WO1993013227A1 - Cmv probes for use in solution phase sandwich hybridization assays - Google Patents

Cmv probes for use in solution phase sandwich hybridization assays Download PDF

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Publication number
WO1993013227A1
WO1993013227A1 PCT/US1992/011170 US9211170W WO9313227A1 WO 1993013227 A1 WO1993013227 A1 WO 1993013227A1 US 9211170 W US9211170 W US 9211170W WO 9313227 A1 WO9313227 A1 WO 9313227A1
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Prior art keywords
seq
segment
oligonucleotide
cmv
nucleic acid
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PCT/US1992/011170
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French (fr)
Inventor
Janice A. Kolberg
Lu-Ping Shen
Michael S. Urdea
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Chiron Corporation
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Priority to JP5511874A priority Critical patent/JPH07502655A/en
Priority to EP93902723A priority patent/EP0625214A4/en
Priority to CA002124796A priority patent/CA2124796A1/en
Publication of WO1993013227A1 publication Critical patent/WO1993013227A1/en

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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
    • C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster

Definitions

  • This invention is in the field of nucleic acid hybridization assays. More specifically, it relates to novel nucleic acid probes for detecting Cytomegalovirus (CMV).
  • CMV Cytomegalovirus
  • Cytomegalovirus is a member of the herpes virus family, causing clinical illness of particular importance in neonates, immunocompromised, and
  • U.S. No. 4,762,780 filed 5 January, 1987, discloses the use of restriction fragments of CMV DNA which do not cross-hybridize with human DNA for use in hybridization assays for CMV.
  • EPA 0271201 filed 2 November 1987, discloses the use of cloned restriction fragments of CMV DNA for use in hybridization assays for CMV.
  • PCT WO 91/02091 filed 9 August 1990, discloses probe sequences to detect and distinguish herpesviruses, including a 31 residue oligonucleotide probe sequence for CMV detection.
  • Spector et al. disclose a 32 P-labeled RNA probe and three single-stranded DNA oligomer capture probes complementary to mRNA encoding the CMV late matrix protein for use in a "target cycling" assay, and four 20-oligonucleotide primers and two 40-oligonucleotide probes for PCR
  • analyte-containing complex to be detected via a signal generated directly or indirectly from the label.
  • capturing probe sets in a first vessel and transferring the complex to another vessel containing immobilized nucleic acid that will hybridize to a segment of the capturing probes are followed.
  • the multimer is then hybridized to the immobilized complex and the labeled probes in turn hybridized to the second segment
  • the signal is amplified.
  • Amplifier and capture probe sequences are disclosed for Hepatitis B virus, Neisseria gonorrhoeae, penicillin and tetracycline resistance in N. gonorrhoeae, and Chlamydia trachomatis.
  • One aspect of the invention is a synthetic oligonucleotide useful as an amplifier probe in a sandwich hybridization assay for CMV comprising
  • Another aspect of the invention is a synthetic oligonucleotide useful as a capture probe in a sandwich hybridization assay for CMV comprising a first segment having a nucleotide sequence substantially complementary to a segment of CMV nucleic acid, and a second segment having a nucleotide sequence substantially complementary to an oligonucleotide bound to a solid phase.
  • Another aspect of the invention is a solution sandwich hybridization assay for detecting the presence of CMV in a sample, comprising
  • step (b) contacting the product of step (a) under hybridizing conditions with said oligonucleotide bound to the solid phase;
  • step (d) contacting the bound product of step (c) under hybridization conditions with the nucleic acid multimer, said multimer comprising at least one
  • oligonucleotide unit that is substantially complementary to the second segment of the amplifier probe polynucleotide and a multiplicity of second
  • step (f) contacting under hybridizing conditions the solid phase complex product of step (e) with the labeled oligonucleotide;
  • step (h) detecting the presence of label in the solid phase complex product of step (g).
  • kits for the detection of CMV comprising a kit for the detection of CMV in a sample comprising in combination
  • a set of amplifier probe oligonucleotides wherein the amplifier probe oligonucleotide comprises a first segment having a nucleotide sequence substantially complementary to a segment of CMV nucleic acid and a second segment having a nucleotide sequence substantially complementary to an oligonucleotide unit of a nucleic acid multimer;
  • a set of capture probe oligonucleotides wherein the capture probe oligonucleotide comprises a first segment having a nucleotide sequence that is substantially complementary to a segment of CMV nucleic acid and a second segment that is substantially
  • nucleic acid multimer comprising at least one oligonucleotide unit that is substantially complementary to the second segment of the amplifier probe polynucleotide and a multiplicity of second oligonucleotide units that are substantially complementary to a labeled oligonucleotide;
  • Solution phase nucleic acid hybridization assay intends the assay techniques described and claimed in commonly owned U.S. Patent No. 4,868,105 and EPA
  • a "modified nucleotide” intends a nucleotide monomer that may be stably incorporated into a
  • the modified nucleotide is a 5'- cytidine in which the N 4 -position is modified to provide a functional hydroxy group.
  • An “amplifier multimer” intends a branched polynucleotide that is capable of hybridizing
  • the branching in the multimers is effected through covalent bonds and the multimers are composed of two types of oligonucleotide units that are capable of hybridizing, respectively, to analyte nucleic acid or nucleic acid hybridized to analyte nucleic acid and to a multiplicity of labeled probes.
  • amplifier probe is intended as a branched or linear polynucleotide that is constructed to have a segment that hybridizes specifically to the analyte nucleic acid and iterations of a second segment that hybridize specifically to an amplifier multimer.
  • capture probe is intended as an oligonucleotide having a segment substantially
  • “Large” as used herein to describe the comb- type branched polynucleotides of the invention intends a molecule having at least about 15 branch sites and at least about 20 iterations of the labeled probe binding sequence.
  • invention intends a polynucleotide having a linear backbone with a multiplicity of sidechains extending from the backbone.
  • a "cleavable linker molecule” intends a
  • polynucleotide chain and which includes a covalent bond that may be broken or cleaved by chemical treatment or physical treatment such as by irradiation.
  • the general protocol for the solution phase sandwich hybridizations is as follows.
  • the analyte nucleic acid is placed in a microtiter well with an excess of two single-stranded nucleic acid probe sets: (1) a set of capture probes, each having a first binding sequence substantially complementary to the analyte and a second binding sequence that is substantially
  • a solid support for example, the well surface or a bead
  • a set of amplifier probes branched or linear
  • the resulting product is a three component nucleic acid complex of the two probes hybridized to the analyte by their first binding sequences.
  • the second binding sequences of the probes remain as single-stranded segments as they are not substantially complementary to the analyte. This complex hybridizes to the immobilized probe on the solid surface via the second binding
  • the resulting product comprises the complex bound to the solid surface via the duplex formed by the oligonucleotide bound to the solid surface and the second binding sequence of the capture probe. Unbound materials are then removed from the surface such as by washing.
  • the amplification multimer is then added to the bound complex under hybridization conditions to permit the multimer to hybridize to the available second binding sequence (s) of the amplifier probe of the complex.
  • the resulting complex is then separated from any unbound multimer by washing.
  • the labeled oligonucleotide is then added under conditions which permit it to hybridize to the substantially complementary oligonucleotide units of the multimer.
  • the resulting immobilized labeled nucleic acid complex is then washed to remove unbound labeled oligonucleotide, and read.
  • the analyte nucleic acids may be from a variety of sources, e.g., biological fluids or solids, and may be prepared for the hybridization analysis by a variety of means, e.g., proteinase K/SDS, chaotropic salts, etc. Also, it may be of advantage to decrease the average size of the analyte nucleic acids by enzymatic, physical or chemical means, e.g., restriction enzymes, sonication, chemical degradation (e.g., metal ions), etc.
  • the fragments may be as small as 0.1 kb, usually being at least about 0.5 kb and may be 1 kb or higher.
  • the analyte sequence is provided in single-stranded form for
  • the sequence should be denatured. Denaturation can be carried out by various techniques, such as alkali, generally from about 0.05 to 0.2 M hydroxide, formamide, salts, heat, enzymes, or
  • the first binding sequences of the capture probe and amplifier probe that are substantially identical
  • complementary to the analyte sequence will each be of at least 15 nucleotides, usually at least 25 nucleotides, and not more than about 5 kb, usually not more than about 1 kb, preferably not more than about 100 nucleotides.
  • the first binding sequences may be selected based on a variety of considerations.
  • a consensus sequence a sequence associated with polymorphisms, a particular phenotype or genotype, a particular strain, or the like.
  • the number of different amplifier and capture probes used influences the sensitivity of the assay, because the more probe sequences used, the greater the signal provided by the assay system. Furthermore, the use of more probe sequences allows the use of more stringent hybridization conditions, thereby reducing the incidence of false positive results.
  • the number of probes in a set will be at least one capture probe and at least one amplifier probe, more preferably two capture and two amplifier probes, and most preferably 5-100 capture probes and 5-100 amplifier probes.
  • Oligonucleotide probes for CMV were designed by aligning the DNA sequences of the glycoprotein B gene of 12 clinical strains of CMV and the laboratory-adapted strains AD169 and Towne. Additional oligonucleotide probes were designed by aligning the UL56 regions of Adl69 and Towne. Regions of greatest homology were chosen for capture probes, while regions of lesser homology were chosen as amplifier probes. Thus, as additional strains or isolates of CMV are made available, appropriate probes made be designed by aligning the sequence of the new strain or isolate with the nucleotide sequences used to design the probes of the present invention, and choosing regions of greatest homology for use as capture probes, with regions of lesser homology chosen as amplifier probes. The set of presently
