WO1994002645A1 - Rapid detection of biopolymers in stained specimens - Google Patents

Rapid detection of biopolymers in stained specimens Download PDF

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Publication number
WO1994002645A1
WO1994002645A1 PCT/US1993/006732 US9306732W WO9402645A1 WO 1994002645 A1 WO1994002645 A1 WO 1994002645A1 US 9306732 W US9306732 W US 9306732W WO 9402645 A1 WO9402645 A1 WO 9402645A1
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Prior art keywords
biopolymer
group
probe
cells
specimen
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PCT/US1993/006732
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French (fr)
Inventor
William Dugald Weber
Joel Bresser
Mark Blick
Shyh-Chen Ju
Michael Lee Cubbage
Morteza Asgari
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Aprogenex, Inc.
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Application filed by Aprogenex, Inc. filed Critical Aprogenex, Inc.
Priority to AU47752/93A priority Critical patent/AU4775293A/en
Publication of WO1994002645A1 publication Critical patent/WO1994002645A1/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6841In situ hybridisation

Definitions

  • the present invention relates to the field of in situ hybridization. More specifically, the present invention relates to the use of in situ hybridization assays as a means to detect as few as 1 copy of cellular biopolymer in a stained cell.
  • in situ hybridization provides a technique for the determination and quantitation of biopolymers such as nucleic acids (DNA and RNA) and proteins in tissues at the single cell level. Such hybridization techniques can detect the presence or absence of specific genes in tissues at the single cell level. In situ hybridization procedures may also be utilized to detect the expression of gene products at the single cell level.
  • RNA or DNA probes genetic markers for infection and other disease states may be detected. Certain genetic diseases are characterized by the presence of genes which are not present in normal tissue. Other diseased conditions are characterized by the expression of RNAs or RNA translation produ ⁇ ts (i.e. peptides or proteins) which are not expressed in normal cells. Some disease states are characterized by the absence of certain genes or gene portions, or the absence or alteration of expression of gene products or proteins. Current methods allow the detection of these markers but are relatively time consuming and of limited sensitivity. Hybridization techniques are based on the ability of single stranded DNA or RNA to pair, i.e., hybridize, with a complementary nucleic acid strand. This hybridization reaction allows the development of specific probes that can identify the presence of specific genes (DNA), or polynucleotide sequences or the transcription and expression of those genes (mRNA).
  • DNA specific genes
  • mRNA polynucleotide sequences or the transcription and expression of those genes
  • In situ hybridization allows the detection of RNA or DNA sequences within individual cells. In situ hybridization yields greater sensitivity than solution hybridization by means of eliminating the dilution of a particular target gene, nucleic acid, or protein by the surrounding and extraneous RNA and DNA of other cells. In situ hybridization also allows for the simultaneous detection of multiple substances, i.e. genes, nucleic acids or proteins within individual cells, permitting the identification of a particular cell expressing a cellular gene or viral sequence. In addition, since in situ hybridization analysis is performed and quantitated for single cells, minimal sample and reagent is required.
  • in situ hybridization procedures were only capable of detecting nucleic acids present at greater than ten copies per cell.
  • 16S DNA sequence segments can serve as molecular probes, which will allow for species identification of any bacterium, whether viable, actively growing or non-viable, dormant organisms.
  • the papanicolaou stain is a common stain used on cells obtained from cervical specimens. Generally, only one slide is prepared from a patient and this slide is stained. If, for example, there are cells present on the stained slide which one suspects for HPV infection, the prior art does not provide a method for removing the coverslip and analyzing those suspicious cells by using in situ hybridization. Accordingly, an additional specimen must be obtained presenting the potential for missing the lesion in a subsequent sampling.
  • a method of detecting the presence of a biopolymer in a previously stained specimen having substantially intact cellular membranes comprises assaying for the presence of cellular biopolymers.
  • the specimen may be heat fixed or fixed with a medium comprising a precipitating agent and/or a cross-linking agent.
  • the fixed cells are contacted with a hybridization solution.
  • the hybridization solution consists of a denaturing agent, hybridized stabilizing agent, buffering agent, a selective membrane performing agent and at least probe having a nucleotide sequence at least substantially complementary to a specific target nucleotide to be detected.
  • the sample of cells is then incubated with the hybridization solution in the presence of at least one detectable label.
  • This method is capable of detecting as few as a single biopolymer per cell.
  • a "one-step" method for the detection of the presence of biopolymers in a previously stained specimen having substantially intact membranes comprising assaying for the presence of cellular biopolymers. Initially, the specimen is contacted with a medium comprising a denaturing agent, hybridized stabilizing agent, buffering agent, a membrane pore forming agent and at least one probe. Optimally, the medium may contain a fixative agent. The specimen is contacted under hybridizing conditions. Subsequently, the sample of cells is incubated with the medium in the presence of at least one detectable label. Duplex or triplex formation is detected by detecting the label without performing a pre-hybridization step for blocking non ⁇ specific binding of the probe and facilitating probe entry. This method is capable of detecting a single copy of a target biopolymer per cell.
  • kits for determining the presence of a biopolymer in a previously stained specimen having substantially intact cellular membranes may be used for assaying cellular biopolymers.
  • the kit comprises a hybridization solution comprising a denaturing agent, a hybrid stabilizing agent, a buffering agent, a membrane pore forming agent and optimally, a fixative agent.
  • the kit may comprise a supply of a probe selected so that the probe will hybridize with the suspect biopolym to form a hybridized complex.
  • the kit may comprise a means for contacting said suspect specimen with the probe to form a hybridized complex and a means for measuring the presence of the labeled probe.
  • Figure 1 demonstrates the detection of HPV in a cervical cell previously stained with a papanicolaou stain.
  • Figure 2 demonstrates the simultaneous detection and differentiation of HPV types 16 and 18, in cells previously stained with a papanicolaou stain.
  • Figure 3 demonstrates the detection of HPV in cervical tissue previously stained with hematoxylin and eosin.
  • Figure 4 demonstrates the detection of chromosomal DNA within cells previously stained by the Diff-Quick method.
  • novel methods of the present invention may be used to detect a wide variety of microorganisms that are the cause of various pathophysiological state
  • the methods of the present invention may be used to detect microorganisms that are the cause of pathophysiological states such as bacteremias, sexually transmitted diseases, diarrhea and respiratory diseases.
  • the novel methods of the present invention may be used to detect a wide variety of bacteria, viruses and fungi.
  • bacteria detectable by the methods of the present invention include Streptococcus, Staphylococcus, Clostridium, Bacillus, Pseudomonas, Salmonella, Klebsiella, Bacteroides, Escherichia coli, Neisseria gonorrhea, and Chlamydia.
  • fungi include Candida, Cryptococcus neoformans, Blastomyces dermatitides, Histoplasma capsulatum, Coccidioides immitis and Paracoccidioides brasiliensis.
  • detectable viruses include human papilloma herpes simplex virus II, hepatitis, human immunodeficiency virus, influenza virus, parainfluenza virus and rota virus.
  • spirochetes include Treponema pallidum,
  • protozoa include Entamoeba histolytic, Balantidium coli, Giardia lamblia, Leishmania tropica, specie of Plasmodium, Trichomonas vaginalis and species or Trypanosoma.
  • the present methods may also be used to detect and quantitate the presence of cellular genes from previously stained mammalian specimens.
  • Representative examples of such cellular genes include oncogenes, tumor suppressor genes and growth stimulating factors.
  • oncogenes are neu (c-erb-B-2), c-ras, c-myc, and c-myb.
  • the present methods may be used to detect and quantitate tumor suppressor genes an stimulating growth factors. Examples of tumor suppressor genes include p53 and retinoblastoma.
  • growth factors include colony simulatin factor-granulocyte/macrophage (CSF-GM), transforming growth factor (TGF- ⁇ ), an epidermal growth factor (EGF).
  • CSF-GM colony simulatin factor-granulocyte/macrophage
  • TGF- ⁇ transforming growth factor
  • EGF epidermal growth factor
  • the cell specimens include the following representative examples: cervi cells, bone marrow cells, hepatocytes, cerebrospinal fluid cells, blood cells, oral mucosa cells, lung cells and skin cells.
  • Representative examples of tissue specimen include lymph node tissue, mammary tissue, cervical tissue, colon tissue, prostrate tissue, cardiac tissue and brain tissue.
  • fluids and exudates
  • the cells and tissue specimens used in the method of the present invention may have been stained by any of the commonly used stains in cytology or histology.
  • Examples of representative stains include a papanicolaou stain, a Wright stain, a Hematoxylin and Eosin stain and Diff-Quick.
  • the first step in the in situ hybridization procedures could be the deposition of specimens onto a solid support.
  • Specimens constitute any material which is composed of or contains cells or portions of cells.
  • the cells may be livin or dead, so long as the target biopolymer, i.e., DNA or mRNA, is largely unaltere and undamaged to the extent that it is capable of detection.
  • the specimen should contain cells with substantially intact membranes. Although it is not necessary that a membranes of the cellular structure be intact, the membranes must be sufficiently preserved to allow: retention of the target biopolymer and introduction of the detecting probe to the site of the target biopolymer.
  • Techniques for depositing the specimens on the solid support will depend upon the cell or tissue type, and they may include, for example, standard sectioning of tissue or smearing or cytocentrifugation of single cell suspensions.
  • Supports which may be utilized include, but are not limited to, glass, Scotch tape
  • tissue samples are broken apart by physical, chemical or enzymatic means into singl cell suspension.
  • a single solution is added to the cells/tissues (hereafter referred to as the specimen).
  • This solution contains the following: optionally, a mild fixative, a chaotrope or other denaturing agent, a synthetic oligonucleotide probe (RNA or DN probe which is prelabeled) and/or antibody probe, salts, detergents, buffers, and blocking agents.
  • the incubation in this solution is carried out at 40°C to 60°C for to 30 minutes.
  • the hybridization procedure is carried out utilizing a single hybridization solution.
  • a mild fixative is included in the solution which also fixes the cells. This fixation is accomplished in the same solution and along with the hybridization reaction.
  • the fixative is one which has been found to b optimal for the particular cell type being assayed (eg., there is one optimal fixative f bone marrow and peripheral blood even though this "tissue" contains numerous distinct cell types).
  • the fixative may be a combination of precipitating fixatives (suc as alcohols) and cross-linking fixatives (such as aldehydes), with the concentration of the cross-linking fixatives kept very low (less than 10%).
  • concentration and typ of precipitating agent and crosslinking agent may be varied depending upon the prob and the stringency requirements of the probe, as well as the desired temperature of hybridization.
  • the precipitating fixative can affect cellular constituents and cellular morphology; such effects can be tissue specific.
  • the precipitating fixative and has the following characteristics: fixes the cellular constituents through a precipitating action; the effect is reversible, the cellular morphology is maintained, the antigenicity of desired cellular constituents is maintained, the nucleic acids are retained in the appropriate location in the cell, the nucleic acids are not modified in such a way that they become unable to form double or triple stranded hybrids, and cellular constituents are not affected in such a way so as to inhibit the process of nucleic acid hybridization to all resident target sequences.
  • fixatives for use in the invention are selected from the group consisting o ethanol, ethanol-acetic acid, methanol, and methanol-acetone.
  • Fixatives most preferable for practicing the one-step procedure include 10-40% ethanol, 10-40% methanol, 10-40% acetone or combinations thereof. These fixatives provide good preservation of cellular morphology and preservation and accessibility of antigens, a high hybridization efficiency.
  • the solution contains 1-40% ethanol, and 5 formalin. Fixation of Cells/Tissues in the Two-Step Method
  • a fixative may be selected from the group consisting of any precipitating agent or cross-linking agent used alone or in combination, and may be aqueous or non-aqueous.
  • the fixative may be selected from the group consisting of formaldehyde solutions, alcohols, salt solutions, mercuric chloride sodium chloride, sodium sulfate, potassium dichromate, potassium phosphate, ammonium bromide, calcium chloride, sodium acetate, lithium chloride, cesium acetate, calcium or magnesium acetate, potassium nitrate, potassium dichromate, sodium chromate, potassium iodide, sodium iodate, sodium thiosulfate, picric acid, acetic acid, paraformaldehyde, sodium hydroxide, acetones, chloroform, glycerin and thymol.
  • the fixative will comprise an agent which fixes the cellular constituents through a precipitating action and has the following characteristics: the effect is reversible, the cellular morphology is maintained, the antigenicity of desired cellular constituents is maintained, the nucleic acids are retained in the appropriate location in the cell, the nucleic acids are not modified in such a way that they become unable to form double or triple stranded hybrids, and cellular constituents are not affected in such a way so as to inhibit the process of nucleic acid hybridization to all resident target sequences.
  • fixatives for use in the invention are selected from the group consisting of ethanol, ethanol-acetic acid, methanol, and methanol-acetone which fixatives afford the highest hybridization efficiency with good preservation of cellular morphology.
  • the fixative may contain a compound which fixes the cellular components by cross-linking these materials together, for example, glutaraldehyde or formaldehyde.
  • the cross-linking agent is generally more "sticky” and causes the cells and membrane components to be secured or sealed, thus, maintaining the characteristics described above for fixatives.
  • the cross linking agents when used are preferably less than 10% (v/v).
  • Cross-linking agents while preserving ultrastructure, often reduce hybridization efficiency by forming networks trapping nucleic acids and antigens and rendering them inaccessible to probes and antibodies. Some also covalently modify nucleic acids preventing later hybrid formation. Examples of cross-linking agents include paraformaldehyde, formaldehyde, dimethylsilserimidate and ethyldimethylamino-propylcarbodimide. Storage of Cells/Tissues
  • microscope slides and other solid supports containing specimens may be stored air dried at room temperature for up to six months, in cold (4°C) 70% ethanol in water for 6-12 months. If specimens are handled under RNAse free conditions, they can be dehydrated in graded alcohols and stored for at least 12 months at room temperature.
  • Nucleic acid hybridization is a process where two or more duplex or triplex mirror images or opposite strands of naturally occurring or synthetic DNA, RNA, oligonucleotides, polynucleotides, or any combination thereof recognize one another and bind together through the formation of some form of either spontaneous or induced chemical bond, usually a hydrogen bond.
  • the degree of binding can be controlled based on the types of nucleic acids coming together, and the extent of "correct" binding as defined by normal nucleic acids coming together, and the extent of "correct” binding as defined by normal chemical rules of bonding and pairing. For example, if the binding of two strands forms 9 out of 10 correct matches along a chain of length 10, the binding is said to be 90% homologous.
  • Cellular nucleic acid sequences are detected by the process of molecular hybridization.
