios Chip Reader. Amperometric current is proportional to the rate of TMB reduction on the sensor surface.
Example 7
Detection of Bacterial DNA by Mass Spectrometry
[0257] Mass spectra are acquired using an Ultraflex I MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). Alternatively, a simpler MALDI-TOF instrument such as the benchtop Microflex (Bruker Daltonics) can be used without losing data quality. Measurements in linear positive ion detection mode are performed, using a Nd:YAG laser at maximum frequency of 66 Hz. Pulsed ion extraction (PIE) is set to zero. Acceleration voltage (IS1) is set to 20 kV. The mass range of spectra is from 2,000 to 20,000 m/z. The final resolution in the mass range of 7,000-10,000 m/z is optimized to be higher than 600 and absolute signal intensities are about 103. Automated spectrum acquisition is performed using the Auto Execute software with fuzzy control of laser intensity. At least 10.sup.7 bacterial cells are required for high quality mass spectra. For reference spectra six spots on the MALDI target are measured. On each spot, four spectra with 10 times 100 laser shots are accumulated. Twenty spectra are stored for the reference spectra library. For identification spectra is acquired by accumulating 1000 laser shots in ten 100 shot portions.
[0258] Factors influencing the intensities of signal peaks comprise concentration and location of proteins in the bacterial cell and biophysical properties of proteins such as solubility, hydrophobicity, basicity, and compatibility with MALDI. In general, most of the proteins detected by MALDI protein bacterial profiling derive from highly abundant, basic ribosomal proteins.
[0259] Data analysis: Mass spectra are analyzed with Flex Analysis software 2.4 (Bruker Daltonics). The mass spectral input data can be listed in generic data formats such as the extensible markup language (XML) to make them independent from the hardware used. Spectra are pre-processed using default parameters for reference spectra libraries also known as call main spectra libraries (MSPs). A maximum of 100 peaks with a signal-to-noise (S/N) ratio of 3 are selected in the range of 3,000-15,000 Da. Afterwards the main spectra are generated as a reference using all spectra given for a single microorganism. In general, 75 peaks are picked automatically, which occur in at least 25% of the spectra and with a mass deviation of 200 ppm.
[0260] For the evaluation of mass spectra reproducibility, the spectra are loaded into the ClinProTools 2.1 software (Bruker Daltonics). Through this process mass spectra are firstly normalized before baseline subtraction, peak detection, realignment, and peak-area calculation are applied. The optimal settings resulted in an S/N ratio of 5, a Top Hat baseline subtraction with 10% as the minimal baseline width, and a 3-cycle Savitsky-Golay smoothing with a 10 Da-peak width filter. For the example shown in
Example 8
Detection of Bacteria by Atomic Force Microscopy
[0261] A Nanoscope IIIA AFM (Digital Instruments, Santa Barbara, Calif., USA) operating in contact mode in air is used to image cells and to measure interaction forces. The relative humidity was 50-60% and no a spring constant of k50.06 N/m (Digital Instruments). The radius of curvature of the AFM tip is approximately 50 nm. The Digital Nanoscope software (version 4.23) is used to analyze the topographic images of the surface, as well as the forcedistance over the sample surface. During the forcedistance measurements, the scanning rate in z-direction is maintained at 30 Hz. Each map of sample surface consisted of 64 3 64 grid points.
Example 9
Detection of Bacteria by Near-Field Scanning Optical Microscope
[0262] NSOM measurements were acquired using a SNOM 210 in the beta-type of the commercially available standard instrumentation equipment with a piezo scanning unit integrated into a microscope condenser (Carl Zeiss Jena GmbH, Digital Instruments Veeco GmbH). Micro-fabricated probes with silicon nitride tips coated with aluminum, with a typical aperture of 100nm, are mounted in a shear-force sensor support. The instrument is equipped with an argon ion laser (1=458 nm, 488 nm) and two HeNe lasers (1=543 nm, 633 nm) for near field illumination. The illumination intensity is independently tuned by an AOTF (Acousto Optical Tunable Filter). The laser light is coupled into the NSOM tip by glass fibers. The topographic scan is controlled by modulation of the lateral shear-force oscillation of the NSOM probe. Fluorescence and absorption are detected in air by an Achroplan long distance objective 40/NA 0.6corr. and transferred to a photo-multiplier or an avalanche photodiode, respectively, using appropriate filter settings. The instrument is controlled by the NanoScope IIIc controller. The scans are performed using a probe velocity of less than 1 m/s. Images 11 m2 up to 1010 m2 are registered and visualized in 3D topographic false color plots using the NanoScope Ma software (version 4.42r1) running under Windows on a PC.
