JP3140045B2 - Improved amplified nucleic acid hybridization assay for HBV - Google Patents

Description

The present invention belongs to the field of nucleic acid chemistry and biochemical assays. More particularly, the present invention relates to novel nucleic acid multimers and nucleic acid hybridization assays.

BACKGROUND ART Nucleic acid hybridization methods are currently commonly used for gene research, biomedical research and clinical diagnosis. In a basic nucleic acid hybridization assay, a single-stranded nucleic acid (both DNA and RNA) of an analyte is hybridized to a labeled nucleic acid probe and the resulting labeled duplex is detected. Both are used.

Modified versions of this basic mechanism have been developed to facilitate the separation of the duplex to be detected from foreign substances and / or to amplify the signal to be detected.

European Patent Application Publication No. 0225807 describes a solution phase nucleic acid hybridization assay.
In this method, an analyte nucleic acid is hybridized to a labeled probe set and a capture probe set. The probe analyte complex is bound by hybridization to a solid-borne capture probe complementary to the capture probe set. According to this method, the analyte nucleic acid can be removed from solution as a solid phase complex. When the analyte is obtained in the form of a solid phase complex, the next separation step in the assay is facilitated. The labeled probe set is complementary to the labeled probe that has been bound to the solid phase / analyte complex by hybridization.

PCT Patent Application No. 84/03520 and European Patent Application Publication No. 124221 describe the following DNA hybridization assays. (1) annealing the analyte with a single-stranded DNA probe having a tail complementary to the enzyme-labeled oligonucleotide; and
(2) A method in which the obtained tailed double strand is hybridized with an enzyme-labeled oligonucleotide. Enzo
The "Bio-Bridge" labeling system from Biochem appears to be similar to the system described in these two patent applications. The "Bio-Bridge" system uses an unmodified 3'-polyT
Add a tail to the DNA probe. The resulting poly-T-tailed probe is hybridized to a labeled DNA sequence and then to biotin-modified poly A.

European Patent Application No. 204510 contains the following DNA:
A hybridization assay has been described. That is, when the analyte DNA is contacted with a probe having a tail, such as a poly-T tail, an amplifier chain having a sequence, such as a poly-A sequence, will hybridize to the probe tail and form a plurality of labeled strands. Can be combined.

European Patent Application No. 0317077, published May 24, 1989 (after the priority date of the present application), describes a hybridization assay using oligonucleotide multimers to achieve amplification. These multimers were used in the solution phase hybridization assay of the aforementioned European Patent Application Publication No. 0225807. These multimers contain one oligonucleotide unit that hybridizes to the labeled probe and a plurality of other oligonucleotide units that bind to the labeled probe. Assays for various analytes, including hepatitis B virus (HBV) nucleic acids, are described in European Patent Application 0317077. The HBV assay exemplified in European Patent Application No. 0317077 uses a set of capture probes and a set of labeled probes from a single HBV subtype.

The present invention is directed to European Patent Application No. 03 with few false negatives.
An improved version of the HBV assay exemplified in 17077 is provided.

DISCLOSURE OF THE INVENTION One aspect of the present invention is a solution sandwich DNA hybridization method for detecting HBV DNA in an analyte, comprising: (a) under hybridizing conditions, an excess of (i) The oligonucleotide comprising: a first segment having a nucleotide sequence complementary to a segment of the HBV chromosome; and a second segment having a nucleotide sequence complementary to the oligonucleotide unit of the multimer of nucleic acids; An aprobe oligonucleotide, and (ii) each oligonucleotide; a first segment having a nucleotide sequence complementary to another segment of the HBV chromosome, and a nucleotide sequence complementary to the oligonucleotide bound to the solid phase A second segment having a set of capture probes Contacting said analyte with said oligonucleotide; and (b) contacting, under hybridizing conditions, the product of step (a) with said oligonucleotide bound to said solid phase; Separating the substance not bound to the solid phase after step (b); (d) bringing the solid phase complex product of step (c) into contact with a nucleic acid multimer under hybridizing conditions Wherein said multimer comprises: (i) at least one oligonucleotide unit complementary to a second segment of said amplifier probe oligonucleotide; and (ii) a plurality of oligonucleotides complementary to a labeled oligonucleotide. (E) removing unbound multimers; (f) subjecting the solid phase complex product of step (e) under hybridizing conditions to: Contacting with a labeled oligonucleotide; (g) removing unbound labeled oligonucleotide; and (h) detecting the presence of the label in the solid phase complex product of step (g). Wherein the first segment of the amplifier probe oligonucleotide and the capture probe oligonucleotide is characterized by having a sequence complementary to a sequence of a consensus HBVds region based on HBV subtype diversity. A method comprising:

Another aspect of the invention is a synthetic oligonucleotide useful as an amplifier probe in a hybridization assay for HBV, wherein the oligonucleotide is selected from the group consisting of: In the parentheses, synthetic oligonucleotides indicating double degenerate positions are shown.

Another aspect of the invention encompasses a set of synthetic oligonucleotides useful as amplifier probes in a sandwich hybridization assay for Hepatitis B virus, comprising an oligonucleotide comprising the sequence: In this, brackets are a set of synthetic oligonucleotides, indicating positions of double degeneracy.

Another aspect of the invention is a synthetic oligonucleotide useful as a capture probe in a sandwich hybridization assay for hepatitis B virus (HBV), wherein the oligonucleotide is selected from the group consisting of: In this, parentheses are synthetic oligonucleotides indicating double degenerate positions.

Another aspect of the invention is a set of synthetic oligonucleotides useful as capture probes in a hepatitis B virus sandwich hybridization assay, comprising an oligonucleotide comprising the sequence: In this, parentheses indicate a set of synthetic oligonucleotides indicating double degenerate positions.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic illustration of the process for enzymatic preparation of linear nucleic acid multimers described in Example 1.

2A and 2B are schematic illustrations of the chemical preparation process of linear and branched nucleic acid multimers described in Example 2.

FIG. 3 (Parts A-F) shows the procedure used in making multimers with “comb” and / or bifurcated structures.

FIG. 4 is a schematic explanatory diagram of the sandwich hybridization assay described in Example 3.

FIG. 5 is an autoradiogram showing the results of the dot blot screening test described in the examples.

6-8 show the authentic HBV DN described in Example 3B.
4 is a bar graph showing test results for Sample A.

FIG. 9 shows partial nucleotide sequences of a capture probe and an amplifier probe used in the HBV assay described in Example 4.

Embodiments of the Invention Description of the Multimers The nucleic acid multimers of the present invention are linear or branched polymers of the same repeating single-stranded oligonucleotide unit or different single-stranded oligonucleotide units. At least one of the units has a sequence, length and composition that can specifically bind to a segment of the HBV amplifier probe of the present invention. To achieve such specificity and stability, this unit is usually 15-50 nucleotides in length, preferably 15-30 nucleotides in length, and has a GC content in the range of 40-60%. . In addition to such units, the multimer can be a second single-stranded nucleotide of interest,
That is, it typically contains a large number of units capable of specifically and stably hybridizing to a labeled oligonucleotide or other multimer. These units are usually 15 to 50 nucleotides in length, preferably
15 to 30 nucleotides with a GC content ranging from 40% to 60%. When a multimer is designed to hybridize to another multimer, the first and second oligonucleotide units are heterogeneous (different).

The total number of oligonucleotide units in the multimer usually ranges from 3 to 50, more usually from 10 to 20. For multimers in which the unit that hybridizes to the amplifier differs from the unit that hybridizes to the labeled oligonucleotide, the latter: former usually has a ratio of the number of 2: 1 to 30: 1, more usually 5: 1. ~ 20: 1, preferably 10: 1
~ 15: 1.

Multimeric oligonucleotide units may be linked via phosphodiester bonds or via intervening binders such as nucleic acids, amino acids, carbohydrates or polyol bridges, or via other crosslinkers which can crosslink nucleic acids or modified nucleic acid strands. And may be directly covalently bonded to each other. The linking site may be at the end of the unit (normal 3'-5 'orientation or random orientation) and / or at one or more internal nucleotides in the chain. In a linear multimer, the ends of the individual units are linked together to form a linear polymer. In one type of division multimer, three or more oligonucleotide units originate from the origin to form a division structure. Said origin can be another oligonucleotide unit or a multifunctional molecule to which at least three units can be covalently linked. In another type, there is an oligonucleotide unit backbone having one or more associated oligonucleotide units. These latter types of monomers are in the form of “fork”, “comb” or a combination of “fork” and “comb”. Ancillary units are usually branched from modified nucleotides or other organic moieties having appropriate functionalities to which the oligonucleotides are conjugated or otherwise linked. Multimers may be all linear, all branched, or a combination of linear and branched moieties. Preferably, the multimer comprises:
There is at least one branch point, more preferably at least 3 and preferably 5-10. Multimers may include one or more segments of a double-stranded sequence.

Multimer Synthesis Multimers can be prepared by cloning (if linear), enzymatic construction, chemical crosslinking, direct chemical synthesis, or a combination thereof. In the case of linear multimers prepared by coulombation, the nucleic acid sequence encoding the whole multimer or a fragment thereof can be made in single-stranded or double-stranded form by conventional cloning methods. When made in double-stranded form, the multimer / fragment is ultimately denatured to a single-stranded multimer / fragment. Multimers can be cloned in single-stranded form using conventional single-stranded phage vectors such as M13. Fragments can be joined together enzymatically or chemically to form multimers. When constructed enzymatically, the individual units are optionally linked by a ligase such as T4 DNA ligase or RNA ligase. When prepared by chemical cross-linking methods, the individual units have been derived to have a functional group that provides a linking site, or have been derived to provide such a linking site after the oligonucleotide has been synthesized. It can be synthesized with one or more nucleic acids. A preferred method of chemical cross linking, as described in European Patent Application Publication No. 0225807, a method of incorporating N ⁴ modified cytosine bases into nucleotides.