  • the probe must be “labeled” in some way so to allow “detection” of any complementary cellular nucleic acid sequences present within the individual cells.
  • hybridization also means the binding of an antibody to a target antigen.
  • the hybridization cocktail contains a denaturing agent, usually formamide, but other chaotropic agents such as Nal, urea, thiocyanate, guanidine, trichloroacetate, tetramethylamine and perchlorate may also be used. Furthermore, several precipitating and/or cross-linking fixatives also have mild denaturing properties; these properties can be used in conjunction with the primary denaturant in either an additive or synergistic fashion.
  • the hybridization cocktail may be constructed to preferentially allow only the formation of RNA-RNA or RNA-DNA hybrids.
  • the hybridization solution may typically comprise a chaotropic denaturing agent, a buffer, a pore-forming agent, a hybrid stabilizing agent.
  • the chaotropic denaturing agents include formamide, urea, thiocyanate, guanidine, trichloroacetate, tetramethylamine, perchlorate, and sodium iodide. Any buffer whic maintains pH at least between 7.0 and 8.0 may be utilized.
  • the pore-forming agent is, for instance, a detergent such as Brij 35,
  • the pore-forming agent is chosen to facilitate probe entry through plasma', nuclear membranes or cellular compartmental structures. For instance, 0.05% Brij 35 or 0.1 % Triton X-100 will permit probe entry through the plasma membrane but not the nuclear membrane. Alternatively, sodium deoxycholat will allow probes to traverse the nuclear membrane. Thus, in order to restrict hybridization to the cytoplasmic biopolymer targets, nuclear membrane pore-formin agents are avoided. Such selective subcellular localization contributes to the specificity and sensitivity of the assay by eliminating probe hybridization to complementary nuclear sequences when the target biopolymer is located in the cytoplasm. Agents other than detergents, such as fixatives, may serve this function
  • Hybrid stabilizing agents such as salts of mono- and divalent cations included in the hybridization solution to promote formation of hydrogen bonds between complementary sequences of the probe and its target biopolymer.
  • Preferab sodium chloride at a concentration from 0.15 M to 1 M is used.
  • nucleic acids unrelated to the target biopolymers are added to the hybridization solution.
  • Representative examples of hybrid stabilizing agents include sodium chloride, lithium chloride and magnesium.
  • a probe is defined as genetic material DNA, RNA, or oligonucleotid or polynucleotides comprised of DNA or RNA and antibodies.
  • the DNA or RNA may be composed of the bases adenosine, uridine, thymidine, guanine, cytosine, or any natural or artificial chemical derivatives thereof.
  • the probe is capable of dupl or triplex hybrid formation by binding to a complementary or mirror image target cellular genetic sequence. Binding occurs through one or more types of chemical bonds, usually through hydrogen bond formation. The extent of binding is referre as the amount of mismatch allowed in the binding or hybridization process.
  • the extent of binding of the probe to the target naturally occurring or synthetically produced cellular sequences also relates to the degree of complementarity to the tar sequences.
  • the size of the probe is adjusted to be of such size that it forms stable hybrids at the desired level of mismatch; typically, to detect a single base mismatch requires a probe of approximately 12-50 bases. Larger probes (from 50 bases up to tens of thousands of bases) are more often used when the level of mismatch is measured in terms of overall percentage of similarity of the probe to the target gen sequence.
  • the size of the probe may also be varied to allow or prevent the probe from entering or binding to various regions of the genetic material or of the cell. Similarly, the type of probe (for example, using RNA versus DNA) may accomplis these objectives.
  • the size of the probe also affects the rate of probe diffusion, probability of finding a cellular target match, etc.
  • double-stranded DNA dsDNA
  • single-stranded DNA ssDNA
  • RNA probes are used in a hybridizatio reaction when nucleotide sequences are the target. Preparation of Probes.
  • RNA or DNA probes useful in the present invention may be prepare according to methods known to those of skill in the art or may be obtained from an commercial source. RNA probes may be prepared by the methods described by
  • DNA probes may be prepared by methods known those of skill in the art such as described by Rigby et al. (1977) J. Mol. Biol. 113:237. Synthetic oligonucleotide probes may be prepared as described by Wallac et al (1979) Nucleic Acids Research 6:3543 or as recommended by the suppliers (e. ABI) of the nucleic acid synthesizers.
  • the probes useful in the present invention m be prepared according to the methods disclosed in copending patent applications, U. Serial No. 784,690, filed October 28, 1991, and Serial No. 668,751, filed March 1 1991.
  • Nucleic acid probes can be prepared by a variety of methods known those of skill in the art. Purified double-stranded sequences of DNA (dsDNA) can labeled intact by the process of nick translation or random primer extension. The ability of double-stranded probes to hybridize to nucleic acids immobilized within c is compromised by the ability of the complementary strands to hybridize to each ot in solution prior to hybridization with the cellular nucleic acids. Single-stranded D (ssDNA) probes do not suffer this limitation and may be produced by the synthesis oligonucleotides, by the use of the single-stranded phage M13 or plasmid derivative ⁇ ⁇
  • ssRNA single-stranded RNA
  • Antibody probes are known to those skilled in the art.
  • the term "antibody probe” means an antibody that is specific for and binds to any target antigen.
  • a target antigen may be a peptide, protein, carbohydrate or any other biopolymer to which an antibody will bind with specificity.
  • Antibody probes specific for antigens such as viruses or specific determinants thereof, peptides and proteins derived from a variety of sources, carbohydrate moieties and a wide variety of biopolymers are known to those of skill in the art.
  • the methods for preparation of such antibodies are also known to those of skill in the art.
  • polyclonal antibodies may be prepared by immunization of an animal host with an antigen.
  • the antigen is administered to the host subcutaneously at weekly intervals followed by a booster dose one month after the final week dose.
  • the serum is harvested, antibodies precipitated from the serum and detectably labeled by techniques known to those of skill in the art.
  • Monoclonal antibodies my be prepared according to any of the methods known to those in the art. Fusion between myeloma cells and spleen cells from immunized donors has been shown to be a successful method of producing continuous cell lines of genetically stable hybridoma cells capable of producing large amounts of monoclonal antibodies against target antigens such as, for instance, tumors and viruses. Monoclonal antibodies may be prepared, for instance, by the method described in U.S. Patent No. 4,172,124 to Koprowski, et al. or according to U.S Patent No. 4,196,265 to Koprowski, et al. Procedures for labeling antibodies are known to those skilled in the art. Detection Systems
  • Probes may be detectably labeled prior to addition to the hybridization solution. Alternatively, a detectable label may be selected and added after hybridization is completed and binds to the hybridization product. Probes may be labeled with any detectable group for use in practicing the invention. Such detectabl group can be any material having a detectable physical or chemical property. Such detectable labels have been well-developed in the field of immunoassays and in general most any label useful in such methods can be applied to the present invention Particularly useful are enzymatically active groups, such as enzymes (see Clin. Chem.. 22: 1243 (1976)); enzyme substrates (see British Pat. Spec. 1,548,741), coenzymes (see U.S. Patents Nos.
  • Biotin labeled nucleotides can be incorporated into DNA or RNA by nick translation, enzymatic, or chemical means. The biotinylated probes are detected after hybridization using avidin/streptavidin, fluorescent, enzymatic or colloidal gold conjugates. Nucleic acids may also be labeled with other fluorescent compounds, wi immunodetectable fluorescent derivatives or with biotin analogues. Nucleic acids cross-linked to radioactive or fluorescent histone HI, enzymes (alkaline phosphatase and peroxidases), or single-stranded binding (ssB) protein also may be used. To increase the sensitivity of detecting the colloidal gold or peroxidase products, a number of enhancement or amplification procedures using silver solutions may be used.
  • the length of a probe affects its diffusion rate, the rate of hybrid formation, and the stability of hybrids.
  • small probes (15-100 bases) yield the most sensitive, rapid and stable system.
  • a mixture short probes (15-100 bases) are prepared which span the entire length of the target biopolymer to be detected. For example, if the target biopolymer were 1000 bases long, about 40 "different" probes of 25 bases would be used in the hybrid solution t completely cover all regions of the target biopolymer.
  • the concentration of the probe affects several parameters of the in sit hybridization reaction. High concentrations are used to increase diffusion, to reduce the time of the hybridization reaction, and to saturate the available cellular sequence To achieve rapid reaction rates while maintaining high signal-to-noise ratios, probe concentrations of 0.01 to 100 ⁇ g/ml of hybridization solution are preferable. Most preferable is use of probes at a concentration of 2.5 ⁇ g/ml.
  • the hybridization solution of the one-step in situ method consists of 25% formamide, 5X SSC, 15 X Ficoll/PVP, .4M guanidinium isothiocyanate, about 50 mM sodium phosphate (Ph 7.4), 50 mM DTT, about 1 mg/ml salmon sperm DNA, 5% Triton X-100, 50 mM EDTA and 21% PE
  • a synthetic oligonucleotide probe is added to this solution. The probe may be at lea
  • the most preferable, optimal temperature of hybridization is 40°-45°C. However, temperatur ranging from 15 °C to 80°C may be used.
  • the probe in the hybridization cocktail may be labeled before the hybridization reaction.
  • the label may be one of the many types described above.
  • the hybrids may be detected by use of a Streptavidin/Avidin (S/A) conjugated to a fluorescent molecule such as FITC, rhodamine, Texas RedTM; to S/A conjugated to a enzyme; or to S/A labeled with a heavy metal such as colloidal gold.
  • S/A Streptavidin/Avidin
  • a fluorescent molecule such as FITC, rhodamine, Texas RedTM
  • S/A conjugated to a enzyme or to S/A labeled with a heavy metal such as colloidal gold.
  • solution containing the streptavidin conjugate is added directly to the hybridization cocktail over the cells after the end of the hybridization reaction. The cells are incubated in this solution for 5 to 30 minutes at 45 °C.
  • the time of hybridization reaction will vary depending on the composition of the hybridization cocktail containing the fixative (type and concentrations of precipitating agents and/or cross-linking agents), buffering agents, pore forming agents, denaturing agents and hybrid stabilizing agents. Similarly, the temperature may be varied as described.
  • the probes may be directly labeled with the fluorescent dye or molecules such as Pontamine Sky BlueTM by incubating the nucleic acid probe and dye together (1:10 weight: weight proportions) and allowing the dye to bind/intercalate. The probe is then precipitated out of the dye solution and the exces unbound dye is removed by repeated washing with 70% ethanol. Probes also are labeled directly and covalently by incubation of double stranded molecules (RNA-RNA, RNA-DNA, or DNA-DNA) with labels which will covalently bind to nucleic acids. After incubation conditions under which the reaction will take place, the strands are separated and each separate strand is used as a probe.
  • the fluorescent dye or molecules such as Pontamine Sky BlueTM
  • the concentration of the probe in the solution is typically 2.5 ⁇ g/ml although a range of 0.01-100 ⁇ g/ml is useful.
  • the probe concentration will affect the reaction kinetics and may affect the sensitivity of the assay along with the signal-to-noise ratio.
  • this reaction may be carried out before or after any wash steps.
  • the slide is incubated with the substrates for the enzyme under conditions specified by the manufacturer or supplier of the enzyme.
  • Cells may be deposited onto slides or centrifuged into a pellet following the fixation/hybridization/ detection reaction(s).
  • the unbound probe washed away from the cells by one wash step using a solution of 0.1 x SSC with 0.1 % Triton X-100TM.
  • a total of 1-200 ml of wash solution may be used per microscope slide (i.e., per about 100,000 separated cells or per tissue section of abo 1 square centimeter).
  • concentration and type of the hybrid stabilizing/denaturin agents and pore forming agents may be varied depending on the type of cells, the ty of probe and the acceptable level of mismatch of the hybrid. Results obtained using the one-step or two-step method.
  • results are visualized manually on a fluorescent microscope when direct or indirectly labeled fluorescent probes are utilized.
  • the results may be automatically analyzed on a fluorescence-based image analysis system.
  • results may be obtained using a flow cytometer to record the amount of fluorescence per cell, which represents the amount of hybrid per cell.
  • the total signal within a cellular sample may be determined using a device such as a liquid scintillation counter (for radioactivity) or a chemluminescent/fluorescent microtiter plate reader for these labels.
  • the present invention requires as little as 5 minutes to complete with a sensitivity of as few as 1 molecule of a cellular biopolymer per cell.
  • the speed and sensitivity result from the combination of at least four factors: 1) cellular constituents are not irreversibly precipitated or fixed onto the nucleic acids, 2) if fixed, the fixation was optimized for the particular tissue used 3) the kinetics of the reaction proceed more rapidly at high probe concentrations and at elevated temperatures, and 4) probes were constructed to facilitate rapid entry and exit through cellular membranes and cellular components.
  • the number of copies of mRNA or DNA per cell can be estimated from the number of grains over cells when radioactive probes are used.
  • the sensitivity of the in situ hybridization techniques of the present invention permit the visual and photographic detection of a single copy of a genetic sequence present within a single cell.
  • a genetic sequence of approximately 6,000 bases can be reliably detected by viewing the results through a standard fluorescent microscope.
  • four fluorophors are attached to a single probe, the presence of absence of a particular genetic sequence of approximately 1500 bases can be reliably detected, using the period of incubation taught herein.
  • probes for corresponding sequences from both the "sense” and the "anti-sense” strands of a two-stranded nucleic acid when using probes for corresponding sequences from both the "sense” and the "anti-sense” strands of a two-stranded nucleic acid, one can detect the presence or absence of a sequence as short as approximately 750 base paris using such periods of incubation described herein. Alternatively, one may detect as few as 75 to 150 base pairs using an image analysis system. Simultaneous Detection of Three mRNAs
  • Both the manual and one-step in situ hybridization procedures allow simultaneous detection of different substances (mRNAS, DNAs and proteins) within the same cells. This may be accomplished in one of two ways. First, multiple probes each containing a unique label (for example, fluorescent tags "A”, "B” and “C" which each emit light at a different detectable wave length) are all added together in the hybridization solutions. Alternatively, a hybridization and detection reaction may be carried out with one probe and label, residual unreacted probe and label washed away, and another hybridization reaction is carried out. This process is repeated as many times as desired. Alternatively, probes A, B and C all contain the same tag.
  • a unique label for example, fluorescent tags "A”, "B” and "C" which each emit light at a different detectable wave length
  • a hybridization and detection reaction may be carried out with one probe and label, residual unreacted probe and label washed away, and another hybridization reaction is carried out. This process is repeated as many times as desired.