Example 9
Detection of Bacteria by Calorimetry
[0263] Samples (10-15 mg) are weighed to 0.01 mg, sealed in volatile aluminum pans and heated in a Perkin-Elmer DSC-2C at a rate of 10 C. min.sup.1 from about 10 C. to 120 C. Samples are weighed before and after calorimetric measurements to check for loss of mass during heating and the results of samples showing signs of leakage are discarded. An empty pan is used as a reference and, after heating, the sample is rapidly cooled to its initial temperature. Selected samples are then re-run in the DSC to investigate the reversibility of the thermograms. The sample dry mass is determined by piercing the pan and drying it overnight in an oven at 105 C. Data are collected and the calorimeter is controlled with a Perkin-Elmer data station but thermogram scans are transferred to a VAX-11/750 computer for high-resolution plotting and peak area determined by trapezoidal integration. The differences between the data collected during the first run and those collected on re-running the DSC are proportional to that component of the specific heat capacity caused by irreversible processes taking place during the first heating. The difference thermogram is useful for precise determination of the onset temperature of irreversible denaturation. Temperature and power scales are calibrated according to the manufacturer's instructions using the melting of indium and ice as standards.
Example 10
Non-Specific Binding of the Fluorescent Dyes to Magnetic Beads and Blocking of Non-Specific Signals from Other Dyes by PMA
[0264] 1% magnetic beads were washed and suspended in 1 PBS. luM SYTOX Orange, 1 SYBR Green, or 0.4 mM PMA was added to the beads and the fluorescence at 577/488/600 nm were recorded. To the SYTOX stained beads, 0.2 mM PMA was added, and the fluorescence recorded at 600 nm. The order of the addition of the SYTOX Orange and PMA were reversed and the fluorescence recorded at the respective emission wavelengths.
TABLE-US-00006 AMS40 (4-5 Ex/Em uM) AM80 (8-10 Treatment Reading Well, B1 uM) Well, B3 No dye 492/600 27 47 No dye 530/577 28 28 Add Sytox Orange to 1 530/577 103 52 uM 492/600 49 46 Add PMA to 0.2 mM 530/577 27 30 SYTOX Orange Dye (530/577); PMA (492/600).
Experiment II. Add PMA to Beads First, Then Add SYTOX Orange.
[0265]
TABLE-US-00007 Ex/Em AMS40 (4-5 AM80 (8-10 Treatment Reading uM) Well, B2 uM) Well, B4 No dye 492/600 30 34 No dye 530/577 21 26 Add PMA to 0.2 mM 530/577 24 27 492/600 39 50 Add Sytox Orange to 1 492/600 45 49 uM 530/577 26 30 Sytox Orange Dye (530/577); PMA (492/600).
Experiment III. Test Absorbance, Scattering of Dyes with Beads
TABLE-US-00008 Sybr Green PMA Sytox Orange Beads 492/530 492/600 530/577 Bead 1 19, 14, 14 42, 39, 37 27, 28, 23 No Dye Bead 2 16, 25, 27 108, 106, 102 83, 92, 88 With Dye Bead 2 11, 12, 8 44, 40, 38 29, 26, 33 No Dye Bead 2 13, 12, 12 98, 96, 101 67, 68, 67 With Dye Bead 3 15, 12, 10 29, 29, 32 22, 20, 21 No Dye Bead 3 23, 8, 10 75, 77, 77 18, 17, 20 With Dye
[0266] Thus, Results: PMA effectively blocks SYTOX Orange from fluorescing.