  • preferred probes and their capture or amplifier overhang regions i.e., the regions which hybridize to sequences immobilized on solid support or to an oligonucleotide multimer, are listed in the examples.
  • the second binding sequences of the capture probe and amplifier probe are selected to be substantially complementary, respectively, to the
  • the second binding sequence may be contiguous to the first binding sequence or be spaced therefrom by an
  • the probes may include other noncomplementary sequences if desired.
  • the capture probe and amplifier probe may be prepared by oligonucleotide synthesis procedures or by cloning, preferably the former.
  • sequences need not have perfect complementarity to provide homoduplexes. In many situations, heteroduplexes will suffice where fewer than about 10% of the bases are mismatches, ignoring loops of five or more nucleotides. Accordingly, as used herein the term "complementary" intends exact complementarity wherein each base within the binding region corresponds exactly, and
  • substantially complementary intends 90% or greater homology.
  • the labeled oligonucleotide will include a sequence substantially complementary to the repeated oligonucleotide units of the multimer.
  • the labeled oligonucleotide will include one or more molecules
  • labels which directly or indirectly provide a detectable signal.
  • the labels may be bound to individual members of the substantially complementary sequence or may be present as a terminal member or terminal tail having a plurality of labels.
  • the labels may be bound either covalently or non-covalently to the substantially complementary sequence. Labels which may be employed include radionuclides, fluorescers,
  • chemiluminescers dyes, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, enzyme subunits, metal ions, and the like.
  • Illustrative specific labels include fluorescein, rhodamine, Texas red, phycoerythrin, umbelliferone, luminol, NADPH, ⁇ - ⁇ -galactosidase, horseradish peroxidase, alkaline phosphatase, etc.
  • the ratio of capture probe and amplifier probe to anticipated moles of analyte will each be at least stoichiometric and preferably in excess. This ratio is preferably at least about 1.5:1, and more preferably at least 2:1. It will normally be in the range of 2:1 to
  • Concentrations of each of the probes will generally range from about 10 -5 to 10 -9 M, with sample nucleic acid concentrations varying from 10 -21 to 10 -12 M.
  • the hybridization steps of the assay will generally take from about 10 minutes to 20 hours, frequently being completed in about 1 hour. Hybridization can be carried out at a mildly elevated temperature, generally in the range from about 20oC to 80°C, more usually from about 35°C to 70°C, particularly 65°C.
  • the hybridization reactions are usually done in an aqueous medium, particularly a buffered aqueous
  • Additives which may be employed include low concentrations of detergent (0.01 to 1%), salts, e.g., sodium citrate
  • solvents may be added to the aqueous medium, such as dimethylformamide, dimethylsulfoxide, alcohols, and formamide. These other solvents are generally present in amounts ranging from 2 to 50%.
  • the stringency of the hybridization medium may be controlled by temperature, salt concentration, solvent system, and the like. Thus, depending upon the length and nature of the sequence of interest, the stringency will be varied.
  • various techniques can be employed for detecting the presence of the label. For fluorescers, a large number of different fluorometers are available. For chemiluminescers, luminometers or films are available. With enzymes, a fluorescent, chemiluminescent, or colored product can be provided and determined fluorometrically, luminometric- ally, spectrophotometrically or visually.
  • the various labels which have been employed in immunoassays and the techniques applicable to immunoassays can be employed with the subject assays.
  • Kits for carrying out amplified nucleic acid hybridization assays will comprise in packaged combination the following reagents: the amplifier probe or set of probes; the capture probe or set of probes; the amplifier multimer; and an
  • kits may also include a denaturation reagent for denaturing the analyte, hybridization buffers, wash solutions, enzyme substrates, negative and positive controls and written instructions for carrying out the assay.
  • This example illustrates the synthesis of a comb-type branched polynucleotide having 15 branch sites and sidechain extensions having three labeled probe binding sites.
  • This polynucleotide was designed to be used in a solution phase hybridization as described in EPA 883096976. All chemical syntheses of oligonucleotides were performed on an automatic DNA synthesizer (Applied to the synthesis of a comb-type branched polynucleotide having 15 branch sites and sidechain extensions having three labeled probe binding sites.
  • This polynucleotide was designed to be used in a solution phase hybridization as described in EPA 883096976. All chemical syntheses of oligonucleotides were performed on an automatic DNA synthesizer (Applied
  • a comb body of the following structure was first prepared:
  • X' is a branching monomer
  • R is a periodate cleavable linker
  • the portion of the comb body through the 15 (TTX') repeats is first synthesized using 33.8 mg
  • CPG aminopropyl-derivatized thymidine controlled pore glass
  • the branching site nucleotide was of the formula:
  • R 2 represents
  • cyanoethylphosphoramidite monomers was 0.1 M for A, C, G and T, 0.15 M for the branching site monomer E, and 0.2 M for PhostelTM reagent. Detritylation was done with 3% trichloroacetic acid in methylene chloride using stepped flowthrough for the duration of the deprotection. At the conclusion the 5 ' DMT was replaced with an acetyl group.
  • Cleavable linker R and six base sidechain extensions of the formula 3'-RGTCAGTp were synthesized at each branching monomer site as follows .
  • the base protecting group removal (R 2 in the formula above) was performed manually while retaining the CPG support in the same column used for synthesizing the comb body.
  • R 2 levulinyl
  • a solution of 0.5 M hydrazine hydrate in pyridine/glacial acetic acid (1:1 v/v) was introduced and kept in contact with the CPG support for 90 min with renewal of the liquid every 15 min, followed by extensive washing with pyridine/glacial acetic acid (1:1 v/v) and then by acetonitrile.
  • the cleavable linker R and six base sidechain extensions were added using a 6.4 cycle.
  • the crude comb body was purified by a standard polyacrylamide gel (7% with 7 M urea and 1X TBE running buffer) method.
  • the 3' backbone extension and the sidechain extensions were ligated to the comb body as follows.
  • the comb body (4 pmole/ ⁇ l), 3' backbone extension (6.25 pmole/ ⁇ l), sidechain extension (93.75 pmole/ ⁇ l),
  • sidechain linking template 75 pmoles/ ⁇ l
  • backbone linking template 5 pmole/ ⁇ l
  • the mixture was incubated at 37°C for 2 hr, then heated in a water bath to 95°C, and then slowly cooled to below 35°C over a 1 hr period.
  • the mixture was adjusted to 1 mM ATP, 5 mM DTT, 14% polyethylene glycol, 50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 2 mM spermidine, 0.5 units/ ⁇ l T4 polynucleotide kinase, and 0.21 units/ ⁇ l T4 ligase were added, and the mixture incubated at 23°C for 16-24 hr. Ligation products were then purified by polyacrylamide gel electrophoresis.
  • a "15 X 3 amplified solution phase nucleic acid sandwich hybridization assay format was used in this assay.
  • the "15 x 3" designation derives from the fact that the format employs two multimers: (1) an amplifier probe having a first segment (A) that binds to CMV and a second segment (B) that hybridizes to (2) an amplifier multimer having a first segment (B*) that hybridizes to the segment (B) and fifteen iterations of a segment (C), wherein segment C hybridizes to three labeled
  • CTGCAGCCGCTTGTTCARCGAGCGGCCCTGATT CMV.UL.26 (SEQ ID NO:27)
  • CTTTACCCGCTGCTTGTACGAGTTGAAYGCGCG CMV.GB.20 (SEQ ID NO:44) CGGYARCGGGTCGACTACCTTGTCCTCCACGTA
  • Each amplifier probe contained, in addition to the sequences substantially complementary to the CMV sequences, the following 5' extension complementary to a segment of the amplifier multimer,
  • amplifier probes used in this assay also contained a sequence at their 3' end, 5'-TTTGTGGG-3' (SEQ ID NO:52), for use in other configurations of the
  • hybridization assay This sequence was not required for the hybridization assay as described here.
  • Each capture probe contained, in addition to the sequences substantially complementary to CMV DNA, a downstream sequence complementary to DNA bound to the solid phase (XT1*),
  • Microtiter plates were prepared as follows.
  • microtiter plates 96 wells/plate
  • 96 wells/plate were purchased from Dynatech Inc. Each well was filled with 200 ⁇ l 1 N HCl and incubated at room temperature for 15-20 min. The plates were then washed 4 times with 1X PBS and the wells aspirated to remove liquid. The wells were then filled with 200 ⁇ l 1 N NaOH and incubated at room temperature for 15-20 min. The plates were again washed 4 times with 1X PBS and the wells aspirated to remove liquid.
  • Poly(phe-lys) was purchased from Sigma
  • This polypeptide has a 1:1 molar ratio of phe:lys and an average m.w. of 47,900 gm/mole. It has an average length of 309 amino acids and contains 155 amines/mole.
  • a 1 mg/ml solution of the polypeptide was mixed with 2M NaCl/lX PBS to a final concentration of 0.1 mg/ml (pH 6.0). 100 ⁇ L of this solution was added to each well. The plate was wrapped in plastic to prevent drying and incubated at 30°C overnight. The plate was then washed 4 times with 1X PBS and the wells aspirated to remove liquid.
  • NAP-25 column was equilibrated with 10 mM sodium
  • Sample preparation consisted of delivering 12.5 ⁇ l P-K Buffer (2 mg/ml proteinase K in 10 mM Tris-HCl, pH 8.0/0.15 M NaCl/10 mM EDTA, pH 8.0/1% SDS/40 ⁇ g/ml sonicated salmon sperm DNA) to each well, followed by 10 ⁇ l of each sample in DMEM/10% FCS to be tested per well.
  • P-K Buffer 2 mg/ml proteinase K in 10 mM Tris-HCl, pH 8.0/0.15 M NaCl/10 mM EDTA, pH 8.0/1% SDS/40 ⁇ g/ml sonicated salmon sperm DNA
  • Neutralization buffer was then added to each well (0.77 M 3-(N-morpholino)propane sulfonic acid/1.845 M NaCl/0.185 M sodium citrate). Plates were covered and incubated for 12-18 hr at 65°C.
  • Amplifier multimer was then added to each well (40 fmoles/well in 4X SSC/ 0.1% SDS / 0.5% Blocking Reagent (Boehringer Mannheim, catalog No . 1096 176)). After covering plates and agitating to mix the contents in the wells, the plates were incubated for 15 min at 65°C.
  • Alkaline phosphatase label probe disclosed in EP 883096976 was then added to each well (50 fmoles in 40 ⁇ l/well). After incubation at 55°C for 15 min, and 5- 10 min at room temperature, the wells were washed twice as above and then 3X with 0.015 M ⁇ aCl/0.0015 M sodium citrate.
  • Results are shown in the Table below. Results for each standard sample are expressed as the difference between the mean of the negative control plus two

