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EP1017856A1 - Procede et ensemble de materiel d'evaluation de mutations vih - Google Patents

Procede et ensemble de materiel d'evaluation de mutations vih

Info

Publication number
EP1017856A1
EP1017856A1 EP98944941A EP98944941A EP1017856A1 EP 1017856 A1 EP1017856 A1 EP 1017856A1 EP 98944941 A EP98944941 A EP 98944941A EP 98944941 A EP98944941 A EP 98944941A EP 1017856 A1 EP1017856 A1 EP 1017856A1
Authority
EP
European Patent Office
Prior art keywords
seq
primer
sequencing
hiv
pair
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98944941A
Other languages
German (de)
English (en)
Inventor
James M. Dunn
Jean-Michel Lacroix
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayer Healthcare LLC
Original Assignee
Visible Genetics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/938,641 external-priority patent/US6007983A/en
Application filed by Visible Genetics Inc filed Critical Visible Genetics Inc
Publication of EP1017856A1 publication Critical patent/EP1017856A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • 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
    • C12Q1/6869—Methods for sequencing
    • 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
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS

Definitions

  • SSCP 'single-stranded conformational polymorphism
  • ddF dideoxy-fingerprinting
  • WO 97/23650 discloses the evaluation of the allelic type of a polymorphic genetic locus by evaluation of less than all four of the bases of a nucleotide sequence.
  • the invention of that application is exemplified with respect to determination of allelic type for HLA and typing o ⁇ Chlamydial strains. There is no specific disclosure of probes for use in evaluation of HIV types.
  • This application relates to a particular series of tests which can be useful alone or as part of a hierarchical testing protocol for the detection and characterization of human immunodeficiency virus (HIV).
  • HAV human immunodeficiency virus
  • the method of the invention provides a streamlined, hierarchical method for obtaining information about the allelic type of a sample of genetic material derived from an HIV-infected sample. It has been determined that 93 to 95% of the known mutational variants of the protease and reverse transcriptase genes of HIV can be determined by evaluating the positions of the A and T nucleotides within the sample. Thus, a substantial fraction of all mutational variations can be unequivocally identified by performing two initial sequencing reactions on the sample in which only ddA and ddT are used as chain terminators.
  • a second test is performed in which the sequence is determined in the 3'-direction for all four bases. This test will identify substantially all of the remaining samples. For those for which an ambiguity remains, however, a final test in which the sequence of the sample is determined in both the 3' and 5 -direction for all four bases is performed.
  • reagents suitable for performing the three tests within the hierarchy are suitably packaged as a kit containing two or more sub- kits.
  • the first sub-kit contains reagents for performing A and T sequencing.
  • the addition sub-kit(s) contains reagents for performing a four-base sequence determination on one or both strands of the target DNA.
  • Fig. 1 shows a schematic representation of the invention
  • Figs. 2A and 2B shows the bases which are changed in known mutations of the protease and reverse transcriptase genes of HIV- 1.
  • Allele refers to a specific version of a nucleotide sequence at a polymorphic genetic locus.
  • Polymorphic site means a given nucleotide location in a genetic locus which is variable within a population.
  • Gene or “Genetic locus” means a specific nucleotide sequence within a given genome.
  • location or “position” of a nucleotide in a genetic locus means the number assigned to the nucleotide in the gene, generally taken from the cDNA sequence or the genomic sequence of the gene.
  • the nucleotides Adenine. Cytosine. Guanine and Thymine are sometimes represented by their designations of A, C, G or T, respectively. Dideoxynucleotides which are used as chain terminators are abbreviated as ddA. ddC. ddG and ddT.
  • each of the four sequencing reactions generates a plurality of primer extension products, all of which end with a specific type of dideoxynucleotide.
