Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6841—In situ hybridisation

Definitions

• This invention relates generally to uncharged probes and more particularly, relates to the use of uncharged probes such as peptide nucleic acids (PNAs) and morpholinos in fluorescence in situ hybridization assays.
• PNAs peptide nucleic acids
• morpholinos in fluorescence in situ hybridization assays.
• FISH fluorescence in situ hybridization
• the assay comprises the steps of contacting said test sample with a ppeptide nucleic acid (PNA) probe capable of attaching to said rRNA in said test sample conjugated to an indicator reagent comprising signal generating compound capable of generating a measurable signal; and detecting said measurable signal as an indication of the presence of rRNA in the test sample.
• PNA ppeptide nucleic acid
• the assay preferably is performed by flow cytometry. Quantitation is performed by exciting fluorescence and measuring said signal by using a light selection filter.
• the signal generating compound preferably is fluorescein or rhodamine.
• the rRNA in the test sample can be fixed prior to performing the assay.
• the assay further comprises hybridizing said test sample in situ.
• the assay described hereinabove also can be performed using morpholino compounds as probes in place of the PNAs as probes. Also, an improved fluorescence in situ hybridization assay is provided wherein the improvement comprises hybridizing said test sample with a PNA or morpholino probe.
• the assay comprises the steps of contacting said test sample which may contain drug resistance gene(s) with a peptide nucleic acid (PNA) probe or a mo ⁇ holino probe capable of attaching to said drug resistance gene(s) in said test sample conjugated to an indicator reagent comprising signal generating compound capable of generating a measurable signal; and detecting said measurable signal as an indication of the presence of the drug resistance gene(s) in the test sample.
• the assay preferably is performed by flow cytometry. Quantitation is performed by exciting fluorescence and measuring said signal by using a light selection filter.
• the signal generating compound preferably is fluorescein or rhodamine.
• the drug resistance gene(s) in the test sample can be fixed prior to performing the assay.
• the assay further comprises hybridizing said test sample in situ.
• test kits for detecting the presence of rRNA or drug resistance gene(s) which may be present in a test sample which comprise a container containing a PNA or morpholino probe conjugated to a signal generating compound capable of generating a measurable signal.
• FIGURE 1 presents a histogram wherein a 25 mer DNA oligo and a 15 mer PNA oligo probe (sequence within the 25 mer of DNA sequence) complementary to 28S rRNA were directly labeled with fluorescein, wherein: A is a negative sample with DNA probe labeled at both ends and B is a positive sample with DNA probe labeled at both ends.
• FIGURE 2 presents a histogram wherein a 25 mer DNA oligo and a 15 mer PNA oligo probe (sequence within the 25 mer of DNA sequence) complementary to 28S rRNA were directly labeled with fluorescein, wherein: C is a negative sample with PNA probe labeled at the amino end and and D is a positive sample with PNA probe labeled at the amino end.
• FIGURE 3 presents a photograph of stained E. coli bacteria in mouse PMNs.
• In situ hybridization was introduced in the late 1960's. J. G. Gall and M. Pardue, Proc. Natl. Acad. Sci. USA 63:378-383 (1969). Generally, it involves taking morphologically intact tissues, cells or chromosomes through the nucleic acid hybridization process to demonstrate not only the presence of a particular piece of genetic information but also its specific location within individual cells. It does not require the homogenization of cells and extraction of the target sequence, and therefore, provides precise localization and distribution of a sequence in cell populations. Secondly, the homogenization of tissues can result in a loss of sensitivity if the target is present in only a small fraction of the cells and at a low copy number.
• In situ hybridization circumvents this problem by identifying the sequence of interest concentrated in the cells containing it.
• in situ hybridization methods can identify the type and the fraction of the cells containing the sequence of interest.
• DNA as well as RNA can be detected with the same assay reagents.
• HSH techniques have improved greatly during the past 15 years.
• the procedure has been simplified from its original tedious and laborious form.
• the assay has become more reproducible and easy to perform.
• this type of assay can be done with multiple oligos in a one-step protocol that can be carried out in about two hours or less (J. Bresser et al., U. S. Patent No. 5,225,326.
• Attempts have also been made to automate in situ hybridization procedures. See, for example, C. Park et al., J. Histotechnology 14:219-229 (1991); E. R. Unger et al., J. Histotechnology 11:253-258 (1988).