  • probes A, B and C all
  • the one-step in situ hybridization technique for the detection of DNA or mRNA may be provided as a kit.
  • a kit includes the following:
  • a solution containing a fixation/hybridization cocktail and one or mor labeled probes will contain 50 mM guanidinium isothiocyante, 25-40% formamide, 21% PEG, 0.4 M
  • the kit may also include: 1. A second detectable reporter system which would react with the probe or the probe-target hybrid.
  • Any mechanical components which may be necessary or useful to practice the present invention such as a solid support (e.g. a microscope slide), an apparatus to affix cells to said support, or a device to assist with any incubations or washings of the specimens.
  • a photographic film or emulsion with which to record results of assa carried out with the present invention may include a solution of probes encapsulated in liposom or microspheres.
  • Cervical cells obtained using a cervical brush were placed in the transpo medium. Upon arrival in the laboratory, the cells were vortexed for 1-3 seconds, the spun at 1500 rpm for 10 minutes. The supernatant was discarded and the cell resuspended in 1 ml of the transport medium. The cells were spotted directly o organosilane treated slides at the desired density. Cells were then stained using th standard papanicolaou staining procedure. Dip slides 5 times in H 2 O. Stain 10 seconds in Gills formulation # followed by 10 dips in deionized water. Dip 10 times in 95% ETO
  • the positive control probe consisted of a human alpha-centromeric repe DNA, known to hybridize to all human chromosomes.
  • the negative probe designate NR, was derived from the nitrogen reductase gene found in bacteria and was known not hybridize to nucleic acid within eukaryotic cells.
  • the sequences for HPV type 16 a HPV type 18 were obtained from the published sequences and were accessed via t
  • the oligodeoxynucleotides were synthesized (Applied Biosystems D Synthesizer Model 380 B using the recommended A.B.I, reagents), and in the last st an aminohexyl phosphate linker was attached to the 5' end.
  • the 5 '-aminohexyl oligodeoxynucleotides were then coupled to a fluorescent rhodamine derivative (Cat. # L-20) from Molecular Probes, Inc. and purified by Waters HPLC using a baseline 810 chromatography work station.
  • hybridization procedure 50 ⁇ l of an hybridization cocktail consisting of 22% PEG, 30% formamide, 5X SSC, .3 mg/ml salmon sperm DNA, 15X Ficoll/PVP, .4M guanidinium isothiocyanate, 50mM DTT, 5% Triton X-100, 50mM EDTA, 7.5% Tween 20, 50mM Na 2 PO 4 , and probe at a concentration of .02 ⁇ g/ ⁇ l was added to the slide. A coverslip was applied and the slide was heated to 95° C for 5 minutes, allowed to cool to 42 °C and incubated for 25 minutes at that temperature. Washing
  • Photomicrographs were taken on an Olympus BH10 microscope with fluorescence capabilities, using Kodak Ektachrome EES-135 (PS 800/1600) film, exposed, and push processed at 1600 ASA. A 50-second exposure time was consistently used, so that direct comparisons could be made between all photomicrographs taken.
  • Figure 1 demonstrates a typical result when the HPV probes (labeled with Rhodamine) and the positive control probe (labeled with FITC) are added together in the same hybridization cocktail.
  • the nucleus of the smaller cell has stained positive for the positive control probe (in color this is green) while the larger cell nucleus is positive for
  • HPV in color this is bright yellow.
  • the probes for the first strand will be "out of phase" relative to the second strand probes as regards how they map on a map of the HPV genome.
  • one half (15 nucleotides) of each first strand probe will be complementary (in nucleotide sequence) to one half of one second strand probe and the other half (15 nucleotides) o that first strand probe will be complementary to a portion of another second strand probe. Staggering of the probes means that, because of the shortness of the overlap (15 nucleotides), probes of the first strand will not hybridize significantly to probes of th second strand. On the other hand, about twice as much hybridization is detected as compared to the situation where only probes corresponding to one strand are used.
  • Probes are made as phosphorothioate oligonucleotides, each 30-me having four sulfur atoms, using an Applied Biosystem (ABI) DNA Synthesizer, Mode 380B and the recommended ABI reagents.
  • the sulfur atoms are located as follows: on is at the extreme 5' end of the probe, a second is between the 7th and 8th nucleoside (counting from the 5' end), the third is between the 22nd and 23rd nucleosides, and th fourth is between the 29th and 30th nucleosides.
  • the sulfur atoms of the polysulfurize oligonucleotides are then coupled to a fluorescent dye, iodoacetamido-fluorescein, a follows (smaller amounts can be synthesized by adjusting the volumes): 200 ⁇ g of drie oligonucleotide is dissolved in 100 ⁇ l of 250 mM Tris buffer, pH 7.4 to form a firs solution. Then one mg of iodoacetamido-fluorescein is combined with 100 ⁇ l of dr dimethylformamide (i.e., 100 percent DMF) in a second solution. The two solutions ar mixed together and shaken overnight.
  • dr dimethylformamide i.e., 100 percent DMF
  • the hybridization cocktail is modified as follows: 1.5% PEG is used instead of 21 % PEG, 30% formamide is used instead of 21 % formamide, 10% DMSO (10% v/v) is included, and 5% (v/v) of vitamin E is included. Also instead of adding
  • solution B is dodecy alcohol.
  • Cervical cells obtained using a cervical brush are placed in the transpor medium. Upon arrival in the laboratory the cells are vortexed for 1-3 seconds, then spu at 1500 rpm for 10 minutes. The supernatant is discarded and the cells are resuspende in 1 ml of the transport medium. The cells are spotted directly on organosilane treate slides at the desired density. Cells are then stained using the standard papanicolao staining procedure as described in Example 1.
  • the positive control probe is as described in Example 1.
  • the sequence for a 7.4 kb plasmid that is a common component of the C genome and occurs in approximately 10 copies per genome is accessed via the Geneti Sequence Data Bank, GenBank, version 69.0. This entire 7.4 kb sequence is cut into 29 separate oligonucleotide probes consisting of 25 bases each. These 296 oligoniers a synthesized and labeled as described below.
  • GC For GC, three repetitive DNA elements are accessed via the Gene Sequence Data Bank, GenBank, version 69.0.
  • oligonucleotide probes consisting 25 bases each and are synthesized and labeled as described below.
  • oligodeoxynucleotides are synthesized (Applied Biosystems DNA
  • the 5 '-aminohexyl oligodeoxynucleotides are then coupled to a fluorochrome from Molecular Probes, Inc. and purified by Waters HPLC using a baseline 810 chromatography work station.
  • GC and CT probes can be labeled with different fluorescent moieties.
  • GC can be labeled with fluorescein while CT can be labeled with a rhodamine derivative.
  • Alternative fluorochromes, with catalogue numbers are listed above.
  • an hybridization cocktail consisting of 30% PEG, 30% formamide, 5X SSC, .3 mg/ml salmon sperm DNA, 15X Ficoll/PVP, .4M guanidinium isothiocyanate, 50mM DTT, 5% Triton X-100, 50mM EDTA, 50mM Na PO 4 , and probe at a concentration of .02 ⁇ g/ul is added to the slide.
  • a coverslip is applied and when DNA is the target, the slide is heated to 95 °C for 5 minutes, allowed to cool to 42°C and incubated for 25 minutes at that temperature. If mRNA is the target, the 95 °C heating step is omitted. Washing Post-hybridization, the slides are placed in a coplin jar to which is added
  • Photomicrographs are taken on an Olympus BH10 microscope wit fluorescence capabilities, using Kodak Ektachrome EES-135 (PS 800/1600) film exposed, and push processed at 1600 ASA. A 50-second exposure time is consistentl used so that direct comparisons could be made between all photomicrographs take Alternatively, commercially available low light monochrome and color cameras allow t operator to both visualize the results on a TV screen and make a permanent record in t form of a color print.
  • the CT and GC organisms stain brightly positive when the CT and GC speci probes are used, respectively.
  • HSV-ID Specific Sequences The sequence for HSV-II is accessed via the Genetic Sequence Data Ban
  • GenBank version 69.0.
  • GenBank version 69.0.
  • the sequences for the immediate early and early genes f HSV-II are cut into separate oligonucleotide probes consisting of 25 bases each.
  • the oligomers are synthesized and labeled as described in Example 1.
  • Hybridization. Washing and Fluorescence Detection Hybridization, washing and detection are crried out as described
  • HSV-II containing cells stain brightly positive when the HSV-II probe used
  • the C-33A (ATCC HTB #31), Ca Ski (ATCC CRL #1550) and Hela Ce were grown, trypsonized, mixed at a 1:1:1 ratio, and spotted directly on organosila treated slides at the desired density. Cells were then stained using the standar papanicolaou staining procedure and prepared for in situ hybridization as outlined i
  • HPV type 16 and HPV type 18 were obtained from th published sequences and were accessed via the Genetic Sequence Data Bank, GenBank, version 69.0 as described in Example 1.
  • Example 1 Fluorescein (Cat. # F-143) was attached to the HPV 16 probe and rhodamine derivative (L-20) to the HPV 18 probe.
  • Example 1 As in Example 1, the hybridization, washing and fluorescent detectio steps were performed.
  • Figure 2 depicts the three different cell types in a single photograph.
  • FITC labeled HPV-16 probes resulted in a positive signal (dots of light in the nucleus in CaSki cells in the upper left corner of the "2A” photo while the rhodamine labele HPV- 18 probe resulted in a positive signal (dots of light in the nucleus) in the lower righ corner of this same "2A” photo.
  • the cell in the center of the photo is a C-33A cell an is appropriately negative.
  • "2B" is a photo of the Hoechst stained nuclei of all three cells
  • the cell lines C-33A (ATCC HTB #31), Caski (ATCC CRL #1550), an SiHa (ATCC HTB #35) are grown, trypsonized and spotted directly on organosilan treated slides at the desired density. Cells are then stained using the standar papanicolaou staining procedure and prepared for in situ hybridization as described above in Example 1. Preparation Of Probes
  • the oligodeoxynucleotide probes are synthesized and labeled as described in Example 1.
  • Hybridization Washing and Fluorescent Detection As described in Example 1, the hybridization, washing and detection steps are performed.
  • Formaldehyde fixed tissue that has been embedded in paraffin can be treated in the following fashion to remove the coverslip and stain with H & E:
  • HPV type 16 and HPV type 18 were obtained as described in
  • Figure 3 demonstrates the detection of HPV types 16 and 18 in cervical tissue previously stained with Hematoxylin and Eosin (H & E).
  • Panel 3 A is a photo
  • the positive control probe consists of the probes described in Example 1.
  • sequences for c-erb-B-2 and MDR1 are obtained from published sequences and are accessed via GenBank version 69.0.
  • oligodeoxynucleotides are designed and synthesized and labeled as described in Example 1. Hybridization and Washing and Fluorescent Detection
  • Example 1 As in Example 1, the hybridization, washing and fluorescent detection steps are performed.
  • Cells containing several copies of the c-erb-B2 or MDR1 gene have a nuclear and/or cytoplasmic stain that is significantly greater than the normal controls, i.e., cell with a single DNA copy of the gene.
  • the amount of signal can be quantitated with any number of image analysis systems, e.g., ACAS 570 by Meridian Instructions, Okemos, Michigan.
  • Example 1 As in Example 1, the hybridization, washing and fluorescent detecti steps were performed.
  • Figure 4 depicts the results obtained in cervical cells stained by the D Quick method (4C is 10X magnification while 4D is 40X) followed by hybridizati containing the positive control probes (4A is 10X magnification while 4B is 10X). bright positive signal is clearly seen in the nucleus of these cells and demonstrates t the chromosomal probes hybridize to DNA within these previously stained cells.
  • Cervical cells obtained using a cervical brush are prepared and stained in Example 1. Scrapings of vagina lesions are applied to clean glass slides in standard fashion. Positive Control Probe
  • the positive control probe is as described in Example 1.
  • Candida albicans. Trichomonas vaginalis and Treponema pallidum
  • Candida albicans Trichomonas vaginalis an Treponema pallidum are accessed via a Sequence Data Bank or from published sequence
  • Example 1 As in Example 1, the hybridization, washing and detection steps ar performed.
  • the Candida, Trichomonas and Treponema microorganisms stain brightl positive when the specific probes designed to detect them are used respectively.

Abstract

The present invention provides methods to detect the presence of a biopolymer in a previously stained specimen using novel in situ hybridization techniques. The method comprising assaying for the presence of cellular biopolymers, specifically RNA and DNA. The novel in situ hybridization procedure may be accomplished either in one or two (fixation followed by hybridization) steps. The methods of the present invention may be used to detect the presence of microorganisms, e.g., bacteria, viruses, fungi or cellular genes, e.g. oncogenes, tumor suppressor factors, or growth stimulatory factors in either previously stained cells or tissue.

Description

RAPID DETECTION OF BIOPOLYMERS IN STAINED SPECIMENS
BACKGROUND OF THE INVENTION
1. Field of the invention.
The present invention relates to the field of in situ hybridization. More specifically, the present invention relates to the use of in situ hybridization assays as a means to detect as few as 1 copy of cellular biopolymer in a stained cell. 2. Description of the related art. In situ hybridization provides a technique for the determination and quantitation of biopolymers such as nucleic acids (DNA and RNA) and proteins in tissues at the single cell level. Such hybridization techniques can detect the presence or absence of specific genes in tissues at the single cell level. In situ hybridization procedures may also be utilized to detect the expression of gene products at the single cell level.
By the use of specific nucleic acid (RNA or DNA) probes, genetic markers for infection and other disease states may be detected. Certain genetic diseases are characterized by the presence of genes which are not present in normal tissue. Other diseased conditions are characterized by the expression of RNAs or RNA translation produςts (i.e. peptides or proteins) which are not expressed in normal cells. Some disease states are characterized by the absence of certain genes or gene portions, or the absence or alteration of expression of gene products or proteins. Current methods allow the detection of these markers but are relatively time consuming and of limited sensitivity. Hybridization techniques are based on the ability of single stranded DNA or RNA to pair, i.e., hybridize, with a complementary nucleic acid strand. This hybridization reaction allows the development of specific probes that can identify the presence of specific genes (DNA), or polynucleotide sequences or the transcription and expression of those genes (mRNA).
Solution hybridization methods which require the destruction of the cell and the isolation of the nucleic acids from the cell prior to carrying out the hybridization reaction sacrifice the cellular integrity, spatial resolution and sensitivity of detection. In situ hybridization allows the detection of RNA or DNA sequences within individual cells. In situ hybridization yields greater sensitivity than solution hybridization by means of eliminating the dilution of a particular target gene, nucleic acid, or protein by the surrounding and extraneous RNA and DNA of other cells. In situ hybridization also allows for the simultaneous detection of multiple substances, i.e. genes, nucleic acids or proteins within individual cells, permitting the identification of a particular cell expressing a cellular gene or viral sequence. In addition, since in situ hybridization analysis is performed and quantitated for single cells, minimal sample and reagent is required.