  • Each lane on the electrophoresis gel thus reflects the positions of one type of base in the extension product, but does not reveal the order and type of nucleotides intervening between the bases of this specific type.
  • the information provided by the four lanes is therefore combined in known sequencing procedures to arrive at a composite picture of the sequence as a whole.
  • the sequence of a good portion of the diagnostically relevant protease and reverse transcriptase genes is obtained in three steps: 1) cDNA is generated from the RNA present in the sample, and amplified, preferably across a region extending from 6 codons before the protease up to codon 335 of the reverse transcriptase of HIV- 1 (the primer regions are not included in this range). 2) Sequencing reactions are performed at one or more of several hierarchical levels . 3) Finally, the sequencing ladders are analyzed, preferably using the OpenGeneTM System: the Micro GeneBlasterTM DNA Sequencer, GeneObjectsTM and GeneLibrarianTM Softwares.
  • Fig. 1 shows one embodiment of the method of the invention schematically. As shown, an RNA sample is obtained and treated by reverse transcriptase-PCR (RT-PCR).
  • RT-PCR reverse transcriptase-PCR
  • This amplicon is then combined with a master sequencing mixture containing buffer (260 mM Tris-HCL, pH 8.3; 32.5 mM MgCl 2 ) and a polymerase enzyme such as
  • Taq FS Perkin Elmer/Applied Biosystems Cat No. 402070
  • This polymerase has a high rate of incorporartion of dideoxynucleotide relateive to the incorporation rate of, for example, conventional Taq polymerase.
  • This mixture is used as stock in the subsequent reactions.
  • the first sequencing reaction performed in the method of the invention is a single-base sequencing reaction performed using either ddA or ddT in the sequencing mixture. This reaction is performed on the protease gene using the following primers: forward primer:
  • reverse primers which may be used are:
  • both the forward and reverse primers are preferably each labeled with one of the two dyes.
  • Forward and reverse sequencing fragments are then generated by thermally cycling the sample through multiple thermal cycles in the presence of either ddA or ddT. Analysis of the sequencing fragments produced using gel electrophoresis will allow the determination of the positions of both A and T bases. As shown in Figs. 2A and B, knowledge of the position of the A and T bases will identify 95% of all known mutational variants within the reverse transcriptase gene and 93% of the variants within the protease gene.
  • the allelic type of majority of samples can be identified. If the sequencer employed is only capable of evaluating a single base, then two reaction need to be employed. These may be a forward and backwards sequencing reaction both employing the same chain terminator (ddA or ddT), or two reaction performed in the same direction, one with ddA and one with ddT so that the positions of A and T bases are determined. These sequencing reactions can be employed using the same primers discussed above.
  • a further sequencing reaction is performed on the sample stock to determine the positions of all four bases.
  • this is a sequencing reaction of intermediate complexity, involving the sequencing of one of the two strands of the
  • DNA or a combination of the two strands making up one complete linear sequence This can be done using the same primers identified above to obtain sequencing fragments.
  • sequencing of both strands may be performed. Again, the same sequencing primers identified above are used. Forward and reverse sequencing fragments can be produced in a single reaction using distinctively labeled forward and reverse primers, or in separate reactions depending on the nature of the detection system being employed.
  • kits suitable for practicing the method of the invention are suitably packaged in kit form.
  • the invention provides a kit for analyzing the genetic type of an HIV-1 gene in a sample using a hierarchical assay comprising, in separately packed combinations:
  • a first subkit for performing A and T sequencing on HIV- 1 comprising a plurality of A or T terminations mixtures, or both A and T termination mixtures, but no G termination mixture or C termination mixture, each of said A and T termination mixtures including one of a plurality of primer pairs, each pair flanking a different region of the HIV-1 genome, the pairs together flanking substantially all of the protease and reverse transcriptase genes, and at least one member of each pair being labeled with a detectable label;
