EP0785996A1 - Sequences de nucleotides et procede d'amplification et de detection du virus de l'hepatite b - Google Patents

Sequences de nucleotides et procede d'amplification et de detection du virus de l'hepatite b

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Publication number
EP0785996A1
EP0785996A1 EP94923386A EP94923386A EP0785996A1 EP 0785996 A1 EP0785996 A1 EP 0785996A1 EP 94923386 A EP94923386 A EP 94923386A EP 94923386 A EP94923386 A EP 94923386A EP 0785996 A1 EP0785996 A1 EP 0785996A1
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EP
European Patent Office
Prior art keywords
probe
probes
downstream
upstream
seq
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EP94923386A
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German (de)
English (en)
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EP0785996A4 (fr
Inventor
Uwe Spies
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Abbott Laboratories
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Abbott Laboratories
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Publication of EP0785996A1 publication Critical patent/EP0785996A1/fr
Publication of EP0785996A4 publication Critical patent/EP0785996A4/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6862—Ligase chain reaction [LCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • NUCLEOTIDE SEQUENCES AND PROCESS FOR AMPLIFYING AND DETECTION OF HEPATITIS B VIRAL DNA
  • This invention relates generally to hepatitis B virus and a method and test kit for the detection of hepatitis B virus. More particularly, the invention relates to nucleotide sequences complementary to segments of the hepatitis B virus genome which can be amplified and/or used to determine the presence of hepatitis B virus DNA in a test sample.
  • HBV hepatitis B virus
  • the complete virion of hepatitis B consists of a complex double- layered structure with an overall diameter of 42 nm.
  • An electron dense core of 27 nm contains a circular double-stranded DNA with a molecular weight of 1.6 x 10 6 .
  • a 7-nm thick outer envelope surrounding the core comprises a biochemically heterogeneous complex designated HBsAg.
  • HBsAg is produced in excess by infected hepatocytes and is released into the blood as spherical particles with a size range of 17 to 25 nm and as tubular filaments with similar diameters but various lengths.
  • Antibodies to the core and surface antigen are designated anti- HBc and anti-HBs, respectively.
  • HBeAg/anti-HBe A third antigen-antibody system has also been observed in hepatitis B infection and is designated HBeAg/anti-HBe.
  • Serum HBV-DNA and HBeAg are considered reliable markers for monitoring HBV replication.
  • the adw and ayr subtypes predominate in most parts of the world except in Southeast Asia and the Far East, where adr is also common.
  • the ayr subtype is rarely observed.
  • the group-reactive determinant a 5 is cross-reactive among all four types, and antibody to this determinant protects against re-infection by a second subtype.
  • Hybridization techniques such as the Southern blot or Dot Blot
  • hybridization methods is not sufficient to recognize a minimal virus replication and can therefore not distinguish infectious from non-infectious patients.
  • viral DNA sequences can be amplified by using, for example, the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the level of sensitivity is about 50 to 100 copies per sample.
  • LCRTM ligase chain reaction
  • LCR has not been used in the detection and/or quantitation of HBV. It therefore would be advantageous to provide oligonucleotide strands of DNA which could be amplified and used to detect the presence, if any, of HBV in a test sample by using LCR. The combined use of oligonucleotide strands would be advantageous for allowing for the specific and sensitive in vitro diagnosis of the presence and specific type of HBV present in test samples. It also would be advantageous to provide a method which provides a degree of quantitation of HBV in a test sample to monitor the success of drug therapy of patients with chronic active hepatitis.
  • Oligonucleotide probes of from about 10 to about 60 nucleotides having a nucleotide sequence hybridizable under hybridizing conditions to a target nucleic acid sequence of hepatitis B virus are provided.
  • the target HBV sequences SEQ ID Nos. 21, 22, 23, 24 and 25
  • the oligonucleotide probes may be selected from at least one of the following: SEQ ID Nos. 21, 22, 23, 24 and 25
  • 16 CCCCGTACCTGTAACTGGGAATATTTC-5' x is hydroxyl unless otherwise specified.
  • Single oligonucleotide probes are selected from the following group: SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 or their complements.
  • compositions for detecting hepatitis B virus DNA present in a test sample containing non-target DNA in which the composition
  • each probe comprising from about 10 to about 60 nucleotides hybridizable under hybridizing conditions to the same strand of a target nucleic acid sequence of hepatitis B virus, the 3' end of the upstream probe being hybridized proximate to the 5' end of the downstream probe, and l o wherein the target nucleic acid sequence is at least one sequence selected from the group consisting of SEQ Id Nos. 21, 22, 23, 24, and 25 as described above or their complements.
  • the composition is further described as having the 3' end of the upstream probe and the 5' end of the downstream probe ligation incompetent absent corrections of the ends.
  • the ligation incompetent end is corrected by extension of the 3' end of the upstream probe with nucleotides complementary to the intervening unhybridized portion of the target nucleic acid sequence so that the ends are ligation competent.
  • compositions may be selected from the following pair sets or their complements:
  • the ligation-incompetent ends are corrected by removal of a non-phosphorylated or mismatched base from the terminus of the 5' end of the downstream probe by a target-dependent exonucleolytic agent, followed by extension of the corresponding upstream probe with nucleotides complementary to the intervening unhybridized portion of
  • compositions may be selected from the following pair sets or their complements: Pair Set SEO ID No
  • the downstream probe forms a 5' overhang when hybridized to its target, and the correction comprises removal of the overhang such that the 5' end of the corrected downstream probes abuts the
  • compositions for detecting the DNA of hepatitis B virus present in a test sample comprising a first and second oligonucleotide probe of from about 10 to about 60 nucleotides capable of hybridizing to a target nucleic acid sequence of hepatitis B virus, wherein the l o target nucleic acid sequence is selected from at least one sequence selected from the group consisting of SEQ Id Nos. 21, 22, 23, 24, and 25 as described above or their complements, and wherein the probes are hybridizable to opposite strands at opposite ends of the same target nucleic acid sequence of hepatitis B virus DNA.
