WO1998011253A2 - Procede et trousse de diagnostic et/ou de quantification par hybridation de type sandwich de sequences d'acides nucleiques sur support solide - Google Patents
Procede et trousse de diagnostic et/ou de quantification par hybridation de type sandwich de sequences d'acides nucleiques sur support solide Download PDFInfo
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- WO1998011253A2 WO1998011253A2 PCT/BE1997/000102 BE9700102W WO9811253A2 WO 1998011253 A2 WO1998011253 A2 WO 1998011253A2 BE 9700102 W BE9700102 W BE 9700102W WO 9811253 A2 WO9811253 A2 WO 9811253A2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
Definitions
- the present invention relates to a method and a kit comprising reagents for the detection and / or quantification by sandwich-type hybridization of nucleic acid sequences on a solid support.
- the labeled nucleotide sequence will be immobilized if the target or standard nucleotide sequence is present and immobilized on a trapping nucleotide sequence.
- the target or standard nucleotide sequence is then sandwiched between the nucleotide sequence trapper and the nucleotide sequence marked, by a double recognition, which increases the specificity, reduces the background noise, and allows a quantification of the nucleotide sequences. This is only possible if the hybridization is carried out quantitatively and reproducibly. This is all the more true as the yield from this hybridization is high.
- nucleic acids coming from "biological agents”, pathogenic or not, such as viruses, fungi, bacteria, mycoplasmas, animal and plant cells or tissues often amplified by a step of amplification, such as PCR (Polymerase Chain Reaction (US Patent 4965188), LCR (Landegren et al., 1988, Science, 241, 1077-1080), NASBA (Kievits et al. 1991, J. Virol. Methods 35 . 273-286), the CPR (Cycling Probe Reaction (WO Patent 95/14106) or the ICR.
- the detection of the amplified sequences requires carrying out a specific sensitive detection which is easily adapted to a large number of samples.
- a first solution consists in using beads, as described in patent application EP-0205532, in which Sephacryl beads of 5 to 50 ⁇ m are activated by diazotasion of aromatic souls.
- multi-well plates already serve as the basis for many tests, in particular ELISA and numerous reading devices in photometry, fluorescence, luminescence already exist for reading these plates.
- This fixation can be obtained by simple adsorption (non-covalent reaction), and makes it possible to measure amplicons present in solution (Dahlem et al. 1987, Mol. Cell. Probes 1.
- Kellér et al. describe a sandwich hybridization method using as trapper sequence a fragment of 3300 base pairs used for the detection of a target sequence of 190 base pairs which is also complementary to another labeled sequence, the single strand trapper sequence being fixed by an NH2 function covalently to a solid support.
- Polsky-Cinky et al. describe a method in which the trapper sequence comprises 4800 base pairs of which 800 base pairs are complementary to the target sequence of 1600 base pairs to be detected. As it appears in FIG. 1 of this document, the target sequence is complementary in the hybridization of another labeled nucleotide sequence.
- Japanese patent application JP-8089300 describes a trapper sequence having a length less than 30,000 base pairs and comprising at least 10 repeating units of 100 base pairs capable of hybridizing in series in the same orientation with target sequences to be detected and increases the sensitivity of traditional methods.
- Patent application EP-0079139 describes a single-strand trapper sequence of 1200 to 1500 base pairs hybridizing with target sequences of 600 to 700 base pairs, detected by sandwich hybridization by means of a labeled and complementary sequence d 'another portion of the target sequence.
- the trapping nucleotide sequence When the trapping nucleotide sequence is very short, there is a poor overlap of the target sequence and a folding of the target sequence onto itself or a rehybridization of target sequences between them. In addition, the low percentage of hybridization of the target sequence on the trapper sequence decreases the sensitivity.
- Patent application EP-0205532 describes a trapper sequence of 341 base pairs fixed so covalent on microbeads, this trapper sequence being capable of reacting with a target sequence to obtain an overlap of the trapper sequence on the target sequence of 175 base pairs, the target sequence reacting with a marked sequence of 201 base pairs by sandwich hybridization .
- this document does not describe an optimal recovery of the single-stranded trapper sequence by the target sequence.
- a free portion of 166 base pairs at the level of which replays of the single-stranded trapper sequence on itself or the pairing with other sequences present in the sample. are likely to decrease the percentage of target sequence hybridizing on the trapper sequence, and the sensitivity of detection and / or quantification.
- amplicons 1) quantitative (often complex) extraction of the nucleic acid from the biological sample; 2) quantitative amplification of the sequence studied; and 3) quantitative measurement of the number of amplified sequences (called amplicons).
- the amplification step in particular by the Polymerase Chain Reaction (PCR) (US Pat. No. 4,965,188), poses great difficulties with regard to the control of quantification and detection.
- PCR Polymerase Chain Reaction
- the first step is to mismatch the double strands of DNA, often very long (and possibly stabilized by various proteins or molecules), and to raise the temperature so that the two strands are separated.
- the second step is the pairing
- Patent application WO96 / 09407 describes an amplification method comprising the use of an internal standard having a specific part different from the target nucleotide sequence to be quantified by 17 animated acids. In this case, the target and standard sequences of the same length are quantified by fixing them to biotins reacting on a streptavidin fixed on a solid support.
- Patent application WO93 / 10257 describes a method for quantifying a DNA fragment by adding an internal standard different from the target DNA fragment to be quantified by less than 10% in sequence and / or in size.
- the standard nucleotide sequence differs from the target DNA fragment by a specific sequence comprising a deletion, mutation or addition at a site of 1 to 5 nucleotides allowing the incorporation of a restriction or cut site achievable by an enzyme or any other way.
- the quantification is carried out by specific recognition of the target DNA fragment or of the standard nucleotide sequence by different specific primers. The use of different primers hybridizing with the fragments in different sites will generate labeled fragments of different sizes and sequences, which can be separated by electrophoresis.
- This process is based on a double check of the specificity of identification.
- methods and devices based on selective identification of the standard sequence in one step do not guarantee a sufficient specificity and sensitivity, which may result in the presence of false positives or false negatives when quantifying a target nucleotide sequence.
- the present invention aims to provide a new method and a kit allowing detection and / or quantification of nucleic acid sequence which would not have the drawbacks of the cited state of the art.
- a particular object of the present invention is to provide a process EDS and a kit for an optimal hybridization of nucleic acid sequences, in particular a high percentage of hybridization of the trapper sequence from a target sequence or standard, low risk of folding back these sequences or of the trapper sequence onto itself and a low risk of reappearance of these sequences by complementary sequences present in the sample.
