+

WO1994026867A1 - Tampon de lyse directe et detection de la presence de vih-1 dans le plasma - Google Patents

Tampon de lyse directe et detection de la presence de vih-1 dans le plasma Download PDF

Info

Publication number
WO1994026867A1
WO1994026867A1 PCT/US1994/004676 US9404676W WO9426867A1 WO 1994026867 A1 WO1994026867 A1 WO 1994026867A1 US 9404676 W US9404676 W US 9404676W WO 9426867 A1 WO9426867 A1 WO 9426867A1
Authority
WO
WIPO (PCT)
Prior art keywords
hiv
pcr
plasma
rna
assay
Prior art date
Application number
PCT/US1994/004676
Other languages
English (en)
Inventor
Denis R. Henrard
Jack Phillips
Original Assignee
Abbott Laboratories
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
Application filed by Abbott Laboratories filed Critical Abbott Laboratories
Priority to EP94915927A priority Critical patent/EP0698082A4/fr
Priority to JP6525476A priority patent/JPH08510004A/ja
Priority to AU67769/94A priority patent/AU6776994A/en
Publication of WO1994026867A1 publication Critical patent/WO1994026867A1/fr

Links

Classifications

    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present invention involves a process to disrupt virions and isolate the nucleic acid of the virus.
  • the invention presents a direct lysis buffer involving a combination of detergent and Proteinase K to isolate nucleic acids.
  • HIV-1 human immunodeficiency virus type 1
  • Robert Gallo and members of his laboratory at the National Institutes of Health, reported having isolated the causative agent of AIDS.
  • diagnostic assays were developed and used to identify persons infected with HIV-1. These assays, which were developed to be both highly sensitive and specific, detected the presence of antibodies to HIV-1 in serum or plasma.
  • HIV-1 virus was mostly detected in specific populations, such as homosexual men and hemophiliacs, and that the main mode of transmission was through sexual contact or receipt of infected blood products. It was later found that IV drug users were a group at high risk of transmitting HIV because of the practice of sharing used needles (i.e., cross contamination of blood).
  • Retroviruses are characterized as having RNA as their genetic material and contain the unique enzyme reverse transcriptase (RT), which catalyzes the reverse transcription of the RNA genome into a DNA copy (cDNA).
  • RT reverse transcriptase
  • retroviral genomes are composed of between 9,000 and 10,000 base pairs and contain three structural genes that are characteristic to all retroviruses (gag, pol, and env). They contain unique sequences located at the 3' terminus of pol and env that code for regulatory proteins. Located at both the 5' and 3' ends of the genome are two identical sequences called long terminal repeats (LTR). The 5' LTR is critical for the expression of proviral DNA by the host's cellular transcription machinery .
  • LTR long terminal repeats
  • the HIV-1 RNA genome is composed of a total of 9,749 nucleotides, representing 9 genes (Haseltine, W.A., Wong-Stall, F. The Molecular Biology Of The AIDS Virus. Scientific American 1988;259:52-62).
  • the genome contains the three characteristic structural genes and an additional six regulatory genes (tat, rev, vif, vpr, nef, and vpu).
  • the gag and pol proteins are translated from full length transcript, while the env protein is translated from a spliced transcript.
  • the gag gene is transcribed to give a full length RNA and translated to give a precursor polyprotein that is subsequently cleaved into three capsid proteins, which make up the major structural proteins of the virus core.
  • the pol protein is actually part of a gag-pol precursor.
  • the pol portion of the gene encodes the enzymes associated with the RNA inside the core of the virus, the protease, reverse transcriptase and integrase.
  • the reverse transcriptase actually has three enzymatic functions, RNA dependent DNA polymerase, DNA dependent DNA polymerase and ribonuclease activity.
  • the envelope gene (env) encodes a precursor protein, gp160, that is cleaved by a protease to make the extracellular glycoprotein gp120 and the transmembrane protein gp41.
  • the gp120 protein is responsible for binding the virus to the cell surface CD4 receptor.
  • the gp41 protein mediates syncytia formation and also assists in the penetration of the virus core into the interior of the cell (Sodrowski, J., Goh, W.C., Resen, S., Campbell, K., and Haseltine, W.A. Role Of The HTLV-III/LAV Envelope In Syncytium Formation And Cytopathicity. Nature 1986;322:470-474; and McCune, J.M., Rabin, L.B., Feinburg, M.B., Lieberman, M., Kosek, J.C., Reyes, G.R., and Weissman, I.L.
  • HIV-1 resembles that of all retroviruses. It contains a cylindrical core which is made up of two gag proteins. Inside the core are two identical single stranded RNA molecules. Associated with the RNA genome are the enzymes reverse transcriptase, protease and integrase. The core is surrounded by an envelope derived from the host cell's plasma membrane. The surface of the membrane is studded with copies of the HIV-1 specific protein, gp120, which are noncovalently associated with the gp41 transmembrane protein.
  • the infectious cycle of HIV begins when viral envelope proteins bind to the CD4+ molecule that is found on the host cell surface.
  • the CD4+ molecule is typically found on T lymphocytes and macrophage/monocytes.
  • the membranes of the virus and host cell fuse, and the core of the virus is injected into the host cell.
  • the viral RNA genome is reverse transcribed into a cDNA copy.
  • the RNA genome is then destroyed by the RT-associated enzyme Ribonuclease H, and the polymerase makes a second DNA copy using the cDNA copy as a template.
  • This double stranded viral DNA migrates into the nucleus where it is integrated into the host cell's DNA by way of the viral protein integrase. Once integrated, the viral DNA is termed a provirus.
  • RNA molecules are used as genetic material while others are used as mRNAs to be translated into new viral proteins.
  • the env proteins are postranslationally processed in the cell's Golgi apparatus and are transported into the host's cell membrane. Proteins that will be used for the core structure of the virus contain a fatty acid and these attach to the inside of the cell membrane. As all the components for the new virus accumulate, they bind to one another and form a spherical structure that bulges outward from the cell membrane. Two RNA molecules are placed into the developing virus particle. Lastly, the core associated enzymes (RT, integrase and protease) are postranslationally processed and the protease cleaves the core precursor proteins. The viral core proteins surround the viral RNA genome, the nearly completed virus encloses itself with a portion of the host cell membrane, and eventually the virus buds from the cell and is released.
  • RT integrase and protease
  • HIV-1 disease progression Infection by HIV, or other lentiviruses, is persistent and is usually characterized by a continuous, although relatively low level of virus production. A progressive increase in productive viral replication occurs and probably contributes to disease progression. This has led investigators to look for biological markers that may be associated with HIV-1 disease progression.
  • Beta2 microglobulin is part of the histocompatability complex (HLA) and is released from a T cell during immune activation and cell turnover. Normal levels measured in healthy individuals are less than 1.9 ⁇ g/ ⁇ (Hofmann, B., Wang, Y., Cumberland, W.G., Detels, R., Bozorgioloi, M., and Fahey, J.L. Serum Beta2- microglobulin Level Increases In HIV Infection: Relation To Seroconversion, CD4 T-cell Fall And Prognosis.
  • HLA histocompatability complex
  • Neopterin is a product of macrophage activation when these cells are stimulated by gamma interferon and reflects immune activation. Normal levels measured in healthy individuals are 6.62 nmol/L or lower (Fuchs, D., Hausen, A., Reibnegger, G., Werner, E.R., Dierich, M.P., and Wachter, H. Neopterin As A Marker For Activated Cell-Mediated Immunity: Application In HIV Infection. Immuno. Today 1988: 9:150-154). An increase above the normal levels of each marker reflects both lymphocyte and macrophage activation.