When prepared by direct chemical synthesis, derived nucleic acids,
Alternatively, oligonucleotides containing equivalent multifunctional molecules whose functional groups are protected are produced by conventional oligonucleotide synthesis methods. Remove the protection of the functional group,
Then, an oligonucleotide unit is synthesized from the unprotected site.

The general structure of a molecule used to generate a breakpoint in a multimer is as follows:

Wherein R is an organic moiety, preferably a nucleic acid,
¹ is a hydroxyl protecting group that can be removed under conditions that do not remove synthetic nucleic acids from the solid phase and do not remove groups that protect exocyclic nitrogen or phosphate, and X is a protected phosphoramidite group A phosphorus-containing group that facilitates nucleic acid synthesis, such as a phosphonate group or a phosphate group;
Is a radical derived from a nucleophilic group such as an amino group, a hydroxyl group, a sulfhydryl group or a protected phosphate group, R ² is removed without affecting R ¹ or R ¹ and hydrogen And a protecting group that can be substituted. In the molecule used to generate a bifurcated or “forked” technique, R ¹ and R ² are the same, whereas in the molecule used to generate a “comb” technique, , R ² is R ¹
It is a protecting group that is stable in the presence of a deprotecting agent. FIG. 3 shows a schematic illustration of a method used to synthesize multimers having a “comb” technique, a “fork” technique or a combination thereof.

Part A of FIG. 3 shows an automated phosphoramidite method (Warn
er et al., DNA, (1984) 3 , p. 401) shows a conventional oligonucleotide synthesis method for preparing linear oligonucleotides. Black squares indicate solid supports,
N represents a _{nucleotide, p-N-OR 1 (} R 1 is equal to R ¹ described below) is a conventional nucleotide derivative having appropriate protecting groups.

Part B shows a method for producing comb-like multimers. Compound Represents a modified base of the following formula (2). Oligomeric units of the desired size and sequence are synthesized and left on the support. Then one or its N ⁴ - modified cytosine bases are incorporated into the chain by automated methods described above. Preferably, the modified base has the formula: Wherein Z is -O-, -NH-, -S-, PO ₄ and R ¹ is a blocking or protecting group, such as dimethoxytrityl (DMT) or pixyl, which is generally stable to bases and sensitive to acids, and R ² is hydrogen. Or methyl and R ³ is the following group: A blocking or protecting group that can be removed without affecting R ¹ and replaced with hydrogen, such as
R ⁵ is a phosphoramidite or other phosphorus derivative (eg, phosphodiester, phosphotriester, etc.) that can add a nucleotide at the 5 ′ position of the oligonucleotide chain during chemical synthesis, and R ⁶ is methyl, hydrogen, I, Br or F, and X is an integer ranging from 1 to 8, including 1 and 8. If more than one modified base is incorporated, they are separated by an intermediate base in the chain, most preferably by a -TT-dimer. Additional oligonucleotide units can be incorporated into the backbone, followed by additional modified bases and the like.

Next, deprotection of the N ⁴ nucleophilic base (to remove the R ³⁾ Then, to create an automated process from which additional oligonucleotide units. Except for residues of R ¹ groups in the chain end, it is cut and divided tricks "comb-like" multimers from the support.

Part C of FIG. 3 shows the general procedure for producing a “forked” multimer. Again, oligomer units of the desired size and sequence are synthesized in a conventional manner and remain on the support.
Next, a protected bifunctional phosphorus-containing group (represented by XP in FIG. 3, Part C), such as a protected phosphoramidite group, is incorporated into the chain by an automated method. Preferred bifunctional phosphorus-containing groups are protected phosphosamidite groups, as represented by the formula:

Wherein R is the hydroxyl protecting group, iPr is isopropyl, and R ¹ is methyl or β-cyanoethyl. Most preferably, R is DMT and R ¹ is β
It is cyanoethyl.

Alternatively, R ₁ = R ₂ a is N ⁴ - modified cytosine bases (e.g., DMT) can be used to.

Next, the two protecting groups are removed and additional oligonucleotide units are generated therefrom by automated methods. The R ¹ residue is removed and the forked multimer is cleaved from the support.

Parts D and E illustrate procedures in which two or more bimeric multimers, "comb-like" multimers, or combinations thereof, are enzymatically or chemically spliced. Uniformly, bifurcated and / or "combed" multimers are prepared as described above and removed from the support. The multimers are then coupled in solution using the enzymatic or chemical ligation methods described above.

Part F shows how to synthesize multiple “comb” multimers.
This method is a modification of the method described in Part B, in which a modified base is incorporated into a branched side chain and a second oligonucleotide side chain is generated therefrom.

Suitable, cleavable, linker molecules are predetermined for the purpose of analyzing the structure of the multimer or as a means of releasing a predetermined segment (eg, the portion of the multimer attached to the labeled oligonucleotide). At the designated site. Following multimer synthesis and purification, these linkers can be specifically cleaved without further degrading the nucleotide structure of the multimer. A preferred type of linker molecule is a 1,2-diol group (which can be selectively cleaved by periodate) so that it can be incorporated into any DNA fragment by standard phosphoramidite chemistry methods, and It was designed to contain protected hydroxyl groups and hydroxyl groups from phosphoramidite. Preferred examples of such linkers are compounds: Wherein DMT and: iPr are the same as defined above. When incorporated into a DNA fragment and completely deprotected, the linker-containing fragment has the following formula: Wherein DNA ₁ and DNA ₂ represent subfragments of DNA which may be the same or different. When this fragment is reacted with sodium periodate, this fragment is cleaved,
The following sub cutting is performed.

Alternatively, the 1,2-diol group can be replaced with a linker group containing a hydroxylamine sensitive bond, a base sensitive sulfone bond, or a thiol sensitive disulfide bond. Such linker groups can be derived from conventional cross-linking agents used to link proteins to other substances. Similarly, protecting groups other than DMT may be used.

Hybridization Assay A solution phase sandwich hybridization assay for HBV is performed as follows. The analyte single-stranded nucleic acid is hybridized under hybridizing conditions to an excess of
Incubate with one set of single-stranded nucleic acid probes: (1) a first binding sequence complementary to a consensus HBV ds region sequence based on HBV subtype diversity, and a single-stranded oligonucleotide bound to a solid phase (2) a consensus HBV ds region sequence based on HBV subtype diversity and a first binding sequence complementary to a second binding sequence complementary to the HBV subtype, and an oligonucleotide of an amplified multimer A set of amplifier probes having a second binding sequence complementary to the unit. The resulting product was generated when two probes were hybridized to the analyte by their first binding sequence, 3
It is a nucleic acid complex of two components. The second binding sequence of the probe is not complementary to the analyte and therefore remains as a single-stranded tail. Multiple probes of each type can be used.

Next, the complex is added to a solid phase to which a single-stranded oligonucleotide complementary to the second binding sequence of the capture probe has been bound under hybridizing conditions.
The resulting product is a complex bound to the solid phase via a duplex formed by the oligonucleotides bound to the solid phase,
It has the second binding sequence of the capture probe.
Next, the solid phase with the bound complex is separated from unbound material.

Then, under conditions of hybridization such that the multimer is capable of hybridizing with the available second binding sequence of the amplifier probe in the solid-analyte-probe complex, the multimer is Add to the above complex. The resulting solid phase complex is then separated from unbound multimers by washing. The labeled oligonucleotide is then added under conditions that allow the oligonucleotide to hybridize to the multimeric complementary oligonucleotide unit. The resulting solid phase labeled nucleic acid complex is then washed, separated from excess labeled oligonucleotide by removing unbound labeled oligonucleotide, and read.

Amplification can be increased by using one or more multimers in the assay. In such a case, the first multimer functions as an amplifier probe or is designed to bind to the amplifier probe and the second multimer, and
Are designed to bind to the first multimer and the labeled oligonucleotide. Any number of multimers can be linked sequentially in such a manner so that greater amplification can be achieved.

The analyte HBV nucleic acid is generally obtained from a biological fluid and can be prepared for hybridization analysis by various means, for example, proteinase K / SDS, chaotropic salts, and the like. Also, for example, restriction enzymes, sonication,
Advantageously, the average nucleic acid size of the analyte is reduced by enzymatic, physical or chemical means such as chemical degradation (eg metal ions). Fragments may be as small as 0.1 kb, but are usually at least about 0.5 kb and 1 kb
It may be the above. Analyte sequences are provided in single stranded form for analysis. If this sequence is naturally single-stranded, it need not be denatured. However, if the analyte sequence is present in double-stranded form, the sequence will be denatured.
Denaturation can be performed in various ways. For example, generally about 0.05
~ 0.2 M alkali hydroxide, formamide, salts, heat or a combination thereof.

The first binding sequence of the capture probe and the amplifier probe complementary to the HBV nucleic acid of the analyte is each at least 15 nucleotides (nt), usually at least 15 nucleotides (nt).
25 nt, and does not exceed about 5 kb, usually does not exceed about 1 kb, and preferably does not exceed about 100 nt. The first binding sequence is generally about 30 nt. The first binding sequence is selected to bind to a different sequence of the consensus sequence for the conserved ds region of the HBV genome.

