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WO1999043849A1 - Procede pour mettre en evidence la presence d'acides nucleiques a haute concentration - Google Patents

Procede pour mettre en evidence la presence d'acides nucleiques a haute concentration Download PDF

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Publication number
WO1999043849A1
WO1999043849A1 PCT/EP1999/001258 EP9901258W WO9943849A1 WO 1999043849 A1 WO1999043849 A1 WO 1999043849A1 EP 9901258 W EP9901258 W EP 9901258W WO 9943849 A1 WO9943849 A1 WO 9943849A1
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nucleic acid
primers
nucleic acids
analyte
detection
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PCT/EP1999/001258
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German (de)
English (en)
Inventor
Stefanie KÖHLER
Peter Ebert
Volker SCHLÜTER
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Roche Diagnostics Gmbh
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Publication of WO1999043849A1 publication Critical patent/WO1999043849A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—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

Definitions

  • the invention relates to a method for the detection of highly concentrated analyte nucleic acids in a sample by means of asymmetrical amplification and the use of asymmetrical nucleic acid amplification for reducing or avoiding false negative results, for the quantitative determination of nucleic acids and for increasing the dynamic measuring range in nucleic acid detection reactions.
  • the detection of nucleic acids is becoming increasingly popular for the detection of viral and bacterial pathogens.
  • the detection of a DNA / RNA section comprises 3 steps, namely sample preparation, amplification of the target sequence by means of the target amplification method and detection of the amplicon.
  • the sample preparation usually involves the release of the nucleic acids from compartments in which they are contained, eg. B. cell lysis, but also pre-cleaning, e.g. B. to remove PCR inhibitors of the sample.
  • PCR polymerase chain reaction
  • Metric PCR is described in WO 91/13174.
  • the main advantage is an increased sensitivity in the detection of fluorescence.
  • the authors cite a decrease in competition with the complementary strand as the reason for this improved sensitivity.
  • the detection takes place here by means of gel shift analysis.
  • this method is insufficiently quantifiable and practically cannot be automated.
  • the detection can be carried out using various methods, e.g. B. Analysis by gel electrophoresis (+ Southern blot), dot blot analysis or ELISA techniques.
  • the object of the present invention was to provide a detection method for nucleic acids with an increased dynamic measuring range.
  • the signal dynamics in the detection can be massively improved and a "hook effect" at high amplification concentrations of the detection can be shifted to higher concentrations or even avoided.
  • the signal position can surprisingly be improved up to a Fal tor of 10.
  • the entire measuring range of the detection can be used. A drop in the signals at a high analyte concentration, which makes a quantitative assessment of the concentration of the analyte impossible, can thus be avoided.
  • the invention therefore relates to a method for the detection of a highly concentrated analyte nucleic acid in a sample, comprising the steps of adding at least two primers, of which the extension product of one primer can serve as a template for the extension of the other primer, to the sample and incubation of the formed mixture under conditions in which, depending on the presence of the analyte nucleic acid, extension products are formed for each of the primers, binding of a labeled single-stranded probe to one of the two extension products to form a binding product and immobilization of the extension products on a solid surface, the two types of Primers are used in a stoichiometric ratio of between 1.5: 1 and 10: 1.
  • the method according to the invention is a special embodiment of the so-called hybridization tests, the basic features of which are known to the person skilled in the art in the field of nucleic acid diagnostics. Insofar as experimental details are not given below, the full content of this is "Nucleic acid hybridization", publisher B.D. Harnes and S.J. Higgins, IRL Press, 1986, e.g. B. in Chapters 1 (Hybridization Strategy), 3 (Quantitative Analysis of Solution Hybridization) and 4 (Quantitative Filter Hybridization), Current Protocols in Molecular Biology, Ed. F.M. Ausubel et al., J. Wiley and Son, 1987, and Molecular Cloning, Ed. J.