Prior to the present invention, in situ hybridization procedures were only capable of detecting nucleic acids present at greater than ten copies per cell.
Such procedures required at least 8 hrs. to over 14 days to perform. Prior in situ procedures were neither quantitative nor capable of performing multiple simultaneous detections. More recently, in situ hybridization procedures, disclosed in copending United States patent applications Serial No. 784,690, filed October 28, 1991, and Serial No. 668,751, filed March 13, 1991, have reduced the time to under four hours and increased the sensitivity to a single copy per cell.
Traditional laboratory methods for identifying bacteria have relied heavily on the use of either simple or differential microbiological tests such as the Gram stain, the growth on common (or exotic) diverse substrates, and, subsequently, the specific use of biochemical tests. In recent years a strong trend in microbial systematics has emerged for the use of direct molecular characterizations, e.g., DNA base composition, DNA/DNA hybridization, gene sequencing, etc., to define taxa. These methods have revolutionized studies in bacterial evolution, but did little to identify a particular organism in a given environment or specimen. Thus, the processes of isolating pure cultures, and conducting the traditional procedures for culture identification, has continued largely unabated.
Recent efforts have attempted to develop methodologies for rapid identification of microorganisms. One of these has been the use of DNA probes specific for an organisms' 16S rRNA. However, the prior art is deficient in the lack of methods sensitive enough to detect 16S DNA, i.e., detect as few as seven copies of 16S DNA. S. Jinks-Robertson et al., In Cellular and Molecular Biology. 2: 1367-1385
(1987). One advantage of using the 16S DNA is the huge data base of sequence information currently available. Almost 1000 16S DNA sequences have been documented for bacterial phylogenetic studies and this database includes a large variety of bacteria, including pathogens. From a technical perspective the 16S DNA sequence segments can serve as molecular probes, which will allow for species identification of any bacterium, whether viable, actively growing or non-viable, dormant organisms.
A number of different stains are used in cytologic and histologic specimens, for example, the papanicolaou stain is a common stain used on cells obtained from cervical specimens. Generally, only one slide is prepared from a patient and this slide is stained. If, for example, there are cells present on the stained slide which one suspects for HPV infection, the prior art does not provide a method for removing the coverslip and analyzing those suspicious cells by using in situ hybridization. Accordingly, an additional specimen must be obtained presenting the potential for missing the lesion in a subsequent sampling.
The prior art remains deficient in its inability to teach the use of in situ hybridization procedures in the detection of bacterial or viral DNA or mRNA or cellular genes in stained specimens. The application of in situ hybridization techniques to detect DNA or forms of RNA and mRNA and thus identify bacterial or viral strains and cellular genes present in stained histological or cytological samples is a long felt need and desire in this art. SUMMARY OF THE INVENTION
In one embodiment of the present invention, there is provided a method of detecting the presence of a biopolymer in a previously stained specimen having substantially intact cellular membranes. This "two-step" method comprises assaying for the presence of cellular biopolymers. Initially, the specimen may be heat fixed or fixed with a medium comprising a precipitating agent and/or a cross-linking agent. Subsequently, the fixed cells are contacted with a hybridization solution. The hybridization solution consists of a denaturing agent, hybridized stabilizing agent, buffering agent, a selective membrane performing agent and at least probe having a nucleotide sequence at least substantially complementary to a specific target nucleotide to be detected. The sample of cells is then incubated with the hybridization solution in the presence of at least one detectable label. This method is capable of detecting as few as a single biopolymer per cell.
In another embodiment of the present invention, there is provided a "one-step" method for the detection of the presence of biopolymers in a previously stained specimen having substantially intact membranes. The method comprising assaying for the presence of cellular biopolymers. Initially, the specimen is contacted with a medium comprising a denaturing agent, hybridized stabilizing agent, buffering agent, a membrane pore forming agent and at least one probe. Optimally, the medium may contain a fixative agent. The specimen is contacted under hybridizing conditions. Subsequently, the sample of cells is incubated with the medium in the presence of at least one detectable label. Duplex or triplex formation is detected by detecting the label without performing a pre-hybridization step for blocking non¬ specific binding of the probe and facilitating probe entry. This method is capable of detecting a single copy of a target biopolymer per cell.
In yet another embodiment of the present invention, there is provided a kit for determining the presence of a biopolymer in a previously stained specimen having substantially intact cellular membranes. The kit may be used for assaying cellular biopolymers. The kit comprises a hybridization solution comprising a denaturing agent, a hybrid stabilizing agent, a buffering agent, a membrane pore forming agent and optimally, a fixative agent. Optionally, the kit may comprise a supply of a probe selected so that the probe will hybridize with the suspect biopolym to form a hybridized complex. Additionally, the kit may comprise a means for contacting said suspect specimen with the probe to form a hybridized complex and a means for measuring the presence of the labeled probe. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 demonstrates the detection of HPV in a cervical cell previously stained with a papanicolaou stain.
Figure 2 demonstrates the simultaneous detection and differentiation of HPV types 16 and 18, in cells previously stained with a papanicolaou stain. Figure 3 demonstrates the detection of HPV in cervical tissue previously stained with hematoxylin and eosin.
Figure 4 demonstrates the detection of chromosomal DNA within cells previously stained by the Diff-Quick method.
DETAILED DESCRIPTION OF THE INVENTION The novel methods of the present invention may be used to detect a wide variety of microorganisms that are the cause of various pathophysiological state For example, the methods of the present invention may be used to detect microorganisms that are the cause of pathophysiological states such as bacteremias, sexually transmitted diseases, diarrhea and respiratory diseases. The novel methods of the present invention may be used to detect a wide variety of bacteria, viruses and fungi. Representative examples of bacteria detectable by the methods of the present invention include Streptococcus, Staphylococcus, Clostridium, Bacillus, Pseudomonas, Salmonella, Klebsiella, Bacteroides, Escherichia coli, Neisseria gonorrhea, and Chlamydia. Representative examples of fungi include Candida, Cryptococcus neoformans, Blastomyces dermatitides, Histoplasma capsulatum, Coccidioides immitis and Paracoccidioides brasiliensis. Representative examples of detectable viruses include human papilloma herpes simplex virus II, hepatitis, human immunodeficiency virus, influenza virus, parainfluenza virus and rota virus. Representative examples of spirochetes include Treponema pallidum,
Borrelia burgdorfeή and Leptospira. Representative examples of protozoa include Entamoeba histolytic, Balantidium coli, Giardia lamblia, Leishmania tropica, specie of Plasmodium, Trichomonas vaginalis and species or Trypanosoma.
In contrast to the detection of a microorganism, the present methods may also be used to detect and quantitate the presence of cellular genes from previously stained mammalian specimens. Representative examples of such cellular genes include oncogenes, tumor suppressor genes and growth stimulating factors. Preferably, such oncogenes are neu (c-erb-B-2), c-ras, c-myc, and c-myb. Similarl the present methods may be used to detect and quantitate tumor suppressor genes an stimulating growth factors. Examples of tumor suppressor genes include p53 and retinoblastoma. Representative examples of growth factors include colony simulatin factor-granulocyte/macrophage (CSF-GM), transforming growth factor (TGF-α), an epidermal growth factor (EGF). A person having ordinary skill in the art would comprehend that other cellular biopolymers may be detected and quantitated from a previously stained specimen by the methods of the present invention. It is specifically contemplated that a wide variety of cell samples and tissue specimens may be employed in the techniques described in the present invention. The cell specimens include the following representative examples: cervi cells, bone marrow cells, hepatocytes, cerebrospinal fluid cells, blood cells, oral mucosa cells, lung cells and skin cells. Representative examples of tissue specimen include lymph node tissue, mammary tissue, cervical tissue, colon tissue, prostrate tissue, cardiac tissue and brain tissue. In addition, fluids and exudates, e.g., urine, stool, sputum may be detected.
The cells and tissue specimens used in the method of the present invention may have been stained by any of the commonly used stains in cytology or histology. Examples of representative stains include a papanicolaou stain, a Wright stain, a Hematoxylin and Eosin stain and Diff-Quick. Mounting Cells/Tissues
The first step in the in situ hybridization procedures could be the deposition of specimens onto a solid support. Specimens constitute any material which is composed of or contains cells or portions of cells. The cells may be livin or dead, so long as the target biopolymer, i.e., DNA or mRNA, is largely unaltere and undamaged to the extent that it is capable of detection. The specimen should contain cells with substantially intact membranes. Although it is not necessary that a membranes of the cellular structure be intact, the membranes must be sufficiently preserved to allow: retention of the target biopolymer and introduction of the detecting probe to the site of the target biopolymer.
Techniques for depositing the specimens on the solid support will depend upon the cell or tissue type, and they may include, for example, standard sectioning of tissue or smearing or cytocentrifugation of single cell suspensions.
Many types of solid supports may be utilized to practice the invention. Supports which may be utilized include, but are not limited to, glass, Scotch tape
(3M), nylon, Gene Screen Plus (New England Nuclear) and nitrocellulose. Most preferably glass microscope slides are used. The use of these supports and the procedures for depositing specimens thereon will be obvious to those of skill in the art. The choice of support material will depend upon the procedure for visualization of cells and the quantitation procedure used. Some filter materials are not uniformly thick and, thus, shrinking and swelling during in situ hybridization procedures is not uniform. In addition, some supports which autofluoresce will interfere with the determination of low level fluorescence. Glass microscope slides are most preferabl as a solid support since they have high signal-to-noise ratios and can be treated to better retain tissue.
Cell Preparation for One-Step Method
In the one-step in situ hybridization procedure of the present invention tissue samples are broken apart by physical, chemical or enzymatic means into singl cell suspension. A single solution is added to the cells/tissues (hereafter referred to as the specimen). This solution contains the following: optionally, a mild fixative, a chaotrope or other denaturing agent, a synthetic oligonucleotide probe (RNA or DN probe which is prelabeled) and/or antibody probe, salts, detergents, buffers, and blocking agents. The incubation in this solution is carried out at 40°C to 60°C for to 30 minutes. In the one-step procedure, regardless of whether the specimen is in suspension or on solid supports, the hybridization procedure is carried out utilizing a single hybridization solution. Optionally, a mild fixative is included in the solution which also fixes the cells. This fixation is accomplished in the same solution and along with the hybridization reaction. The fixative is one which has been found to b optimal for the particular cell type being assayed (eg., there is one optimal fixative f bone marrow and peripheral blood even though this "tissue" contains numerous distinct cell types). The fixative may be a combination of precipitating fixatives (suc as alcohols) and cross-linking fixatives (such as aldehydes), with the concentration of the cross-linking fixatives kept very low (less than 10%). The concentration and typ of precipitating agent and crosslinking agent may be varied depending upon the prob and the stringency requirements of the probe, as well as the desired temperature of hybridization.
Choice of fixatives and fixation procedures can affect cellular constituents and cellular morphology; such effects can be tissue specific. Preferably, the precipitating fixative and has the following characteristics: fixes the cellular constituents through a precipitating action; the effect is reversible, the cellular morphology is maintained, the antigenicity of desired cellular constituents is maintained, the nucleic acids are retained in the appropriate location in the cell, the nucleic acids are not modified in such a way that they become unable to form double or triple stranded hybrids, and cellular constituents are not affected in such a way so as to inhibit the process of nucleic acid hybridization to all resident target sequences. Preferably, fixatives for use in the invention are selected from the group consisting o ethanol, ethanol-acetic acid, methanol, and methanol-acetone. Fixatives most preferable for practicing the one-step procedure include 10-40% ethanol, 10-40% methanol, 10-40% acetone or combinations thereof. These fixatives provide good preservation of cellular morphology and preservation and accessibility of antigens, a high hybridization efficiency. Typically, the solution contains 1-40% ethanol, and 5 formalin. Fixation of Cells/Tissues in the Two-Step Method
After depositing specimens on solid supports or leaving them in solution, the specimens are fixed. A fixative may be selected from the group consisting of any precipitating agent or cross-linking agent used alone or in combination, and may be aqueous or non-aqueous. The fixative may be selected from the group consisting of formaldehyde solutions, alcohols, salt solutions, mercuric chloride sodium chloride, sodium sulfate, potassium dichromate, potassium phosphate, ammonium bromide, calcium chloride, sodium acetate, lithium chloride, cesium acetate, calcium or magnesium acetate, potassium nitrate, potassium dichromate, sodium chromate, potassium iodide, sodium iodate, sodium thiosulfate, picric acid, acetic acid, paraformaldehyde, sodium hydroxide, acetones, chloroform, glycerin and thymol.
Preferably, the fixative will comprise an agent which fixes the cellular constituents through a precipitating action and has the following characteristics: the effect is reversible, the cellular morphology is maintained, the antigenicity of desired cellular constituents is maintained, the nucleic acids are retained in the appropriate location in the cell, the nucleic acids are not modified in such a way that they become unable to form double or triple stranded hybrids, and cellular constituents are not affected in such a way so as to inhibit the process of nucleic acid hybridization to all resident target sequences. Preferably, fixatives for use in the invention are selected from the group consisting of ethanol, ethanol-acetic acid, methanol, and methanol-acetone which fixatives afford the highest hybridization efficiency with good preservation of cellular morphology.
Simultaneously, the fixative may contain a compound which fixes the cellular components by cross-linking these materials together, for example, glutaraldehyde or formaldehyde. The cross-linking agent is generally more "sticky" and causes the cells and membrane components to be secured or sealed, thus, maintaining the characteristics described above for fixatives. The cross linking agents when used are preferably less than 10% (v/v). Cross-linking agents, while preserving ultrastructure, often reduce hybridization efficiency by forming networks trapping nucleic acids and antigens and rendering them inaccessible to probes and antibodies. Some also covalently modify nucleic acids preventing later hybrid formation. Examples of cross-linking agents include paraformaldehyde, formaldehyde, dimethylsilserimidate and ethyldimethylamino-propylcarbodimide. Storage of Cells/Tissues
After fixation, microscope slides and other solid supports containing specimens may be stored air dried at room temperature for up to six months, in cold (4°C) 70% ethanol in water for 6-12 months. If specimens are handled under RNAse free conditions, they can be dehydrated in graded alcohols and stored for at least 12 months at room temperature.