Abstract

Novel DNA probe sequences for detection of CMV in a sample in a solution phase sandwich hybridization assay are described. Amplified nucleic acid hybridization assays using the probes are exemplified.

Description

CMV PROBES FOR USE IN SOLUTION PHASE
SANDWICH HYBRIDIZATION ASSAYS
DESCRIPTION Technical Field
This invention is in the field of nucleic acid hybridization assays. More specifically, it relates to novel nucleic acid probes for detecting Cytomegalovirus (CMV).
Background Art
Cytomegalovirus (CMV) is a member of the herpes virus family, causing clinical illness of particular importance in neonates, immunocompromised, and
immunosuppressed patients.
Chou and Dennison (J. Infect. Pis. 163:1229- 1234, 1991) disclose the nucleotide sequence of part of the envelope glycoprotein B gene of human CMV that encodes epitopes recognized by virus-neutralizing
monoclonal antibodies for 12 distinct clinical isolates of CMV.
U.S. No. 4,731,325, filed 24 January, 1985, discloses restriction fragments of CMV DNA in sandwich hybridizations assays.
U.S. No. 4,762,780, filed 5 January, 1987, discloses the use of restriction fragments of CMV DNA which do not cross-hybridize with human DNA for use in hybridization assays for CMV. EPA 0271201, filed 2 November 1987, discloses the use of cloned restriction fragments of CMV DNA for use in hybridization assays for CMV.
PCT WO 91/02091, filed 9 August 1990, discloses probe sequences to detect and distinguish herpesviruses, including a 31 residue oligonucleotide probe sequence for CMV detection.
Spector et al. (Clin. Chem. 35/8:1581- 1587,1989) disclose a 32P-labeled RNA probe and three single-stranded DNA oligomer capture probes complementary to mRNA encoding the CMV late matrix protein for use in a "target cycling" assay, and four 20-oligonucleotide primers and two 40-oligonucleotide probes for PCR
amplification of CMV.
Commonly owned U.S. 4,868,105 describes a solution phase nucleic acid sandwich hybridization assay in which analyte nucleic acid is first hybridized in solution to a labeling probe set and to a capturing probe set in a first vessel. The probe-analyte complex is then transferred to a second vessel that contains a solid- phase-immobilized probe that is substantially
complementary to a segment of the capturing probes. The segments hybridize to the immobilized probe, thus
removing the complex from solution. Having the analyte in the form of an immobilized complex facilitates
subsequent separation steps in the assay. Ultimately, single stranded segments of the labeling probe set are hybridized to labeled probes, thus permitting the
analyte-containing complex to be detected via a signal generated directly or indirectly from the label.
Commonly owned European Patent Application
(EPA) 883096976 discloses a variation in the assay
described in U.S. 4,868,105 in which the signal generated by the labeled probes is amplified. The amplification involves the use of nucleic acid multimers. These multimers are branched polynucleotides that are
constructed to have a segment that hybridizes
specifically to the analyte nucleic acid or to a nucleic acid (branched or linear) that is bound to the analyte and iterations of a second segment that hybridize specifically to the labeled probe. In the assay
employing the multimer, the initial steps of hybridizing the analyte to label or amplifier probe sets and
capturing probe sets in a first vessel and transferring the complex to another vessel containing immobilized nucleic acid that will hybridize to a segment of the capturing probes are followed. The multimer is then hybridized to the immobilized complex and the labeled probes in turn hybridized to the second segment
iterations on the multimer. Since the multimers provide a large number of sites for label probe attachment, the signal is amplified. Amplifier and capture probe sequences are disclosed for Hepatitis B virus, Neisseria gonorrhoeae, penicillin and tetracycline resistance in N. gonorrhoeae, and Chlamydia trachomatis.
Commonly owned copending application U.S.
Serial No. 558,897, filed 27 July 1990, describes the preparation of large comb-type branched polynucleotide multimers for use in the above-described solution phase assay. The combs provide greater signal enhancement in the assays than the smaller multimers.
Disclosure of the Invention
One aspect of the invention is a synthetic oligonucleotide useful as an amplifier probe in a sandwich hybridization assay for CMV comprising
a first segment having a nucleotide sequence
substantially complementary to a segment of CMV nucleic acid, and a second segment having a nucleotide sequence substantially complementary to an oligonucleotide multimer.
Another aspect of the invention is a synthetic oligonucleotide useful as a capture probe in a sandwich hybridization assay for CMV comprising a first segment having a nucleotide sequence substantially complementary to a segment of CMV nucleic acid, and a second segment having a nucleotide sequence substantially complementary to an oligonucleotide bound to a solid phase.
Another aspect of the invention is a solution sandwich hybridization assay for detecting the presence of CMV in a sample, comprising
(a) contacting the sample under hybridizing conditions with an excess of (i) an amplifier probe oligonucleotide comprising a first segment having a nucleotide sequence substantially complementary to a segment of CMV nucleic acid and a second segment having a nucleotide sequence substantially complementary to an oligonucleotide unit of a nucleic acid multimer and (ii) a capture probe oligonucleotide comprising a first segment having a nucleotide sequence that is
substantially complementary to a segment of CMV nucleic acid and a second segment that is substantially
complementary to an oligonucleotide bound to a solid phase;
(b) contacting the product of step (a) under hybridizing conditions with said oligonucleotide bound to the solid phase;
(c) thereafter separating materials not bound to the solid phase;
(d) contacting the bound product of step (c) under hybridization conditions with the nucleic acid multimer, said multimer comprising at least one
oligonucleotide unit that is substantially complementary to the second segment of the amplifier probe polynucleotide and a multiplicity of second
oligonucleotide units that are substantially
complementary to a labeled oligonucleotide;
(e) removing unbound multimer;
(f) contacting under hybridizing conditions the solid phase complex product of step (e) with the labeled oligonucleotide;
(g) removing unbound labeled oligonucleotide; and
(h) detecting the presence of label in the solid phase complex product of step (g).
Another aspect of the invention is a kit for the detection of CMV comprising a kit for the detection of CMV in a sample comprising in combination
(i) a set of amplifier probe oligonucleotides wherein the amplifier probe oligonucleotide comprises a first segment having a nucleotide sequence substantially complementary to a segment of CMV nucleic acid and a second segment having a nucleotide sequence substantially complementary to an oligonucleotide unit of a nucleic acid multimer;
(ii) a set of capture probe oligonucleotides wherein the capture probe oligonucleotide comprises a first segment having a nucleotide sequence that is substantially complementary to a segment of CMV nucleic acid and a second segment that is substantially
complementary to an oligonucleotide bound to a solid phase;
(iii) a nucleic acid multimer, said multimer comprising at least one oligonucleotide unit that is substantially complementary to the second segment of the amplifier probe polynucleotide and a multiplicity of second oligonucleotide units that are substantially complementary to a labeled oligonucleotide; and
(iv) a labeled oligonucleotide. These and other embodiments will occur to one of ordinary skill in the art in view of the disclosure herein. Modes for Carrying out the Invention
Definitions
"Solution phase nucleic acid hybridization assay" intends the assay techniques described and claimed in commonly owned U.S. Patent No. 4,868,105 and EPA
883096976.
A "modified nucleotide" intends a nucleotide monomer that may be stably incorporated into a
polynucleotide and which has an additional functional group. Preferably, the modified nucleotide is a 5'- cytidine in which the N4-position is modified to provide a functional hydroxy group.
An "amplifier multimer" intends a branched polynucleotide that is capable of hybridizing
simultaneously directly or indirectly to analyte nucleic acid and to a multiplicity of polynucleotide iterations (i.e., either iterations of another multimer or
iterations of a labeled probe). The branching in the multimers is effected through covalent bonds and the multimers are composed of two types of oligonucleotide units that are capable of hybridizing, respectively, to analyte nucleic acid or nucleic acid hybridized to analyte nucleic acid and to a multiplicity of labeled probes. The composition and preparation of such
multimers are described in EPA 883096976 and U.S. Serial No. 558,897 filed 27 July 1990, the disclosures of which are incorporated herein by reference.
The term "amplifier probe" is intended as a branched or linear polynucleotide that is constructed to have a segment that hybridizes specifically to the analyte nucleic acid and iterations of a second segment that hybridize specifically to an amplifier multimer.
The term "capture probe" is intended as an oligonucleotide having a segment substantially
complementary to a nucleotide sequence of the target DNA and a segment that is substantially complementary to a nucleotide sequence of a solid-phase-immobilized probe.
"Large" as used herein to describe the comb- type branched polynucleotides of the invention intends a molecule having at least about 15 branch sites and at least about 20 iterations of the labeled probe binding sequence.
"Comb-type" as used herein to describe the structure of the branched polynucleotides of the
invention intends a polynucleotide having a linear backbone with a multiplicity of sidechains extending from the backbone.
A "cleavable linker molecule" intends a
molecule that may be stably incorporated into a
polynucleotide chain and which includes a covalent bond that may be broken or cleaved by chemical treatment or physical treatment such as by irradiation.
All nucleic acid sequences disclosed herein are written in a 5' to 3' direction. Nucleotides are
designated according to the nucleotide symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Solution Phase Hybridization Assay
The general protocol for the solution phase sandwich hybridizations is as follows. The analyte nucleic acid is placed in a microtiter well with an excess of two single-stranded nucleic acid probe sets: (1) a set of capture probes, each having a first binding sequence substantially complementary to the analyte and a second binding sequence that is substantially