  • a second subkit for performing four base sequencing on HIV-1 comprising a plurality of A, C, G and T terminations mixtures, each of said termination mixtures including one of a plurality of primer pairs, each pair flanking a different region of the HIV-1 genome, the pairs together flanking substantially all of the protease and reverse transcriptase genes, and at least one member of each pair being labeled with a detectable label. Additional subkits for performing four base sequencing may be included when intermediate and final assays on one strand and both strands are desired.
  • termination mixture refers to a mixture containing a mixture of the four deoxunucleotide triphosphates (dATP, dCTP, dGTP, and dTTP), one species of chain terminating dideoxynucleotide (ddATP. ddCTP, ddGTP or ddTTP) and the appropriate sequencing primers.
  • the subkit for performing A and T sequencing on HIV-1 may also be provided separately for performing the initial determination of only the A and T nucleotides.
  • a preferred kit of this type whether provided separately or as part of a kit for performing a hierarchical assay has primer pairs in which each primer is labeled with a different an spectroscopically distinguishable fluorescent dye, such as Cy5.0 and Cy5.5 and includes only one of the two possible types of termination mixtures, for example just the T termination mixture.
  • Example 1 The variety or sub-type of HIV can be determined by Single Track Sequencing of a sample which has been amplifed by RT-PCR.
  • a reaction mixture is prepared as follows: 3 ul bound beads
  • the sequencing primer employed is the non-biotinylated primer of the sequencing template amplification reaction, but this time it is labeled with a detectable label.
  • the preferred label for detection on the MicroGene Blaster is Cy5.5 linked to the 5' nucleotide of the primer.
  • a single chain termination reaction mixture in this case for the T nucleotide, is prepared by combining 750 uM of each of dATP, dCTP, dGTP and dTTP; and 2.5 uM of ddTTP. 3 ul of the termination reaction mix is place in a tube. 3 ul of the sequencing reaction mixture is added. An oil overlay is added and the single track reaction mixture is heated to 95 C for 2 mins in a PTC- 100 Programmable Thermal Controller (MJ Research, Inc.) or Robocycler Gradient 96 (Stratagene) before being thermally processed for 25 cycles (or fewer if found to be satisfactory) as follows:
  • the sample After a final extension at 70 °C for 5 min the sample is denatured at 95 °C for 30 sees and left on ice.
  • the sample is mixed with 6 ul of STOP/Loading buffer containing 100%) formamide and 5 mg/ml dye such as dextran blue. 1.5 ul of the mixture is loaded on a single lane of a MICROGENE BLASTER
  • reaction products are separated by electrophoresis through a denaturing polyacrylamide gel.
  • the reaction products are detected and presented with GENEOBJECTS software (Visible Genetics Inc., Toronto).
  • GENEOBJECTS software Visible Genetics Inc., Toronto.
  • the finger-print or barcode of the reaction products is compared to all known varieties of the pathogen nucleic acid sequence. An exact match is sought. If only one match is found, that subtype or variety is positively identified. If the patient sample had mixed varieties the result may show a heterogenous mix. The members of the heterogenous mix and relative quantities may be determined.
  • EXAMPLE 2 The variety or sub-type of the pathogen can be determined using CLIPTM sequencing methodology. In this method the sequence of both the sense strand and antisense strand of the protease gene of HIV- 1 may be obtained in a one step reaction as follows.
  • 13X reaction buffer consists of Tris-HCL 260 mM pH 8.3, MgCl 2 39 mM.
  • PR526 CCATTCCTGG CTTTAATTTT ACTGG Seq ID No. 22
  • results from each primer were compared to the known protease gene sequences of HIV- 1 and -2 by GENELIBRARIAN (a component of GENEOBJECTS (Visible Genetics Inc., Toronto).
  • the sub-type of HIV- 1 or HIV-2 is determined, and the presence of drug resistance codons is determined. Once the sequence of the HIV sub-type(s) is determined, it is reported to the patient file along with the quantitation data.
  • EXAMPLE 3 The RT-PCR is done on the HIV-1 RNA using the TitanTM One Tube RT-PCR System from Boehringer Mannheim. This RT-PCR is done on the RNA preparation obtained using the AmplicorTM HIV Monitor Test from Roche Diagnostic. It can also be done on the RNA extract for the NucliSenseTM (formerly known as NASBA) HIV Viral