  • the pairs may be selected from the following pair sets or their
  • composition for detecting the DNA of hepatitis B virus present in a test sample the composition defined as:
  • a first set of oligonucleotides comprising a first upstream probe and a 20 first downstream probe, each probe comprising from about 10 to about 60 nucleotides hybridizable under hybridizing conditions to the same strand of a target nucleic acid sequence of hepatitis B virus, the 3' end of the first upstream probe being hybridized proximate to the 5' end of the first downstream probe; and wherein the target nucleic acid sequence is at least one sequence selected from the group consisting of SEQ Id Nos. 21, 22, 23, 24, and 25 as described above or their complements; and
  • a second set of oligonucleotides comprising a second upstream probe 5 and a second downstream probe; both probes hybridizable to the first set of oligonucleotides of step (a), the 5' end of the second upstream probe being hybridized proximate to the 3' end of the second downstream probe.
  • the composition has ligation incompetent ends which are corrected by extension of the 3' end of the first upstream probe with nucleotides complementary to the l o intervening unhybridized portion of the target nucleic acid sequence so that the ends are ligation competent.
  • the four oligonucleotide probes are selected from the group consisting of:
  • composition has four oligonucleotide probes are selected from the group consisting of: (a) Set 403E, wherein SEQ Id Nos. 1 and 28 are the first upstream and first downstream probes, respectively and SEQ Id Nos 27 and 4 are the second downstream and second upstream probes, respectively;
  • the composition comprises a downstream probe having a 5' overhang when hybridized to its target, and the correction comprises removal of the overhang such that the 5' end of the corrected downstream probes abuts the 3' end of the upstream, so that the ends of the probes are ligation competent
  • a method for determining the presence of hepatitis B virus DNA in a test sample wherein the target sequence is selected from at least 5 one sequence selected from the group consisting of SEQ Id Nos. 21, 22, 23, 24, and 25 and their complements, comprising hybridizing the DNA in the test sample with at least one oligonucleotide probe of the present invention wherein the hybridized probe is capable of differentiation from the unhybridized probe, and detecting the presence of the hybridized probe.
  • ligatable pair sets are utilized in a method for determining the presence of hepatitis B virus DNA in a test sample wherein the target sequence is selected from at least one sequence selected from the group consisting of SEQ Id Nos. 21, 22, 23, 24, and 25 and their complements, comprising: (a) hybridizing the DNA in the test sample with at least one an
  • upstream oligonucleotide probe and at least one downstream oligonucleotide probe according to the present invention to the same strand of a target nucleic acid sequence of hepatitis B virus, said hybridization resulting in ligation- inco petent ends, absent correction; (b) correcting the 3' end of the upstream probe in a target dependent manner to render the probes ligatable; (c) ligating the
  • hybridized upstream probe 20 3' end of the hybridized upstream probe to the 5' end of the hybridized downstream oligonucleotide probe, wherein the hybridized probe is capable of differentiation from the unhybridized probe; and (d) detecting the presence of the hybridized probe.
  • hepatitis B virus DNA in a test sample by PCR wherein the target sequence is selected from at least one sequence selected from the group consisting of SEQ Id Nos. 21, 22, 23, 24, and 25 and their complements, comprising: (a) hybridizing the first oligonucleotide probe and the second oligonucleotide probe to opposite strands at opposite ends of the same target nucleic acid sequence of
  • hepatitis B virus DNA (b) extending the hybridized first and second oligonucleotide probes to be contiguously complementary to the target sequence, wherein the hybridized probes are capable of differentiation from the unhybridized probes; and (c) detecting the presence of the hybridized probes.
  • LCR pairs are also used in a method of detecting the presence, absence or quantity of hepatitis B virus DNA in a test sample wherein the target sequence is selected from at least one sequence selected from the group consisting of SEQ Id Nos.
  • a kit for detecting hepatitis B virus comprising at least one oligonucleotide according to the present invention targeted to at least one sequence selected from the group consisting of SEQ Id Nos. 21, 22, 23, 24, and 25 and their complements , said oligonucleotide being labeled so as to be capable of detection; and means for detecting said oligonucleotide and further comprises
  • FIG. 1 is a graphic representation of the process of ligase chain reaction as it is known in the prior art.
  • FIG. 2 is a standard curve relating the number of HBV DNA target molecules to the IMx® rate (counts/sec/sec) with probe set 403G (SEQ ID Nos. 1, 2, 3, and 4) 10-*** HBV genome copies/mL of patient's serum.
  • HBV DNA positive samples were diluted in human serum negative for HBV markers. The serum diluent also served as the negative control. The dilution of the positive controls indicated in the graphs as molecules of HBV DNA per mL serum, were tested in duplicates and the mean values are shown as IMx® rates.
  • a detection limit of 5-10 fg HBV-DNA/mL sample, equivalent to about 2000-3000 genome copies/mL or 10-15 copies per assay, respectively was obtained.
  • LCR results were obtained from Example 1, below.
  • FIGS. 3a-3c are graphs of results obtained from three patients (designated A, B, C) with HBV infection.
  • the data (Example 7) obtained with LCR clearly paralleled the alanine aminotransferase ("ALT') levels semi- quantitatively (HG.3a).
  • HBV-DNA levels could also be determined in samples of serial bleeds of patients receiving interferon treatment (3b-3c). In these patients' sera, HBV-DNA was clearly detectable by conventional dot blot hybridization test before starting interferon treatment but became negative after therapy. In the LCR detection system, serum HBV-DNA could be detected even after successful treatment for several weeks longer than with the conventional hybridization assay.
  • “Assay Conditions” refers to the conditions of LCR with regard to temperature, ionic strength, probe concentration and the like. These are generally known in the art. LCR involves essentially two states or conditions: annealing or hybridization conditions, and denaturation conditions. "Hybridization conditions” is defined generally as conditions which promote annealing and hybridization. It is well known in the art, however, that such annealing and hybridization is dependent in a rather predictable manner on several parameters, including temperature, ionic strength, probe length and G:C content of the probes. For example, lowering the temperature of the reaction promotes annealing. For any given set of probes, melt temperature, or Tm, can be estimated by any of several known methods.