complementary to nucleic acid bound to a solid support, for example, the well surface or a bead, and (2) a set of amplifier probes (branched or linear), each having a first binding sequence that is capable of specific binding to the analyte and a second binding sequence that is capable of specific binding to a segment of the multimer. The resulting product is a three component nucleic acid complex of the two probes hybridized to the analyte by their first binding sequences. The second binding sequences of the probes remain as single-stranded segments as they are not substantially complementary to the analyte. This complex hybridizes to the immobilized probe on the solid surface via the second binding
sequence of the capture probe. The resulting product comprises the complex bound to the solid surface via the duplex formed by the oligonucleotide bound to the solid surface and the second binding sequence of the capture probe. Unbound materials are then removed from the surface such as by washing.
The amplification multimer is then added to the bound complex under hybridization conditions to permit the multimer to hybridize to the available second binding sequence (s) of the amplifier probe of the complex. The resulting complex is then separated from any unbound multimer by washing. The labeled oligonucleotide is then added under conditions which permit it to hybridize to the substantially complementary oligonucleotide units of the multimer. The resulting immobilized labeled nucleic acid complex is then washed to remove unbound labeled oligonucleotide, and read.
The analyte nucleic acids may be from a variety of sources, e.g., biological fluids or solids, and may be prepared for the hybridization analysis by a variety of means, e.g., proteinase K/SDS, chaotropic salts, etc. Also, it may be of advantage to decrease the average size of the analyte nucleic acids by enzymatic, physical or chemical means, e.g., restriction enzymes, sonication, chemical degradation (e.g., metal ions), etc. The fragments may be as small as 0.1 kb, usually being at least about 0.5 kb and may be 1 kb or higher. The analyte sequence is provided in single-stranded form for
analysis. Where the sequence is naturally present in single-stranded form, denaturation will not be required. However, where the sequence may be present in
double-stranded form, the sequence should be denatured. Denaturation can be carried out by various techniques, such as alkali, generally from about 0.05 to 0.2 M hydroxide, formamide, salts, heat, enzymes, or
combinations thereof.
The first binding sequences of the capture probe and amplifier probe that are substantially
complementary to the analyte sequence will each be of at least 15 nucleotides, usually at least 25 nucleotides, and not more than about 5 kb, usually not more than about 1 kb, preferably not more than about 100 nucleotides.
They will typically be approximately 30 nucleotides.
They will normally be chosen to bind to different
sequences of the analyte. The first binding sequences may be selected based on a variety of considerations.
Depending upon the nature of the analyte, one may be interested in a consensus sequence, a sequence associated with polymorphisms, a particular phenotype or genotype, a particular strain, or the like.
The number of different amplifier and capture probes used influences the sensitivity of the assay, because the more probe sequences used, the greater the signal provided by the assay system. Furthermore, the use of more probe sequences allows the use of more stringent hybridization conditions, thereby reducing the incidence of false positive results. Thus, the number of probes in a set will be at least one capture probe and at least one amplifier probe, more preferably two capture and two amplifier probes, and most preferably 5-100 capture probes and 5-100 amplifier probes.
Oligonucleotide probes for CMV were designed by aligning the DNA sequences of the glycoprotein B gene of 12 clinical strains of CMV and the laboratory-adapted strains AD169 and Towne. Additional oligonucleotide probes were designed by aligning the UL56 regions of Adl69 and Towne. Regions of greatest homology were chosen for capture probes, while regions of lesser homology were chosen as amplifier probes. Thus, as additional strains or isolates of CMV are made available, appropriate probes made be designed by aligning the sequence of the new strain or isolate with the nucleotide sequences used to design the probes of the present invention, and choosing regions of greatest homology for use as capture probes, with regions of lesser homology chosen as amplifier probes. The set of presently
preferred probes and their capture or amplifier overhang regions, i.e., the regions which hybridize to sequences immobilized on solid support or to an oligonucleotide multimer, are listed in the examples.
The second binding sequences of the capture probe and amplifier probe are selected to be substantially complementary, respectively, to the
oligonucleotide bound to the solid surface and to a segment of the multimer and so as to not be encountered by endogenous sequences in the sample/analyte. The second binding sequence may be contiguous to the first binding sequence or be spaced therefrom by an
intermediate noncomplementary sequence. The probes may include other noncomplementary sequences if desired.
These noncomplementary sequences must not hinder the binding of the binding sequences or cause nonspecific binding to occur.
The capture probe and amplifier probe may be prepared by oligonucleotide synthesis procedures or by cloning, preferably the former.
It will be appreciated that the binding
sequences need not have perfect complementarity to provide homoduplexes. In many situations, heteroduplexes will suffice where fewer than about 10% of the bases are mismatches, ignoring loops of five or more nucleotides. Accordingly, as used herein the term "complementary" intends exact complementarity wherein each base within the binding region corresponds exactly, and
"substantially complementary" intends 90% or greater homology.
The labeled oligonucleotide will include a sequence substantially complementary to the repeated oligonucleotide units of the multimer. The labeled oligonucleotide will include one or more molecules
("labels"), which directly or indirectly provide a detectable signal. The labels may be bound to individual members of the substantially complementary sequence or may be present as a terminal member or terminal tail having a plurality of labels. Various means for
providing labels bound to the oligonucleotide sequences have been reported in the literature. See, for example, Leary et al., Proc. Natl. Acad. Sci. USA (1983) 80:4045: Renz and Kurz, Nucl. Acids Res. (1984) 12:3435:
Richardson and Gumport, Nucl. Acids Res. (1983) 11:6167: Smith et al., Nucl. Acids. Res. (1985) 13:2399; Meinkoth and Wahl, Anal. Biochem. (1984) 138:267. The labels may be bound either covalently or non-covalently to the substantially complementary sequence. Labels which may be employed include radionuclides, fluorescers,
chemiluminescers, dyes, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, enzyme subunits, metal ions, and the like. Illustrative specific labels include fluorescein, rhodamine, Texas red, phycoerythrin, umbelliferone, luminol, NADPH, α-β-galactosidase, horseradish peroxidase, alkaline phosphatase, etc.
The ratio of capture probe and amplifier probe to anticipated moles of analyte will each be at least stoichiometric and preferably in excess. This ratio is preferably at least about 1.5:1, and more preferably at least 2:1. It will normally be in the range of 2:1 to
106:1. Concentrations of each of the probes will generally range from about 10-5 to 10-9 M, with sample nucleic acid concentrations varying from 10-21 to 10-12 M. The hybridization steps of the assay will generally take from about 10 minutes to 20 hours, frequently being completed in about 1 hour. Hybridization can be carried out at a mildly elevated temperature, generally in the range from about 20ºC to 80°C, more usually from about 35°C to 70°C, particularly 65°C.
The hybridization reactions are usually done in an aqueous medium, particularly a buffered aqueous
medium, which may include various additives. Additives which may be employed include low concentrations of detergent (0.01 to 1%), salts, e.g., sodium citrate
(0.017 to 0.17 M), Ficoll, polyvinylpyrrolidone, carrier nucleic acids, carrier proteins, etc. Nonaqueous
solvents may be added to the aqueous medium, such as dimethylformamide, dimethylsulfoxide, alcohols, and formamide. These other solvents are generally present in amounts ranging from 2 to 50%.
The stringency of the hybridization medium may be controlled by temperature, salt concentration, solvent system, and the like. Thus, depending upon the length and nature of the sequence of interest, the stringency will be varied. Depending upon the nature of the label, various techniques can be employed for detecting the presence of the label. For fluorescers, a large number of different fluorometers are available. For chemiluminescers, luminometers or films are available. With enzymes, a fluorescent, chemiluminescent, or colored product can be provided and determined fluorometrically, luminometric- ally, spectrophotometrically or visually. The various labels which have been employed in immunoassays and the techniques applicable to immunoassays can be employed with the subject assays.
Kits for carrying out amplified nucleic acid hybridization assays according to the invention will comprise in packaged combination the following reagents: the amplifier probe or set of probes; the capture probe or set of probes; the amplifier multimer; and an
appropriate labeled oligonucleotide. These reagents will typically be in separate containers in the kit. The kit may also include a denaturation reagent for denaturing the analyte, hybridization buffers, wash solutions, enzyme substrates, negative and positive controls and written instructions for carrying out the assay.
The following examples further illustrate the invention. These examples are not intended to limit the invention in any manner.
EXAMPLES
Example I
Synthesis of Comb-type Branched Polynucleotide
This example illustrates the synthesis of a comb-type branched polynucleotide having 15 branch sites and sidechain extensions having three labeled probe binding sites. This polynucleotide was designed to be used in a solution phase hybridization as described in EPA 883096976. All chemical syntheses of oligonucleotides were performed on an automatic DNA synthesizer (Applied
Biosystems, Inc., (ABI) model 380 B). Phosphoramidite chemistry of the beta cyanoethyl type was used including 5' -phosphorylation which employed Phostel™ reagent (ABN). Standard ABI protocols were used except as indicated.
Where it is indicated that a multiple of a cycle was used (e.g., 1.2 cycle), the multiple of the standard amount of amidite recommended by ABI was employed in the specified cycle. Appended hereto are the programs for carrying out cycles 1.2 and 6.4 as run on the Applied Biosystems Model 380 B DNA Synthesizer.