  • RNA sample from the Amplicor HIV Monitor Test and keep on ice. This is the material obtained at step 14 of the section B "Specimen Preparation”. If using RNA prepared for the NucliSense Assay, proceed the same way: thaw it and keep it on ice.
  • RNA sample
  • EXAMPLE 4 To determine the sequence of amplicon, 7 ⁇ l of each terminator mix (32 mixes when using a single dye instrument, 4 when using a two dye instrument) are combined with a 5 ul of a master mix as follows: MASTER MIX (single dye system))
  • MASTER MIX two-dye system 18.5 ⁇ l of buffer 72.5 ⁇ l of sterile water
  • thermocylcing reaction The following is the programming for the PTC-200:
  • Termination mix for the protease - one dye system 1.07 ⁇ M ddATP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP;
  • C-Mix 2.14 ⁇ M ddCTP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP;
  • G-Mix 2.14 ⁇ M ddGTP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP; 330 nM each of forward and reverse primers
  • T-Mix 2.14 ⁇ M ddTTP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP;
  • Termination mix for the first region of reverse transcriptase - (one dye system)
  • A-Mix 1.07 ⁇ M ddATP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP;
  • C-Mix 2.14 ⁇ M ddCTP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP;
  • each of forward and reverse primers G-Mix 2.14 ⁇ M ddGTP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP;
  • each of forward and reverse primers T-Mix 2.14 ⁇ M ddTTP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP; 330 nM each of forward and reverse primers One primer of eachpair is labeled.
  • A-Mix 1.07 ⁇ M ddATP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP;
  • C-Mix 2.14 ⁇ M ddCTP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP;
  • each of forward and reverse primers G-Mix 2.14 ⁇ M ddGTP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP;
  • T-Mix 2.14 ⁇ M ddTTP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP;
  • One primer is each pair is labeled.
  • A-Mix 1.07 ⁇ M ddATP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP;
  • C-Mix 2.14 ⁇ M ddCTP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP; 330 nM each of forward and reverse primers
  • G-Mix 2.14 ⁇ M ddGTP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP;
  • T-Mix 2.14 ⁇ M ddTTP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP;
  • A-Mix 1.07 ⁇ M ddATP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP; 330 nM each of forward and reverse primers
  • C-Mix 2.14 ⁇ M ddCTP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP; 330 nM each of forward and reverse primers
  • G-Mix 2.14 ⁇ M ddGTP; 643 ⁇ M dATP; 643 ⁇ M dCTP: 643 ⁇ M dGTP; 643 ⁇ M dTTP; 330 nM each of forward and reverse primers
  • T-Mix 2.14 ⁇ M ddTTP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP; 330 nM each of forward and reverse primers
  • Both primers are labeled, for example with Cy5.0 and Cy5.5, respectively.
  • A-Mix 1.07 ⁇ M ddATP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP; 330 nM each of forward and reverse primers
  • C-Mix 2.14 ⁇ M ddCTP; 643 ⁇ M dATP; 643 ⁇ M dCTP: 643 ⁇ M dGTP; 643 ⁇ M dTTP;
  • G-Mix 2.14 ⁇ M ddGTP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP;
  • each of forward and reverse primers T-Mix 2.14 ⁇ M ddTTP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP;
  • Both primers are labeled, for example with Cy5.0 and Cy5.5, respectively.
  • Second reverse transcriptase region A-Mix 1.07 ⁇ M ddATP; 643 ⁇ M dATP; 643 ⁇ M dCTP: 643 ⁇ M dGTP; 643 ⁇ M dTTP;
  • C-Mix 2.14 ⁇ M ddCTP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP;
  • G-Mix 2.14 ⁇ M ddGTP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP; 330 nM each of forward and reverse primers
  • T-Mix 2.14 ⁇ M ddTTP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP;
  • Both primers are labeled, for example with Cy5.0 and Cy5.5, respectively.
  • A-Mix 1.07 ⁇ M ddATP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP; 330 nM each of forward and reverse primers
  • C-Mix 2.14 ⁇ M ddCTP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP;
  • G-Mix 2.14 ⁇ M ddGTP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP;
  • T-Mix 2.14 ⁇ M ddTTP; 643 ⁇ M dATP; 643 ⁇ M dCTP; 643 ⁇ M dGTP; 643 ⁇ M dTTP;
  • Both primers are labeled, for example with Cy5.0 and Cy5.5, respectively.