- Another object of the present invention is to provide a detection and / or quantification method and kit having improved specificity and sensitivity compared to the methods and devices of the prior art, in particular for the detection and / or the quantification of any type of nucleic acids present in a biological sample and possibly obtained after genetic amplification
- An additional aim of the present invention is to obtain a method and a kit for detection and / or quantification of said target nucleic acid sequence which allow the amplification of a sequence. internal or external standard regardless of the number of cycles of genetic amplification.
- the present invention relates to a method for detecting and / or quantifying a nucleotide sequence called "target " "present in a biological sample, characterized in that it comprises a type of contacting "sandwich” of said target nucleotide sequence 2 with a nucleotide sequence called “trapper” 5 fixed on an insoluble solid support 3, said trapping nucleotide sequence being complementary to part 7 of the target nucleotide sequence, contacting type " sandwich “also being carried out with one or more other nucleotide sequence (s) (6, 11) of which at least one (6) is marked, the said nucleotide sequence (s) (6 , 11) (of which at least one (6) is marked) being complementary (s) to another part 8 of the target nucleotide sequence 2 (another part than that 7 hybridized by the nucleotide sequence "trapper”5); in that the trapping nucleotide sequence 5 is covalently attached by one of its ends to the solid support 3; in
- the parts 13 of the target nucleotide sequence 2 do not hybridize with the trapping nucleotide sequence 5 and with the nucleotide sequence (s) (6) , 11) (of which at least one (6) is marked), is less than 60 bases, preferably less than 40 bases, or even zero.
- nucleotide sequence (s) (6, 11) (of which at least one (6) is marked) will take the name of nucleotide sequences "helper" 11 when the said sequence (s) do not are not marked, and nucleotide sequences "labeled” 6 when c * c ⁇ -they are likely to be recognized directly or indirectly by a system for detecting and / or quantifying, preferably selected from the group consisting of fluorescence, chemolummescence, electroluminescence, staining, detection by radioactive labeling, bioluminescence, electrochemistry, light reflection, an optical process or a mixture of them.
- nucleotide sequences "helper” 11 are used to stabilize the "sandwich” and obtain the most complete possible coverage of the target nucleotide sequence 2 on the marked nucleotide sequence 6 and on the trapper nucleotide sequence 5, which increases the sensitivity and the specificity of the process according to the invention.
- the sandwich hybridization is preferably carried out in two stages, that is to say that the first stage consists in the hybridization of the target nucleotide sequence 2 to the nucleotide sequence trapper 5 and that the second step is the hybridization of the target nucleotide sequence 2 with one or more nucleotide sequences (6, 11) of which at least one (6) is marked.
- the two stages are preferably separated by a washing stage.
- the sequences are chosen so that the conditions (temperature, salt concentration, reaction time) are compatible for the two hybridizations, which makes it possible to carry out the hybridization in a single step.
- the target and standard nucleotide sequences to be detected and / or quantified consist of any type of nucleic acid, DNA or RNA.
- the trapped, "helper”, labeled and standard nucleotide sequences used according to the present invention consist of DNA so as to avoid any destruction of these sequences by RNase possibly present in the biological sample. .
- the target 2 and standard 1 nucleotide sequences are the result of a prior amplification by a genetic amplification method, preferably chosen from the group consisting of PCR, LCR, CPR, NASBA or ICR.
- the 5 'terminal part 9 of the target nucleotide sequence 2 can be left uncovered by the labeled sequence (s) (s) 6 and the nucleotide sequence (s) "helper” 11, and is then covered by a complementary "primer” nucleotide sequence 12 (also called “primer”), used for amplification of the sequence target nucleotide 2.
- this "primer" 12 nucleotide sequence can be considered to be a "helper" type sequence.
- the invention also relates to a method for quantifying a target nucleotide sequence 2 present in a biological sample, which comprises the following steps:
- - a sandwich type contact of the target 2 and standard 1 nucleotide sequences with a trapping nucleotide sequence 5, preferably as described above, the trapping nucleotide sequence 5 being complementary to the common part of the target nucleotide sequence and the standard nucleotide sequence, the sandwich type contact also taking place with one or more sequences nucleotides (6, 11) of which at least one (6) is labeled and complementary to the specific part B of the target nucleotide sequence 2 or to the specific part B of the standard nucleotide sequence 1, - said method also comprising a quantification of the ratio between the specific labeling of the target nucleotide sequence 2 and the specific labeling of the standard nucleotide sequence 1.
- the term “content in close GC / AT bases” is understood to mean that the ratio of GC / AT bases of the standard is less than 20% of the ratio of GC / AT bases of the target nucleotide sequence.
- the contacting of the "sandwich" type as advantageously described, whether or not previously described, is advantageously combined with a device for quantification by a standard internal or external nucleotide sequence.
- the specific parts B of the standard and target nucleotide sequences preferably correspond to part 8 of the target or standard nucleotide sequences.
- the common part A preferably corresponds to the part 7 of this target or standard nucleotide sequence hybridizing with the trapper nucleotide sequence 5.
- the specific part B of the standard nucleotide sequence 1 is different from the specific part B of the target nucleotide sequence by 5 to 500 nucleotides, preferably by 20 to 40 nucleotides.
- the internal standard nucleotide sequence 1 comprises on either side of the specific sequence B and common A common parts 15 to parts 15 of the target nucleotide sequence 2, and which can serve in whole or in part as sequences complementary to primer sequences 12 for genetic amplification.
- the method according to the invention is particularly suitable for the use of internal standard nucleotide sequences amplified jointly with the target nucleotide sequence 2 or external standards amplified in parallel with the target nucleotide sequences 2.
- the method according to the invention comprises a high number of cycles of genetic amplification, preferably by PCR or LCR, preferably greater than 30.
- the internal standard is added in quantity variable to the starting sample and the ratio obtained between the specific labeling of the target nucleotide sequence 2 and the specific labeling of the standard nucleotide sequence 1 is plotted as a function of known quantities added to the starting sample, allowing to determine on the line thus obtained, for a ratio equal to 1, what is the quantity of target nucleotide sequence 2 present in the sample.
- the standard nucleotide sequence is added in an identical amount to a sample having undergone various dilutions, and the ratio between the specific labeling of the target nucleotide sequence and the specific labeling of the nucleotide sequence standard is plotted as a function of the dilutions of the sample, and the line obtained allows: to determine, for a ratio equal to 1, what is the quantity of target nucleotide sequence 2 present in the sample. Quantification can also be carried out by comparison of the ratio obtained with a single determined quantity of standard added to a single quantity of sample and a calibration line.