  • nucleoside triphosphates as described in EP-B-0 324474, the chemical synthesis of modified and unmodified oligonucleotides, the cleavage of nucleic acids by means of restriction enzymes, the selection of hybridization conditions by which a Specificity can be achieved, which depends on the extent of the homology between the nucleic acids to be hybridized, their GC content and their length, and the formation of nucleic acids from nucleoside triphosphates with the aid of polymerases, optionally using so-called primers.
  • a label in the sense of the present invention consists of a directly or indirectly detectable group L.
  • Directly detectable groups are, for example, radioactive ( 32 P), colored or fluorescent groups or metal atoms.
  • Indirectly detectable groups are, for example, immunologically or enzymatically active compounds such as antibodies, antigens, haptens or enzymes or enzymatically active partial enzymes. These are detected in a subsequent reaction or reaction sequence. Haptens are particularly preferred, since nucleoside triphosphates labeled with them can generally be used particularly well as substrates for polymerases and a subsequent reaction with a labeled antibody against the hapten or the haptenized nucleoside can easily be carried out.
  • nucleoside triphosphates are, for example, bromine nucleoside triphosphates or digoxigenin, digoxin or fluorescein-coupled nucleoside triphosphates.
  • the steroids mentioned in EP-A-0 324 474 and their detection have proven to be particularly suitable.
  • Nucleoside triphosphates are ribo (rNTP) - or deoxyribonucleoside triphosphates (dNTP).
  • An analyte nucleic acid is understood to mean a nucleic acid which is the aim of the detection.
  • a highly concentrated analyte nucleic acid is a nucleic acid that is or can be contained in the reaction mixture to more than 10 5 genome equivalents. The upper limit of the concentration is approx. 10 12 genome equivalents / ml.
  • the indication of the concentration in so far as it is referred to as highly concentrated analyte nucleic acids, refers to the concentration of the analyte nucleic acid in the PCR reaction mixture before the start of the amplification, which is available for carrying out the amplification without being diluted again.
  • Analyte nucleic acids are to be understood as meaning nucleic acids of any origin, for example nucleic acids of viroid, viral, bacterial or cellular origin. They can be present in solution, suspension, but also fixed to solids or in cell-containing media, cell swabs, fixed cells, tissue sections or fixed organisms. The nucleic acids are preferably in solution.
  • a template nucleic acid is a nucleic acid to which an essentially complementary nucleic acid strand is newly formed. With regard to the sequence information, the template nucleic acid serves as a template for the description.
  • Denaturation of nucleic acids means separation of nucleic acid double strands into single strands.
  • a multitude of possibilities are available to the person skilled in the art, e.g. B. Treatment by alkali hydroxides, by heat, or by chemicals.
  • a nucleic acid or nucleic acid sequence that is essentially complementary to a nucleic acid is understood to mean nucleic acids or sequences that can hybridize with the corresponding nucleic acid, the nucleotide sequence of which in the hybridizing region is in each case either exactly complementary to the other nucleic acid for directly successive bases or in a few bases is different from that distinguishes exactly complementary nucleic acid.
  • the specificity depends on both the degree of complementarity and the hybridization conditions.
  • the method according to the invention is a special embodiment of an amplification, in particular the polymerase chain reaction (PCR) according to US Pat. No. 4,683,202, but one of the two primers is used in excess.
  • this ratio is chosen to be far lower than is described, for example, in WO 91/13174 or WO 90/03442. It has been found that higher ratios result in a considerable loss of sensitivity and, in addition, a larger dynamic measuring range can be achieved with the ratio according to the invention.
  • the dynamic measuring range is understood to be the measuring range in which an unambiguous assignment of the measured signals to the concentrations of the analyte nucleic acid present in the sample is possible.
  • the dynamic measuring range is limited at the bottom by the measurement error, and at the top by the plateau formation.
