WO1992016654A1

WO1992016654A1 - Non-radioactive determination of the presence of a given nucleic acid in a biological sample

Info

Publication number: WO1992016654A1
Application number: PCT/FR1992/000251
Authority: WO
Inventors: Jacques Cohen; Thierry Tabary
Original assignee: Universite De Reims Champagne-Ardenne
Priority date: 1991-03-20
Filing date: 1992-03-19
Publication date: 1992-10-01
Also published as: AU1646092A; FR2674254B1; FR2674254A1

Abstract

A method for exposing one or more synthetically-produced nucleotide RNA or DNA sequences in liquid phase and in the presence of excess deoxynucleotides or nucleotides and a predetermined polymerase respectively, from a primer oligonucleotide hybridized with a nucleic acid which is to be detected in a biological sample. Said method includes exposing in the sample the production of pyrophosphate (PPi) resulting from the synthesis reaction of the specific nucleotide sequence(s).

Description

Non-radioactive detection of the presence of a specific nucleic acid in a biological sample.

The invention relates to the in vitro revelation of the presence of a determined nucleic acid, or the detection of a particular event within a nucleic acid present in a biological sample. More precisely, the subject of the invention is a method for revealing in a biological sample a nucleotide sequence synthesized from a nucleic acid sought in the sample, if necessary after amplification.

The invention provides means of revealing the presence of a desired nucleic acid in a sample, more efficient than those currently available. In this sense, the detection (or revelation) technique according to the invention provides greater reliability with equal detection sensitivity and possibly a higher sensitivity than the techniques known up to now. It is also simpler to implement and therefore lends itself to automation. This aptitude for automation is of particular interest for routine use in the medical sector but also in various types of industries such as the food industry.

The technique of the invention is all the more appreciable since it does not require the use of radioactive products.

This revelation method also has the advantage of being able to be integrated into various methods usually intended for the detection of nucleic acid after synthesis of nucleotide sequences. which derive therefrom (neosynthesized nucleotide sequences), by intervening at the stage of revealing the products of synthesis. The revelation method advantageously allows increased automation of such detection methods.

The invention also provides a kit for the in vitro detection in a biological sample of a determined nucleic acid, this detection involving a step of demonstrating a nucleotide sequence neo-synthesized from this nucleic acid.

A process for the liquid phase revelation of one or more DNA or RNA nucleotide sequences produced by synthesis in the presence respectively of deoxynucleotides or excess nucleotides and of a determined polymerase, starting from a primer nucleotide hybrid with a nucleic acid which one seeks to detect in a biological sample, is according to the invention characterized in that it comprises the demonstration in the sample, of the production of pyrophosphate (also designated hereinafter by PPi), resulting from the synthesis reaction of the specific nucleotide sequence (s).

The specific sequence or nucleotide sequences neosynthesized from the biological sample can be obtained whatever the nature of the nucleic acid sought in the sample, whether it is DNA, RNA or DNA synthesized from the RNA or DNA sought in the sample.

It is sufficient to add the excess nucleotides or deoxynucleotides when the amount of PPi formed by the synthesis reaction is not modified by the presence of these nucleotides or deoxynucleotides in the free state in the mixture.

Note also that the neo-synthesized nucleotide sequences are DNA, cDNA or RNA sequences depending on the nature of the nucleic acid initially present and according to the reaction conditions chosen for the synthesis, in particular according to the nature of the polymerases and nucleotides.

The method according to the invention allows the detection of nucleotide sequences produced by synthesis either of a single copy, or of several nucleotide sequences possibly at the end of several synthesis steps. The disclosure process described above is particularly advantageous suitable for the disclosure of nucleotide sequences produced by synthesis in solid phase or also by synthesis in liquid phase.

The choice to carry out a single cycle or several cycles of synthesis, is determined according to parameters usually considered according to the type of nucleic acid sought and in particular according to the presumed quantity more or less large of copies of the cide nucleic acid that one seeks to detect in the biological sample.

For example, when looking for a rare event such as a viral genome, it is necessary to carry out amplification by several cycles of synthesis beforehand.

On the other hand, upon detection of an infection by a pathogenic organism, amplification may or may not be necessary depending on the stage of the infection.

Amplification can also be avoided when seeking to detect a mutation in the human genome. The deoxynucleotides used for this DNA synthesis reaction are also generally designated by the abbreviation dXTP which designates the four deoxynucleotides dATP, dGTP, dCTP, dTTP.

The polymerase chosen to carry out the synthesis reaction is a polymerase usually used in this type of reaction, whether for the elongation of a single copy from a nucleic acid present or for the multiplication of the number of copies for example for the extension of an exponential number of copies for example by means of a PCR type reaction (Polymerase Chain Reaction). Such polymerases are for example thermostable polymerases of the Taq polymerase type or any enzyme having a polymerase activity 3 ', 5,' capable of synthesizing under determined conditions the complementary strand of a nucleic acid present in the sample.