  • Positivity for HIV-1 p24 reflects increased viral activity and production, and has been shown to be associated with poor prognosis (Allain, J.P., Laurian, Y., Paul, D.A., Verroust, F., Leuther, M., Gazengel, C., Senn D., Larrieu, M.J., and Bosser, C. Long-Term Evaluation Of HIV Antigen And Antibodies To p24 And gp41 In Patients With Hemophilia. - En ⁇ l. J. Med. 1987;317:1 1 14-1 121 ) .
  • CD4+ lymphocytes As expressed as absolute numbers, was found to be the best predictor of HIV-1 progression. This was followed by the levels of neopterin or beta microglobulin, and finally the p24 antigen (Fahey, J.L., Taylor,
  • PCR Polymerase Chain Reaction
  • Plasma HIV-1 viremia was monitored by immunocapture-cDNA/PCR in which the reverse transcription and amplification steps were carried out in a single tube.
  • the direct lysis buffer of the present invention was formulated to isolate plasma HIV-1 RNA for direct use in the RT and PCR reactions without inhibiting enzymatic reactions, thus eliminating the need for organic solvent extraction and ethanol precipitation normally required to isolate nucleic acids. This resulted in a significant saving of time needed to complete the assay (saving approximately 16 hours) and may have decreased the likelihood of contamination due to decreased handling steps.
  • a viral capture assay involving latex microparticles (0.1 ⁇ m) coated with monoclonal antibodies directed to the gp41 and gp120 envelope proteins of HIV-1 was used to capture cell free virions from serum/plasma.
  • the standard parameters of the assay require that the plasma sample be incubated in the presence of the microparticles for three hours. In this study, the time was varied in order to determine whether the incubation time can be reduced without decreasing the sensitivity of the assay.
  • the conventional method of extraction and purification of HIV-1 RNA from viral proteins requires several hours, excluding a final overnight ethanol precipitation. The method also requires multiple tube changes, which makes it relatively prone to contamination.
  • the present invention involves a series of buffers and conditions for the direct lysis of HIV-1 virions bound to the particles.
  • Compatibility of the direct lysis buffer components with the reverse transcription and PCR enzymes is a major concern and particular attention was devoted to the formulation of a buffer that met this requirement.
  • Lysis buffers included a single detergent at various concentrations. Ionic and non-ionic detergents were investigated as components of the direct lysis buffer. Also, the effectiveness of adding low concentrations of Proteinase K in combination with the various detergents was studied. Once the formulation of a direct lysis buffer was determined, the time and temperature conditions required for disruption of the viral membranes was determined.
  • the standard method used to reverse transcribe the HIV- 1 RNA into a cDNA copy and its amplification by PCR requires two separate procedures. Maximum sensitivity was obtained by optimizing the assay components used in the two procedures (i.e., concentration of buffer, salt, primers, dNTPs and enzymes). However, there could possibly be a significant gain in assay sensitivity and time by combining the two procedures. A series of experiments was done to determine whether the reverse transcription and amplification procedures could be combined into one procedure. Compatibility of the direct lysis buffer with the RT and PCR assay components was maintained. The assay sensitivity was maintained by optimizing the MgC and dNTP concentrations, and other components if necessary.
  • Figure 1 Assay Controls.
  • Figure 2 Effect of various detergent concentrations on sensitivity of detection. Each detergent concentration was evaluated using the RNA controls R-5 and R-6, respectively. The standard RT/PCR procedures were used in the evaluation. The RNA controls were processed using the standard extraction procedure. Autoradiography was done for three hours at -80°C.
  • Figure 3 Effect of direct lysis buffer on immunocapture controls. A). Organic solvent extraction. Direct lysis buffer containing B). 0.005% Triton X-100, C). 0.0045% Tween 20, D).
  • Figure 6 Effect Of MgCl2 concentration on the single step RT/PCR procedure at a dNTP concentration of 50 mM.
  • the controls were assayed using the standard RT/PCR procedure.
  • the MgC concentrations ranged from 1.5 to 2.0 mM.
  • Autoradiography was done for three hours at -80°C.
  • Figure 7 Effect of MgCl2 concentration on the single step RT/PCR procedure at a dNTP concentration of 100 ⁇ M.
  • the controls were assayed using the standard RT/PCR procedure.
  • the MgC-2 concentrations ranged from 1.5 to 2.0 mM.
  • HIV-1 Determining the factors that relate to disease transmission and progression is a major concern in HIV-1 research. Recently, several studies have focused on identifying factors that may correspond to the transmission of HIV-1. In this regard, the correlation of HIV-1 viral load with transmission may be important. Two of the types of HIV-1 transmission include that from mother to child (vertical), and from blood donor to recipient (horizontal). Although the number of infections through vertical transmission of HIV-1 from a mother to her child is relatively low in the U.S., it does account for approximately 2% of new infections per year. Similarly, the current number of transfusion-associated HIV-1 transmissions is extremely low. However, determining whether there is a correlation between HIV-1 viral load and transmission of HIV-1 in these two cases will increase the understanding of the pathogenicity of HIV- 1 .
  • Samples were collected from transmitting and non-transmitting pregnant women, determined retrospectively by the loss or persistence of H1V-1 antibodies after 15 months in the infant (provided by Case Western Reserve University and the University of Washington, Seattle). Duplicate viral capture and RT/PCR for each sample were performed on coded samples. A consensus semi-quantitative value (3+, 2+, 1+, negative) was assessed. The semi-quantitative plasma RNA viral load was compared to other possible virologic markers of transmission (p24 antigenemia, CD4+ count and beta2 microglobulin levels) to determine its clinical usefulness for the prediction of
  • Plasma samples from HIV-1 seropositive blood donors were obtained (Transfusion Safety Study Repository, San Francisco, CA), and HIV-1 plasma viral load was determined retrospectively. Samples were selected from a pool of 78 samples known to have infected the recipient and 12 that did not infect the recipient. A total of twenty two samples were tested, with an equal number of samples coming from those that did infect and those that did not infect the recipient. Duplicate viral capture and RT/PCR for each sample were performed. A consensus semi-quantitative value was assessed. The semi- quantitative plasma RNA viral load was compared to other virologic markers of transmission (p24 antigenemia, CD4+ count and beta2 microglobulin) to determine the association of HIV-1 viral load in transfusion-associated transmission of HIV-1.
  • RNA samples were prepared from the HIV-1 NIB chronically infected H9 cell line (Abbott Laboratories). Total cellular nucleic acids were extracted with guanidinium thiocyanate, and the RNA was purified by centrifugation through a cesium chloride (CsCI) cushion (Chirgwin, J. M., Prsybla, G., MacDonald, P.J., and Rutter, W. J. Isolation Of Total Cellular RNA. Biochem.
  • CsCI cesium chloride
  • RNA pellet was dissolved to 0.5 ⁇ g/ml with dd ⁇ O and serially diluted 10 fold into ddH 0 containing 20 ⁇ g/ml yeast tRNA (GIBCO-BRL, Gaithersburg, MD). Samples diluted 10 5 and 10 6 fold, referred to as R *5 and R-* 3 , were the two lowest dilutions that consistently gave positive results after reverse transcription and amplification. These samples were used as controls.