• Fisher Code-OnTM available from Fisher Scientific, Pittsburgh, PA
• Ventana Medical Systems, Inc. (Tucson, Arizona) recently introduced a completely automated in situ hybridization system capable of handling 40 slides.
• FISH FISH with cell suspensions and flow cytometric detection
• the procedure also can be automated.
• in situ hybridization is particularly useful in the analysis of infections.
• In situ hybridization being an anatomic method of diagnosis, complements amplification-based assays such as PCR and LCR which are extremely sensitive but do not provide information on the cell types that are infected and quantitative information on the number of infected cells.
• In situ hybridization methods can determine the cells which are infected, the pathogen which is infecting the cells, and also it can determine (by quantitative measures) the extent of the infection.
• Labeled PNA probes directed against rRNA targets of microorganisms offer the potential to achieve the desired sensitivity and specificity with a practical protocol.
• fluorescence detection offers the fastest detection technology, the ability to multiplex and the potential for good quantitation in an automated format.
• FISH fluorescence in situ hybridization
• PNA peptide nucleic acids
• the backbone of PNA is made from repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. Unlike natural peptides which have amino acids attached to the backbone, PNA contains purine (A, G) and pyrimidine (C, T) bases attached to the backbone by methylene carbonyl linkage. Unlike DNA or other DNA analogs, PNAs do not contain any pentose sugar moieties or phosphate groups. The molecules are neutral at physiological conditions. By convention, PNAs are depicted like peptides, with the N-terminus at the first (left) position and the C-terminus at the right.
• PNA is biochemically different from both peptides and nucleic acids in that they are resistant to all known proteinases and nucleases.
• PNAs have been shown to bind more strongly in solution to both DNA and RNA than the same length DNA oligonucleotides.
• the use of PNAs in in situ hybridization assays has not been demonstrated heretofore.
• Various factors have contributed to the uncertainty of the use of PNAs in in situ hybridization. For example, all cells have not only a large pool of variaty of macromolecules but also a very complex morphological structure.
• a probe that can be used successfully in fixed cells should have the ability to penetrate layers of cellular memberane under suitable conditions and, at the same time, should only bind to the designated target(s) but not to other components of a cell.
• fluorescently labeled PNA probes can be used for in situ hybridization, and that they provide remarkably improved (i.e., six times better) signal-t ⁇ -noise than the corresponding DNA oligo.
• FIGURES 1 and 2 show a histogram of cell counts v.
• PNA probes are speed and the possibility of targeting short but specific regions of bacterial nucleic acids. Longer plasmid probes generally require overnight incubation, but 30 minutes to two hours is a sufficient time for short probes to penetrate cells and bind to the target.
• PNA probes can be used as a general screen of all bacterial types when the probes are complementary to a consensus region of the target (so- termed "universal probe").
• the probes are designed to hybridize to sequence specific regions of a particular bacterial species, subtyping of bacteria can be achieved.
• the synthesis and labeling of probes are reproducible and the labelled probes are stable for years.
• PNA probes are chemically very stable substituents.
• An additional advantage for using PNA probes is that PNAs are resistant to all known enzymes since they are not molecules which exist in nature.
• FISH methods targeting rRNA for the identification of microbial cells were first introduced by DeLong et al., Science 243:1360-1363 (1989). Due to the abundance of rRNA (10 ⁇ and 10-5 CO j e s per cell), phylogenetic identification of microbes was demonstrated by FISH using fluorescence microscopy and fluorescently labeled oligonucleotide probes (17-34 mers) complementary to bacterial 16S rRNA. In that study, it was shown that oligo probes could distinguish a bacterium dubbed "son-killer" (Nasonia vilripennis) and its closest known relative, Proteus vulgaris.
• son-killer Nasonia vilripennis
• PCR polymerase chain reactions
• LCR ligase chain reaction
• NASBA nucleic acid sequence based amplification
• the present invention provides an assay which has a fast turn around time and further provides information on the specific type of bacteria present.
• the assay disclosed herein also will work when more than one pathogenic species is present, as is detailed hereinbelow in the examples. Rapid identification of the bacterial species provides extremely valuble information for appropriate patient treatment.
• PNA-FISH The assay disclosed herein (PNA-FISH) potentially can be used for simultaneous identification of microorganisms and microbial drug resistance testing.
• PNA-FISH The assay disclosed herein
• More drug resistance genes are being identified and isolated, which allows the development of probe-based assay for direct detection of drug resistant strains at the genetic level.
