CMV PROBES FOR USE IN SOLUTION PHASE SANDWICH HYBRIDIZATION ASSAYS

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 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 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 cronorrhoeae. penicillin and tetracycline resistance in ΪL_ 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 --^-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 icrotiter 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 - 8 - 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 frag- ments 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 sub¬ stantially 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, horse- radish 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 10^:1. Concentrations of each of the probes will gener¬ ally range from about 10 "5 to 10 ' - 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 20 q C 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'T 18 (TTX') 15 GTTTGTGG-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: where R represents 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:l) 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. In the case of 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. 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 38OB 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 r 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 N0:2) Sidechain extension 3' -GATGCG(TTCATGCTGTTGGTGTAG) 3 -5' (SEQ ID NO:3) Ligation template for linking 3' backbone extension 3' -AAAAAAAAAAGCACCTp-5' (SEQ ID N0:4) Ligation tem¬ plate for link¬ ing 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 IX 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 MgCl 2 / 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 MgCl 2 , 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 32 P and subjected to cleavage at the site of R achieved by oxidation with aqueous NaI0 4 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.l (SEQ ID N0:6) CCGRTTGATGTARCYGCGCAACGTRTCRTAGGT CMV.GB.Z.2 (SEQ ID NO:7) CACACACCARGCYTCKGCGATYTGYGYYARCGC CMV.GB.Z.3 (SEQ ID N0:8) TTCCYTGAAGACCTCYAGGGWGCGCCGTTGATC CMV.GB.Z.4 (SEQ ID NO:9) YGAGAGAATRGCTGAYGGRTTGATCTTGCTRAG CMV.GB.Z.5 (SEQ ID N0:10) GAAACGCGCGGC--AATCGGTTTGTTGTARATGGC CMV.GB.Z.6 (SEQ ID NO:11) C^CGCAGCTGGCCARRCCC--ARRACATCACCCAT CMV.GB.Z.7 (SEQ ID NO:12) ACGC-AGCACCTTRACGCTKGTTTGGTTRATRGT CMV.GB.Z.8 (SEQ ID NO:13) GCAGCGTCCTGGCGAYTCYTTCACRTTCATATC CMV.GB.Z.9 (SEQ ID N0:14) GRCGAAATTAAAGATGACCACKGGTCGYGAGTA CMV.GB.Z.IO (SEQ ID NO:15) RCCCAGTTGACCGTACTGCACRTACGAGCTGTT CMV.GB.Z.ll (SEQ ID NO:16) GCGGTGGTTGCC(---AACAGGATTTCGTTRTCCTC CMV.GB.Z.12 (SEQ ID NO:17) GATCTTGAGGCTGGGAARCTGACATTCCTCAGT CMV.GB.Z.13a (SEQ ID N0:18) CACGTACTCGTAGGCCGAGTTSCCGGCGATGAA CMV.GB.Z.14 (SEQ ID N0:19) GCTGAGGTC^T(---ATGCGTTTGAAGAGGTAGTC 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 N0:24) GCATGTCGTCCCTTCGACGTACACTTCCTGACG CMV.UL.24 (SEQ ID N0:25) CGGGATGATGGTC--AGCTCCTCGTAG--ATTGGGC CMV.UL.25 (SEQ ID N0:26) CTGCAGCCGCTTGTTC-ARCGAGCGGCCCTGATT CMV.UL.26 (SEQ ID N0:27) ACGGTGGACCGCTATATGGTTGCACAGCAAGCC -20 - 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 N0:31) 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) CTGGTGATAC--ACATTTAGCTGCTGGATGGTGAT 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: 0) GGGGGCGGCAAAGACCGACCCCACGAACATGCG CMV Capture