METHOD FOR AMPLIFYING AND DETECTING NUCLEIC ACID IN A TEST LIQUID

Abstract: Method for qualitatively and/or quantitatively detecting a double-stranded nucleic acid in a test liquid, preferably after an amplification of single-stranded or double-stranded nucleic acid. The 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.

Description: Method for amplifying and detecting nucleic acid in a test liquid. 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. First of all, 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. For this purpose, after the PCR technique has been carried out, it is often necessary to separate the reaction mixture on an agarose gel by means of electrophoresis, separation taking place on the basis of the size of the DNA. Then said gel is transferred with the DNA using the so-called Southern blotting technique to a nitrocellulose filter. Subsequently the probe, a single-stranded nucleic acid provided with a label, which nucleic acid is complementary to a specific section in the DNA to be detected, is allowed to hybridize with the DNA present on the nitrocellulose filter. If hybridization occurs, the label has still to be detected subsequentl . 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. It is one of the essential features of the invention that the 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. According to the invention, 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.. As a marker substance biotin, N-acetoxy-N-2- acetylaminofluorene (AAAF) or the 7-iodo derivative thereof, or a suitable hapten can be used. Preferably use is made of N-acetoxy-N-2-acetyl- aminofluorene as marker substance while, for example, monoclonal anti-Guo-AAF (N-2-acetylaminofluorene bound to guanosine) is used as binding partner. If several molecules of the same marker substance are incorporated into the amplificate, 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. In the detection method according to the invention 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. As 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. As 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. Preferably, 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. The invention is explained by means of the following example. Example Detection of a double-stranded CMV DNA sequence (cytomegalovirus) . 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. PCR technique (in summary) Firstly, primers (for the first strand and for the second strand) are modified using AAAF (AAAF in dimethyl sulphoxide; DNA in a Tris-EDTA buffer) by a standard procedure, by which AAF-marked primers were obtained. Subsequently, a reaction mixture is made up and contains: - target DNA (1 μg of genomic DNA) - 1 μM AAF-marked primers - 1.5 M dNTPs (all four of the deoxy- ribonucleotides) - 10 mM Tris.HCl (pH 8.8) - 50 mM NaCl - 10 mM MgCl 2 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 - Polystyrene microtitration plates are coated with 1 μg/ml of monoclonal antibodies directed against AAF (McAb- -Guo-AAF) by standard procedures at 4 °C, and incubated overnight in 50 mM Tris.HCl, pH 7.5. - The plates are subsequently drained and washed with a PBST/EDTA buffer in order to wash off excess McAb-α- Guo-AAF (PBST/EDTA = phosphate buffered saline, 1% Tween-20 R , 5 mM EDTA pH 8.0) . - 50 μl of AAF-marked PCR amplificate is incubated at 37 °C for 1 hour with McAb-α-Guo-AAF, which has already been coated onto the microtitration plate. - Unbound AAF-marked PCR amplificate and unbound AAF- marked primers are- washed off with 4 x 250 μl of buffer (PBST/EDTA) . - The bound AAF-marked PCR amplificate is detected with McAb-α-Guo-AAF conjugated with a peroxidase enzyme by incubation of said conjugate at 37 °C for 1 hour in the presence of PBST/EDTA and 5% normal sheep serum. - The unbound conjugate is washed off with 4 x 250 μl of buffer (PBST/EDTA) . - The bound McAb-α-Guo-AAF-peroxidase conjugate is detected with tetramethylbenzidine (TMB) and urea peroxide in a citrate buffer at room temperature for 30 minutes. - The reaction is stopped with 50 μl of 1 N H 2 0 4 and the extinction is measured at 450 n (E 450 nm) . Result

Claims: Claims 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, characterized in that the 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. Method for qualitatively and/or quantitatively detecting a double-stranded nucleic acid in a test liquid according to claim 1, characterized in that the insolubilized component is an insolubilized antibody directed against said first marker substance present in the amplified nucleic acid and that the labelled reagent is a labelled antibody directed against said second marker substance. Method for qualitatively and/or quantitatively detecting a double-stranded nucleic acid in a test liquid according to claims 1 or 2 , characterized in that an enzyme is used as label. 4. Test kit for use in the detection according to claim 1, which contains - an insolubilized component, which component has a binding affinity for a first marker substance present in the amplified nucleic acid; - a labelled reagent, which reagent has a binding affinity for a second marker substance, that is identical to said first marker substance. 5. Test kit for use in the detection according to claim 2, which contains .- an insolubilized antibody which is directed against a first marker substance present in the amplified nucleic acid; - an antibody directed against a second marker substance, that is identical to said first marker substance and which antibody is provided with a label. 6. Test amplification kit for use in the detection according to claim 1, containing - oligonucleotides provided with a marker substance, which oligonucleotides are complementary to a section of the original strand of the nucleic acid to be detected; - reagents for carrying out the amplification reaction, - an insolubilized component, which component has a binding affinity for said first marker substance present in the amplified nucleic acid; - a labelled reagent, which reagent has a binding affinity for said second marker substance that is identical to said first marker substance.

Inventors: WIELAARD FRITS, WIELAARD, FRITS
Applicants: AKZO NV, AKZO N.V.
Application year: 1989
Main classification: C12Q1
IPC codes: C12Q1