EP0338067A1 - Diagnostic pour la detection de papillomavirus humains avec sondes de nucleotides - Google Patents

Diagnostic pour la detection de papillomavirus humains avec sondes de nucleotides

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Publication number
EP0338067A1
EP0338067A1 EP88909874A EP88909874A EP0338067A1 EP 0338067 A1 EP0338067 A1 EP 0338067A1 EP 88909874 A EP88909874 A EP 88909874A EP 88909874 A EP88909874 A EP 88909874A EP 0338067 A1 EP0338067 A1 EP 0338067A1
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EP
European Patent Office
Prior art keywords
hpv
probe
dna
sample
assay according
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP88909874A
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German (de)
English (en)
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EP0338067A4 (en
Inventor
Dennis E. Schwartz
Trevor H. Adams
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Nanogen Inc
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MicroProbe Corp
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Publication of EP0338067A1 publication Critical patent/EP0338067A1/fr
Publication of EP0338067A4 publication Critical patent/EP0338067A4/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/708—Specific hybridization probes for papilloma
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation

Definitions

  • the present invention relates generally to the diagnosis of viral infections and, more specifically, to the detection of Human Papillomavirus using nucleic acid in situ hybridization assays.
  • BACKGROUND OF THE INVENTION Prior to the 1940's, the leading cause of cancer death in women was cervical carcinoma. In 1943, Papariicolaou demonstrated that the cytology of stained exfoliated cells could be used for the detection of the early and late forms of cervical carcinoma. Since 1954, the "Pap smear" has been used as a cancer screen ⁇ ing method and has been largely responsible for de- creasing the mortality rate in women by cervical cancer in the Western World, such that it now ranks behind carcinoma of the breast, bowel, gastrointestinal tract, and ovary.
  • Cervical cancer is the result of a spectrum of morphological changes.
  • the pre-invasive forms are mild dysplasia, severe dysplasia, and carcinoma in situ (these forms are also termed cervical intraepithelial neoplasia [CIN] and graded in their order of increasing severity as CIN I, CIN II and CIN III) .
  • the subsequent invasive forms are micro-invasive carcinoma and inva ⁇ sive carcinoma.
  • the Pap smear detects both pre-invasive and invasive forms of cervical cancer in a test which is inexpensive, easy to use, and for which cells are read- ily obtained. This test rarely gives false-positive results, but unfortunately gives high false-negative results; approximately 10-20% of abnormal Pap smears are incorrectly reported as normal (Benedet, John L. , and Murphy, Katherine J. , "Cervical Cancer Screening," Postgraduate Medicine, 78:69-79 (1985)).
  • HPV Types 31, 33, and 35 are found in the remaining 10% of cervical cancers (Lorincz, A.T. , Lan ⁇ caster, W.D., Kurman, R.J., Jenson, A.B., and Temple, G.F., "Characterization of Human Papillomaviruses in Cervical Neoplasias and Their Detection in Routine Clinical Screening," "Viral Etiology of Cervical Can ⁇ cer,” Cold Spring Harbor Laboratory Publications, Cold Spring Harbor, Branbury Report, 21:225-237 (1986) and Cole, S.T., and Streeck, R.E., "Genome Organization and Nucleotide Sequence of Human Papillomavirus Type 33, Which is Associated with Cervical Cancer," J.
  • HPV-6 and HPV-11 are associated with benign growths (wart lesions) within the cervix (or vulva or vagina)
  • HPV Types 16, 18, 31, 33, and 35 are implicated as etiological agents of cervical cancer.
  • Cervical cancer may be a sexually transmitted disease.
  • the disease is absent in sexually inactive women, but does occur with high frequency among sexual- ly active women, particularly those who are sexually active at an early age and who have or have had multi ⁇ ple sex partners. Consistent with these observations, HPV-induced warts are observed, although rarely, on the penile shaft or urethra of males, and HPV is found in sperm (Ostrow, R.S., Zachow, K.R. , Niimura, M. , Okaga- ki, T., Muller, S., Bender, M. , and Faras, A.J. , "De ⁇ tection of Papillomavirus DNA in Human Semen," Science, 231:731-733 (1986)).
  • HPV proteins Recently have been prepared in E ⁇ coli expres ⁇ sion systems, thus allowing the generation of HPV-spe- cific polyclonal or monoclonal antibodies (Matlashew- ski, G. , Banks, L. , Wu-Liao, J. , Spence, P., Pirn, D. , and Crawford, L. , "The Expression of Human Papilloma ⁇ virus Type 18 E6 Protein in Bacteria and the Production of Anti-E6 Antibodies," J. Gen.