Probes CMV.GB.17 (SEQ ID NO:41) ACGCARYTCTTTCTGCGAGTAAAGTTCCAGTAC CMV.GB.18 (SEQ ID NO: 2) CATGATCTCYTCGAGRTCAAAAACGTTGCTGGA CMV.GB.19a (SEQ ID NO: 3) CTTTACCCGCTGCTTGTACGAGTTGAAYTCGCG CMV.GB.20 (SEQ ID NO:44) -21- CGGYARCGGGTCGACTACCTTGTCCTCCACGTA CMV.GB.21 (SEQ ID N0:45) 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) TTTGGTGTAC--ATCTCGTTGCTTTCGTGGAGCTT 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 HC1 and incubated at room temperature for 15-20 min. The plates were then washed 4 times with IX 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 IX 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 IX 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 OD 260 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 OD 260 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 IX 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 IX 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) . Table I SAMPLE delta 0.23 0.27 1.26 0.07 0.58 7.99 18.96 4.40 8.09 0.82 11.85 5.01 8.44 1.82 9.14 2.33 1.24 0.24 11.13 1.72 2.51 0.25 -0.06 -0.04 -0.04 -0.05 -27- 152 -0.05 152 (.1) -0.04 153 -0.06 153 (.1) -0.05 154 -0.04 154 (.1) -0.04 HSV+ 117 -0.19 117 (.1) -0.19 Host cells 118 -0.24 118 (.1) -0.16 Modifications of the above-described 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:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear -29 - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: TGACTGR (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 (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 (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 (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 : 5 CAGTCACTAC GC (2) INFORMATION FOR SEQ ID NO : 6 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ■ J-O (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6 : CCGRTTGATG TARCYGCGCA ACGTRTCRTA GGT 3 5 (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 0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CACACACCAR GCYTCKGCGA TYTGYGYYAR CGC 3 (2) INFORMATION FOR SEQ ID NO:8: 5 (i) SEQUENCE ' CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: TTCCΪTGAAG ACCTCYAGGG WGCGCCGTTG ATC 33 (2) INFORMATION FOR SEQ ID NO:9: 5 (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 3 (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 3 (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: - 33 - GCGGTGGTTG CCCAACAGGA TTTCGTTRTC CTC (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 (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 (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 (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 ( i) SEQUENCE DESCRIPTION: SEQ ID NO : 20 : YARGGCGATC ATGCTGTCGA CDGTRGAGAT RCT (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 3 (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 3 (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 3 (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 3 (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 3 (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 3 (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 (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 3 (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 3 (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 3 (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 (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 (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 (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 (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 3 (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 3 (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 (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 (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 (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 (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: 3 : CTTTACCCGC TGCTTGTACG AGTTGAA TC GCG (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 (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 (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 3 (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 (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 (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 (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 (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 CCCGTGT TT (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 (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 2 FILE TYPE: BE5IN PROCEDURE STD PREP 08 27 , 1991 08 27, 1991 phos003 07 01 . 1985 07 01 . 1 FILE TYPE: SHUT-DOWN PROCEDURE clβan003 07 01 , 1986 07 01 , 1986 FILE TYPE: DNA SEQUENCES ISX-2 08 27. 1991 08 27. 1991 _ 15X-I 08 27. 1991 08 27, 1 {Continued next page.) (Continued next page. > STEP ACTIVE FOR BASES A 5 C T 5 S 7 Yes Yea Yea Yes Yea Yea Yes Y Yea Yes Yes Yes Yes Yea Yea Y Yea Yea Yea Yea Yea Yea Yea Y Yes Yes Yea Yea Yes Yea Yea Y Yea Yea Yea Yea Yea Yea Yea Y Yes Yes Yes Yes Yes Yes Yea Y Yes Yes Yes Yes Yes Yes Yes Y Yes Yes Yes Yes Yes Yes Yea Y Yes Yes Yes Yes Yes Yes Yes Y Yes Yes Yes Yes Yes Yes Yea Y Yea Yes Yes Yes Yes Yes Yea Y Yea Yes Yes Yes Yes Yes Yes Y Yes Yea Yes Yes Yes Yes Yes Y Yes Yes Yes Yes Yea Yes Yes Y Yes Yes Yes Yes Yes Yes Yes Y Yes Yes Yes Yes Yes Yes Yes Y Yes Yes Yes Yes Yes Yes Yes Y YOs Yes Yes Yes Yes Yes Yes Y Yes Yes Yes Yes Yes Yes Yes Y Yes Yes Yes Yes Yes Yes Yes Y Yes Yes Yes Yes Yes Yes Yes Y Yes Yes Yes Yes Yes Yes Yes Y Yes Yes Yes Yes Yes Yes Yes Y Yes Yes Yes Yes Yes Yes Yes Y Yes Yes Yes Yes Yes Yes Yes Y Yes Yes Yes Yes Yes Yes Yes Y Yes Yes Yes Yes Yes Yes Yes Y Yes Yes Yes Yes Yes Yes Yes Y Yes Yes Yes Yes Yes Yes Yes Y Yes Yes Yes Yes Yes Yes Yes Y Yes Yes Yes Yes Yes Yes Yes Y Yes Yes Yes Yes Yes Yes Yes Y Yes Yes Yes Yes Yes Yes Yes Y Yes Yes Yes Yes Yes Yes Yes Y Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes- Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yea Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes (Continued next page. ) , >Λ - * - X \ f- * N ¬ STEP A ", P CTIVE FOR BASES S STEP FUNCTION STE S NUMBER a NAME SHE __≤ c T ~ s__ Yes Yea Yea Yes Yes 1 10 $18 To Uaste Yes Yes Yes Yes Yes Yes Yes 2 9 $18 To Column 10 Yes Yes Yes Yes Yea Yes Yes 3 2 Reverae Flush 5 Yea Yes Yes Yes Yes Yea Yes 4 1 Block Flush 3 Yea Yes Yes Yes Yea Yea Yes 5 5 Advance FC 1 Yea Yes Yes Yes Yea Yes Yes 6 28 P os Prep 3 Yes Yes Yes Yes Yes Yes Yes 7 +45 Group 1 On 1 Yes Yes Yes Yes Yes Yes Yes 8 90 TET To Column 10 Yes Yes Yes Yes Yea Yea Yes 9 19 B+TET To Col 1 8 Yes Yes es Yes Yea Yea Yea Yes 10 90 TET To Column 4 Yea Y Yea Yes Yes Yes Yes Yea Yes 11 -46 Group 1 Off I Yes Yes Yes Yea Yea Yes 12 +47 Group 2 