- the present invention also relates to the detection and / or quantification kit comprising the reagents for carrying out the methods described above.
- the kit for detection and / or quantification by hybridization of the “sandwich” type of a nucleotide sequence - target 2 comprises a trapping nucleotide sequence 5 fixed on a solid insoluble support 3 and complementary to part 7 of the target nucleotide sequence 2 and one or more other nucleotide sequence (s) (6, 11) (of which at least one (6) is marked), the said nucleotide sequence (s) (6, 11) being complementary (s) ) of another part 8 of the target nucleotide sequence 2.
- said trapping nucleotide sequence 5 is covalently attached by one of its ends to the solid support 3 and has a length of between 50 and 500 bases, preferably between 100 and 300 bases, more particularly between 120 and 250 bases, and part 10 of the trapper nucleotide sequence 5 which does not hybridize with part 7 of the naked sequence target key 2 is less than 40 bases, preferably less than 20 oases, or even zero.
- the nucleotide sequence labeled has a length sufficient to specifically recognize the target or standard nucleotide sequence to be detected and / or quantified, this specificity depending on the type of target or standard nucleotide sequence to be detected and / or quantified, and can be characterized by recognition by hybridization a specific complementary portion of at least 10 bases, preferably more than 20 bases, of the target or standard nucleotide sequence.
- the kit according to the invention will also include the reagents necessary for the specific identification of the marked sequence, for detection and / or quantification by a * method preferably chosen from the group consisting of fluorescence, chemolummescence, electroluminescence , staining, detection by radioactive labeling, bioluminescence, electrochemistry, light reflection, an optical process or a mixture of them.
- a * method preferably chosen from the group consisting of fluorescence, chemolummescence, electroluminescence , staining, detection by radioactive labeling, bioluminescence, electrochemistry, light reflection, an optical process or a mixture of them.
- the trapper nucleotide sequence 5 is covalently attached to the insoluble solid support 3 and is preferably produced via a 5 ′ phosphate of the trapper nucleotide sequence 5 on one or more amino functions of the solid support 3 insoluble by reaction with ide fuel.
- the insoluble solid support 3 is preferably chosen from the group consisting of tubes, filters, beads, possibly magnetic, multi-well plates, a plate or a mixture of them.
- the invention also relates to a detection and / or quantification kit which comprises a standard internal or external nucleotide sequence 1 as described below and all the elements necessary for extraction, amplification, detection and / or quantification according to the methods described above.
- FIG. 1 represents a schematic example of sandwich hybridization according to the invention.
- FIG. 2 represents a concentration DNA target curve of CMV obtained after sandwich hybridization and detection in bioluminescence.
- FIG. 3 represents a DNA sensitivity curve for Chlamydia trachomatis measured after amplification by PCR, sandwich hybridization and detected in colorimetry by the streptavidin-peroxidase conjugate.
- FIG. 4 represents a diagram illustrating the nucleotide structure of a standard competitive standard, compared to that of a target DNA to be measured during an amplification by PCR.
- FIG. 5 represents a diagram of a competitive standard (A) for the measurement of CMV viral DNA and its comparison with the target DNA (B).
- FIG. 6 represents a diagram of a competitive standard (A) for the measurement of the RNA of the HIV virus and its comparison with the target RNA (3).
- the numbering corresponds to the sequence nucleotide of viral RNA ("Los Alamos de
- FIG. 7 is a schematic description of the principle of the quantification of a target DNA by using a competitive standard according to the invention and their respective measurement by specific probes after capture on a common immobilized sequence.
- FIG. 8 represents a curve of the concentration of CMV target DNA and of the corresponding standard by sandwich hybridization and measured spectrophotometrically.
- Figure 9 shows a target DNA concentration curve of CMV and corresponding standard after PCR amplification and hybridization sandwich using a probe common trapper and a specific biotinylated probe to the target DNA or standard.
- FIG. 10 represents the measurements of the target DNA of CMV and of the internal standard after an amplification by 40 cycles of PCR when 1000 copies of standard have been added to increasing amounts of target DNA and measured after PCR by sandwich hybridization as defined in Figure 7.
- FIG. 11 represents the calibration line of a target DNA of CMV by the use of an internal standard according to the invention.
- the abscissa represents the ratios of the target signals and the standard obtained in FIG. 10.
- FIG. 12 represents the quantification of a target CMV DNA as a function of the number of cycles of the PCR thanks to the use of a competitive standard according to the invention.
- a competitive CMV sequence standard as described in FIG. 5 was used and added in constant quantity to the sample having undergone 4 different dilutions.
- FIG. 13 represents a competitive RT-PCR carried out on a target RNA of HIV and a standard RNA (FIG. 6) and a detection in bioluminescence after sandwich hybridization.
- the graph represents for each RT-PCR the ratio between the detection obtained using " a target specific probe and that of the standard.
- the results show a competition between the amplification of the target added in constant quantity (10 8 copies) compared to the standard added in decreasing quantity ranging from 10 10 (1), 10 8 (2) to 10 6 (3) copies.
- the trapper nucleotide sequence 5 preferably a DNA sequence, is covalently attached to multiwell plates 3. This covalent attachment is obtained by the binding of a phosphate located at the 5 'terminal position of the trapper sequence on an amine located on the support in the presence of carbodii ide, as described by Rasmussen et al. (1991, Anal.
- the attached trapper 5 nucleotide sequences can hybridize a target DNA 2, which will then be sandwich sandwich with a labeled nucleotide sequence 6.
- An advantage of sandwich hybridization is a very low background noise which makes it possible to carry out a massive analysis of clinical samples with a minimum of DNA purification.
- the quantity of trapping nucleotide sequences fixed on the multiwells can reach 1.2 pmoles for small oligonucleotides, but as regards the larger nucleotide sequences, the quantity fixed is less important (of the order of 300 fmoles for nucleotide sequences about 500 bases).
- the quantity of " trapped nucleotide sequences fixed is sufficient not to be limiting in the hybridization process of the invention.
- the fixing of the trapping nucleotide sequence on microbeads also makes it possible to increase the number of trapping nucleotide sequences in the reaction solution by using a larger quantity of beads.
- the trapper nucleotide sequences according to the invention chosen are complementary over their entire length to the target nucleotide sequence to be detected. This advantageously allows easy production of these trapping nucleotide sequences by PCR from target nucleotide sequences cloned into plasmids and can thus be used for a reproducible industrial production of these trapping nucleotide sequences.