  • a DNA probe test could be used as a sec ⁇ ondary test, after a positive Pap smear, to identify the type of HPV within a clinical sample.
  • a test could be used in conjunction with the Pap smear as a screening tool for the routine concurrent detec ⁇ tion of abnormal and HPV infected cervical cells.
  • the DNA probe test could ultimately displace the Pap smear test, if HPV is proven unequivocally to be the etiological agent of cervical cancer and a rapid, reliable and economical HPV assay capable of utilizing a cervical smear format were commercially available.
  • the present invention provides a rapid in situ hybridization assay for detecting the presence of or typing of Human Papillomavirus (HPV) in a biological sample, such as a non-frozen cellular smear that has been fixed on a support in the absence of aldehyde- based cross-linking reagents, the assay including the steps of: combining the nucleic acids from the fixed biological sample with at least one detectable probe, preferably nick-translated with a detectable nucleotide of about 50 or more nucleotides, or analogs thereof, wherein the probe is capable of specifically hybridiz ⁇ ing with a substantially complementary region from one or more HPV types; and detecting the presence or absence of probe hybridization complexes.
  • HPV Human Papillomavirus
  • the entire assay can be complet ⁇ ed in less than about 4 hours, preferably about 2 hours, and most preferably less than about 2 hours.
  • the assay is particularly useful for the detection of one, two or more different HPV types, including 6, 11, 16, 18, 31, 33, and 35.
  • the biological sample for example a cervical smear
  • a support such as a glass slide.
  • the assay utilizing a cervical smear sample fixed on a glass slide will comprise the steps of: (i) inactivating competing endogenous enzyme activity; (ii) denaturing nucleic acids in the sample; (iii) hybridizing a detectable probe to the target nucleic acids, wherein the probe comprises a sequence of 200 to 600 or more nucleotides complementary to one HPV type DNA or mRNA; (iv) washing the sample to remove unbound probes; (v) incubating the sample with the detection agents; and (vi) visually inspecting the sample, such as with the aid of a microscope.
  • kits will include a first probe reagent component comprising a biotin-labelled probe of at least 50 nucleotides com- plementary to a nucleic acid sequence of HPV types 6 or 11 and/or a second probe of at least about 50 nucleo ⁇ tides complementary to nucleic acids sequence of HPV types 16, 18, 31, 33, or 35; a denaturation reagent for converting double stranded DNA to single stranded DNA; and a hybridization reaction mixture.
  • the kit can also include an avidin-labelled or streptavidin-labelled enzyme and a substrate for the enzyme.
  • FIGURE 1 shows partial nucleotide sequence comparisons between a Human Papillomavirus (HPV) Type associated with benign cervical warts (HPV-6) and two HPV Types believed to be the etiologic agents of cervi ⁇ cal carcinoma (HPV-16 and HPV-33) .
  • HPV-16 and HPV-33 Two HPV Types believed to be the etiologic agents of cervi ⁇ cal carcinoma.
  • Figure 1 shows partial nucleotide sequence comparisons between a Human Papillomavirus (HPV) Type associated with benign cervical warts (HPV-6) and two HPV Types believed to be the etiologic agents of cervi ⁇ cal carcinoma (HPV-16 and HPV-33) .
  • About 700 nucleo ⁇ tides of sequence at the 5' end of each HPV genome are shown as follows: Figure la shows HPV-6 compared with HPV-11; Figure lb shows HPV-6 compared with HPV-16; Figure lc shows HPV-6 compared with HP
  • nucleic acid probes of at least about 50 nucleotides are used in rapid, reliable and economical in situ hy ⁇ bridization assays for detecting the existence and type of HPV present in biological samples, such as cervical or other cellular smears.
  • the assays can be stream- lined such that the entire test can be conducted under preselected hybridization conditions with a non- isotopic format in less than about four hours, and as little as about two hours or less, with few steps.
  • all steps may be performed at room tempera- ture, thus alleviating the need for temperature-con ⁇ trolled incubators.
  • the in situ hybridization tests of the pres ⁇ ent invention when using, e.g. , unstained smears of cervical cells, are often more sensitive than the Southern blot "gold standard," particularly when con ⁇ ducted on slides, which allows for the detection of a • few positive cells in a milieu of negative cells. Furthermore, these assays can be conducted in large batches in short time periods, rather than the several days typically required in hybridization formats using the Southern blot or dot blot methods.