The second binding sequences of the capture probe and the amplifier probe are complementary to the solid phase bound oligonucleotide and multimeric oligonucleotide units, respectively, and are selected such that they do not encounter endogenous sequences in the sample / analyte. Is done. The second binding sequence is
It may be adjacent to the binding sequence or separated from the first binding sequence by an intermediate non-complementary sequence. These probes may have other non-complementary sequences as necessary. These non-complementary sequences must not interfere with the binding of the binding sequence or non-specific binding will occur.

The capture probe and the amplifier probe can be produced by an oligonucleotide synthesis method or cloning, but the former is preferable.

The solid phase used in the assay may be a particle,
Alternatively, it may be a solid wall surface of various containers (for example, a centrifuge tube, a column, a microtiter plate well, a filter, a tube, and the like). If particles are used, their size preferably ranges from about 0.4 to 200 microns, more usually from about 0.8 to 4.0 microns. The particles can be any convenient substance, such as latex or glass. Microtiter plates are a preferred solid surface. An oligonucleotide complementary to the second binding sequence of the capture probe can be stably bound to the solid surface via a functional group by a known method.

The second binding sequence of the capture probe and the oligonucleotide bound to the solid phase can be replaced by a suitable ligand receptor that forms a stable bond that binds the solid phase to the first binding sequence of the capture probe. It is clear that. Examples of such pairs are biotin / avidin, thyroxine / thyroxine binding globulin,
Antigen / antibody, sugar / lectin and the like.

The labeled oligonucleotide has a sequence complementary to the multimeric second oligonucleotide unit. The labeled oligonucleotide contains one or more molecules that directly or indirectly provide a detectable signal ("label"). The labels can be attached to individual members of the complementary sequence or can be present as terminal members or terminal tails with multiple labels. Various means of providing labels attached to sequences have been reported in the literature (eg, Leary
Proc Natl Acad Sci USA, 80, 4045, 1983; R
enz and Kurz, Nucl Acids Res, 12, 3435, 1984
Year; Richardson and Gumport, Nucl Acids Res, Volume 11, 6
167, 1983; Smith et al., Nucl Acids Res, 13, 2399
1985, Meinkoth and Wahl, anal Biochem, 138,
267, 1984). The label can be attached covalently or non-covalently to the complementary sequence. Labels that can be used include radioactive nucleic acids, fluorophores,
Includes chemiluminescers, dyes, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, enzyme subunits, metal ions, and the like. Illustrative examples of specific labels include fluorescein, rhodamine, Texas Red, phycoerythrin, umbeciferone, luminol, NADPH, α-β-galactosidase, horseradish radish peroxidase, and the like.

Labeled oligonucleotides may be conveniently prepared by chemical synthesis methods as described in EP-A-0225807. By providing a terminal group with a convenient functional group, various labels can be attached via the functional group. Thus, carboxy, thiol, amine, hydrazine or other functional groups can be provided that can bind various labels without detrimentally affecting the duplex formation by the sequence. As already mentioned, molecules with multiple labels linked to sequences complementary to the sequence to be labeled can be obtained. Alternatively, a ligand can be obtained that binds to the sequence to be labeled, and a labeled receptor that binds to the ligand can be used to provide a labeled analyte complex.

The ratio of capture probe and amplifier probe to the expected number of moles of analyte is each at least a stoichiometric ratio, with excess ratios being preferred. This ratio is preferably at least about 1.5: 1, more preferably at least 2: 1. This ratio usually ranges from 2: 1 to 10,000: 1. The concentration of each probe generally ranges from about 10 ^-9 M to 10 ^-6 M, and the concentration of nucleic acid in the sample varies from 10 ^-21 M to 10 ^-12 M. Assay hybridization steps generally take from about 10 minutes to 2 hours and are often completed in about 1 hour. Hybridization can be performed at moderately elevated temperatures, generally from about 20 ° C to 80 ° C, more usually about 35 ° C.
To 70 ° C, especially 65 ° C.

The rehydration reaction is usually performed in an aqueous medium, especially a buffered aqueous medium, which may contain various additives. Additives that can be utilized include low concentrations of surfactants (0.1-1%), salts such as sodium citrate (0.017-0.170M), Ficoll, polyvinylpyrrolidine, carrier nucleic acids, carrier proteins, and the like. It is. Non-aqueous solvents such as dimethylformamide, dimethylsulfoxide, alcohols and formamide can be added to the aqueous solvent. These other solvents are 2-50
%.

The stringency of the hybridization medium can be controlled by temperature, salt concentration, solvent system and the like. Therefore, stringency can be varied depending on the length and nature of the sequence of interest.

The method used in the separation step of the assay depends on the nature of the solid phase. In the case of particles, the particles are separated by centrifugation or filtration, and the supernatant is discarded or separated. When assaying particles, a suitable buffering medium, e.g., PBS containing a detergent such as SDS
The particles are usually thoroughly washed 1 to 5 times. If the separation means is a wall or support, the supernatant is separated or discarded and the wall is washed in the same way as described for the particles.

Depending on the type of label, various methods can be used to detect the presence of the label. In the case of fluorophores, a number of different fluorometers can be utilized. For chemiluminescers, a photometer or film may be utilized. In the case of enzymes, fluorescent, luminescent or colored products can be provided, which can be measured by fluorescence, luminescence, spectroscopy or visually. Various labels that have been used in immunoassays and methods applicable to immunoassays can be used in the assays of the present invention.

The kit for performing the amplified nucleic acid hybridization assay of the present invention includes the following reagents in combination and packaged: multimers, appropriate labeled oligonucleotides, which bind to the analyte. The resulting solid phase, a set of capture probes, and a set of amplifier probes. In the kit, these agents are generally in separate containers. The kit may also include a denaturing agent for denaturing the analyte, hybridization buffer, washing solution, enzyme substrate, negative and positive controls, and instructions for performing the assay.

The following examples of the invention are provided by way of illustration and not limitation.

Example 1 Enzymatic Preparation of Linear Multimer An 18-mer linear multimer complementary to the amplifier probe (see 3.F. below) was prepared by the method shown in FIG.

LLA-1 and LLA-2, two 18-mers, were purchased from Warner
DNA, vol. 3, p. 401, 1984, using the automated phosphoramidite method. Purification was performed using the Sanchez-Pescado
r, and Urdea, DNA, 3, 339, 1984. Phosphorylation of the 5 'end of LLA-2 was performed by the chemical phosphorylation method described in Example 1 of Applicant's co-pending European Patent Application No. 0304215. The disclosure of EP-A-0304215 is incorporated herein by reference. The sequences of LLA-1 and LLA-2 were as follows.

Linear LLA-2 polymer was produced using T4 DNA ligase. The product of this ligation is double-stranded. Single-stranded polymers can be obtained by gel-purifying the product under denaturing conditions.

10 ⁵ pmole of each sequence was placed in a 1.5 ml test tube and evaporated to dryness under reduced pressure.

 The following solutions were added to the dried array below.

100 μl KBTS buffer 100 μl 10 mM DTT 100 μl 10 mM ATP 50 μl H ₂ O 50 μl 1 M NaCl 500 μl 30% PEG * 10 × (50 mM Tris HCl, pH 7.6, 10 mM MgCl ₂ , 1 mg / ml spermidine) The test tube was shaken by vortexing. Then 30 at 55 ° C
Heated for minutes. Next, the test tube was cooled to room temperature, and the following solution was added.

6.7 μl of T4 DNA ligase (New England Nuclear
18.8 μl of 5 × ligase dilution buffer (NEN) 74.5 μl of H ₂ O The tubes were again vortexed and incubated overnight at room temperature. The resulting reaction mixture is
-Extract with butanol to a volume of 100 μl and add 100 μl of 3 × stop mixture (25% glycerol, 0.05% bromophenol blue, 0.5% sodium dodecyl sulfate, 25 mM E
DTA) was added, followed by heating at 100 ° C. for 5 minutes to denature the sample. Next, 20 μl of this solution was added to each well of a 7% denaturing polyacrylamide gel (10 cm × 10 cm × 1.5 mm).
Was added. The gel was electrophoresed at 10 V / cm until the bromophenol blue dye was within 0.5 cm of the bottom of the gel. The resulting gel was placed on a thin layer chromatography plate containing an ultraviolet fluorescent dye and covered with Saran Wrap and illuminated with a long wavelength UV lamp to visualize the product. The product absorbs ultraviolet radiation and forms a visible shadow. Larger bands were observed and the polymer product larger than 20 oligomer units long was cut off. Maxam Gilbert's buffer (0.1 M Tris HCl, pH 8, 0.5 M NaCl, 5 mM EDTA)
These products were eluted from the gel by immersion in the gel.

2. Chemical preparation of linear and branched multimers A. Production of linear and branched multimers Linear and branched multimers of 18-mers complementary to amplifier probes ( See Figure 3A below)
Prepared by the method described in 2B.

As shown in the figure, phenyldiisothiocyanate (DI
An oligonucleotide having a base derivatized by crosslinking with TC) is used.

The oligonucleotide used to produce the linear multimer had the following sequence.

Here, X is N ⁴ of cytidine - shows the (6-aminocaproyl-2-aminoethyl) derivative.

The oligonucleotide used to produce the split multimer had the following sequence:

Here, X is as defined above.

Cytidine N ⁴ - (6- aminocaproyl-2-aminoethyl) derivative, EP 0225807
It was prepared in the same manner as described in the above item. The oligomer was synthesized and purified as described in Example 1.

