WO1996003526A1

WO1996003526A1 - Nucleic acid amplification method using a modified nucleoside, and detection of the amplification product using antibodies

Info

Publication number: WO1996003526A1
Application number: PCT/FR1995/000985
Authority: WO
Inventors: Alain Niveleau
Original assignee: Parteurop Developpement
Priority date: 1994-07-21
Filing date: 1995-07-21
Publication date: 1996-02-08
Also published as: EP0770144A1; FR2722799B1; FR2722799A1; AU3081495A; CA2195480A1; JPH10505230A

Abstract

A nucleotide sequence amplification method wherein a polynucleotide containing the sequence is contacted with oligonucleotide primers, nucleoside triphosphates and/or deoxynucleoside triphosphates containing the various purine and pyrimidine bases needed to synthesise the amplification product, and an enzyme system having DNA polymerase and/or RNA polymerase and/or reverse transcriptase activity, under conditions enabling the sequence of interest to be amplified in a per se known manner. One of the nucleoside triphosphates and/or deoxynucleoside triphosphates is present either in a modified form or in both a modified form and an unmodified form, and, in said modified form, the purine or pyrimidine base is substituted by at least one methyl, hydroxymethyl or acetyl grouping.

Description

A method of amplifying nucleic acid using a modified nucleoside, and detecting the amplification product using antibodies.

The subject of the invention is a method of amplifying a nucleic acid sequence using a nucleoside triphosphate with a modified purine or pyrimide base which is incorporated into the strands of nucleic acid neosynthesized during the amplification operation. . Using antibodies specifically recognizing the modified base, it is possible to detect and / or quantify the amplification product.

In addition, the amplification product can be used in the preparation of multimers containing several copies of the amplified sequence with modified base, and further containing at least one sequence specific for a sequence of interest. Such a multimer can be used for the detection and / or quantification of any polynucleotide sequence, with amplification of the signal.

It is known that the in vitro amplification methods by enzymatic route aim in particular to obtain a large number of copies of a sequence of a polynucleotide present in a sample in small quantity, without it being necessary to isolate and cloning said polynucleotide.

The enzymatic amplification method which was first described is the polymerase chain amplification, better known under the designation PCR (Polymerase Chain Reaction). In this method, two oligonucleotide primers are used, each of which is complementary to a segment of one of the strands of a DNA containing the sequence to be amplified, so that said segments frame said sequence. After heating to denature the DNA, the primers hybridize with their complementary sequence and, in the presence of a thermostable DNA polymerase and of nucleoside triphosphates in excess, a strand of DNA, complementary to the template strand, is synthesized by elongation from the 3 'end of the hybridized primer. The mixture is then denatured again by heat, and on cooling the primers hybridize on the DNA strands originally present or newly synthesized, a new elongation of the primers takes place, and so on. These successive cycles of pairing of primers, elongation and denaturation lead to a doubling of the number of copies of the sequence of interest in each cycle.

A method analogous to PCR, called RT-PCR consists in using as starting material to amplify an RNA, the method then comprising a prior step of reverse transcription of the RNA into complementary DNA. This method is useful in particular when the target is the genome of a retrovirus or when a messenger RNA is used as target to avoid amplifying the introns. Various other amplification methods have been described, and in particular those which consist in using a primer containing an RNA polymerase promoter sequence, so as to obtain by elongation a double strand of DNA whose transcription in the presence of RNA polymerase will lead to l '' obtaining several tens of copies, in the form of transcribed RNA which can enter the amplification cycle. The method includes reverse transcription of the RNA to form a DNA-RNA heteroduplex, then denaturation of the heteroduplex formed, hybridization of a primer to the DNA strand and elongation of the primer, followed by transcription of the double strand obtained, and so on. The strands of RNA formed previously can each give rise to a double strand of DNA which can in turn be transcribed.