Example 11
Detection of Bacteria by Monitoring the Reduction of Resazurin and Resorufin
[0267] To 1-1000 l of samples (milk, whey, salt whey, culture, milk culture, broth), 1-1000 l of bacterial cells with OD0.01-1 were added. In some cases, bacteriophages specific for the bacteria are added. In some embodiments, the bacteriophages were bound by a compound. To this mixture, Resazurin and/or Resorufin and/or Dihydroresorufin were added as a fluorescent tag. The fluorescence of Resorufin was measured between 27-37 C. In some experiments, the sample was incubated with the cells before the addition of the fluorescent tag. In some samples, the fluorescent tags were incubated with the sample. In some experiments fluorescent tags were incubated with cells. In some experiments, the incubated sample and cells were diluted one-fold before the addition of the tag. In some experiments, the incubated sample and cells were diluted one order before the addition of the tag. The fluorescence of Resorufin was measured at or above 577 nm over a period of time. The kinetic profile of the reduction of Resazurin to Resorufin to Dihydroresorufin of bacterial cells infected with bacteriophage were compared to corresponding uninfected standard bacterial samples. The above conditions were tested in the following experiments.
[0268] 1. Incubating Cultures and Bacteriophages Together at 32 C., and adding Resazurin to the Diluted and Undiluted Samples
[0269] In one case, wild-type phages (10.sup.5 pfu/ml) and cocci and/or bacillus (gram positive) bacterial culture were added to milk. The mixture was incubated for 45 min. The incubation mixture was diluted 1-fold. To the diluted mixture, Resazurin was added and mixed. The absorbance and/or absorbance, scattering and/or fluorescent intensity of the mixture was measured at 577 nm for at least 30 min. The fluorescence intensity was plotted as a function of time as shown in
[0270] In another case, wild-type phages (10.sup.4 pfu/ml) and cocci and/or bacillus bacterial culture were added to of bacterial culture medium. The mixture was incubated for 90 min. To the incubation mixture, Resazurin was added and mixed. The absorbance and/or absorbance, scattering and/or fluorescent intensity of the mixture was measured at 577 nm for at least 20 min. The fluorescence intensity was plotted as a function of time as shown in
[0271] In another case, wild-type phages (10.sup.3 pfu/ml) and cocci and/or bacillus bacterial culture were added to bacterial culture medium. The mixture was incubated for 75 min. To the incubation mixture, Resazurin was added and mixed. The absorbance and/or absorbance, scattering and/or fluorescent intensity of the mixture was measured at 577 nm for at least 40 min. The fluorescence intensity was plotted as a function of time as shown in
[0272] Additionally, inventors identified an unexpected and surprising difference in the kinetic profile of the reduction of Resazurin to Resorufin to Dihydroresorufin between phage infected bacterial cells and uninfected bacterial cells. The uninfected bacterial cells reduced the Resazurin (weakly fluorescent) to Resorufin (fluorescent) and finally to Dihydroresorufin (non-fluorescent) at a faster rate as compared to phage infected bacterial cells. The reduction of Resazurin to Resorufin is irreversible while the reduction of Resorufin to Dihydroresorufin is reversible. After some time, the predominant metabolic product of Resazurin is Dihydroresorufin (non-fluorescent) for uninfected bacterial cells. As a result, the fluorescence peaks faster for uninfected bacterial cells, and the fluorescence diminishes with time.
[0273] In contrast, the reduction of Resazurin (weakly fluorescent) to Resorufin (fluorescent) is slower for phage infected bacterial cells. As a result, the fluorescence peak for Resorufin appears later in phage infected bacterial cells. Additionally, phage infected bacterial cells preferably oxidize Dihydroresorufin (non-fluorescent) to Resorufin. Accordingly, using a phage specific for bacteria, a kinetic profile of infected bacteria is indicative of the presence of the bacteria in a sample.