  • the biological sample to be tested includes cell smears obtained by standard techniques, such as scraping (e.g., a cervical smear) or biopsy samples converted to smears (such as with the Cyto-Trac system. Medical Packaging Corpora- tion. Panorama City, CA) . Sources of cells include cervical, vaginal, vulval, oral, prostate, lung, rectal or any body tissue suspected of containing HPV.
  • cellular smear samples are col ⁇ lected and fixed to a support, such as a glass surface (e.g., a glass slide), plastics (e.g., polycarbonate), or other transparent inert substrates.
  • Fixing agents may be precipitants, such as picric and mecuric acid, ethanol, ethanol/acetic acid, methanol and methanol- acetone mixtures.
  • Most preferred precipitating fixa- tion solutions include ethanol and Carnoy's B solution. Standard aldehyde-based fixation is generally unnecessary.
  • Pretreatment of the cells to increase probe diffusion may be helpful, and can include acid treat- ment or protease treatment.
  • this pretreatment may also serve to inactivate endogenous enzyme activity.
  • in situ hybridization protocols for detecting viral infections are known in the art, and can be utilized in accordance with the teachings of the present invention to assay for HPV in the cellular smears.
  • the following two review articles provide an overview of in situ hybridization technology: Singer, R.H. , et al., Biotechniques, 4(3):230-250 (1986), and Haase, A., et al., Methods in Virology, Vol. VII, pp. 189-226 (1984) , and are both incorporated by reference herein.
  • Target polynucleotides can be obtained from a wide variety of sources, depending upon the particular HPV type to be detected.
  • such probes can be any HPV specific nucleic acid sequences integrated or otherwise present in a biological sample, including mutations of the wild-type virus populations, regardless of whether they are phenotypically expres ⁇ sed.
  • Probes are DNA or RNA polynucleotides or oligonucleotides, or their analogs, which have suffi ⁇ cient complementarity with the target polynucleotides so that stable binding occurs between target and probe. Homoduplexing is preferred, i.e., a perfect base match, but such is often not achieved when using cocktails of long probes and/or when detecting multiple HPO types. The degree of homology required for detectable binding varies with the stringency of the hybridization medium- and/or wash medium.
  • the lengths of the probes which are useful for the given invention are at least 15 bases, but may be 50 to 100 bases long, preferably 200 to 600 bases or more.
  • Essentially whole HPV virus genomes of about 8,000 nucleotides, with or without plasmid vector se ⁇ quences, are commonly utilized for nick-translation in the presence of labelled nucleotides to yield an aver ⁇ age probe length of about 400 bases.
  • DNA probes may be cloned in bacterial host cells following insertion into appropriate replication vectors, such as pBr322 or M13, or vectors containing R A polymerase specific promoters, such as the SP6 promoter, and purified from the host cell by cell lysis, DNA extraction. Further purification, if desired, may be achieved by digestion with selected restriction enzymes, and further separation by gel or column fractionation techniques.
  • appropriate replication vectors such as pBr322 or M13
  • R A polymerase specific promoters such as the SP6 promoter
  • the probes used in the present invention may also be synthesized, chemically or enzymatically, using commercially available methods and equipment.
  • the solid phase phosphoramidite methods are particularly useful for producing smaller probes.
  • DNA probes can also be synthesized, for example, by reverse transcription of mRNA or produced by nick-translation of cloned HPV genes.
  • Nucleotide analogs within HPV specific se ⁇ quences that can be inserted during chemical or enzymatic synthesis include: l-(2-deoxy-a-D-ribo- furanosyl)-2-pyrimidinone, 2'-deoxyinosine, 2'-deoxy-7-deazaguanosine, 2'-deoxy-5-substituted uri- dine, or the appropriately blocked phosphoramidites thereof. These can be substituted for naturally occur ⁇ ring nucleotides, e.g., during labelling or synthesis of the probe, yet maintain acceptable hybridization specificity of the probe. Other analogs having similar functionality can, of course, be produced in accordance with well known teachings in the art.
  • sequence When synthesizing a probe for a specific tar ⁇ get, the choice of sequence will determine the speci- ficity of the test. For example, by comparing DNA se ⁇ quences from several virus isolates, one can select a sequence for virus detection that is either type speci ⁇ fic or genus specific. Comparisons of DNA regions and sequences can be achieved using commercially available computer programs. Generally, the more unique the sequence selected to be probed, the less background noise will be generated.