One of the disadvantages of the previous methods is the need to denature the duplexes. This operation, which is generally done at a temperature above 90 ° C, requires the use of thermostable enzymes. To avoid the denaturation steps, amplification methods have been devised which can be applied isothermally. An isothermal method consists of a target RNA sequence and a primer of

DNA. Using reverse transcriptase, an RNA-DNA heteroduplex is obtained. By the action of a ribonuclease, the RNA is hydrolyzed. The single stranded DNA is transformed into a double strand by elongation of an appropriate primer in the presence of a DNA polymerase. The double stranded DNA obtained can then be transcribed and several copies of the target RNA are thus obtained, which can in turn enter the amplification cycle. Another isothermal method consists in using the phenomenon of strand displacement using two primers capable of hybridizing with the same strand of target nucleic acid: the product of extension of a first primer located on the 5 'side relative to a second primer, displaces the extension product of the second primer which is thus released in the form of a single strand, while the extension product of the first primer forms a duplex with the starting target strand. The single strand thus formed can in turn enter an amplification cycle using a similar system of two primers. In addition, the first primer may contain on the 5 ′ side, relative to the target recognition site, a recognition site for an RNA polymerase or for a restriction enzyme, which makes it possible to use different routes for the d cycle. 'amplification; see EP-054 3612.

All specialists in the field believe that amplification techniques are called upon to play a considerable role, in particular in the diagnosis of bacterial or viral infections or contaminations.

There is therefore a need for simple and rapid techniques allowing a quantitative evaluation of the amplification products, to replace the techniques of gel electrophoresis and of membrane transfer (blotting). The most sensitive techniques currently are to use a modified nucleoside triphosphate, namely a biotinylated deoxyuridine triphosphate (dUTP) or a dUTP linked to digoxigenin. Amplification products containing the modified nucleotide are detected or quantified by an avidin-enzyme conjugate in the first case and by an anti-digoxigenin monoclonal antibody in the second case. We have now discovered that it is possible to carry out the amplification methods using a nucleoside triphosphate, or NTP (NTP designating here both a deoxyribonucleoside triphosphate and a ribonucleoside triphosphate) whose purine or pyrimide base is modified by a simple substitution such as a substitution with a methyl, hydroxymethyl or acetyl group. It has been discovered that the modified NTPs can be used by the polymerases to synthesize in vitro new copies of a nucleotide matrix, without any efficiency. With antibodies which specifically recognize a modified base nucleoside and which do not recognize a nucleoside containing said base in unmodified form, it is thus possible to detect an amplified sequence of interest in which the modified nucleoside has been incorporated. In addition, surprisingly, the detection technique with antibodies directed against these particular modified nucleosides is, as will be seen in the experimental part below, much more sensitive than conventional detection techniques using a avidin-enzyme conjugate (when using biotinylated primers) or using the digoxigenin / anti-digoxigenin antibody system. The subject of the invention is therefore a method of amplification, by the enzymatic route, of a nucleotide sequence, in which a polynucleotide containing said sequence is brought into contact, oligonucleotide primers, the nucleoside triphosphates and / or deoxynucleoside triphosphates containing the various purine and pyrimidine bases necessary for the synthesis of the amplification product, and an enzymatic system having DNA polymerase and / or RNA polymerase and / or reverse transcriptase activity, under conditions making it possible to carry out, in a manner known per se , an amplification of said sequence of interest, characterized in that one of said nucleoside triphosphates and / or deoxynucleoside triphosphates is present either in a modified form, or both in a modified form and in an unmodified form, and that, in said modified form, the pyrimidine or purine base is substituted by at least one methyl group, hydr oxymethyl or acetyl.

The subject of the invention is also a method for amplifying a nucleotide sequence, and for detecting and / or quantifying the amplification product, in which a polynucleotide containing said sequence is brought into contact, oligonucleotide primers, nucleoside triphosphates and / or deoxynucleoside triphosphates containing the various purine and pyrimidine bases necessary for the synthesis of the amplification product, and an enzymatic system having an activity of DNA polymerase and / or RNA polymerase and / or reverse transcriptase, under conditions making it possible to carry out, in a manner known per se, an amplification of said sequence of interest, characterized in that one of said nucleoside triphosphates and / or deoxynucleoside triphosphates is present either in a modified form, or both in a modified form and in a non-form modified, and that, in said modified form, the pyrimidine or purine base is substituted by at least one methyl, hydroxymethyl or acetyl group, and that the amplification product is detected and / or quantified, according to methods known per se , using an antibody specifically recognizing said modified nucleoside and not recognizing said unmodified nucleoside.

The amplification can be carried out by any known method based on the extension of primers to synthesize a double-stranded DNA, a complementary DNA, or to synthesize a transcribed RNA. Amplification methods can be used, for example, which have been described above, and whose implementation details, which are well known, will not be repeated here.