[0274] 2. Adding Resazurin at Different times of Incubation
[0275] Adding Resazurin at the start of incubation (T0) resulted in an increased efficiency of the assay by reducing a step. The infected cells having higher levels of fluorescence compared to the uninfected control. All samples rapidly become pink as the Resazurin is reduced irreversibly to Resorufin. The sample then became white in color as the Resorufin is reduced to Dihydroresorufin, a reversible step. After this step occurs, based on the phage concentration, phage infected cell samples start to have an increased pink color (Resorufin) while readings on the uninfected controls continue to decrease. The results with different concentration phages are shown in
[0276] When varying concentration of wild-type phages (10.sup.5 pfu/ml-0 pfu/ml), bacterial culture (cocci and bacillus) and Resazurin was added and mixed with milk and incubated for 4 hours, the kinetic profile of the reduction of Resazurin was significantly different than of the phage infected cells as shown in
[0277] 3. Effect of Temperature on the Length of Time Required to Detect Infection of Bacteria with Phages.
[0278] Increasing the temperature beyond 29 C. decreased the length of time needed to detect differences between the infected and uninfected cells by about 10-20 minutes and increased sensitivity, allowing detection of bacteria using bacteriophage concentration as low as 10.sup.2 pfu/mL of phage (
[0279] 4. Difference Between Starting Incubations with Resorufin and Resazurin.
[0280] To see if the times to detect the bacteria could be decreased further, Resorufin instead of Resazurin was added to eliminate the time required for the cells to convert Resazurin to Resorufin. This did not significantly shorten the length of time to detection of phage infected bacteria using phage at higher concentrations but did shorten times by about 5-10 minutes for lower concentrations of virus. When the initial compound is Resazurin, the fluorescence and/or absorsance intensity of Resorufin shows a correlation with the phages. It is observed that in additon to the change in kinetics of change from Dihydroresorufin to Resorufin in the presence of infectious phages, the amount of Resorufin oxidised from Dihydroresorufin is proportional to the phage concentration when the reagent used is Resazurin. When the initial reagent is Resorufin, in some experiments, the final oxidized fluorescence shows little correlation to the amount of phage present (
[0281] Difference in Time to Detection of Bacteria with Different Bacteria and Phages.
[0282] The time to detection of phage infected bacteria varied with different cells and phage. cocci and/or bacillus cells and wild-type phage required almost twice as long for the assay to detect phage infected cocci and/or bacillus bacteria as compared to different wild-type phage Detection of wild-type phage infection of the cocci and/or bacillus bacteria culture was also faster at 37 C. than 32 C. as shown in
[0283] Binding of PMA and PMAX to bacteriophage renders the phage non-lytic.
[0284] Two strains of bacteriophages, DT1 and T7 with and without PMA were tested for lytic activity. Streptococcus thermophilus with (
[0285] Resazurin Assay with E. coli Only E. coli with T7, and E. coli with PMA Treated T7.
[0286] Biotinylated T7 bacteriophages were immobilized onto streptavidin magnetic beads. The T7-beads with (
[0287] Resazurin assay with capture of E. coli from a mixture of bacteria with T7 treated with PMA
[0288] Biotinylated T7 bacteriophages were immobilized onto streptavidin magnetic beads. T7 beads were treated with PMA and was introduced into sample containing E. coli and Streptococcus thermophilus as a negative control. The beads were then washed to remove excess and unbound bacteria. After a 4.5-hour incubation for bacteria outgrowth, the binding and capturing ability of the T7 coated beads were measured using Resazurin. The results shown in
Example 12
Detection of Antibiotic Resistant Bacteria and Capturing Them Using Bacteriophages Immobilized on a Solid Support
[0289] Biotinylated T7 bacteriophages were immobilized onto streptavidin magnetic beads. The T7-beads were stained with PMA and then incubated with E. coli and antibiotic resistant E. coli (ER2508). The beads were then washed to remove excess and unbound bacteria. Fresh LB media with and without the antibiotic tetracycline (10 ug/mL) were added followed by a 4.5-hour period for bacterial outgrowth. Using Resazurin, the results show that the T7-beads were able to capture both E. coli and antibiotic resistant E. coli (ER2508). In addition, this assay shows that antibiotic resistant E. coli (ER2508) can be captured and selected over non-antibiotic resistant strains from a broth containing antibiotics, providing a novel approach to testing of drug-resistance bacteria.