WO1993023573A1 - Quantification de l'arn viral par amplification competitive de chaine par polymerase - Google Patents

Quantification de l'arn viral par amplification competitive de chaine par polymerase Download PDF

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WO1993023573A1
WO1993023573A1 PCT/US1993/004548 US9304548W WO9323573A1 WO 1993023573 A1 WO1993023573 A1 WO 1993023573A1 US 9304548 W US9304548 W US 9304548W WO 9323573 A1 WO9323573 A1 WO 9323573A1
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rna
dna
pcr products
sample
standard
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PCT/US1993/004548
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David T. Scadden
Zhengyu Wang
Jerome E. Groopman
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New England Deaconess Hospital
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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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • 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

Definitions

  • HIV-1 human immunodeficiency virus type 1
  • circulating CD4+ lymphocyte cell numbers, serum HIV-l p24 antigen, serum neopterin, serum beta-2 microglobulin, soluble CD8 and soluble interleu in-2 receptor levels have been proposed to be clinical indicators of HIV-1 disease activity, they are limited by sensitivity and specificity.
  • PCR of PBMC DNA provides the investigator with an estimate of the number of infected cells, but does not provide any direct information regarding changes in viral replicative activity, such as is induced by drug therapies or during the natural progression of the disease.
  • An assay which could rapidly and accurately evaluate the concentration of circulating virus in patient serum would provide a useful means of directly measuring virus activity.
  • the invention described herein is a method for quantitating virus RNA of interest in a biological sample.
  • the invention is a method for measuring the concentration of HIV-l virus RNA in a biological sample, such as in the blood plasma of HIV-l-infected individuals.
  • the method described takes advantage of the polymerase chain reaction (PCR) method of amplifying small amounts of DNA.
  • the method also employs an internal standard RNA, which allows for control of the variability in the amount of end product produced by PCR.
  • the internal standard RNA is reverse transcribed to produce DNA which is amplified as efficiently as the DNA transcription product of the viral RNA from the biological sample; the internal standard RNA transcription product (DNA) effectively competes with the transcription product of the RNA of interest during the polymerase chain reaction.
  • internal standard RNA and sample RNA are combined in a dilution series in which each member contains the same amount of sample RNA; the amount of standard RNA varies among the members of the series and is at least 10 2 copies.
  • the range is generally 10 2 to 10 8 copies and in one embodiment is 10 2 copies to 10 7 copies.
  • internal standard RNA and sample RNA are combined in a dilution series in which each member contains the same amount of standard RNA within the range described above and the amount of sample RNA varies among the members of the series.
  • RNA of interest and the standard RNA are then reverse transcribed in the presence of either random nucleotide hexamers or DNA primers which are specific to RNA sequences characteristic of both the RNA of interest and the standard RNA.
  • Reverse transcription results in production of DNA transcripts of the RNA of interest, referred to as sample DNA, and DNA transcripts of the standard RNA, referred to as standard DNA.
  • the DNA transcripts are then amplified by the polymerase chain reaction (PCR) method in the presence of detectable oligonucleotide primers which are specific for nucleic acid sequences characteristic of DNA sequences in both sample DNA and standard DNA.
  • PCR polymerase chain reaction
  • This PCR reaction results in the production of a mixture of two species of double-stranded DNA in each member of the dilution series: one species of DNA derived from the internal standard RNA, and one species of DNA derived from the RNA of interest.
  • detectable oligonucleotide primers are incorporated into both DNA species produced.
  • the number of copies of each species of double-stranded DNA in a given member of the dilution series after PCR depends on the relative number of copies of sample DNA and standard DNA in the mixture prior to PCR.
  • the PCR products resulting from amplification of sample DNA and standard DNA are separated on the basis of size, and the relative amount of primer incorporated into each species is determined for each member of the dilution series.
  • the relative copy numbers of PCR end products representing the standard DNA and the sample DNA are dependent upon the relative copy numbers of each of the two DNA species prior to initiation of the PCR reaction. Therefore, a competition curve can be generated for each dilution series, and the point at which standard DNA and sample DNA compete equally in the PCR is extrapolated.
  • the amount of internal standard RNA in the reaction mix which results in equivalent copy numbers of sample DNA and standard DNA after PCR can be calculated, and the amount of sample RNA extrapolated therefrom.