  • FIG. 1 (1A and 1B) graphically shows for HBV which measured values for certain known concentrations are obtained with symmetrical PCR (both primers are present in the same concentration, dark) or asymmetrical PCR (lighter). The signal intensities are plotted against the concentration of HBV in geq / ml. It can be seen that the curve for the asymmetric PCR allows a clear assignment to a measurement signal even at higher concentrations.
  • FIG. 1A This comparison is shown in FIG. 1A for test conditions when no sodium chloride is present in the PCR mixture (low salt conditions).
  • Figure IB the case is shown that 625 mM sodium chloride is present (normal salt conditions). From the comparison between Figure 1A and Figure IB it is clear that it is preferred if relatively little salt is contained in the reaction mixture. At lower salt concentrations, the hook effect is surprisingly shifted to higher concentrations, ie the dynamic measuring range is increased compared to higher salt concentrations.
  • the two types of primers are used in a stoichiometric ratio of between 1.5: 1 and 10: 1, preferably between 2: 1 and 5: 1.
  • the narrower range has the advantage that the sensitivity of the detection is particularly high. Care is taken to ensure that the nucleic acid strand to be detected is formed in excess and is marked immobilized.
  • the concentration of the primers is usually selected from a range between 200 mM and 1 ⁇ M.
  • one of the primers preferably the one that is immobilized, preferably from the concentration range of between 150 to 400, particularly preferably between 150 to 200 mM
  • the other primer preferably one that is not (immobilizable) labeled, from a concentration range between 200 and 1000 , particularly preferably selected between 300 to 800 mM.
  • a concentration of approximately 400 mM has proven particularly useful for the second primer.
  • FIG. 2 shows that the dynamic measuring range for HGV ranges from approximately 10 3 to between 10 8 and 10 9 in the case of asymmetrical PCR, while for symmetrical PCR it is only between 10 3 and 10 6 .
  • an added internal control which in the present case is a competitive template (HGV with a modified nucleotide sequence in the intermediate primer range). It is therefore clearly recognizable that the dynamic measuring range with asymmetrical PCR (AI and B1) is much larger than with symmetrical PCR (A2 and B2).
  • the reaction sequence is usually started by making the analyte nucleic acid available with appropriate reagents. This can include changes in pH (alkaline), heat, repetition of extreme temperature changes - 7 -
  • the nucleic acids to be detected are in a sample to which the reagents required for the amplification are added. This is preferably done by adding a solution which contains all the reagents, such as the polymerase, the deoxyribonucleoside triphosphates, which contain a set of primers and buffers. After the reaction mixture has been prepared, the mixture formed is incubated under conditions in which, depending on the presence of the analyte nucleic acid, extension products are formed for each of the primers, in particular using thermocyclers. For denaturing double strands, hybridizing the primers to one or both of the strands of the Analyte nucleic acid or the extension products of the primers formed and enzymatic extension of the primers. The thermocycles are continued until sufficient amplification products have been formed, preferably between 20 and 40 cycles. The amplification products essentially represent copies of part of the analyte nucleic acid to be detected.
  • the analyte nucleic acid can be both DNA and RNA.
  • Preferred analyte nucleic acids are viral nucleic acids.
  • DNA viruses are e.g. HBV and Parvo B 19.
  • RNA viruses such as HGV, is advantageously carried out a so-called RT-PCR, d. H.
  • a cDNA is formed for the RNA, which is then available for amplification. This procedure is also known for symmetrical PCR and is modified according to the present invention by using the specific primer ratio.
  • a primer is understood to mean a compound which binds the analyte nucleic acid and which can be extended by at least one NTP by the enzymatic activity of a DNA polymerase, preferably at its 3 'end, the bound nucleic acid as template nucleic acid for the base sequence of the extension product of the primer serves.
  • Primers are preferably oligonucleotides. - 8th -
  • a set of primers for the PCR contains at least two primers which are complementary to different strands of the analyte nucleic acids. These are usually referred to as “forward" and “reverse” primers.