Prehybridization Treatments
According to the present invention no formal prehybridization step is necessary. Blocking nonspecific binding of probe and facilitating probe entry can be accomplished in the hybridization solution. If short hybridizations are to be done (less than 30 minutes) slides may be preheated to hybridization temperature before addition of the hybridization solution. Hybridizations
Nucleic acid hybridization is a process where two or more duplex or triplex mirror images or opposite strands of naturally occurring or synthetic DNA, RNA, oligonucleotides, polynucleotides, or any combination thereof recognize one another and bind together through the formation of some form of either spontaneous or induced chemical bond, usually a hydrogen bond. The degree of binding can be controlled based on the types of nucleic acids coming together, and the extent of "correct" binding as defined by normal nucleic acids coming together, and the extent of "correct" binding as defined by normal chemical rules of bonding and pairing. For example, if the binding of two strands forms 9 out of 10 correct matches along a chain of length 10, the binding is said to be 90% homologous.
Cellular nucleic acid sequences are detected by the process of molecular hybridization. The probe must be "labeled" in some way so to allow "detection" of any complementary cellular nucleic acid sequences present within the individual cells. In the present invention, the term "hybridization" also means the binding of an antibody to a target antigen.
In the one-step hybridization procedure, the hybridization cocktail contains a denaturing agent, usually formamide, but other chaotropic agents such as Nal, urea, thiocyanate, guanidine, trichloroacetate, tetramethylamine and perchlorate may also be used. Furthermore, several precipitating and/or cross-linking fixatives also have mild denaturing properties; these properties can be used in conjunction with the primary denaturant in either an additive or synergistic fashion. The hybridization cocktail may be constructed to preferentially allow only the formation of RNA-RNA or RNA-DNA hybrids. This is accomplished by adjusting the concentration of the denaturing agents along with the concentration of salts (primarily monovalent cations of the Group I series of metals along with the ammonium ion) and along with the temperature of hybridization which is used. This allows for the selective hybridization of probe to either cellular RNA or DNA or both RNA and DNA simultaneously with distinct probes. This further allows the probes to be supplied in premixed solution which presents the optimal conditions for generating a signal and minimizing noise while simultaneously optimally "fixes" the morphology of the cells/tissues. Hybridization Solution Components The hybridization solution may typically comprise a chaotropic denaturing agent, a buffer, a pore-forming agent, a hybrid stabilizing agent. The chaotropic denaturing agents include formamide, urea, thiocyanate, guanidine, trichloroacetate, tetramethylamine, perchlorate, and sodium iodide. Any buffer whic maintains pH at least between 7.0 and 8.0 may be utilized. The pore-forming agent is, for instance, a detergent such as Brij 35,
Brij 58, sodium dodecyl sulfate, Tween, CHAPS or Triton X-100. Depending on th location of the target biopolymer, the pore-forming agent is chosen to facilitate probe entry through plasma', nuclear membranes or cellular compartmental structures. For instance, 0.05% Brij 35 or 0.1 % Triton X-100 will permit probe entry through the plasma membrane but not the nuclear membrane. Alternatively, sodium deoxycholat will allow probes to traverse the nuclear membrane. Thus, in order to restrict hybridization to the cytoplasmic biopolymer targets, nuclear membrane pore-formin agents are avoided. Such selective subcellular localization contributes to the specificity and sensitivity of the assay by eliminating probe hybridization to complementary nuclear sequences when the target biopolymer is located in the cytoplasm. Agents other than detergents, such as fixatives, may serve this function
Hybrid stabilizing agents such as salts of mono- and divalent cations included in the hybridization solution to promote formation of hydrogen bonds between complementary sequences of the probe and its target biopolymer. Preferab sodium chloride at a concentration from 0.15 M to 1 M is used. In order to preve non-specific binding of nucleic acid probes, nucleic acids unrelated to the target biopolymers are added to the hybridization solution. Representative examples of hybrid stabilizing agents include sodium chloride, lithium chloride and magnesium. Preparation of Cells for Flow Cytometry
When cells are analyzed by flow cytometry, the entire procedure is performed while the specimen is maintained in solution. Briefly, cells are suspend in PBS and then pelleted by centrifugation. To the cell pellet is added either a fixative or a hybridization solution containing a fixative. After resuspension, cells incubated as described below. After pelleting, resuspension and washing, cells are resuspended in the mounting medium and analyzed on a flow cytometer. Types of Probes
A probe is defined as genetic material DNA, RNA, or oligonucleotid or polynucleotides comprised of DNA or RNA and antibodies. The DNA or RNA may be composed of the bases adenosine, uridine, thymidine, guanine, cytosine, or any natural or artificial chemical derivatives thereof. The probe is capable of dupl or triplex hybrid formation by binding to a complementary or mirror image target cellular genetic sequence. Binding occurs through one or more types of chemical bonds, usually through hydrogen bond formation. The extent of binding is referre as the amount of mismatch allowed in the binding or hybridization process. The extent of binding of the probe to the target naturally occurring or synthetically produced cellular sequences also relates to the degree of complementarity to the tar sequences. The size of the probe is adjusted to be of such size that it forms stable hybrids at the desired level of mismatch; typically, to detect a single base mismatch requires a probe of approximately 12-50 bases. Larger probes (from 50 bases up to tens of thousands of bases) are more often used when the level of mismatch is measured in terms of overall percentage of similarity of the probe to the target gen sequence. The size of the probe may also be varied to allow or prevent the probe from entering or binding to various regions of the genetic material or of the cell. Similarly, the type of probe (for example, using RNA versus DNA) may accomplis these objectives. The size of the probe also affects the rate of probe diffusion, probability of finding a cellular target match, etc. Typically, double-stranded DNA (dsDNA), single-stranded DNA (ssDNA) or RNA probes are used in a hybridizatio reaction when nucleotide sequences are the target. Preparation of Probes.
RNA or DNA probes useful in the present invention may be prepare according to methods known to those of skill in the art or may be obtained from an commercial source. RNA probes may be prepared by the methods described by
Green et al. (1981) Cell 32:681. DNA probes may be prepared by methods known those of skill in the art such as described by Rigby et al. (1977) J. Mol. Biol. 113:237. Synthetic oligonucleotide probes may be prepared as described by Wallac et al (1979) Nucleic Acids Research 6:3543 or as recommended by the suppliers (e. ABI) of the nucleic acid synthesizers. The probes useful in the present invention m be prepared according to the methods disclosed in copending patent applications, U. Serial No. 784,690, filed October 28, 1991, and Serial No. 668,751, filed March 1 1991.
Nucleic acid probes can be prepared by a variety of methods known those of skill in the art. Purified double-stranded sequences of DNA (dsDNA) can labeled intact by the process of nick translation or random primer extension. The ability of double-stranded probes to hybridize to nucleic acids immobilized within c is compromised by the ability of the complementary strands to hybridize to each ot in solution prior to hybridization with the cellular nucleic acids. Single-stranded D (ssDNA) probes do not suffer this limitation and may be produced by the synthesis oligonucleotides, by the use of the single-stranded phage M13 or plasmid derivative ^~
-14- of this phage, or by reverse transcription of a purified RNA template. The use of single-stranded RNA (ssRNA) probes in hybridization reactions potentially provides greater signal-to-noise ratios than the use of either double or single-stranded DNA probes. Regardless of whether a dsDNA, a ssDNA, or a ssRNA triplex or circular probe is used in the hybridization reaction, there must be some means of detecting hybrid formation. The means of detecting hybrid formation utilizes a probe "labeled" with some type of detectable label.
Antibody probes are known to those skilled in the art. The term "antibody probe" means an antibody that is specific for and binds to any target antigen. Such a target antigen may be a peptide, protein, carbohydrate or any other biopolymer to which an antibody will bind with specificity. Preparation of Antibody Probe
Antibody probes specific for antigens such as viruses or specific determinants thereof, peptides and proteins derived from a variety of sources, carbohydrate moieties and a wide variety of biopolymers are known to those of skill in the art. The methods for preparation of such antibodies are also known to those of skill in the art.
Briefly, polyclonal antibodies may be prepared by immunization of an animal host with an antigen. Preferably, the antigen is administered to the host subcutaneously at weekly intervals followed by a booster dose one month after the final week dose. Subsequently, the serum is harvested, antibodies precipitated from the serum and detectably labeled by techniques known to those of skill in the art.
Monoclonal antibodies my be prepared according to any of the methods known to those in the art. Fusion between myeloma cells and spleen cells from immunized donors has been shown to be a successful method of producing continuous cell lines of genetically stable hybridoma cells capable of producing large amounts of monoclonal antibodies against target antigens such as, for instance, tumors and viruses. Monoclonal antibodies may be prepared, for instance, by the method described in U.S. Patent No. 4,172,124 to Koprowski, et al. or according to U.S Patent No. 4,196,265 to Koprowski, et al. Procedures for labeling antibodies are known to those skilled in the art. Detection Systems
Probes may be detectably labeled prior to addition to the hybridization solution. Alternatively, a detectable label may be selected and added after hybridization is completed and binds to the hybridization product. Probes may be labeled with any detectable group for use in practicing the invention. Such detectabl group can be any material having a detectable physical or chemical property. Such detectable labels have been well-developed in the field of immunoassays and in general most any label useful in such methods can be applied to the present invention Particularly useful are enzymatically active groups, such as enzymes (see Clin. Chem.. 22: 1243 (1976)); enzyme substrates (see British Pat. Spec. 1,548,741), coenzymes (see U.S. Patents Nos. 4,230,797 and 4,238,565); enzyme inhibitors (see U.S. Patent No. 4,134,792); fluorescers (see Clin. Chem.. 25:353 (1979); chromophores; luminescers such as chemiluminescers and bioluminescers (see Clin. Chem.. 25:512 (1979)); specifically bindable ligands; and proximal interacting pairs; and radioisotopes such as 3H, 35S, 32P, 125I and 14C.
Biotin labeled nucleotides can be incorporated into DNA or RNA by nick translation, enzymatic, or chemical means. The biotinylated probes are detected after hybridization using avidin/streptavidin, fluorescent, enzymatic or colloidal gold conjugates. Nucleic acids may also be labeled with other fluorescent compounds, wi immunodetectable fluorescent derivatives or with biotin analogues. Nucleic acids cross-linked to radioactive or fluorescent histone HI, enzymes (alkaline phosphatase and peroxidases), or single-stranded binding (ssB) protein also may be used. To increase the sensitivity of detecting the colloidal gold or peroxidase products, a number of enhancement or amplification procedures using silver solutions may be used.
An indirect fluorescent immunocytochemical procedure also may be utilized (Rudkin and Stollar (1977) Nature 265: 472; Van Prooijen, et al. (1982) Exp.Cell.Res. 141: 397). In that procedure, polyclonal antibodies were raised again RNA-DNA hybrids by injecting animals with poly(Ra)-poly(dT). DNA probes were hybridized to cells in situ and hybrids were detected by incubation with the antibody to RNA-DNA hybrids. Probe Size and Concentration
The length of a probe affects its diffusion rate, the rate of hybrid formation, and the stability of hybrids. According to the present invention, small probes (15-100 bases) yield the most sensitive, rapid and stable system. A mixture short probes (15-100 bases) are prepared which span the entire length of the target biopolymer to be detected. For example, if the target biopolymer were 1000 bases long, about 40 "different" probes of 25 bases would be used in the hybrid solution t completely cover all regions of the target biopolymer.
The concentration of the probe affects several parameters of the in sit hybridization reaction. High concentrations are used to increase diffusion, to reduce the time of the hybridization reaction, and to saturate the available cellular sequence To achieve rapid reaction rates while maintaining high signal-to-noise ratios, probe concentrations of 0.01 to 100 μg/ml of hybridization solution are preferable. Most preferable is use of probes at a concentration of 2.5 μg/ml. Hybridization Solution and Temperature
In a preferred embodiment, the hybridization solution of the one-step in situ method consists of 25% formamide, 5X SSC, 15 X Ficoll/PVP, .4M guanidinium isothiocyanate, about 50 mM sodium phosphate (Ph 7.4), 50 mM DTT, about 1 mg/ml salmon sperm DNA, 5% Triton X-100, 50 mM EDTA and 21% PE A synthetic oligonucleotide probe is added to this solution. The probe may be at lea
15-20 bases, preferably, about 25 bases, and labeled with Photobiotin™. The most preferable, optimal temperature of hybridization is 40°-45°C. However, temperatur ranging from 15 °C to 80°C may be used.
In the one-step and two-step methods, the probe in the hybridization cocktail may be labeled before the hybridization reaction. The label may be one of the many types described above. If the probe is labeled with Photobiotin™, the hybrids may be detected by use of a Streptavidin/Avidin (S/A) conjugated to a fluorescent molecule such as FITC, rhodamine, Texas Red™; to S/A conjugated to a enzyme; or to S/A labeled with a heavy metal such as colloidal gold. Specifically, solution containing the streptavidin conjugate is added directly to the hybridization cocktail over the cells after the end of the hybridization reaction. The cells are incubated in this solution for 5 to 30 minutes at 45 °C. Longer times of hybridization however, may be used along with both higher or lower temperatures. The time of hybridization reaction will vary depending on the composition of the hybridization cocktail containing the fixative (type and concentrations of precipitating agents and/or cross-linking agents), buffering agents, pore forming agents, denaturing agents and hybrid stabilizing agents. Similarly, the temperature may be varied as described.
Alternatively, the probes may be directly labeled with the fluorescent dye or molecules such as Pontamine Sky Blue™ by incubating the nucleic acid probe and dye together (1:10 weight: weight proportions) and allowing the dye to bind/intercalate. The probe is then precipitated out of the dye solution and the exces unbound dye is removed by repeated washing with 70% ethanol. Probes also are labeled directly and covalently by incubation of double stranded molecules (RNA-RNA, RNA-DNA, or DNA-DNA) with labels which will covalently bind to nucleic acids. After incubation conditions under which the reaction will take place, the strands are separated and each separate strand is used as a probe. The concentration of the probe in the solution is typically 2.5 μg/ml although a range of 0.01-100 μg/ml is useful. The probe concentration will affect the reaction kinetics and may affect the sensitivity of the assay along with the signal-to-noise ratio.