  • any viral RNA in a biological sample can be quantit ted by the method of the present invention.
  • the method can be used to quantitate human immunodeficiency virus RNA, human T-cell lymphotropic virus RNA, simian immunodeficiency virus RNA, avian leukosis virus RNA, murine leukemia virus RNA, and feline leukemia virus RNA.
  • Internal standard RNA is RNA homologous to two distinct and separate sequences within a region of viral RNA characteristic of the virus of interest, and is a different size than the corresponding region of the virus of interest.
  • Internal standard RNA may be a deletion mutant or an insertion mutant of a region characteristic of the virus of interest.
  • the internal standard RNA may be an RNA polymerase product of a DNA clone of an approximately 115 bp deletion/insertion mutant of a region of the HIV ⁇ a ⁇ gene.
  • the invention described herein is also a method of monitoring levels of HIV or other RNA viruses in the blood plasma of infected individuals.
  • the method involves obtaining a blood sample by phlebotomy, separating the plasma from the whole blood, and extracting RNA from the plasma.
  • the RNA from blood plasma is then used as the RNA of interest in the PCR strategy already described, wherein a constant amount of RNA of interest is added to each member of a dilution series of internal standard RNA, reverse transcribed, amplified by PCR, separated from standard DNA, and the amount of RNA of interest in the initial mixture calculated as described above.
  • the method of this invention allows for control over the tube to tube variability in the amount of end product produced by PCR.
  • the method of this invention is useful for accurate quantitation of the concentration of virus in blood plasma or other biological samples.
  • the dynamic range of the assay is at least four orders of magnitude, which is substantially higher than that of other reported PCR-based strategies (Holodniy, M. , e_t al « , J. Infect. Diseases 162:862-866 (1991)).
  • the wide dynamic range of the assay makes it particularly attractive for use in evaluating . anti-viral therapies where large individual differences may be anticipated in the concentrations of circulating virus, both before and during treatment.
  • Figure 1A is a graphic representation of the sample processing and analysis schema.
  • Figure IB is competitive PCR results of the wild type HIV-l (upper band) or K4 standard (lower band) at different ratios of wild-type:K4 RNA.
  • Figure 1C shows the ratio of wild-type:K4 RNA represented by lanes 4 and 5 of (B) , and an intermediate ratio, subjected to sequential three-fold dilutions prior to reverse transcription and ampli ication.
  • Figure 2A shows equivalent amplification of wild type (H9/IIIB) or K4 RNA at different PCR cycle numbers.
  • Figure 2B depicts the relationship between the amount of DNA produced in a PCR, and the initial amount of K4 cRNA in the reaction mixture.
  • Figure 2C is an analysis of a single AIDS patient's plasma sample divided at the time of phlebotomy and independently analyzed.
  • Evaluation of the therapeutic benefit of compounds used to treat HIV infection may be based on a number of clinical and laboratory parameters.
  • the laboratory assays widely in use are indirect measures of the activity of the virus, such as T lymphocyte subsets, neopterin or beta-2 microglobulin or direct measurements of a virus product such as the HIV-l p24 antigen assay.
  • the latter assay is limited by the high frequency of p24 antigen-negative patients.
  • Improved direct measures of the infectious burden of HIV are desirable because of the nonspecificity of the indirect laboratory parameters and because of the potential time delay for indirect measures to reflect alterations in circulating virus. Methods which rapidly and accurately measure the circulating levels of free virus, as opposed to virus incorporated into circulating cells, would be useful to evaluate therapies directed at the viral replicative cycle.
  • monitoring the concentrations of circulating virus could be used to measure the effects on viral replication of inhibitors of reverse transcriptase, Tat or protease, or inhibitors of virus assembly or release.
  • Described herein is a method of quantitating the amount of viral RNA in a biological sample. Because the claimed method makes use of an internal standard, it permits control of the variability in the amount of end product produced by PCR.
  • the method of the present invention involves the steps of t obtaining viral RNA of interest from a biological sample; adding approximately the same amount of that RNA to each member of a dilution series of internal standard RNA; reverse transcribing the mixture to yield sample DNA and standard DNA; amplifying that mixture by PCR; separating the two major PCR amplification products from each other, measuring the relative amount of each produced, and extrapolating the amount of RNA of interest in the biological sample by calculating the amount of standard DNA necessary to compete equivalently with sample DNA in the PCR reaction.
