US20130115597A1 - Method for detecting specific nucleic acid sequences - Google Patents

Method for detecting specific nucleic acid sequences Download PDF

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Publication number
US20130115597A1
US20130115597A1 US13/639,774 US201113639774A US2013115597A1 US 20130115597 A1 US20130115597 A1 US 20130115597A1 US 201113639774 A US201113639774 A US 201113639774A US 2013115597 A1 US2013115597 A1 US 2013115597A1
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oligonucleotide
nucleic acid
label
detection
reaction
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Elmara Graser
Timo Hillebrand
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AJ Innuscreen GmbH
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • 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
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present invention relates to a method and a test kit for detecting specific nucleic acid sequences, comprising the steps of 1. matrix-dependent de novo synthesis of the target nucleic acid, 2. target-specific probe hybridization and 3. detection of the hybridization event.
  • the detection of the hybridization reaction may be accomplished both fluorimetrically in the form of a homogeneous assay and thereafter immunologically for verification of the result. The detection reaction always takes place chronologically after the matrix-dependent de novo synthesis has ended.
  • a widely used method for detecting specific nucleic acids is the LightCycler technology of Roche.
  • Roche Company developed special hybridization probes, consisting of two different oligonucleotides, each of which is labeled with only one fluorophore.
  • the acceptor is located at the 3′-end of the one probe, while the other oligonucleotide has a donor at the 5′-end.
  • the probes are chosen such that they both bind to the same DNA strand, wherein the distance between acceptor and donor is permitted to be only 1 to 5 nucleotides at most, so that the so-called FRET effect can develop.
  • the fluorescence is measured during the annealing step, in which light of this wavelength is detectable only as long as both probes are bound to the DNA.
  • the melting points of both probes should be identical.
  • Double-dye probes carry two fluorophores on one probe.
  • the reporter dye is located at the 5′-end and the quencher dye at the 3′-end.
  • a phosphate group may also be located at the 3′-end of the probe, so that the probe cannot function as a primer during elongation. As long as the probe is intact, the released light intensity is low, since almost the entire light energy produced after excitation of the reporter is absorbed and converted due to the spatial proximity of the quencher.
  • the emitted light of the reporter dye is “quenched”, i.e. extinguished.
  • This FRET effect continues to exist after the probe has bound to the complementary DNA strand.
  • the polymerase encounters the probe and hydrolyzes it.
  • the ability of the polymerase to hydrolyze an oligonucleotide (or a probe) during strand synthesis is known as 5′-3′ exonuclease activity. Not all polymerases have 5′-3′ exonuclease activity (Taq and Tth polymerase).
  • Taq and Tth polymerase This principle was described for the first time for Taq polymerase. The principle is known as the TaqMan principle.
  • the reporter dye is no longer in the spatial proximity of the quencher.
  • the emitted fluorescence is now no longer converted and this fluorescence increase is measured.
  • reporter and quencher are coupled on the individual dNTPs (deoxyribonucleotides) added to the reaction.
  • dNTPs deoxyribonucleotides
  • the fluorescence of the sample is reduced by the FRET effect.
  • a disadvantage of the method is the incorporation of the labeled nucleotides even in the case of mispriming and of primer dimers. Thus such a method cannot be used for diagnostic applications.
  • the respectively labeled probes are hybridized with one another.
  • one of the probes is able to form a duplex with the target nucleic acid, whereby the FRET interaction is canceled.
  • a further option for specific detection of amplification products by means of real-time PCR technology consists in the use of intercalating dyes (ethidium bromide, Hoechst 33258, Yo-Pro-1 or SYBR GreenTM, etc.). After excitation by high-energy UV light, these dyes emit light in the visible lower-energy wavelength range (fluorescence). If the dye is present as a free dye in the reaction mixture, the emission is very weak. It is only by the intercalation of the dye, i.e. by incorporation into the small furrows of double-strand DNA molecules, that the light emission is greatly intensified.
  • the dyes are inexpensive and universally usable, since in principle any PCR reaction can be followed in real time with them.
  • the fluorescence is measured continuously.
  • the point at which the double-strand DNA melts is characterized by a drop (peak) of the fluorescence of the intercalating dye, since the intercalating dye dissociates from the single-strand DNA.
  • a sharply accentuated melting-point peak should be expected. This melting point represents the specific product to be expected. Products of different sizes and products from different sequences have different melting points.
  • the described methods satisfy the requirement of specific detection of an amplification product.
  • less expensive methods for detecting nucleic acids include, for example, PCR ELISA.
  • the DNA sequence to be investigated is amplified and the produced DNA fragment is then covalently immobilized on a solid phase (e.g. microtiter plate or strip), subsequently denatured to a single strand and hybridized with a sequence-specific probe.