A 0.2 unit (OD260) sample of each fragment was dissolved in 0.5 μl of 0.1 M sodium borate pH 9.3, and 9.5 μl of dimethylformamide (2 mg / ml) containing DITC was added to the solution. The solution was vortexed and left at room temperature overnight in the dark. After adding and mixing 300 μl of n-butanol, 300 μl of water was added. The resulting mixture was vortexed and centrifuged to separate into layers. The sample was extracted several times until the volume of the aqueous layer was reduced to about 50 μl and then dried under reduced pressure. The resulting polymer was then treated with 10 μl of 1 M glycine, pH 9.5, for 2 hours to modify the remaining isothiocyanate groups.
The resulting mixture was injected onto a 10 ml Sephadex G-25 column, eluted with water, collected, evaporated to dryness, dissolved in 1% SDS and run on a 7% polyacrylamide gel (vertical). 2% agarose gel is preferred for preparative electrophoresis). The gel was electrophoresed at 60ma and stained with ethidium bromide. Bands estimated to contain 10-25 units of oligomer were excised, electroeluted, and precipitated.

B. Preparation of Comb and Bifurcated Multimers The abbreviations used in this section have the following meanings:
DMT = dimethoxytrityl; T = deoxythymidine; DMF =
Dimethylformamide; BDMS = t-butyldimethylsilyl; C = deoxycytidine; TLC = thin layer chromatography; D
MAP = N, N-dimethylaminopyridine; THF = tetrahydrofuran; DIPEA = diisopropylethylamine; LEV = levulinic acid ester; DCA = dichloroacetic acid; DCC = dicyclohexylcarbodiimide; DCHU = dicyclohexylurea; TEA
= Triethylamine; TMS = trimethylsilyl; FMOC = 9
-Fluorenylmethoxycarbonyl.

B.1A. Nucleotide synthesis for comb-like branch point formation 5-DMT-T-OH (27.3 g, 50 mmole) and imidazole (10 g, 150 mmole) were co-evaporated with 200 ml of DMF. Dissolve the residue in 250 ml of DMF and use BDMS chloride (75 mmol)
Was added. The reaction mixture was stirred at 20 ° C. for 18 hours. Methanol (50 ml) was added and after 30 minutes the solvent was removed in vacuo.
The oily residue was dissolved in 50 ml of ethyl acetate, the organic phase was extracted twice with 500 ml of a 5% aqueous solution of NaHCO _{3 and} 500 ml of 80% saturated brine, and finally dried over solid Na ₂ SO ₄ . The solvent was removed by vacuum, and 5'-DMT-3'-BDMS T35 g (50 mm
ole) was obtained (100% yield). This material was used without further purification.

25.6 g of triazole was suspended in 400 ml of CH ₃ CN (0 ° C.), and POCl ₃ (8 ml) was added with rapid stirring. Then 0
Triethylamine (60 ml) was added dropwise over 15 minutes to the slurry stirred at 30 ° C. for 30 minutes. CH ₃ CN100ml
5'-DMT-3'-BDMS T (25 mmole crude) dissolved in water
Was added dropwise to the above slurry stirred at 0 ° C.
The ice water bath was removed and stirring was continued at 20 ° C. for 1 hour. Dilute the reaction mixture with 800 ml of ethyl acetate, 500 ml of 5% aqueous NaHCO ₃
The organic phase was extracted twice with l and with 500 ml of 80% saturated saline. After drying over solid Na ₂ SO ₄ , the solvent was removed in vacuo. The resulting residue was dissolved in toluene (400 ml) and CH ₃ CN (400 m
l) Co-evaporation with 1), 5'-DMT-3'-BDMS-5
-Methyl-4-triazoyl β-D-2-deoxyribofuranosyl-2 (1H) -pyrimidinone was obtained as a white foam in quantitative yield. This material was used without further purification.

6-aminohexanol dissolved in 400 ml of CH ₃ CN (1
1.7 g, 100 mmole) was added 5 dissolved in CH ₃ CN100ml '
-DMT-3'-BDMS-5-methyl-4-triazoyl β
-D-2-deoxyribofuranosyl-2 (1H) -pyrimidinone (8.7 g, 12 mmole) was added and the reaction mixture was added for 20 minutes.
Stirred at ° C. The progress of the reaction was monitored by TLC (every 30 minutes) and when the starting material had completely disappeared (usually 1 min).
２2 hours), dilute the reaction mixture with 500 ml of ethyl acetate,
This was extracted with 5% aqueous NaHCO ₃ and 80% saturated saline as described above. The organic phase was dried over Na ₂ SO ₄ solids, and the solvent removed in vacuo, 5'-DMT-3'-BDMS -5- methyl -N ^{4-6-hydroxyhexyl} deoxycytidine 7.
0 g (9.2 mmole) was obtained (77% yield). This material was used without further purification.

5'-DMT-3'-BDMS-5 dissolved in 100 ml of THF
- methyl -N ^{4-6-hydroxyhexyl} deoxycytidine (7g, 9.2mmole) was added to a solution of was dissolved in _{THF100ml (CH 3 COCH 2 CH 2} CO) 2 O and (50 mmole) was added, followed 2,6- 10 ml of 6.5% DMAP in lutidine / THF was added. The reaction mixture was kept stirring for 30 minutes. Upon analysis, the starting material was completely consumed. The reaction mixture was diluted with 700 ml of ethyl acetate, diluted with 700 ml of ethyl acetate, and this was
3 times with 500 ml of CO ₃ 5% aqueous solution and 80% saturated saline (500m
Extracted in l). After drying over solid Na ₂ SO ₄ , the solvent was removed and the residue was washed with toluene (200 ml) and CH ₃ CN (200 ml).
Co-evaporation with l) gave 12.3 g of crude product.

This crude product was dissolved in 100 ml of THF and 10 ml of a 1.1 M solution of tetrabutylammonium fluoride in THF was added. The progress of the reaction was monitored by TLC. This is usually 30
It ends in minutes, but can take longer. When the starting material had been consumed, the reaction mixture was diluted with 700 ml of ethyl acetate and extracted with NaHCO ₃ and brine as described above. Upon removal of the solution, 5'-DMT-5- methyl -N ⁴ (O-levulinyl-6-oxy-hexyl) -
8.5 g of a crude product of 2'-deoxycytidine were obtained. This material was chromatographed on silica gel. CH ₂ C
elution with 4% methanol in ₂ to separate the purified product,
5.0 g of a slightly brownish foam was obtained (6.7 mmole, 73% yield).

5'-DMT-5-methyl-N purified on silica gel
⁴ (O-levulinyl-6-oxy-hexyl) -2'-deoxycytidine and (7.7Mmole) was coevaporated twice with CH ₃ CN. The resulting dry powder was dissolved in 70 ml of CH ₂ Cl ₂ containing 4.9 ml of DIPEA in a flask under argon. 0 ° C
After cooling, 1.65 ml (8.5 mmole) of N, N-diisopropylaminomethoxychlorophosphine was added with a syringe,
The reaction mixture was stirred at 0 ° C. for 30 minutes. Ethyl acetate 40
Diluted with 0 ml, and the organic phase was diluted with 400 ml of 80% saturated saline solution.
Washed twice, then dried over solid NaSO ₄ and filtered. The solvent was removed in vacuo and the resulting residue was co-evaporated twice with toluene to give an oil. Dissolved in 30 ml of this oily toluene and cooled hexane (about -20 ° C)
It was added dropwise to 400 ml. The precipitate was quickly collected by filtration and dried in vacuo for 18 hours to give phosphoramidite 5.
45 g (6.0 mmole, 78% yield) were obtained.

B.1B. Comb min Technical points forming 5 prepared as described above dissolved in the synthesis CH ₂ Cl ₂ 200 ml of a desired another nucleotide for '
-DMT-3-BDMS-5- methyl -N ^{4-6-hydroxyhexyl-deoxy} cytidine (34g, 50 mmole) was added, N, N-
1.5 g of dimethylaminopyridine and 25 ml of triethylamine
Was added. To this solution at 0 ° C., DMT-Cl (75 mmole, 25.5 g) dissolved in CH ₂ Cl ₂ (100 ml) was added dropwise. The reaction mixture was stirred for 1 hour. Analysis showed that the starting material had been completely consumed. Next, 50 ml of methanol was added. After 30 minutes the reaction was diluted with 800 ml of ethyl acetate and extracted twice with 500 ml of a 5% aqueous solution of NaHCO ₃ and with 80% saturated saline (500 ml) as described above. Solid Na ₂ SO ₄
After in the drying, after the solvent was removed in vacuo and the residual material was co-evaporated with toluene (200ml) and CH ₃ CH (200ml).

This crude product was dissolved in 200 ml of THF and 200 ml of a 1.1 M solution of tetrabutylammonium fluoride in THF was added. The progress of the reaction was monitored by TLC. This is usually 30
It ends in minutes, but can take longer. When the starting material had been consumed, the reaction mixture was diluted with 700 ml of ethyl acetate and extracted with NaHCO ₃ and brine as described above. Upon removal of the solution, 5'-DMT-5- methyl -N ⁴ (O-DMT-6- oxy-hexyl) - crude deoxycytidine 36g was obtained. This material was chromatographed on silica gel. 2-4% in CH ₂ Cl ₂ to
The purified product was separated by elution with methanol to give 32.7 g of purified product (yield: 69% based on 34 mmole, 5'-DMT-T-OH).