• a hybridization cocktail could contain probes for microbial identification and probes targeting drug resistance genes or their transcripts (rnR ⁇ A). Each of these probes could be attached to a spectrally distinct fluorescent dye to allow multiparameter assay on one test sample.
• the present invention also encompasses utilizing a capture reagent comprising a solid support having PNAs directly immobilized thereon which can can be contacted with a test sample, or, having the test sample directly immobilized on the solid support.
• the test sample can be any liquid suspected of containing a nucleic acid sequence which can specifically hybridize with the immobilized oligopeptides.
• the capture reagent can be contacted for a time and under conditions suitable for allowing nucleic acids in the test sample, if any, and the PNAs to hybridize and thereby form hybridization complexes.
• the hybridization complexes if any, can be contacted with a conjugate for a time and under conditions sufficient to enable the conjugate to specifically bind any hybridization complexes.
• a signal can then be detected as an indication of the presence or amount of any nucleic acid sequences which may be present in the test sample.
• Immobilized PNAs as taught herein can also be employed in a "one-step" assay configuration. According to such a configuration, a test sample suspected of containing nucleic acids which are complementary to the immobilized PNAs can be contacted with a conjugate for a time and under conditions suitable for allowing the conjugate to bind any nucleic acid sequences which may be present in the test sample to form conjugate/nucleic acid complexes.
• the nucleic acids which may be present in a test sample may comprise a detectable moiety.
• Nucleic acid sequences can be labeled or conjugated with a detectable moiety through, for example, nick translation whereby labeled nucleotides are incorporated into a target sequence.
• Conjugate/nucleic acid complexes or nucleic acids which comprise a detectable moiety can then be contacted with the support bound PNAs to form conjugate/nucleic acid/PNA complexes or nucleic acid/PNA complexes.
• a signal can then be detected as an indication of the presence or amount of any nucleic acid sequences present in the test sample.
• a method for quickly detecting the presence of an nucelic acids in a test sample is provided.
• a sample which is suspected of containing nucleic acids can be contacted with a support material and the nucleic acids which may be present in the test sample can be immobilized to the support material.
• a conjugate can then be contacted with the immobilized nucleic acids for a time and under conditions for allowing the conjugate to bind the immobilized nucleic acids.
• a signal generating compound comprising PNAs can then be detected as an indication of the presence or amount of any nucleic acids which may have been present in the test sample.
• nucleic acids which are immobilized as taught herein are contacted with, for example, a test sample, conjugate/nucleic acid complexes, or a conjugate is not important. However, it is preferred that such a contact period be kept to a minimum, for example, less than 30 minutes, more preferably less than 15 minutes and most preferably less than 10 minutes.
• a conjugate may comprise a signal generating compound capable of generating a measurable signal attached to specific binding pair member .
• Signal generating compound (detectable moieties) may include any compound or conventional detectable chemical group having a detectable and measurable physical or chemical property variably referred to as a signal.
• detectable groups can be, but are not intended to be limited to, enzymatically active groups such as enzymes and enzyme substrates, prosthetic groups or coenzymes; spin labels; fluorescent molecules such as fluorescers and fluorogens; chromophores and chromogens; luminescent molecules such as luminescers, chemiluminescers and bioluminescers; phosphorescent molecules; specifically bindable ligands such as biotin and avidin; electroactive species; radioisotopes; toxins; drugs; haptens; polysaccharides; polypeptides; liposomes; colored or fluorescent particles; colored or fluorescent microparticles; colloidal particles such as selenium colloid or gold colloid; and the like.
• a detectable moiety can comprise, for example, a plurality of fluorophores immobilized to a polymer such as that described in co-owned and co-pending U.S. Patent Application Serial No. 08/091,149 filed on July 13, 1993, which is herein incorporated by reference.
• the detectable physical or chemical property associated with a detectable moiety can be detected visually or by an external means.
• Specific binding member is a well known term and generally means a member of a binding pair, i.e., two different molecules where one of the molecules through chemical or physical means specifically binds to the other molecule.
• binding pairs include, but are not intended to be limited to, avidin and biotin, antibody and hapten, complementary nucleotide sequences or complementary nucleic acid sequences such as DNA or RNA, or PNAs, or morpholino compounds, an enzyme cofactor or substrate and an enzyme, a peptide sequence and an antibody specific for the sequence or an entire protein, dyes and protein binders, peptides and specific protein binders (e. g., ribonuclease, S-peptide and ribonuclease S-protein), and the like.