  • the successful binding of the probe can be visualized by an antibody-mediated color reaction.
  • Another variant is based on immobilizing the probe on a solid phase and then bringing the PCR product after the end of denaturing into contact with the immobilized probe. The detection of a completed hybridization event takes place by analogy with the first method variant.
  • PCR ELISA techniques are simple to perform, but nevertheless comprise multiple process steps, so that several hours of working time to perform the subsequent detection method are also needed in addition to the time needed to perform the PCR. Such a method usually needs 8 hours and therefore is also not suitable as a rapid test.
  • lateral-flow method is used to detect nucleic acids. This method also depends on the technology of hybridization of nucleic acids on a solid phase.
  • One advantage of lateral flow methods is that they represent a small, manual test format (strip test).
  • a very rapid detection method that also depends on the detection of amplification products by means of a test strip and is commercially available is in turn based on a completely different principle from that in the above publication.
  • the PCR reaction is performed with one biotinylated primer and one non-biotinylated primer. After completion of the PCR, a PCR product labeled with biotin at one end is therefore obtained.
  • a test strip e.g. of Millenia, Amodia, etc.
  • a streptavidin site for coupling the biotin-labeled DNA strand and an FITC binding site for checking the function of the test strip.
  • the PCR product is detected by denaturing the PCR batch at the end of the PCR and hybridizing with a probe complementary to the biotin-labeled DNA strand.
  • the probe is FITC-labeled.
  • the PCR hybridization batch is mixed with a run buffer and applied on the test strip.
  • the biotinylated DNA strand binds to the streptavidin binding site of the strip.
  • Detection is accomplished via the FITC label of the probe hybridized with the DNA strand.
  • a typical signal in the form of a stripe appears. This signal is supposedly the specific detection of the amplification product.
  • the method does not combine the hybridization of the probe with the PCR process but instead performs it as a separate method step.
  • Unexamined application WO 2009/000764 A2 permits the performance of amplification and hybridization of the PCR product in one reaction batch.
  • a doubly labeled amplificate-probe dimer which can then be visualized, e.g. by means of a lateral-flow strip.
  • This is a very inexpensive way of visualizing a PCR hybridization product, and in particular is independent of instrumentation.
  • a disadvantage of this method is that primarily it is not a homogeneous assay, since the reaction vessel must be opened after the amplification/hybridization reaction has taken place, in order that the amplification mixture can be transferred to a lateral-flow strip.
  • the method does not permit any numerical display of the detection result, but is always limited merely to a YES-NO decision based on visual observation. Quantification of the reaction products is also not possible on a lateral-flow strip, since the upper limit is dictated by the binding capacity of the strip.
  • WO 03/072051 A2 is a fluorescence energy transfer (FET) labeled probe with a nucleic acid intercalator, which contains a polycyclic compound bound to an FET-labeled oligonucleotide, wherein the nucleic acid intercalator is covalently bound at the 3′-end of the FET-labeled oligonucleotide, and wherein the FET-labeled oligonucleotide represents a dark quencher, which is positioned at its 3′-end, and wherein the FET-labeled probe is resistant to 3′-5′-exonuclease.
  • FET fluorescence energy transfer
  • the objective of the present invention is to provide a universally usable method for specific detection of target nucleic acids, which method also makes it possible to perform the detection reaction in the form of a homogeneous assay, meaning that the detection of the detection reaction already takes place in the reaction cavity, in which the actual amplification/hybridization reaction is also occurring.
  • Another objective was to permit diagnostic certainty of a detection reaction by quasi dual detection, since a series of test procedures necessitates not only a first detection reaction (such as real-time PCR) but also a second control detection (such as application of the amplification products on an agarose gel).
  • such a novel method could also be used in such a way that the detection reaction takes place as a homogeneous assay (instrument-dependent) or else, as an alternative thereto, even as a test based on a lateral-flow strip (instrument-independent).
  • the double-labeled probe has an integrated cut site for a nuclease. During hybridization of the probe, the formed double strand is cut by the nuclease and thereby the fluorescence is released. After having been cut with the nuclease, the labeled probe is able to function as a primer. Together with the other labeled primer, there is then formed a double-labeled PCR product, which can be detected on a LFA strip.
  • This method is chemically very complex and difficult.
  • the probe must be prepared at a minimum of four sites: 1. reporter fluorophore, 2. quencher, 3. cut site of the nuclease, 4. amplification blockade at the 3′-end.
  • the present object was solved surprisingly simply according to the features of the claims.
  • the inventive method combines a matrix-dependent DNA de novo synthesis with a hybridization step.
