EP0406280A1 - Procede d'amplification et de detection d'acide nucleique dans un liquide test - Google Patents

Procede d'amplification et de detection d'acide nucleique dans un liquide test

Info

Publication number
EP0406280A1
EP0406280A1 EP89903732A EP89903732A EP0406280A1 EP 0406280 A1 EP0406280 A1 EP 0406280A1 EP 89903732 A EP89903732 A EP 89903732A EP 89903732 A EP89903732 A EP 89903732A EP 0406280 A1 EP0406280 A1 EP 0406280A1
Authority
EP
European Patent Office
Prior art keywords
marker substance
nucleic acid
insolubilized
component
binding affinity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP89903732A
Other languages
German (de)
English (en)
Inventor
Frits Wielaard
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Akzo Nobel NV
Original Assignee
Akzo NV
Akzo Nobel NV
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 Akzo NV, Akzo Nobel NV filed Critical Akzo NV
Publication of EP0406280A1 publication Critical patent/EP0406280A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—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
    • C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
    • 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
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • 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
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the invention relates to a method for qualitatively and/or quantitatively detecting a double-stranded amplified nucleic acid in a test liquid, which nucleic acid is provided on the 5 ' side of the first strand with a first marker substance which is available for a later binding reaction, and is provided on the 5' side of the second strand with a second marker substance that is identical to said first marker substance.
  • the invention also refers to a test kit for use in the said detection.
  • a problem which often occurs in the examination for the possible presence of specific nucleic acids in, for example, chromosomal DNA (deoxyribonucleic acid) is that said specific nucleic acids occur in a small quantity in the chromosomal DNA to be analysed.
  • a technique which has recently become known for overcoming said problem of the small quantity is the so-called PCR technique (polymerase chain reaction) ; said technique is described in detail in Patent Application EP 201,184 by Cetus Corporation (USA) . After said PCR technique has been carried out, which results in a multiplication of the specific nucleic acids if they are present in the chromosomal DNA, these multiplied nucleic acids have to be determined.
  • a much used technique for such a specific determination is the hybridization of the DNA with a specific probe, which is followed by a detection of the hybridized probe.
  • This detection method is described in Patent Application EP 200,362 by Cetus Corporation (USA) .
  • a number of disadvantages are associated with these detection methods used.
  • a specific oligo- nucleotide sequence has to be provided with a label, which label has to be determined after a hybridization has taken place with the nucleic acid to be determined.
  • This laborious detection method has furthermore the disadvantage that only a limited number of test liquids can be analysed for the presence of the specific nucleic acids. Moreover, such detection method also fails to provide a simple possibility for automating the method.
  • test liquid is brought into contact with an insolubilized component, which component has a binding affinity for said first marker substance present in said amplified nucleic ⁇ acid, after which the insolubilized phase thus obtained is brought into contact with a labelled reagent, which reagent has a binding affinity for said second marker substance, after which the determination of said label provides a measure of the quantity and/or presence of the double-stranded nucleic acid to be detected.
  • the invention also relates to a test kit for use in the abovementioned detection which contains an insolubilized component, which component has a binding affinity for a first marker substance present in the amplified nucleic acid and a labelled reagent, which reagent has a binding affinity for a second marker substance used, that is identical to said first marker substance.
  • the method for qualitatively and/or quantitatively detecting a double-stranded nucleic acid in a test liquid is preferably used for that test liquid that is the result of an amplification of a single-stranded or double-stranded nucleic acid.
  • Single-stranded nucleic acid is understood to mean single-stranded DNA or single-stranded RNA.
  • Double-stranded nucleic acid is understood to mean any hybrid nucleic acid such as DNA:DNA or DNA:RNA or even RNArRNA configurations.
  • a test amplification kit according to the invention for use in said detection mentioned should contain oligonucleotides provided with a marker substance, which oligonucleotides are complementary to a section of the original strand of the nucleic acid to be detected, and reagents for carrying out the amplification reaction.
  • the test amplification kit should, moreover, contain an insolubilized component, which component has a binding affinity for a first marker substance present in the amplified nucleic acid as well as a labelled reagent, which reagent has a binding affinity for a second marker substance that is identical to said first marker substance.
  • the method according to the invention for qualitatively and/or quantitatively detecting a double- stranded nucleic acid in a test liquid will hereinafter be explained in more detail.
  • the detection according to the invention preferably takes place after an amplification reaction of the nucleic acid to be detected, e.g. the PCR technique, or any other appropriate amplification technique in which an amplificate is produced.