  • the PCR can use other primers, e.g. B. in a multiplex PCR, d. H. with simultaneous amplification of several sequences of an analyte nucleic acid or several analyte nucleic acids, or with simultaneous amplification of the analyte nucleic acid and a standard nucleic acid.
  • the primers of a set compete with the analyte nucleic acid and the standard nucleic acid.
  • Competitive (symmetrical) amplification is e.g. B. from WO 91/02817 and can be applied analogously to the invention (asymmetrical) format according to the invention. This allows the use of relatively high and therefore more precisely metered concentrations of standard nucleic acids, preferably between 100 and 10 million genome equivalents (geq).
  • a standard nucleic acid is a nucleic acid that is added to the sample in one or more known and defined concentrations, an aliquot of the sample or a reference liquid for the sample.
  • the standard nucleic acid behaves similarly to the analyte nucleic acid in terms of its separability and amplification, but differs from the analyte nucleic acid in a defined manner in its sequence and / or length.
  • a partial sequence in the standard nucleic acid is preferably exchanged for the analyte nucleic acid with the overall length of the amplificate produced remaining essentially the same, although the primer binding sites are essentially identical.
  • HCV the construction of such standard nucleic acids is described, for example, in J. Clin. Microb. 32/8: 1887-1893.
  • One or both of the primers can be modified by linking to a modifying group, for example a detectable or immobilizable group.
  • a modifying group for example a detectable or immobilizable group.
  • An immobilizable primer is understood to mean an oligonucleotide which is structurally modified compared to normal nucleic acid which is essentially complementary to the nucleic acid to be detected in such a way that it has one or more immobilizable groups I.
  • Groups I which can be used for immobilization are, for example, chemical groups which are normally not present in natural nucleic acids and which can be covalently bound to a solid phase, for example by means of a chemical or a photoreaction, or groups or parts of molecules which are different from one another via group-specific interactions Molecule or part of the molecule can be recognized and bound. Such groups are therefore z. B.
  • haptens antigens and antibodies, nucleotide sequences, receptors, regulatory sequences, glycoproteins, for example lectins, or also the binding partners of binding proteins, such as biotin or iminobiotin.
  • Vitamins and haptens are preferred, and biotin, fluorescein or steroids, such as digoxigenin or digoxin, are particularly preferred.
  • the liquid which contains the nucleic acid hybrid of the amplified product and the detector probe when the nucleic acid to be detected was present in the sample is brought into contact with a solid phase which can specifically bind the hybrid via the immobilizable group of the built-in primer.
  • the type of this solid phase depends on the group I capable of immobilization. It preferably has an immobilizing group R which can enter into a binding interaction with I. If the immobilizable group is, for example, a hapten, then a solid phase can be used which has antibodies against this hapten on its surface. Is the immobilizable group a vitamin, such as. B. biotin, then the solid phase can contain binding proteins such as avidin or streptavidin immobilized.
  • residues I and R are biotin and streptavidin. Immobilization via a group on the modified nucleic acid is particularly advantageous since it can take place under milder conditions than, for example, hybridization reactions.
  • the reaction mixture is preferably filled into a vessel before, during or after the formation of the nucleic acid hybrids, the surface of which can react with the immobilizable group.
  • the hybridization reaction with the probe preferably takes place essentially simultaneously with the immobilization. It is also possible to use a solid phase in the form of a porous material, - 10 - such as a membrane, a fabric or a nonwoven, to which the reaction mixture is applied.
  • beads, so-called beads, or latex particles is also possible.
  • the vessel for carrying out the immobilization on such porous materials is preferably a cuvette, a tube or a microtiter plate.
  • the solid phase should have at least as many binding sites for the immobilizable group of the probe as there are nucleic acid hybrids and thus nucleic acids to be detected.