  • the method of the present invention uses known laboratory techniques in conjunction with a mutant internal standard RNA template to evaluate the quantity of HIV-l RNA in the plasma of AIDS patients.
  • This method requires only a small quantity of patient plasma (e.g., obtained through the phlebotomy of a single tube of blood) which can be processed and analyzed within a relatively short period of time (e.g., 48 hours).
  • An internal RNA standard that is equivalently amplified during PCR is included in the analysis in order to avoid inaccurate quantitation of the sample material based on intrasample variability.
  • Use of an external standard does not control for this variability, which may result in alterations in final product quantitation of up to 600% (Gilliland, G.
  • the method presented here has an average intrasample variability of 26.
  • This relatively simple assay for quantitating plasma levels of HIV-l RNA provides a rapid tool for measuring changes in circulating virus in vivo.
  • the method can be modified to apply to other RNA viruses without undue experimentation.
  • the modifications would include use of an appropriate standard RNA, and primers designed to be specific for nucleic acid sequences which occur both in the sample RNA of interest, and in the standard RNA.
  • the method of the present invention is described in detail below.
  • Sample RNA The method described herein may be used to detect viral RNA from a variety of biological samples, and from a variety of RNA viruses. For example, because it measures unincorporated viral genomic RNA, the method can be used to quantitate genomic RNA of any replicating RNA virus in any tissue. The method is particularly useful for measuring the concentration of circulating virus particles or RNA in body fluid, such as in blood plasma, mucous, saliva or semen. In particular, the present method is useful to determine blood plasma levels of HIV in infected individuals, as shown herein.
  • RNA is separated from the sample by any of a variety of well-known methods.
  • the RNA may be isolated by precipitation with guanidine isothyocyanate r followed by ultracentrifugation through 5.7M cesium chloride.
  • the lower limit of sensitivity of the present method is on the order of 10 copies of RNA of interest per reaction, and the upper limit is at least 10 6 copies per reaction. Accordingly, the amount of sample required for analysis by this method depends on the concentration of viral RNA in the sample to be tested and may vary over a wide range.
  • RNA containing between 10 3 and 8.3 x 10* copies of HIV RNA per ml is sufficient for one reaction mixture, and, if four dilutions of internal standard RNA are used for an assay, then a total of 2 ml of blood plasma is sufficient for an assay by the present method.
  • the amount of biological sample of other types, for example semen or mucous, may be easily determined.
  • the internal standard RNA of the present method is an RNA with nucleic acid sequence homology to regions near the 5' and 3' ends of a nucleic acid sequence characteristic of the viral RNA of interest.
  • DNA transcripts of the internal standard RNA are amplified by PCR with an efficiency substantially equal to that of DNA transcripts of the viral RNA of interest, and the PCR products of the two RNAs are separable on the basis of size.
  • the same DNA primers are used for PCR amplification of DNA transcripts from both the internal standard RNA and the sample RNA of interest.
  • the internal standard RNA must share nucleic acid sequence homology with sequences near the 5' and 3' ends of a region characteristic of the viral RNA of interest, and must be of a different size than the region of the RNA of interest with which it shares homology.
  • the internal standard RNA may be an RNA polymerase product of a cloned deletion, insertion or deletion/insertion mutant of a region of the virus of interest, wherein the insertion or deletion occurs internal to the cloned viral nucleic acid sequences, rather than at the 5' or 3' ends.
  • the internal standard RNA shares substantial sequence homology with sequences near the 5' and 3' ends of a highly conserved region of a retroviral genome, such as a portion of the ⁇ a ⁇ region.
  • the internal standard RNA is an approximately 175 base deletion/insertion mutant of a portion of the ⁇ a ⁇ region between the sequences corresponding to the primers SK100 and SK104.
  • the internal standard RNA is approximately 115 bases smaller than the corresponding region in the wild- type virus.
  • RNA from the biological sample is added to each member of a dilution series of mixtures, each containing a known amount of an internal standard RNA; each member of the dilution series contains the same amount of sample RNA, and the amount of standard RNA varies among the members of the series and is at least 10 2 copies. The range is generally between 10 2 and 10 s copies. Quantitation in this assay is dependent on determining the amount of internal standard RNA required for equivalent amplification of the DNA transcripts resulting from reverse transcription of a mixture containing internal standard RNA and sample RNA.