Non-thermostable polymerases such as Klenow polymerase can also be used, in particular when the detection of PPi is carried out on a simple copy of the sought nucleic acid. An RNA polymerase will be chosen to synthesize RNA.

The nature of the polymerase enzyme as well as the quantity of deoxynucleotides present in the synthesis reaction can also advantageously be determined as a function of the length of the nucleic acid from which one or more copies are sought to be synthesized and as a function of the nature of the neo-synthesized sequence (s) depending on whether it is DNA, in particular cDNA, or RNA.

Furthermore, the synthesis of RNA or DNA can be carried out for example from a specific primer of a given gene capable of hybridizing with an acid. nucleic acid, in particular RNA sought after or from non-specific primers such as polynucleotides (poly A for example).

The synthesis of cDNA can be carried out by reacting a reverse transcriptase (or reverse transcriptase, RT) from a primer capable of specifically hybridizing or from a primer of polynucleotides (poly A, poly T), with an acid. nucleic acid sought.

The stringency conditions used to carry out the hybridization are the usual conditions used in particular in PCR reactions, in particular strong stringency conditions to ensure the specific hybridization of the primers on the nucleic acids used as templates.

Conditions of lower stringency are acceptable when the nucleic acids which one seeks to detect are of such a nature or in such a quantity that mismatches of the primers used do not modify the result of the synthesis cycle (s) or allow '' simultaneously amplify the different members of a gene family.

It is understood that the syntheses referred to above are carried out either from double-stranded nucleic acid, after a denaturation step, or from single-stranded nucleic acid.

It is quite interesting and fundamental in the context of carrying out the invention, to note that the quantity of pyrophosphate produced within the framework of the reaction for the synthesis of particular nucleotide sequences, coincides in a directly proportional manner with the quantity of nucleotides incorporated into the sequence synthesized during the reaction. Consequently, the quantity of PPi produced is directly proportional to the quantity of nucleotide sequences, the length of which is known, produced during the synthesis. The amount of PPi produced therefore makes it possible to deduce the weight of nucleotide sequences obtained and consequently, as a function of the number of synthesis steps, it is possible to deduce the amount by weight of nucleic acid present in the sample.

One can also express the quantity of PPi produced in molar quantity, that is to say in molar quantity of incorporated nucleotides which corresponds to the molar quantity of synthesized nucleotide sequences (also designated by neo-synthesized nucleic acids or neo-synthesized nucleotide sequences ).

The revelation method according to the invention therefore makes it possible to very quickly obtain results as to the quantity of neo-synthesized nucleic acids, and this for example by reference to a preset curve which would translate the quantity of nucleotides incorporated in a nucleic acid (for example on the ordinate) as a function of the quantities of pyrophosphate produced during this incorporation (for example on the abscissa).

Once established, such a curve can be used as a reference, whatever the composition of the nucleic acid sought, since the production of pyrophosphate is independent of the nature of the nucleic acid (DNA, RNA), of the nature and of the order (sequence) of the nucleotides.

The implementation of the revelation method, the definition of which is given above, can be adapted as required. We can indeed measure the amount of pyrophosphate produced, at the end of the synthesis when it is unique or at the end of all the synthesis steps, for example at the end of a determined number of PCR cycles.

It is also possible to measure the quantity of pyrophosphate produced at a determined time or at determined time intervals during the synthesis reaction.

According to a first embodiment of the disclosure process of the invention, the demonstration and, where appropriate, the quantitative assay of the pyrophosphate produced by synthesis of the nucleotide sequence (s) from a nucleic acid present in a sample, are carried out after a determined number of gene amplification cycles, in particular by chain polymerization (PCR).

In certain cases it may be advantageous to demonstrate the sole production of pyrophosphate in a sample placed under conditions specific to the synthesis of particular nucleotide sequences, this demonstration not requiring any subsequent use of the assay. Indeed, depending on the nature of the nucleic acid that one seeks to determine, it is not necessarily essential to obtain a quantitative result.

On the contrary in other cases the (quantitative) assay of PPi must be carried out to determine exactly the quantity of neosynthesized nucleic acids.

The compatibility of the method of the invention with the realization of the revelation at different times during several synthesis stages, makes it possible in particular in the case of gene amplification, for example by PCR, to carry out this revelation at regular predetermined intervals and possibly also after all the stages of gene amplification. This makes it possible to determine the kinetics of the synthesis and thus to differentiate the background noise generated by the presence of contaminants, in particular the PPi contaminating the reagents, from the specific reaction insofar as the kinetics of the background noise is different from that of the specific reaction.

According to another embodiment of the disclosure process of the invention, the use of gene amplification prior to disclosure is not necessary. This is particularly the case when the nucleic acid which it is sought to detect is present in an amount sufficient to be detected on the basis of a single synthesis cycle. In this case, each nucleic acid present in the biological sample is copied during a single step.

In addition to the efficiency of the revealing method, due to the nature of the marker chosen for revealing the presence of a nucleic acid in a given biological sample, the method described in the preceding pages also has the advantage of allowing detection and if necessary, an easy dosage of the pyrophosphate produced. Indeed, all the appropriate chemical or physical means making it possible to detect and if necessary to dose pyrophosphate can be used within the framework of the invention.

In addition, the detection or quantification of the pyrophosphate produced is advantageously carried out in one time.

By way of example, the measurement of the quantity of pyrophosphate produced corresponding to an quantity of neo-synthesized nucleic acids can be determined by a physical method, for example by NMR. This determination results from the application of the principle according to which the nuclear magnetic resonance spectrum of pyrophosphate is different from that of a nucleotide triphosphate. The means used to carry out the measurement are the conventional NMR means.

According to another embodiment of the disclosure process of the invention, the amount of pyrophosphate produced is determined by chemical means, this comprising suitable enzymatic reactions, and generally any chemical reaction dependent on PPi, for example when it acts as a reactant, the reaction giving rise to a colorimetric, luminescent or other type of signal. Advantageously, one or more linked enzymatic reactions will be used, in particular any cascade leading to a colorimetric signal or a bioluminescence reaction.

For example, the quantity of pyrophosphate produced within the framework of the revelation process can be determined at the end of a cascade of enzymatic reactions comprising the following steps: the sample likely to contain the desired nucleic acid and consequently likely to contain the pyrophospahte produced at the end of the synthesis or syntheses of nucleotide sequences is brought into contact with Adenosine 5'-Phosphosulfate (APS), under conditions allowing the formation of ATP,

the ATP obtained at the end of the previous step is placed in the presence of luciferin, under conditions such that it is degraded by producing a bioluminescent signal in the form of photons, - if necessary, the intensity of the bioluminescent signal is read and correlated to the quantity of PPi that has reacted.

The amount deduced from pyrophosphate having reacted in the above steps can then be correlated to the amount of neo-synthesized nucleic acid according to what has been explained above.

Before measuring the amount of PPi produced during the reaction for synthesizing the nucleotide sequences, a measurement of the background noise can be carried out. For this, the reagents used for the revelation and if necessary the quantification of the PPi produced during the synthesis of the nucleotide sequences are brought into contact with the medium in which this synthesis has taken place, with the exception of at least one reagent necessary to trigger the reaction with PPi. The background noise (or "white") reading is then performed.

Another example of chemical revelation leads to a colorimetric signal as obtained with a reaction such as the following: the sample capable of containing the desired nucleic acid, previously subjected to one or more synthesis cycles, according to the methods described in the preceding pages, and therefore likely to contain PPi produced at the end of these syntheses, is brought into contact with a fructose 6-phosphokinase, pyrophosphate dependent (aldolase). The development system for highlighting the reaction product can be the NADH / NAD system using a glycerophosphate dehydrogenase. As before, the colorimetric signal obtained is then correlated to the amount of PPi formed in the sample.

These measurements can, as has already been said, be carried out at the end of the reaction or during sampling. sequential at regular intervals during gene amplification.

The results of the measurement can be expressed in different ways: it can be the measurement of the raw signal which is linear on a logarithmic scale, the molar quantity or by weight of PPi detected as equivalent to the incorporation of nucleotides, the number of molecules or number of copies of a synthesized nucleic acid sequence, of given length (neo-synthesized sequence).

The revelation technique for the detection and the possible assay of PPi not being analytical, the specificity of the reaction can for example be ensured by carrying out the synthesis of the nucleotide sequences starting from the sought nucleic acid, by gene amplification by a nested PCR type system. This system has in particular been applied to improving the specificity of gene amplifications by Porter-Jordan J. Med. Virol 90 30: 85-91. Another possibility for verifying the specificity of the synthesis reaction consists in carrying out a hybridization with a probe specific for the desired nucleic acid, according to the usual solid phase techniques, at the end of the syntheses and then in copying the retained probe after hybridizations.

Another interesting feature in the context of the invention is that the developing process described above can be carried out in homogeneous phase and therefore without any purification or separation of the pyrophosphate product in 1 ^• biological sample. This revelation in homogeneous phase can be carried out even when the source nucleic acid (specific nucleic acid that one seeks to detect in the sample) is fixed on a solid phase.

The homogeneous phase development according to the invention is a liquid phase development without physical separation of the incorporated nucleotides on the one hand from the unincorporated nucleotides and / or their degradation products on the other hand.

Taking into account the characteristics set out above, the process of the invention can be designated by the expression HPPPIM or H3PIM (English abbreviation for Homogen Phase PPi Measurement).

When it is preferable or necessary for the sensitivity of the method, the step of highlighting and optionally dosing the pyrophosphate is nevertheless preceded by a treatment of the reaction medium under conditions making it possible to eliminate the ATP present in the medium and dATP when it is present, for example by adding a hexokinase leading to the formation of ADP. The PPi contaminating reagents, in particular deoxynucleotides not used during the synthesis, can also be removed by adding a pyrophosphatase, itself neutralized following the removal of the deoxynucleotides, for example by heating.

The elimination of the contaminating PPi makes it possible to attenuate or even eliminate the background noise generated by the reagents used during the synthesis of the nucleotide sequences, the sensitivity of the detection of the presence of PPi then being able to reach 10 ^{* 12} M PPi.

In addition, it may be important to ensure, before carrying out the reaction, the specificity thereof since any neo-synthesized nucleic acid is taken into account in the detection reaction of PPi produced by the method of the invention.

If necessary, if necessary depending on the nature of the sample, the skilled person can choose according to conventional techniques, very specific primers and parameters for amplification, also specific. Measuring kinetics or measuring the background noise level of a control sample can also be used.

The invention also relates to a method for the quantitative determination of the content of nucleotide sequences synthesized from a nucleic acid whose presence is sought to be detected in a biological sample, characterized by:

- carrying out a determined number of cycles of synthesis of nucleotide sequences (neo-synthesized nucleotide sequences) by extension from a primer oligonucleotide hybridizing with the desired nucleic acid when it is present in the test sample, presence of an excess of deoxynucleotides or nucleotides depending on whether the synthesis of DNA or RNA and of a determined polymerase is sought,

the assay of the quantity of pyrophosphate produced during the synthesis of the nucleotide sequences and, where appropriate, the ratio of this measurement to the background noise, the determination of the quantity of neo-synthesized nucleotide sequences present in the biological sample.

Advantageously, the amount of neo-synthesized nucleic acid present in the sample is determined by matching the measurement of the quantity of pyrophosphate produced during synthesis from the desired nucleic acid initially present in the sample, with quantities of pyrophosphate obtained by synthesis correlated beforehand with known quantities of given nucleotide sequences, reported for example on a preset curve or any preset diagram or table.

The applications of the invention are numerous and relate to very different sectors. One can in particular have recourse to the methods described in the preceding pages for the detection in vitro and if necessary the quantitative assay of a nucleic acid characteristic of an infection by a foreign organism for example viral or bacterial in a biological sample, in view the diagnosis of a pathological condition or the characterization of contamination in a given biological environment.

It is therefore possible to use the methods of the invention to carry out a method for diagnosing the presence of a determined nucleic acid in a biological sample, for example a blood or serum sample, this method comprising the steps of synthesizing at least one copy of the nucleic acid which it is sought to detect and of revealing the presence of neo-synthesized nucleic acids, by highlighting and if necessary the assay according to the protocol described above for the pyrophosphate produced by the synthesis reaction.

The means of the invention are suitable for demonstrating any type of nucleic acid whether it is rare (that is to say in the form of a very low number of copies) or not in a given biological sample. In particular any diagnosis including advantageously, steps of gene amplification in particular by PCR can benefit from the means of disclosure described above. By way of example, the above means are suitable for diagnosing an infection with a virus responsible for AIDS, such as the HIV virus (also known as HIV).

These means also allow the diagnosis of hepatitis B, hepatitis C, viruses such as CMV or HPV, and also the diagnosis of the presence of mycobacteria.

Another medical application of the invention is the detection within the genome of genetic anomalies, which may result from substitution, addition, deletion, or inversion of nucleotides within a nucleic acid present in a biological sample.

This technique can, for example, be used in the case of prenatal diagnosis of certain conditions usually using PCR techniques.

Another application of the method according to the invention is tissue typing.

This typing can be carried out from a copy of the DNA of an individual produced when this DNA is specifically retained by an oligonucleotide on a solid phase. Gene amplification specific for alleles or cross-reactive groups can also be used with a specific nucleotide and a non-allele nucleotide. Histocompatibility typing can then be carried out using a series of reactions making it possible to determine which alleles the subject in which the research is carried out is carrying, at each locus of the Major Histocompatibility Complex. Another type of application of the techniques of the invention, in different fields such as for example experimentation on a pharmacological model or within the framework of a physiopathology examination, is the quantification of the expression of a given gene. in a cell.

For example, one can quantify the RNA coding for a chosen interleukin by determining the quantity of copies of this RNA which can be produced by the cell, in the form of cDNA or RNA.

The technique of the invention offers in particular the completely advantageous advantage of not requiring purification to separate the RNA produced by the specific gene studied from other cellular RNAs.

However, the medical field is not the only one concerned by the applications of the method according to the invention. On the contrary, it is possible to put into practice the means described above in any process of checking, with a biological material, the presence of contamination by microorganisms, for example in the food industry, to detect the presence of contamination. particular in food samples.

In general, the invention provides means for carrying out the quality control of reagents, whether or not used in the medical field. For example, the invention offers means for any quality control operation.

By way of example, mention may be made of the search for bacteria of the genus Listeria in milk products.

The invention also provides means enabling the control, for example, of the transfer of a determined heterologous nucleic acid into given cell layers with a view to their recombination; the Demonstration of the recombination can be carried out by detection of neosynthesized nucleic acids from the heterologous nucleic acid which has been integrated into the cell and this by means of the pyrophosphate produced by the synthesis reaction.

The invention further relates to a kit for the in vitro detection of the presence and optionally of the content of a determined nucleic acid in a biological sample by detection of neo-synthesized nucleotide sequences of DNA or cDNA, characterized in that 'He understands:

the deoxynucleotides dATP, dTTP, dCTP, dGTP, a polymerase determined preferably a polymerase, for example a thermostable polymerase suitable for gene amplification by PCR, such as Taq polymerase,

a reagent for revealing in a liquid phase medium the presence of pyrophosphate, this reagent can comprise one or more enzymes dependent on pyrophosphate, capable of behaving as bioluminescent marker (s) or as colorimetric marker (s) ).

The invention also relates to a kit for the in vitro detection of the content of a determined nucleic acid in a biological sample by the detection of neo-synthesized sequences of RNA, characterized in that it comprises:

- ATP, UTP, CTP, GTP nucleotides,

- a determined RNA polymerase, for example a polymerase suitable for gene amplification by PCR,

a reagent for revealing the presence of pyrophosphate in a liquid phase medium by implementing the method according to the invention, for example one or more enzymes dependent on pyrophosphate, likely to act as a bioluminescent marker (s) or as a colorimetric marker (s).

This kit can for example be used to detect cDNA.

As was specified above, the revelation method according to the invention makes it possible to envisage the production of automatic systems for the detection of nucleic acids in determined samples, such automatic systems being able to include the following means:

- a device making it possible to take, if necessary sequentially, microvolumes in the reaction medium, to incubate these microvolumes in a reading cell for example a luminometer in the presence of the reagents allowing the characterization of the presence of PPi and optionally to assay the quantity of PPi, with the exception of at least one reagent necessary for the reaction with the PPi (missing reagent), and this for carrying out the measurement of the blank (or control),

- Means making it possible to add the missing reagent, to incubate the time necessary for the reaction and to measure the signal again in the reaction medium after the initiation of the reaction using the PPi, according to predetermined times.

Possibly after the microvolumes have been removed, the device makes it possible to incubate this sample with a reaction mixture for destroying certain constituents, in particular for destroying dATP or ATP and to heat again to destroy the enzyme which acted on dATP and / or ATP.

The examples which follow describe the implementation of the method of the invention for detecting an amplified DNA. Other characteristics and advantages of the invention also appear in the figures.

'F I G U R E S

- Figure 1 :

Homogeneous, non-radioactive phase, quantitative detection of gene amplification

Evolution of the bioluminescence signal as a function of the quantity of neosynthesized HIV nucleic acid, in homogeneous phase.

Detection of HIV DNA diluted in normal human genomic DNA. The DNA of the ACH2 cells containing a copy of HIV per cell was diluted in series to a constant quantity of DNA of 1 μlG, then subjected to a PCR reaction for the detection of HIV. The abscissa data are expressed in the form of log (1 / G DNA). The ordinate data are expressed in the form of log (signal). A single cell contains up to 10 ^"12 μlG of DNA and a single copy of HIV. A log / log representation has led to a linear relationship between the number of copies of HIV available and the amount of synthesized DNA detected using the HPPPIM system.

- Figure 2:

Gene amplification (one copy per genome) - Revelation in bioluminescence in homogeneous phase Kinetic curve obtained by PPi measurements by bioluminescence during a determined number of cycles of gene amplification. The number of PCR cycles is indicated on the abscissa with the bioluminescence signal on the ordinate. The signal is expressed in nannomoles of nucleotides per PCR tube. - Figure 3:

Calibration curve representing bioluminescence signals as a function of given quantities of PPi.

- Figure 4:

Detection and quantification of PPi. The abscissa represents the concentration of PPi detected. The scale is given in the form of a log (l / (mole PPi / luminescence tube)). The ordinate represents the bioluminescence signal on a logarithmic scale.

- Figure 5:

Quantification of the expression of the DQ gene of the HLA complex from mRNA. The photo made it possible to detect a DNA fragment of 271 bases amplified among the mRNAs extracted from the cells, subjected to a stimulating agent for different periods of time consisting of a monoclonal antibody D1.12 anti-HLA DR for 1, 2, 4 , 8 and 24 hours on lines 2 and 6 respectively. Line 1 from the top is the unstimulated control sample representing the basic level of DR HLA expression.

The signal obtained using the H3PIM system is reported in front of each line and makes it possible to compare the intensity of the ethidium bromide staining with the quantitative signal measured. The signal is expressed both as an arbitrary unit of luminescence / second (AU) and as the molar concentration of neosynthesized nucleotides. The background noise has been subtracted. - Figure 6:

Quantification of DNA synthesized using random primer hybridization on phage lambda DNA. The amount of lambda DNA introduced into the hybridization reactions of the primers at random is reported on the abscissa. The ordinate shows the amount of newly synthesized DNA expressed as moles of incorporated nucleotides.

E X E M P L E S

I Example of detection of gene amplification (Saiki et al _/ 1988, Science 239: 487) by bioluminescence

1) Serial dilution of viral DNA in human DNA

The DNA of the ACH2 line (described by K. Clouse J. Immunol 1989, vol. 142: 431-438), containing a copy of the HIV virus by human genome (Schnittman et al, 1990, Ann. Int. Med., 113: 438) was diluted serially in a constant amount (1 μg) of DNA from a healthy individual. The HIV genome was detected using the 25-base LAV1 probe (D'AURIOL L., Genset, Paris) and the 28-base SK primers (Ou et al, 1988, Science, 239: 295) for amplification of genes for 30 cycles. The parameters of the PCR were: heating for 2 minutes at 94 ° C. then hybridization at 60 ° C. for 3 minutes and elongation at 72 ° C. for 3 minutes. A final elongation step was carried out for 7 minutes at 72 ° C. .

After amplification by PCR the signal was measured and represented in the form of a Log base 10 of the number of events (each event corresponding to a bioluminescence signal) per minute on the ordinate, against the Log base 10 of the dilution of the DNA from the HIV positive line. I. l0 ^"12 g of DNA corresponds approximately to an equivalent genome and a copy of the virus. The signal is detectable above the background noise up to 10 copies by manual manipulation. The reagents used in the reaction have the following characteristics:

- PPi: PPI 10 H ₂ 0: Merck Ref. n ^β 6591 (PM 446.06)

- Tris: Merck Ref. n ^β 8382 (PM 121.14)

- High resistivity water (> 10 MOhmC): millipore ion exchange (MilliQ)

- Glacial acetic acid 96%: Merck (PM 60.05; 1 liter = 1.06 kg)

- APS: adenosine 5'-phosphosulfate (sodium salt) (PM 427.3) 96% purity preserved at -80 ^β C:

Sigma Ref. n ° A9266 (delivered in dry ice) 0.1 mM solution in 0.1 M Tris-AcOH buffer pH 7.75

- Sulfurylase: adenosine 5'-triphosphate sulfurylase (ATP: sulfate adenylytransferase: EC 2.7.7.4): Sigma Ref. n ^β A5761

Solution at 1.8 U / ml of 0.1 M tris-AcOH buffer pH 7.75

- Bioluminescence reaction medium: Sigma kit: adenosine 5'-triphosphate (ATP) Bioluminescent Assay Kit, Ref. FL-AA. The reaction mixture (FL-AAM) contains luciferase, luciferin, MgSO, EDTA, DTT, BSA in tricin buffer. It is diluted to 1/200 in FL-AAB buffer.

Reaction:

Sample

First, 100 μl of AAM diluted to 1/200, 68 μl of water, 12 μl of sulfurylase and 5 μl of the sample containing the HIV DNA and the neo-synthesized nucleic acid are brought into contact. gene amplification by PCR.

The blank is read after at least 3 minutes of contact. Then add 10 μl of PSA which triggers the reaction with the PPi produced in the sample during gene amplification. Reading is carried out after exactly one minute of luminescence (average of the emission for 2 seconds over 30 seconds).

It is possible to carry out an internal calibration by successive addition of 10 " ¹¹ , ^10" , 10 ^"9" mole of PPi in the reaction tube under 10 μl and read 30 seconds after 1 mm of incubation.

Establishment of an external reference curve

Another possibility is to perform an external calibration in the following ways:

100 μl of diluted AAM, 68 μl of water, 12 μl of sulfurylase and 10 μl of APS are brought into contact and then the blank is read. Then add 10 μl of diluted PPi solution to have an amount of

10 "1 ¹ 1 ¹ (10 ^{* 6} M in the luminescence reading tube) mole of PPi in the tube and the reading is carried out after exactly one minute, over 30 seconds.

Add 10 μl of diluted PPi solution to have a quantity of 10 ^"10 (10 M in the luminescence reading tube) ole of PPI in the tube and continue in the same way with an addition of 10 μl of PPi to have 10 " ⁹ mole of PPi in the tube.

The following table summarizes the results obtained:

2) Kinetic of the detection of a unique genomic copy

An 800 base fragment of the human esterase C1 gene (Tosi et al, 1987, Biochemistry 26>: 8516) was amplified using primers of 24 bases AS1-AS2 of human DNA (1 μg / tube) . The tampon was prepared according to the manufacturer's instructions (PERKIN ELMER CETUS, Norwalk, CT, USA). The PCR steps were as follows: heat to 94 ° C for 2 minutes, hybridize to 60 ^β C for 3 minutes, extend at 72 "C for 3 minutes. The samples were tested after, 20, 25, 26, 27, 28, 30 and 35 cycles PCR was performed on a Techne PHC-1 device (Osi-Paris, France) (Saiki et al, 1988).

The samples were prepared according to the technique described in part 1) by replacing the HIV DNA with the gene of interest.

II - Quantitative determination of the expression of a gene, using mRNA

The expression of the HLA class II system DQ gene products in normal human marrow B cells, after stimulation with different concentrations of anti-HLA class II monoclonal antibodies or interferon alpha, was tested according to the following technique: mRNA was extracted using the RNAzol kit (Bioprobe Systems, Montreuil sous Bois, France), cDNA was synthesized using reverse transcriptase (BRL, Cergy Pontoise, France) and oligodT (Promega, Coger, Paris, France) according to the manufacturer's instructions and then the gene amplification was carried out using primers GH-46, GH-50 (Sharf et al, 1988, Science 233: 1076) in Cetus PCR buffer on a Perkin-Elmer / Cetus PCR apparatus. The PCR parameters were: 24 heating cycles at 94 ° C for 1 minute, hybridization at 57 ° C for 1 minute and extension at 72 ° C for 1 minute.

III- Quantification of the DNA synthesized by implementing one-step procedures, hybridization of primers at random

The phada lambda DNA was subjected to random hybridization using hexanucleotides allowing the synthesis of DNA in a time (Feinberg et al, 1983, Anal. Biochem., 132: 6). Random hybridization was performed on 8.75, 25 and 87.5 nanograms of lambda phage DNA according to the manufacturer's instructions (Boehringer-Mannhei, Penzberg, FRG).

IV - Results

1) Detection of PPi and quantification

Using the standard conditions reported in Part I, the PPi could be detected and quantified at a concentration level of 10 ^"9 to 10 ^{" 12} moles per 200 μl / luminescence reading tube (2.10 ^"4 to 2.10 ^{" 7} M in the PCR reaction tube). As shown in Figure 4, the signal was linear for 10 ^"9 to 10 ^{" 11} moles / tube (2.10 ^"4 to 2.10" ^) using the logarithmic scale.

No interference in the measurement of PPI was observed by addition of heated urine or serum samples. The heated serum added a significant amount of ATP to the measurement of the blank but did not prevent the subsequent measurement of the PPi formed.

2) Kinetics of gene amplification of a single copy of human DNA

The kinetics of gene amplification as a function of the number of PCR cycles is shown in FIG. 2. The PCR products were detected from the 20th cycle. After an exponential phase, the production of the reaction decreased to a plateau phase.

3) Detection of HIV DNA diluted in series in normal human genomic DNA

As shown in Figure 1, the amount of amplified HIV DNA is proportional to the number of copies of HIV present, leading to a linear signal according to the logarithmic scale. The threshold for detection of HIV was approximately 10 copies in this experiment. Ethidium bromide staining and hybridization with a ³² P-labeled probe led to the same threshold for detection in this experiment.

4) Quantitative detection of the expression of the DQ gene of the HLA system, from the mRNA

The expression of the DQ gene of the HLA complex (11 ^th HLA Workshop, 1991) was between 1 and 20 times as shown in Figure 5, while the background noise level was 163 AU (AU: arbitrary unit corresponding to the signal of bioluminescence of the machine), leading to a signal to noise ratio (signal / noise) of 5 to 40. This signal can be standardized using a range of PPi produced by means of an external reference curve, according to the technique described in Part I.

The intensity of the bands stained with ethidium bromide was parallel to the signals detected using the HPPPIM system (H3PIM) as shown in Figure 5.

5) Quantification of the DNA Synthesized by the Techniques of Hybridization of Random Primers and of Synthesis in a Time

The H3PIM system was entirely suitable for obtaining quantitative signals using standard protocols both for hybridization of the primers at random and for the H3PIM procedure itself as shown in Figure 6.

Claims

1. Method for the revelation in liquid and homogeneous phase of one or more nucleotide sequences of DNA or RNA, produced by synthesis in the presence respectively of deoxynucleotides or excess nucleotides and of a determined polymerase, from a primer oligonucleotide specifically hybridized with a nucleic acid which it is sought to detect in a biological sample, characterized in that it comprises the demonstration in the sample, of the production of pyrophosphate (PPi), resulting of the synthesis reaction of the specific nucleotide sequence (s).

2. Disclosure method according to claim 1, characterized in that the quantity of pyrophosphate produced is metered at a determined time of the synthesis.