Among the modified nucleosides, there may be mentioned in particular 5-methyl cytidine, N4-acetyl cytidine, 3-methyl cytidine, N6-methyl adenosine, N6, N6-dimethyl adenosine, 1-methyl guanosine, N2- methyl guanosine, N2, N2-dimethyl guanosine, 7-methyl guanosine, 3-methyl uridine, 5-hydroxymethyl uridine, and 1-methyl inosine.

The triphosphates of these modified nucleosides are known or can be prepared according to known methods.

Antibodies capable of specifically recognizing these modified nucleosides can be prepared according to known methods. They are preferably monoclonal antibodies.

Some of these antibodies have already been described; see C. Reynaud et al., Cancer Letters, 255-262 (1991). During the preparation of the monoclonal antibodies, it has been found that it is generally possible to obtain antibodies which specifically recognize the modified nucleoside. Thus, for example, in the case of anti-5-methyl cytidine antibodies, it was observed that approximately 3% of the hybridoma clones tested secreted specific antibodies recognizing 5-methyl cytidine and not recognizing the cytidine. Similarly, in the case of anti-7-methyl guanosine antibodies, 4% of the hybridoma clones tested secreted antibodies recognizing the modified nucleoside, but not guanosine. It follows from the studies carried out that the nucleoside triphosphate can be used in the same way as the corresponding nucleoside modified in the elongation of a nucleotide primer. When the sequence to be amplified contains only a small proportion of the nucleoside which it is desired to replace with the incorporation of the modified nucleoside phosphate, it is in principle possible to use the nucleoside triphosphate considered in modified form only. However, when the nucleoside under consideration is present at several neighboring positions in the sequence to be amplified, the incorporation of the modified nucleoside triphosphate alone may prove difficult. This is why it is generally preferable to use, for the nucleoside considered, a mixture of the modified form and the unmodified form.

The proportion of modified nucleoside triphosphate relative to the total proportion of the modified nucleoside and of the corresponding unmodified nucleoside can generally vary from 10 to 100 mol% and in particular from 20 to 75 mol%.

Of course, when a mixture of modified nucleoside and unmodified nucleoside is used, the amplification product will contain a variable proportion of the corresponding nucleoside in modified form and in unmodified form, this proportion depending on the nature of the composition of the composition. sequence to be amplified and proportions of triphosphates (modified and unmodified) present during the amplification. It has been found that, for a given sequence to be amplified, and for a given proportion of modified and unmodified triphosphates, the respective proportions of modified and unmodified nucleosides in the amplification product are substantially constant. As a result, the detection of the amplification product using antibodies specific for the modified nucleoside also allows quantification.

To detect the amplification products, the specific antibodies mentioned above are used. Antibodies prepared by immunization using modified nucleosides, conjugated to a protein, have been found to be able to recognize the modified nucleoside incorporated into a strand of DNA or RNA.

The antibodies can be used in the form of antibodies labeled with a tracer agent, for example with an enzyme such as peroxidase or alkaline phosphatase, which allows development with the formation of a colored product, according to known methods.

The revelation of the binding of the specific antibody to the amplification product can also be made with an antibody labeled anti-immunoglobulin, in known manner. For example if the monoclonal antibody used is a mouse IgG, a labeled anti-mouse IgG antibody will be used for the revelation.

Disclosure and possibly quantification using antibodies are well known operations, the details of which will not be repeated here. Furthermore, by using amplification primers linked, in a known manner, to a ligand (for example biotin), it is possible to carry out the detection and / or the quantification on a microtiter plate on which an anti - corresponding ligand (for example avidin). The method of the invention can therefore be used in particular in the field of medical diagnosis or in the food industry, in the search for infections or bacterial or viral contamination.

Another advantage of the amplification process is that it leads to products which can be used for the manufacture of multimers which can be used as signal amplification agents in hybridization methods using nucleic probes.

The principle of signal amplification is known. When the number of copies of the target polynucleotide is low, it is possible, instead of multiplying the number of copies of the target by amplification, to use a detection probe comprising a large quantity of tracer agent, hence amplification of the signal. The specific detection probe for a target to be detected is grafted to a polymer on which are fixed several patterns containing the tracer agent.

Examples of embodiments of multimers which can be used as signal amplification reagents are described in particular in patent applications EP-0317077, EP-0450594, etc. In the case where it is desired to use the amplification product obtained according to the amplification method of the present application as signal amplification reagent, it is advisable to produce a multimer comprising a plurality of patterns constituted by the product of amplification and at least one polynucleotide motif capable of hybridizing with a polynucleotide of interest. In this case, the sequence of the amplification product can be arbitrary: it is sufficient that it is not capable of hybridizing with the target polynucleotide.