A comb body of the following structure was first prepared:
3'T18(TTX')15GTTTGTGG-5'
|
(RGTCAGTp-5')15
wherein X' is a branching monomer, and R is a periodate cleavable linker.
The portion of the comb body through the 15 (TTX') repeats is first synthesized using 33.8 mg
aminopropyl-derivatized thymidine controlled pore glass (CPG) (2000 A, 7.4 micromoles thymidine per gram
support) with a 1.2 cycle protocol. The branching site nucleotide was of the formula:
Figure imgf000017_0001
where R2 represents
Figure imgf000017_0002
For synthesis of the comb body (not including sidechains), the concentration of beta
cyanoethylphosphoramidite monomers was 0.1 M for A, C, G and T, 0.15 M for the branching site monomer E, and 0.2 M for Phostel™ reagent. Detritylation was done with 3% trichloroacetic acid in methylene chloride using stepped flowthrough for the duration of the deprotection. At the conclusion the 5 ' DMT was replaced with an acetyl group.
Cleavable linker R and six base sidechain extensions of the formula 3'-RGTCAGTp (SEQ ID NO:1) were synthesized at each branching monomer site as follows . The base protecting group removal (R2 in the formula above) was performed manually while retaining the CPG support in the same column used for synthesizing the comb body. In the case of R2 = levulinyl, a solution of 0.5 M hydrazine hydrate in pyridine/glacial acetic acid (1:1 v/v) was introduced and kept in contact with the CPG support for 90 min with renewal of the liquid every 15 min, followed by extensive washing with pyridine/glacial acetic acid (1:1 v/v) and then by acetonitrile. After the deprotection the cleavable linker R and six base sidechain extensions were added using a 6.4 cycle.
In these syntheses the concentration of phosphoramidites was 0.1 M (except 0.2 M R and Phostel™ reagent; R was 2-(4-(4-(2-
Dimethoxytrityloxy)ethyl-)phenoxy 2,3-di(benzoyloxy)- butyloxy)phenyl)ethyl-2-cyanoethyl-N,N- diisopropylphosphoramidite).
Detritylation is effected with a solution of 3% trichloroacetic acid in methylene chloride
using continuous flowthrough, followed by a rinse solution of toluene/chloromethane (1:1 v/v). Branched polynucleotide chains were removed from the solid
supports automatically in the 380B using the cycle "CE NH3." The ammonium hydroxide solution was collected in 4 ml screw-capped Wheaton vials and heated at 60°C for 12 hr to remove all base-protecting groups. After cooling to room temperature the solvent was removed in a Speed- Vac evaporator and the residue dissolved in 100 μl water.
3' backbone extensions (segment A), sidechain extensions and ligation template/linkers of the following structures were also made using the automatic
synthesizer:
3' Backbone
extension 3'-TCCGTATCCTGGGCACAGAGGTGCp-5' (SEQ ID NO:2)
Sidechain
extension 3'-GATGCG(TTCATGCTGTTGGTGTAG)3-5' (SEQ ID NO:3) Ligation
template for
linking 3'
backbone
extension 3'-AAAAAAAAAAGCACCTp-5' (SEQ ID NO:4)
Ligation template for linking sidechain
extension 3'-CGCATCACTGAC-5' (SEQ ID NO:5)
The crude comb body was purified by a standard polyacrylamide gel (7% with 7 M urea and 1X TBE running buffer) method.
The 3' backbone extension and the sidechain extensions were ligated to the comb body as follows. The comb body (4 pmole/μl), 3' backbone extension (6.25 pmole/μl), sidechain extension (93.75 pmole/μl),
sidechain linking template (75 pmoles/μl) and backbone linking template (5 pmole/μl) were combined in 1 mM ATP/ 5 mM DTT/ 50 mM Tris-HCl, pH 8.0/ 10 mM MgCl2/ 2 mM spermidine, with 0.5 units/μl T4 polynucleotide kinase. The mixture was incubated at 37°C for 2 hr, then heated in a water bath to 95°C, and then slowly cooled to below 35°C over a 1 hr period. 2 mM ATP, 10 mM DTT, 14% polyethylene glycol, and 0.21 units/μl T4 ligase were added, and the mixture incubated for 16-24 hr at 23°C. The DNA was precipitated in NaCl/ethanol, resuspended in water, and subjected to a second ligation as follows.
The mixture was adjusted to 1 mM ATP, 5 mM DTT, 14% polyethylene glycol, 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 2 mM spermidine, 0.5 units/μl T4 polynucleotide kinase, and 0.21 units/μl T4 ligase were added, and the mixture incubated at 23°C for 16-24 hr. Ligation products were then purified by polyacrylamide gel electrophoresis.
After ligation and purification, a portion of the product was labeled with 32P and subjected to cleavage at the site of R achieved by oxidation with aqueous NaIO4 for 1 hr. The sample was then analyzed by PAGE to determine the number of sidechain extensions incorporated by quantitating the radioactive label in the bands on the gel. The product was found to have a total of 45 labeled probe binding sites.
Example 2
A "15 X 3 amplified solution phase nucleic acid sandwich hybridization assay format was used in this assay. The "15 x 3" designation derives from the fact that the format employs two multimers: (1) an amplifier probe having a first segment (A) that binds to CMV and a second segment (B) that hybridizes to (2) an amplifier multimer having a first segment (B*) that hybridizes to the segment (B) and fifteen iterations of a segment (C), wherein segment C hybridizes to three labeled
oligonucleotides.
The amplifier and capture probe segments and their respective names used in this assay were as
follows.
CMV Amplifier Probes
CMV.GB.Z.1 (SEQ ID NO:6)
CCGRTTGATGTARCYGCGCAACGTRTCRTAGGT CMV.GB.Z.2 (SEQ ID NO:7)
CACACACCARGCYTCKGCGATYTGYGYYARCGC
CMV.GB.Z.3 (SEQ ID NO:8)
TTCCYTGAAGACCTCYAGGGWGCGCCGTTGATC CMV.GB.Z.4 (SEQ ID NO:9)
YGAGAGAATRGCTGAYGGRTTGATCTTGCTRAG CMV.GB.Z.5 (SEQ ID NO:10) GAAACGCGCGGCAATCGGTTTGTTGTARATGGC CMV.GB.Z.6 (SEQ ID NO: 11)
CACGCAGCTGGCCARRCCCARRACATCACCCAT CMV.GB.Z.7 (SEQ ID NO:12)
ACGCAGCACCTTRACGCTKGTTTGGTTRATRGT
CMV.GB.Z.8 (SEQ ID NO:13)
GCAGCGTCCTGGCGAYTCYTTCACRTTCATATC CMV.GB.Z.9 (SEQ ID NO:14)
GRCGAAATTAAAGATGACCACKGGTCGYGAGTA CMV.GB.Z.10 (SEQ ID NO:15)
RCCCAGTTGACCGTACTGCACRTACGAGCTGTT CMV.GB.Z.11 (SEQ ID NO:16)
GCGGTGGTTGCCCAACAGGATTTCGTTRTCCTC CMV.GB.Z.12 (SEQ ID NO:17)
GATCTTGAGGCTGGGAARCTGACATTCCTCAGT
CMV.GB.Z.13a (SEQ ID NO:18)
CACGTACTCGTAGGCCGAGTTSCCGGCGATGAA CMV.GB.Z.14 (SEQ ID NO:19)
GCTGAGGTCAATCATGCGTTTGAAGAGGTAGTC CMV.GB.Z.15 (SEQ ID NO:20)
YARGGCGATCATGCTGTCGACDGTRGAGATRCT CMV.GB.Z.16 (SEQ ID NO:21)
CCTGAAGTCRGTRTTTTCCAGCGGGTCGATRTC CMV.GB.Z.13b (SEQ ID NO:22)
CACATATTCATAGGCCGAGTTSCCGGCGATGAA
CMV.UL.22 (SEQ ID NO:23)
CGCCACCGGCGAGATGCCGCATAGGCGACGGAG CMV.UL.23 (SEQ ID NO:24)
GCATGTCGTCCCTTCGACGTACACTTCCTGACG CMV.UL.24 (SEQ ID NO:25)
CGGGATGATGGTCAGCTCCTCGTAGCATTGGGC CMV.UL.25 (SEQ ID NO:26)
CTGCAGCCGCTTGTTCARCGAGCGGCCCTGATT CMV.UL.26 (SEQ ID NO:27)
ACGGTGGACCGCTATATGGTTGCACAGCAAGCC CMV.UL.27 (SEQ ID NO:28 )
CGTCTGGATATTCACATCGGACTGGCTTGACGG CMV.UL.38 (SEQ ID NO:29)
GCGCGTTGTCAGGTCCAGCAGGTCCTGCTCCAC CMV.UL.29 (SEQ ID NO:30)
GAGGGCCGAAAGGACTCCAGCCAAGTGGGGGAT CMV.UL.30 (SEQ ID NO:31)
GTGGTAGGCCGATGAAGAAGAGAATAGGCTTTT CMV.UL.31 (SEQ ID NO: 32)
CCTCAGCGCCTCCTCCGCCTCCTGGATGTAGCT
CMV.UL.32 (SEQ ID NO:33)
TCGTTCCGGTATATCCGTAAACAGGTTGTACTC CMV.UL.33 (SEQ ID NO: 34)
GGACCAGTAGGTAAAATCCGACAAGGAATATAT CMV.UL.34 (SEQ ID NO:35)
GCCCACCCGCTTGACGATAACCTCCGAGGTACG CMV.UL.35 (SEQ ID NO:36)
CTGGTGATACACATTTAGCTGCTGGATGGTGAT CMV.UL.36 (SEQ ID NO:37)
GCGACTGATGCCGTTCATGAGCGCCCGGCACAG
CMV.UL.37 (SEQ ID NO:38)
GAAGATGTCCTCCACGTCCTCCCCGTACAGATG CMV.UL.38 (SEQ ID NO:39)
CTCCTCCCCGTCCAACGCCTTTTCCCCGAGCAC CMV.UL.39 (SEQ ID NO:40)
GGGGGCGGCAAAGACCGACCCCACGAACATGCG
CMV Capture Probes
CMV.GB.17 (SEQ ID NO:41)
ACGCARYTCTTTCTGCGAGTAAAGTTCCAGTAC
CMV.GB.18 (SEQ ID NO:42)
CATGATCTCYTCGAGRTCAAAAACGTTGCTGGA CMV. GB .19a (SEQ ID NO : 43)
CTTTACCCGCTGCTTGTACGAGTTGAAYGCGCG CMV.GB.20 (SEQ ID NO:44) CGGYARCGGGTCGACTACCTTGTCCTCCACGTA
CMV.GB.21 (SEQ ID NO:45)
GCTCATGAGGTCGTCCAGACCCTTGAGGTAGGG CMV.GB.19b (SEQ ID NO:46)
CTTTACCCGCTGCTTATACGAATTGAAYTCGCG
CMV.UL.40 (SEQ ID NO:47)
GCTGAGGGATGTGATGAGGTCGATGATCCTGTT CMV.UL.41 (SEQ ID NO:48)
GTTGAACACCGGGTTGTCCTCGAAAGCTTGAAT CMV.UL.42 (SEQ ID NO:49)
TTTGGTGTACATCTCGTTGCTTTCGTGGAGCTT CMV.UL.43 (SEQ ID NO:50)
CGGACGTCGAATCTCCTCGAGAATATGCTTGAT Each amplifier probe contained, in addition to the sequences substantially complementary to the CMV sequences, the following 5' extension complementary to a segment of the amplifier multimer,
AGGCATAGGACCCGTGTCTT (SEQ ID NO:51).
Some of the amplifier probes used in this assay also contained a sequence at their 3' end, 5'-TTTGTGGG-3' (SEQ ID NO:52), for use in other configurations of the
hybridization assay. This sequence was not required for the hybridization assay as described here.
Each capture probe contained, in addition to the sequences substantially complementary to CMV DNA, a downstream sequence complementary to DNA bound to the solid phase (XT1*),
CTTCTTTGGAGAAAGTGGTG (SEQ ID NO:53).
Microtiter plates were prepared as follows.
White Microlite 1 Removawell strips (polystyrene
microtiter plates, 96 wells/plate) were purchased from Dynatech Inc. Each well was filled with 200 μl 1 N HCl and incubated at room temperature for 15-20 min. The plates were then washed 4 times with 1X PBS and the wells aspirated to remove liquid. The wells were then filled with 200 μl 1 N NaOH and incubated at room temperature for 15-20 min. The plates were again washed 4 times with 1X PBS and the wells aspirated to remove liquid.
Poly(phe-lys) was purchased from Sigma
Chemicals, Inc. This polypeptide has a 1:1 molar ratio of phe:lys and an average m.w. of 47,900 gm/mole. It has an average length of 309 amino acids and contains 155 amines/mole. A 1 mg/ml solution of the polypeptide was mixed with 2M NaCl/lX PBS to a final concentration of 0.1 mg/ml (pH 6.0). 100 μL of this solution was added to each well. The plate was wrapped in plastic to prevent drying and incubated at 30°C overnight. The plate was then washed 4 times with 1X PBS and the wells aspirated to remove liquid.
The following procedure was used to couple the oligonucleotide XT1* to the plates. Synthesis of XT1* was described in EPA 883096976. 20 mg disuccinimidyl suberate was dissolved in 300 μl dimethyl formamide
(DMF) . 26 OD260 units of XT1* was added to 100 μl coupling buffer (50 mM sodium phosphate, pH 7.8). The coupling mixture was then added to the DSS-DMF solution and stirred with a magnetic stirrer for 30 min. An
NAP-25 column was equilibrated with 10 mM sodium
phosphate, pH 6.5. The coupling mixture DSS-DMF solution was added to 2 ml 10 mM sodium phosphate, pH 6.5, at 4°C. The mixture was vortexed to mix and loaded onto the equilibrated NAP-25 column. DSS-activated XT1* DNA was eluted from the column with 3.5 ml 10 mM sodium
phosphate, pH 6.5. 5.6 OD260 units of eluted DSS- activated XT1* DNA was added to 1500 ml 50 mM sodium phosphate, pH 7.8. 50 μl of this solution was added to each well and the plates were incubated overnight. The plate was then washed 4 times with 1X PBS and the wells aspirated to remove liquid. Final stripping of plates was accomplished as follows. 200 μL of 0.2N NaOH containing 0.5% (w/v) SDS was added to each well. The plate was wrapped in plastic and incubated at 65°C for 60 min. The plate was then washed 4 times with 1X PBS and the wells aspirated to remove liquid. The stripped plate was stored with desiccant beads at 2-8°C.