  • the concentration of HIV RNA observed in the HIV-infected individuals described in the Examples below ranged from 10 3 to 8.3 x 10* copies in 1 ml of plasma.
  • a dilution series may be provided wherein the sample RNA from 0.5 ml of blood plasma is added to each of four members of a dilution series which contain, respectively, 10 3 , 10*, 10 5 or 10 6 copies of the internal standard RNA.
  • the method described herein would work as well if the dilution series was comprised of mixtures containing a series of dilutions of the viral RNA of interest, and a constant amount of internal standard RNA in each member of the series.
  • the mixtures containing sample RNA and internal standard RNA are to be reverse transcribed into DNA before amplification by PCR, the mixtures are maintained under conditions appropriate for reverse transcription by methods well recognized in the art and described in the Methods Section below.
  • the polymerase chain reaction is performed under conditions well recognized in the art. The conditions used in the Examples provided herein are listed in the Methods section below. The reaction is terminated while amplification is in log phase, for example, at approximately 30 cycles or less.
  • Separation of the two species of DNA produced by PCR amplification of sample DNA and standard DNA can be accomplished by a variety of known chromatographic methods such as agarose gel electrophoresis, acrylamide gel electrophoresis, affinity chromatography and chromatographic separation on the basis of size or charge.
  • the PCR products may be quantitated by any of a variety of known methods.
  • the primers, which are incorporated into the PCR products may be radiolabeled before incorporation and quantitated after separation of the PCR products by counting in a scintillation counter.
  • the primers may be labeled with a fluorescent compound and the amount of primer incorporated into the PCR products determined by measuring fluorescence of the two types of PCR products.
  • the amount of PCR product resulting from each of the two species depends on the relative molar concentrations of the two templates present at the start of the amplification reaction. For example, if the molar concentration of sample DNA is greater than the molar concentration of standard DNA, then the amount of PCR product of the sample DNA will be relatively greater than the amount of PCR product of the standard DNA. Conversely, if the molar concentration of standard DNA is greater than the molar concentration of sample DNA, then the amount of PCR product of the standard DNA will be relatively greater than the amount of PCR product of the sample DNA.
  • RNA from the blood plasma of an HIV-infected individual may be quantitated in this manner, and the effect of anti-viral therapies thereby monitored.
  • the method of the present invention can, for example, be used to monitor changes in blood plasma levels of HIV in infected individuals, as described below.
  • the relationship between the two PCR products varies linearly with changes in the relative molar amounts of the DNA templates before amplification. Accordingly, differences in the relative amounts of sample DNA and standard DNA among members of a dilution series, as previously described, will result in differences in the relative amounts of PCR end product among the members of the dilution series.
  • the ratio of the two PCR products for each of the several members of the dilution series is determined and together will define a relationship from which the quantity of RNA of interest in the original sample can be determined (e.g., using linear regression analysis) .
  • RNA pellets 10-15 ml of blood was obtained by phlebotomy either from HIV-l infected individuals with an AIDS defining diagnosis or from normal controls. Blood was collected in heparinized tubes (Becton-Dickenson, Rutherford, NJ) and plasma harvested following centrifugation at 1200 x g for 10 minutes. 2.0 ml of plasma was mixed with 4.0 ml of 5M quanidine isothyocyanate (Bethesda Research Laboratories, Gaithersburg, MD) and 100 ug yeast tRNA and ultracentrifuged through 5.7 M cesium chloride (BRL) at 180,000 + g for 18 hours at 25 ⁇ C. The resultant RNA pellet was resuspended in 0.3M sodium acetate and ethanol precipitated. The RNA pellet was washed with 70% ethanol and resuspended in water or stored under ethanol at -80°C.
  • 5M quanidine isothyocyanate Bethesda Research
  • the DNA clone used to produce the internal standard RNA was derived from a mututated ⁇ a ⁇ region comprising the nucleic acid sequence between bases 1377 and 1667 of HIV-l _ 2 (GenBank #K02007) and including the intact primer sites for the primers SKI00 and SK104 (Rayfield, M. , ________ l-, J. Infect. Diseases 158:1170-1176 (1988); Ou, C.Y. , e__ al «. Science 239:295-297 (1988)).