5'-DMT-5-methyl-N purified on silica gel
⁴ (O-DMT-6- oxy-hexyl) -2'-deoxycytidine (34 mmol) was coevaporated twice with CH ₃ CN. The obtained dry powder is placed in a flask under argon under DIPEA.
Was dissolved in CH ₂ Cl ₂ 100 ml containing 7.5 ml. After cooling to 0 ° C., 7.37 ml (38 mmole) of N, N-diisopropylaminomethoxychlorophosphine was added via syringe and the reaction mixture was stirred at 0 ° C. for 30 minutes. After dilution with 800 ml of ethyl acetate, the organic phase was washed four times with 800 ml of 80% saturated saline, dried over solid Na ₂ SO ₄ and filtered.
The solvent was removed in vacuo and the resulting residue was co-evaporated twice with toluene to give an oil. This oil was dissolved in 80 ml of toluene and cooled with hexane (about -20 ° C).
0 ml was added dropwise. The precipitate was quickly collected by filtration and dried in vacuo for 18 hours to yield 31.8 g of phosphoramidite (28.
7 mmol, 84% yield).

5′-DMT-T-OH (16.4 ml, 30 mmole) was added to dry CH ₃ C
Dissolved in 200 ml of N and 1- (TMS) imidazole (14.6 ml, 1
00mmole). After 60 minutes, the solvent was removed in vacuo. The oily residue was dissolved in ethyl acetate 700ml, NaHCO ₃ 5
The mixture was extracted twice with 500 ml of a 50% aqueous solution and with 80% saturated saline (500 ml), and finally dried over solid Na ₂ SO ₄ . Removal of the solvent in vacuo gave 30 mmole of 5'-DMT-3'-TMS-T (100% yield). This material was used without further purification.

Triazole (37.8 g) was suspended in 450 ml of CH ₃ CN (0 ° C.) and POCl ₃ (12 ml) was added with rapid stirring. Next, triethylamine (90 ml) was added dropwise over 15 minutes to the slurry stirred at 0 ° C. for 30 minutes. CH ₃ CN1
5′-DMT-3′-TMS-T (30 mmol,
(Purified) was added dropwise to the above stirred slurry at 0 ° C. The ice water bath was removed and stirring was continued at 20 ° C. for 1 hour. Dilute the reaction mixture with 800 ml of ethyl acetate and add 5% NaHCO
_{3 The} organic phase was extracted twice with 500 ml and with 80% saturated saline (500 ml). After drying the organic phase over solid Na ₂ SO ₄ , the solvent was removed in vacuo. The obtained residue was dissolved in toluene (400 m
l) and co-evaporation with CH ₃ CN (400 ml) give 5′-D
MT-3'-TMS-5-methyl-4-triazoyl β-D
2-Deoxyribofuranosyl-2 (1H) -pyrimidinone was obtained as a white foam in quantitative yield. This material was used without further purification.

CH ₃ CN400ml mixture of 6-amino hexanol (23 g, 200 mm
the ole) solution, 5'-DMT-3'- dissolved in CH ₃ CN100ml
TMS-5-methyl-4-triazoyl β-D-2-deoxyribofuranosyl-2 (1H) -pyrimidinone (20 g, 3
0 mmol) was added and the reaction mixture was stirred at 20 ° C. The progress of the reaction was monitored by TLC (every 30 minutes) and when the starting material had completely disappeared (usually 1-2 hours), the reaction mixture was diluted with 800 ml of ethyl acetate and this was added as described above. N
Extracted with a 5% aqueous solution of aHCO ₃ and 80% saturated saline. Na ₂ SO ₄
After drying the organic phase with, the solvent is removed by vacuum,
-DMT-3'-TMS-5- methyl -N ^{4-6-hydroxyhexyl} deoxycytidine 20.3 g (about 30 mmole) was obtained. This material was used without further purification.

5'-DMT-3'-TMS- 5- methyl -N ⁴ in methanol 250 ml - to (6-hydroxyhexyl) deoxycytidine solution was added concentrated aqueous ammonia 25 ml, and stirring in a closed round bottom flask The reaction mixture was left for one hour. Next, the solvent is removed in a vacuum, and ethanol 1 × 200 m
obtained in quantitative yield (6-hydroxyhexyl) deoxycytidine - l, toluene 1 × 100 ml and CH ₃ CN1 × 100 ml with co evaporated to a 5'-TMT-5-methyl -N ^4. This material was used without further purification. This material is dissolved in 200 ml of CH ₂ Cl ₂ , 4 ml of pyridine is added and then CH ₂ C
FMOC-Cl (7.8 g, 30 mmole) dissolved in l ₂ (50 ml) was added dropwise. The reaction mixture was stirred for 30 minutes. Upon analysis, the starting material was completely consumed. Dilute the reaction mixture with 500 ml of ethyl acetate and add it to a 5% aqueous NaHCO ₃ solution as described above.
The mixture was extracted three times with 500 ml and with 500 ml of 80% saturated saline. After drying over solid NaSO ₄ , the solvent was removed and the residue was co-evaporated with toluene (200 ml) and CH ₃ CN (200 ml) to give 23.7 g of crude product. This crude product was subjected to silica gel chromatography. About 4 in CH ₂ Cl
% Of the separated methanol elution to purified product, pure 5'-DMT-5-methyl ^{-N 4 - (O-FMOC-} 6- oxy-hexyl) deoxycytidine 13.3g (15.3mmole) was obtained ( Yield 50% based on 5'-DMT-TOH).

5'-DMT-5-methyl-N purified on silica gel
⁴ (O-FMOC-6- oxy-hexyl) -2'-deoxycytidine (15.3Mmole) was coevaporated twice with CH ₃ CN. The resulting dry powder was placed in a flask under argon,
Dissolved in 60 ml of CH ₂ Cl ₂ containing 4.1 ml of DIPEA. After cooling to 0 ° C., 3.19 ml (16.5 mmole) of N, N-diisopropylaminomethoxychlorophosphine was added via syringe and the mixture was stirred at 0 ° C. for 30 minutes. Ethyl acetate 40
After dilution with 0 ml, the organic phase was washed four times with 400 ml of 80% saturated saline, then dried over solid Na ₂ SO ₄ and filtered. The solvent was removed in vacuo and the resulting residue was co-evaporated twice with toluene to give an oil. This oil is dissolved in 50 ml of toluene and cooled in hexane (about -20 ° C).
0 ml was added dropwise. The precipitate was quickly collected by filtration,
After drying in vacuum for 18 hours, phosphoramidite 12.15 g
(11.8 mmole, 77% yield) was obtained. O during solid phase synthesis
The removal of the -FMOC group was performed using t-butylamine / pyridine (1:10
v / v) at 20 ° C. for 1 hour. Removal of the O-levulinyl group was performed with 0.5 M hydrazine hydrate in pyridine / glacial acetic acid (4: 1 v / v) at 20 ° C. for 15 minutes.

B.2. Synthesis of polyfunctional phosphoramidites for bifurcated branch point formation Glycerol (10 mmole) was co-evaporated with pyridine and dried. The obtained oil was dissolved in 50 ml of pyridine, and DMT-Cl
(6.8 g, 20 mmole) and the reaction mixture
Stirred at C for 18 hours. After adding methanol (10 ml)
The solvent was removed on a rotary evaporator. The obtained oil was dissolved in 250 ml of ethyl acetate, and a 5% aqueous solution of NaHCO ₃ (2 ×
The organic phase was washed with (250 ml) and 80% saturated saline (1 × 250 ml) and then dried over solid Na ₂ SO ₄ . The solvent was removed in vacuo and the residue was co-evaporated with 100 ml of toluene and 100 ml of CH ₃ CN. The product was separated by silica gel chromatography and O, O-bis DMT glycerol 2.5 g (3.6 m
mole) (yield 35%). The product eluted at 0-1% methanol.

This bis-DMT glycerol (2.3 mmole) was dissolved in CH ₂ Cl ₂ (10 ml) containing DIPEA (0.8 ml) under argon, and N, N-diisopropylamino-2-cyanoethoxychlorophosphine (1 ml) was added to the syringe. Added in. After 30 minutes, the mixture was diluted with ethyl acetate (200 ml) and 80%
The organic phase was washed with 200 ml of a saturated saline solution. After drying over solid Na ₂ SO ₄ , the solvent is removed in vacuo and toluene 10
When coevaporated the residue with CH ₃ CN100ml free of 0ml and moisture, bis -DMT glycerol phosphoramidite 2.15g (2.3mmole, 100% yield).
The septum lid of the vial (septum-capped vial), water distributes the product with CH ₃ CN100ml containing no, after removal of the solvent, was stored under argon at -20 ° C..

B.3. Synthesis of Periodically Oxidized Cleavable Linker Phosphoramidite O, O-dibenzoyltartaric acid monohydrate (18.8 g, 50 mmole) is dissolved in 250 ml of CH ₃ CN and the solvent is removed in vacuo did. This step was repeated. The obtained oil was dissolved in 250 ml of THF, and DCC (10.6
g, 50 mmole). A precipitate began to form in a few minutes.
After stirring at 20 ° C. for 18 hours, the reaction mixture was filtered and further treated with TH.
The precipitate was washed with F. Dry the precipitate under high vacuum, 10.8 g DCHU
(50mmole). 2- (N-methyl) aminoethanol (4.0 ml, 50 mmole) was added to the mixed filtrate, and the reaction mixture was stirred at 20 ° C for 1 hour. Next, DCC (1
0.6 g, 50 mmole). A small precipitate formed. After about one hour, 2- (N-methyl) aminoethanol (4.0 m
l, 50 mmole) was added and the reaction mixture was stirred at 20 ° C for 18 hours.