  • assay conditions typically include temperatures which are slightly below the melt temperature. Ionic strength or "salt" concentration also impacts the melt temperature, since small cations tend to stabilize the formation of duplexes by negating the negative charge on the phosphodiester backbone. Typical salt concentrations depend on the nature and valency of the cation but are readily understood by those skilled in the art. Similarly, high G:C content and increased probe length are also known to stabilize duplex formation because G:C pairings involve 3 hydrogen bonds where A:T pairs have just two, and because longer probes have more hydrogen bonds holding the bases together. Thus a high G:C content and longer probe lengths impact the "assay conditions" by lowering the melt temperature.
  • the G:C content and length will be known and can be accounted for in determining precisely what "assay conditions" will encompass. Since ionic strength is typically optimized for enzymatic activity, the only parameter left to vary is the temperature and obtaining suitable "assay conditions" for a particular probe set and system is well within ordinary skill.
  • “Denaturation conditions” is defined generally as conditions which promote dissociation of double stranded oligonucleotides to the single stranded form. These conditions include high temperature and/or low ionic strength; essentially the opposite of the parameters described above as is well understood in the art.
  • nucleic acid sequences refers to the following base pairs in the case of DNA: A and T; C and G; in the case of RNA: A and U and C and G. Thus, G is complementary to C and vice versa.
  • Complementary bases are "matching", non-complementary bases are "mismatched”.
  • nucleic acid sequences a nucleic acid sequence or probe that is “complementary” to a probe or target means the sequence can hybridize to the complementary probe or target under assay conditions. Thus, they include sequences that may have mismatched base pairs in the hybridizable region, provided the sequences can be made to hybridize under assay conditions. As defined below, probe A is complementary to probe A' and probe B is complementary to probe B'.
  • “Correction” refers to the process of rendering, in a target dependent manner, the upstream probes ligatable to their downstream partners. Thus, only those probes hybridized to target, target complement or polynucleotide sequences generated therefrom are “corrected.” Preferably, the hybridized probes are enzymatically corrected in a manner which is dependent upon the sequence information contained within the target to render them ligatable to each 5 other.
  • the preferred enzyme is a DNA polymerase exhibiting 5' to 3' target dependent exonuclease activity. A 5' to 3' target dependent exonuclease activity can also be used in combination with a reagent with 5' to 3' target dependent polymerase activity.
  • Correction can be accomplished by several procedures, depending on the type of modified end used. For example, some of the probes l o of the present invention, were designed to be "corrected” by gap filling as described in U.S. Serial No.07 769,743 filed October 1, 1991 or by exonuclease cleavage as described in U.S. Serial No. 07/925,402 filed August 3, 1992, both of which are herein incorporated bt reference.
  • Exo format refers to correction of ligatable-incompetent ends by
  • Exonucleolytic refers to the excising activity, preferably of an enzyme, from the 5 '-end of a DNA or RNA substrate. Exonucleolytic activity may be associated with an exonuclease or the 5 '-3' exonuclease activity traditionally associated with some DNA polymerases. Generally, exonucleolytic activity is template-dependent, as is discussed in more detail below.
  • Gap format refers to a method for correcting ligation incompetent ends by extending the 3' end of an upstream oligonucleotide probe hybridized to target with nucleotides complementary to the intervening unhybridized portion of the target nucleic acid sequence so that the ends are capable of ligation.
  • Ligation is a general term and refers to any method of covalently
  • Ligating reagents useful in the present invention include T4 ligase, and prokaryotic ligases such as E. coli ligase, and Thermus ihermophilus ligase (e.g., ATCC 27634) as taught in ⁇ P-320 308. This latter ligase is presently preferred for its ability to maintain activity during the thermal cycling of LCRTM. Absent a thermally stable ligase, the ligase must be added again each time the cycle is 5 repeated. Also useful are eukaryotic ligases, including DNA ligase of
  • “Ligation incompetent absent correction” describes the 3' end of an l o upstream probe or the 5 ' end of a downstream probe which is incapable of being ligated to another probe, absent correction in a target dependent manner.
  • the correction can be the removal, replacement, or further modification of this end to render it ligatable.
  • An example of a ligation incompetent end is a non- phosphorylated 5' terminus of a downstream probe, which cannot be ligated to
  • Proximate is refers to the positioning of the upstream and downstream probes, as defined below, which are hybridized proximately to the same target strand so that their 3' and 5' ends are within about 1-20 nucleotides, more preferably about 1-10 nucleotides apart. Proximate may include gaps and
  • adjacent probes by definition are hybridized proximately so their respective 3' and 5' ends are 0 nucleotides apart. Proximate probes become adjacent upon correction.
  • Upstream and downstream probes refer to two different non- overlapping oligonucleotides hybridized to different regions of the same target 0 nucleic acid strand, the 3' end of one oligonucleotide pointing to the 5' end of the other.
  • the former is termed the "upstream” probe and the latter the “downstream” probe.
  • the "prime” (') designation is used to indicate a complementary base or sequence.
  • probe A can be complementary , as defined above, to A' even though it may have ends not co-terminal with A'. The same is true of B and B'.
  • Suitable and/or appropriate deoxynucleotide triphosphates refer to nucleotides needed to fill gaps in proximate probes. The type and quantity of nucleotide required are dependent on the target DNA. Further discussion of gap filling is found below. Typical nucleotides involve guanine (G), cytosine (C), adenine (A) and thymine (T) when the context is that of DNA; in the case of RNA, the base uracil (U) replaces thymine. The term also includes analogs and derivatives of the bases named above such as described in 37 CFR 1.822(p)(l). Although the degenerate base inosine (I) may be employed with this invention, it is not preferred to use I within modified portions of the probes according to the invention.
  • the oligonucleotide sequences of the present invention identify specific positions on the gene coding for the hepatitis B virus antigen. Different HBV strains having known genomic sequences were compared to the wild type HBV strain (strain adw ) to find conserved regions and the conserved regions tested as consensus sequences. Oligonucleotide probes covering homologous regions are of the HBV genome are first tested which can then be amplified in a target dependent manner. In a preferred embodiment, the oligonucleotide sequences identify specific loci on the gene coding for the surface antigen ("S") of HBV.