If the probe is labeled directly with an enzymatic label or is detected using an enzymatic or secondary detectable system, then this reaction may be carried out before or after any wash steps. Following the incubation of the specimen with th appropriate buffer for the enzyme, the slide is incubated with the substrates for the enzyme under conditions specified by the manufacturer or supplier of the enzyme. Cells may be deposited onto slides or centrifuged into a pellet following the fixation/hybridization/ detection reaction(s). Next, the unbound probe washed away from the cells by one wash step using a solution of 0.1 x SSC with 0.1 % Triton X-100™. A total of 1-200 ml of wash solution may be used per microscope slide (i.e., per about 100,000 separated cells or per tissue section of abo 1 square centimeter). The concentration and type of the hybrid stabilizing/denaturin agents and pore forming agents may be varied depending on the type of cells, the ty of probe and the acceptable level of mismatch of the hybrid. Results obtained using the one-step or two-step method.
When cells are deposited onto slides, results are visualized manually on a fluorescent microscope when direct or indirectly labeled fluorescent probes are utilized. Alternatively, the results may be automatically analyzed on a fluorescence-based image analysis system.
When cells are maintained in solution, results may be obtained using a flow cytometer to record the amount of fluorescence per cell, which represents the amount of hybrid per cell. Alternatively, the total signal within a cellular sample may be determined using a device such as a liquid scintillation counter (for radioactivity) or a chemluminescent/fluorescent microtiter plate reader for these labels.
Speed. Sensitivity and Quantification of In situ Hybridizations
In the one-step embodiment, the present invention requires as little as 5 minutes to complete with a sensitivity of as few as 1 molecule of a cellular biopolymer per cell. The speed and sensitivity result from the combination of at least four factors: 1) cellular constituents are not irreversibly precipitated or fixed onto the nucleic acids, 2) if fixed, the fixation was optimized for the particular tissue used 3) the kinetics of the reaction proceed more rapidly at high probe concentrations and at elevated temperatures, and 4) probes were constructed to facilitate rapid entry and exit through cellular membranes and cellular components. The number of copies of mRNA or DNA per cell can be estimated from the number of grains over cells when radioactive probes are used. With fluorescent or enzymatic detections a relative estimate of fluorescence or precipitated colored products allows estimation of mRNA or DNA copy number. Usually, the approximation of copy number is easier after manual photography, film processing and comparisons of photographic prints. The quantitation of radioactive or fluorescent signals obtained after in situ hybridizations may be automated by use of an image analysis system, such as the Meridian AC AS 570 workstation.
The sensitivity of the in situ hybridization techniques of the present invention permit the visual and photographic detection of a single copy of a genetic sequence present within a single cell. For example and not by way of limitation, using a single fluorophor on each probe, with each probe being about 30 bases in length, a genetic sequence of approximately 6,000 bases can be reliably detected by viewing the results through a standard fluorescent microscope. When four fluorophors are attached to a single probe, the presence of absence of a particular genetic sequence of approximately 1500 bases can be reliably detected, using the period of incubation taught herein. Moreover, when using probes for corresponding sequences from both the "sense" and the "anti-sense" strands of a two-stranded nucleic acid, one can detect the presence or absence of a sequence as short as approximately 750 base paris using such periods of incubation described herein. Alternatively, one may detect as few as 75 to 150 base pairs using an image analysis system. Simultaneous Detection of Three mRNAs
Both the manual and one-step in situ hybridization procedures allow simultaneous detection of different substances (mRNAS, DNAs and proteins) within the same cells. This may be accomplished in one of two ways. First, multiple probes each containing a unique label (for example, fluorescent tags "A", "B" and "C" which each emit light at a different detectable wave length) are all added together in the hybridization solutions. Alternatively, a hybridization and detection reaction may be carried out with one probe and label, residual unreacted probe and label washed away, and another hybridization reaction is carried out. This process is repeated as many times as desired. Alternatively, probes A, B and C all contain the same tag.
When DNA and RNA were both detected, the selection of the type of probe became important. When the cellular target biopolymer is RNA, an anti-sense, single stranded DNA probe was used in the assay. If the cellular target DNA is the biopolymer to be detected, a sense-strand, single-stranded RNA probe would be used in the assay. This probe selection, and the selection and concentration of components of the fixation/hybridization solution would allow only RNA-DNA hybrids to be formed. Therefore, the probe could only bind to the desired target cellular biopolymer; other nucleic acids would inherently be prevented from interfering with the reaction assay. The one-step in situ hybridization technique for the detection of DNA or mRNA may be provided as a kit. Such a kit includes the following:
1. A solution containing a fixation/hybridization cocktail and one or mor labeled probes. Preferably, this solution will contain 50 mM guanidinium isothiocyante, 25-40% formamide, 21% PEG, 0.4 M
DTT, 15X Ficoll/PVP, 50 mM EDTA, 1 mg/ml salmon sperm DNA, 50 mM Tris-acetate (Ph 7-8), about 5% Triton X-100, and about .06 μg/μl of a synthetic oligonucleotide probe directly labeled with a reporter molecule. This solution and the probes would have measurable predefined and identified characteristics and reactivities with cells and target sequences.
2. Means and instructions for performing the said in situ hybridization reaction of the present invention.
Alternatively, the kit may also include: 1. A second detectable reporter system which would react with the probe or the probe-target hybrid.
2. Concentrated stock solution(s) to be used directly or to be diluted sufficiently to form wash solution(s).
3. Any mechanical components which may be necessary or useful to practice the present invention such as a solid support (e.g. a microscope slide), an apparatus to affix cells to said support, or a device to assist with any incubations or washings of the specimens.
4. A photographic film or emulsion with which to record results of assa carried out with the present invention. Another version of this kit may include a solution of probes encapsulated in liposom or microspheres.
The following examples are offered by way of illustration and are not intended to limit the invention in any manner. In all examples, all percentages are b weight if for solids and by volume if for liquids, and all temperatures are in degrees Celsius unless otherwise noted. All literature citations are expressly incorporated by reference. EXAMPLE 1
In situ Hybridization Procedure Using DNA Probes To Detect HPV On A Previously Stained Papanicolaou Smear
Cervical cells obtained using a cervical brush were placed in the transpo medium. Upon arrival in the laboratory, the cells were vortexed for 1-3 seconds, the spun at 1500 rpm for 10 minutes. The supernatant was discarded and the cell resuspended in 1 ml of the transport medium. The cells were spotted directly o organosilane treated slides at the desired density. Cells were then stained using th standard papanicolaou staining procedure. Dip slides 5 times in H2O. Stain 10 seconds in Gills formulation # followed by 10 dips in deionized water. Dip 10 times in 95% ETO
Stain 1 minute in OG-50. Dip 10 times in 95% ETOH. Stain 2 minut in EA-50. Destain in 95% ETOH; 10 dips. On a standard frosted en slide add 50 ul GEL/mount (Biomeda Corp., Foster City, CA.- Catalo M01). Cover with a 24 by 50 mm coverslip and let set for 10 minutes
Prior to in situ hybridization, the slides were soaked in DDH2O until th coverslips fell off, usually about 10 minutes. The slides were soaked an additional minutes in fresh H2O. Then dehydrated in graded (50%, 70%, & 95%) ethan solutions. Preparation Of Probes
The positive control probe consisted of a human alpha-centromeric repe DNA, known to hybridize to all human chromosomes. The negative probe, designate NR, was derived from the nitrogen reductase gene found in bacteria and was known not hybridize to nucleic acid within eukaryotic cells. The sequences for HPV type 16 a HPV type 18 were obtained from the published sequences and were accessed via t
Genetic Sequence Data Bank, GenBank, version 69.0.
Figure imgf000024_0001
Five hundred separate probes (250 for type 16 and 250 for type 18) were designed 25-bases in length and were synthesized.
Several 25-base synthetic oligonucleotide probes were prepared from ea of the DNA sequences listed below.
Figure imgf000024_0002
Probe Synthesis and Labeling
The oligodeoxynucleotides were synthesized (Applied Biosystems D Synthesizer Model 380 B using the recommended A.B.I, reagents), and in the last st an aminohexyl phosphate linker was attached to the 5' end. The 5 '-aminohexyl oligodeoxynucleotides were then coupled to a fluorescent rhodamine derivative (Cat. # L-20) from Molecular Probes, Inc. and purified by Waters HPLC using a baseline 810 chromatography work station. Hybridization
For the hybridization procedure, 50 μl of an hybridization cocktail consisting of 22% PEG, 30% formamide, 5X SSC, .3 mg/ml salmon sperm DNA, 15X Ficoll/PVP, .4M guanidinium isothiocyanate, 50mM DTT, 5% Triton X-100, 50mM EDTA, 7.5% Tween 20, 50mM Na2PO4, and probe at a concentration of .02 μg/μl was added to the slide. A coverslip was applied and the slide was heated to 95° C for 5 minutes, allowed to cool to 42 °C and incubated for 25 minutes at that temperature. Washing
Post-hybridization, the slides were placed in a coplin jar to which was added 100 ml of a wash solution, consisting of .IX SSC and .4M guanidinium isothiocyanate and .1 % Triton X-100. The solution was agitated until the coverslip fell off and held in this solution for 4 minutes. This wash solution was removed and a second wash solution, consisting of .IX SSC and .1% Triton X100 was added. This solution was agitated for 1 minute, poured off and the wash was repeated 5 times. Following the washes, 8 μl of antifade/Hoechst counterstain was added. The slide was coverslipped and viewed under the fluorescent microscope.
Fluorescence Detection
Photomicrographs were taken on an Olympus BH10 microscope with fluorescence capabilities, using Kodak Ektachrome EES-135 (PS 800/1600) film, exposed, and push processed at 1600 ASA. A 50-second exposure time was consistently used, so that direct comparisons could be made between all photomicrographs taken.
Results
Figure 1 demonstrates a typical result when the HPV probes (labeled with Rhodamine) and the positive control probe (labeled with FITC) are added together in the same hybridization cocktail. The nucleus of the smaller cell has stained positive for the positive control probe (in color this is green) while the larger cell nucleus is positive for
HPV (in color this is bright yellow). The procedure of Example may be modified as follows:
1) Four hundred and sixteen (400) separate probes (200 for type 16 and 200 for type 18) each designed as 30-bases in length, are synthesized. However, in addition to making probes corresponding to those 400 separate oligonucleotides that together comprise probes for one strand of each of the two HPV targets, one also makes
400 additional oligonucleotide probes for the second strands of both of the two HPV targets. The probes for the first strand will be "out of phase" relative to the second strand probes as regards how they map on a map of the HPV genome. As a result, one half (15 nucleotides) of each first strand probe will be complementary (in nucleotide sequence) to one half of one second strand probe and the other half (15 nucleotides) o that first strand probe will be complementary to a portion of another second strand probe. Staggering of the probes means that, because of the shortness of the overlap (15 nucleotides), probes of the first strand will not hybridize significantly to probes of th second strand. On the other hand, about twice as much hybridization is detected as compared to the situation where only probes corresponding to one strand are used.
2) Probes are made as phosphorothioate oligonucleotides, each 30-me having four sulfur atoms, using an Applied Biosystem (ABI) DNA Synthesizer, Mode 380B and the recommended ABI reagents. The sulfur atoms are located as follows: on is at the extreme 5' end of the probe, a second is between the 7th and 8th nucleoside (counting from the 5' end), the third is between the 22nd and 23rd nucleosides, and th fourth is between the 29th and 30th nucleosides. The sulfur atoms of the polysulfurize oligonucleotides are then coupled to a fluorescent dye, iodoacetamido-fluorescein, a follows (smaller amounts can be synthesized by adjusting the volumes): 200 μg of drie oligonucleotide is dissolved in 100 μl of 250 mM Tris buffer, pH 7.4 to form a firs solution. Then one mg of iodoacetamido-fluorescein is combined with 100 μl of dr dimethylformamide (i.e., 100 percent DMF) in a second solution. The two solutions ar mixed together and shaken overnight. After the overnight incubation, the labele oligonucleotide is precipitated with ethanol and 3M sodium acetate. This crude materia is then loaded on to a PD-10 column to remove free dye. The desired fractions are the collected. The liquid phase is then removed under vacuum. The crude material is the purified with HPLC (high performance liquid chromatography). 3) Negative and positive control probes are constructed in analogy to steps (1) and (2).
4) The hybridization cocktail is modified as follows: 1.5% PEG is used instead of 21 % PEG, 30% formamide is used instead of 21 % formamide, 10% DMSO (10% v/v) is included, and 5% (v/v) of vitamin E is included. Also instead of adding
50 μl of the hybridization cocktail to the slide, 40 μl of the cocktail is added to 5 μl o squalene plus 5 μl of pyrrolidinone and the combined 50 μl is added to the slide. It can
I be useful to add 5 μl of 1 M (1 molar) DTT and 5 μl of Proteinase K (1 mg/ml) solution per 100 μl of hybridization cocktail and run the hybridization reaction at, for example, 42°C for 5 minutes, then at 95 °C for 5 minutes. It can also be useful to add about
0.50% or 0.10% aurintricarboxylic acid in the hybridization cocktail.
5) Instead of adding 8 μl of antifade/Hoechst to the slide, 8 μl of th following solution is added: 9 volumes of solution A plus one volume of solution B wher solution A is 0.01 % 1,4 diphenylamine (antifade) plus nuclear stain Hoechst (#33258; 1 μg/ml) plus 0.0025 % Evans Blue in 50% (v/v) glycerol plus 50% (v/v) 1 x PBS (0.13
M NaCl, 0.003 M KC1, 0.008 M Na2HPO4, 0.001 M KH2PO4) and solution B is dodecy alcohol.
EXAMPLE 2
In situ Hybridization Procedure Using DNA Probes To Detect Chlamydia trachomatou and Neisseria gonorrhea DNA or mRNA On A Previously Stained Papanicolaou Smea
Preparation of Cells
Cervical cells obtained using a cervical brush are placed in the transpor medium. Upon arrival in the laboratory the cells are vortexed for 1-3 seconds, then spu at 1500 rpm for 10 minutes. The supernatant is discarded and the cells are resuspende in 1 ml of the transport medium. The cells are spotted directly on organosilane treate slides at the desired density. Cells are then stained using the standard papanicolao staining procedure as described in Example 1.
Preparation of Probes:
The positive control probe is as described in Example 1. The sequence for a 7.4 kb plasmid that is a common component of the C genome and occurs in approximately 10 copies per genome is accessed via the Geneti Sequence Data Bank, GenBank, version 69.0. This entire 7.4 kb sequence is cut into 29 separate oligonucleotide probes consisting of 25 bases each. These 296 oligoniers a synthesized and labeled as described below.
For GC, three repetitive DNA elements are accessed via the Gene Sequence Data Bank, GenBank, version 69.0.
These sequences are cut into separate oligonucleotide probes consisting 25 bases each and are synthesized and labeled as described below.