- trappers can be used, part of the nucleotide sequence of which comprises one or more parts of sequences which are not complementary to the sequence of the ciole.
- the inventors have tested sequences trapping nucleotides having a 20 or 40 base sequence not complementary to the target nucleotide sequence and located in the 5 'terminal position used for attachment to the multiwells. These sequences serve as "spacers" between the trapper sequence proper and the solid support. In this experiment, the sequence carrying a 20 base spacer is more efficient than the 40 and 60 base spacers for hybridizing small target sequences.
- Another property of the method and of the kit of the invention is the advantageous use of trapping nucleotide sequences having a minimum size of 50 bases if possible 100 and at best 150 or more bases. This observation is unexpected based on the following considerations.
- the stability of the hybrids in the case where the ionic strength is constant and the size sufficient (beyond 50 cells), only depends on the composition (% G + C i and not on the size of the hybrids
- the importance of the size to favor or not the hybridization depends on the speed of the hybridization and not on their stability. Indeed, in solution, the size of the nucleic acid strands influences the rate of re-pairing. This is proportional to the square root of the length (Wetmur, JG and Davidson, N. 1968, J. Mol. Biol.
- the speed is proportional at the square root of the shortest strand, be it DNA (Wetmur, JG 1971, Biopolymers 10, 601) or RNA (Hutton, JR and Wetmur, JG 1973, J. Mol. Biol. 77, 495).
- the other factor which influences the speed of hybridization is the respective concentration of the target nucleotide sequences and the trapping nucleotide sequences.
- the situation in this case is complex because two reactions are possible: on the one hand the target nucleotide sequences to be measured being usually double strands in the starting solution, they will be able to reassociate with each other and on the other hand they will be able to hybridize on the trapper nucleotide sequence. These are therefore two competitive reactions, one taking place in solution (reassociation of the double strands of the target nucleotide sequence) and the other on a solid support (hybridization on the trapping nucleotide sequence).
- k2 kinetic constant of DNA reassociation
- k] _ kinetic constant of hybridization with the nucleotide sequence
- Cf concentration of the nucleotide sequence which hybridizes on DNA.
- the trapper is made up of a single strand because a single phosphorylated primer in the 5 'terminal position is used during its construction by PCR. This phosphate is the only one able to react on the amino support.
- the plate is washed in the presence of 0.4 N NaOH (cf. example 1) in order to remove the second strand. So we end up with a single strand fixed on the support. This strand being complementary to only one of the two strands of target DNA, it will fix this strand. After washing, this hybrid strand can be easily dehybnded for example by heating or with 0.4 N NaOH. A single strand of DNA is thus obtained in the solution.
- This technique can be used on a large scale, for example by using beads on which the trapping nucleotide sequences are fixed.
- This single strand preparation can have multiple applications as reagents using a chemically labeled strand for example.
- Detection of unlabeled target sequences is carried out by using their hybridization on trapping nucleotide sequences fixed on an insoluble support using one or more nucleotide sequences of which at least one is labeled (detection nucleotide sequences) which can hybridize on the part of the target nucleotide sequence not recognized by the trapper nucleotide sequence.
- This sequence can be chemically labeled and detected according to the various methods known to those skilled in the art. It is possible to obtain a fixation of at least 80% of the detection nucleotide sequence relative to the sequence target nucleotide hybridized to the trapper nucleotide sequence.
- the target nucleotide sequence dissociated in single strand is found in the solution in the presence of its strand of complementary target nucleotide sequence but also of the detection nucleotide sequence (and possibly primers) and on the support of the trapping nucleotide sequence.
- the target nucleotide sequence can either react first with the detection nucleotide sequence before binding to the trapper, or it can bind to the trapper before fixing the detection nucleotide sequence. In both cases, the use of large nucleotide sequences will promote the reaction speed as well as the stability of the hybrids formed.
- the target nucleotide sequence has a part of its unpaired sequence which can then be recognized by a complementary target nucleotide sequence which will be able to move the intermediate hybrid and redo a target nucleotide sequence double strand.
- the target nucleotide sequence will be hybridized and completely covered by the trapping nucleotide sequence and by the detection nucleotide sequence without having any (or too little) free sequence, the pairing (annealmg) of another strand of the sequence complementary target nucleotide can no longer take place and the sandwich hybrid will be stable.
- the target nucleotide sequence even after sandwich hybridization keeps a free sequence, this can always serve as a pairing site for the other strand of the complementary target nucleotide sequence, which will destabilize the hybrid and may even dissociate according to the experimental conditions (temperature, salts, etc.), because once the pairing has started, the propagation of the formation of the double strand is very rapid and thermodynamically favorable.
- Another characteristic of the sandwich hybridization method according to the invention is that not only the target sequence can be entirely covered by the trapping nucleotide sequence and the labeled nucleotide sequence, but that the trapping sequence can be entirely covered by the target sequence.
- This therefore makes it possible " to use as marked nucleotide sequence large double-stranded nucleotide sequences produced for example easily by PCR amplification in the presence of dUTP-biotin. Indeed, once one of these biotinylated strands paired with the target nucleotide sequence , it will be completely covered and can no longer be redeployed by its complementary strand, which is not the case in this work reported by Keller et al. (1989, Anal. Bioch. 177, 27-32).
- the trapper nucleotide sequences by using a primer carrying a phosphate in 5 'terminal which will allow the covalent fixing of only one of these strands on the amino microplates and on the other hand of the sequences labeled nucleotides.
- the labeled sequences are small, 20-30 or 40 bases, they will be chemically synthesized and will be single-stranded. ndes, they can be produced by amplification. Exceeding the target nucleotide sequence outside the hybridized part on the sensor and the detection nucleotide sequence by its 5 'terminal end resulted in a reduction in the hybridization yield if this uncovered part became important.
- a target nucleotide sequence of 20 bases is fixed on a trapping nucleotide sequence of 20 bases which is complementary to it with a yield which can be 25 times greater compared to a sequence which has the same 20 nucleotides but which additionally has 20 additional nucleotides on the 3 'terminal side.
- a yield which can be 25 times greater compared to a sequence which has the same 20 nucleotides but which additionally has 20 additional nucleotides on the 3 'terminal side.
- the invention relates to the construction of one or more sequences of oligonucleotides (DNA or RNA) having specific specificities, as described below, and their use as standards for the measurement of target sequence (s). DNA or RNA by sandwich hybridization using oligonucleotide probes labeled according to the method described above or not. The construction represented in FIG.