  • the HIV-1 LAV infected cell line 8E5 (Memorial Sloan Kettering Institute, New York), which contains one copy of proviral HIV-1 per cell, was used to prepare amplification controls.
  • the lysate was then extracted with an equal volume of phenol (pH 7.0), then with chloroform (adjusted to 0.3 M NaOAc) and was then precipitated with twice the volume of absolute ethanol at -20°C for 16 hours.
  • the DNA was centrifuged (Hill Scientific mv13) at 10,000 rpm for 10 minutes, the supernatant removed, and the pellet washed with ice cold 70% ethanol.
  • the DNA pellet was dissolved in 0.5 ml 10 mM TRIS, pH 8.0, 100 mM NaCI.
  • the purified DNA corresponding to approximately 10 6 HIV-1 provirus copies, was serially diluted 10 fold into dd ⁇ O containing 20 ⁇ g/ml salmon sperm DNA (Sigma, St. Louis, MO). Fifty microliters of a 10 "3 and 10 "4 dilution (corresponding to 100 and 10 HIV-1 proviral copies, respectively) were used as amplification controls.
  • Plastic disposable, single use beakers were used to prepare reagents.
  • Bottled distilled water, free of any RNase (Abbott Laboratory, Catalog #NDC 0074-7139-09) was used to prepare all reagents. All reagents were aliquoted into single use tubes and were stored at -20°C until used.
  • Sample handling, amplification, and detection were done in three separate laboratories. Sample handling was done in a laminar flow hood. Latex gloves were always worn and were changed numerous times during the assay. Each laboratory contained a separate set of pipetmen, and barrier pipet tips were used throughout the procedure. Amplification was done in an acrylic biosafety cabinet.
  • the primers SK38/SK39 (representing nucleotides 1551-1578 and 1638- 1665, respectively) and the probe SK19, representing nucleotides 1597-1635 of the HIV-1 (HIVSF2, Genebank K02007) gag region were synthesized at Abbott Laboratories using an Applied Biosciences, Inc. 380 Synthesizer (Foster City, CA) and HPLC purified with a Waters Photodioarray 990 (Milford, MA).
  • microparticles were washed two times with equal volumes of wash buffer (PBS containing 2% Tween 20) and were then resuspended to volume with overcoat buffer (150 mM TRIS, pH 8.0, 100 mM NaCI, 0.5% porkskin gelatin, 0.1% Tween 20, 9.5% sucrose and 0.02% NaN ⁇ ). After incubating in overcoat buffer for 16 hours at
  • microparticles were pelleted, and the supernatant was discarded.
  • the microparticles were resuspended to 50% of their original volume with storage buffer (65.5 mM TRIS, 84.5 mM TRIS HCI, pH 8.0, 100 mM NaCI, 0.4 M sucrose, 1% porcine skin gelatin, and 0.1% Tween 20), and the percentage of solid was determined by comparing the A500 of a diluted fraction to a standard curve of known solids.
  • the microparticles were adjusted to a predetermined percent solids with storage buffer and were stored at 2-8°C until used.
  • the SK19 oligonucleotide (5'-ATCCTGGGATTAAATAAAATAGAA GAATGTATAGCCCTAC) was labeled with 32 P ⁇ 4 at the 5' terminus by using T4 polynucleotide kinase.
  • the reaction consisted of 5.0 mM TRIS HCI, pH 8.0, 1.0 mM MgCI 2 , 5.0 mM NaCI, 1.0 ⁇ g SK19, 50 uCi gamma 32 P ATP (Amersham, 3000 Ci/mmol) and 10 Units of T4 kinase (New England BioLabs, Beverly, MA), in a total volume of 10 ⁇ l.
  • reaction was carried out for 30 minutes at 37°C, followed by inactivation of the T4 kinase for 5 minutes at 95°C.
  • the reaction mixture was electrophoresed through a 10% polyacrylamide gel (29.25 ml H2O, 2.25 ml 10x TBE [Sondergard-Anderson, J., Lauritzen, E., Lind, K., and Holm, A. Covalently Linked Peptides For Enzyme-Linked
  • the tube was rotated (Labquake Shaker, Berkley, CA) for 16 hours at room temperature. Three microliters of eluted probe, corresponding to approximately 6.0 ng, was used to determine the labeling efficiency.
  • the specific activity of the labeled SK19 probe was routinely between . 1x10 7 and 2x10 7 cpm/ ⁇ g.
  • the labeled probe was stored at -20°C until used.
  • Viral Capture Immunocapture of HIV-1 virions was carried out in a 1.5 milliliter Eppendorf tube containing phosphate buffered saline (150 ⁇ l; PBS, 137 mM NaCI, 2.68 mM KCI, 12 mM Na2HP0 , 1.76 mM KH2PO4), anti-HIV-1 antibody coated microparticles (50 ⁇ l) and plasma (50 ⁇ l). The mixture was incubated for three hours at room temperature on a rocking platform (20 rpm, Thermolyne VariMix), then centrifuged for 10 minutes at 5000 rpm, and the supernatant was either saved or discarded.
  • Genomic HIV-1 RNA was extracted by the addition of a Proteinase K SDS solution (200 ⁇ l; 10 M TRIS, pH 7.4, 0.25% SDS, 0.5 mg/ml Proteinase K, and 10 ⁇ g/ml yeast tRNA) and further incubation (one hour at 56°C).
  • the HIV-1 RNA was purified by extraction with an equal volume of phenol, followed by extraction with an equal volume of chloroform (adjusted to 0.3 Ivf NaAOc) and was precipitated with twice the volume of absolute ethanol at -20°C for 16 hours.
  • the RNA was pelleted by centrifugation (Hill Scientific mv13) for 10 minutes at 12,000 rpm, and the supernatant was discarded. The RNA pellet was washed with ice cold 70% ethanol, centrifuged as before, the supernatant discarded, and the RNA dissolved in 30 ⁇ l of dd ⁇ O).
  • Viral RNA was reverse transcribed into cDNA using the enzyme reverse transcriptase, from Avian Myeloblastosis Virus (AMV-RT, Gibco-BRL). Duplicate aliquots (15 ⁇ l) of isolated HIV-1 genomic RNA were placed into 0.5 ml centrifuge tubes containing 5.0 ⁇ l of RT mix. The final RT reaction contained 10 mM TRIS, pH 8.3, 2 mM MgCI 2 , 50 mM KCI, 20 mM DTT, 0.001% gelatin, 25 ⁇ M each dNTP (Pharmacia,
  • ATAATCCACCTATCCCAGTAGGAGAAAT, SK395'-TTTGGTCCTTGTCTTATGTCCAGAATGC The tubes were heated to 95°C for five minutes, centrifuged, and reverse transcriptase (2.0 U; Gibco-BRL) containing 8.0 U of RNasin (Promega, Madison, Wl) was added. The RT reaction was carried out for 30 minutes at 42°C. Thirty microliters of water and two drops of mineral oil (Sigma, St. Louis, MO) were then added, and the reverse transcriptase was heat inactivated at 95°C for five minutes. Polymerase Chain Reaction
  • Amplification of HIV-1 DNA proceeded by addition of 50 ⁇ l of PCR mix to each sample.
  • the final PCR reaction contained 10 mM TRIS, pH 8.3, 1.5 mM MgC.2, 50 mM
  • Amplified HIV-1 DNA was detected by hybridization of 5 ⁇ l of 32 P SK 19 probe (2.5 ⁇ l probe plus 2.5 ⁇ l R3 buffer [50 mM TRIS HCI, pH 8.0, 10 mM Mgbl 2 , 100 mM NaCI]) (1-2x10 7 cpm/ ⁇ g) to 15 ⁇ l of amplified material.
  • the amplified material and probe were mixed and heated for 10 minutes at 100°C (to separate the double stranded amplified fragments), centrifuged to return the condensation to the bottom of the tube, and allowed to anneal for 30 minutes at 56°C.
  • Five microliters of loading dye (0.25% bromophenyl blue, 40% sucrose) was added to each sample, and the entire volume was loaded onto a 10% polyacrylamide gel.
  • Eiectrophoresis was done at 150 V for 30 minutes, followed by 250 V for 1.5 hours.
  • the gel was placed onto a piece of Whatman blot paper, covered with plastic wrap, and autoradiographed for one and four hours at -80°C using an intensifying screen (DuPont Cronex). Development of the autoradiograph was done with a Kodak M35A X-OMAT Processor.
  • Immunocapture controls consisted of the two lowest positive serial dilutions of a tissue culture supernatant from an HIV-1 IIIB infected H9 cell line.
  • Reverse transcription controls consisted of the two lowest positive serial dilutions of a purified preparation of RNA extracted from HIV-1 IIIB infected H9 cell line.
  • Amplification controls consisted of purified DNA obtained from the HIV-1 LAV infected 8E5 cell line, which contains one copy of HIV-1 per cell.
  • the viral capture assay uses latex microparticles (0.1 ⁇ m) covalently coupled with monoclonal antibodies directed to the gp41 and gp120 envelope proteins of HIV-1 , which capture cell free virions from serum/plasma.