• binding pairs can include members that are analogs of the original binding member, for example, an analyte-analog or a binding member made by recombinant techniques or molecular engineering.
• PNAs and morpholino compounds are specific binding members for DNA or RNA.
• the binding member is an immunoreactant it can be, for example, a monoclonal or polyclonal antibody, a recombinant protein or recombinant antibody, a chimeric antibody, a mixture(s) or fragment(s) of the foregoing.
• Signal generating compounds can be attached to specific binding pair members through any chemical means and/or physical means that do not destroy the specific binding properties of the specific binding member or the detectable properties of the detectable moiety.
• a method provided herein can be employed to immobilize oligons to a glass surface which is then employed in further analysis, such as a waveguide configuration as that taught in co-owned and co-pending U.S. Patent Application Serial No. 08/311,462 entitled "Light Scattering Optical Waveguide Method for Detecting Specific Binding Events" which is herein incorporated by reference.
• a waveguide device's ability to be employed in an immunoassay type format is based upon a phenomenon called total internal reflection (TIR). TIR operates upon the principle that light traveling in a denser medium (i.e. having the higher refractive index, Ni) and striking the interface between the denser medium and a rarer medium (i.e.
• TIR Scattered Total Internal Reflectance
• a beam of light is scanned across the surface of a TIR element at a suitable angle and the light energy is totally reflected except for the evanescent wave.
• Particles such as red blood cells, colloidal gold or latex specifically bound within the penetration depth will scatter the light and the scattered light is detected by a photodetection means.
• Immobilizing oligos contained to support materials comprises contacting a support material with an oligo solution and drying the solution upon the support material. If the oligo is suspected of being contained within a test sample, the test sample is dried upon the support material.
• Support materials or solid supports to which oligos can be immobilized are well known in the art and include materials that are substantially insoluble.
• Porous materials can serve as solid supports and may include, for example, paper; nylon; and cellulose as well as its derivatives such as nitrocellulose.
• Smooth polymeric and nonpolymeric materials are also suitable support materials and include, but are not intended to be limited to, plastics and derivatized plastics such as, for example, polycarbonate, polystyrene, and polypropylene; magnetic or non-magnetic metal; quartz and glass.
• plastics and derivatized plastics such as, for example, polycarbonate, polystyrene, and polypropylene; magnetic or non-magnetic metal; quartz and glass.
• quartz, glass or nitrocellulose is employed as a support material.
• Solid supports can be used in many configurations well known to those skilled in the art including, but not limited to, test tubes, microtiter wells, sheets, films, strips, beads, microparticles, chips
• Oligonucleotides according to the invention will be understood to mean a sequence of DNA or RNA, whereas the term oligopeptides will be understood to mean a sequence of PNA or mo ⁇ holino compounds. All may be generally termed as oligos herein. Both PNAs and mo ⁇ holino compounds have a higher binding affinity, better penetrability and lower susceptibilityt o enzymatic digestion than nucleic acid probes.
• the length of an oligo which is immobilized to a support material is largely a matter of choice for one skilled in the art and is typically based upon the length of a complementary sequence of, for example, DNA, RNA, or PNA or mo ⁇ holino compound which will be captured.
• an immobilized oligo is typically between about 5 and about 50 base pairs, preferably, the length of an immobilized oligo is between about 5 and about 30 base pairs, more typically between about 10 and about 25 base pairs.
• a "capture reagent”, as used herein, refers to an unlabeled specific binding member which is specific either for the analyte as in a sandwich assay, for the indicator reagent or analyte as in a competitive assay, or for an ancillary specific binding member, which itself is specific for the analyte, as in an indirect assay.
• the capture reagent can be directly or indirectly bound to a solid phase material before the performance of the assay or during the performance of the assay, thereby enabling the separation of immobilized complexes from the test sample .
• Test samples which can be tested by the methods of the present invention described herein include human and animal body fluids which can contain nucleic acids such as whole blood, serum, plasma, cerebrospinal fluid, urine, biological fluids such as cell culture supernatants, fixed tissue specimens and fixed cell specimens. It also is within the scope of the present invention that a variety of non- human or non-animal body fluids which can contain nucleic acids also can be analyzed according to the present invention.
• Synthesis of oligos is routine using automated synthesizers. If desired, automated synthesizers can produce oligoss which are modified with terminal amines or other groups. A useful review of coupling chemistries is found in Goodchild, Bioconiugate Chemistry. 1(3): 165-187 (1990).