  • the preparation of a preferred solid phase is described in EP-A-0 344 578, to which reference is made in full.
  • a labeled single-stranded probe is preferably used to detect one of the two extension products.
  • the probe is complementary to one of the two strands of the nucleic acid to be detected.
  • a detectable or immobilizable group is provided on the probe.
  • the variety of primer present in excess preferably has an immobilizable group, so that the extension product is labeled immobilizable.
  • the complementary labeled single-stranded probe is then preferably detectably mocked.
  • the labeled single-stranded probe is also preferably used in an excess compared to the expected amount of extension products.
  • the liquid phase from the vessel becomes porous Material or removed from the pelleted beads.
  • the solid phase can then be washed with a suitable buffer, since the binding of the hybrids to the solid phase is very efficient.
  • the amount of detectable label bound to the solid phase is then measured and determined as a measure of the amount of nucleic acid to be detected on the sample. In the case of directly detectable groups, for example fluorescent labels or electrochemiluminescent labels, such as bispyridine-ruthenium complexes, the amount is increased - 11 -
  • the modified nucleic acid is preferably reacted with a mock antibody against the hapten, as described in EP-A-0 324 474.
  • the label on the antibody can be, for example, a color or fluorescent label or, preferably, an enzyme label, such as ⁇ -galactosidase, alkaline phosphatase or peroxidase.
  • enzyme labeling the amount of nucleic acid is measured via the mostly photometric chemiluminometric or fluorometric monitoring of a reaction of the enzyme with a chromogenic, chemiluminogenic or fluorogenic substrate.
  • the measurement signal is a measure of the amount of originally present nucleic acid to be detected and thus possibly of organisms to be detected.
  • FIG. 3 A particularly preferred embodiment is shown in FIG. 3 when the immobilizable group I biotin and the detectable group D is a ruthenium bispyridyl complex.
  • the immobilizable labeled primer 2 is amplified with the aid of PCR (DNA) or RT-PCR (RNA).
  • RNA RT-PCR
  • nn extension products of primer 1 and extension products of primer 2 where m is greater than n.
  • a denaturation step with alkali preferably follows.
  • the detectably labeled probe which is complementary to a part of the extension product of the second primer, which was newly formed from mononucleotides, is added and hybridized with the immobilizable labeled extension product.
  • the probe and the unlabeled strand of the PCR product compete for the marked strand of the PCR product. Since more marked strand of the PCR product is formed in the asymmetric PCR, the competition reaction is reduced and a larger amount of the desired hybridization product is formed for the detection.
  • the hybrid formed from the probe and the biotin-labeled extension product is bound to streptavidin-coated magnetic particles.
  • the buffer for the generation of electrochemiluminescence is added to the magnetic particles and the electrochemiluminescent signal is applied by applying a sp. generated.
  • the flash signal is measured and used for evaluation for the detection of the analyte nucleic acid.
  • the method according to the invention is excellently suitable for a qualitative determination (presence of the analyte nucleic acid in the sample) but also excellent for quantitative determination (determination of the amount of analyte nucleic acid in the sample).
  • the method according to the invention has the advantage that false negative analysis results are reduced or even avoided with highly concentrated analyte nucleic acids. This is probably due to the fact that the high-dose hook effect, which is common in liquid hybridization processes, is either avoided by the process according to the invention or is shifted to even higher concentrations.
  • a high dose high effect is understood to mean the phenomenon that the measured signal initially increases as expected with increasing analyte concentrations, but surprisingly decreases again with increasing analyte concentrations.
  • the method according to the invention can also be automated for routine work as is required in clinical diagnostics.
  • the dynamic measuring range is particularly high in the method according to the invention.
  • RNA transcripts were approximately 900 nucleotides in length, which contained sequences of the 5 'non-coding region of HGV.