  • Probes may be labeled by any one of several methods typically used to detect the presence of hybrid polynucleotides.
  • a common method of detection is the use of autoradiography with 3 H, 125 I, 34 s, 14 C, or 32 P labeled probes or the like.
  • Other labels include directly conjugated fluorophores, chemiluminescent agents, enzymes, and enzyme substrates.
  • the same components may be indirectly bonded through a ligand-antiligand complex, such as antibodies reactive with a liquid conjugated with label.
  • the choice of label depends on sensitivity required, ease of conjugation with the probe, stability requirements, and available instrumentation.
  • Radio ⁇ active probes are typically made using commercially available nucleotides containing the desired radioac ⁇ tive isotope.
  • the radioactive nucleotides can be in ⁇ corporated into probes, for example, by using DNA synthesizers, by nick-translation, by tailing of radioactive bases to the 3' end of probes with terminal transferase, by copying M13 plasmids having specific inserts with the Klenow fragment of DNA polymerase in the presence of radioactive dNTP's, or by transcribing RNA from templates using RNA polymerase in the presence of radioactive rNTP's.
  • Non-radioactive probes can be labeled direct ⁇ ly with a signal (e.g., fluorophore, chemiluminescent agents or enzyme) or labelled indirectly by conjugation with a ligand.
  • a signal e.g., fluorophore, chemiluminescent agents or enzyme
  • This ligand then binds to a receptor molecule which is either inherently detectable or covalently bound to a detectable signal, such as an enzyme or photoreactive compound.
  • Ligands and antiligands may be varied widely. Where a ligand has a natural "antiligand", namely ligands such as biotin (which is recognized by avidin or streptavidin) , thyroxine, and cortisol, it can be used in conjunction with labeled, naturally occurring receptors.
  • any haptenic or antigenic compound can be used in combination with a suitably labelled antibody.
  • a preferred labelling method utilizes biotin labelled analogs of polynucleotides, as disclosed in Langer, P. and Waldrop, A. (Proc. Nat. Acad. Sci. U.S.A. 78: 6633-6637 1981) , which is incorporated herein by reference.
  • Another preferred labelling method utilizes a direct conjugation of enzyme (e.g., horseradish peroxidase or alkaline phosphatase) to the DNA in DNA probe (either double-stranded or single- stranded) . See, Reng, M. and Kurz, C. "A Colorimetric Method for DNA Hybrization," Nucl. Acids Res. , 12:3435-3444 (1984), which is incorporated herein by reference.
  • Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases, ureases and glycosidases, or oxidoreductases, particu ⁇ larly peroxidases.
  • Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
  • Chemilumines- cers include luciferin, and 2,3-dihydrophthalazinedion- es, e.g., luminol.
  • the amount of labeled probe present in the hybridization solution may vary widely, depending upon the nature of the label, the amount of the labeled probe that can reasonably bind to the cellular target nucleic acid, and the precise stringency of the hybrid ⁇ ization medium and/or wash medium. Generally, substan- tial probe excesses over the stoichiometric amount of the target will be employed to enhance the rate of binding of the probe to the target nucleic acids.
  • Hybridization solutions and procedures are generally described by Gall and Pardue (1969) , Proc. Natl. Acad. Sci., U.S.A., 63:378-383; John, Burnsteil and Jones (1969) Nature, 223:582-587; and "Nucleic Acid Hybridization: A Practical Approach", Eds. Hames, B. and Higgins, S. IRL Press (1985) Washington, D.C., all of which are incorporated herein by reference. As im ⁇ provements are made in hybridization techniques, they can readily be applied.
  • hybridization solutions may be em ⁇ ployed provided they provide rapid hybridization in accordance with the present invention, i.e., typically less than two to three hours, preferably less than one hour, and most preferably, 20 to 30 minutes.
  • These solutions can comprise from about 20 to 60% volume, preferably about 30%, of a polar organic solvent.
  • a common stringent hybridization solution employs about 50% formamide, about 0.5 to IM sodium chloride, about 0.05 to 0.1M buffers, such as sodium.citrate, Tris, HEPES or PIPES, about 0.05 to 0.2% nonionic or ionic detergent, such as Tween 20 or sodium dodecylsulfate, or minor amounts of EDTA, Ficoll (about 300-500 kilodaltons) , polyvinylpyrrolidone (about 250-500 kdal) , and serum albumin.