  • the inventive choice of labels of the oligonucleotides participating in the reactions permits not only a reaction-dependent fluorescence measurement and associated therewith a numerical and possibly quantitative evaluation but also instrument-independent visualization of the reaction, e.g. on a lateral-flow strip. It is also particularly advantageous that the detection of the fluorescence reduction following a FRET effect is possible with the inventive method in the form of end-point detection. From the instrumental viewpoint, such a measurement principle can therefore also dispense with the use of expensive real-time instrumental systems.
  • This inventive method is based on the following steps:
  • the term “partly complementary” means that sufficient complementarity must be present. In the present case, at least 50%, preferably 70% of the labeled oligonucleotide must be complementary to the target nucleic acid.
  • matrix-dependent means that the de novo synthesis of the target nucleic acid is controlled by the primers being used.
  • the labels of the two oligonucleotides are chosen such that together they form a FRET pair (such as FITC/TAMRA, FAM/TAMRA, FAM/BHQ1, etc.) and, in relation to the inventive dual detection, are also capable of having complementary binding partners on a lateral-flow strip.
  • a FRET pair such as FITC/TAMRA, FAM/TAMRA, FAM/BHQ1, etc.
  • RNA reverse transcriptase reaction
  • DNA amplification
  • RNA a rare RNA, such as mRNA, samples with a small number of particles
  • oligo type 1 participates in this first reaction.
  • the oligo type 1 functions as a primer either in an RNA-dependent reverse transcription (whereby a labeled cDNA strand is formed) or in amplification of the target DNA or cDNA (whereby a labeled PCR product is formed).
  • One-step RT-PCR can also be performed. In this process a second unlabeled primer oligonucleotide increases the yield of the PCR reaction.
  • the oligo type 2 does not participate in the DNA de novo synthesis. Thereafter the reaction batch is heated to a temperature of >90° C. This step leads to thermal separation of the strands. After the end of this thermal denaturing reaction, the reaction batch is cooled to the hybridization temperature of the oligo type 2. During this step, the oligo type 2 binds specifically to the complementary DNA strand. This strand then carries label 1, which was incorporated into the reaction product by the oligo type 1.
  • the detection reaction can take place in two variants, but according to the invention the two detection variants may also be used in parallel or else may even be combined as a verification reaction.
  • the labels incorporated by the two oligos type 1 and type 2 form a FRET pair.
  • the hybridization of the oligo type 2 with the synthesis product of the oligo type 1 that takes place in the inventive method leads to a FRET effect between labels 1 and 2.
  • This effect now leads to a measurable decrease of the fluorescence. This reduction of the fluorescence is numerically evaluated, thus permitting unambiguous detection of the reaction.
  • the solid phase (e.g. a lateral-flow strip, microtiter plate, microparticle) contains a binding site for one of the labels of oligo type 1 or type 2 and/or antibodies or other binding molecules against the labeling molecules of oligos type 1 or type 2 that are able to bind to the labeling molecules of type 1 or type 2 (for example, covalent bonds or hydrogen bonds or via bridging molecules).
  • a detection molecule for visualization or measurement of the hybridization event is located on the solid phase, or such a detection molecule is added to the detection reaction.
  • the detection of a diagnostically relevant target nucleic acid to be detected takes place in the form of a homogeneous assay via the end-point fluorescence measurement of fluorescence quenching.
  • the result can be acquired numerically and it also permits quantification of the target nucleic acid to be detected (using an internal standard) detected and quantified.
  • the inventive method also permits highly specific dual detection, since after fluorescence detection has been achieved the result can be verified on a lateral-flow strip. From the diagnostic viewpoint, such a verification is therefore very much more exact than the detection that has been possible heretofore of real-time PCR products on an agarose gel. If necessary, the method also makes it possible to perform the tests independently of one another (test by means of fluorescence detection or test by means of detection on, for example, a lateral-flow strip).
  • the inventive integration of a hybridization probe into the reaction provides the certainty that the amplified fragment actually contains the target sequence. Thereby false-positive results caused by mispriming are excluded.
  • the use of the chemically modified probe preferably phosphorylation of the last nucleotide of the probe
  • the detection of the specific detection signal takes place not during amplification, where the fluorescence is released either due to the probe hydrolysis caused by the Taq polymerase (EP 0972848 A2) or is reduced by the FRET effect (EP 1384789 B1), but only after the end of the amplification-hybridization reaction.
  • This also causes the positive effect that the method is independent of instrumental equipment. Measurements may be made both in a real-time PCR instrument and after the end of the reaction with a fluorescence reader (see exemplary embodiments).
  • the inventive method also differs from the patent (EP 0826066 B1) that also describes a combination of PCR and hybridization.