  • An approximately 290 bp portion of the HIV-l ⁇ a ⁇ region of HlV-l ⁇ y . j was PCR amplified using the oligonucleotide primer pair
  • the resultant fragment was filled in using Klenow (BRL) and blunt end ligated into the Smal site in the multiple cloning site of the plasmid vector pGEM-3Z (Promega, Madison, WI) .
  • the gag-specific insert was mutated to produce a ⁇ a ⁇ -related fragment with an insertion and an approximately 115 bp deletion internal to the region cloned, and to also include sequences specific for the primers SK100/SK104 at the 5' and 3' ends of the insert (SEQ ID NO:l).
  • the resultant plasmid (K4) was determined, on the basis of size by agarose gel electrophoresis, to be a satisfactory template for PCR directed by the primer pair, SK100/SK104.
  • Single-standard RNA was made using single-stranded K4 DNA as the template for T7 polymerase (Promega) using random nucleotide hexamers as primers.
  • K4 cRNA This RNA is referred to as K4 cRNA or as internal standard RNA.
  • the K4 cRNA was purified by ultracentrifugation through a 5.7M cesium chloride cushion, precipitated and resuspended, and the concentration determined by both spectrophotometry and incorporation of 32 P-uridine triphosphate (UTP) .
  • UTP 32 P-uridine triphosphate
  • RRIS Moloney murine leukemia virus reverse transcriptase
  • Example 1 Production of a DNA Clone of a Standard RNA A portion of the HIV-l gag region, delimited by sequences specific to the oligomers SK100 and SK104,
  • K4 was amplified by PCR using the SK100/SK104 primer pair as described in the methods section. PCR amplification of K4 yielded a PCR product which was readily distinguished from wild-type HIV-l by either acrylamide or agarose gel electrophoresis ( Figure 1B,C). In Figure IB, a fixed amount of K4 cRNA was maintained in the samples corresponding to lanes 1-9.
  • Lane 1 contained K4 cRNA alone (10* copies) and lane 10 contained H_V-l mB infected H9 RNA alone (1 meg) .
  • the K4 cRNA was evaluated as a template for PCR, as described in the Methods, using the SK100/SK104 primer pair and directly compared with the wild-type HIV-l genome represented by RNA derived from HIV-1 driving
  • 32 P-end labeled SK104 was added to the PCR reaction mixture and the PCR product was analyzed by electrophoresis. The yield of DNA was quantitated at different cycle numbers of PCR by the amount of 32 P, measured as counts per minute, incorporated into the appropriate bands isolated from the electrophoretic gels.
  • H9/IIIB RNA or K4 cRNA (10* copies) were independently analyzed for incorporation of 32 P-labeled oligonucleotide primer (SK104) following reverse transcription and amplification by scintillation counting of isolated agarose gel slices of the electrophoresed reaction products.
  • Figure 2A indicates that the rate of incorporation of 32 P-labeled primer is the same when either K4 or wild-type HIV-l IIIB is used as the PCR template.
  • K4 is amplified with an efficiency equal to that of wild-type virus and is, therefore, an appropriate template to use in a quantitative assay for wild-type HIV-l.
  • K4 can be readily distinguished from wild-type HIV-l, and that it is amplified by PCR as efficiently as wild-type HIV-l, the sensitivity and range of the method remained to be demonstrated. Because the range of concentrations of infectious virus particles detected in the plasma of HIV-l infected individuals has been reported to be quite variable (Holodniy, M. , ejt al « , J. Infect. Diseases 163:862-866 (1991)), and because the range of concentrations of virus particles containing detectable viral RNA (not necessarily infectious virus) may vary over an even broader range, a wide dynamic range for the assay is desirable. Known amounts of K4 cRNA were diluted and used as starting material for the reverse transcription and PCR reactions.
  • the resulting incorporation of 32 P- labeled tracer SKI04 primer for each starting concentration of K4 cRNA is indicated in Figure 2B.
  • known amounts of K4 cRNA were reverse transcribed and amplified for 30 cycles in the presence of 32 P labelled SK104 primer.
  • the reaction products were subjected to agarose gel electrophoresis, the gel slices were isolated and scintillation counted.
  • the number of K4 cRNA indicated is the log of copies per assay.
  • the intrasample coefficient of variation (CV) ranged from 0.05 to 0.26 for the samples with 10 3 to 10 6 copies of K4 cRNA respectively.