• the amount of oligo solution (which can be a test sample) that is applied or "spotted" upon a solid support need be large enough only to capture sufficient complementary sequences to enable detection of, for example, a captured sequence or conjugate. This is dependent in part on the density of support material to which the capture oligo is immobilized. For example, areas of as little as 150 ⁇ m in diameter may be employed. Such small areas are preferred when many sites on a support material are spotted with oligonucleotide solution(s).
• the practical lower limit of size is about l ⁇ m in diameter. For visual detection, areas large enough to be detected without magnification are desired; for example at least about 1 to about 50 mm 2 ; up to as large as 1 cm 2 or even larger. There is no upper size limit except as dictated by manufacturing costs and user convenience.
• evaporation is the preferred drying method and may be performed at room temperature (about 25°C). When desired, the evaporation may be performed at an elevated temperature, so long as the temperature does not significantly inhibit the ability of the oligos to specifically hybridize with complementary sequences.
• the process of immobilizing oligos to a solid support may further comprise "baking" the support material and the oligo solution thereon.
• Baking may include subjecting the solid phase and oligonucleotide solution residue, to temperatures between about 60°C and about 95°C, preferably between about 70°C and about 80°C.
• the baking time is not critical and preferably lasts for between about 15 minutes and about 90 minutes. Baking is particularly preferred when porous support materials such as, for example, nitrocellulose are employed.
• An overcoating step may optionally be employed in the method herein provided. Overcoating typically comprises treating the support material so as to block non-specific interactions between the support material and complementary sequences which may be in a fluid sample.
• the overcoating or blocking material is applied after the oligo solution has been dried upon the support material.
• the blocking material should be applied after the baking step.
• Suitable blocking materials are casein, zein, bovine serum albumin (BSA), 0.5% sodiumdodecyl sulfate (SDS), and IX to 5X Denhardt's solution (IX Denhardt's is 0.02% Ficoll, 0.02% polyvinylpyrrolidone and 0.2 mg/ml BSA).
• BSA bovine serum albumin
• SDS 0.5% sodiumdodecyl sulfate
• IX to 5X Denhardt's solution IX Denhardt's is 0.02% Ficoll, 0.02% polyvinylpyrrolidone and 0.2 mg/ml BSA.
• Other blockers can include detergents and long-chain water soluble polymers.
• Casein has been found to be a preferred blocking material and is available from Sigma Chemical, St. Louis, MO. Casein belongs to a class of proteins known as "meta-soluble" proteins (see, e.g., U.S. Patent 5,120,643 to Ching, et al, inco ⁇ orated herein by reference) which are preferably treated to render them more soluble. Such treatments include acid or alkaline treatment and are believed to perform cleavage and/or partial hydrolysis of the intact protein.
• Casein is a milk protein having a molecular weight of about 23,600 (bovine beta-casein), but as used herein, "casein” or “alkaline treated” casein both refer to a partially hydrolyzed mixture that results from alkaline treatment as described in example 1 of US Patent 5,120,643.
• An electrophoresis gel (20% polyacrylamide TBE) of the so-treated casein shows a mixture of fragments predominantly having molecular weight less than 15,000, as shown by a diffused band below this marker.
• a preferred assay method for the detection of nucleic acids in a test sample according to the present invention includes flow cytometric procedures and particle counting procedures.
• particle counting analytes which are members of specific binding pairs are quantified by mixing an aliquot of test sample with microparticles coated with a capture reagent capable of binding to the nucleic acid of interest as the other member of the specific binding pair. If the nucleic acid is present in the test sample, it will bind to some of the microparticles coated with the capture reagent and agglutinates will form.
• the analyte concentration is inversely proportional to the unagglutinated particle count. See, for example, Rose et al., eds., Manual of Clinical Laboratory Immunology. 3rd edition, Chapter 8, pages 43-48, American Society for Microbiology, Washington, D. C. (1986).
• Flow cytometry methods that sense electronic and optical signals from cells which are illuminated allows dete ⁇ nination of cell surface characteristics, volume and cell size.
• Nucleic acids present in, for example, bacteria present in a test sample are bound to the PNA or como ⁇ holino compound and detected with a fluorescent dye which is either directly conjugated to the PNA or mo ⁇ holino compound or added via a second reaction.