The signal-amplifying reagent can be produced, for example, in the form of a polymer carrying in branching a plurality of nucleotide sequences in which the modified nucleoside is incorporated. Polymers bearing branching in a fork, comb, etc. can be prepared in a known manner. ; see for example the patent applications mentioned above.

Since the signal amplification reagent thus contains numerous molecules of the modified nucleoside, detection using antibodies specifically recognizing the modified nucleoside will lead to the obtaining of a signal of high intensity.

The invention therefore also relates to the use of an amplification product which can be obtained according to the amplification method described above.

This use consists in the preparation, in a manner known per se, of a multimer comprising a plurality of units constituted by said amplification product and at least a polynucleotide motif capable of hybridizing with a polynucleotide of interest.

A subject of the invention is also the use of an antibody in the detection and / or quantification, according to a method known per se, of an amplification product obtained according to the method described above, or in the detection and / or the quantification, according to a method known per se, of a multimer obtainable as described above, said multimer being hybrid with said polynucleotide of interest. This use is characterized in that the antibody used is an antibody capable of recognizing the modified nucleoside without recognizing the corresponding unmodified nucleoside. The following example illustrates the invention.

EXAMPLE 1 Amplification of the Epstein-Barr Virus (EBV) Eenoma

a) Primers

Two primers were synthesized located at the ends of a segment of 186 base pairs chosen from the BamHIW region of the EBV genome which is a highly conserved region among the different strains of EBV. Each primer was biotinylated at the 5 ′ end during the synthesis according to the method described by P.R. Langer et al., P.N.A.S. USA Vol. 78 No. 11, 6633-6637 (1981).

The sequences of the primers are as follows: Primer 1: 5 'TTT GTC CCC ACG CGC GCA TA 3' Primer 2: 5 'AGG TGG CGT AGC AAC GCG AA 3' The position of these primers relative to the BamHIW region of the genome d EBV is described in the article by R. Griffais et al., Nucleic Acids Research, Vol. 19, no.14,

3887-3891 (1991).

b) Preparation of the DNA matrix

Burkitt lymphoma cells of the Namalwa line are used, which have intact EBV DNA in a chromosome; see Matsuo et al., Science 226, 1322-1325 (1984) and Gargano et al., Genes Chrom. Cancer, 4, 205-210 (1992). In fact, it has been shown that two EBV genomes are integrated at a known site on chromosome 1; see Lawrence et al, Cell 52, 51-61 (1988). The cells are lysed by sodium hydroxide in a known manner.

Serial dilutions of cell lysates are prepared and used as matrices for amplification according to the PCR method.

c) Amplification

Aliquots of cell lysates (10 microliters) are added to 100 μl of a buffer solution containing 50 mM KC1, 10 mM HC1 (pH 8.4) 1.5 mM MgCl ₂ , 100 μg / ml of gelatin, 200 μM of each of the nucleoside triphosphates dATP, dTTP and dGTP, 40 μM of dCTP, 40 μM of 5-methyl dCTP (hereinafter: 5-MedCTP) (Boehringer Mannheim) 0.5 μM of each primer and 1 unit of Taq polymerase. A Perkin-Elmer 180 thermal cycler is used.

The cycles are programmed as follows: - 1 cycle consisting of 10 minutes at 99 ° C, 2 minutes at 62 ° C and 2 minutes at

72 ° C,

- then 35 cycles consisting of 30 seconds at 95 ° C, 1 minute at 62 ° C and 2 minutes at 72 ° C. At the end of the operation, the samples are kept for 5 minutes at 72 ° C.

d) Characterization

Aliquot parts (10 μl) of each amplification product are subjected to electrophoresis on agarose gel (3% NueSieve agarose and 1% SeaKem agarose).

After migration, the DNA is visualized by fluorescence of ethidium bromide under UV radiation.

The DNA is then transferred to nylon membranes (Hybond N +, Amersham International).