On 1 Yea Yes Yes Yea Yea Yea Yes 13 90 TET To Column 10 Yea Yes Yea Yea Yea Yea Yes 14 20 B+TET To Col 2 8 Yes Yes Yea Yea Yea Yea Yes 15 90 TET To Column 4 Yes Yes Yes Yes Yes Yes Yes 16 -48 βroup 2 Off 1 Yes Yes Yes Yes Yes Yes Yes 17 +49 Group 3 On 1 Yes 90 TET To Column Yes Yes Yes Yes Yes Yes 18 10 Yes Yes Yes Yes Yes Yes Yes 19 21 B+TET To Col 3 8 Yes Yes Yes Yes Yes Yes Yes 20 90 TET To Column 4 Yes Yes Yes Yes Yes Yes Yes 21 -50 Group 3 Off 1 Yes Yes Yes Yes Yes Yes Yes 22 4 Uait IS Yes Yes Yes Yes Yes Yes Yes 23 +45 Group I On 1 Yes Yes Yes Yes Yes Yes Yes 24 90 TET To Column 10 Yes Yes Yes Yes Yes Yes Yes 25 19 B+TET To Col I 8 Yes Yes Yes Yes Yes Yes Yes 25 90 TET To Column 4 Yes Yes Yes Yes Yes Yes Yes 27 -46 Group 1 Off 1 Yes Yes Yes Yes Yes Yes Yes 23 +47 Group 2 On 1 Yes Yes Yes Yes Yes Yes Yes 29 90 TET To Column 10 Yes Yes Yes Yes Yes Yes Yes 30 20 B+TET To Col 2 8 Yes Yes Yes Yes Yes Yes Yes \ 1 90 TET To Column 4 Yes Yes Yes Yes Yes Yes Yes 32 -48 Group 2 Off 1 Yes Yes Yes Yes Yes Yes Yes 33 +49 Group 3 On I Yes Yes Yes Yes Yes Yes Yes 34 98 TET To Column 10 Yes Yes Yes Yes Yes Yes Yes 35 21 B+TET To Col 3 8 Yes Yes Yes Yes Yes Yes Yes 35 90 TET To Column 4 Yes Yes Yes Yes Yes Yes Yes 37 -50 - Group 3 Off 1 Yes Yes Yes Yes Yes Yes Yes 38 4 Uait 30 Yes Yes Yes Yes Yes Yes Yes 39 +45 Group 1 On 1 Yes Yes Yes Yes Yes Yes Yes 40 90 TET To Column 10 Yes Yes Yes Yes Yes Yes Yes 41 19 B+TET To Col 1 8 Yes Yes Yes Yes Yes Yes Yes 42 90 TET To Column 4 Yes Yes Yes Yes Yes Yes Yes 43 -48 θroup 1 Off 1 Yes (Continued next page-. ) (Continued next page. ) (Continued next page. ) (Continued next page.) STEP ACTIVE FOR BASES A S C T 5 6 7 Yea Yes Yes Yes Yes Yes Yea Yea Yea Yes Yea Yes Yea Yes Yes Yes Yes Yes Yea Yea Yea Yea Yes Yes Yes Yes Yes Yea (Continued next page.) (Continued next page. ) STEP STEP ACTIVE FOR BASES 5 C T 5 S 7 2 Yes Yes Yes Yes Yes Yes Yes 9 Yes Yes Yes Yea Yes Yea Yea 5 Yea Yea Yea Yea Yes Yea Yea 3 Yea Yea Yes Yes Yes Yes Yes 1 Yes Yes Yes Yes Yes Yes Yes 3 Yes Yes Yes Yes Yes Yes Yes 1 Yes Yes Yes Yes Yes Yes Yes 5 Yes Yes Yes Yes Yes Yes Yes 5 Yes Yes Yes Yes Yes Yes Yes 3 Yes Yes Yes Yes Yes Yes Yes 3 Yes Yes Yes Yes Yea Yes Yes 3 Yes Yes Yes Yes Yes Yes Yes 3 Yes Yes Yes Yes Yes Yes Yes 1 Yes Yes Yes Yes Yes Yes Yes 1 Yes Yes Yes Yes Yes Yes Yes 1 Yes Yes Yes Yes Yes Yes Yes 4 Yes Yes Yes Yes Yes Yes Yes 3 Yes Yes Yes Yes Yes Yes Yes 6 Yes Yes Yes Yes Yes Yes Yes 6 Yes Yes Yes Yes Yes Yes Yes 3 Yes Yes Yes Yes Yes Yes Yes 3 Yes Yes Yes Yes Yes Yes Yes 3 Yes Yes Yes Yes Yes Yes Yes 3 Yes Yes Yes Yes Yes Yes Yes 1 Yes Yes Yes Yes Yes Yes Yes 1 Yes Yes Yes Yes Yes Yes Yes 1 Yes Yes Yes Yes Yes Yes Yes 4 Yes Yes Yes Yes Yes Yes Yes 3 Yes Yes Yes Yes Yes Yes Yes 5 Yes Yes Yes Yes Yes Yes Yes S Yes Yes Yes Yes Yes Yes Yes 3 Yes Yes Yes Yes Yes Yes Yes 3 Yes Yes Yes Yes Yes Yes Yes 3 Yes Yes Yes Yes Yes Yes Yes 3 Yes Yes Yes Yes Yes Yes Yes 1 Yes Yea Yes Yes Yes Yes Yes t Yes Yes Yes Yes Yes Yes Yes 20 Yes Yes Yes Yes Yes Yes Yes 3 Yes Yes Yes Yes Yes Yes Yes 5 Yes Yes Yes Yes Yes Yes Yes 3 Yes Yes Yes Yes Yes Yes Yes 12 Yes Yes Yes Yes Yes Yes Yes 3 Yes Yes Yes Yes Yes Yes Yes (Continued next page.