  • the PCR-technique described in Patent Application EP 201,184, makes use of so-called primers, which are oligonucleotides having a specific nucleic acid sequence, which nucleic acid sequence can hybridize with at least a section of a nucleic acid to be detected.
  • the primers are provided with a marker substance which is available for a later binding reaction before they participate in the amplification reaction.
  • a marker substance which is available for a later binding reaction is understood to mean a marker substance, preferably one molecule of marker substance, which is incorporated in the amplificate on the 5* side of the first strand and on the 5' side of the second strand, which marker substance is recognized in a later reaction by a binding partner which is suitable for the marker substance. Any suitable receptor-ligand combination can be used for such a binding reaction.
  • biotin N-acetoxy-N-2- acetylaminofluorene (AAAF) or the 7-iodo derivative thereof, or a suitable hapten can be used.
  • AAAF N-acetoxy-N-2- acetylaminofluorene
  • N-acetoxy-N-2-acetyl- aminofluorene is used as marker substance while, for example, monoclonal anti-Guo-AAF (N-2-acetylaminofluorene bound to guanosine) is used as binding partner.
  • these marker substances should be incorporated so near to each other on the 5' side of the first strand and also on the 5' side of the second strand that only one marker substance is recognized on the 5' side of the first strand and the 5' side of the second strand by a binding partner.
  • the test liquid will probably still contain oligo ⁇ nucleotides provided with the marker substance. After the amplification reaction oligonucleotides, which did not react with the nucleic acid to be detected, will remain in the test liquid because said oligonucleotides are present in excess in the test liquid.
  • the test liquid is brought into contact with an insolubilized component, which component has a binding affinity for said first marker substance present in the amplified nucleic acid.
  • An insolubilized component may be understood to mean any component which is bound to carrier material.
  • carrier materials may be used for example test tubes, microtitration plates, small rods, beads or small discs manufactured from, for example, glass or plastic.
  • the insolubilized component used is preferably made of an antibody which is directed against said first marker substance and which antibody has been insolubilized by coating onto a solid phase e.g. the inner wall of a well in a microtitration plate.
  • Both the unreacted oligonucleotides which are present in excess and which oligonucleotides are provided with the marker substance, and the amplificate in which one and the same marker substance is incorporated on the 5' side of the first strand and on the 5' side of the second strand will be bound by the insolubilized component which has a binding affinity for said first marker substance. Subsequently, the insolubilized phase thus obtained is brought into contact with a labelled reagent, which reagent has a binding affinity for said second marker substance.
  • a first marker substance present in said amplified nucleic acid and "a second marker substance that is identical to said first marker substance” are understood to mean one and the same type of marker substance with the difference that said first marker substance designates that marker substance having a binding affinity for an insolubilized component, whereas a second marker substance designates that marker substance having a binding affinity for a labelled reagent.
  • a labelled reagent is understood to mean a reagent provided with a label to be determined directly or indirectly, which reagent is directed against said second marker substance.
  • labels may be used for example enzymes, gold sols, dyestuff sols or fluorescent compounds, while the reagent may, for example, be an antigen or fragment thereof, an antibody or fragment thereof or a hapten such as avidin.
  • use is made of an enzyme as label, while as reagent use is preferably made of an antibody directed against the second marker substance. Determination of said label then takes place by using a substrate solution suitable for the enzyme, as a result of which the colour of the solution may change, which provides a measure of the quantity and/or presence of the double-stranded nucleic acid to be detected.
  • Target DNA Isolation by standard procedures (Maniatis et al., Molecular Cloning, 1982) from leukocytes of patients for the purpose of CMV diagnosis.
  • As a control use is made of DNA isolated from leukocytes of CMV-seronegative donors.
  • primers for the first strand and for the second strand
  • AAAF AAA in dimethyl sulphoxide; DNA in a Tris-EDTA buffer
  • This mixture is denatured for 5 minutes at a temperature of 95 °C. Subsequently, 1 unit of Taq DNA polymerase (Thermus aquaticus) is added. For the primer annealing, the mixture is incubated for 1 minute at 37 °C, after which the chain extension subsequently takes place at 55 °C for 5 minutes. Then the strands are again denatured and the subsequent cycle takes place. In total, 30 cycles are carried out in this manner and a PCR amplificate (AAF-marked) is obtained. Detection of the PCR amplificate by the ELISA technique
  • PBST/EDTA phosphate buffered saline
  • Unbound AAF-marked PCR amplificate and unbound AAF- marked primers are- washed off with 4 x 250 ⁇ l of buffer (PBST/EDTA) .
  • McAb- ⁇ -Guo-AAF-peroxidase conjugate is detected with tetramethylbenzidine (TMB) and urea peroxide in a citrate buffer at room temperature for 30 minutes.
  • TMB tetramethylbenzidine