To verify the specificity of the amplification products, hybridization was carried out with a detection probe marked with digoxigenin and capable of hybridizing with a sequence of a DNA strand located inside the sequence framed by the primers 1 and 2. This detection probe was obtained by PCR, in the presence of nucleoside triphosphates and of a dUTP labeled with digoxigenin (digoxigenin 1 1 dUTP - Boehringer

Mannheim), with the following primers:

- primer 3: 5 'CCA GGA AGC GGG TCT ATG 3'

- primer 4: 5 ^* CCT TTG GTG AAG TCA CAA ACA 3 '. An amplimèrc of 106 nucleotides is thus obtained, the sequence of which is included in the sequence of 186 nucleotides amplified previously.

It has been verified that the probe thus obtained specifically hybridizes with the PCR amplification products obtained in c) above. The revelation of the digoxigenin-labeled hybridized probe is carried out using anti-digoxigenin antibodies conjugated with alkaline phosphatase, in the presence of NBT / X-phosphate which provides a blue precipitate (nucleic acid detection kit, Boehringer Mannheim).

e) Immunodetection of 5-methyl cytidine

The incorporation of 5-methyl cytidine into the amplified DNA was demonstrated as follows. After electrophoresis, then transfer to nylon membranes as mentioned above, the membranes are immersed for 30 minutes in a 0.5% solution of blocking reagent (Boehringer Mannheim) diluted in 0.05 M TRIS buffer pH 7.4.

The membranes are washed three times with PBST (phosphate buffer containing 0.2% Tween 20) and then allowed to incubate for one hour at room temperature with an anti-5-methyl cytidine monoclonal antibody. The monoclonal antibody is that described by C. Reynaud et al., Cancer Letters, Elsevier Scientific Publishers Ireland Ltd, 255-262 (1991). The antibody solution is an undiluted supernatant of hybridoma culture containing between 10 and 15 μg / ml of antibody. The membranes are then incubated for one hour at room temperature with anti-IgG goat antibodies

(H + L) of mice conjugated to peroxidase and purified by affinity chrom atography (Biorad Laboratories). Wash again three times with PBST and then rinse with phosphate buffer (PBS).

The substrate is then added (60 ml of a solution of 4-chloro-1-naphthol at 0.3% in methanol, in 100 ml of phosphate buffer containing 0.02% of hydrogen peroxide) and allowed to incubate at room temperature for 30 minutes.

f) Preparation of microplates

Microtiter plates made of carboxylated polystyrene are used; reference:

Niveleau et al. J. Immunol. Methods, 159, 177-189 (1993).

100 μl of a 10 μg / ml solution of N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (Merck) diluted in phosphate buffer pH 7.2 is added to each well. After 10 minutes at room temperature, 100 μl of a solution containing 10 μg / ml of avidin (Sigma) in a phosphate buffer of pH 7.2 are added to each well.

After one hour, the plates are washed with phosphate buffer pH 7.2 containing 0.05% Tween 20, then washed 3 times with phosphate buffer. Amplification products denatured by heat are added at the rate of 50 μl per well. After 15 minutes at room temperature, the wells are washed 3 times with PBST. 50 μl of the anti-5-methyl cytidine monoclonal antibody solution (supernatant of hybridoma culture) are then added. Incubated for 30 minutes at room temperature and then washed 3 times with PBST containing 1% bovine serum albumin

(PBST-BSA).

Then added 50 μl of goat anti-mouse IgG (H + L) antibody conjugated with peroxidase (Biorad Laboratories). After 30 minutes at room temperature, rinsing 3 times with PBST-BSA and then adding the peroxidase substrate which is 3,3 ', 5,5'-tetramethylbenzidine, in solution in dimethylformamide, mixed with 10 % (by volume) of hydrogen peroxide. 100 μl of this mixture are added to each well and the mixture is left to incubate at room temperature for 17-20 minutes, until an intense blue coloration of the reaction product appears. The reaction is stopped by adding IN sulfuric acid (100 μl per well). The optical density at 655 nm is measured by spectrophotometry.

Results

The 10-fold serial dilutions of EBV DNA that were used as templates in PCR amplification contained 2-20,000 copies of the EBV genome.

The PCR amplification results, carried out as in c) above, were compared with a mixture of dCTP and 5-MedCTP and biotinylated primers, and the PCR amplification results carried out in a conventional manner with non-NTP. modified and non-biotinylated primers.

The amplification products were analyzed by agarose gel electrophoresis and visualized by fluorescence of ethidium bromide under ultraviolet irradiation. The electrophoresis analysis shows that in both cases the amplified DNAs have the expected sizes, according to the migration distances. The sensitivity of the reaction, determined by the intensity of the fluorescence on the gel, reaches 200 target molecules.