  • the original protocol for immunocapture included a three hour incubation, followed by Proteinase K/SDS digestion, a phenol-chloroform extraction, an ethanol precipitation to purify the viral RNA, reverse transcription, and amplification by the polymerase chain reaction. The total time required to complete this part of the assay was five to seven hours. Additionally, detection of the amplified material required liquid hybridization, gel electrophoresis, and autoradiography.
  • Direct Lysis Buffer Purification of viral RNA by phenol/chloroform extraction and ethanol precipitation is labor intensive and time consuming. However, this purification is conventionally performed in order to overcome the inhibitory effect of SDS on the polymerase chain reaction and to- remove excess Proteinase K which may interfere with the assay (Erlich, H.A. PCR Technolo gy . Principles and Applications fpr DNA Amplification. Stockton Press, 1989. New York, NY. pp. 17-22). To eliminate the use of organic solvents and the need for ethanol precipitation, a series of direct lysis buffers were examined. The objective was to formulate a buffer that would be compatible with the reverse transcription and amplification procedures, while yielding results comparable to the standard digestion/extraction procedure.
  • the most common reagents used to disrupt cellular and viral membranes include the ionic detergent sodium dodecyl sulfate (SDS), and the non-ionic detergents Triton X- 100 and Tween 20. Proteinase K was used in combination with these detergents in order to digest proteins which may be associated with nucleic acids.
  • the Triton X-100 and Tween 20 concentrations ranged from 0.1-0.5%. These two detergents, when used at concentrations of 0.5% or lower, do not interfere with the Taq polymerase during the amplification procedure.
  • SDS on the other hand, inhibits the polymerase chain reaction 99% and 90% when present in concentrations of 0.1% and 0.01%, respectively. SDS does not inhibit the Taq polymerase when used at concentrations of 0.001% or lower (Erlich, H.A., above).
  • the components used in the standard lysis of the viral membranes included 0.25% SDS and 500 ⁇ g/ml Proteinase K.
  • concentrations needed to lyse the viral membrane in a direct lysis buffer and also be compatible in the reverse transcription/amplification procedures may need to be significantly reduced.
  • lysis buffers containing low concentrations of Triton X-100, Tween 20 or SDS were evaluated.
  • a low concentration of Proteinase K was added to the lysis buffers containing low concentrations of detergent. In this case, the lysis buffer was heated at 95°C for 10 minutes to inactivate the
  • Proteinase K prior to initiating the RT and PCR procedures.
  • 10 mM TRIS, pH 7.0 was chosen and was included in all lysis buffers examined.
  • composition of the various direct lysis buffers which were examined are presented in Table 1.
  • the standard assay using the RNA and DNA controls was initially used to evaluate the lysis buffers at an incubation time and temperature of 56°C for one hour.
  • Buffer 2 TRIS (mM. Tween 20 .%.
  • the buffers containing 10 mM TRIS and various concentrations of Triton X-100 produced signals similar to those obtained with the standard procedure.
  • buffers containing various concentrations of Tween 20 produced signals similar to those obtained with the standard procedure.
  • the lysis buffers containing 0.1% and 0.01% SDS generated no signals. This confirmed the inhibitory effect that SDS has on the Taq polymerase.
  • lysis buffers containing SDS and 1 ⁇ g/ml of Proteinase K also showed a decrease in sensitivity as the SDS concentration increased above 0.001%.
  • Viral Capture Time The minimal time required for immunocapture of the plasma-associated virions by the anti-HIV-1 antibody-coated microparticles was determined. The effect of reducing the capture time from three hours to two hours and one hour at ambient temperature was evaluated. The results obtained after capture for one hour were equivalent to those obtained with the standard procedure (Figure 5).
  • Reverse transcription was carried out for 45 minutes at 42°C, followed by denaturation of the reverse transcriptase at 95°C for three minutes. Amplification was directly initiated using the standard parameters. Each sample was assayed in duplicate, and both RT and PCR procedures were done with a thermocycler. To achieve comparable sensitivity to that of the standard method, the single addition assay component concentrations had to be optimized. The TRIS, KCI, gelatin, and DTT concentrations were identical in both the standard RT and PCR procedures, and their concentrations were chosen for the single addition procedure. Therefore, they were not modified. The two most critical parameters affecting the sensitivity of the reverse transcription and amplification procedures are the MgCl2 and dNTP concentrations
  • the MgC*2 concentrations used to determine optimal sensitivity were 1.5, 1.75, and 2.0 mM.
  • the dNTP concentrations tested were 50, 100 and 200 ⁇ M.
  • the concentration of dNTP was raised to 100 ⁇ M, and the same concentrations of MgCl2 were evaluated. As shown in Figure 7, there were slight decreases in RT and amplification control signals as the MgC.2 concentration increased. However, there did not appear to be the same significant decrease in amplification control signal as in the previous set of experiments.
  • the single step RT/PCR procedure (involving 1.75 mM MgCl2 and 200 ⁇ M dNTP) produced control signals similar to the standard procedure.
  • the final amplification mixture for the single step RT/PCR procedure contained 10 mM TRIS, pH 8.3, 50 mM KCI, 5 mM DTT, 0.001% gelatin, 1.75 mM MgCI 2 , 200 ng primers, 200 ⁇ M each dNTP, 10 U RNasin, 2.5 U AMV RT, and
  • HIV-1 viral load in the plasma of blood donors may play an important role in transfusion-associated transmission of HIV-1.
  • HIV-1 viral load was measured in plasma samples obtained from the Transfusion Safety Study Repository (San Francisco, CA). Twenty two samples were selected from a pool of 78 infectious and 12 non-infectious samples. Of the 22 seropositive samples selected, there were 11 transfusion associated transmissions and 11 non-transmissions (Table 4). The CD4+ lymphocyte counts could not be determined for the original samples, but approximately one year after collection, the average CD4+ lymphocyte count for the transmitting and non-transmitting donors were 470 and 746 per ⁇ l, respectively.
  • HIV-1 infection is a major factor in controlling the spread of the virus.
  • antibody EIA and Western Blot tests are used to identify an HIV-1 infection.
  • interest has focused on monitoring the progression of HIV infection and the response to therapy in HIV infected individuals.
  • a number of methods primarily based on p24 antigen detection, culture and nucleic acid amplification, have been developed to monitor HIV-1 disease progression.
  • PBMC peripheral blood mononuclear cells
  • the number of infected PBMC varied, depending on the clinical stage of the individual, and ranged from 1:50,000 in asymptomatic to 1:400 (infectedmormal) in AIDS individuals (Ho, D.D., Moudgil, T., and Alam, M. Quantitation Of Human Immunodeficiency Virus Type 1 In The Blood Of Infected Persons, ti. Engl. _ ⁇ . Med. 1989;321:1621- 1625).
  • the sensitivity of plasma culture is not sufficient to detect a majority of known infections, and its reproducibility depends on the stimulated donor cells used in the assay (Eschaich, S., Ritter, J., Rougler, P., Lepot, D., Lamelin, J.P., Sepetjan, M., and Trepo, C. Plasma Viremia As A Marker Of Viral Replication In HIV Infected Individuals. AIDS
  • PCR polymerase chain reaction
  • the present inventions is useful in an assay using immunocapture of plasma associated HIV-1 virions for the direct measurement of HIV-1 replication (viremia).
  • This type of assay represents a sensitive and specific method for measurement of plasma associated HIV-1 virus without the need for culture techniques or cumbersome chemical extraction procedures.