  • Internal control RNA is a so-called homologous internal control, which differs from the wild-type (wt) RNA by only 23 nucleotides. This exchanged sequence is used to distinguish between two RNA molecules during the detection, which is done by using different detection probes (see below).
  • the following HGV-specific primers were used for the amplification of both the wt RNA and the internal control:
  • the labeling of the non-coding primer only served the detection method, which is described below.
  • the amplification was carried out with the following temperature profile on a Perkin Elmer Cycler 9600:
  • Perkin Elmer Thermal Cycler PE 9600 cDNA synthesis 30 min 50 ° C initial denaturation 2 mm 94 ° C cycling profile (lOcycles) denaturation 15 sec. At 94 ° C approx. 30 sec. At 55 ° C elongation 30 sec. At 68 ° C following cycling profile (25-30cycles) denaturation 15 sec. at 94 ° C approx. 30 sec. at 55 ° C elongation 30 sec. at 68 ° C + 5s elongation / cycle
  • the PCR products were then used directly in the detection reaction below.
  • the detection was carried out with the help of electrochemilinescence (ECL) on an Elecsys 1010 (Boehringer Mannheim GmbH).
  • ECL electrochemilinescence
  • the reaction temperature for all steps is 37 ° C.
  • 10 ⁇ l of sample are first mixed with 35 ⁇ l of denaturing reagent and incubated for 5 minutes.
  • 120 ⁇ l of the probe solution 50 ng / ml in hybridization buffer
  • 35 ⁇ l of streptavidin-coated magnetic microparticles are added to the reaction mixture and this is incubated again for 10 minutes.
  • the hybrids (bi-labeled HGV strand with Ru-labeled detection probe) are bound to the solid phase (microparticles).
  • the samples are transferred to the measuring cell and the microparticles are bound there by a magnet.
  • the chemiluminescence then takes place in the measuring cell.
  • the Ru complex Under catalysis by TPA, the Ru complex emits flashes of light, the number of which are determined in the measuring cell.
  • the chemistry for electrocheminoluminescence is in J. Electrochem. Soc. 137: 3127-3131.
  • a detection probe with a final concentration of 50 ng / ml is added to the hybridization buffer.
  • the following specific ruthenium bispyride complexes (for the detection of HGV wt or HGV internal control) were used as detection probes:
  • HGV wild type Ru- CCACTATAGGTGGGTCTT SEQ.ID.NO. 3
  • FIG. 2A shows the result using the HGV wild-type St.andard
  • 2B shows the result using the internal control RNA.
  • the key findings are:
  • the asymmetrical use of the primers leads to a significant signal increase.
  • the HGV-specific signal is amplified up to a factor of 7, the signal from the internal control is amplified up to 5.5 times.
  • the number of copies at which the maximum signal is reached is shifted to higher concentrations.
  • the signal maximum is only reached two powers of ten later. As a result, the hook effect only sets in later, false negative results with strongly positive samples can be avoided.
  • Example 1 The general implementation of the experiment is described in Example 1. The changes compared to Example 1 are given below.
  • HBV positive plasma was used as sample material.
  • the virus concentration in the material was determined on the "Eurohep HBV reference plasma sample 1 / genotype A" (Gerlich WH, Heermann KH, Thomssen R et al .: Quantitative assays for hepatitis B virus DNA: standardization and quality control. Virol. Hepatitis Rev. 1995; 1: 53-57). The rehearsal - 17 -
  • the amplification was carried out with the following temperature profile on a Perkin Elmer Cycler 9600:
  • the detection was carried out analogously to Example 1.
  • the symmetrical approach was compared directly with the asymmetrical approach.
  • the following specific ruthenium bispyride complex-labeled probe was used as the detection probe:
  • FIG. 3A shows the result using 625mM NaCl in the hybridization buffer.
  • the hybridization buffer contained no NaCl.
  • the number of copies at which the maximum signal is reached is shifted to higher concentrations.
  • the signal maximum is shifted by at least 3 powers of ten.
  • the hook effect does not occur if there is no salt in the hybridization buffer (Fig. 3B).
  • the avoidance or reduction of the hook effect can also be achieved by reducing the salt concentration during the hybridization.
  • it has often been described in the literature that nucleic acid hybridizations without salt proceed much more poorly and therefore sensitivity is lost (Bryan et al., Pp. 47ff in Nucleic acid hybridization, Editors BD Harnes, SJ. Higgins; IRL press, 1985) .