  • buffers such as sodium.citrate, Tris, HEPES or PIPES
  • nonionic or ionic detergent such as Tween 20 or sodium dodecylsulfate
  • minor amounts of EDTA such as Tween 20 or sodium dodecylsulfate
  • Ficoll about 300-500 kilodaltons
  • polyvinylpyrrolidone about 250-500 kdal
  • unlabelled carrier nucleic acids from about 0.1 to 5 mg/ml, fragmented DNA, e.g., calf thymus or salmon sperm DNA, and/or yeast tRNA or partially fragmented rRNA, and optionally from about 0.5 to 2% wt./vol. glycine.
  • Other additives may also be included, such as volume exclusion agents, which include a variety of water-soluble or swellable agents, such as anionic polymers of polyacrylate or polymethacrylate, charged saccharide polymers, such as dextran sulfate (preferably, about 10%) , and tetra- alkylammonium salts or triethlamine salts.
  • Stringent hybridization conditions are pre ⁇ ferred either during the hybridization or wash step.
  • the precise degree of stringency typically is controlled by ionic strength, partially denaturizing solvents and temperature.
  • the stringency of hy ⁇ bridization or washing is conveniently varied by changing the polarity of the reactant solution through manipulation of the concentration of a partially denaturizing solvent, such as formamide, within the range of about 20% to 50%, typically the higher percentages.
  • Stringency can also be conveniently varied by changing salt concentration, up to 0.5 M or higher, preferably 10 mM to 150 mM.
  • Strigency can also be varied by changing temperatures, which will normally be in the range of about 20* to 75 ⁇ C, but the present invention preferably utilizes temperatures from about 20 ⁇ to 37 ⁇ C.
  • the cello After the cells fixed to the support have been contacted with a hybridization solution, the cello are then typically introduced into a wash solution having predetermined concentrations of salts, buffers, and detergents.
  • the time period for the wash may vary from five minutes to an hour or more. Typically, it is the wash solution that most often determines the stringency and facilitates dissociation of mismatched duplexes.
  • the complex After washing the hybridization complex at room temperature with dilute buffered sodium chloride solution, the complex may be assayed for the presence of duplexes (e.g. , bound probe) in accordance with the nature of the label. This detecting step will typically be completed in from one to three hours, but may be completed in 15 to 30 minutes depending on the detection system.
  • the support is dried and exposed to X-ray film.
  • X-ray film In the alterna- tive, a nuclear track emulsion is.coated upon the sup ⁇ port in the dark, allowed to develop, washed, stained, and viewed under a microscope (Haase, et al., Methods of Virology, Vol. VII, pp. 209-210).
  • Enzymatic detection is typically performed in conjunction with biotin, such as biotinylated peroxi ⁇ dase or alkaline phosphotase. Enzyme-conjugated avidin or streptavidin are then used to bind the enzyme to the probe. After the appropriate enzyme substrate has been added, the cells may be observed visually for the pres- ence of HPV.
  • biotin such as biotinylated peroxi ⁇ dase or alkaline phosphotase.
  • Enzyme-conjugated avidin or streptavidin are then used to bind the enzyme to the probe. After the appropriate enzyme substrate has been added, the cells may be observed visually for the pres- ence of HPV.
  • kits comprising compartments (e.g., vials) containing the means components for performing the as ⁇ says of the present invention.
  • the kit includes a first probe reagent comprising a probe of at least about 200 to 600 nucleotides com ⁇ plementary to a nucleic acid sequence of at least one HPV type (e.g., 6 or 11) and/or a second probe of at least about 200 to 600 nucleotides complementary to a nucleic acid sequence of one or more different HPV types (e.g., 16, 18, 31, 33 an 35); and a denaturation step (preferably heat or a chemical denaturation re ⁇ agent) for converting double stranded DNA in cells to single stranded DNA.
  • a denaturation step preferably heat or a chemical denaturation re ⁇ agent
  • the kit can also include an avi- din or streptavidin labelled enzyme and a substrate for the enzyme, as well known in the art. All of the kit compositions are commonly provided in liquid form or may be lyophilized for subsequent reconstitution with water at the assay site. The following examples are offered by way of illustration and not by limitation.
  • DNA probes were synthesized on an Applied Biosystems DNA Synthesizer (Model 38OB, Foster City, CA) with reagents from the same source.
  • DNA probe pu ⁇ rification was accomplished by: 1) fractionation of the tritylated product with a high pressure liquid chromatography system using a reverse phase column, or 2) fractionation by gel electrophoresis on a 20% poly- acrylamide gel containing 7M urea.