Sample preparation. consisted of delivering 12.5 μl P-K Buffer (2 mg/ml proteinase K in 10 mM Tris-HCl, pH 8.0/0.15 M NaCl/10 mM EDTA, pH 8.0/1% SDS/40 μg/ml sonicated salmon sperm DNA) to each well, followed by 10 μl of each sample in DMEM/10% FCS to be tested per well. A standard curve of CMV DNA was prepared by diluting stock CMV in DMEM/10% fetal calf serum (FCS) and
delivering aliquots of dilutions corresponding to 5, 10, or 50 plaque forming units to wells of microtiter dishes prepared as described above. Cloned CMV DNA was also used to establish a standard curve, using 10-1000 tmoles DNA/well (1 tmole=10-21 Mole or 602 molecules). Plates were covered and agitated to mix samples, then incubated at 65°C to release nucleic acids.
A cocktail of the CMV-specific amplifier and capture probes listed above was added to each well (25 fmoles/well in 1 N NaOH). Plates were covered and gently agitated to mix reagents and then incubated at 65°C for 30 min.
Neutralization buffer was then added to each well (0.77 M 3-(N-morpholino)propane sulfonic acid/1.845 M NaCl/0.185 M sodium citrate). Plates were covered and incubated for 12-18 hr at 65°C.
The contents of each well were agitated to remove all fluid, and the wells washed 2X with washing buffer (0.1% SDS/0.015 M NaCl/ 0.0015 M sodium citrate).
Amplifier multimer was then added to each well (40 fmoles/well in 4X SSC/ 0.1% SDS / 0.5% Blocking Reagent (Boehringer Mannheim, catalog No . 1096 176)). After covering plates and agitating to mix the contents in the wells, the plates were incubated for 15 min at 65°C.
After a further 5-10 min period at room
temperature, the wells were washed as described above.
Alkaline phosphatase label probe, disclosed in EP 883096976, was then added to each well (50 fmoles in 40 μl/well). After incubation at 55°C for 15 min, and 5- 10 min at room temperature, the wells were washed twice as above and then 3X with 0.015 M ΝaCl/0.0015 M sodium citrate.
An enzyme-triggered dioxetane (Schaap et al., Tet. Lett. 191987) 28:1159-1162 and EPA Pub. No. 0254051, obtained from Lumigen, Inc., was employed. The detection procedure was as follows. 30 μl Lumiphos 530 (Lumigen) was added to each well. The wells were tapped lightly so that the reagent would fall to the bottom and gently swirled to distribute the reagent evenly over the bottom. The wells were covered and incubated at 37°C for 40 min.
Plates were then read on a Dynatech ML 1000 luminometer. Output was given as the full integral of the light produced during the reaction.
Results are shown in the Table below. Results for each standard sample are expressed as the difference between the mean of the negative control plus two
standard deviations and the mean of the sample minus two standard deviations (delta). If delta is greater than zero, the sample is considered positive. In this study, standard curves using both CMV viral particles and cloned CMV DNA were generated to demonstrate sensitivity.
Additionally, host cells, CMV-positive, Herpes simplex virus (HSV) - positive, and adenovirus-positive clinical samples were tested in the assay. Each clinical sample was also diluted 10X and tested. These results indicate the ability of these probes to distinguish CMV DNA from heterologous organisms and a sensitivity of about 5 CMV plaque forming units (pfu).
Figure imgf000028_0001
Figure imgf000029_0001
Mod f cat ons of the above-descrbed modes for carrying out the invention that are obvious to those of skill in biochemistry, nucleic acid hybridization assays, and related fields are intended to be within the scope of the following claims.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Kolberg, Janice A.
Shen, Lu-Ping
Urdea, Michael S.
(ii) TITLE OF INVENTION: CMV PROBES FOR USE IN SOLUTION
PHASE SANDWICH HYBRIDIZATION ASSAYS
(iii) NUMBER OF SEQUENCES: 53
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Morrison & Foerster
(B) STREET: 755 Page Mill Road
(C) CITY: Palo Alto
(D) STATE: California
(E) COUNTRY: USA
(F) ZIP: 94304-1018
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 07/813,590
(B) FILING DATE: 23-DEC-1991
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Thomas E. Ciotti
(B) REGISTRATION NUMBER: 21,013
(C) REFERENCE/DOCKET NUMBER: 22300-20236.00
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 415-813-5600
(B) TELEFAX: 415-494-0792
(C) TELEX: 706141 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
TGACTGR 7
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2
CGTGGAGACA CGGGTCCTAT GCCT 24
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :
GATGTGGTTG TCGTACTTGA TGTGGTTGTC GTACTTGATG TGGTTGTCGT ACTTGCGTAG 60
(2) INFORMATION FOR SEQ ID NO : 4 :
(i) SEQUENCE CHARACTERISTICS :
(A) LENGTH: 16 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
TCCACGAAAA AAAAAA 1 6
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs
(B) TYPE : nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5 :
CAGTCACTAC GC 1 2
(2) INFORMATION FOR SEQ ID NO : 6 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6 :
CCGRTTGATG TARCYGCGCA ACGTRTCRTA GGT 33 (2) INFORMATION FOR SEQ ID NO: 7 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CACACACCAR GCYTCKGCGA TYTGYGYYAR CGC 33
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS :
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
TTCCYTGAAG ACCTCYAGGG WGCGCCGTTG ATC 33
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
YGAGAGAATR GCTGAYGGRT TGATCTTGCT RAG 33
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GAAACGCGCG GCAATCGGTT TGTTGTARAT GGC 33
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 11 :
CACGCAGCTG GCCARRCCCA RRACATCACC CAT 33
(2 ) INFORMATION FOR SEQ ID NO : 12 :
(i) SEQUENCE CHARACTERISTICS :
(A) LENGTH: 33 base pairs
(B) TYPE : nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12 :
ACGCAGCACC TTRACGCTKG TTTGGTTRAT RGT 33 (2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
GCAGCGTCCT GGCGAYTCYT TCACRTTCAT ATC 33 (2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 14 :
GRCGAAATTA AAGATGACCA CKGGTCGYGA GTA 33
(2 ) INFORMATION FOR SEQ ID NO : 15 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
RCCCAGTTGA CCGTACTGCA CRTACGAGCT GTT 33
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GCGGTGGTTG CCCAACAGGA TTTCGTTRTC CTC 33
(2) INFORMATION FOR SEQ ID NO: 17 :
(i) SEQUENCE CHARACTERISTICS :
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17 :
GATCTTGAGG CTGGGAARCT GACATTCCTC AGT 33
(2) INFORMATION FOR SEQ ID NO: 18 :
(i) SEQUENCE CHARACTERISTICS :
(A) LENGTH: 33 base pairs
(B) TYPE : nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18 :
CACGTACTCG TAGGCCGAGT TSCCGGCGAT GAA 33
(2) INFORMATION FOR SEQ ID NO: 19 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
GCTGAGGTCA ATCATGCGTT TGAAGAGGTA GTC 3 33
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 20 :
YARGGCGATC ATGCTGTCGA CDGTRGAGAT RCT 3 33
(2) INFORMATION FOR SEQ ID NO : 21 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
CCTGAAGTCR GTRTTTTCCA GCGGGTCGAT RTC 33
(2) INFORMATION FOR SEQ ID NO :22 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
CACATATTCA TAGGCCGAGT TSCCGGCGAT GAA 33
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO :23 :
CGCCACCGGC GAGATGCCGC ATAGGCGACG GAG 33
(2 ) INFORMATION FOR SEQ ID NO : 24 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
GCATGTCGTC CCTTCGACGT ACACTTCCTG ACG 33 (2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
CGGGATGATG GTCAGCTCCT CGTAGCATTG GGC 33
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
CTGCAGCCGC TTGTTCARCG AGCGGCCCTG ATT 33
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
ACGGTGGACC GCTATATGGT TGCACAGCAA GCC 33
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
CGTCTGGATA TTCACATCGG ACTGGCTTGA CGG 33
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
GCGCGTTGTC AGGTCCAGCA GGTCCTGCTC CAC 33
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
GAGGGCCGAA AGGACTCCAG CCAAGTGGGG GAT 33 (2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31 :
GTGGTAGGCC GATGAAGAAG AGAATAGGCT TTT 33
(2) INFORMATION FOR SEQ ID NO: 32 : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
CCTCAGCGCC TCCTCCGCCT CCTGGATGTA GCT 33
(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
TCGTTCCGGT ATATCCGTAA ACAGGTTGTA CTC 33
(2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
GGACCAGTAG GTAAAATCCG ACAAGGAATA TAT 33
(2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 35 :
GCCCACCCGC TTGACGATAA CCTCCGAGGT ACG 3 33 (2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
CTGGTGATAC ACATTTAGCT GCTGGATGGT GAT 333 (2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37 :
GCGACTGATG CCGTTCATGA GCGCCCGGCA CAG 33
(2) INFORMATION FOR SEQ ID NO: 38 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 38 :
GAAGATGTCC TCCACGTCCT CCCCGTACAG ATG 33
(2) INFORMATION FOR SEQ ID NO:39 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: CTCCTCCCCG TCCAACGCCT TTTCCCCGAG CAC 33
(2 ) INFORMATION FOR SEQ ID NO : 40 :
(i) SEQUENCE CHARACTERISTICS :
(A) LENGTH: 33 base pairs
(B) TYPE : nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:
GGGGGCGGCA AAGACCGACC CCACGAACAT GCG 33
(2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
ACGCARYTCT TTCTGCGAGT AAAGTTCCAG TAC 33
(2) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
CATGATCTCY TCGAGRTCAA AAACGTTGCT GGA 33
(2) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 43 :
CTTTACCCGC TGCTTGTACG AGTTGAAYTC GCG 3 33
(2) INFORMATION FOR SEQ ID NO : 44 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
CGGYARCGGG TCGACTACCT TGTCCTCCAC GTA 33
(2) INFORMATION FOR SEQ ID NO:45 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 45 :
GCTCATGAGG TCGTCCAGAC CCTTGAGGTA GGG 33
(2) INFORMATION FOR SEQ ID NO:46 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46 :
CTTTACCCGC TGCTTATACG AATTGAAYTC GCG 33
(2) INFORMATION FOR SEQ ID NO: 47 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47 :
GCTGAGGGAT GTGATGAGGT CGATGATCCT GTT 33
(2) INFORMATION FOR SEQ ID NO:48 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
GTTGAACACC GGGTTGTCCT CGAAAGCTTG AAT 33
(2) INFORMATION FOR SEQ ID NO:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:
TTTGGTGTAC ATCTCGTTGC TTTCGTGGAG CTT 33
(2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:
CGGACGTCGA ATCTCCTCGA GAATATGCTT GAT 3 33
(2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:
AGGCATAGGA CCCGTGTCTT 2 20
(2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:
TTTGTGGG 8 (2) INFORMATION FOR SEQ ID NO: 53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:
CTTCTTTGGA GAAAGTGGTG 20
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
5'- GGT GTT TGG TTG TTG TTG TTG TTG TTG TTG TTG TTG
TTG TTG TTG TTG TTG TTT TTT TTT TTT TTT TTT TT -3'
DNA SEQUENCE VERSION 2.00
SEQUENCE NAME: 15X-2
SEQUENCE LENGTH: 10
DATE: Aug 27, 199
TIME: 14:06
COMMENT: 5'- 77T SAC T65 T -3'