Abstract

Le procédé décrit sert à la détection qualitative et/ou quantitative d'un acide nucléique à toron double dans un liquide test, de préférence après amplification d'acide nucléique à toron unique ou à toron double. L'acide nucléique est situé sur le côté 5' du premier toron avec une première substance marqueur utilisable pour une réaction de liaison ultérieure et est situé sur le côté 5' du second toron avec une seconde substance marqueur identique à la première substance marqueur.
EP89903732A 1988-03-25 1989-03-22 Procede d'amplification et de detection d'acide nucleique dans un liquide test Withdrawn EP0406280A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NL8800750 1988-03-25
NL8800750 1988-03-25

Publications (1)

Publication Number Publication Date
EP0406280A1 true EP0406280A1 (fr) 1991-01-09

Family

ID=19852001

Family Applications (1)

Application Number Title Priority Date Filing Date
EP89903732A Withdrawn EP0406280A1 (fr) 1988-03-25 1989-03-22 Procede d'amplification et de detection d'acide nucleique dans un liquide test

Country Status (2)

Country Link
EP (1) EP0406280A1 (fr)
WO (1) WO1989009281A1 (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5858652A (en) * 1988-08-30 1999-01-12 Abbott Laboratories Detection and amplification of target nucleic acid sequences
FR2648152A1 (fr) * 1989-06-12 1990-12-14 Oris Ind Cie Procede de detection et/ou d'identification d'acides nucleiques par amplification et hybridation en solution et ses applications
DE4001154A1 (de) * 1990-01-17 1991-07-18 Boehringer Mannheim Gmbh Verfahren zur herstellung von modifizierten nukleinsaeuren
FR2722799B1 (fr) * 1994-07-21 1996-10-04 Parteurop Procede d'amplification d'acide nucleique a l'aide d'un nucleoside modifie, et detection du produit d'amplification a l'aide d'anticorps
GB9823005D0 (en) * 1998-10-22 1998-12-16 Harbron Stuart Detection of amplified products in nucleic acid assays

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK171161B1 (da) * 1985-03-28 1996-07-08 Hoffmann La Roche Fremgangsmåde til påvisning af forekomst eller fravær af mindst én specifik nukleinsyresekvens i en prøve eller til skelnen mellem to forskellige nukleinsyresekvenser i denne prøve
CA1290664C (fr) * 1986-03-05 1991-10-15 Nanibhushan Dattagupta Essai d'hybridation pour la detection de sequences de polynucleotides
AU622104B2 (en) * 1987-03-11 1992-04-02 Sangtec Molecular Diagnostics Ab Method of assaying of nucleic acids, a reagent combination and kit therefore

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8909281A1 *

Also Published As

Publication number Publication date
WO1989009281A1 (fr) 1989-10-05

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Owner name: AKZO NOBEL N.V.