* xty** 4 * ^' S6T STT TSS TTS TTS TTS TTS TTS TTS TTS TTB TTS TT5 TTS TT6 TTS TT6 TTT TTT TTT TTT TTT .TTT TT -3' DNA SEQUENCE VERSION 2.00 SEQUENCE NAME: 15X-2 SEQUENCE LENGTH: 10 OftTE: Aug 27, 199 TIME: 14:06 COMMENT: 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 CCG TTGATGTARCYGCGCAACGTRTCRTAGGT (SEQ ID NO:6), CACACACCARGCYTCKGCGATYTGYGYYARCGC (SEQ ID NO:7), TTCCYTGAAGACCTCYAGGGWGCGCCGTTGATC (SEQ ID NO:8), YGAGAGAATRGCTGAYGGRTTGATCTTGCTRAG (SEQ ID NO:9) , GAAACGCGCGGC-AATCGGTTTGTTGTARATGGC (SEQ ID NO:10), CΑCGC-AGCTGGCl-^^RCCαi RACATCACCCAT (SEQ ID NO:11) , ACGCΑGC-ACC TRACGCTKGTTTGG'I-TRATRGT (SEQ ID NO:12), GCAGCGTCCTGGCGAYTCYTTCACRTTCATATC (SEQ ID NO:13), GRCGAAATTAAAGATGACCACKGGTCGYGAGTA (SEQ ID NO:14), RCCCAG TGACCGTACTGCACRTACGAGCTGTT (SEQ ID NO:15), GCGGTGG TGCCCAACAGGATTTCGTTRTCCTC (SEQ ID NO:16), GATCTTGAGGCTGGGAARCTGACATTCCTCAGT (SEQ ID NO:17), CACGTACTCGTAGGCCGAGTTSCCGGCGATGAA (SEQ ID NO:18) , GCTGAGGTC-aATCATGCGTTTGAAGAGGTAGTC (SEQ ID NO:19), YARGGCGATCATGCTGTCGACDGTRGAGATRCT (SEQ ID NO:20) , CCπΩAAGTCRGTRTTTTCCAGCGGGTCGATRTC (SEQ ID NO:21), CACATATΓCATAGGCCGAGTΓSCCGGCGATGAA (SEQ ID NO:22) , CGCCACCGGCGAGATGCCGCATAGGCGACGGAG (SEQ ID NO:23), GCATGTCGTCCCTTCGACGTACACTTCCTGACG (SEQ ID NO:24) , CGGGATGATGGTCAGCTCCTCGTAGCATTGGGC (SEQ ID NO:25) , C GCAGCCGCTTGTTCARCGAGCGGCCCTGATT (SEQ ID NO:26) , ACGGΕ-røACCGCTATATGGTTGCACAGCΑAGCC (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 ACGC--ARYTC-TTTCTGCGAGTAAAGTTCC-AGTAC (SEQ ID NO:41) , CΑTGATCTCYTCGAGRTC-AAAAACGTTGCTGGA (SEQ ID NO:42) , CTTTACCCGCTGCTTGTACGAGTTGAAYTCGCG (SEQ ID NO:43) , • CGGYARCGGGTCGACTACCTTGTCCTCCACGTA (SEQ ID NO:44) , GCTCATGAGGTCGTCCAGACCCTTGAGGTAGGG (SEQ ID NO:45) , CTTTACCCGCTGCTTATACGAATTGAAYTCGCG (SEQ ID NO: 6) , GCTGAGGGATGTGATGAGGTCGATGATCCTGTT (SEQ ID NO:47) , GTTGAACACCGGGTTGTCCTCGAAAGCTTGAAT (SEQ ID NO:48) , TTTGGTGTA(---ATCTCGTTGCTTTCGTGGAGCTT (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, coiπprising 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), CΑCGC-AGCTGGCCaRRCCC--ARRAC-ΑTCACCCAT (SEQ ID NO:11) , ACGC--AGC-ACCTTRACGCTKGTTTGGTTRATRGT (SEQ ID NO:12) , GCAGCGTCCTGGCGAYTCYTTCACRTTCATATC (SEQ ID NO:13) , GRCGAAATTAAAGATGACCACKGGTCGYGAGTA (SEQ ID NO:14), RCCC--AGTTGACCGTACTGCACRTACGAGCTGTT (SEQ ID NO:15) , GCGGTGGTTGCCCAACAGGATTTCGTTRTCCTC (SEQ ID NO:16) , GATCTTGAGGCTGGGAARCTGACATTCCTCAGT (SEQ ID NO:17 CACGTACTCGTAGGCCGAGTTSCCGGCGATGAA (SEQ ID NO:18 GCTGAGGTC-.AATCATGCGTTTGAAGAGGTAGTC (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 GCGCGTTGTC-AGGTCCAGCAGGTCCTGCTCCAC (SEQ ID NO:29 GAGGGCCGAAAGGACTCCAGCCAAGTGGGGGAT (SEQ ID NO:30 GTGGTAGGCCGATGAAGAAGAGAATAGGCTTTT (SfiQ 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 CTCCTCCCCGTCC^CGCCTTTTCCCCGAGCAC (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 ACGC^YTCTTTCTGCGAGTAAAGTTCCAGTAC (SEQ ID NO:41) , CATGATCTCYTCGAGRTCAAAAACGTTGCTGGA (SEQ ID NO:42) , CTTTACCCGCTGCTTGTACGAGTTGAAYTCGCG (SEQ ID NO:43) , CGGY-^CGGGTCGACTACCTTGTCCTCC-ACGTA (SEQ ID NO:44) , GCTCATGAGGTCGTCCAGACCCTTGAGGTAGGG (SEQ ID NO:45) , CTTTACCCGCTGCTTATACGAATTGAAYTCGCG (SEQ ID NO: 6) , GCTGAGGGATGTGATGAGGTCGATGATCCTGTT (SEQ ID NO:47) , GTTGAACACCGGGTTGTCCTCGAAAGCTTGAAT (SEQ ID NO:48) , TTTGGTGTAC^TCTCGTTGCTTTCGTGGAGCTT (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.

Inventors: KOLBERG JANICE A, SHEN LU-PING, URDEA MICHAEL S, KOLBERG, JANICE, A., SHEN, LU-PING, URDEA, MICHAEL, S.
Applicants: CHIRON CORP, CHIRON CORPORATION
Application year: 1992
Main classification: C07H21
IPC codes: C07H21, C12N15, C12Q1