3. Disclosure method according to any one of claims 1 or 2, characterized in that the demonstration and if necessary the quantitative assay of the pyrophosphate produced by synthesis of the nucleotide sequence (s) starting from a nucleic acid present in a sample, are carried out after a determined number of cycles of gene amplification, in particular by chain polymerization (PCR).

4. Method according to claim 3, characterized in that the demonstration and if necessary the quantitative dosage of pyrophosphate are carried out at several times during the gene amplification, in particular at predetermined regular intervals and / or at resulting from gene amplification, in particular in order to determine the kinetics of the synthesis.

5. Method according to any one of claims 1 to 3, for revealing the synthesis of one or several nucleotide sequences, characterized in that the detection of the pyrophosphate produced is carried out on a given biological sample subjected to a single synthesis step.

6. Disclosure method according to any one of claims 1 to 5, characterized in that the measurement of the quantity of pyrophosphate produced is carried out by NMR.

7. Disclosure method according to any one of claims 1 to 5, characterized in that the measurement of the quantity of pyrophosphate produced is carried out at the end of one or more enzymatic reactions dependent on PPi, in particular of colorimetric type or by bioluminescence.

8. Disclosure method according to claim 1, characterized in that the quantity of pyrophosphate is determined at the end of a cascade of enzymatic reactions comprising the following steps: the sample likely to contain the desired nucleic acid and consequently likely to contain the pyrophosphate produced at the end of the synthesis or syntheses of nucleotide sequences is brought into contact with Adenosine 5'-Phosphosulfate (APS), under conditions allowing the formation of ATP, the ATP obtained at the end of the previous step, luciferin is placed in conditions such that it is degraded by producing a bioluminescent signal in the form of photons, if necessary reading the intensity of the bioluminescent signal and the correlation with the amount of reacted PPi.

9. Disclosure method according to any one of claims 1 to 8, characterized in that the pyrophosphate highlighted and if necessary quantitatively measured is produced by synthesis of one or more specific nucleotide sequences from DNA, RNA or cDNA present in the sample.

10. Method according to any one of claims 1 to 9, characterized in that the step of highlighting and optionally metering the pyrophosphate is preceded by a treatment of the reaction medium under conditions allowing on the one hand d eliminating the ATP present in the medium and the dATP, for example by adding a hexokinase leading to the formation of ADP, and on the other hand eliminating the PPi contaminating the other deoxynucleotides by adding a pyrophosphatase, itself neutralized following the elimination of deoxynucleotides, for example by heating.

11. Method for the quantitative assay of the content of nucleotide sequences synthesized from a nucleic acid whose presence is sought to be detected in a biological sample, characterized by: - carrying out a determined number of gene amplification cycles by synthesis of nucleotide sequences by extension from a primer oligonucleotide hybridizing with the desired nucleic acid when it is present in the test sample, in the presence of an excess of deoxynucleotides or nucleotides depending on whether one seeks respectively the synthesis of DNA or RNA and a specific polymerase, - the determination of the quantity of pyrophosphate produced during gene amplification and the ratio of this measurement to the background noise. determining the quantity of nucleotide sequences present in the biological sample.

12. Detection method according to claim 11 characterized in that the determination of the quantity of nucleic acid present in the sample is carried out after comparison of the measurement of the quantity of pyrophosphate produced during the synthesis of nucleotide sequences from nucleic acid, with known quantities of pyrophosphate obtained by synthesis of known quantities of given nucleotide sequences, reported for example on a preset calibration curve.

13. Use of the method according to any one of claims 1 to 12 for the in vitro detection and if necessary the quantitative assay of a viral nucleic acid or a bacterial nucleic acid in a biological sample, for the diagnosis of '' a pathological condition or the characterization of a contamination in a biological medium.

14. Use of the method according to any one of claims 1 to 12 for the detection of a genetic anomaly, for example by substitution, addition, deletion, inversion of nucleotides, within a nucleic acid present in a biological sample.

15. Use of the method according to any one of claims 1 to 12, for tissue typing.

16. Kit for the in vitro detection of the content of a determined nucleic acid in a biological sample by detection of neosynthesized nucleotide sequences of DNA or of cDNA, characterized in that it comprises: the deoxynucleotides dATP, dTTP, dCTP, dGTP, a specific polymerase, for example a thermostable polymerase suitable for gene amplification by PCR, such as Taq polymerase,

- a reagent for revealing in a liquid phase medium the presence of pyrophosphate by implementing the method according to any one of claims 1 to 11, for example one or more enzymes dependent on pyrophosphate, capable of behaving as a marker (s ) bioluminescent (s) or as colorimetric marker (s).

17. Kit for the in vitro detection of the content of a determined nucleic acid in a biological sample by detection of neo-synthesized nucleotide sequences of RNA, characterized in that it comprises: the nucleotides ATP, UTP, CTP, GTP,