  • Computer analysis of HPV nucleotide sequences was done with the Micro- Genie Program (Beckman Instruments, Palo Alto, CA) .
  • SSC buffer is 0.015M sodium citrate (pH 7.0), 0.15M NaCl; PBS is 0.01M sodium phosphate (pH 7.4), 0.13M NaCl; and Denhardt's solution is 0.02% ficoll 400, 0.02% polyvmylpyrolidone (MW 360,000), 0.02% BSA.
  • Carnoy's B solution is 10% HOAc, 30% chloroform, 60% EtOH.
  • HRP substrate solution is 0.4 mg/ml aminoethyl carbazole, 0.025% hydrogen peroxide, 0.1M NaOAc (pH 4.5) .
  • Oligonucleotides contained approximately 50% G and C residues.
  • Hybridizations were carried out at 43°C, 15 h, in oligo hybridization solution (0.5 to 1.0 ng/ml oligo, 0.6M NaCl, 90 mM Tris HC1, pH8, 10 mM EDTA, 0.5% 5 x Denhardts, 30% forma- ide, 0.1 mg/ml hydrolyzed yeast RNA). Filters were washed 15 min, room temperature, in lx oligo wash solu- tion (0.09M NaCl. 0.009 M Tris, pH8, 0.6 mM EDTA, 0.1% SDS) , 15 min at 50*C in lx wash solution, and then 2 min, room temperature, in lx wash solution. Probe hybridization was detected by autoradiography. In almost all cases, the HPV genomes detected were full length and complementary to the appropriate oligonucletides.
  • P2392 a host which selects for recombinant phage.
  • Approximately 3 x 10 5 plaque forming units were screen ⁇ ed by filter hybridization (Maniatis, T., Fritsch, E.F., and Sambrook, J. , Molecular cloning: a laboratory manual. 1982 by Cold Spring Harbor Laboratory) .
  • Hy ⁇ bridization and washing conditions were as established above for Southern blots. Positive clones were detect-
  • DNA containing Type 31 or 35 were cut with EcoRl: DNA containing Type 33 was cut with Bglll. DNAs were extracted with phenol:chlo- reform (1:1), chloroform, and then ethanol precipitat ⁇ ed. Approximately half of each sample was analyzed by Southern blot for hybridization to nick-translated HPV16 DNA. Hybridizations were carried out at low stringency (30% formamide, 0.6M NaCl, 90 mM Tris, pH8, 10 mM EDTA, 0.5% SDS, 5 x Denhardts, 0.1 mg/ml yeast RNA, at 43 ⁇ C) .
  • Bacteriophage DNA was isolated from positive clones and analyzed by Southern blot following restric- tion with PstI, BamHl, or EcoRl. In almost all cases, the clones also contained additional inserts which con ⁇ sisted of human genomic DNA. The DNA's were then cut with restriction enzymes which excised the HPV genomes away from the lambda and human DNA. EcoRl was used for Types 18, 31, and 35; BamHl was used for Types 6, 11, and 16; Bglll was used for Type 33. The restricted DNA's were electrophoresed on agarose gels and the HPV DNA isolated by eluting onto NA-45 paper (Schleicher and Schuell, Keene, NH) , according to standard proto ⁇ col.
  • Bluescript Ml3 DNA (Stratagene) which had been EcoRl or BamHl digested and treated with calf intestinal phosphatase (Boeringer Mannheim, Indianapolis, IN) .
  • Bluescript vectors have large polylinkers with 26 unique restriction sites. The polylinkers are flanked by T7 and T3 polymerase promoters present at the NH 2 -terminal portion of a LacZ gene fragment.
  • the vectors also contain a 454 nucleo ⁇ tide intergenic region (M13 related) .
  • the ligation reactions were transformed into E_ ; _ coli BB-4 (Strata ⁇ gene) and recombinant colonies identified by restric- • tion analysis of their DNA (Maniatis et al., above).
  • HPV6 With the exception of HPV6, all clones matched publish ⁇ ed restriction maps. The HPV6 clone did not have the expected 5.3 kb PstI fragment, but instead had 3.6 and 1.7 kb fragments. Recombinant plasmids contained full length HPV genomes and were free of human chromosomal DNA.
  • Mammalian cells (about 10,000) were spotted on glass slides in 100 ul of the appropriate culture media and grown at 37'C for 18-24 hrs. This method allowed the generation of a large number of slides, each containing about 50,000 cells that had tenaciously adhered to the glass surface.
  • Method #1 A conjugate of horse radish peroxidase (HRP) with streptavidin (75 ul) was added to the sample and incubated for 30 min. at room"tempera- ture. The sample was washed in 2 x SSC for 5 min. , then in 2 x SSC, 0.1% triton X-100 for 5 min. The sam ⁇ ple was very lightly blotted, and 75 ul HRP substrate solution was added and incubated for 30 min. at room temperature. The sample then was washed with PBS for one minute and mounted for viewing under a microscope. Method #2: (About twofold more sensitive than Method #1, but requires more steps) .
  • the sample is treated with 75 ul of blocking solution (10% normal rabbit serum, 4% BSA, 2 x SSC) for 10 min. at 37°C and washed twice in 2 x SSC at room temperature.
  • Bio- tinylated goat anti-avidin IgG (75 ul at mg/ml in 0.1% BSA) is added, incubated for 30 min. at 37'C, and re ⁇ moved by three washes (3 min./wash) in 2 x SSC at room temperature.
  • 75 ul of an avidin-biotinylated HRP complex (ABC complex. Vector labs) was added and incubated for 15 min. at 37*C.
  • a model system for demonstration and optimi- zation of in situ hybridization is the CaSki cell line which is derived from a cervical tumor and contains multiple copies of HPV-16 (Yee, et al., "Presence and Expression of Human Papillomavirus Sequences in Human Cervical Carcinoma Cell Lines," Am. J. Pathology, 119:361-365 (1985) and Pater, M.M. , and Pater, A., "Hu ⁇ man Papillomavirus Types 16 and 18 Sequences in Carcinoma Cell Lines of the Cervix," Virology, 145:313-318 (1985)).
  • CaSki cells are easily cultured and therefore ideal for the production of a large num ⁇ ber of "standardized" glass slides containing immobil ⁇ ized cells. Such standardized samples are extremely useful for optimization of in situ hybridizations. It was found that a large number of standardized glass " slides could be prepared conveniently by growing cells directly on glass slides overnight. During this growth, the cells adhered tightly to the slides and subsequently could be processed using a variety of pro ⁇ cedures without cell loss.
  • DNA was chosen as probe, since this will hybridize rap ⁇ idly with the highly repetitive DNA sequences of human cells, thus allowing optimal in situ hybridization conditions to be quickly defined.
  • a detectable non-isotopic signal can be picked up during in situ hybridization with cervical smears using 10 ng of biotinylated human placental DNA.
  • a preferred minimal treatment to achieve an in situ hybridization signal from cervical smear samples was derived in the follow ⁇ ing experiments. Four cervical smear samples were taken by a gynecologist and immersed in 95% ethanol by the stan ⁇ dard procedure:
  • Sample 2 was immersed in Carnoy's B solution and processed.
  • Sample 3 was fixed in paraformalde- hyde, washed and processed.
  • Sample 4 was fixed in paraformalde- hyde, treated with Triton X-100, fixed again in para- formaldehyde, washed and processed (this procedure pre ⁇ viously was developed for cervical smear iji situ hy ⁇ bridization) .
  • the present invention provides a hybridization probe-based HPV diagnostic test that can be used in conjunction with traditional cellular smear methodol ⁇ ogy, particularly for identifying the presence and/or type of HPV in an infected sample of a cervical smear.
  • the assays of the present invention are rapid, reliable and economical, and can be readily formatted for clini- cal laboratories.

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Abstract

La présente invention se rapporte à une analyse d'hybridation in situ rapide qui permet de détecter la présence de Papillomavirus humains (HPV) dans un frottis cellulaire non congelé fixé sur un porte-échantillon ou autre support en l'absence de réactifs de réticulation à base d'aldéhyde. L'analyse peut être effectuée en moins de quatre heures et utilise des sondes d'acides nucléiques immédiatement détectables, généralement marquées par translation par incision avec de la biotine, ce qui produit une liaison avec une enzyme marquée par de l'avidine ou de la streptavidine, ou avec un isotope pour une détection visuelle.