  • a direct lysis buffer was formulated that was used directly with a simplified method of reverse transcription and amplification of HIV-1 genomic RNA. These changes considerably reduced the time required to monitor HIV-1 viral load.
  • a particle size of 0.1 to 0.3 ⁇ was chosen because of a general increase in surface area obtained per unit volume. Theoretically, this would maximize the quantity of antibody coupled onto the particles and this should, in turn, increase the sensitivity of the immunocapture.
  • High affinity monoclonal antibodies rather than polyclonal antibodies, were used to capture the HIV-1 virions in order to maximize sensitivity.
  • Both the gp120 and gp41 proteins are components of the virus outer membrane and are present on every infectious HIV-1 virus particle. Thus, anti-gp120 and anti-gp41 specific monoclonal antibodies were selected for immunocapture.
  • the method of attachment of the antibody to the particle may affect both the sensitivity and specificity of the assay.
  • the specific attachment of the antibody depends on the chemical composition of the particle. Passive adsorption is achieved by relying on ionic and hydrophobic interactions between the particle and antibody.
  • Covalent attachment relies on production of a covalent bond between the particle and the antibody.
  • Carboxylated particles were used for the covalent attachment of the monoclonal antibodies. A covalent bond is formed, with the aid of EDC, between the carboxyl groups on the particle and the amino group(s) on the antibody. Both methods have been shown to be highly sensitive. While not all of the antibody attached to the particles is done covalently, this method offers superior stability, and because of this the covalent method for attachment of antibodies was used.
  • the original procedure for isolating the genomic RNA was cumbersome and time consuming because it used conventional molecular biology techniques.
  • the viral membranes were disrupted using relatively high concentrations of SDS.
  • Proteinase K was included to digest any proteins associated with the viral RNA.
  • Purification of the HIV-1 RNA was accomplished using a combination of organic solvent extractions, followed by an overnight ethanol precipitation.
  • a direct lysis buffer was formulated to extract genomic HIV-1 RNA from the intact virion thereby providing for the direct use of the genomic HIV-1 RNA in a reverse transcription and amplification procedure. This eliminated both the organic solvent extraction and the ethanol precipitation procedures that are required in the conventional procedure. At a minimum, a total of 16 hours was eliminated from the assay by not using the organic solvent purification procedure.
  • the approximate detergent and/or Proteinase K concentrations required to disrupt an equivalent amount of cellular and viral material were determined.
  • the disruption of 10 6 human cells requires one milliliter of a solution containing 0.25% SDS and 0.5 mg/ml Proteinase K (19).
  • the theoretical binding capacity of the immunoparticles was determined in an earlier study to be approximately 10,000 virions (Henrard, D.R., Mehaffey, W.F., and Allain, J.P. A Sensitive Viral Capture Assay For The Detection Of
  • the concentrations of SDS and Proteinase K needed to lyse 10 6 cells should be at least 100 fold greater than that needed to lyse 10,000 virions.
  • the volume of a cell is approximately 1000 times greater than that of a virus (i.e., the average diameters of a cell and a virus are 3 ⁇ and 0.1 ⁇ , respectively). Therefore, a total 10 5 fold reduction in the amount of detergent and Proteinase K should be sufficient to lyse an equivalent amount of viral material.
  • the volume of direct lysis buffer that is added to the immunocaptured virions is, however, 30 ⁇ l, which corresponds to a 33 fold decrease in the net amount of detergent and Proteinase K compared to that needed for lysing cells.
  • concentrations are 10 fold lower than the actual concentrations tested, which gave results comparable to the organic extraction procedure.
  • the concentrations of SDS and Proteinase K that were used to disrupt the HIV-1 virions were at least 10 fold greater than is required based on these calculations.
  • the glycerol concentration, resulting from the addition of the stock enzymes, in the 50 ⁇ l procedure increased over two fold (2.2% vs 0.97%) compared to the 100 ⁇ l procedure. According to the manufacturers, no significant inhibitory effect on the enzymes occur when the glycerol concentration is within this range.
  • the decrease in assay volume did, however, concentrate the particles that were associated with the immunocapture. It is possible that the particles somehow either interfered or inhibited the enzyme reactions.
  • the major difference in the actual laboratory time required to complete the standard assay procedure and the modified procedure was in the overnight RNA precipitation step. The elimination of this step from the modified procedures saved, at a minimum, 15 hours. Overall, the modified procedure reduced the immunocapture time by two hours, the time for lysis and isolation of the HIV-1 genomic RNA by 15 hours, and the time to set up the reverse transcription and PCR mixes by another one hour. While at least two working days were required to obtain the results of a sample using the standard procedure, it took only nine hours to get that same result using the modified procedure.
  • a woman with a low viral load but extensive blood loss maybe more likely to transmit HIV than a woman with high viral load but minimal blood loss during delivery. This may explain why, in our study, an asymptomatic mother who had no detectable HIV RNA in her peripheral blood, and a very high CD4+ count, could still transmit HIV-1 to her child.
  • HIV-1 transmission Unlike vertical HIV-1 transmission, the horizontal transmission of HIV-1 from a blood donor to a recipient was highly correlated with plasma viremia. All twenty-two blood recipients in this study were transfused with HIV-1 seropositive blood. A significant proportion (64%) of the donor samples that transmitted an HIV-1 infection to the recipient had detectable HIV-1 viremia, while the individuals that did not infect the recipient had no detectable HIV-1 viremia. These results suggest that HIV infection depends primarily on the level of plasma viremia in the context of blood transfusions. As the number of HIV-1 infected individuals increases, it is becoming more important to monitor their status during the course of infection. The development of an assay that can rapidly and efficiently identify the progression of the disease will aid in monitoring the infection, and the efficacy of various therapeutics.
  • the immunocapture -cDNA/PCR assay which is highly sensitive, may be particularly useful for these purposes.
  • the assay described here can differentiate log differences in plasma HIV-1 viral load and efforts are currently underway to develop a more precise way of detecting and quantitating the level of plasma HIV-1 viremia. These include the development of a quantitative detection system based on an amplification system other than the polymerase chain reaction.
  • LCR Ligase Chain Reaction
  • the ligase chain reaction uses a thermostable DNA ligase to covalently join adjacent 3' hydroxyl and 5' phosphoryl termini of the oligo primers that are complementary to the target DNA, and Taq polymerase is not required.
  • the ligase chain reaction amplifies the target DNA by use of a series of annealing and denaturation steps.
  • the oligonucleotide products from each round serve as substrates for each successive round. This makes it possible to increase the number of target molecules of DNA by a factor of over 10 5 fold.
  • the primers are modified to include a fluorophore. An automated fluorimetric assay system is then used where 24 samples can be processed in 45 minutes.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Mycology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Virology (AREA)
  • Immunology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Detergent Compositions (AREA)