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Abstract

L'invention concerne un procédé rationalisé et hiérarchique permettant d'obtenir des informations sur le type allélique d'un échantillon de matériel génétique dérivé d'un échantillon infecté par VIH. On peut déterminer entre 93 et 95 % des variants mutationnels connus des gènes de transcriptase inverse et de protéase de VIH-1 en évaluant les positions des nucléotides A et T à l'intérieur de l'échantillon. Ainsi, on peut identifier de manière univoque une portion considérable de toutes les variations mutationnelles en effectuant deux réactions de séquençage initiales sur l'échantillon dans lequel seuls les ddA et ddT sont utilisés comme agents de terminaison de chaîne. Pour la petite proportion d'échantillons qui ne sont pas identifiables en fonction des positions de ces deux bases, on effectue une seconde épreuve au cours de laquelle on détermine la séquence dans la direction 3' pour les quatre bases. Cette épreuve permet d'identifier sensiblement tous les échantillons restants. Pour ceux pour lesquels une ambiguïté subsiste, on effectue une épreuve finale dans laquelle la séquence de l'échantillon est déterminée dans les directions 3' et 5' pour les quatre bases. Pour mettre en oeuvre ce procédé, les réactifs convenant à la réalisation de ces trois épreuves dans l'ordre hiérarchique sont convenablement emballés sous forme d'ensemble de matériel contenant deux ou plusieurs sous-ensembles. Le premier sous-ensemble renferme des réactifs destinés à effectuer le séquençage A et T. Les sous-ensembles supplémentaires renferment des réactifs destinés à effectuer une détermination d'une séquence à quatre bases sur un ou plusieurs brins de l'ADN cible.
EP98944941A 1997-09-26 1998-09-28 Procede et ensemble de materiel d'evaluation de mutations vih Withdrawn EP1017856A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US938641 1986-12-05
US08/938,641 US6007983A (en) 1995-12-22 1997-09-26 Method and kit for evaluation of HIV mutations
PCT/CA1998/000913 WO1999016910A1 (fr) 1997-09-26 1998-09-28 Procede et ensemble de materiel d'evaluation de mutations vih

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EP1017856A1 true EP1017856A1 (fr) 2000-07-12

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EP (1) EP1017856A1 (fr)
JP (1) JP2001518313A (fr)
AU (1) AU751471B2 (fr)
CA (1) CA2302201A1 (fr)
WO (1) WO1999016910A1 (fr)

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US6265152B1 (en) * 1995-12-22 2001-07-24 Visible Genetics Inc. Method and kit for evaluation of HIV mutations
JP4824236B2 (ja) 1999-07-09 2011-11-30 ジェン−プローブ・インコーポレーテッド 核酸増幅によるｈｉｖ−１の検出
GB2395008B (en) * 1999-10-15 2004-07-21 Visible Genetics Inc Method and kit for evaluation of HIV mutations
GB2395787A (en) * 1999-10-15 2004-06-02 Visible Genetics Inc Method and kit for evaluation of HIV mutations
US6582920B2 (en) 2000-09-01 2003-06-24 Gen-Probe Incorporated Amplification of HIV-1 RT sequences for detection of sequences associated with drug-resistance mutations
AU7104200A (en) * 2000-09-01 2002-03-22 Gen Probe Inc Amplification of hiv-1 sequences for detection of sequences associated with drug-resistance mutations
US9085807B2 (en) 2004-09-14 2015-07-21 Argos Therapeutics, Inc. Strain-independent amplification of pathogens and vaccines thereto

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AU9249398A (en) 1999-04-23
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AU751471B2 (en) 2002-08-15
WO1999016910A1 (fr) 1999-04-08

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