The resulting precipitate was filtered and washed with THF. Dry DCHU
The weight of the precipitate was 10.8 g. Evaporation of the combined filtrate gave an oil. In chromatography using silica,
8g (17mmole) of O, O-dibenzoyltartaric di (N
- methyl-2-hydroxyethyl) amide (this product elutes with _{6% MeOH / CH 2 Cl 2} ) was obtained.

CH ₂ Cl ₂ 5 containing DMAP (0.11 g) and TEA (2.4 ml)
Amide product in 0ml to (8.6mmole), was added DMT-Cl (8.6mmole) the dropwise dissolved in CH ₂ Cl ₂ 50ml. DMT−
After addition of Cl, the reaction mixture was stirred at 20 ° C for 1 hour,
Then the solvent was removed by evaporation. The residue was ethyl acetate 60
The solution was dissolved in 0 ml, and the organic phase was washed with 400 ml of a 5% aqueous solution of NaHCO ₃ and 400 ml of an 80% saturated saline solution. This organic phase is solid NaS
And dried over O _4. After 30 minutes, the Na ₂ SO ₄ was filtered and the supernatant was concentrated to an oil then co-evaporated with toluene and CH ₃ CN. Elution using n- butanol / CH ₂ Cl _2, was subjected to crude material chromatographed on silica gel. 2-3%
Elution with n-butanol / CH ₂ Cl ₂ of pure mono-D
MT product, O, O-dibenzoyltartaric 2- (O-
Dimethoxytrityl) hydroxyethyl-N, N-dimethyl, N-methyl-2-hydroxyethyldiamide 1.53
g (2 mmole) was obtained.

This material was dissolved in 20 ml of CH ₂ Cl ₂ containing DIPEA (3 mmole). After cooling to 10 ° C., 2.2 mmole of methoxy-N, N-diisopropylaminochlorophosphine was added under argon. After 15 minutes, ethyl acetate is added and 80%
Wash the organic phase with saturated saline, dry over solid Na ₂ SO ₄ ,
Evaporated to dryness. CH ₃ C without toluene and water
After co-evaporation with N, the phosphoramidite residue was dissolved in 10 ml of water-free CH ₂ CN. This solution was aliquoted into 19 dry Weaton vials and the solvent was removed in vacuo. Put the small bottle into a screw-type diaphragm lid (septum screw c
ap) and stored at -20 ° C.

The phosphoramidite can be coupled to the oligonucleotide using standard DNA synthesis methods and equipment. As described above, after deprotection, the resulting DNA containing the linker can be cleaved at the 1,2-diol site.

B.4. Synthesis of four-site branched amplified multimers To synthesize the branched fragments, a 2000 A control pore glass (CPG) carrier (30 mg, 7.3 μmole nucleoside / g) was used. CPG carrier, European Patent No. 030
Synthesized as described in 4215. 2.5% (v / v) in toluene (instead of CH ₂ Cl ₂ ) for detritylation during the addition of branched oligonucleotide segments
Except for using DCA, an automated methyl phosphoramidite coupling method was used for the above DNA synthesis.

Fragment LLA-2 (GACACGGGTCCTATGCCT, see above) was synthesized with CPG, removed from the automatic synthesizer and placed in a sintered glass funnel. The following description (Urdea,
The phosphoramidite coupling was performed manually as per MS, Methods in Enzymol (1987) 146 : 22-41). 100 μmole of DMT ₂ -glycerol phosphoramidite (2-cyanoethyl form) and 250 mole of tetrazole were added to the 5 ′ deprotected fragment and incubated for 15 minutes. This step was then repeated. Then two T
Methyl phosphoramidite was added by hand and further glycerol phosphate. Next, the CPG is returned to the automatic synthesizer, and each unbranched fragment GATGTGGTT
GTCGTACTTTT was synthesized. This step was then repeated. Next, standard deprotection and purification were performed as described above.

B.5. Synthesis of Four or Five Site Comb Amplified Multimers Fragments TTOTTOTTOTTGACACGG using the method described above
GTCCTATGCCT (O = 5'-DMT- N 4 - [LevO (CH 2) 6]
-5-Me cytidine methyl phosphoramidite) was synthesized. The fragment was then removed from the machine and treated with 8: 2 (v / v) 0.5 M hydrazine monohydrate in pyridine / concentrated acetic acid for 1 hour at room temperature. Next, the CPG was washed with pyridine followed by 1: 1: 2 (v / v / v) H ₂ O / lutidine / THF. Next, the carrier was returned to the automatic synthesizer, and a fragment without O in the above formula was synthesized. Next, standard deprotection and purification were performed as described above. Note that one of the copies of the labeled probe (LLA-2) associated with the fragment is at the 5 'end of the multimer, and three of the copies of the fragment are O residues.

 Five-site multimers were also prepared in a similar manner.

3. Sandwich Hybridization Assay of Hepatitis B Virus (HBV) DNA Using Multimers FIG. 4 illustrates the procedure of the assay.

A. Standard analyte HBV DNA Consists of the complete 3.2 kb HBV genome cloned into the EcoR I site of plasmid pBR325 linearized with EcoR I, using plasmid pHE63 diluted in normal human serum as the standard analyte. did. The analyte is shown in FIG.

B. Solid-Phase Oligonucleotide Complex (FIG. 4C) Synthesize 21 base oligomer 5′-XCACCACTTTCTCCAAAGAAG-3 ′ (where X is as defined above) as described in Example I. Biotinylated using N-hydroxysuccinimidyl biotin in 0.1 M sodium phosphate, pH 7.5. 5 μl (800 pmole) of this biotinylated fragment was added to 0.25% (w / v) in 1 × PBS.
Was added to a 1.5 ml Eppendorf tube containing 500 μl of 2.8 micron avidin polystyrene beads. 37
After incubating for 1 hour at 500 ℃, 0.1% SDS 500ml, 4X
The beads were washed three times by centrifugation using SSC, then resuspended and stored in the same solution until use.

C. Labeled Oligomer (E in FIG. 4) Oligomers 5'-XGGTCCTAGCCTGACAGC-of 18 bases
3 ′ (where X is as defined above),
95: 5 (v / v) dimethylformamide: modified with DITC in 0.1 M sodium borate pH 9.3, extracted with n-butanol and conjugated with horseradish peroxidase (HRP).

D. Capture probe (A in FIG. 4) Each oligomer has a 30 base long variable portion complementary to the specific sequence of the HBV genome, and a base complementary to the oligonucleotide bound to the solid phase. One set of twelve single-stranded oligomers having a constant 3 'portion of 20 lengths was synthesized according to the procedure described in Example 1.

E. Amplifier Probe (FIG. 4B) Each oligomer has a 30 base long variable portion complementary to the specific sequence of the HBV genome and a 20 base long complementary to the multimer. Consisting of the 3 'stationary part of
One set of single-stranded oligomers (D in FIG. 4) was synthesized according to the procedure described in Example 1.

Both the capture probe and the amplifier probe were designed for the constant ds region of the HBV virus.

F. Bead assay A 10 μl sample of the analyte is placed in a 1.5 ml Eppendorf tube and the 12.51 proteinase K / SDS (Gene (198
7) 61: 254) for 30 minutes at 37 ° C. One of 48 HBV oligonucleotide probes (12 capture probes and 36 amplifier probes) was added to each sample with 5 μl of 1 M NaOH containing 50 fmoles, and the tubes were heated to 100 ° C.
Heated for 10 minutes. Place the sample on ice for 5 minutes,
Centrifuge briefly for 0.38 M acetic acid, 12.3 X SSC
(Final 4 X SSC). Annealing of the probe to the analyte was performed at 55 ° C. for 1 hour. Subsequently, 25 μl of capture beads were added, and the solution was further added at 55 ° C. for 15 minutes.
Left. 500 μl of a washing solution (0.1% SDS, 4 × SSC) was added, vortexed, centrifuged for 1 minute, and further decanted twice. HM buffer (0.1% SDS, 4 X
20 μl containing 50 fmole of multimer (D in FIG. 4) in SSD, sonicated 1 mg / ml salmon sperm DNA, 1 mg / ml poly A, 10 mg / ml BSA) was added. After vortexing, place the test tube in 55
C. for 15 minutes and washed twice as above. HM
Labeling was performed at 37 ° C. for 1 hour using 20 μl containing 250 fmoles of the E-type probe. After washing three times as described above, the beads were completely removed by inversion over a Kimwipe, treated with an appropriate substrate and measured as described below. The total time required for the analysis from the addition of the proteinase K / SDS solution to the reading of the data is 3 for the chemiluminescence method.
The time was 50 minutes.

An enhanced chemiluminescence method for HRP detection reported in Matthews et al., Anal Biochem (1985) 151 : 205-209 was used. The beads were collected in 15 μl of a chemiluminescent substrate solution (luminol containing p-hydroxycinnamic acid) and
Transferred to an 8 × 50 mm evergreen polypropylene test tube containing 5 μl of M H ₂ O ₂ . After 30 seconds, turner TD-20e
Test tubes were measured with a luminometer (delay 10 seconds, integrated
20 seconds, smoothing 20 seconds). Output was expressed as the total integral of light generated during the reaction.

Fresh o-phenylenediamine solution (OPD; Sigma Chem
icals tablets; 50 mg dissolved in 5 ml of 50 mM sodium citrate, pH 5.1, containing 3 μl of 30% H ₂ O ₂ ), 100 μl was added to each tube. After 20 minutes at 37 ° C., the reaction was stopped by adding 50 μl of 4N H ₂ SO ₄ . The beads were then pelleted by centrifugation and the supernatant was transferred to microtiter dish wells. The dish was then read at 490 nm using a Biotek EL310 plate reader. Longer incubations did not improve the signal to noise ratio.