- Standard 1 is compatible with possible prior amplification and quantitative detection of a DNA sequence (or RNA) of which part A of the sequence is identical to the target DNA and part B, at least (the smallest possible) is different.
- these two parts will be flanked by two sequences 3 identical to those of the target DNA or RNA which serve as matrix for the attachment of oligonucleotide primers
- the length of the standard will be the same or very close to that of the target DNA or RNA. Care will also be taken to introduce into the specific part B an AT and GC base content close to or identical to that of the target DNA or RNA.
- An example of a standard used for the quantification of a DNA fragment of the CMV virus is given in FIG. 5, and an example of an RNA standard for the quantification of a RNA sequence of the HIV virus is shown in FIG. 6.
- Such a standard also allows use as an external standard or as an internal standard for PCR amplification.
- their similarity and partial identity allows an amplification rate very close to or identical to that of the target DNA or RNA.
- They have matrix sequences 3 for primers 4 identical, therefore which will carry out their fixation (annealing) in the same proportions.
- Their identical length, their sequence identity over a large distance and the similarity of the non-common part in length and in number of GC bases / number of AT bases ratio means that reading by DNA polymerase will be carried out with the same efficiency.
- it also has a particular property.
- slowing down and stopping the amplification of the standard sequence results in the same slowing down for the target sequence and vice versa.
- This slowdown in efficiency at the end of the amplification process can be due to several reasons: the decrease in the number of primers, the number of free nucleotides, the decrease in DNA polymerase activity or the too rapid rehybridization of the amplify with each other rather than with the primers during the step of fixing the primers (annealing). All these reasons are equivalent for the 2 sequences and will therefore have the same influence on their amplification.
- the inventors have observed that by sandwich hybridization of the invention, the limiting factor is the binding of amplicons 1 and 2 to the immobilized trapper sequence 5. Since this is present on a surface, the reaction speed is much slower due to the slower diffusion of the reagents when approaching a rigid surface and with steric hindrances. Experimentally, we observe that less than 50% of amplicons 1 and 2 are fixed on the trapper sequences 5. However, even if this percentage of fixation is low, we observe that it is the same for standard 1 and for standard target 2. This property is explained by the fact that the 2 sequences are the same size and that they are fixed on a trapper 5 sequence of the same type.
- this sandwich hybridization also allows quantification of these two amplicons with the same efficiency by operating as follows: the preparation containing the 2 amplicons is diluted adequately and is added to 2 or a double series of wells
- the fixation of the two amplicons on the trapper DNA will be identical in the two tuos since they will be present at the same concentration.
- the trapper since the trapper is common to the two amplicons and they have an identical size, their fixing will be carried out with the same yield, that is to say that the proportion of the 2 fixed amplicons will be identical to that present in the preparation. after PCR.
- the sandwich hybridization can be carried out either in two stages or in one stage, by carrying out the fixation on the trapper sequence then by adding the specific marked sequence or by adding together the specific marked sequence to the amplicons during the immobilization on the trapper sequences. .
- results indicate the possibility of quantitatively detecting between 30 and a million copies of these target sequences by using a thousand copies of standard sequences at the start.
- This property constitutes an important practical advantage because the biological samples can contain very variable quantities of target nucleotide sequences to be measured and in this case, a linear assay of more than 4 orders of magnitude makes it possible to greatly reduce, if not totally, the number of dilutions to be made in order to be in the zone of quantification.
- RNA for example messenger RNAs or viral RNAs.
- a standard must be used which consists of an RNA chain having the same specificities as the DNA standard explained above.
- the amplification and measurement approach requires a preliminary step which is the transformation of RNA into a DNA chain by an enzyme having reverse transc ⁇ ptase activity.
- the rest of the operations and the quantification are identical to that explained above.
- An example of a standard used for the quantification of the RNA of the HIV virus is given in FIG. 6 and the quantification of HIV presented in Example 11 and in FIG. 13.
- oligonucleotides as external standards is less advantageous than their use as internal standards and it requires special controls and conditions. It must be certain that the efficiency of the amplification is identical for the sample and for the tube containing the external standard; it is also necessary to work in the linear amplification zone in order to keep a proportionality between the number of amplicons obtained and the quantity of sequences present in the sample and the standard at the start. It will also be necessary to carry out the tests in several copies in order to minimize the variations observed from tube to tube during amplification.
- the sample containing the target DNA or RNA is processed in parallel with tubes containing increasing concentrations of external standard. All the amplification and detection conditions are identical and made with the same solutions in order to minimize processing variations. The results are compared with the calibration curve obtained with the standards. In the invention, a series of dilutions of the target sample are preferred in order to obtain values corresponding to the area covered by the standards.
- a more adequate method but which requires more tests consists in diluting the sample (for example 4 dilutions of 10 in 10) and adding to it a constant amount of internal standard.
- the sample for example 4 dilutions of 10 in 10.
- the internal standard is normally added to the initial sample. It may possibly, for practical reasons, be added after extraction if it is quantitative. This is the case where the nucleic acids are extracted from a sample and then made 4 dilutions before adding a constant amount of internal standards before quantifying as in Example 10 and Figure 12. This procedure requires only one sample extraction.
- Example 1 Influence of the length of the trapper for a simple hybridization of a target DNA of HPV-18
- Hybridization was carried out from these amplicons labeled at the 5 'terminal end via the T4 polynucleotide kinase in the presence of [ ⁇ ATP-at 32 P].
- Two types of trappers were used, corresponding to 180 and 360 bases complementary to the amplicon sequence.
- the hybridizations were carried out in the presence of increasing concentrations of target amplicons at 45 ° C. for 2 hours in a solution consisting of SSC 2x concentrated, ⁇ e Denhart 5x concentrated, Denatured salmon sperm DNA at 0.1 mg / ml.
- the experiment was carried out for hybridization of amplicons of 435 base pairs corresponding to a CMV sequence at position 171075 of the genome (strain AD169, reference GENBANK X17403).
- MIE5 antisense: 5 'CAGCACCATCCTCCTCTTCCTCTGG as described by Demmler et al. (J. Infect. Dis., 158, pp;
- the trapper 50, 100, 150 and 250 bases were produced by
- Hybridization was performed at 60 ° C for
- the experiment was carried out for the hybridization of amplicons of 586 base pairs corresponding to a sequence of HPV-18 located at position 6193 to 6779 of the viral DNA. This sequence was amplified by the primers described in Example 2.