• Different dyes which may be excitable at different wavelengths, can be used with more than one PNA or mo ⁇ holino compound specific to different nucleic acids such that more than one type of nucleic acid can be detected from one sample.
• a suspension of particles is transported through a flowcell where the individual particles in the sample are illuminated with one or more focused light beams.
• One or more detectors detect the interaction between the light beam(s) and the labeled particles flowing through the flowcell. Commonly, some of the detectors are designed to measure fluorescence emissions, while other detectors measure scatter intensity or pulse duration.
• each particle that passes through the flowcell can be mapped into a feature space whose axes are the emission colors, light intensities, or other properties, i.e., scatter, measured by the detectors.
• the different particles in the sample map into distinct and non-overlapping regions of the feature space, allowing each particle to be analyzed based on its mapping in the feature space.
• the operator manually pipettes a volume of test sample from the sample tube into an analysis tube. A volume of the desired fluorochrome labeled PNA or mo ⁇ holino compound is added. The sample/PNA or mo ⁇ holino compound mixure then is incubated for a time and under conditions sufficient to allow nucleic acid PNA or mo ⁇ holino compound bindings to take place. After incubation, and if necessary, the operator adds a volume of RNS lyse to destroy any RBCs in the sample.
• the sample After lysis, the sample is centrifuged and washed to remove any left-over debris from the lysing step. The centrifuge/wash step may be repeated several times. The sample is resuspended in a volume of a fixative and the sample then passes through the fluorescence flow cytometry instrument.
• a method and apparatus for performing flow automated analysis is described in co-owned U.S. Patent application Serial No. 08/283,379, which is inco ⁇ orated herein by reference. It is within the scope of the present invention that microspheres can be utilized in the methods described herein, tagged or labeled, and employed for in vitro diagnostic applications.
• kits with one or more containers such as vials or bottles, with each container containing a separate reagent such as a PNA or mo ⁇ holino compound, or a cocktail of these compounds, employed in the assay (s).
• kits also could contain vials or containers of other reagents needed for performing the assay(s), such as washing, processing and indicator reagents.
• the flourescent signal intensity of the PNA (SEQUENCE I.D. NO. 4) and DNA probes (SEQUENCE I.D. NO. 1) were compared.
• the DNA oligo probe (SEQUENCE I.D. NO. 1) was a 25mer that amplified 28S rRNA of mammalian cells. It was labeled with fluorescein at both the 3' and 5' ends.
• the PNA probe (SEQUENCE I.D. NO. 4) was a 15mer whose sequence resided within that of (SEQUENCE I.D. NO. 1).
• the negative control DNA probe (SEQUENCE I.D. NO.
• the negative control PNA probe (SEQUENCE I.D. NO. 5) was a 15mer complimentary to Hepatitis B viral DNA (positions 330-344).
• the cells were washed with PBS (0.14 M NaCl, 2.7 mM KC1, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) twice and fixed immediately with 4% paraformaldehyde for 15 min at room temperature. The cells then were washed twice with PBS and stored either in PBS at 4°C for up to a week or in 70% ethanol for months at -20°C.
• PBS 0.14 M NaCl, 2.7 mM KC1, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4
• probes (20 pmol PNA [SEQUENCE I.D. NO. 4 ] or DNA [SEQUENCE I.D. NO. 1] per million cells) were resuspended in 15 ⁇ l hybridization mixture (2X SSC, 50% formamide, 0.5% SDS, 100 ⁇ g/ml Salmon sperm DNA [available from Boehringer Mannhein, Indianapolis, IN) to form a probe mixture.
• the probe mixture was heated in a boiling water bath for 2 min and cooled down rapidly on ice.
• Cells were spun down at 450 x g for 10 min and the probe mixture was hybridized to the cells. Probes then were hybridized for 3 hours at 40°C. The cells were washed first with 2 X SSC, 50% formamide and 0.5% SDS, then 2X SSC with 0. IX SSC, each for 30 min at 40°C. The cells which were resuspended in PBS were analyzed by flow cytometry.