  • S surface antigen
  • some exemplary nucleotide sequences and their corresponding positions on the gene coding for the surface antigen ("3") of HBV are set forth below:
  • the methods of the invention comprise repeated steps of (a) hybridizing the selected primary probes to the target HBV DNA (and, if double stranded so that target complement is present, to the target complement); (b) correcting the selected probes in a target dependent manner to render the primary probes ligatable; (c) ligating the corrected probe to its partner to form a fused or ligated product; and (d) dissociating the fused product from the target and repeating the hybridization, correction and ligation steps to amplify the desired target sequence.
  • probe length e.g. EP-320 308, US 5185243, and US 4883750.
  • Probe length, probe concentration and stringency of conditions all affect the degree and rate at which hybridization will occur.
  • the probes are sufficiently long to provide the desired specificity; i.e., to avoid being hybridizable to nontarget sequences in the sample.
  • probes on the order of 15 to 100 bases serve this purpose.
  • hybridization of single probes and pair sets is effected at a temperature selected to give effective hybridization selectivity, preferably maximum hybridization selectivity for the specific length of the linked probe.
  • moderate temperatures are normally employed for probes and probe pairs of the present invention. Temperatures will generally range from about 20 °C to 90°C, more usually from about 30°C to 70 ⁇ C, preferably 37°C to 60 ⁇ C. Modified PCR
  • Hybridization for a modified form of PCR herein called “short PCR” or “sPCR” primers will be as is generally known in the art. Typical conditions are given in example 11 and 12, below. Hybridizing conditions should enable the binding of probes to the single nucleic acid target strand. As is known in the art, the probes are selected so that their relative positions along a duplex sequence are such that an extension product synthesized from one probe, when the extension product is separated from its template (complement) serves as a template for the extension of the other probe to yield a replicate chain of defined length.
  • Ligase Chain Reaction Ligase Chain Reaction
  • LCRTM probe sets to their targets and optionally to the target complements is adequately explained in the prior art; e.g., EP 320,308 and EP-439,182.
  • the probes are added in approximately equimolar concentration since they are expected to react stoichiometrically.
  • Each probe is present in a concentration ranging from about 5 nanomolar (nM) to about 90 nM; preferably from about 10 nM to about 35 nM.
  • nM nanomolar
  • the optimum quantity of probe used for each reaction also varies depending on the number of cycles which must be performed and, of course, the reaction volume. Probe concentrations can readily be determined by one of ordinary skill in this art to provide optimum signal for a given number of cycles.
  • the stringency of conditions is generally known to those in the art to be dependent on temperature, solvent and other parameters. Perhaps the most easily controlled of these parameters is temperature and thus it is generally the stringency parameter varied in the performance of LCR. Since the stringency conditions required for practicing this invention are not unlike those of ordinary LCR, further detail is deemed unnecessary, the routine practitioner being guided by the examples which follow.
  • reactions were performed in LCR Buffer (50 mM EPPS pH 7.8, 20 mM KC1, 30 mM MgCl2, 10 mM NH4CI and optionally 0.5 mM
  • NAD NAD
  • acetylated BSA acetylated BSA
  • Temperature cycling was achieved with a e.g. thermal cycler from Coy Laboratory Products (Ann Arbor, MI) or the Programmable Cycler ReactorTM (available from Ericomp, San Diego, CA).
  • Oligonucleotide probes of the present invention may be corrected by a "gap-fill” format or "exo" format. Both types of correction are described below. Correction bv Gap-Fill Format
  • Probes which are corrected by a gap-fill method have modified ends which are created by eliminating from one or more of the probes a short sequence of bases, thereby leaving a recess or gap between the 5' end of one probe and the 3' end of the other probe when they are both hybridized to the target (or target complement, or polynucleotide generated therefrom).
  • the gaps between the probes must be filled in (i.e., the modification must be "corrected”).
  • this can be done using a polymerase or a reverse transcriptase and an excess of deoxyribonucleotide triphosphates which are complementary to the target strand opposite the gap.
  • the invention involves repeated steps of (a) hybridizing the probes to the target HBV (and, if double stranded so that target complement is present, to the target complement); (b) extending at least one probe to fill in at least one gap; (c) ligating the extended probe to the adjacent probe to form a fused or ligated product; and (d) dissociating the fused product from the target and repeating the hybridization, extension and ligation steps to amplify the desired target sequence.
  • extension of a probe hybridized to a HBV DNA target is accomplished by a DNA polymerase or a Klenow fragment as is known in the art.
  • extension is accomplished by a reverse transcriptase.
  • exemplary reverse transcriptases include those from avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (M-MuLV) generally available to those skilled in the art.
  • AMV avian myeloblastosis virus
  • M-MuLV Moloney murine leukemia virus
  • Certain DNA polymerases will also recognize RNA as template under certain conditions. It is, of course, preferable to utilize extension reagents which are thermally stable and can withstand the cycling of high temperatures required for LCR.
  • thermostable polymerases presently include AmpliTaq ' T ⁇ , (available from Cetus-Perkin Elmer), Thermits polymerase (available from Molecular Biology Resources, Inc. Milwaukee, WI, "MBR") and recombinant or purified native polymerases from Thermits aquaticus, Thermus thermophilus or other species known to be thermostable.
  • dNTP's deoxyribonucleotide triphosphates
  • dX'TP deoxyribonucleotide triphosphates
  • the dNTP's are commercially available from a number of sources, including Pharmacia (Piscataway, NJ) and Bethesda Research Laboratories (Gaithersburg, MD).
  • Extension must be terminated precisely at the point of ligation so that the extended probe abuts the adjacent probe and can be ligated to it.
  • Stopbase designated Q', (see Figure 1) is defined in terms of its complement, Q and is accomplished by omitting from the reaction mixture, dNTP's that are complementary to Q; i.e. by omitting dQ'TP from the reaction mixture.
  • Extension by polymerase or transcriptase proceeds in a 5' to 3' direction. Consequently, the 3' ends of both upstream probe ( Figure 1, probes A and B') will be extendible by polymerase in the absence of anything to prevent extension. Extension is terminated when the next base called for by the template is absent from the reaction mixture.
  • probe A is extended through gap X n until stopbase complement (Q) is encountered along the target strand.
  • probe B' is extended through gap Ym until stopbase complement (Q) is encountered (either on the target complement or on the A half of reorganized A:B).