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Abstract

L'invention concerne un procédé selon lequel l'utilisation d'un surnageant déterminé d'amorces dans une amplification d'acide nucléique et la mise en évidence de la présence des produits de prolongement formés, à l'aide d'une sonde marquée monobrin, permettent d'obtenir une plage de mesure dynamique nettement augmentée, ce qui rend également possible la mise en évidence d'acides nucléiques d'analysat à haute concentration.
PCT/EP1999/001258 1998-02-28 1999-02-26 Procede pour mettre en evidence la presence d'acides nucleiques a haute concentration WO1999043849A1 (fr)

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DE19808534.6 1998-02-28
DE19808534A DE19808534A1 (de) 1998-02-28 1998-02-28 Verfahren zum Nachweis hochkonzentrierter Nukleinsäuren

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Cited By (1)

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JP2002515261A (ja) * 1998-05-15 2002-05-28 アボット・ラボラトリーズ 核酸増幅産物を得るために異なるプライマー濃度を用いるための方法

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US9340833B2 (en) 2010-04-30 2016-05-17 Arkray, Inc. Method for adjusting amplification efficiency of target polynucleotide
EP2383348B1 (fr) * 2010-04-30 2015-03-04 ARKRAY, Inc. Procédé pour le réglage de l'efficacité d'amplification de polynucléotides cibles

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Publication number Priority date Publication date Assignee Title
WO1990003444A1 (fr) * 1988-09-23 1990-04-05 Cetus Corporation Procede de production d'adn monobrin par une reaction en chaine de polymerase
EP0370694A2 (fr) * 1988-11-21 1990-05-30 Eastman Kodak Company Trousse de diagnostic et procédé utilisant un moyen de capture en phase solide pour la détection d'acides nucléiques
EP0418960A2 (fr) * 1989-09-22 1991-03-27 Eastman Kodak Company Procédé pour la dÀ©tection des acides nucléiques utilisant une concentration de primer impartie dans la réaction de polymérase en chaîne
WO1991013174A1 (fr) * 1990-02-26 1991-09-05 The United States Of America, Represented By The Secretary, United States Department Of Commerce Procede de detection par fluorescence d'une sequence d'adn en temps reel

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990003444A1 (fr) * 1988-09-23 1990-04-05 Cetus Corporation Procede de production d'adn monobrin par une reaction en chaine de polymerase
EP0370694A2 (fr) * 1988-11-21 1990-05-30 Eastman Kodak Company Trousse de diagnostic et procédé utilisant un moyen de capture en phase solide pour la détection d'acides nucléiques
EP0418960A2 (fr) * 1989-09-22 1991-03-27 Eastman Kodak Company Procédé pour la dÀ©tection des acides nucléiques utilisant une concentration de primer impartie dans la réaction de polymérase en chaîne
WO1991013174A1 (fr) * 1990-02-26 1991-09-05 The United States Of America, Represented By The Secretary, United States Department Of Commerce Procede de detection par fluorescence d'une sequence d'adn en temps reel

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002515261A (ja) * 1998-05-15 2002-05-28 アボット・ラボラトリーズ 核酸増幅産物を得るために異なるプライマー濃度を用いるための方法
US7037688B2 (en) 1998-05-15 2006-05-02 Abbott Laboratories Method for using unequal primer concentrations for generating nucleic acid amplification products
EP1076721B1 (fr) * 1998-05-15 2007-07-25 Abbott Laboratories Generation de produits d'amplification de l'acide nucleique par utilisation de concentrations inegales d'amorces

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