• Example 1 A. Experimental Protocol. In this experiment, different salt buffers in the hybridization cocktail were tested using CHO cells as described in Example 1. The FISH protocol as described in Example 1 was followed. Twenty (20) pmol of the EuB338 probe (SEQUENCE I.D. No.7 ) was dissolved per cell sample in various hybridization cocktails for comparison. A negative control sample (treated with 100 ⁇ g/ml RNase A [available from Sigma, St. Louis, MO] in PBS at room temperature for 1 hour) was included for each hybridization cocktail tested. The compositions of hybridization cocktails compared were (1) Bl: 2X SSC, 50% formamide (Molecular Biology Grade, available from Fisher Scientific, Pittsburgh, PA), 0.5% SDS (available from Sigma, St. Louis, MO) and 0.1% BSA (available from Sigma, St. Louis, MO); (2) B2: PBS, 50% formamide, 0.5% SDS and 0.1 % BSA; (3) B3: TE (10 mM Tris, 1 mM EDTA), 50% formamide, 0.5% SDS and 0.1% BSA
• This example was designed to determine the optimal concentration of denaturing reagents for PNA in situ hybridization.
• E. coli available from the A.T.C.C, 12301 Parklawn Drive, Rockville, MD 20852 as ATCC deposit number 8739
• TLB trypticase soy broth
• the cells were fixed with 4% paraformaldehyde for 15 min at room temperature.
• the cells next were washed with PBS twice and stored in PBS at 4°C for up to a week.
• E. coli cells in PBS [A.T.C.C. deposit number 8739] lml per sample, 10 ⁇ cells/ml), prepared as described in this Example hereinabove, were pelleted by centrifugation at 1000 x g for 10 min. The cells were resuspended in 20 mM Tris + 2 mM CaCl2- Proteinase K was added to 1 ⁇ g/ml and incubated at 37°C for 7.5 min. The cells then were washed with PBS + 0.02% glycine, followed by two more washes with PBS. For negative control samples, E.
• coli cells in PBS were incubated with 100 ⁇ g/ml RNase A for 1 hour at room temperature. The cells then were washed twice with PBS. All samples were spun down and resuspended in 100 ⁇ l of hybridization cocktail without probe and allowed to pre-hybridize for 10 min at room temperature. The cells next were pelleted, and 20 ⁇ l hybridization cocktail with 20 pmol of Probe 7 (SEQUENCE ID. NO. 7 ) was added to the cell pellet and allowed to hybridize for 3 hours at 38°C.
• Probe 7 SEQUENCE ID. NO. 7
• Hybridization cocktails with 50% formamide (standard for DNA hybridization), various concentrations of urea or no denaturing agents were tested by the methods described herein.
• the compositions of various hybridization cocktails are shown in TABLE 3.
• the pu ⁇ ose of using denaturing agent in DNA probe hybridizations is to make the target accessible to the probe.
• the targets double stranded or single stranded but with complicated secondary structure, have low hybridization efficiencies if denaturing agents such as formamide are not used.
• PNAs have a much stronger binding efficiency to the target and can unwind double stranded DNA upon strand-displacement (Egholm, M. et al., _ Am. Chem. Soc. 114:9677-9678, (1992); Cherny, D. Y. el al., Proc. Natl. Acad. Sci. U. S. A. 90:1667, (1993)).
• BSA + lOOug/ml Salmon sperm DNA Eub338 probe (SEQUENCE I. D. NO 7), dissolved in different compositions described in TABLE 5 was added to E. coli and the negative controls (E. coli treated with RNase). After hybridization as described in Experiment 3, samples were analyzed on flow cytometer.
• Yeast cells were grown in yeast peptone dextrose (YPD, DIFCO Laboratories, Detroit, MI) medium overnight at 29°C and S. aureus was grown in trypticase soy broth medium (TSB) (DIFCO Laboratoreis, Detroit, MI) overnight at 35°C
• TLB trypticase soy broth medium
• the cells were collected by centrifugation at 2500 ⁇ m for 8 min and washed with PBS three times. Cells were fixed and treated as described in Example 3.
• One (1) x 10* cells were used in each sample.
• Twenty (20) pmol of the universal probe (SEQUENCE I.D. NO. 6 ) which hybridized to all 16S like rRNA was dissolved in hybridization buffer (PBS + 0.5% SDS), applied to each sample (10 8 cells) and allowed to hybridize. After post-hybridization washes with PBS, the cells were analyzed as described in Example 3.