Abstract

L'immunocapture des VIH-1 plasmiques couplée à une lyse directe des virions et une procédure simplifiée de transcription inverse (TI) et d'amplification de l'ADN complémentaire des HIV-1 par PCR constituent une méthode rapide et hautement sensible pour suivre la progression de la maladie causée par les VIH-1. Cette méthode demande également moins de temps et de travail que la culutre quantitative. En plus, la procédure mise au point permettant de lyser directement les virions immunocaptés et la procédure simplifée TI/PCR en une seule étape éliminent la nécessité d'une extraction par un solvant organique et diminuent le nombre d'étapes de la méthode. Un tampon de lyse directe a été formulé pour isoler l'ARN des VIH-1 plasmiques en vue d'une utilisation directe dans la TI et la PCR, ce qui évite d'utiliser une extraction par des solvants organiques et une précipitation par de l'éthanol. Ceci diminue considérablement le temps nécessaire pour effectuer l'analyse et les risques de contamination associés avec les PCR. La méthode immunocapture - TI/PCR a été utilisée pour montrer que la contagion verticale des VIH-1 de la mère à son enfant dépendant largement de facteurs autres que la charge virale. Inversement, la charge virale plasmique joue un rôle significatif dans les infections par les VIH-1 lors de transfusions. Finalement, la détection et la quantification de charges virales associées avec le plasma par immunocapture-TI/PCR peut servir d'outil additionnel pour suivre la progression de la maladie et déterminer l'efficacité de différentes thérapies contre le VIH.
PCT/US1994/004676 1993-05-06 1994-04-28 Tampon de lyse directe et detection de la presence de vih-1 dans le plasma WO1994026867A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP94915927A EP0698082A4 (fr) 1993-05-06 1994-04-28 Tampon de lyse directe et detection de la presence de vih-1 dans le plasma
JP6525476A JPH08510004A (ja) 1993-05-06 1994-04-28 直接溶解緩衝液およびｈｉｖ−１血漿ウィルス血症の検出
AU67769/94A AU6776994A (en) 1993-05-06 1994-04-28 Direct lysis buffer and the detection of hiv-1 plasma viremia