- Three types of trappers were used, corresponding to a sequence of 25, 180 and 360 bases complementary to the amplicon sequence.
- the various trappers recognized the part of the amplicons located on the 3 'terminal side.
- the probes included either 360 bases for hybridization on the 180-base trapper, or 180 bases for hybridization on the 360-base trapper, or 21 bases for hybridization on the 25-base trapper. They were labeled with 32 P by phosphorylation in the 5 ′ terminal position by T4 kmase in the presence of [ 32 P] ATP. They corresponded to the 5 'terminal end of the target sequence.
- the 21 base probes were single strand, while the 180 and 360 base probes were double strand.
- the sandwich sandwiches have been optimized with regard to temperature, salt concentration and concentration of amplicons.
- the hybridization carried out at 45 ° C.
- Hybridization on the 180 and 360 base trappers was carried out in a single step and under the following conditions: the amplicons were added respectively at a rate of 2.5 fmoles in the presence of 15 fmoles of labeled probe. Hybridization took place for 20 hours of a 2x SSC solution concentrated at 45 ° C.
- the quantity of labeled hybridized probe is 0.54 fmoles for the 180 base trapper and 0.69 fmoles for the 360 base trapper.
- the difference in probe fixation was due to a higher efficiency of hybridization of the target sequence on the large trapper (cf. example 2).
- the DNA extracted from Mycobacterium tuberculosis was amplified by its sequence Mt 308 using the following primers: T2MT3 '(primer 5' -3 '): GTCGACACGCCTCTGCACGGAAGTCCTT DMT3' (antisense primer 5 '-3'): GCTCGACTTCTGGTCACGACGTCC
- the trapper was obtained using the T2MT3 probe 'phosphorylated in position 5' and an antisense primer SMT5 ': GGGCATCCGCGAGTTGAAGACCTGAAGTGG.
- the sandwich hybridization was performed in a j outant each well 54 fmoles DNA amplified target by PCR in the presence of 50 ng of 40 base probe and 25 ng for the 135 base sequence. Hybridization was carried out for 2 hours at 60 ° C.
- the reading is done for an hour. After subtracting the white, a value of 1.5 million RLU (Relative Light Unit) for the 40 base probe and 2.7 million RLU for the 135 base probe are obtained.
- RLU Relative Light Unit
- the 135 base probe was double stranded and lowered. Despite these two unfavorable conditions, the signal obtained is almost twice as high, which clearly indicates the importance of making a large detection probe which covers the entire target sequence.
- the amplicons originating from the CMV DNA were obtained from a PCR carried out with the primers MIE4 and MIME5 described in example 2. They make it possible to obtain amplicons of 435 base pairs.
- the amplicons were used to produce the sandwich hybridization curve described below. This was carried out on multi-well plates on which were fixed trappers complementary to this target sequence and a biotinylated probe of 185 bases also produced by PCR. The size of the trapper was 257 bases. They were produced from a PCR using primers MIE4 described in Example 3 and MEI- ⁇ ⁇ or.t the sequence was GTACAGGGGACTCTGGGGGTGAC
- the trapper once produced is purified on spin column G25.
- the total hybridization volume per well is 110 ⁇ l, containing:
- hybridization buffer SSC 4 4x, Denhart lOx, salmon sperm DNA 200 ⁇ g / ml denatured 10 minutes at
- Hybridization lasts 2 hours at 70 ° C. After hybridization, the wells are washed twice with 200 ⁇ l of 0.1 x SSC then with 200 ⁇ l of 100 mM maleate buffer, 150 mM NaCl, 0.3% Tween pH 7.5 for 15 minutes.
- the kinase activity is revealed in 20 mM Tris buffer pH 7.75 containing 60 ⁇ M DTT, 100 ⁇ M EDTA, 5 mM MgCl 2 , 8 ⁇ M Luciferine, 6 mU luciferase per 20 mM KCl well, 1 mM Phosphoenolpyruvate and ADP 3.2 ⁇ M.
- the light emission is followed for one hour with a luminometer (Lum oskan, Labsystem, Finland) and the results are expressed in RLU. mm.
- Example 6 Differential hybridization of a small and a
- the marked initial probes are large and are cut into two pieces which must be able to be detected and measured.
- the problem is therefore to be able to measure by hybridization a small probe in the presence of its mother sequence which is larger.
- the inventors have chosen in this example a mother sequence (OL1) biotinylated with 40 bases which have the following sequence: 5 'CCGCGACTATCCCTCTGTCCTCAGTAATTGTGGCTGAGAA 3'
- This sequence corresponds to a specific sequence of the CMV genome located on the "Major Immediate Early Gene” (Akrigg et al., Virus Res. 2, p. 107).
- the inventors wanted to detect a biotinylated probe (OL2) corresponding to the first 20 bases of this probe in the presence of the mother sequence (OL1).
- the trapper consisted of an oligonucleotide of 20 bases complementary to the OL2 probe and terminated by a phosphate group at 5 '. This trapper was attached to the amino-wells by covalent reaction.
- the wells were washed with 0.1 x concentrated SSC solution at 45 ° C. After four washes, the wells are incubated with the streptavidin-kinase conjugate and its fixation is estimated by measuring the kinase activity in bioluminescence. Under these conditions, the wells having the large fragment (OLA) showed an RLU x mm of 9 while the small fragments showed a RLU x mm of 210.
- OLA large fragment
- the target DNA to be measured consists of a double brm oligonucleotide of 314 base pairs corresponding to position 171193 of the CMV genome (strain 10169, reference GENBANK X17403).
- the internal standard consists of a double brm oligonucleotide of 314 base pairs whose nucleotide sequence is identical to that of the target DNA to be measured except for the 40 nucleotides ranging from 3217 to 3256 which constitute a random sequence but whose Dourcenta ⁇ e in GC is similar to that of DNA target.
- the composition of these nucleotides is presented in FIG. 5.
- These nucleotides are first heated at 100 ° C. for 10 mm and then incubated in increasing concentration in wells to which have been attached sensor probes of the invention and corresponding to a portion. of the common sequence and supplied by Lambdatech (Namur-Belgium).
- the 0.06 ml hybridization solution per well contains a 2 times concentrated SSC solution, 5 times concentrated Denhardt, 100 ⁇ g / ml of denatured salmon sperm DNA and 50 ng of biotinylated probe of 40 single brm nucleotides corresponding either to the sequence complementary to the target DNA, or to that of the standard DNA presented in FIG. 7.