• Probe 4 (SEQUENCE I.D. No. 4) complementary to mammalian 28S rRNA
• Probe 9 (SEQUENCE I.D. No. 9) complementary to S. Aureus 16S rRNA found only in (position 78-93)
• Probe 6 (SEQUENCE I.D. No. 6) complementary to the conserved region of microbial 16S rRNA (position 1392-1406)
• Probe 7 (SEQUENCE I.D. No. 7) complementary to conserved region of eubacterial 16S rRNA (338-352)
• Probe 8 (SEQUENCE I.D. No. 8) complementary to conserved region of eukaryotic 16S rRNA (position 1209-1223)
• Probe 7 0.106 1.074 10.1 (SEQ I.D. No. 7)
• A. In vitro infection Three (3) ml of 6% dextran was added to 10 ml of human blood obtained from a healthy donor and incubated at 37°C for 30 min. The PMN- rich layer then was collected. It was incubated with a preparation of£. coli previously prepared by incubating 10 ml of an E. coli solution (having an absorbance of 0.1 at 600 nm wavelength, incubated for 30 min at 37°C, having the supernatant decanted and using the remaining layer of bacteria at the bottom of the flask) in PBS for 10 min at 37°C in a tissue culture flask.
• the cell monolayer was collected, washed with PBS and deposited onto microscopic slides using a Cytospin centrifuge at 800 x g for 5 min. Cells deposited on the slides were fixed with 4% paraformaldehyde for 15 min at room temperature and then stored in 70% ethanol at 4°C until use.
• mice obtained from Charles River Laboratory, Wilmington, MA
• Blood from mice was collected by cardiac puncture at various times (5 min, 15 min, 1 hour, 2 hours, 3 hours, 6 hours, 8 hours and 12 hours) after inoculation.
• the blood was separated using 6% dextran gradient, washed and fixed as described in the above text.
• In situ hybridization was performed using Probe 7 (SEQUENCE I.D. NO. 7) as described above.
• PMNs isolated from infected mice were hybridized with fluorescein-labeled PNA probes targeting bacterial 16S rRNA.
• Fluorescein signal was collected with FTTC visual filter cube for Nikon microscope (Fryer Co.Inc, Huntley, EL) with 480/30 nm exiciter filter, 505 DCLP dichroic mirror and 535/40 nm emitter filter.
• FIGURE 3 clearly shows that bacteria E. coli was brightly stained with FTTC-labeled PNA probe (SEQUENCE I.D. NO. 7) six hours after the mice had been inoculated with bacteria E. coli. Also, at the inoculum level of 1 x 10*5 E. coli per mouse, bacteria in the bloodstream were detectedwith the PNA-FTSH technique five minutes through 12 hours after inoculation, when the mice started to die.
• the PNA-FISH assay thus can be used for the identification of gram positive, gram negative bacterial or fungal infections, in a very short turn-around time (4-5 hours). This technique thus is very sensitive and provides valuble information to the physicians for the diagnosis of becteremia and sepsis.
• A. In situ Hybridization As described in Example 1, mo ⁇ holino oligos can be used as probes for fluorescence in situ hybridization.
• a mo ⁇ holino oligo (SEQUENDE I.D. NO. 4) is labeled with fluorescein at the amino-end of the peptide and is used to detect the complimentary sequence of 28S rRNA following the FISH assay as disclosed hereinabove in Example 1.
• the optimal mo ⁇ holino hybridization conditions are determined as described in Examples 1, 2, 3, 4 and 5.
• EPICS®Profile II Coulter Co ⁇ ., Hialeah, Florida
• flow cytometer equipped with an Argon laser, is used to detect the probe. The laser is set to 15mW at 488nm, with fluorescein fluorescence acquired using the light selection filter of 525/30 nm (central wavelength/full bandwidth at half-maximal transmission).
• Mo ⁇ holino oligos e.g., SEQUENCE I.D. NO. 4
• DNA or RNA complementary nucleic acid
• Bacteremia is a serious and urgent disease caused by the emergence and habitancy of various bacteria in blood. An individual may die within several hours to several days. Clinicians need rapid test results to confirm blood infection, to identify the invading organism and to administer an effective antibiotic to the individual.
• blood infections are tested using blood culture bottle method followed by gram stain. The current procedures take a day to a week, and sometimes even longer, for organisms that are hard to culture. Furthermore, false negative culture data can result in individuals who receive antibiotic therapy prior to or during sample collection.
• PNA-FISH technique we developed, we can detect bacteria in phagocytic cells in peripheral blood. Polymo ⁇ honuclear neutrophils (PMNs) form our first line of defense against invading bacteria.
• PMNs Polymo ⁇ honuclear neutrophils
• NAME POREMBSKI, PRISCILLA E.
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)

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