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US5862393A 1993-05-06 1993-05-06
US08/058,623 1993-05-06

Publications (1)

Publication Number Publication Date
WO1994026867A1 true WO1994026867A1 (fr) 1994-11-24

Family

ID=22017952

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1994/004676 WO1994026867A1 (fr) 1993-05-06 1994-04-28 Tampon de lyse directe et detection de la presence de vih-1 dans le plasma

Country Status (5)

Country Link
EP (1) EP0698082A4 (fr)
JP (1) JPH08510004A (fr)
AU (1) AU6776994A (fr)
CA (1) CA2161337A1 (fr)
WO (1) WO1994026867A1 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0671473A1 (fr) * 1994-03-10 1995-09-13 Gen-Probe Incorporated Procédé pour supprimer l'inhibition de réactions enzymiques par des détergents ioniques
WO1998054306A1 (fr) * 1997-05-29 1998-12-03 Bio Merieux Procede pour isoler un materiel intracellulaire
WO1999028742A1 (fr) * 1997-11-28 1999-06-10 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Procede et dispositif de desintegration de cellules biologiques en vue de l'extraction et de l'analyse de leur contenu
WO2001042456A3 (fr) * 1999-12-10 2002-01-03 Genespan Corp Isolation et purification d'acides nucleiques
EP1000174A4 (fr) * 1997-07-28 2004-08-04 New York Blood Ct Inc Technique de purification d'acides nucleiques viraux
WO2005123960A1 (fr) * 2004-06-11 2005-12-29 Ambion, Inc. Derives biologiques bruts adaptes pour la detection des acides nucleiques
WO2006103094A2 (fr) 2005-04-01 2006-10-05 Qiagen Gmbh Procede pour traiter un echantillon contenant des biomolecules
US7964350B1 (en) 2007-05-18 2011-06-21 Applied Biosystems, Llc Sample preparation for in situ nucleic acid analysis
WO2011028887A3 (fr) * 2009-09-03 2011-07-28 Becton, Dickinson And Company Procédés et compositions de lyse chimique directe
US8211637B2 (en) 2008-12-19 2012-07-03 Life Technologies Corporation Proteinase K inhibitors, methods and compositions therefor
US9611497B2 (en) 2002-01-28 2017-04-04 Applied Biosystems, Llc Crude biological derivatives competent for nucleic acid detection
EP3910067A4 (fr) * 2019-01-08 2022-03-16 Sansure Biotech Inc. Agent de libération d'acide nucléique, procédé d'amplification pcr d'acide nucléique et kit d'amplification pcr

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4144863A4 (fr) * 2020-04-30 2024-07-31 Takara Bio Inc. Procédé de détection de virus à arn

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5175094A (en) * 1991-08-01 1992-12-29 Becton Dickinson And Company Increased expression of HBcAg
US5182377A (en) * 1988-09-09 1993-01-26 Hoffmann-La Roche Inc. Probes for detection of human papillomavirus
US5213796A (en) * 1991-05-06 1993-05-25 Dana Farber Cancer Institute Assay for polyomavirus in humans and uses thereof

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0338591A3 (fr) * 1988-04-21 1991-09-04 Microprobe Corporation Procédé pour l'extraction des acides nucléiques
US5231015A (en) * 1989-10-18 1993-07-27 Eastman Kodak Company Methods of extracting nucleic acids and pcr amplification without using a proteolytic enzyme
EP0488243A1 (fr) * 1990-11-30 1992-06-03 Sanwa Kagaku Kenkyusho Co., Ltd. Procédé d'extraction du génome du virus d'un échantillon derivé d'un corps vivant infecté par le virus et procédé de détection du génome

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5182377A (en) * 1988-09-09 1993-01-26 Hoffmann-La Roche Inc. Probes for detection of human papillomavirus
US5213796A (en) * 1991-05-06 1993-05-25 Dana Farber Cancer Institute Assay for polyomavirus in humans and uses thereof
US5175094A (en) * 1991-08-01 1992-12-29 Becton Dickinson And Company Increased expression of HBcAg