  • the sensitivity of the assay is limited to 10-100 copies of K4 in the starting material, but the dynamic range of the assay is at least four orders of magnitude.
  • Example 4 The Competitive Polymerase Chain Reaction In a competitive PCR assay, the relationship of end products of PCR to the starting quantities is dependent upon the ratio of the two starting templates at the time of initiation of the amplification protocol (Gilliland, G., __al., Proc. Natl. Acad. Sci. 87:2725-2729 (1990); Want, A.M., s£ al- , Proc. Natl. Acad. Sci.. USA 86:9717- 9721 (1989)). At ratios of sample template to standard template of less than or greater than 1:1 the amplification of the two templates is not equal.
  • Figure IB represents PCR products of reactions containing a fixed amount of the K4 standard with increasing concentrations of sample HIV-l RNA.
  • the variable intensity of the visible lower band (K4) indicates the change in amplification of the K4 template in the presence of changing concentrations of competing wild-type HIV-l RNA.
  • dilutions of RNA of three fixed ratios of wild-type RNA to K4 RNA were added to the PCR reaction mixture and amplified.
  • the series of lanes for each numbered set (1-3) represent the ethidium bromide stained, UV illuminated products of a thirty cycle amplification.
  • MOLECULE TYPE DNA (genomic)

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Abstract

L'invention concerne un procédé de quantification de l'ARN viral dans un échantillon biologique au moyen de l'amplification compétitive de chaîne par polymérase. Ledit procédé utilise un ARN interne standard qui est contenu dans la réaction d'amplification de chaîne par polymérase afin de contrôler la variabilité du volume du produit d'amplification de chaîne par polymérase de réaction en réaction. Ledit procédé est particulièrement utile pour contrôler les niveaux plasmiques de l'ARN viral. L'invention se rapporte également à une séquence d'acides nucléiques clonés utile pour produire l'ARN standard utilisé dans le procédé selon l'invention.
PCT/US1993/004548 1992-05-15 1993-05-13 Quantification de l'arn viral par amplification competitive de chaine par polymerase WO1993023573A1 (fr)

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Cited By (8)

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EP0714987A2 (fr) * 1994-09-26 1996-06-05 IMMUNO Aktiengesellschaft Procédé pour quantifier d'ADN génomiques
EP0714988A2 (fr) * 1994-09-26 1996-06-05 IMMUNO Aktiengesellschaft Procédé pour quantifier d'acides nucléiques
WO1997012058A1 (fr) * 1995-09-26 1997-04-03 Dynal A/S Procede de quantification d'acide nucleique consistant a utiliser plusieurs acides nucleiques concurrents
WO1997017465A1 (fr) * 1995-11-06 1997-05-15 Microdiag Procede et kit pour la quantification et la detection des micro-organismes
US5858732A (en) * 1995-05-19 1999-01-12 Abbott Laboratories Wide dynamic range nucleic acid detection using an aggregate primer series
WO1999012948A2 (fr) * 1997-08-22 1999-03-18 Olfert Landt Acides polyribonucleiques enveloppes de proteines, procede de production correspondant et leur utilisation
EP1000174A1 (fr) * 1997-07-28 2000-05-17 The New York Blood Center, Inc. Technique de purification d'acides nucleiques viraux
EP0769954B2 (fr) † 1995-05-08 2011-03-09 Baxter Aktiengesellschaft Medicament de qualite garantie contenant un ou plusieurs derives plasmatiques

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0714988A2 (fr) * 1994-09-26 1996-06-05 IMMUNO Aktiengesellschaft Procédé pour quantifier d'acides nucléiques
EP0714987A3 (fr) * 1994-09-26 1997-03-26 Immuno Ag Procédé pour quantifier d'ADN génomiques
EP0714987A2 (fr) * 1994-09-26 1996-06-05 IMMUNO Aktiengesellschaft Procédé pour quantifier d'ADN génomiques
EP0714988A3 (fr) * 1994-09-26 1997-04-16 Immuno Ag Procédé pour quantifier d'acides nucléiques
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WO1997017465A1 (fr) * 1995-11-06 1997-05-15 Microdiag Procede et kit pour la quantification et la detection des micro-organismes
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WO1999012948A3 (fr) * 1997-08-22 1999-07-22 Olfert Landt Acides polyribonucleiques enveloppes de proteines, procede de production correspondant et leur utilisation

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