G. Microtiter dish assay A microtiter dish assay was performed. Microtiter dish wells were prepared as described below. Two types of microtiter dish wells were prepared. The two types of microtiter wells are:
(1) N wells for sample preparation and negative control and (2) S wells for capture of probe and analyte complexes from samples and positive controls.

The N well was prepared as follows. 300 μl of HM buffer
Was added to Immulon II Remov-a-wells (Dynatech Inc.). The well plate was covered and left at room temperature for 1 hour. Aspirate the HM buffer and remove 1 ×
The wells were washed three times with 400 μl of PBS. The platelets were covered with plastic wrap and stored at 4 ° C. until use. Alternatively, wells were covered with polyphenylalanyl lysine and then with HM buffer as described below.

S-wells were prepared from Immulon II as described below. Polyphenylalanyl lysine (Sigma Chemical Inc.) 2
200 μl of a 00ug / ml aqueous solution was added to each well. The covered platelets were left at room temperature for 30 minutes to 2 hours and then washed as described above. A 10 OD ₂₆₀ sample of 3B oligonucleotide in 60 μl of 1 × PBS described below was prepared using ethylene glycol bis (succinimidyl succinate) (Pierce Chemicals
Inc.) was treated with 140 μl of DMF containing 10 mg. The mixture was vortexed and incubated in the dark at room temperature. Fifteen
After one minute, the solution was washed with a Sephadex G-25 column (Pharmacia PD-10) previously equilibrated with 30 ml of 1X PBS.
Passed through. The excluded volume of the column was diluted with 1 × PBS to a final volume of 35 ml. 50 μl of capture probe solution
Was added to each well. After being covered with plastic wrap, the wells were incubated at room temperature in the dark for 30 minutes to overnight. Wells were washed with 1 × PBS, then covered with H buffer, washed and stored as above.

The labeled oligonucleotide was induced with alkaline phosphatase (AP) as described below. Calf intestinal AP (3 mg in buffer; immunoassay grade, Boehringer-Mannhe
im) was placed in a Centricon 30 Microconcentrator.
Add about 2 ml of 0.1 M sodium borate, pH 9.5, and add
The device was spun at 3500 rpm until 40 μl was obtained. Next, alkylamino oligonucleotides (Section 3C)
Was activated with DITC, extracted with butanol, and coupled to the protein as described for the HRP probe. PAGE and elution described for HRP binding (0.1 M pH 7.5
Of _{Tris, 0.1M NaCl, 10mM MgCl 2} , using 0.1 mM ZnCl ₂₎
And a concentration method.

To perform the analysis twice, each sample was placed in two N wells of 20 μl and treated with 25 μl of proteinase K / SDS solution. The wells were covered with a Linbro-Titertek microtiter plate sealer, rocked gently and incubated in a water bath at 65 ° C for 30 minutes. 10 μl of a capture probe and amplifier probe set in 1 M NaOH was added to each well. After sealing, the plate was incubated at 65 ° C to 72 ° C for 10 to 30 minutes as described above.
26 μl of 0.38 M acetic acid (or 0.76 M 3- [N-morpholino] propanesulfonic acid (MOPS), free acid), 12.3 XS
Neutralize this solution with SC, cover at 65 ° C and cover another 15-30
Incubated for minutes. A 40 μl sample was transferred from each N-well to a new S-well containing a solid support-bound capture probe. The wells were sealed and placed at 65 ° C for 1-2 hours. Next, 0.1% SDS, 0.1XS
Each well was washed twice by aspiration using SC. Amplifier multimer in HM buffer (N.go
norrhoeae, penicillin-resistant N. gonorrhoeae, tetracycline-resistant N. gonorrhoeae and chlamydia tests: use a 5-site comb-like structure (Section 2.B.5)) solution and add the sample to a 55 ° C. water bath for 15 minutes Sealed and left for ~ 1 hour. After washing as described above, 20 μl of enzyme-labeled probe in HM buffer was added. 42 ° C for HRP probe
For 15 minutes, during which time an alkaline phosphatase probe was used at 55 ° C. for 15 minutes. After washing as described above, an appropriate detection reagent was added.

As described above, HRP contains an enhanced luminol reagent (3F
See).

For AP detection, enzyme-induced dioxetane obtained from Lumigen Inc. (Schaap et al., Tet Lett (198
7) 28 : 1159-1162 and EP 0 254 051) were used. The detection method was as follows. For the labeling step, 20 μl of MM buffer containing the AP probe was added to each well and the wells were incubated at 55 ° C. for 15 minutes. The supernatant was removed and the wells were washed twice with 380 μl of 0.1 × SSC-0.1% SDS. Next, the wells were washed twice with 380 μl of 0.1 × SSC to remove any remaining SDS. 3.3 x 10 ^-4 M dioxetane 20μ in CTAB buffer
1 was added to each well. The wells were tapped lightly to allow the reagent to drop to the bottom and vortexed gently to distribute the reagent evenly to the bottom. The wells were covered with a microtiter plate sealer and incubated in a 37 ° C. oven for 1 hour. The wells were then read on a luminometer.

Results A. Test on Standard Analyte Using the multimer of Example 1-2A, a known amount of the standard analyte
The assay described above was performed on samples containing HBV DNA. For comparison, a three-part assay (bound analyte; oligonucleotide complementary to both analyte and labeled probe; HRP-labeled probe) was performed.
In the comparative assay, the obtained value was 1. Acquisition is the ratio of the signal obtained by the amplifier assay to the signal obtained by the unamplified three-part assay.

The results of these assays are reported in the table below. S / N indicates the signal-background ratio.

B. Testing on Authentic HBV DNA Samples Authentic HBV DNA samples were identified as follows.

To determine the presence or absence of HBV DNA in 49 HBV surface antigen-positive samples, dot blot screening was performed using the protein-DNA complex extraction method of Zyzik et al. (Eur J Chem Microbiol (1986) 5 : 330-335). Carried out. FIG.
It is, ³² P nick-translated p as a probe
FIG. 6 shows the analysis of six DNA-positive sera identified in a set of 49 samples using HE63. Each sample was blotted, (10 ⁰⁾ without dilution, the normal human pooled sera
Dilute 1:10 (10 ¹ ) and 1: 100 (10 ² )
Probed twice. Pooled normal human serum was also blotted twice in pHE63 samples in buffer (10X SSC). Five separate blotting experiments with dilutions and plasmid standards were performed on these sera to confirm the calculated range. These samples were used in the evaluation of the sandwich hybridization assay of the present invention.

FIG. 6 shows the results obtained by reading the chemiluminescence of the bead capture assay using the above-described HBV DNA-positive sample in a read format. Analysis of a 4 pg sample of pHE63 is also shown. Each sample has two values with a line. The bar with a line indicates the absolute signal obtained for each sample and represents the average of two sets of three samples (six samples in total). White bars indicate the same number of control samples for the same serum using beads without the C-type catcher probe. This control was used to determine non-specific binding or noise (N) of each sample matrix that would lead to false positive signals, which is not possible with a blotting assay.
The specific signal of each sample is S and N as defined
It can be expressed by the ratio of A S / N ratio of 1 indicates no specific binding. The S / N minimum ratio indicating a positive sample is discussed below.

S / N ratios of sera containing HBV DNA between 0.2 and 8 pg ranged from 3.9 to 49. Serum in pooled healthy human serum
Assays performed with 2 and 4 fold dilutions of 4825 and 3657 resulted in an approximately linear decrease in the absolute signal of each sample, demonstrating the particular nature of the hypothetical method assumed. Excellent reproducibility when repeated was confirmed for all samples using different lots of beads and reagents. Samples were read only 30 seconds after the addition of the chemiluminescent substrate solution, but comparable results were obtained up to 45 minutes. S /
The N ratio was not improved. The serum used in the apparently turbid to clear range was cryopreserved and repeatedly freeze-thawed. No attempt was made to clarify the sample prior to the hybridization assay. Increasing solution hybridization or bead capture time (up to 18 hours) did not significantly increase S / N ratio.

In FIG. 7, analysis of sub-picogram HBV DNA samples is compared to known negative sera and abundant heterologous nucleic acids in pooled sera. The S value of the lowest positive serum used (upper panel, 1:10 dilution of serum 3657) is slightly higher than the S value of negative serum (0092) or non-HBV DNA (HIV), but true positive A clear demarcation between false positives below the and pg levels is not possible based on absolute S alone. However, comparing the S / N ratios of these same samples (lower panel), a clear distinction can be made at the S / N = 2.5 break. None of the negative samples showed high values above 1.7, while the lowest positive sample at 0.2 pg showed S / N = 4.3. This clearly shows the value of using a non-specifically bound control for each sample.

FIG. 8 shows a comparison between assays performed on HBV DNA positive and negative sera using both iminol-p-hydroxycinnamic acid (shown as relative luminescence RL) and o-phenylenediamine (OPD) detection. Was done. Although both methods were comparable in sensitivity, the chemiluminescence method was more desirable for several reasons. First, using the bead method in an Eppendorf tube
The OPD reaction was performed and the best colorimetric results were obtained when the solution was transferred to a microtiter dish for reading with an ELISA reader. This is because scattering from the beads was a significant background source. Second, the chemiluminescence method was fairly fast. The chemiluminescence reaction could be read quickly 30 seconds after adding the substrate, whereas waiting 30 minutes after addition of OPD before detection. Finally, using RL for OPD, SM
Increased 1.5 to 2 times.