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP0698082A4 *

Cited By (33)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0671473A1 (fr) * 1994-03-10 1995-09-13 Gen-Probe Incorporated Procédé pour supprimer l'inhibition de réactions enzymiques par des détergents ioniques
WO1995024499A1 (fr) * 1994-03-10 1995-09-14 Gen-Probe Incorporated Procede permettant de reprimer l'inhibition de reactions a mediation enzymatique dues a des detergents ioniques
US5766890A (en) * 1994-03-10 1998-06-16 Gen-Probe Incorporated Kits for suppressing inhibition of enzyme-mediated reactions by ionic detergents using high concentrations of non-ionic detergents
US5846701A (en) * 1994-03-10 1998-12-08 Gen-Probe Incorporated Method for suppressing inhibition of enzyme-mediated reactions by ionic detergents using high concentration of non-ionic detergent
US5871975A (en) * 1994-03-10 1999-02-16 Gen-Probe Incorporated Method for suppressing inhibition of enzyme-mediated nucleic acid amplification reactions by ionic detergents
WO1998054306A1 (fr) * 1997-05-29 1998-12-03 Bio Merieux Procede pour isoler un materiel intracellulaire
FR2763957A1 (fr) * 1997-05-29 1998-12-04 Bio Merieux Procede pour isoler un materiel intracellulaire et procede de traitement d'un materiel nucleique
EP1000174A4 (fr) * 1997-07-28 2004-08-04 New York Blood Ct Inc Technique de purification d'acides nucleiques viraux
WO1999028742A1 (fr) * 1997-11-28 1999-06-10 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Procede et dispositif de desintegration de cellules biologiques en vue de l'extraction et de l'analyse de leur contenu
WO2001042456A3 (fr) * 1999-12-10 2002-01-03 Genespan Corp Isolation et purification d'acides nucleiques
US9611497B2 (en) 2002-01-28 2017-04-04 Applied Biosystems, Llc Crude biological derivatives competent for nucleic acid detection
US10590411B2 (en) 2002-01-28 2020-03-17 Applied Biosystems, Llc Crude biological derivatives competent for nucleic acid detection
US10233444B2 (en) 2002-01-28 2019-03-19 Applied Biosystems, Llc Crude biological derivatives competent for nucleic acid detection
US10011831B2 (en) 2002-01-28 2018-07-03 Applied Biosystems, Llc Crude biological derivatives competent for nucleic acid detection
US8114594B2 (en) 2004-06-11 2012-02-14 Applied Biosysyems, LLC Crude biological derivatives competent for nucleic acid detection
WO2005123960A1 (fr) * 2004-06-11 2005-12-29 Ambion, Inc. Derives biologiques bruts adaptes pour la detection des acides nucleiques
WO2006103094A2 (fr) 2005-04-01 2006-10-05 Qiagen Gmbh Procede pour traiter un echantillon contenant des biomolecules
EP1869179B1 (fr) * 2005-04-01 2009-10-28 Qiagen GmbH Procédé pour traiter un échantillon contenant des biomolécules
US7838233B2 (en) 2005-04-01 2010-11-23 Qiagen Gmbh Method for the treatment of a sample containing biomolecules
US8076069B2 (en) 2005-04-01 2011-12-13 Qiagen Gmbh Method for the treatment of a sample containing biomolecules
US8785120B2 (en) 2005-04-01 2014-07-22 Qiagen Gmbh Method for the treatment of a sample containing biomolecules
US8828664B2 (en) 2007-05-18 2014-09-09 Applied Biosystems, Llc. Sample preparation for in situ nucleic acid analysis, methods and compositions therefor
US9279152B2 (en) 2007-05-18 2016-03-08 Applied Biosystems, Llc Sample preparation for in situ nucleic acid analysis, methods and compositions therefor
US8288106B2 (en) 2007-05-18 2012-10-16 Applied Biosystems, Llc Sample preparation for in situ nucleic acid analysis, methods and compositions therefor
US10501780B2 (en) 2007-05-18 2019-12-10 Applied Biosystems, Llc Compositions for in situ nucleic acid analysis
US7964350B1 (en) 2007-05-18 2011-06-21 Applied Biosystems, Llc Sample preparation for in situ nucleic acid analysis
US9114104B2 (en) 2008-12-19 2015-08-25 Life Technologies Corporation Proteinase K inhibitors, methods and compositions therefor
US8211637B2 (en) 2008-12-19 2012-07-03 Life Technologies Corporation Proteinase K inhibitors, methods and compositions therefor
US10190152B2 (en) 2009-09-03 2019-01-29 Becton, Dickinson And Company Methods and compositions for direct chemical lysis
WO2011028887A3 (fr) * 2009-09-03 2011-07-28 Becton, Dickinson And Company Procédés et compositions de lyse chimique directe
US10323267B2 (en) 2009-09-03 2019-06-18 Becton Dickinson And Company Methods and compositions for direct chemical lysis
US11434519B2 (en) 2009-09-03 2022-09-06 Becton, Dickinson And Company Methods and compositions for direct chemical lysis
EP3910067A4 (fr) * 2019-01-08 2022-03-16 Sansure Biotech Inc. Agent de libération d'acide nucléique, procédé d'amplification pcr d'acide nucléique et kit d'amplification pcr

Also Published As

Publication number Publication date
EP0698082A4 (fr) 1997-12-17
AU6776994A (en) 1994-12-12
CA2161337A1 (fr) 1994-11-24
JPH08510004A (ja) 1996-10-22
EP0698082A1 (fr) 1996-02-28

Similar Documents

Publication Publication Date Title
Van Gemen et al. Quantification of HIV-1 RNA in plasma using NASBATM during HIV-1 primary infection
Psallidopoulos et al. Integrated proviral human immunodeficiency virus type 1 is present in CD4+ peripheral blood lymphocytes in healthy seropositive individuals
Jurriaans et al. The natural history of HIV-1 infection: virus load and virus phenotype independent determinants of clinical course?
Sonza et al. Human immunodeficiency virus type 1 replication is blocked prior to reverse transcription and integration in freshly isolated peripheral blood monocytes
Bagnarelli et al. Molecular profile of human immunodeficiency virus type 1 infection in symptomless patients and in patients with AIDS
Chowers et al. The growth advantage conferred by HIV-1 nef is determined at the level of viral DNA formation and is independent of CD4 downregulation
WO1994026867A1 (fr) Tampon de lyse directe et detection de la presence de vih-1 dans le plasma
Stieger et al. Competitive polymerase chain reaction assay for quantitation of HIV-1 DNA and RNA
Duvall et al. Dendritic cells are less susceptible to human immunodeficiency virus type 2 (HIV-2) infection than to HIV-1 infection
JP3759226B2 (ja) Ｈｉｖグル−プに属するレトロウイルスおよびその用途
Clementi et al. Clinical use of quantitative molecular methods in studying human immunodeficiency virus type 1 infection
US6136534A (en) Methods for sensitive detection of reverse transcriptase
Vicenzi et al. Hemophilia and nonprogressing human immunodeficiency virus type 1 infection
Kaplan et al. Human immunodeficiency virus type 1 virions composed of unprocessed Gag and Gag-Pol precursors are capable of reverse transcribing viral genomic RNA
Hearps et al. Stability of dried blood spots for HIV-1 drug resistance analysis
Curran et al. Concordance between semen‐derived HIV‐1 proviral DNA and viral RNA hypervariable region 3 (V3) envelope sequences in cases where semen populations are distinct from those present in blood
EP1210465B1 (fr) Procede de production d'echantillon biologique de qualite controlee et composition contenant cet echantillon
US20020090611A1 (en) Detection of human immunodeficiency virus
Rasheed Detection, Quantitation, and Characterization of Human Retroviruses
US6525173B1 (en) Process for expression and production of recombinant protein hybrid protein (p24/p17) derived from human immunodeficiency viruses (HIV-1)
Bournique et al. Detection of HIV-1 infections by PCR: evaluation in a seropositive subject population
FEVEREIRO et al. Antibody response to reverse transcriptase in cats infected with feline immunodeficiency virus
Wattel et al. No evidence of HTLV-I infection in French patients with multiple sclerosis using the polymerase chain reaction.
CA2211165C (fr) Procedes de detection sensible de transcriptase inverse
Malmsten Reverse Transcriptase Activity Assays for Retrovirus Quantitation and Characterization

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP KR

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2161337

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 1994915927

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1994915927

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1994915927

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载