Claims

Claims
1. A synthetic oligonucleotide useful as an amplifier probe in a sandwich hybridization assay for CMV comprising
a first segment comprising a nucleotide
sequence substantially complementary to a segment of CMV nucleic acid; and
a second segment comprising a nucleotide sequence substantially complementary to an
oligonucleotide multimer,
wherein the CMV nucleic acid segment is selected from the group consisting of
CCGRTTGATGTARCYGCGCAACGTRTCRTAGGT (SEQ ID NO:6), CACACACCARGCYTCKGCGATYTGYGYYARCGC (SEQ ID NO:7), TTCCYTGAAGACCTCYAGGGWGCGCCGTTGATC (SEQ ID NO:8), YGAGAGAATRGCTGAYGGRTTGATCTTGCTRAG (SEQ ID NO:9), GAAACGCGCGGCAATCGGTTTGTTGTARATGGC (SEQ ID NO:10), CΑCGCAGCTGGCCARRCCCARRACATCACCCAT (SEQ ID NO:11), ACGCΑGCACCTTRACGCTKGTTTGGTTRATRGT (SEQ ID NO:12), GCAGCGTCCTGGCGAYTCYTTCACRTTCATATC (SEQ ID NO:13), GRCGAAATTAAAGATGACCACKGGTCGYGAGTA (SEQ ID NO:14), RCCCAGTTGACCGTACTGCACRTACGAGCTGTT (SEQ ID NO:15), GCGGTGGTTGCCCAACAGGATTTCGTTRTCCTC (SEQ ID NO:16), GATCTTGAGGCTGGGAARCTGACATTCCTCAGT (SEQ ID NO:17), CACGTACTCGTAGGCCGAGTTSCCGGCGATGAA (SEQ ID NO:18), GCTGAGGTCAATCATGCGTTTGAAGAGGTAGTC (SEQ ID NO: 19), YARGGCGATCATGCTGTCGACDGTRGAGATRCT (SEQ ID NO:20), CCTGAAGTCRGTRTTTTCCAGCGGGTCGATRTC (SEQ ID NO:21), CACATATTCATAGGCCGAGTTSCCGGCGATGAA (SEQ ID NO:22), CGCCACCGGCGAGATGCCGCATAGGCGACGGAG (SEQ ID NO:23), GCATGTCGTCCCTTCGACGTACACTTCCTGACG (SEQ ID NO:24), CGGGATGATGGTCAGCTCCTCGTAGCATTGGGC (SEQ ID NO:25), CTGCAGCCGCTTGTTCARCGAGCGGCCCTGATT (SEQ ID NO:26), ACGGTGGACCGCTATATGGTTGCACAGCΑAGCC (SEQ ID NO:27), CGTCTGGATATTCACATCGGACTGGCTTGACGG (SEQ ID NO:28), GCGCGTTGTCAGGTCCAGCAGGTCCTGCTCCAC (SEQ ID NO:29),
GAGGGCCGAAAGGACTCCAGCCAAGTGGGGGAT (SEQ ID NO:30),
GTGGTAGGCCGATGAAGAAGAGAATAGGCTTTT (SEQ ID NO:31),
CCTCAGCGCCTCCTCCGCCTCCTGGATGTAGCT (SEQ ID NO:32), TCGTTCCGGTATATCCGTAAACAGGTTGTACTC (SEQ ID NO:33),
GGACCAGTAGGTAAAATCCGACAAGGAATATAT (SEQ ID NO:34),
GCCCACCCGCTTGACGATAACCTCCGAGGTACG (SEQ ID NO:35),
CTGGTGATAC-ACΑTTTAGCTGCTGGATGGTGAT (SEQ ID NO:36),
GCGACTGATGCCGTTCATGAGCGCCCGGCACAG (SEQ ID NO:37), GAAGATGTCCTCCACGTCCTCCCCGTACAGATG (SEQ ID NO:38),
CTCCTCCCCGTCCAACGCCTTTTCCCCGAGCAC (SEQ ID NO:39),
GGGGGCGGCAAAGACCGACCCCACGAACATGCG (SEQ ID NO:40) .
2. The synthetic oligonucleotide of claim 1, wherein said second segment comprises
AGGCATAGGACCCGTGTCTT (SEQ ID NO: 51).
3. A synthetic oligonucleotide useful as a capture probe in a sandwich hybridization assay for CMV comprising
a first segment comprising a nucleotide sequence substantially complementary to a segment of CMV nucleic acid; and
a second segment comprising a nucleotide sequence substantially complementary to an
oligonucleotide bound to a solid phase,
wherein said CMV nucleic acid segment is selected from the group consisting of
ACGCARYTCTTTCTGCGAGTAAAGTTCCAGTAC (SEQ ID NO:41),
CΑTGATCTCYTCGAGRTCAAAAACGTTGCTGGA (SEQ ID NO:42),
CTTTACCCGCTGCTTGTACGAGTTGAAYTCGCG (SEQ ID NO:43), CGGYARCGGGTCGACTACCTTGTCCTCCACGTA (SEQ ID NO:44), GCTCATGAGGTCGTCCAGACCCTTGAGGTAGGG (SEQ ID NO:45), CTTTACCCGCTGCTTATACGAATTGAAYTCGCG (SEQ ID NO:46), GCTGAGGGATGTGATGAGGTCGATGATCCTGTT (SEQ ID NO:47), GTTGAACACCGGGTTGTCCTCGAAAGCTTGAAT (SEQ ID NO:48), TTTGGTGTACATCTCGTTGCTTTCGTGGAGCTT (SEQ ID NO:49), CGGACGTCGAATCTCCTCGAGAATATGCTTGAT (SEQ ID NO:50) .
4. The synthetic oligonucleotide of claim 3, wherein said second segment comprises CTTCTTTGGAGAAAGTGGTG (SEQ ID NO:53).
5. A set of synthetic oligonucleotides useful as amplifier probes in a sandwich hybridization assay for CMV, comprising two oligonucleotides, wherein each member of the set comprises
a first segment comprising a nucleotide
sequence substantially complementary to a segment of CMV nucleic acid; and
a second segment comprising a nucleotide sequence substantially complementary to an
oligonucleotide multimer,
wherein the CMV nucleic acid segments are CCGRTTGATGTARCYGCGCAACGTRTCRTAGGT (SEQ ID NO:6), CACACACCARGCYTCKGCGATYTGYGYYARCGC (SEQ ID NO:7), TTCCYTGAAGACCTCYAGGGWGCGCCGTTGATC (SEQ ID NO:8), YGAGAGAATRGCTGAYGGRTTGATCTTGCTRAG (SEQ ID NO:9), GAAACGCGCGGCLΑATCGGTTTGTTGTARATGGC (SEQ ID NO:10), CΑCGCAGCTGGCCARRCCCARRACΑTCACCCAT (SEQ ID NO:11), ACGCAGCACCTTRACGCTKGTTTGGTTRATRGT (SEQ ID NO:12), GCAGCGTCCTGGCGAYTCYTTCACRTTCATATC (SEQ ID NO:13), GRCGAAATTAAAGATGACCACKGGTCGYGAGTA (SEQ ID NO:14), RCCCAGTTGACCGTACTGCACRTACGAGCTGTT (SEQ ID NO:15), GCGGTGGTTGCCCAACAGGATTTCGTTRTCCTC (SEQ ID NO:16), GATCTTGAGGCTGGGAARCTGACATTCCTCAGT (SEQ ID NO:17), CACGTACTCGTAGGCCGAGTTSCCGGCGATGAA (SEQ ID NO:18), GCTGAGGTCAATCATGCGTTTGAAGAGGTAGTC (SEQ ID NO:19), YARGGCGATCATGCTGTCGACDGTRGAGATRCT (SEQ ID NO:20), CCTGAAGTCRGTRTTTTCCAGCGGGTCGATRTC (SEQ ID NO:21), CACATATTCATAGGCCGAGTTSCCGGCGATGAA (SEQ ID NO:22), CGCCACCGGCGAGATGCCGCATAGGCGACGGAG (SEQ ID NO:23), GCATGTCGTCCCTTCGACGTACACTTCCTGACG (SEQ ID NO:24), CGGGATGATGGTCAGCTCCTCGTAGCATTGGGC (SEQ ID NO:25), CTGCAGCCGCTTGTTCARCGAGCGGCCCTGATT (SEQ ID NO:26), ACGGTGGACCGCTATATGGTTGCACAGCAAGCC (SEQ ID NO:27), CGTCTGGATATTCACATCGGACTGGCTTGACGG (SEQ ID NO:28), GCGCGTTGTCAGGTCCAGCAGGTCCTGCTCCAC (SEQ ID NO:29), GAGGGCCGAAAGGACTCCAGCCAAGTGGGGGAT (SEQ ID NO:30), GTGGTAGGCCGATGAAGAAGAGAATAGGCTTTT (SEQ ID NO:31), CCTCAGCGCCTCCTCCGCCTCCTGGATGTAGCT (SEQ ID NO:32), TCGTTCCGGTATATCCGTAAACAGGTTGTACTC (SEQ ID NO:33), GGACCAGTAGGTAAAATCCGACAAGGAATATAT (SEQ ID NO:34), GCCCACCCGCTTGACGATAACCTCCGAGGTACG (SEQ ID NO:35), CTGGTGATACACATTTAGCTGCTGGATGGTGAT (SEQ ID NO:36), GCGACTGATGCCGTTCATGAGCGCCCGGCACAG (SEQ ID NO:37), GAAGATGTCCTCCACGTCCTCCCCGTACAGATG (SEQ ID NO:38), CTCCTCCCCGTCCAACGCCTTTTCCCCGAGCAC (SEQ ID NO: 39), GGGGGCGGCAAAGACCGACCCCACGAACATGCG (SEQ ID NO:40).
6. The set of synthetic oligonucleotides of claim 5, wherein said second segment comprises
AGGCATAGGACCCGTGTCTT (SEQ ID NO:51).
7. A set of synthetic oligonucleotides useful as capture probes in a sandwich hybridization assay for CMV, comprising two oligonucleotides, wherein each member of the set comprises a first segment comprising a nucleotide sequence substantially complementary to a segment of CMV nucleic acid; and
a second segment comprising a nucleotide sequence substantially complementary to an
oligonucleotide bound to a solid phase,
wherein said CMV nucleic acid segments are
ACGCARYTCTTTCTGCGAGTAAAGTTCCAGTAC (SEQ ID NO:41), CATGATCTCYTCGAGRTCAAAAACGTTGCTGGA (SEQ ID NO:42), CTTTACCCGCTGCTTGTACGAGTTGAAYTCGCG (SEQ ID NO:43), CGGYARCGGGTCGACTACCTTGTCCTCCACGTA (SEQ ID NO:44), GCTCATGAGGTCGTCCAGACCCTTGAGGTAGGG (SEQ ID NO:45), CTTTACCCGCTGCTTATACGAATTGAAYTCGCG (SEQ ID NO:46), GCTGAGGGATGTGATGAGGTCGATGATCCTGTT (SEQ ID NO:47), GTTGAACACCGGGTTGTCCTCGAAAGCTTGAAT (SEQ ID NO:48), TTTGGTGTACATCTCGTTGCTTTCGTGGAGCTT (SEQ ID NO:49), CGGACGTCGAATCTCCTCGAGAATATGCTTGAT (SEQ ID NO:50).
8. The set of synthetic oligonucleotides of claim 7, wherein said second segment comprises
CTTCTTTGGAGAAAGTGGTG (SEQ ID NO:53).
9. A solution sandwich hybridization assay for detecting the presence of CMV in a sample, comprising
(a) contacting the sample under hybridizing conditions with an excess of (i) amplifier probes
comprising the set of synthetic oligonucleotides of claim 5 and (ii) a set of capture probe oligonucleotides wherein the capture probe oligonucleotide comprises a first segment comprising a nucleotide sequence that is substantially complementary to a segment of CMV nucleic acid and a second segment that is substantially complementary to an oligonucleotide bound to a solid phase;
(b) contacting the product of step (a) under hybridizing conditions with said oligonucleotide bound to the solid phase;
(c) thereafter separating materials not bound to the solid phase;
(d) contacting the bound product of step (c) under hybridization conditions with the nucleic acid multimer, said multimer comprising at least one
oligonucleotide unit that is substantially complementary to the second segment of the amplifier probe
polynucleotide and a multiplicity of second
oligonucleotide units that are substantially
complementary to a labeled oligonucleotide;
(e) removing unbound multimer;
(f) contacting under hybridizing conditions the solid phase complex product of step (e) with the labeled oligonucleotide;
(g) removing unbound labeled oligonucleotide; and
(h) detecting the presence of label in the solid phase complex product of step (g).
10. A solution sandwich hybridization assay for detecting the presence of CMV in a sample, comprising
(a) contacting the sample under hybridizing conditions with an excess of (i) a set of amplifier probe oligonucleotides wherein the amplifier probe
oligonucleotide comprises a first segment comprising a nucleotide sequence substantially complementary to a segment of CMV nucleic acid and a second segment
comprising a nucleotide sequence substantially
complementary to an oligonucleotide unit of a nucleic acid multimer and (ii) capture probes comprising the set of synthetic oligonucleotides of claim 7;
(b) contacting the product of step (a) under hybridizing conditions with said oligonucleotide bound to the solid phase;
(c) thereafter separating materials not bound to the solid phase;
(d) contacting the bound product of step (c) under hybridization conditions with the nucleic acid multimer, said multimer comprising at least one
oligonucleotide unit that is substantially complementary to the second segment of the amplifier probe
polynucleotide and a multiplicity of second
oligonucleotide units that are substantially
complementary to a labeled oligonucleotide;
(e) removing unbound multimer;
(f) contacting under hybridizing conditions the solid phase complex product of step (e) with the labeled oligonucleotide;
(g) removing unbound labeled oligonucleotide; and
(h) detecting the presence of label in the solid phase complex product of step (g).
11. A kit for the detection of CMV in a sample comprising in combination
(i) a set of amplifier probe oligonucleotides wherein the amplifier probe oligonucleotide comprises a first segment comprising a nucleotide sequence
substantially complementary to a segment of CMV nucleic acid and a second segment comprising a nucleotide
sequence substantially complementary to an
oligonucleotide unit of a nucleic acid multimer;
(ii) a set of capture probe oligonucleotides wherein the capture probe oligonucleotide comprises a first segment comprising a nucleotide sequence that is substantially complementary to a segment of CMV nucleic acid and a second segment that is substantially
complementary to an oligonucleotide bound to a solid phase;
(iii) a nucleic acid multimer, said multimer comprising at least one oligonucleotide unit that is substantially complementary to the second segment of the amplifier probe polynucleotide and a multiplicity of second oligonucleotide units that are substantially complementary to a labeled oligonucleotide; and
(iv) a labeled oligonucleotide.
12. The kit of claim 11, wherein said set of said amplifier probe oligonucleotides is the set of claim
5.
13. The kit of claim 11, wherein said set of said capture probe oligonucleotides is the set of claim 7.
14. The kit of claim 11, said kit further comprising instructions for the use thereof.
PCT/US1992/011170 1991-12-23 1992-12-22 Cmv probes for use in solution phase sandwich hybridization assays WO1993013227A1 (en)

Priority Applications (3)

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JP5511874A JPH07502655A (en) 1991-12-23 1992-12-22 CMV probe for use in solution phase sandwich hybridization assays
EP93902723A EP0625214A4 (en) 1991-12-23 1992-12-22 Cmv probes for use in solution phase sandwich hybridization assays.
CA002124796A CA2124796A1 (en) 1991-12-23 1992-12-22 Cmv probes for use in solution phase sandwich hybridization assays

Applications Claiming Priority (2)

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US81359091A 1991-12-23 1991-12-23
US07/813,590 1991-12-23

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995016055A1 (en) * 1993-12-08 1995-06-15 Chiron Corporation Solution phase nucleic acid sandwich assays having reduced background noise
WO1996006191A2 (en) * 1994-08-18 1996-02-29 Akzo Nobel N.V. Primers and probes for the amplification and detection of cmv nucleic acid
US5780227A (en) * 1995-06-07 1998-07-14 Sheridan; Patrick J. Oligonucleotide probe conjugated to a purified hydrophilic alkaline phosphatase and uses thereof
US5853974A (en) * 1995-06-07 1998-12-29 Chiron Corporation Enhancement of alkaline phosphatase with SDS in chemiluminescent substrates
US6010847A (en) * 1994-08-18 2000-01-04 Akzo Nobel N.V. Oligonucleotides that can be used in the amplification and detection of CMV nucleic acid
WO2012097105A1 (en) * 2011-01-12 2012-07-19 Abbott Laboratories Materials and method for detecting cytomegalovirus (cmv)

Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE246244T1 (en) * 1988-01-29 2003-08-15 Aventis Pasteur RECOMBINANT CMV NEUTRALIZATION PROTEINS
US5744298A (en) * 1991-08-29 1998-04-28 Behringwerke Aktiengesellschaft DNA sequences which encode immunoreactive HCMV-specific peptides
US5846717A (en) 1996-01-24 1998-12-08 Third Wave Technologies, Inc. Detection of nucleic acid sequences by invader-directed cleavage
US5994069A (en) * 1996-01-24 1999-11-30 Third Wave Technologies, Inc. Detection of nucleic acids by multiple sequential invasive cleavages
US6872816B1 (en) 1996-01-24 2005-03-29 Third Wave Technologies, Inc. Nucleic acid detection kits
EP1382386A3 (en) * 1992-02-19 2004-12-01 The Public Health Research Institute Of The City Of New York, Inc. Novel oligonucleotide arrays and their use for sorting, isolating, sequencing, and manipulating nucleic acids
WO1997014812A2 (en) * 1995-10-16 1997-04-24 Chiron Corporation Method of screening for factors that modulate gene expression
US5770176A (en) * 1995-12-08 1998-06-23 Chiron Diagnostics Corporation Assays for functional nuclear receptors
US6875572B2 (en) 1996-01-24 2005-04-05 Third Wave Technologies, Inc. Nucleic acid detection assays
US20080160524A1 (en) * 1996-01-24 2008-07-03 Third Wave Technologies, Inc. Methods and Compositions for Detecting Target Sequences
US7195871B2 (en) * 1996-01-24 2007-03-27 Third Wave Technologies, Inc Methods and compositions for detecting target sequences
US6706471B1 (en) 1996-01-24 2004-03-16 Third Wave Technologies, Inc. Detection of nucleic acid sequences by invader-directed cleavage
US5985557A (en) 1996-01-24 1999-11-16 Third Wave Technologies, Inc. Invasive cleavage of nucleic acids
US6103535A (en) * 1996-05-31 2000-08-15 University Of Maryland Optical fiber evanescent field excited fluorosensor and method of manufacture
US6768005B2 (en) 2000-12-20 2004-07-27 Avecia Limited Process
WO2002046205A2 (en) * 2000-12-05 2002-06-13 Avecia Limited Process for the preparation of phosphorothionate oligonucleotides
US20080311564A1 (en) * 2004-08-06 2008-12-18 Fort Thomas L Sequences and Methods for Detection of Cytomegalovirus
US20090253142A1 (en) * 2008-03-15 2009-10-08 Hologic, Inc. Compositions and methods for analysis of nucleic acid molecules during amplification reactions

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989003891A1 (en) * 1987-10-15 1989-05-05 Chiron Corporation Nucleic acid multimers and amplified nucleic acid hybridization assays using same

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4762780A (en) * 1984-04-17 1988-08-09 The Regents Of The University Of California Method and composition for screening and diagnosing "HCMV"
FI71768C (en) * 1984-02-17 1987-02-09 Orion Yhtymae Oy Enhanced nucleic acid reagents and process for their preparation.
US5175270A (en) * 1986-09-10 1992-12-29 Polyprobe, Inc. Reagents for detecting and assaying nucleic acid sequences
ZA878031B (en) * 1986-11-04 1988-04-28 Scripps Clinic And Research Foundation Nucleic acid probes for the detection of latent human cytomegalovirus in blood products
AU622104B2 (en) * 1987-03-11 1992-04-02 Sangtec Molecular Diagnostics Ab Method of assaying of nucleic acids, a reagent combination and kit therefore
US5124246A (en) * 1987-10-15 1992-06-23 Chiron Corporation Nucleic acid multimers and amplified nucleic acid hybridization assays using same
ATE246244T1 (en) * 1988-01-29 2003-08-15 Aventis Pasteur RECOMBINANT CMV NEUTRALIZATION PROTEINS
US4888105A (en) * 1988-02-16 1989-12-19 Mobil Oil Corporation Process for the dehydrocyclization of acyclic hydrocarbons and catalyst composition therefor
AU6164890A (en) * 1989-08-10 1991-03-11 Northwestern University Method of identifying herpesviruses and oligonucleotides for use therein

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989003891A1 (en) * 1987-10-15 1989-05-05 Chiron Corporation Nucleic acid multimers and amplified nucleic acid hybridization assays using same
EP0317077A1 (en) * 1987-10-15 1989-05-14 Chiron Corporation Nucleic acid multimers and amplified nucleic acid hybridization assays using same

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of EP0625214A4 *
Virology, Volume 167, issued 1988, SPAETE et al., "Human Cytomegalovirus Strain Towne Glycoprotein B is Processed by Proteolytic Cleavage", pages 207-225, see especially p. 209, column 1. *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU694468B2 (en) * 1993-12-08 1998-07-23 Chiron Diagnostics Corporation Solution phase nucleic acid sandwich assays having reduced background noise
US5635352A (en) * 1993-12-08 1997-06-03 Chiron Corporation Solution phase nucleic acid sandwich assays having reduced background noise
US5681697A (en) * 1993-12-08 1997-10-28 Chiron Corporation Solution phase nucleic acid sandwich assays having reduced background noise and kits therefor
WO1995016055A1 (en) * 1993-12-08 1995-06-15 Chiron Corporation Solution phase nucleic acid sandwich assays having reduced background noise
JP2006055170A (en) * 1993-12-08 2006-03-02 Bayer Corp Solution phase nucleic acid sandwich assay having reduced background noise
WO1996006191A2 (en) * 1994-08-18 1996-02-29 Akzo Nobel N.V. Primers and probes for the amplification and detection of cmv nucleic acid
WO1996006191A3 (en) * 1994-08-18 1996-03-14 Akzo Nobel Nv Primers and probes for the amplification and detection of cmv nucleic acid
US6010847A (en) * 1994-08-18 2000-01-04 Akzo Nobel N.V. Oligonucleotides that can be used in the amplification and detection of CMV nucleic acid
US6306602B1 (en) 1994-08-18 2001-10-23 Akzo Nobel N.V. Oligonucleotides that can be used in the amplification and detection of CMV nucleic acid
US5780227A (en) * 1995-06-07 1998-07-14 Sheridan; Patrick J. Oligonucleotide probe conjugated to a purified hydrophilic alkaline phosphatase and uses thereof
US5853974A (en) * 1995-06-07 1998-12-29 Chiron Corporation Enhancement of alkaline phosphatase with SDS in chemiluminescent substrates
WO2012097105A1 (en) * 2011-01-12 2012-07-19 Abbott Laboratories Materials and method for detecting cytomegalovirus (cmv)
US9085808B2 (en) 2011-01-12 2015-07-21 Abbott Molecular Inc. Materials and method for detecting cytomegalovirus (CMV)

Also Published As

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TW294721B (en) 1997-01-01
JPH07502655A (en) 1995-03-23
CA2124796A1 (en) 1993-07-08
EP0625214A1 (en) 1994-11-23
EP0625214A4 (en) 1997-07-16
US5407795A (en) 1995-04-18

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