DNA, both with the PCR according to the invention and with conventional PCR.

Furthermore, by way of comparison, the amplification products obtained by conventional PCR were analyzed with the specific probe conjugated to digoxigenin (obtained in d) above), by Southern hybridization and revelation with an anti-digoxigenin antibody (Boehringer Mannheim). The sensitivity of the reaction reaches 20 molecules of target DNA. The PCR amplification product according to the invention was analyzed in one section after electrophoresis and Southern transfer (see e) above) and in another section on a microtiter plate coated with avidin (see f) above. above).

In the first case (electrophoresis and Southern transfer) the amplification product is denatured by sodium hydroxide and the detection is carried out using the anti-5 MeC monoclonal antibody, the binding of which is revealed as indicated previously by a anti-mouse IgG antibody, labeled with peroxidase. The sensitivity threshold of the method is then two target DNA molecules.

When the analysis is made by depositing the amplification product on a microtitration plate coated with avidin, and detection using the anti-5 monoclonal antibody

MeC as before, it was found that the detection threshold is this time lower than two target DNA molecules.

The intensity of the signal increases with the amount of amplification product, which allows quantification.

Claims

1. Method for amplifying a nucleotide sequence, in which a polynucleotide containing said sequence is brought into contact, oligonucleotide primers, the nucleoside triphosphates and / or deoxynucleoside triphosphates containing the various purine and pyrimidine bases necessary for the synthesis of the product d amplification, and an enzymatic system having DNA polymerase and / or RNA polymerase and / or reverse transcriptase activity, under conditions making it possible to carry out, in a manner known per se, an amplification of said sequence of interest, characterized by the fact that one of said nucleoside triphosphates and / or deoxynucleosides triphosphates is present either in a modified form, or both in a modified form and in an unmodified form, and that, in said modified form, the pyrimidine base or purine is substituted by at least one methyl, hydroxymethyl or acetyl group.

2. Method for amplifying a nucleotide sequence, and for detecting and / or quantifying the amplification product, in which a polynucleotide containing said sequence is contacted, oligonucleotide primers, nucleoside triphosphates and / or deoxynucleoside triphosphates containing the various purine and pyrimidine bases necessary for the synthesis of the amplification product, and an enzymatic system having an activity of DNA polymerase and / or RNA polymerase and / or reverse transcriptase, under conditions making it possible to carry out, in a manner known per se, an amplification of said sequence of interest, characterized in that one of said nucleoside triphosphates and / or deoxynucleoside triphosphates is present either in a modified form, or both in a modified form and in a non-form modified, and that, in said modified form, the pyrimidine or purine base is substituted by at least one group t methyl, hydroxymethyl or acetyl, and that the amplification product is detected and / or quantified, according to methods known per se, using an antibody specifically recognizing said modified nucleoside and not recognizing said non-nucleoside amended.

3. Method according to claim 1 or 2, characterized in that said modified nucleoside is chosen from 5-methyl cytidine, N4-acetyl cytidine, 3-methyl cytidine, N6-methyl adenosine, N6, N6- dimethyl adenosine, 1-methyl guanosine, N2-methyl guanosine, N2, N2-dimethyl guanosine, 7-methyl guanosine, 3-methyl uridine, 5-hydroxymethyl uridine, and 1-methyl inosine.

4. Method according to any one of the preceding claims, characterized in that the proportion of nucleoside triphosphate modified with respect to the total proportion of modified nucleoside and corresponding unmodified nucleoside is from 10 to 100 mol%.

5. Method according to claim 4, characterized in that said propoπion is from 20 to 75 mol%.

6. Use of an amplification product obtainable according to the method of any one of claims 1 and 3 to 5, in the preparation, in a manner known per se, of a multimer comprising a plurality of units consisting of said amplification product and at least one polynucleotide motif capable of hybridizing with a polynucleotide of interest.

7. Use of an antibody in the detection and / or quantification, according to a method known per se, of an amplification product obtained according to the method of any one of claims 1 to 3, or in the detection and / or the quantification, according to a method known per se, of a multimer obtainable according to claim 6, said multimer being hybrid with said polynucleotide of interest, characterized in that said antibody is an antibody capable of recognizing said nucleoside modified without recognizing the corresponding unmodified nucleoside.