- To these 60 ⁇ l are added 40 ⁇ l of target or standard DNA. Hybridization takes place at
- the wells are washed 1 time with 0.2 ml of SSC solution 0.1 times, then once with 0.2 ml of maleate buffer pH 7.5 containing 0.15 M NaCl and 0.3% Tween and finally with Maleate buffer pH 7.5 0.15 M NaCl and containing 1% milk powder.
- a streptavidme-peroxidase conjugate is added in 0.1 ml at a 1/1000 dilution as recommended by the supplier (BIOSOURCE - Fleurus - Belgium) then washed 3 times with 0.2 ml of 0.1 M Maleate buffer pH 7.5 containing 0.15 M NaCl and 0.3% Tween then 1 time with a 500 mM glycine solution pH, 1 containing 100 mM KC1 and 2 lM MgCl.
- the peroxidase activity is measured by the oxidation of TMB in the presence of K2 ⁇ 2 in a 1.1 ml of tarcor. 0.2 M acetate citrate pH 7.5 After 10 mm of reaction, 0.22 ml of a 1.2 M solution of H2SO4 is added to each well and the optical density is measured at 450 mM.
- the results of the example are shown in Figure 8.
- Example 8 Measurement of the Quantity of CMV Target DNA by Using an External Sample by Sandwich Hybridization After Amplification by
- tubes are prepared containing increasing concentrations of external standard as defined in FIG. 5. All the tubes have undergone a PCR of 40 cycles using primers corresponding to the sequence
- the PCR starts with 1 passage 3 mm at 94 ° C and continues with 40 cycles defined as follows: Each PCR cycle included a denaturation temperature at 94 ° C for 30 sec, a period of pairing of primers at 65 ° C for 30 sec and 30 sec polymerization at 72 ° C. PCR solution from
- 0.1 ml included 100 pmol of each of the 2 primers, 200 mM of the various dNTPs and 2.5 U of Taq DNA polymerase in a 10 mM TRIS-HCl buffer pH 8.4 with 1.5 mM MgCl 2 and KCl
- the PCR tubes pass 10 mm at 72 ° C.
- Each point represents the average of 3 measurements. There is a correlation between the number of starting copies and the signal obtained. In addition, the curves obtained for the target and the standard are very close, which makes it possible to use the curve of the standard as a reference for determining the number of copies of the target DNA CMV in the starting sample.
- Example 9 Measurement of the Quantity of CMV Target DNA Using an Internal Standard for Sandwich Hybridization After Amplification by PCR
- a standardization curve is established in the following manner. Increasing amounts of ur were added to each of the tubes. plasmid containing part of the CMV virus genome and a constant amount of standard internal is equivalent to 1000 copies.
- the CMV target DNA quantities were 30, 100, 300, 1,000, 3,000, 10,000, 30,000, 100,000, 300,000, and 1 million copies.
- the composition of the internal standard is given in FIG. 5.
- Each tube was subjected to amplification by 40 cycles of PCR under the conditions presented in example 15 using the primers corresponding to the sequence 3191-3217 and 3504-3481 of CMV gene ( Figure 5). After amplification, 314 base pair amplicons corresponding to the CMV target and to the standard are therefore obtained. From each of the PCR solutions, 0.04 ml are taken and incubated in 2 wells containing an identical trapper for the common sequences " of the 2 amplicons. In one of the wells, is added the biotinylated 40 base probe specific for the CMV target and in the other, the 40-base biotinylated probe specific for the standard.
- a streptavidme-peroxidase conjugate is added to each tube and the peroxidase activity measured as in Example 7 by measuring the optical density (OD) corresponding to the absorbance of the peroxidase reaction product (figure 10).
- OD optical density
- the ratio between the OD corresponding to the detection of target amplicons and that corresponding to the detection of standard amplicons is determined and plotted as a function of the number of copies of CMV present at the start of the experiment.
- the results are presented in FIG. 11 on a logarithmic scale in order to cover all the concentrations.
- Each of the points represents the average of 3 experiences. There is very good linearity with a regression coefficient of 0.97 for the straight line, which means the possibility of determining the quantity of target CMV in a sample of starting from 30 to 1 million copies.
- Example 9 This experiment was carried out essentially according to Example 9 and the diagram in FIG. 7, that is to say using at the start a sample containing 100,000 copies of CMV DNA diluted 10-fold and 10 times to which we added 1,000 copies of the standard.
- Each of these tubes was subjected to a PCR as in Example 5 except that certain tubes were stopped after 25, 30, 35, or 40 cycles of PCR.
- the amplicons were used for sandwich hybridization on the same trapper but with either a biotinylated probe specific for the target CMV or the standard.
- the revelation was made in bioluminescence using a streptavidme-k ase conjugate and measurement of the light emitted by luciferase.
- the target RNA to be measured consists of a 267 base single brm nucleic acid corresponding to the nucleotide sequence ranging from 4235 to 4481 of the AIDS virus (HIV) polymerase (Pol) gene (numbering of HIV virus (HIV) polymerase (Pol) gene (numbering of HIV virus (HIV) polymerase (Pol) gene (numbering of HIV virus (HIV) polymerase (Pol) gene (numbering of HIV virus (HIV) polymerase (Pol) gene (numbering of HIV virus (HIV) polymerase (Pol) gene (numbering of HIV virus (HIV) polymerase (Pol) gene (numbering of HIV virus (HIV) polymerase (Pol) gene (numbering of HIV virus (HIV) polymerase (Pol) gene (numbering of HIV virus (HIV) polymerase (Pol) gene (numbering of HIV virus (HIV) polymerase (Pol) gene (numbering of HIV virus (HIV) polymerase (Pol)
- HIV-LAI Los Alamos HIV data bank ACK02013).
- the standard consists of a single brm RNA nucleic acid whose nucleotide composition is identical to that of the target RNA to be measured with the exception of 38 nucleotides ranging from position 4367 to 4404, constituting a random sequence whose percentage in GC is similar to that of target RNA.
- the composition of these RNAs is presented in FIG. 8.
- the RNA standard is prepared by transcription m vi tro from a plasmid having in 5 'the promoter of an RNA polymerase and in 3' of a poly A sequence After purification and quantification of the latter, an increasing number of copies of RNA standard is placed in each tube in the presence of a fixed quantity of target RNA.