  • probe A' nor B will serve as a template for extension of A or B', so probes A and B' are extended only if hybridized to the target (or to reorganized polynucleotide products from previous cycles).
  • the gaps X n and Ym can be any number of bases long, i.e., n can be any integer greater than or equal to 1, and m is any integer greater than 0. It is to realized, however, that the choice of which gap is X n and which is Y m is arbitrary in the first place; but n and m cannot both be zero.
  • the gaps need not be the same length, i.e., m need not equal n.
  • m need not equal n.
  • the double gap variation degenerates into the specialized case of the single gap, which is not used in the embodiment being claimed herein.
  • the only restriction on the bases X is that they be selected from a set N which consists of from 1 to any 3 of the four bases.
  • the bases Y are drawn from set M. Since at least one stopbase Q' must be maintained, the combined sets N and M which represent the possible bases for X and Y, respectively, must include no more than three of the four bases. Accordingly, Y can be from zero to any three of the four bases provided that at least one base remains in the set "not N and not M". If set N constitutes less than three of the four bases, then Y can be a base that is not within N so long as there is at least one base remaining, the complement of which can serve as the stopbase Q' for termination of probe extension. A single stopbase can serve to terminate extension in both the X n and Y m gaps.
  • a second limitation on sequence Y m occurs if m equals n. If the gaps are the same length, the sequence Y m should not be complementary to the sequence X n or the 3' ends of probes A and B' would constitute "sticky ends". "Sticky ends" would permit a target independent double stranded complex to form wherein probe A hybridizes to probe B' such that ligations and amplification would proceed. Rather, when m equals n it is preferred that Y m not be complementary to X n . In other words, the ends of probes A and B 1 should at least be “slippery ends" which may be the same length, but are not complementary.
  • the fourth probe B' includes a 3' terminal sequence of X n , identical in length to the X n sequence gap in the target.
  • the 3' terminus of the fourth probe B' may stop short of the 3' end of sequence X n , provided there is no 3' recessed end with respect to the second probe B. Since extension occurs in a 5' to 3' direction and dX'TPs must be present anyway (to extend through X n ), probe B' would be extended through the gap, (both Y m and any remainder of X n ) just as the first probe A is extended through the X n gap. Correction by the "Exo Format"
  • a ligation incompetent end may be a non- phosphorylated 5' terminus of a downstream probe, which cannot be ligated to the 3' end of the upstream probe but which can be corrected in a target dependent manner to render it ligatable.
  • Another example of a ligation incompetent end may be terminal or internal mismatches of the probe with respect to the terminus of the target.
  • these mismatches are corrected in a target dependent manner by modification of the 5' end of one or more of the probes by eliminating a short sequence of bases, thereby leaving a recess or gap between the 5' end of one probe and the 3' end of the other probe when they are both hybridized to the target (or target complement, or polynucleotide generated therefrom).
  • This modification is corrected by an exonucleolytic activity, preferably the 5' to 3' exonuclease activity associated with a DNA polymerase (Gelfand, D., Tag DNA Polymerase in PCR Technology: Principles and Applications for DNA Amplification. Erlich, H. A., Ed., Stockton Press, N.Y. (1989)).
  • these DNA polymerases will initiate synthesis from the 3' hydroxyl end of a probe hybridized to a target DNA, proceed along the DNA target template, hydrolyzing hybridized DNA sequences and replacing them in the process.
  • the exonucleolytic degradation of the DNA sequences results in the release of mono, di, and larger nucleotide fragments.
  • this exonuclease activity is synthesis dependent. It therefore follows that the termination of synthesis should result in the termination of 5' to 3' exonuclease activity.
  • One way to terminate synthesis in a controlled manner is to limit the dNTP pool by leaving out one or several of the four dNTPs required for DNA synthesis.
  • LCR LCR-Likrase mediated reverse transcriptase clease clease clease clease .
  • a DNA polymerase with synthesis dependent, strand replacement 5' to 3' exonuclease activity is used to extend the upstream probe and hydrolyze the downstream probe using the target nucleic acid as a template.
  • the extension of the upstream probe and thereby the hydrolysis of the downstream probe could be controlled such that when a template base in the target is encountered to which no complementary dNTP is present, synthesis and hydrolysis would stop.
  • the resultant downstream probe would terminate with a 5' phosphate which would be adjacent to the 3' hydroxyl end of the extended upstream probe. Adjacent DNA sequences in this orientation represent a suitable substrate for ligation by DNA ligase.
  • the resulting gap between the probes must be filled in (i.e., the modification must be "corrected”). This correction proceeds as described above for the gap-filling format.
  • each corrected first upstream (or primary) probe is ligated to its associated first downstream probe and each corrected second downstream (or secondary) probe is ligated to its associated secondary upstream probe.
  • An "adjacent" probe is either one of two probes hybridizable with the target in a contiguous orientation, one of which lies with its phosphorylated 5' end in abutment with the 3' hydroxyl end of the partner probe. "Adjacent" probes are created upon correction of the modified end(s) in a target dependent manner, as described above. Enzymatic ligation is the preferred method of covalently attaching two adjacent probes; however, "ligation” is a general term and is to be understood to include any method of covalently attaching two probes.
  • Ligating reagents useful in the present invention include T4 ligase, and prokaiyotic ligases such as E coli ligase, and Thermus thermophilus ligase (e.g., ATCC 27634) as taught in EP-320308. This latter ligase is presently preferred for its ability to maintain activity during the thermal cycling of LCR. Absent a thermally stable ligase, the ligase must be added again each time the cycle is repeated. Also useful are eukaryotic ligases, including DNA ligase oi Drosophila, reported by Rabin, et al., J. Biol. Chem. 261:10637-10647 (1986).
  • the fused, reorganized probe is dissociated (e.g. melted) from the target and, as with conventional LCR, the process is repeated for several cycles.
  • the number of repeat cycles may vary from 1 to about 100, although from about 15 to about 70 are preferred presently.
  • ligatable sticky or blunt ends should be avoided. If such ends must be used, then 5' terminal phosphates should be avoided, eliminated or blocked. This can be accomplished either through synthesizing oligonucleotide probes (which normally carry no 5' terminal phosphate groups), or through the use of phosphatase enzymes to remove terminal phosphates (e.g. from oligonucleotides generated through restriction digests of DNA).
  • ligation of the "wrong" outside ends of the probes can be prevented by blocking the end of at least one of the probes with a "hook” or marker moiety as will be described in detail below.
  • the outside ends of the probes can be staggered so that if they are joined, they will not serve as template for exponential amplification.
  • haptens, or "hooks” are attached at the available outside ends of at least two probes (opposite ends of fused product), and preferably to the outside ends of all four probes.
  • a "hook” is any moiety having a specific ligand-receptor affinity. It may be for example a hapten or a segment of a polynucleotide.
  • a hood may be attached to one probe and a label may be attached to the other probe of the same sense. Ligation joins the label to the affinity moiety and separated label can be measured on a solid phase following separation.
  • the presence of amplified target can be detected by any number of methods.
  • One method is to differentiate reaction products of a specific size by means of molecular weight.
  • Methods for molecular weight differentiation include affinity labeling, composition, gel filtration, sedimentation velocity, osmotic pressure, or gel electrophoresis.
  • a particularly preferred method is gel electrophoresis which is particularly useful when the nucleotides used are labeled with a radiolabel, such as 32p.
  • detection is performed after separation, by determining the amount of label in the separated fraction.
  • label in the separated fraction can also be determined subtractively by knowing the total amount of label added to the system and measuring the amount present in the unseparated fraction.
  • Separation may be accomplished by electrophoresis, by chromatography or by the preferred method described below. Typically, detection is performed after separation, by determining the amount of label in the separated fraction. Of course, label in the separated fraction can also be determined subtractively by knowing the total amount of label added to the system and measuring the amount present in the unseparated fraction. Where used, separation may be accomplished by electrophoresis, by chromatography or by the preferred method described below.
  • labeling the nucleotides with a physical label which is capable of generating a detectable signal.
  • the various "signal generating compounds"(labels) contemplated include chromogens, catalysts such as enzymes, luminescent compounds such as fluoroscein and rhodamine, chemiluminescent compounds, radioactive elements, and direct visual labels.
  • enzymes include alkaline phosphatase, horseradish peroxidase, beta-galactosidase, and the like.
  • the selection of a particular label is not critical, but it will be capable of producing a signal either by itself or in conjunction with one or more additional substances.
  • haptens are known, and virtually any hapten can be used 5 with the present invention.
  • the invention requires only that a specific binding partner is known or can be prepared (a definitional property of "hapten") and that the hapten can be coupled to the probe such that it does not interfere with hybridization.
  • Many methods of adding haptens to probes are known in the literature. Enzo Biochemical (New York) and Clontech (Palo Alto) both have l o described and commercialized probe labeling techniques.
  • a primary amine can be attached to a 3' oligo end using 3'-amine-ON CPGTM (Clontech, Palo Alto, CA).
  • a primary amine can be attached to a 5' oligo end using Aminomodifier ⁇ ® (Clontech). The amines can be reacted to various haptens using conventional activation and linking chemistries.
  • haptens include many drugs (e.g. digoxin, theophylline, phencyclidine (PCP), salicylate,
  • Protocols for the detection of more than one target for example HBV
  • haptens may be used as both the common and unique labels. It is, of course, understood that another label is easily substituted for at least one of the haptens, especially the common hapten.
  • a first hapten is used to capture and separate the reorganized molecules.
  • a second hapten is used to couple the reorganized complex with the signaling entity.
  • a fluorescein moiety is attached to the 5' end of the first primary probe and to the 3' end of the first secondary probe.
  • a different hapten say biotin, is attached to the 3' end of the second primary probe and to the 5' end of the second secondary • probe.
  • a solid phase having a coating of anti-fluorescein is used to separate reorganized molecules from unligated probes having biotins. (Unligated probes having fluoresceins are also captured.)
  • the separated complexes are detected by using avidin or anti-biotin labeled with a detectable signaling entity such as an enzyme.
  • the test sample can be any biological material suspected of containing HBV.
  • the test sample can be human body tissue, or a test sample which contains cells suspected of containing HBV.
  • the term can refers to virtually any liquid sample.
  • the test sample can be derived from any desired source, such as a physiological fluid, for example, blood, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk, ascites fluid, mucous, synovial fluid, peritoneal fluid, amniotic fluid or the like.
  • the liquid test sample can be pretreated prior to use, such as preparing plasma from blood, diluting viscous liquids, etc. Methods of pretreatment can also involve separation, filtration, distillation, concentration, inactivation of interfering components, and the addition of reagents.
  • physiological fluids other liquid samples such as water, food products and the like can be used.
  • a solid test sample can be used once it is modified to form a liquid medium.
  • Diagnostic kits may include the labeled oligonucleotides; if the oligonucleotide is unlabeled, the specific labeling reagents may also be included in the kit.
  • the kit may further contain suitably packaged combination nucleoside triphosphates, e.g., dATP, dGTP, dCTP, or dTTP or combinations of up to three of the dNTPs, depending on the particular probe design and the gap full or sexo fill needs.
  • the kit can further include a polynucleotide polymerase and also means for covalently attaching upstream and downstream sequences, such as a
  • kits 5 ligase.
  • These reagents will typically be in separate containers in the kit but can be packaged in one container where reactivity and shelf life permit.
  • the relative amounts of various agents in the kits can vary widely to provide for concentrations of reagents which optimize the reactions needed to occur during the instant invention and to optimize the sensitivity of the assay.
  • the kits may l o further include a denaturation agent for denaturing the analy te, hybridization buffers, wash solutions, enzyme substrates, negative and positive controls and written instructions for carrying out the assay.
  • BSA bovine serum albumin
  • EDTA a metal chelator, ethylenediamine tetraacetic acid
  • EPPS chemical abbreviation for [N-(2-hydroxyethyl)piperazine-
  • FITC chemical abbreviation for fluorescein isothiocyanate, a fluorescent hapten derivative.
  • MES chemical abbreviation for [2-(N- morpholino)ethanesulfonic acid] a buffer.
  • IMx® trademark of Abbott Laboratories for an automated instrument for performing microparticle enzyme immunoassay (MELA). Tris a buffer comprising rm(hydroxymethyl)aminomethane
  • any kind of cells, swabs, smears, blood or tissue may be prepared by centrifugation of the respective cellular suspension in phosphate- buffered saline (PBS). Generally, the pellets are resuspended in 100 ⁇ l of 10 mM NaOH and heated for 5 to 10 minutes at 100 °C. After boiling, the samples are re-centrifuged and the cellular debris removed.
  • PBS phosphate- buffered saline
  • oligonucleotide probe of the present invention An aliquot of the supernatant is added to a reaction mix containing 50 mM EPPS pH 7.8, 30 mM MgCl2, 20 mM KC1, 1 ⁇ M deoxynucleotide triphosphate, and 5 xlO**- * molecules of oligonucleotide probe of the present invention.
  • the capture ligand oligonucleotide A and A' may be derivatized with carbazole and/or FITC.
  • Probe B and B' may be derivatized with adamantane or biotin as signal moieties.
  • Polynucleotide kinase is used for phosphorylation of the selected probes' at their 5' ends.
  • the tube tubes containing the sample and reaction mix are overlaid with mineral oil and boiled for about 3 minutes. Afterwards the tubes are held for 1 minute at 85°C and 50°C for another 1 minute. Thermus thermophilus ligase (Abbott) and Thermus DNA polymerase (Molecular Biology Resources) are added to the reaction mixture. The tubes are then alternated between 85°C and 50°C either in a thermal cycler or between two water baths. Normally 30 LCR cycles are sufficient to amplify the target HBV DNA for assay. For detecting the reaction product, the mix is separated from mineral oil layer and diluted with an equal volume of distilled water. A portion of the reaction mixture is loaded into a disposable reaction cell of the IMx® analyzer.
  • the respective reagent of the test such as sample dilution buffer, methyl umbelliferone phosphate, antibiotin alkaline phosphatase conjugate, anti-fluorescein coated particles, and the like) are also loaded on the IMx® analyzer.
  • the rate of alkaline phosphatase bound is derived from the reaction rate in counts/sec/sec.
  • Quantities of polymerase are expressed in units, defined as follows: 1 unit of enzyme is as defined by the manufacturer (Molecular Biology Resources). Units of ligase enzyme are defined herein as: 1 mg of 95% purified Thermus thermophilus DNA ligase has a specific activity of about 1 x 10 ⁇ units. While this is not precisely standardized and may vary by as much as 20%, optimization is within the skill of the routine practitioner. LCRTM Conditions
  • the gel was autoradiographed, the autoradiograph used as a template to excise the ligated and unligated probes and the amount of radioactivity in each band was measured by liquid scintillation counting. The percentage of radioactivity in the ligated product was calculated as a function of the total counts in each lane.
  • Example 1 LCRTM was performed using probe set 403G consisting of SEQ ID Nos. 1, 2, 3, and 4 (See Table A) in a 0.5 mL polypropylene tube containing LCR Buffer.
  • a test sample containing HBV DNA (2.8 x 10 7 molecules HBV
  • DNA/ml was diluted in human serum negative for HBV markers.
  • the serum diluent also served as the negative control.
  • Each probe was present at 5 x 10 11 molecules/reaction and the final concentration of DNA ligase at 5000 units and DNA polymerase at 1.0 units.
  • the samples were overlaid with mineral oil and the temperature cycle consisted of a 85°C incubation for 30 seconds followed by a 50°C incubation for 20 seconds.
  • the evaluation of the linear range is demonstrated in Figure 2 as a linear graph.
  • the axis values are obtained by multiplying the number of molecules/reaction (Table 1) times the number of mL in the reaction (x200) Table i
  • Example 3 LCR was performed using probe sets 23 IE (SEQ ID Nos. 9, 31, 32, and 33) and 231G (SEQ ID Nos. 9, 10, 11, and 12) under conditions described in Example 1, above.
  • probe set 321E 45 cycles were performed.
  • the results are in Table 3, below.
  • probe set 23 IG 40 cycles at temperature of 85° and 60° for 30 and 20 seconds, respectively, were performed. Results are shown in Table 4.
  • Table 3
  • IMx® (c/s/s) IMx® (c/s/s) IMx® (c/s/s) IMx® (c/s/s) IMx® (c/s/s)
  • Example 4 LCR was performed using probe sets 664G (SEQ ID Nos. 17, 18, 19, and 20) and 664E (SEQ ID Nos. 17, 35, 36 and 37) under conditions described in Example 1, above. The results are illustrated in Table 5 and Table 6 for 664G and 664E, respectively.
  • Example 5 LCR was performed using probe set 403G (SEQ ED Nos. 1, 2, 3, and 4) under conditions described in Example 1, above except that 1350 units of ligase were used. The specificity of these probes for HBV-DNA was demonstrated
  • Example 6 LCR- was performed using probe sets 403G (SEQ ID Nos. 1, 2, 3, and 4) and 184G (SEQ ID Nos. 5, 6, 7, and 8) under conditions described in Example 1 , above using twenty human serum samples from different groups of patients with hepatitis B infections (HBsAg+). The results shown in Table 7, below. The samples were tested in two replicates and mean values are shown as signal-to-noise (S/N) ratio.
  • S/N signal-to-noise
  • IMx® background (bg) rates wherein a 0 - 1.5 x bg rate is defined as negative (-); >1.5 - 3.0 x bg rate as indeterminate (+/-); 3.0 - 20 x bg rate as weak positive (+); and >20 x bg rate as positive (++).
  • HBV DNA test DNA solution hybridization assay
  • PCR Polymerase Chain Reaction
  • All samples were HBsAg positive and the results of HBeAg or Anti-HBe are included.
  • the tested HBV carriers had different clinical backgrounds. High viremia was characterized by the presence of HBV DNA and HBeAg; low viremia by the presence of anti-HBe and HBV DNA; and asymptomatic HBV carriers by the presence of anti-HBe.
  • Example 7 LCR was performed using probe set 403G (SEQ ID Nos. 1, 2, 3, and 4) under conditions described in Example 1 and used to monitor HBV DNA levels in follow-up samples of patients (A, B, C) with chronic liver infections.
  • the LCR data from the IMx® instrument presented as signal-to-noise (S/N) ratio can be compared directly in the graphs with the serum level (m/1) of the liver derived enzyme alanine aminotransaminase (ALT).
  • S/N signal-to-noise
  • ALT liver derived enzyme alanine aminotransaminase
  • LCR was performed using probe set 403G (SEQ ID Nos. 1, 2, 3, and 4) under conditions described in Example 1, except 0.5 units of DNA polymerase and 3400 units of DNA ligase were used. Reactions were set up either with a HBV DNA negative serum (negative control) or serum containing HBV type adw or type ay.
  • probe set 403G is useful for consensus detection of HBV subtypes and for following patients eligible for or undergoing anti viral therapy.
  • Probes SEQ Id Nos. 1 and 4 were used as primers for the detection of HBV DNA using a modification of Polymerase Chain Reaction (PCR) referred to as “short PCR” or “sPCR”.
  • PCR Polymerase Chain Reaction
  • sPCR was performed essentially following the package insert of the commercially available Gene- AMPTM kit available form Perkin-Elmer/Cetus, Emeryville, CA. Controls were used from the Abbott
  • Genostics TM DNA kit PCR was run using primer set SEQ ID nos. 1 and 4 at l l ⁇ l2 molec/reaction, reaction buffer for 30 cycles of: 94°C for 30 seconds and 50°C for 20 seconds and Taq Polymerase (Cetus) at 1.25 units per reaction.
  • the duplex product was isolated and detected in an IMx® instrument as described in EP 357,011 to Laffler, et al. published March 7, 1990, which is herein incorporated in its entirety by reference. The average results of duplicate runs are given below. PCR with SEO Id. Nos.l and 4
  • Example 10 Probes SEQ Id Nos. 5 and 8 were used as primers for the detection of HBV DNA with PCR as described in Example 9, above. The average results of duplicate runs are given below.
  • Example 11 Using probes SEQ ID Nos. 1 and 3, target specific polymerization and ligation is performed as described in U. S. Patent Number 5185243 issued February 9, 1993, which is incorporated in its entirety by reference.
  • the probes are derivatized with FITC and/or fluorescein as described above and mixed with selected target HBV DNA, a modified t7 DNA polymerase available from United States Biochemical, Cleveland, Ohio), buffer and water.
  • the mixture is heated to 80°C for 5 minutes and is allowed to cool slowly to 23°C. After a brief spin, 32 P-labeled dCTP, T4 DNA ligase, and DNA polymerase are added to the reaction.
  • the resulting solution is incubated at 23°C for 12 hours.
  • Denaturing polyacrylamide gel electrophoresis of an aliquot from the above reaction shows a 32 P-labeled product of 48 bases in length. This product corresponds to the fill- in and ligation product of SEQ ID Nos. 1 and 3 after hybridization to target.
  • Example 12 Using probes SEQ ID Nos. 1 and 28 are hybridized to target HBV DNA under conditions described in Example 11, above except dATP and dCTP are added. An aliquot of the reaction mixture shows a 32 P-labeled product of 48 bases in length. This product corresponds to the fill-in and ligation product of SEQ ID Nos.l and 28 after hybridization to target.
  • MOLECULE TYPE Other nucleic acid (synthetic DNA)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 1:
  • MOLECULE TYPE Other nucleic acid (synthetic DNA)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 2:
  • MOLECULE TYPE Other nucleic acid (synthetic DNA)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 3:
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  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 5:
  • MOLECULE TYPE Other nucleic acid (synthetic DNA)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 6: CTGTAACACG AGCAGGGGTC 20
  • MOLECULE TYPE Other nucleic acid (synthetic DNA)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 7:
  • MOLECULE TYPE Other nucleic acid (synthetic DNA)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 8:
  • MOLECULE TYPE Other nucleic acid (synthetic DNA)
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  • MOLECULE TYPE Other nucleic acid (synthetic DNA)
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  • MOLECULE TYPE Other nucleic acid (synthetic DNA)
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  • MOLECULE TYPE Other nucleic acid (synthetic DNA)
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  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 19:
  • MOLECULE TYPE Other nucleic acid (synthetic DNA)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 20: CTACGAACCA CTGAACAAAT GG 22
  • MOLECULE TYPE Other nucleic acid (synthetic DNA)
  • FEATURE FEATURE:
  • MOLECULE TYPE Other nucleic acid (synthetic DNA)
  • FEATURE FEATURE:
  • MOLECULE TYPE Other nucleic acid (synthetic DNA)
  • FEATURE FEATURE:
  • MOLECULE TYPE Other nucleic acid (synthetic DNA)
  • FEATURE FEATURE:
  • MOLECULE TYPE Other nucleic acid (synthetic DNA)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 33:
  • MOLECULE TYPE O ⁇ otth: er nucleic acid (synthetic DNA)
  • MOLECULE TYPE Other nucleic acid (synthetic DNA)
  • FEATURE FEATURE:
  • MOLECULE TYPE Other nucleic acid (synthetic DNA)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 37:

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Abstract

L'invention concerne de courtes séquences de nucléotides du virus de l'hépatite B qui permettent de déterminer la présence et le type d'un virus de l'hépatite B dans un échantillon d'essai. Ces séquences peuvent être amplifiées par diverses techniques d'hybridation de l'ADN, notamment par LCR ou PCR modifiée. Elles peuvent aussi être hybridées par des procédures courantes d'analyse ou de réplique des taches. Des méthodes et des trousses permettant de détecter le virus de l'hépatite B dans un échantillon d'essai et d'en déterminer le type sont également proposées.
EP94923386A 1993-07-13 1994-07-08 Sequences de nucleotides et procede d'amplification et de detection du virus de l'hepatite b Withdrawn EP0785996A1 (fr)

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WO1994002648A1 (fr) * 1992-07-21 1994-02-03 Imclone Systems Incorporated Amplification et detection d'acides nucleiques de remplissage de breches

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CA2167056A1 (fr) 1995-01-26
JPH09501829A (ja) 1997-02-25
AU7326494A (en) 1995-02-13
WO1995002690A1 (fr) 1995-01-26
EP0785996A4 (fr) 1997-07-30

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