EP19880909874 1987-10-02 1988-09-30 Human papillomavirus type diagnosis with nucleotide probes Withdrawn EP0338067A4 (en)

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US10397987A 1987-10-02 1987-10-02
US103979 1987-10-02

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ATE153706T1 (de) * 1988-08-31 1997-06-15 Aprogenex Inc Manuelles in situ hybridisierungsverfahren
US5182377A (en) * 1988-09-09 1993-01-26 Hoffmann-La Roche Inc. Probes for detection of human papillomavirus
US5447839A (en) * 1988-09-09 1995-09-05 Hoffmann-La Roche Inc. Detection of human papillomavirus by the polymerase chain reaction
GB8919145D0 (en) * 1989-08-23 1989-10-04 Royal Postgrad Med School Apparatus for the in situ hybridisation of slide-mounted cell samples
CA2025180A1 (fr) * 1989-10-12 1991-04-13 William G. Weisburg Sondes a l'acide nucleique et methodes pour la detection de levures pathogenes de candida
US5863717A (en) * 1989-11-03 1999-01-26 Abbott Laboratories Use of conserved oligonucleotide primers to amplify human papillomavirus DNA sequences
US5580970A (en) * 1989-12-01 1996-12-03 Amoco Corporation Detection of HPV transcripts
NL9000134A (nl) * 1990-01-19 1991-08-16 Stichting Res Fonds Pathologie Primers en werkwijze voor het detecteren van humaan papilloma virus genotypen m.b.v. pcr.
US5346811A (en) * 1991-07-22 1994-09-13 Cerveceria Polar Method and products for human papillomavirus detection
BE1005174A3 (nl) * 1991-08-13 1993-05-11 Universitaire Instelling Antwe Werkwijze voor het opsporen van een virus in celmateriaal van een afschraapsel of lichaamsvocht.
US5582985A (en) * 1994-08-05 1996-12-10 The Regents Of The University Of California Detection of mycobacteria
WO1997018334A2 (fr) * 1995-11-15 1997-05-22 Gen-Probe Incorporated Sondes d'acides nucleiques complementaires des acides nucleiques du virus du papillome humain, methodes et preparations associees
KR100382703B1 (ko) * 2000-03-15 2003-05-09 주식회사 바이오메드랩 인유두종바이러스의 유전형 진단키트 및 상기 진단키트의제조방법
KR100437626B1 (ko) * 2001-06-21 2004-06-26 주식회사 마이진 인간 유두종바이러스 유전자형 검사 방법과 이를 위한검사 키트
KR100452163B1 (ko) * 2001-09-14 2004-10-12 주식회사 바이오메드랩 인유두종 바이러스의 감염을 진단하기 위한 유전형 분석키트
EP1302550A1 (fr) * 2001-10-10 2003-04-16 King Car Food Industrial Co., Ltd. Procédé et détecteur pour la détection de virus de type de HPV
KR100453207B1 (ko) * 2001-11-19 2004-10-15 킹 카 푸드 인더스트리얼 콤파니 리미티드 다종의 사람 유두종 병원체 서브타입을 동시에 식별 및감별하기 위한 검출기 및 검출방법
WO2005033333A2 (fr) 2003-10-07 2005-04-14 Dako Denmark A/S Methodes et compositions pour le diagnostic de cancer
US20070122909A1 (en) * 2003-10-20 2007-05-31 Syssmex Corporation Method of treating cells
JP5020825B2 (ja) 2004-12-08 2012-09-05 ジェン−プローブ・インコーポレーテッド 複数型のヒトパピローマウイルスに由来する核酸の検出
WO2011109705A2 (fr) * 2010-03-04 2011-09-09 Purdue Research Foundation Essai intégré qui combine la cytométrie en flux et l'identification de génotypes de papillomavirus humain (hpv) multiplex
AU2013205122B2 (en) * 2012-10-11 2016-11-10 Gen-Probe Incorporated Compositions and Methods for Detecting Human Papillomavirus Nucleic Acid
WO2017041005A1 (fr) 2015-09-03 2017-03-09 Abbott Molecular Inc. Tampons d'hybridation comprenant un diester d'alkyle
JP2019502386A (ja) 2016-01-08 2019-01-31 アボツト・モレキユラー・インコーポレイテツド グアニジウムチオシアネートを含むハイブリダイゼーション緩衝液
CN112195287B (zh) * 2020-11-12 2022-03-01 武汉凯德维斯生物技术有限公司 一种用于人乳头瘤病毒hpv分型和整合检测的探针组及其试剂盒
CN112195281A (zh) * 2020-11-12 2021-01-08 武汉凯德维斯生物技术有限公司 一种用于检测人乳头瘤病毒hpv56的探针及其试剂盒

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JPH02501442A (ja) 1990-05-24
EP0338067A4 (en) 1991-05-02
AU2785589A (en) 1989-04-18

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