- Each tube is subjected to a first reverse transcription step (AMV-RT, Avian Myeloblastosis Virus) for the synthesis of the first brm DNA (primer 4481-4501) and in a second step to the synthesis of the second strand of cDNA as well as the amplification of DNA (Tfl DNA polymerase Thermus flavius; System Access RT-PCR Pro ega) (primers 4235-4256; 4481-4501).
- a reaction buffer optimized for the two stages simplifies the procedure and reduces the risk of contamination.
- the amplification procedure comprises in a first step the transcription mverse at 48 ° C for 60 mm, followed by a step of mactivation of the AMV-RT and denaturation of RNA / cDNA hybrids at 94 ° C for 2 mm. Then, the synthesis of the second br of cDNA and the PCR are carried out by an amplification of 40 cycles each comprising a denaturation at 94 ° C. for 30 sec, a pairing of the primers at 50 ° C. for 30 sec and a step of extension. at 72 ° C for 30 sec.
- the RT-PCR reaction takes place in 50 ⁇ l of solution comprising in final concentration 1 ⁇ M of each of the 2 primers, 0.2 mM of dNTPs, reaction buffer AMV / Tfl, 1 mM MgS0, 0.1 u / ⁇ l AMV -RT and 0.1 u / ⁇ l Tfl DNA polymerase.
- the sandwich hybridization is carried out on the trapper probe common to the two amplicons fixed on the multiwells.
- the presence of target and standard amplicons was demonstrated using a biotinylated 38 base probe specific for each of the 2 sequences.
- the revelation is made in bioluminescence using a streptavidme-k ase conjugate and measurement of the light emitted by luciferase (cf. example 11).
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BE9700244A BE1011052A3 (fr) | 1997-03-20 | 1997-03-20 | Procede et trousse de diagnostic et/ou de quantification par hybridation de type sandwich de sequences d'acides nucleiques sur support solide. |
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Cited By (7)
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EP1096024A1 (fr) * | 1999-10-28 | 2001-05-02 | Remacle, José | Méthode et trousse pour le criblage et/ou la quantification de séquences d' acides nucléiques multiples homologues avec des ensembles de sondes |
WO2001077372A2 (fr) * | 2000-03-24 | 2001-10-18 | Facultes Universitaires Notre-Dame De La Paix | Identification de (micro-)organismes biologiques par detection de leurs sequences nucleotidiques homologues sur des jeux ordonnes d'echantillons |
EP1164201A1 (fr) * | 2000-06-14 | 2001-12-19 | Facultés Universitaires Notre-Dame de la Paix | Détection inverse pour l'identification et/ou quantification des nucléotides cibles par des biopuces |
US7202026B2 (en) | 2000-03-24 | 2007-04-10 | Eppendorf Array Technologies Sa (Eat) | Identification of a large number of biological (micro)organisms groups at different levels by their detection on a same array |
US7829313B2 (en) | 2000-03-24 | 2010-11-09 | Eppendorf Array Technologies | Identification and quantification of a plurality of biological (micro)organisms or their components |
US7875442B2 (en) | 2000-03-24 | 2011-01-25 | Eppendorf Array Technologies | Identification and quantification of a plurality of biological (micro)organisms or their components |
EP2441520A1 (fr) | 2010-10-12 | 2012-04-18 | Eppendorf AG | Application en temps réel et détection basée sur un micro-réseau de cibles d'acides nucléiques dans un dosage sur puce de flux |
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Cited By (14)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2001031055A2 (fr) * | 1999-10-28 | 2001-05-03 | Facultes Universitaires Notre-Dame De La Paix | Procede et kit de detection et/ou de quantification de sequences d"acides nucleiques homologues multiples en reseau |
EP1096024A1 (fr) * | 1999-10-28 | 2001-05-02 | Remacle, José | Méthode et trousse pour le criblage et/ou la quantification de séquences d' acides nucléiques multiples homologues avec des ensembles de sondes |
WO2001031055A3 (fr) * | 1999-10-28 | 2001-12-20 | Univ Notre Dame De La Paix | Procede et kit de detection et/ou de quantification de sequences d"acides nucleiques homologues multiples en reseau |
US7205104B2 (en) | 2000-03-24 | 2007-04-17 | Eppendorf Array Technologies Sa (Eat) | Identification of biological (micro) organisms by detection of their homologous nucleotide sequences on arrays |
WO2001077372A2 (fr) * | 2000-03-24 | 2001-10-18 | Facultes Universitaires Notre-Dame De La Paix | Identification de (micro-)organismes biologiques par detection de leurs sequences nucleotidiques homologues sur des jeux ordonnes d'echantillons |
US7875442B2 (en) | 2000-03-24 | 2011-01-25 | Eppendorf Array Technologies | Identification and quantification of a plurality of biological (micro)organisms or their components |
US7829313B2 (en) | 2000-03-24 | 2010-11-09 | Eppendorf Array Technologies | Identification and quantification of a plurality of biological (micro)organisms or their components |
WO2001077372A3 (fr) * | 2000-03-24 | 2002-06-13 | Univ Notre Dame De La Paix | Identification de (micro-)organismes biologiques par detection de leurs sequences nucleotidiques homologues sur des jeux ordonnes d'echantillons |
US7202026B2 (en) | 2000-03-24 | 2007-04-10 | Eppendorf Array Technologies Sa (Eat) | Identification of a large number of biological (micro)organisms groups at different levels by their detection on a same array |
EP1164201A1 (fr) * | 2000-06-14 | 2001-12-19 | Facultés Universitaires Notre-Dame de la Paix | Détection inverse pour l'identification et/ou quantification des nucléotides cibles par des biopuces |
WO2001096592A3 (fr) * | 2000-06-14 | 2002-04-11 | Univ Notre Dame De La Paix | Detection inverse pour l'identification et/ou la quantification des sequences nucleotidiques cibles sur des biopuces |
WO2001096592A2 (fr) * | 2000-06-14 | 2001-12-20 | Facultes Universitaires Notre-Dame De La Paix | Detection inverse pour l'identification et/ou la quantification des sequences nucleotidiques cibles sur des biopuces |
EP2441520A1 (fr) | 2010-10-12 | 2012-04-18 | Eppendorf AG | Application en temps réel et détection basée sur un micro-réseau de cibles d'acides nucléiques dans un dosage sur puce de flux |
WO2012049066A2 (fr) | 2010-10-12 | 2012-04-19 | Eppendorf Ag | Amplification en temps réel et détection basée sur un micro-réseau de cibles d'acide nucléique dans un test sur puce en continu |
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WO1998011253A3 (fr) | 1998-05-07 |
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