4. HBV Subtype Non-specific Sandwich Hybridization Assay For the dot blot using the 32P-labeled strain adwHBV probe containing the whole genome of the sample obtained in the United States, see B of the results of Example 3 above. Extensive comparison of the described assays designed for the adwHBV subtype revealed that the latter exhibited a small number of false negatives. Further comparative testing of samples obtained in Japan also showed larger and more significant false negatives. These results indicate that the relatively short oligonucleotide probe used in the hybridization assay did not bind to some samples due to strain changes at the DNA level (ie, the sample did not bind to the oligonucleotide probe). Included non-adw subtypes that had changed so far as to affect).

Therefore, a catcher probe and amplifier probe pair were redesigned from a computer comparison of the nucleotide sequences of the nine HBV subtypes reported at Gene Bank. These probes (subtype non-specific, S
N) varied in length and could contain up to a 32-fold level of degeneracy (multiple nucleotides at various positions). These 3 'sequences are shown in FIG. As can be seen from the figure, these sequences are spread out, leading to the 20-mer LLA2C
(For amplification assets) and XTI (for catcher set).

Assays using these redesigned probes were compared to those using the probe (adw) and dot blot of Example 3. (The microtiter dish MTD method using a multimer of five site units was used for sandwich high residination.) The results are shown below.

As described, a negative sample in the adw assay was SN
The assay and the genomic dot plot were positive. This indicates that this sample is a subtype other than adw. Samples of known adr subtype were used as controls.

Modifications of the above-described modes for carrying out the invention which are obvious to those skilled in nucleic acid chemistry, biochemical assays and related fields are intended to be within the scope of the following claims.

Continued on the front page (72) Inventor Running, Joyce A. United States California 94519 Concorde, Fitzpatrick Drive 3141 (72) Inventor Colberg, Janice A. United States California 94804 Richmond, Schooner Coat 179 (72) Inventor Klein United States California 94553 Martinez, Alhambra Avenue 1034 (72) Inventor Sanchez-Pescador, United States California 94577 San Lindro, Woodland Avenue 787 (72) Inventor Horn, Thomas United States of America 94708 Berkeley, Arch Street 1583- A. (56) References Gene, 1987, Vol. 61, No. 3, p. 253-264 Nature, 1979, Vol. 281, no. 25, p. 646-650 (58) Field surveyed (Int. Cl. ⁷ , DB name) C12Q 1/68 C12N 15/00 BIOSIS (DIALOG) WPI (DIALOG)

Claims (4)

(57) [Claims] 1. A solution sandwich DNA hybridization method for detecting HBV DNA in an analyte, comprising: (a) under hybridizing conditions, an excess of (i) each oligonucleotide comprising: HBV A set of amplifier probe oligonucleotides, comprising: a first segment having a nucleotide sequence complementary to a segment of the genome and a second segment having a nucleotide sequence complementary to the oligonucleotide unit of the multimeric nucleic acid; And (ii) each oligonucleotide has a first segment having a nucleotide sequence complementary to another segment of the HBV genome, and a second segment having an oligonucleotide sequence complementary to the oligonucleotide bound to the solid phase. Set of capture probe oligonucleotides (B) contacting the product of step (a) with the oligonucleotide bound to the solid phase under hybridizing conditions; After (b), separating a substance not bound to the solid phase; (d) contacting the solid phase complex product of step (c) with a nucleic acid multimer under hybridizing conditions Wherein the multimer comprises: (i) at least one oligonucleotide unit complementary to a second segment of the amplifier probe oligonucleotide; and (ii) a plurality of second oligonucleotides complementary to the labeled oligonucleotide. (E) removing unbound multimers; (f) subjecting the solid phase complex product of step (e) to labeled oligonucleotide under hybridizing conditions. (G) removing unbound labeled oligonucleotide; and (h) detecting the presence of a label in the solid phase complex product of step (g), Wherein the first segment of the amplifier probe oligonucleotide and the capture probe oligonucleotide is characterized by having a sequence complementary to a sequence of a consensus HBVds region based on the diversity of the HBV subtype. Wherein the hybridization method employs a set of amplifier probe oligonucleotides having a first segment comprising the following sequence: Wherein the brackets indicate the position of double degeneracy, and wherein the hybridization method employs a set of capture probe oligonucleotides having a first segment comprising the following sequence: ] In this, parentheses indicate the position of double degeneracy, the method. 2. A set of synthetic oligonucleotides useful as probes in a hybridization assay for HBV, comprising an oligonucleotide comprising the sequence: Wherein the brackets indicate a position of double degeneracy, a set of synthetic oligonucleotides, and wherein the assay comprises the following steps: (a) under hybridizing conditions, Excess amounts of (i) each oligonucleotide having: a first segment having a nucleotide sequence complementary to a segment of the HBV genome, and a second segment having a nucleotide sequence complementary to the oligonucleotide unit of the multimer of nucleic acids. A set of amplifier probe oligonucleotides; and (ii) each oligonucleotide is bound to a first segment having a nucleotide sequence complementary to another segment of the HBV genome, and a solid phase. A second segment having an oligonucleotide sequence complementary to the oligonucleotide; Contacting the analyte with a char-probe oligonucleotide; (b) contacting the product of step (a) with the oligonucleotide bound to the solid phase under hybridizing conditions; c) after step (b), separating the substance not bound to the solid phase; (d) converting the solid phase complex product of step (c) to a nucleic acid multimer under hybridizing conditions. Contacting, wherein the multimer comprises: (i) at least one oligonucleotide unit complementary to a second segment of the amplifier probe oligonucleotide; and (ii) complementary to a labeled oligonucleotide. (E) removing unbound multimers; (f) under hybridizing conditions of step (e). Contacting the solid phase complex product with a labeled oligonucleotide; (g) removing unbound labeled oligonucleotide; and (h) step (g) of labeling in the solid phase complex product. Detecting the presence, wherein the first segment of the amplifier probe oligonucleotide and the capture probe oligonucleotide has a sequence complementary to the sequence of a consensus HBVds region based on the diversity of HBV subtypes. Process, characterized by:
A set of synthetic oligonucleotides, including assays. 3. A set of synthetic oligonucleotides useful as amplifier probes in a solution phase sandwich assay for HBV, comprising an oligonucleotide comprising the sequence: Wherein the brackets indicate a position of double degeneracy, a set of synthetic oligonucleotides, and wherein the assay comprises the following steps: (a) under hybridizing conditions, Excess amounts of (i) each oligonucleotide having: a first segment having a nucleotide sequence complementary to a segment of the HBV genome, and a second segment having a nucleotide sequence complementary to the oligonucleotide unit of the multimeric nucleic acid. A set of amplifier probe oligonucleotides; and (ii) each oligonucleotide is bound to a first segment having a nucleotide sequence complementary to another segment of the HBV genome, and a solid phase. A second segment having an oligonucleotide sequence complementary to the oligonucleotide; Contacting the analyte with a char-probe oligonucleotide; (b) contacting the product of step (a) with the oligonucleotide bound to the solid phase under hybridizing conditions; c) after step (b), separating the substance not bound to the solid phase; (d) converting the solid phase complex product of step (c) to a nucleic acid multimer under hybridizing conditions. Contacting, wherein the multimer comprises: (i) at least one oligonucleotide unit complementary to a second segment of the amplifier probe oligonucleotide; and (ii) complementary to a labeled oligonucleotide. (E) removing unbound multimers; (f) under hybridizing conditions of step (e). Contacting the solid phase complex product with a labeled oligonucleotide; (g) removing unbound labeled oligonucleotide; and (h) step (g) of labeling in the solid phase complex product. Detecting the presence, wherein the first segment of the amplifier probe oligonucleotide and the capture probe oligonucleotide has a sequence complementary to the sequence of a consensus HBVds region based on the diversity of HBV subtypes. Process, characterized by:
A set of synthetic oligonucleotides, the assay comprising: 4. A set of synthetic oligonucleotides useful as capture probes in a solution phase hybridization assay for HBV, comprising an oligonucleotide comprising the sequence: Wherein the brackets indicate a position of double degeneracy, a set of synthetic oligonucleotides, and wherein the assay comprises the following steps: (a) under hybridizing conditions, Excess amounts of (i) each oligonucleotide having: a first segment having a nucleotide sequence complementary to a segment of the HBV genome, and a second segment having a nucleotide sequence complementary to the oligonucleotide unit of the multimer of nucleic acids. A set of amplifier probe oligonucleotides; and (ii) each oligonucleotide is bound to a first segment having a nucleotide sequence complementary to another segment of the HBV genome, and a solid phase. A second segment having an oligonucleotide sequence complementary to the oligonucleotide; Contacting the analyte with a char-probe oligonucleotide; (b) contacting the product of step (a) with the oligonucleotide bound to the solid phase under hybridizing conditions; c) after step (b), separating the substance not bound to the solid phase; (d) converting the solid phase complex product of step (c) to a nucleic acid multimer under hybridizing conditions. Contacting, wherein the multimer comprises: (i) at least one oligonucleotide unit complementary to a second segment of the amplifier probe oligonucleotide; and (ii) complementary to a labeled oligonucleotide. (E) removing unbound multimers; (f) under hybridizing conditions of step (e). Contacting the solid phase complex product with a labeled oligonucleotide; (g) removing unbound labeled oligonucleotide; and (h) step (g) of labeling in the solid phase complex product. Detecting the presence, wherein the first segment of the amplifier probe oligonucleotide and the capture probe oligonucleotide has a sequence complementary to the sequence of a consensus HBVds region based on the diversity of HBV subtypes. Process, characterized by:
A set of synthetic oligonucleotides, the assay comprising: