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WO2000058477A1 - Procede de detection d'une mutation dans le virus de l'hepatite b et kit de detection - Google Patents

Procede de detection d'une mutation dans le virus de l'hepatite b et kit de detection Download PDF

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Publication number
WO2000058477A1
WO2000058477A1 PCT/JP2000/001849 JP0001849W WO0058477A1 WO 2000058477 A1 WO2000058477 A1 WO 2000058477A1 JP 0001849 W JP0001849 W JP 0001849W WO 0058477 A1 WO0058477 A1 WO 0058477A1
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probe
seq
dna
virus
hepatitis
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PCT/JP2000/001849
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English (en)
Japanese (ja)
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Satsuki Kobayashi
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Genome Science Laboratories Co., Ltd.
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Application filed by Genome Science Laboratories Co., Ltd. filed Critical Genome Science Laboratories Co., Ltd.
Priority to AU45198/00A priority Critical patent/AU4519800A/en
Publication of WO2000058477A1 publication Critical patent/WO2000058477A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis

Definitions

  • the present invention relates to the mutation of a gene encoding a reverse transcriptase activity center of hepatitis B virus, which is useful for the detection of hepatitis B therapeutic drug resistant hepatitis B virus such as lamivudine and the like in the field of medicine and clinical testing and for drug monitoring.
  • hepatitis B therapeutic drug resistant hepatitis B virus such as lamivudine and the like in the field of medicine and clinical testing and for drug monitoring.
  • HBV hepatitis B virus
  • Lamivudine is an anti-wizard developed by Glaxo Welcome
  • the site where this mutation occurs is the active center site of the reverse transcriptase of the HBV polymerase, and is an amino acid sequence unique to reverse transcriptase, that is, tyrosine (Y) ⁇ methionine (M) ⁇ aspartic acid (D) ⁇ asparagine. Since it has an amino acid sequence consisting of an acid (D), it is called a YMDD motif (nucleotides 736 to 747) (LA Kohls taedt et al., SCIENCE 256: 1783-1790, 1992).
  • M methionine of this YMDD motif is replaced by V (parin) and I (isoleucine), and HBV acquires lamivudine resistance.
  • V parin
  • I isoleucine
  • HBV acquires lamivudine resistance.
  • the methionine codon ATG in ATG is mutated to G and replaced with valine, or the G in ATG is replaced with T or ⁇ to be replaced with isoleucine.
  • the monitoring of the appearance of lamivudine-resistant virus at the time of lamivudine administration that is, a virus having the above-mentioned mutation in the gene encoding the YMDD motif, will be helpful in determining the effect of lamivudine and in planning a dose, and is very useful. It is believed that there is. .
  • detection of the above mutations in HBV and detection of mutant viruses themselves are performed at the nucleic acid level because it is very difficult to detect them at the amino acid level.
  • One example is the extraction and purification of the HBV virus gene from the serum collected from a patient, followed by amplification of the area around the gene sequence suspected of having a mutation by PCR, and direct amplification of the amplified product (direct sequence method).
  • direct sequence method direct sequence method
  • a sequencing test may be performed by the dideoxy method.
  • the 0101-33? Method which uses a gene amplification primer having a base corresponding to the point mutation at the 3 ′ end and amplifies only the mutated gene by PCR to detect the point mutation.
  • T ⁇ ru (Chayama et al.? 8 1 ( ⁇ 0 ⁇ 27: 171 1-1716, 1998)
  • the examination by HBV gene sequencing as described above is now a large part due to the appearance of auto sequencers and the like. Although it is automated, it takes several days from sample acquisition to detection, which is time-consuming, involves very complicated and skillful operations, and has a large economic burden and is difficult to process many samples.
  • the genetic sequence test has the drawback that when the wild-type and mutant-type viruses coexist, the results that reflect the abundance ratio may not be obtained.
  • the PCR-SSP method requires designing primer sets and setting PCR conditions for each type of point mutation, and has disadvantages such as a complicated detection system.
  • the present invention provides (i) a method that completes the process from obtaining a sample to detection in one day; It can be carried out without special and expensive equipment such as sequencers and skilled techniques, (iii) has high sensitivity and specificity, and (iv) has a wild-type and mutant-type abundance of about 10 times each other.
  • An object of the present invention is to provide a method for detecting a mutation in the gene encoding the reverse transcriptase activity center of hepatitis B virus, which can detect both if present. Disclosure of the invention
  • the present inventors have proposed a method for detecting a mutation in a gene encoding a reverse transcriptase activity center (YMDD motif) of hepatitis B virus,
  • the 3 'end of the amplification product is changed in the vicinity of the 3' end according to the gene mutation that may occur on the gene encoding the YMDD motif during long-term administration of lamivudine.
  • the present inventors have found a method that is simple, can be operated in a short time, and accurately determines a mutation on a gene, thereby completing the present invention.
  • the method of the present invention is based on the fact that the ability of the probe to complement the allele contained in the sample and the degree of complementarity affect the subsequent incorporation of the labeled substrate by the DNA polymerase. Use.
  • the present invention will be described in more detail by taking, as an example, the detection of lamivudine-resistant HBV generated during long-term administration of lamivudine and the detection of a mutation in a gene encoding the YMDD motif on the HBV gene.
  • a similar mutation is caused on the YMDD motif of HBV by administration of a drug other than lamivudine, it goes without saying that it can be detected.
  • FIG. 1 shows the position of the YMDD motif and the positions of the primer sequences of SEQ ID NO: 1 and SEQ ID NO: 2 in HBV-DNA.
  • the DNA in the sample in advance so as to include the gene region encoding the YMDD motif, which is the active center of the reverse transcript of HBV.
  • the type II HBV gene to be analyzed is preferably extracted and purified from serum, tissue, etc., from an HBV infected person.
  • the amplification product used in the present method preferably has a size of 50 b or more and 300 bp or less, more preferably 80 b or more and 130 bp or less.
  • the amplified product is denatured with alkali to form a single strand, and then hybridized with each probe corresponding to the mutation.
  • HBV is a virus that is relatively easily mutated, it is desirable to use a site that is highly conserved between clones when setting primers.
  • primers with high PCR amplification efficiency and high specificity are used. Need to be set.
  • the present invention provides the following primer set:
  • SEQ ID NOs: 1 and 2 are located before and after the YMDD motif, as shown in FIG. HBV-DNA consists of 3215 nucleotides.
  • SEQ ID NO: 2 corresponds to nucleotides 645-664 and SEQ ID NO: 2 corresponds to nucleotides 749-768.
  • the primer set should be inside or near the outside of the amplification product. It goes without saying that it can be set.
  • probes having exactly the same sequence as the target sequence may be used.
  • the length is generally about 10 to 40 bp considering the cost of nucleotide synthesis.
  • the probe should be designed so as to bias the hybridization intensity between the target group and the non-target group. It must be designed, and it becomes more difficult to select the number of mismatched bases in the probe and the position of introduction.
  • the binding between the target sequence and the probe is affected by the stringency under the hybridization conditions.
  • the effect of stringency can be moderated by changing the length of the probe or by adjusting the location and number of mismatches. That is, when the length of the probe cannot be increased for some reason, or when there is a restriction in the site of mismatch introduction, the stringency can be used to adjust the strength of allied groups between alleles.
  • the design of the probe used to identify the mutated gene of the virus during the biopsy should cover all degeneracy of the mutated gene, as well as the wild-type and mutated forms due to complex infection or reversion of the mutated form.
  • reversion there are two types of reversion: genuine reversion that completely returns to the wild-type genotype and repression that reverts phenotypically to a new mutation. Possibilities must be considered.
  • the length of the probe is preferably from 20 bp to 40 bp, more preferably from 25 bp to 35 bp.
  • the probe of the present invention has been designed after finding that suitable results can be obtained by designing such that the mismatch mutation site is located several bases upstream from the 3 ′ end.
  • mutant V mutants of human-derived HBV caused by lamivudine administration have been reported in the case of mutant V; mutants of GTA, GTT, and GTC, which are 2-point mutants compared to wild-type M (ATG), have been reported. Not. In the case of variant I, only mutations to ATT have been reported, but mutations to ATA in nature have been reported to exist independently of lamivudine administration (Stephan Gunther et al., VIROLOGY 235: 104—108, 1997). Probe for detection of wild type (YMDD)
  • Probes for detecting wild-type include the underlined TAT ATG GAT GAT, which is the nucleotide sequence of the wild-type YMDD motif, at the end.
  • This probe can sufficiently detect wild-type (YMDD). However, this probe is a mutant sequence in the underlined methionine codon ATG.
  • YVDD type TAT gTG GAT GAT or YIDD type: TAT ATa GAT GAT, TAT ⁇ ⁇ Only one base is different from GAT GAT and TAT ATc GAT GAT (lower case part). If these mutants are mixed in the sample, it is possible that the amplification product of the mutant is sometimes weakly hybridized.
  • V is A or G or C. This probe artificially introduces a new mutation at the site indicated by underlining and makes it a mismatch of 2-3 bases from the gene sequence of each mutant type, so that the reaction specificity in the wild type is To Non-specific reaction with the mutant can be suppressed without lowering.
  • V may be A or G, but the mutation to C has been introduced,
  • the terminal 7 bases (TATA c GG) of the probe of SEQ ID NO: 5 are (i) a mismatch with the wild-type is a single base in lower case, and (ii) a mismatch with the 7 mutants is Any two to three bases in the underlined part.
  • Mutants can be detected in four base sequences: TAT GTa GAT GAT, TAT GTt GAT GAT, TAT GTc GAT GAT, and TAT GTg GAT GAT. If so, the following 4 sequences containing these first 7 bases at the 3 'end:
  • the probe of SEQ ID NO: 9 or SEQ ID NO: 10 has only one base difference from the wild type gene (I: inosine Is a universal base that binds to any base), and if wild-type is present in the sample, the wild-type amplification product may hybridize though weak.
  • a mixture of probes comprising each sequence of
  • the probes of SEQ ID NOS: 11 to 14 artificially introduce a new mutation (V is A or G or C) at the underlined positions, and are wild-type and mutant (YIDD) genes. Non-specific reactions with wild-type or mutant YIDD can be suppressed without reducing the reaction specificity of the mutant (YVDD type) by making a mismatch of 2 bases in the sequence. it can.
  • a probe of SEQ ID NO: 15 that can detect all YVDD types may be used.
  • V may be A or G, but a mutation to C has been introduced,
  • a mixture of probes comprising each sequence of
  • Mutants can be detected in three base sequences: TAT ATa GAT GAT, TAT AT c GAT GAT, and TAT ATt GAT GAT.
  • a mixture of three types of probes having is used.
  • the sequence reported to date for the mutant (YVDD type) is only TAT £ T £ G, and the underlined portion has a difference of 2 nucleotides from SEQ ID NOS: 21 to 23.
  • Non-specific reaction due to the mutant type (YVDD type) does not occur and does not pose a problem in actual detection.
  • it differs from the wild type (YMDD type) base sequence by only one base (lower case), but in the hybridization reaction at the same temperature as other probes (30 bp), the probe length is shortened by 5 bp. This makes it possible to increase the influence of the mutation introduction site at the 3 ′ end, improve the specificity of hybridization, and prevent nonspecific reactions. Therefore, a probe having SEQ ID NOs: 21, 22, and 23 is used for detecting a gene mutation in a mutant type (YIDD type).
  • the probe of SEQ ID NO: 6, 7, 8, and 9 can be used for the YVDD type mutation. , Or 16, 17, 18 and 19, and in the case of YIDD mutation, the probes of SEQ ID NOs: 21, 22 and 23 are preferably used separately.
  • the reverse transcriptase activity of hepatitis B virus Probe set used to detect the presence or absence of mutations in the gene encoding the sex center
  • a probe set consisting of at least one of the following; and Z or
  • a probe set used to detect the presence or absence of a mutation in a gene encoding the reverse transcriptase activity center of hepatitis B virus
  • a probe set consisting of at least one of:
  • a probe set used for detecting the presence or absence of a mutation in a gene encoding the reverse transcriptase activity center of hepatitis B virus comprises:
  • the gene probe to be used is previously immobilized on a carrier for each type of mutation.
  • a microplate that can be easily operated as a solid-phased carrier is used. It is preferable that the solid phase be immobilized so that the volume of the probe is 3 gel / sample, and an eight-bridging reaction is performed in the well where the probe is immobilized.
  • the hybridization reaction is a reaction for selectively removing by-products during amplification. Furthermore, if the selectivity is to be pursued during the hybridization, it is necessary to optimize the temperature during the hybridization.
  • the temperature during the hybridization of the present invention is preferably from 25: to 65, more preferably from 37 "C to 60.
  • the labeling substrate and DNA polymerase are added to perform the uptake reaction (mini-sequence reaction).
  • the reaction conditions such as the temperature and the composition of the reaction buffer are adjusted so that the incorporation of the labeled substrate occurs only when the probe is hybridized.
  • the reaction temperature is preferably 37 to 80, more preferably 50 to 75 ° C. .
  • reaction buffer used for carrying out the extension synthesis reaction generally, a buffer prepared under conditions so that the enzyme activity of the DNA polymerase used is exhibited may be used.
  • a buffer prepared under conditions so that the enzyme activity of the DNA polymerase used is exhibited may be used.
  • the enzymatic activity of DNA polymerase can be controlled subtly by adding urea so that the final concentration is about 0.1 to 1.5M.
  • an extension synthesis reaction (incorporation reaction) is performed from the 3 'end of the hybridized probe group, so that the base portion corresponding to the unmutated amino acid is located at the end portion of all probes. Then, it is preferable that the first base to be incorporated is common.
  • SEQ ID NOs: 3-23 used in a preferred embodiment of the invention end with a G in the first nucleotide of the codon (GAT) of the unmutated amino acid D, immediately following the mutated amino acid M in the YMDD motif.
  • the first base added to the 3 'end of all probes is the A of the second nucleotide.
  • a color-forming method, a luminescence method, a fluorescence method, or the like is performed using an enzyme-labeled substance capable of specifically binding to the label.
  • an enzyme-labeled substance capable of specifically binding to the label is also possible to directly label the base and directly incorporate them in the extension synthesis reaction.
  • label itself as an enzyme
  • protein enzymes are generally macromolecules and are likely to cause steric hindrance when the labeled base is incorporated, and the enzyme is deactivated when heated during incorporation Since it is highly likely that the labeling is performed, it is desirable to use a substance having a small molecular weight, a relatively stable substance that can be specifically detected, such as piotin, dinitrophenyl (DNP), and digoxigenin (DIG).
  • DNP dinitrophenyl
  • DIG digoxigenin
  • an enzyme-recognized antibody is bound to an antibody that recognizes each, and the enzyme activity is measured by a colorimetric method, a luminescent method, or the like.
  • the abundance ratio of each type of gene before amplification is reflected in the abundance ratio of the amplified product after amplification, the abundance ratio of alleles can also be estimated based on the detection intensity of each X-ray.
  • kits using a method for detecting a point mutation on a gene encoding the YMDD motif in the HBV genomic gene.
  • the kit include: A set of primers for PCR consisting of the primer of SEQ ID NO: 1 and the primer of SEQ ID NO: 2 for obtaining an amplified product in the peripheral region of the encoding gene, (2) a point on the gene encoding the YMDD motif of HBV A gene probe having a base corresponding to the mutation site near the 3 ′ end, preferably a mixed probe of SEQ ID NO: 5, SEQ ID NO: 20, and SEQ ID NOS: 21 to 23, or a solid-phased product of each probe; ) A denaturing solution for converting the double-stranded amplification product into a single strand, (4) a hybridization buffer used for the hybridization reaction, and (5) a DNA polymer. Over zero, and a buffer for mini Sequence for ⁇ (6) labeled substrate.
  • plasmids for specificity evaluation containing a region to be subjected to PCR amplification in the present invention and containing sequences corresponding to wild type and mutant type were prepared. This was subjected to PCR amplification to form III, denatured, hybridized to a probe immobilized on a plate, and the incorporation of the labeled substrate was detected. As a result, the evaluation plasmid having the wild-type sequence was used for the well on which the probe for wild-type was immobilized, and the evaluation plasmid having the mutant-type sequence of norin-type and isoleucine-type was used for each mutant type. Incorporation of the labeled substance was detected only in the gel on which the probe was immobilized. Was not detected.
  • the conventional method in this specification means that the HBV gene is extracted and purified from serum, the area around the gene sequence encoding the reverse transcriptase activity center is amplified by PCR, and the nucleotide sequence of the amplified product is determined by the dideoxy method. Is the way. In this method, it takes 2-3 days to detect the presence or absence of the mutation.
  • Sense primer 5'-AGTGGGCCTC AGNCCGTTTC-3 '(SEQ ID NO: 1) and antisense primer: 5'-AGACTTGGCC CCCAATACCA-3' (SEQ ID NO: 2) were used. However, N in SEQ ID NO: 1 was used for I (inosine).
  • 50 l of a gene amplification solution having the following composition was prepared, and the gene was amplified by PCR.
  • the above solution was set on a Thermal Cycler PJ-9600 (PerkinElmer) .
  • the temperature conditions for PCR were 95T: for 2 minutes, then for 15 seconds to 60 seconds for 20 seconds, followed by 5 cycles of 90 °.
  • Gene amplification was carried out by repeating the cycle of 15 seconds at C-6 (20 seconds at TC 65 times. As a result, when HBV was positive, a 124 bp amplification product was obtained.
  • 3M sodium hydroxide solution 1001 was added and left at room temperature for 5 minutes to perform alkali denaturation of the amplification product (to convert double-stranded DNA to single-stranded). .
  • one set (one sample) of the probe-immobilized probe is provided for the probe for detecting the wild-type (YMDD), the probe for detecting the mutant (YVDD), and the probe for detecting the mutant (YIDD). did.
  • Each well has the composition shown below.
  • the hybridization buffer was dispensed at 100 ⁇ 1 each.
  • 25 1 of the amplification product subjected to alkali denaturation was added to each well into which the above hybridization buffer was dispensed, and mixed well.
  • the mixture was left at 55 ° C. for 30 minutes for hybridization.
  • a gene fragment amplified as a non-specific amplification product is present at the stage of the PCR method, a gene fragment other than the target amplification product is selectively removed.
  • the target amplification product of each type has a difference of about 1 to 2 bases at each end from each type of probe, so hybridization to all probes is somewhat it seems to do.
  • washing was carried out five times at a rate of 350/1 per well using a washing solution having the following composition.
  • a solution 1001 containing the addition of a biotin dATP to which the base A labeled biotin was bound and Taq DNA polymerase as shown below was added to each well, and reacted at 55 for 30 minutes. .
  • peroxidase-labeled streptavidin was added to 20 mM Tris-HCl buffer (pH 7.5) to a concentration of 0.2 U / ml, and 100 1 was added to each well, followed by reaction at 55 ⁇ for 30 minutes. .
  • peroxidase is labeled on the immobilized probe into which the biotin-labeled substrate has been incorporated in the above-mentioned reaction.
  • washing was performed using the above-described washing solution and washing conditions.
  • the results of the mutation detection in this example indicate that the first sample in which methionine type (ATG) was detected in all samples Color development was observed only in Dwell, and it was determined that only wild type was included.
  • the measured absorbance is as shown below.
  • the cutoff value of the absorbance value was 0.1 for the first well, 0.3 for the second well, and 0.3 for the third well. "10" in parentheses indicates positive, and "one" indicates negative.
  • the measured absorbance is as shown in the table below.
  • the cutoff value of the absorbance value was 0.1 for the first level, 0.3 for the second level, and 0.3 for the third level. "10" in parentheses indicates positive, and "one” indicates negative.
  • a specific primer and a gene probe by using a specific primer and a gene probe, a specific primer, a specific gene probe immobilized on a solid support, and an alkali denaturing solution prepared by component preparation and optimized
  • a kit containing a hybridization buffer, a DNA polymerase, and a minisequence buffer containing a labeling substrate point mutation of the gene encoding the YMDD motif on the HBV gene can be easily and easily performed. Can be determined accurately and in a short time. This is useful for judging (monitoring) the effect of medication at the time of administration, especially for long-term continuous administration of lamivudine, and for formulating a dosage plan.

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Abstract

L'invention concerne un procédé permettant de détecter une mutation dans un gène codant pour le centre actif de transcriptase inverse du virus de l'hépatite B, et en particulier, une mutation sur le site YMDD. L'invention concerne également un ensemble amorce à utiliser dans ce procédé et un kit comprenant cet ensemble amorce et un ensemble sonde. Ce procédé consiste à amplifier, au moyen du procédé PCR, une partie de la séquence génique codant pour le centre actif de transcriptase inverse du virus de l'hépatite B subissant apparemment une mutation, au moyen d'un ensemble amorce SEQ ID n° 1 et 2, puis à hybrider le produit d'amplification avec chaque sonde d'un mélange de sondes ou d'un ensemble de sondes contenant la séquence SEQ ID n°3 ou 23, à incorporer un substrat étiqueté, au moyen du procédé de mini-séquence, et à détecter le substrat étiqueté pour pouvoir ainsi détecter la survenue de la mutation dans le centre actif de transcriptase inverse du virus de l'hépatite B.
PCT/JP2000/001849 1999-03-26 2000-03-27 Procede de detection d'une mutation dans le virus de l'hepatite b et kit de detection WO2000058477A1 (fr)

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JP11083930A JP2000270876A (ja) 1999-03-26 1999-03-26 Ｂ型肝炎ウイルス遺伝子の変異検出方法および検出キット
JP11/83930 1999-03-26

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003083094A1 (fr) * 2002-03-29 2003-10-09 Innogenetics N.V. Methodes de detection de resistance medicamenteuse au hbv
USRE40233E1 (en) 1996-11-08 2008-04-08 Melbourne Health Viral variants and methods for detecting same
US7422848B2 (en) 2005-03-15 2008-09-09 Innogenetics N.V. Hepatitis-B viral variants with reduced susceptibility to nucleoside analogs and uses thereof
US7807437B2 (en) 2005-03-15 2010-10-05 Rheinishche Friedrich-Wilhelms-Universitaet Bonn Variants of hepatitis B virus resistant against some nucleoside analogues, but sensitive to others, and uses thereof

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2002249613A1 (en) * 2002-04-18 2003-10-27 Hokkaido Technology Licensing Office Co., Ltd. Method of selecting primer for dna synthesis
JP2008283977A (ja) * 2008-06-09 2008-11-27 Toshiba Corp 標的核酸の遺伝子型、および変異の判定方法、不溶性支持体に核酸断片を固定化する方法、所望の核酸を精製し、かつ増幅するための方法、並びに遺伝子型アッセイキット

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6322098A (ja) * 1986-06-18 1988-01-29 Handai Biseibutsubiyou Kenkyukai Ｂ型肝炎ウイルス抗原とその製造法
JPH0343100A (ja) * 1989-07-11 1991-02-25 Takara Shuzo Co Ltd ヒトｂ型肝炎ウイルスの検出方法
JPH1143100A (ja) * 1996-12-17 1999-02-16 Space Syst Loral Inc 衛星姿勢制御装置

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6322098A (ja) * 1986-06-18 1988-01-29 Handai Biseibutsubiyou Kenkyukai Ｂ型肝炎ウイルス抗原とその製造法
US5693497A (en) * 1986-06-18 1997-12-02 The Research Foundation For Microbial Diseases Of Osaka University Process for expressing the hepatitis B virus antigen using a Saccharomyces cerevisiae transformant
JPH0343100A (ja) * 1989-07-11 1991-02-25 Takara Shuzo Co Ltd ヒトｂ型肝炎ウイルスの検出方法
JPH1143100A (ja) * 1996-12-17 1999-02-16 Space Syst Loral Inc 衛星姿勢制御装置

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KARIN KIDD-LJUNGGREN ET. AL.: "The hepatitis B virus X gene: analysis of functional domain variation and gene phylogeny using multiple sequences", JOURNAL OF GENERAL VIROLOGY, vol. 76, 1995, pages 2119 - 2130, XP002929043 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
USRE40233E1 (en) 1996-11-08 2008-04-08 Melbourne Health Viral variants and methods for detecting same
WO2003083094A1 (fr) * 2002-03-29 2003-10-09 Innogenetics N.V. Methodes de detection de resistance medicamenteuse au hbv
US8278432B2 (en) 2002-03-29 2012-10-02 Innogenetics N.V. HBV drug resistance methods
US8686126B2 (en) 2002-03-29 2014-04-01 Innogenetics N.V. HBV drug resistance methods
US7422848B2 (en) 2005-03-15 2008-09-09 Innogenetics N.V. Hepatitis-B viral variants with reduced susceptibility to nucleoside analogs and uses thereof
US7807437B2 (en) 2005-03-15 2010-10-05 Rheinishche Friedrich-Wilhelms-Universitaet Bonn Variants of hepatitis B virus resistant against some nucleoside analogues, but sensitive to others, and uses thereof

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