TABLE 2
Probe GenBank
Designation Locus Name
CT. CHTP
G.C. NGORPTA NGORPTB NGORPTC
Figure imgf000028_0001
Probe Synthesis and Labeling
The oligodeoxynucleotides are synthesized (Applied Biosystems DNA
Synthesizer Model 380 B using the recommended A.B.I, reagents), and in the last stage an aminohexyl phosphate linker is attached to the 5' end. The 5 '-aminohexyl oligodeoxynucleotides are then coupled to a fluorochrome from Molecular Probes, Inc. and purified by Waters HPLC using a baseline 810 chromatography work station.
For simultaneous detection of both organisms GC and CT probes can be labeled with different fluorescent moieties. For example, GC can be labeled with fluorescein while CT can be labeled with a rhodamine derivative. Alternative fluorochromes, with catalogue numbers are listed above.
Hybridization
For the hybridization procedure, 50 μl an hybridization cocktail consisting of 30% PEG, 30% formamide, 5X SSC, .3 mg/ml salmon sperm DNA, 15X Ficoll/PVP, .4M guanidinium isothiocyanate, 50mM DTT, 5% Triton X-100, 50mM EDTA, 50mM Na PO4, and probe at a concentration of .02 μg/ul is added to the slide. A coverslip is applied and when DNA is the target, the slide is heated to 95 °C for 5 minutes, allowed to cool to 42°C and incubated for 25 minutes at that temperature. If mRNA is the target, the 95 °C heating step is omitted. Washing Post-hybridization, the slides are placed in a coplin jar to which is added
100 ml of a wash solution, consisting of . IX SSC and .4M guanidinium isothiocyanate and .1 % Triton X-100. The solution is agitated until the coverslips fall off and held in this solution for 4 minutes. This wash solution is removed and a second wash solution, consisting of .IX SSC and .1 % Triton X-100 is added. This solution is agitated for 1 minute and poured off and this last wash is repeated 5 times. Following the washes, 25 μl of antifade/Hoechst counterstain is added. The slide is coverslipped and is viewed under the fluorescent microscope. Fluorescence Detection
Photomicrographs are taken on an Olympus BH10 microscope wit fluorescence capabilities, using Kodak Ektachrome EES-135 (PS 800/1600) film exposed, and push processed at 1600 ASA. A 50-second exposure time is consistentl used so that direct comparisons could be made between all photomicrographs take Alternatively, commercially available low light monochrome and color cameras allow t operator to both visualize the results on a TV screen and make a permanent record in t form of a color print. The CT and GC organisms stain brightly positive when the CT and GC speci probes are used, respectively.
EXAMPLE 3 In situ Hybridization Procedure Using DNA Probes To Detect Herpesvirus II DNA mRNA On A Previously Stained Papanicolaou Smear
Preparation of Cells
Previously stained cervical cells are prepared as in Example 1. Positive Control Probe:
The positive control probe is as described in Example 1. Herpesvirus II (HSV-ID Specific Sequences: The sequence for HSV-II is accessed via the Genetic Sequence Data Ban
GenBank, version 69.0. The sequences for the immediate early and early genes f HSV-II are cut into separate oligonucleotide probes consisting of 25 bases each. The oligomers are synthesized and labeled as described in Example 1. Hybridization. Washing and Fluorescence Detection Hybridization, washing and detection are crried out as described
Example 1.
HSV-II containing cells stain brightly positive when the HSV-II probe used
EXAMPLE 4 In situ Procedure Using DNA Probes To Detect HPV In Cells Previously Stained Wi
A Papanicolaou Stain And Demonstrating Simultaneous Detection And Differentiation HPV16 From HPV18 Preparation of Cells
The C-33A (ATCC HTB #31), Ca Ski (ATCC CRL #1550) and Hela Ce were grown, trypsonized, mixed at a 1:1:1 ratio, and spotted directly on organosila treated slides at the desired density. Cells were then stained using the standar papanicolaou staining procedure and prepared for in situ hybridization as outlined i
Example 1.
Preparation of Probes The positive control probe was as described in Example 1.
The sequences for HPV type 16 and HPV type 18 were obtained from th published sequences and were accessed via the Genetic Sequence Data Bank, GenBank, version 69.0 as described in Example 1.
Probe Synthesis and Labelling The oligodeoxynucleotides were synthesized and labeled as described i
Example 1. Fluorescein (Cat. # F-143) was attached to the HPV 16 probe and rhodamine derivative (L-20) to the HPV 18 probe.
Hybridization and Washing and Fluorescent Detection
As in Example 1, the hybridization, washing and fluorescent detectio steps were performed.
Results
Figure 2 depicts the three different cell types in a single photograph. Th
FITC labeled HPV-16 probes resulted in a positive signal (dots of light in the nucleus in CaSki cells in the upper left corner of the "2A" photo while the rhodamine labele HPV- 18 probe resulted in a positive signal (dots of light in the nucleus) in the lower righ corner of this same "2A" photo. The cell in the center of the photo is a C-33A cell an is appropriately negative. "2B" is a photo of the Hoechst stained nuclei of all three cells
This demonstrates the ability of this assay system to simultaneously detect an differentiate the two HPV types 16 and 18, within cells previously stained. EXAMPLE 5
In situ Procedure Using DNA Probes To Detect A Single Copy Of HPV On Cell
Previously Stained With A Papanicolaou Stain
Preparation of Cells •
The cell lines C-33A (ATCC HTB #31), Caski (ATCC CRL #1550), an SiHa (ATCC HTB #35) are grown, trypsonized and spotted directly on organosilan treated slides at the desired density. Cells are then stained using the standar papanicolaou staining procedure and prepared for in situ hybridization as described above in Example 1. Preparation Of Probes
The positive control probe and the sequences for HPV 16 are prepared as described in Example 1
Probe Synthesis and Labeling
The oligodeoxynucleotide probes are synthesized and labeled as described in Example 1.
Hybridization. Washing and Fluorescent Detection As described in Example 1, the hybridization, washing and detection steps are performed.
Since there is a single integrated DNA copy of HPV type 16 in the SiHa cells, there is a single point of light in the SiHa cells. The CaSki cells have several points of light while the C-33A cells have none. EXAMPLE 6
In situ Procedure Using DNA Probes To Detect HPV In Cervical Tissue Sections Previously Stained With Hematoxylin And Eosin (H&E) Preparation of Tissue
Formaldehyde fixed tissue that has been embedded in paraffin can be treated in the following fashion to remove the coverslip and stain with H & E:
1. Xylene, three changes 2 minutes each
2. Absolute alcohol 10 dips
3. 95% alcohol, two changes 10 dips each
4. Tap water rinse until water runs off evenly. 5. Hematoxylin - Delafield's, Ehrlich's, or Harris' without acetic acid . . . . 10 to
15 minutes
6. Tap water, two changes 10 dips each.
7. 1 % Hydrochloric acid in 70% alcohol 5 to 10 dips
8. Running water wash well. 9. Ammonia water, 0.24% or lithium carbonate, 0.5% . . . . 30 seconds.
10. Tap water, two changes . . . .10 dips each. 11. Eosin . . . . 10 to 20 dips. or eosin-phloxine . . . .1 to 3 minutes.
12. 95% alcohol, two changes . . . . 10 dips each.
13. Absolute alcohol, three changes. . . . 10 dips each. 14. Xylene, three changes 10 dips each.
15. 95% alcohol, two changes . . . .10 dips each.
16. 70% alcohol, two changes . . . .10 dips each.
17. 50/% alcohol, two changes . . . .10 dips each.
18. DDH2O, two changes . . . .10 dips each. 19. Proceed with hybridization as described below.
Preparation Of Probes
The sequences for HPV type 16 and HPV type 18 were obtained as described in
Example 1.
Probe Synthesis and Labelling The oligodeoxynucleotide probes were synthesized and labeled as described in
Example 1.
Hybridization. Washing and Fluorescent Detection
As described in Example 1, the hybridization, washing and detection steps were performed. Results
Figure 3 demonstrates the detection of HPV types 16 and 18 in cervical tissue previously stained with Hematoxylin and Eosin (H & E). Panel 3 A is a photo
(magnification = 10X) of the H & E stained cervical tissue, prior to hybridization. Panel
3B shows a photo (magnification = 40X) of the Hoechst stained nuclei while panel 3C shows a photo (magnification 40X) of HPV positively stained cells following hybridization with the HPV containing cocktail. While all cells are brightly positive with the Hoechst stain, only a few cells are brightly positive for HPV. Negative control probes were appropriately negative.
EXAMPLE 7 In situ Procedure Using DNA Probes To Detect The Multidrug Resistance Gene and
ErbB Gene In a Tissue Specimen Previously Stained With Hematoxylin and Eosin Preparation of Cells
Tissue obtained following a biopsy of the human cervix or breast, fixed in formaldehyde, and stained with H & E is analyzed. Preparation Of Probes
The positive control probe consists of the probes described in Example 1.
The sequences for c-erb-B-2 and MDR1 are obtained from published sequences and are accessed via GenBank version 69.0.
Figure imgf000034_0001
The oligodeoxynucleotides are designed and synthesized and labeled as described in Example 1. Hybridization and Washing and Fluorescent Detection
As in Example 1, the hybridization, washing and fluorescent detection steps are performed.
Cells containing several copies of the c-erb-B2 or MDR1 gene have a nuclear and/or cytoplasmic stain that is significantly greater than the normal controls, i.e., cell with a single DNA copy of the gene. In addition, the amount of signal can be quantitated with any number of image analysis systems, e.g., ACAS 570 by Meridian Instructions, Okemos, Michigan. EXAMPLE 8
In situ Procedure Using DNA Probes to Detect chromosomal DNA on a Specim
Previously Stained by the Diff-Quick Method
Preparation of Cells Cervical cells obtained using a cervical brush were placed in the transp medium. Upon arrival in the laboratory the cells were vortexed (1-3 seconds), then sp at 1500 rpm for 10 minutes. The supernatant was disregarded and the cells resuspend in 1 ml of the transport medium. The cells were spotted directly on organosilane trea slides at the desired density. Cells were then stained using the Diff-Quick Meth following the recommendations of supplier. Coverslips were removed as described
Example 1.
Preparation of Probes
The positive control probes were synthesized and labeled as described
Example 1. Hybridization and Washing and Fluorescent Detection
As in Example 1, the hybridization, washing and fluorescent detecti steps were performed.
Results
Figure 4 depicts the results obtained in cervical cells stained by the D Quick method (4C is 10X magnification while 4D is 40X) followed by hybridizati containing the positive control probes (4A is 10X magnification while 4B is 10X). bright positive signal is clearly seen in the nucleus of these cells and demonstrates t the chromosomal probes hybridize to DNA within these previously stained cells.
EXAMPLE 9 In Situ Hybridization Procedure Using DNA Probes To Detect Trichomonas vagina
Candida species, and Treponema pallidum DNA or mRNA On A Previously Stai
Papanicolaou Smear
Preparation of Cells
Cervical cells obtained using a cervical brush are prepared and stained in Example 1. Scrapings of vagina lesions are applied to clean glass slides in standard fashion. Positive Control Probe
The positive control probe is as described in Example 1. Candida albicans. Trichomonas vaginalis and Treponema pallidum
The sequences for Candida albicans, Trichomonas vaginalis an Treponema pallidum are accessed via a Sequence Data Bank or from published sequence
The sequences are cut into separate oligonucleotide probes consisting of 25 bases eac These oligomers are synthesized and labeled as described in Example 1. Hybridization and Washing and Fluorescent Detection
As in Example 1, the hybridization, washing and detection steps ar performed.
The Candida, Trichomonas and Treponema microorganisms stain brightl positive when the specific probes designed to detect them are used respectively.
All patents and publications mentioned in this specification are indicati of the levels of those skilled in the art to which the invention pertains. All patents an publications are herein incorporated by reference to the same extent as if each individu publication was specifically and individually indicated to be incorporated by reference
One skilled in the art will readily appreciate that the present invention well adapted to carry out the objects and obtain the ends and advantages mentioned, well as those inherent therein. The components, methods, procedures and techniqu described herein are presently representative of the preferred embodiments, are intend to be exemplary, and are not intended as limitations on the scope of the prese invention. Changes therein and other uses will occur to those skilled in the art which a encompassed within the spirit of the invention and are defined by the scope of t appended claims. What is claimed is:

Claims

Claims
1. A method of detecting the presence of a biopolymer in a previously staine specimen having substantially intact cellular membranes by assaying cellular biopolymer comprising the steps of: fixing said specimen with a fixation medium comprising at least one agen selected from the group consisting of a precipitating agent and a cross linkin
I agent, contacting said fixed specimen with a hybridization solution comprising o a denaturing agent, a hybrid stabilizing agent, a buffering agent, a selectiv membrane pore-forming agent and at least one probe, said contacting being unde hybridizing conditions, detecting hybrid formation by means of said label.
2. The method of Claim 1 , wherein said hybrid formation is selected from th group consisting of duplex and triplex.
3. The method of Claim 1, wherein said probe has a nucleotide sequence a least substantially complementary to a specific target nucleotide sequence to be detected
4. The method of Claim 3, wherein said target nucleotide sequence is abou 75 bases.
5. The method of Claim 1, wherein said specimen comprises microorganism.
6. The method of Claim 5, wherein said microorganism is selected from th group consisting of bacteria, viruses and fungi.
7. The method of Claim 1 , wherein said specimen comprises a eukaryotic cell
8. The method of Claim 7, wherein said eukaryotic cell is a human cell.
9. The method of Claim 1, wherein said specimen comprises a cellular gen
10. The method of Claim 9, wherein said cellular gene is selected from t group consisting of an oncogene, a tumor suppressor gene and a stimulating grow factor. I
11. The method of Claim 10, wherein said oncogene is selected from the gro consisting of c-erb-B-2, c-myc, c-myb, and c-ras.
12. The method of Claim 10, wherein said tumor suppressor gene is select from the group consisting of P-53 and retinoblastoma gene.
13. The method of Claim 10, wherein said stimulating growth factor is select from the group consisting of transforming growth factor-α, epidermal growth factor a color simulating factor-granulocyte/macrophage.
14. The method of Claim 6, wherein said bacteria in said previously stain specimen is selected from the group consisting of Streptococcus, Staphylococcu Clostridium, Bacillus, Pseudomonas, Salmonella, Klebsiella, Bacteroides, Escherich coli, Neisseria gonorrhoea, and Chlamydia.
15. The method of Claim 6, wherein said bacteria is selected from the gro consisting of Chlamydia trachomatous and Neisseria gonorrhoea.
16. The method of Claim 6, wherein said fungi is selected from the gro consisting of Candida, Cryptococcus neoformans and Blastomyces dermatitid
Histoplasma capsulatum, Coccidioides immitis and Paracoccidiodes brasiliensis.
17. The method of Claim 1, wherein said previously stained specimen selected from the group consisting of a cell sample and a tissue sample.
18. The method of Claim 17, wherein said cell sample is selected from t group consisting of cervical cells, bone marrow cells, hepatocytes, cerebrospinal flui cells, blood cells, oral mucosa, lung cells and skin cells.
19. The method of Claim 17, wherein said tissue sample is selected from th group consisting of lymph node tissue, mammary tissue, cervical tissue, colon tissu prostate tissue, cardiac tissue and brain tissue.
20. The method of Claim 1, wherein said previously stained specimen w stained with a stain selected from the group consisting of a papanicolaou stain, a Wrig stain, a Hematoxylin and Eosin stain and Diff-Quick stain.
21. The method of Claim 6, wherein said virus in said previously staine specimen is selected from the group consisting of human papilloma virus, herpes simpl virus II, hepatitis, human immunodeficiency virus, influenza virus, parainfluenza vir and rota virus.
22. The method of Claim 14, wherein said biopolymer is mRNA.
23. The method of Claim 14, wherein said biopolymer is DNA.
24. The method of Claim 14, wherein said biopolymer is 16S rDNA.
25. The method of Claim 14, wherein said biopolymer is selected from t group consisting of mRNA, DNA, a synthetic biopolymer and 16S rDNA.
26. The method of Claim 14, wherein said biopolymer is a synthet biopolymer.
27. The method of Claim 21, wherein said biopolymer is mRNA.
28. The method of Claim 21, wherein said biopolymer is a syntheti biopolymer.
29. The method of Claim 21, wherein said biopolymer is DNA.
30. The method of Claim 21, wherein said biopolymer is 16S rDNA.
31. The method of Claim 21, wherein said biopolymer is selected from th group consisting of mRNA, DNA, a synthetic biopolymer and 16S rDNA.
32. The method of Claim 14, wherein said specimens are cervical cells.
33. The method of Claim 21, wherein said specimens are cervical cells.
34. The method of Claim 14, wherein said staining is a papanicolaou stain.
35. The method of Claim 21, wherein said stain is a papanicolaou stain.
36. The method of Claim 1, wherein said label is attached to said probe.
37. The method of Claim 1, wherein said label is added after the hybri formation is complete.
38. The method of Claim 1, wherein said label is selected from the grou consisting of radioactive labels, fluorescescers, chemiluminescers, and enzyme labels.
39. The method of Claim 1, wherein said label is a conjugate of avidin an streptavidin.
40. The method of Claim 1, wherein said fixative is selected from the grou consisting of ethanol, methanol, acetone and formaldehyde.
41. The method of Claim 1, wherein said cross-linking agent is selected fro the group consisting of paraformaldehyde, formaldehyde, dimethysilserimidate an ethyldimethylamino-propylcarbodiimide.
42. The method of Claim 1 , wherein said denaturing agent is selected from th group consisting of formamide, urea, sodium iodide, thiocyanate, guanidine, perchlorate, trichloroacetate and tetramethylamine.
43. The method of Claim 1, wherein said hybrid stabilizing agent is selecte from the group consisting of sodium chloride, lithium chloride, magnesium chloride an ferric sulfate.
44. The method of Claim 1, wherein said pore forming agent is selected fro the group consisting of Brij 35, Tween, Brij 58, Triton X-100, CHAPS™, deoxycholat and dodecyl sulfate.
45. The method of Claim 1, wherein at least two biopolymers are assaye simultaneously in the same sample.
46. The method of Claim 1, wherein said temperature is 15°C to 80°C.
47. The method of Claim 1, wherein said temperature is 40°C to 45 °C.
48. The method of Claim 1 , wherein said fixed specimen is contacted with sai hybridization medium for about 5 minutes to about 240 minutes.
49. The method of Claim 1 , wherein said method is accomplished within abou 5 minutes.
50. The method of any of Claim 1, wherein said probe comprises a mixtur of short probes to multiple regions of the target polymer.
51. A method of detecting the presence of a biopolymer in a previously staine specimen having substantially intact cellular membranes by assaying cellular biopolymer comprising the steps of: contacting said sample with a medium comprising a denaturing agent, hybrid stabilizing agent, a buffering agent, a membrane pore-forming agent an at least one probe, said contacting being under hybridizing conditions in th presence of at least one detectable label; and detecting hybrid formation by means of said label, without performing prehybridization step for blocking nonspecific binding of said probe(s) an facilitating probe entry before contacting said sample with said medium.
52. The method of Claim 51, wherein said medium further comprises fixative agent.
53. The method of Claim 51, wherein said specimen comprises microorganism.
54. The method of Claim 51, wherein said hybrid formation is selected fro the group consisting of duplex and triplex.
55. The method of Claim 51, wherein said probe has a nucleotide sequen at least substantially complementary to a specific target nucleotide sequence to detected.
56. The method of Claim 55, wherein said target nucleotide sequence is abo 75 bases.
57. The method of Claim 53, wherein said microorganism is selected from the group consisting of bacteria, viruses and fungi.
58. The method of Claim 1, wherein said specimen comprises a eukaryotic cell.
.
59. The method of Claim 7, wherein said eukaryotic cell is a human cell.
60. The method of Claim 1 , wherein said specimen comprises a cellular gene.
61. The method of Claim 60, wherein said cellular gene is selected from the group consisting of an oncogene, a tumor suppressor gene and a stimulating growth factor.
62. The method of Claim 61, wherein said oncogene is selected from the group consisting of c-erb-B-2, c-myc, c-myb and c-ras.
63. The method of Claim 61, wherein said tumor suppressor gene is selected from the group consisting of P-53 and retinoblastoma gene.
64. The method of Claim 61, wherein said stimulating growth factor is selected from the group consisting of transforming growth factor-α, epidermal growth factor and colony simulating factor-granulocyte-macrophage.
65. The method of Claim 57, wherein said bacteria in said previously stained specimen is selected from the group consisting of Streptococcus, Staphylococcus, Clostridium, Bacillus, Pseudomonas, Salmonella, Klebsiella, Bacteroides, Escherichia coil, Neisseria gonorrhea and Chlamydia.
66. The method of Claim 57, wherein said fungi are selected from the grou consisting of Candida, Cryptococcus neoformans and Blastomyces dermatitides Histoplasma capsulation, Coccidioides immitis and Paracoccidiodes brasiliensis.
67. The method of Claim 51, wherein said previously stained specimen i selected from the group consisting of a cell sample and a tissue sample.
68. The method of Claim 67, wherein said cell sample is selected from th group consisting of cervical cells, bone marrow cells, hepatocytes, cerebrospinal flui cells, blood cells, oral mucosa cells, lung cells and skin cells.
69. The method of Claim 67, wherein said tissue sample is selected from th group consisting of lymph node tissue, mammary tissue, cervical tissue, colon tissue prostate tissue, cardiac tissue and brain tissue.
70. The method of Claim 51, wherein said previously stained specimen wa stained with a stain selected from the group consisting of a papanicolaou stain, a Wrig stain, a Hematoxylin and Eosin stain and Diff-Quick stain.
71. The method of Claim 57, wherein said virus in said previously staine specimen is selected from the group consisting of human papilloma virus, herpes simple virus II, hepatitis, human immunodeficiency virus, influenza virus, parainfluenza viru and rota virus.
72. The method of Claim 65, wherein said biopolymer is mRNA.
73. The method of Claim 65, wherein said biopolymer is DNA.
74. The method of Claim 65, wherein said biopolymer is 16S DNA. _»-
-43-
75. The method of Claim 65, wherein said biopolymer is selected from the group consisting of mRNA, a synthetic biopolymer, DNA and 16S rDNA.
76. The method of Claim 65, wherein said biopolymer is a synthetic biopolymer.
77. The method of Claim 71, wherein said biopolymer is mRNA.
78. The method of Claim 71, wherein said biopolymer is DNA.
79. The method of Claim 71, wherein said biopolymer is 16S DNA.
80. The method of Claim 71, wherein said biopolymer is a synthetic biopolymer.
81. The method of Claim 71, wherein said biopolymer is selected from the group consisting from mRNA, DNA, 16S rDNA and a synthetic biopolymer
82. The method of Claim 65, wherein said specimens are cervical cells.
83. The method of Claim 71, wherein said specimens are cervical cells.
84. The method of Claim 65, wherein said staining is a papanicolaou smear stain.
85. The method of Claim 71, wherein said stain is a papanicolaou stain.
86. The method of Claim 51, wherein said label is attached to said probe.
87. The method of Claim 51, wherein said label is added after the hybrid formation is complete.
88. The method of Claim 51, wherein said label is selected from the gro consisting of radioactive labels, fluorescescers, chemiluminescers and enzyme labels.
89. The method of Claim 51, wherein said label is a conjugate of avidin a streptavidin.
90. The method of Claim 52, wherein said fixative is selected from the gro consisting of ethanol, methanol, acetone and formaldehyde.
91. The method of Claim 51, wherein said denaturing agent is selected fr the group consisting of formamide, urea, sodium iodide, thiocyanate, guanidi perchlorate, trichloroacetate and tetramethylamine.
92. The method of Claim 51 , wherein said hybrid stabilizing agent is select from the group consisting of sodium chloride, lithium chloride, magnesium chloride ferric sulfate.
93. The method of Claim 51 , wherein said pore forming agent is selected fr the group consisting of Brij 35, Tween, Brij 58, Triton X-100, CHAPS™, deoxychol and dodecyl sulfate.
94. The method of Claim 51, wherein at least two biopolymers are assa simultaneously in the same sample.
95. The method of Claim 51, wherein said method if capable of detecting few as one copy of target biopolymer per cell.
96. The method of Claim 51, wherein said hybridizing conditions comp a temperature of 15°C to 80°C.
97. The method of Claim 51, wherein said hybridizing conditions comprise a temperature of 40°C to 45°C.
98. The method of Claim 51, wherein said method is accomplished within about 5 minutes.
99. The method of any of Claim 51, wherein said probe comprises a mixture of short probes to multiple regions of the target polymer.
100. A kit for detecting the presence of a biopolymer in a previously stained specimen having substantially intact cellular membranes by assaying cellular target biopolymers comprising, a hybridization solution comprising a denaturing agent, a hybrid stabilizing agent, a buffering agent, and a membrane pore-forming agent.
101. The kit of Claim 100 further comprising, a supply of a probe, said probe capable of hybridizing with said target biopolymer to form a hybridized complex.
102. The kit of Claim 100 further comprising, means for contacting said suspect specimen with said probe to form said hybridized complex, and means for measuring for the presence of said probe.
103. The kit of Claim 100 further comprising, a detectable label capable of detecting hybrid formation.
104. The kit of Claim 103, wherein said detecting of hybrid formation is quantitative.
105. The kit of Claim 103, wherein said detectable label is an energy emitti label.
106. The method of Claim 51, in which the contacting is carried out in soluti
107. The method of Claim 51, in which the detecting is carried out by fl cytometry.
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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996008582A2 (en) * 1994-09-12 1996-03-21 Bergeron Michel G Specific and universal probes and amplification primers to rapidly detect and identify common bacterial pathogens and antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories
WO1996036734A1 (en) * 1995-05-18 1996-11-21 Abbott Laboratories Polymeric peptide probes and uses thereof
WO1997005282A2 (en) * 1995-07-28 1997-02-13 Rijksuniversiteit Groningen Methods and materials for determining relative abundance of microorganisms in mixed populations
US5888733A (en) * 1995-11-16 1999-03-30 Dako A/S In situ hybridization to detect specific nucleic acid sequences in eucaryotic samples
WO1999031273A2 (en) * 1997-12-12 1999-06-24 Digene Corporation Universal collection medium
US5985563A (en) * 1994-05-19 1999-11-16 Dako A/S Detection of Ribosomal RNA using PNA probes
US5994066A (en) * 1995-09-11 1999-11-30 Infectio Diagnostic, Inc. Species-specific and universal DNA probes and amplification primers to rapidly detect and identify common bacterial pathogens and associated antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories
WO2000065092A2 (en) * 1999-04-22 2000-11-02 Science And Technology Corporation Blocking non-specific binding of granulocytes in microorganism detection
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WO2000068668A1 (en) * 1999-05-11 2000-11-16 Hitachi Software Engineering Co., Ltd. Method and device for fluorescence measurement
US7371518B2 (en) 2000-06-21 2008-05-13 Digene Corporation Universal collection medium
US7943346B2 (en) 1994-09-12 2011-05-17 Geneohm Sciences Canada Inc. Probes and primers for detection of bacterial pathogens and antibiotic resistance genes
US8034588B2 (en) 1997-11-04 2011-10-11 Geneohm Sciences Canada Inc. Species-specific, genus-specific and universal DNA probes and amplification primers to rapidly detect and identify common bacterial and fungal pathogens and associated antibiotic resistance genes from clinical specimens for diagnosis in microbiology laboratories
US8114601B2 (en) 1999-09-28 2012-02-14 Geneohm Sciences Canada Inc. Highly conserved genes and their use to generate probes and primers for detection of microorganisms
US8426137B2 (en) 1996-11-04 2013-04-23 Genohm Sciences Canada, Inc. Methods and probes for detecting a vancomycin resistance gene
CN111492223A (en) * 2017-12-29 2020-08-04 豪夫迈·罗氏有限公司 Tissue sample preparation system
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Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110241179B (en) * 2019-06-28 2023-04-11 南京农业大学 Method for positioning distribution of symbiotic bacteria Cardinium in different tissues of rice planthopper

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4888278A (en) * 1985-10-22 1989-12-19 University Of Massachusetts Medical Center In-situ hybridization to detect nucleic acid sequences in morphologically intact cells

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4888278A (en) * 1985-10-22 1989-12-19 University Of Massachusetts Medical Center In-situ hybridization to detect nucleic acid sequences in morphologically intact cells

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
THE JOURNAL OF HISTOCHEMISTRY AND CYTOCHEMISTRY, Volume 39, No. 6, issued 1991, X. LIANG et al., In Situ Hybridization with Human Papillomavirus using Biotinylated DNA Probes on Archival Cervical Smears", pages 771-775. *
W.K. JOKLIK et al., Eds., "Zinsser Microbiology", Published 1984, APPLETON-CENTURY-CROFTS, (NORWALK, CONNECTICUT), pages 444, 445, 498, 499, 788, 789, 1134, 1135, 1150, 1151, 1184. *

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