  • a FRET-effect-mediated fluorescence signal is again detected. This occurs during the process of amplification by hybridization of a doubly labeled probe having a lower annealing temperature than that of the primer.
  • the release of fluorescence in this case takes place not by hydrolysis of the probe as a result of the exonuclease activity of the polymerase but instead by the fact that the secondary structure of the probe is loosened during hybridization and fluorescence is released by the increase of the distance of the reporter from the quencher.
  • enzymes having no exonuclease activity e.g. Klenow fragments or T4 or T7 polymerases
  • reaction batch contains:
  • the method comprises the following steps:
  • Labels 1 and 2 form a FRET pair.
  • the hybridization of the oligo type 2 with the synthesis product of the oligo type 1 leads to a measurable decrease of the fluorescence caused by the FRET effect between labels 1 and 2.
  • the solid phase contains a binding site for one of the labels of the oligo type 1 or type 2 and/or antibodies or other binding molecules against the labeling molecules of oligos type 1 or type 2, which bind to the labeling molecules of type 1 or type 2 (for example, covalent bonds or hydrogen bonds or via bridging molecules) and at the same time a detection molecule for visualization or measurement of the hybridization event, or such a molecule is added to the sample bound to the solid phase.
  • Oligos type 1 and type 2 may also carry labels other than the labels named in claim 4 .
  • the additional labels may be used for detection of the hybridization event on the solid phase (1e).
  • the melting temperature (T m ) of oligo type 1 is preferably 5° C. to 15° C. higher than the T m of oligo type 2.
  • labels 1 and 2 are preferably 1 to 50 by apart from one another.
  • the oligo with a reporter label can be present in the reaction in a lower concentration than the oligo with the quencher label, preferably in the ratio of 1:10 to 1:20.
  • Negative samples NTC
  • H1N1 cDNA-positive samples POS
  • H1N1 cDNA-positive samples POS
  • the samples that indeed contained human DNA material swab smear of nasal mucous membranes
  • H1N1 NEG
  • H1N1 sense primer (5′-tgg gaa atc cag agt gtg aat cac tct c-3′)
  • H1N1 antisense primer (oligo type 1)
  • oligo type 2 H1N1 probe
  • oligo type 2 (5′-agc aag ctc atg gtc cta cat t-FAM-3′)
  • the PCR was carried out in the SpeedCycler (Analytik Jena) using the rapid cycler technology:
  • Step 1 denaturing 98° C./90 sec
  • Step 2 amplification for 41 cycles (98° C./4 sec; 57° C./4 sec; 72° C./10 sec)
  • Step 3 denaturing 95° C./900 sec
  • Step 4 hybridization: 43° C./600 sec
  • the amplification event/the hybridization reaction was detected by means of an end-point measurement with the SpeedScan fluorescence reader (Analytik Jena AG; FIG. 2 ). At the same time, the change of fluorescence intensity of the sample was measured in real time during the entire reaction ( FIG. 3 ).
  • the measured data of the SpeedScan instrument were subjected to a Pos/Neg determination according to the following formula:
  • x N is the mean value of the NTC values
  • xN min is the smallest NTC value
  • desired percentage deviation of the positive value from the negative value e.g. 20% of x N
  • P is the measured value of the sample to be tested.
  • the sample may be evaluated semi-quantitatively by using a concentration standard.
  • Measured value Value A at Sample ID after the PCR 20% Value B A ⁇ B pos/neg Pos 3447 11485 15506 ⁇ 4021 POS Pos 6533 11485 12420 ⁇ 935 POS Pos 6024 11485 12929 ⁇ 1444 POS Neg 18408 11485 5573 5912 NEG Neg 11764 11485 7189 4296 NEG Neg 18937 11485 16 11469 NEG NTC 11258 11485 7695 3790 NEG NTC 26666 11485 ⁇ 7713 19198 NEG NTC 18937 11485 16 11469 NEG
  • Two batches were prepared: for the inventive method and a real-time PCR batch with a probe labeled with FAM-BHQ1.
  • the cDNAs (see table for particle count/PCR batch) synthesized from Influenza H1N1 virus strains were used as samples.
  • H1N1 RT sense primer (5′-tgg gaa atc cag agt gtg aat cac t-c-3′)
  • H1N1 RT antisense primer (5′- cgt tcc att gtc tga act agr tgt t-3′)
  • H1N1 RT probe (5′-FAM-cca caa tgt agg acc atg agc ttg ctg t-BHQ1-3′)
  • the PCR was carried out in the SpeedCycler (Analytik Jena) using the rapid cycler technology:
  • Step 1 denaturing 98° C./90 sec
  • Step 2 amplification 41 cycles (98° C./4 sec; 57° C./4 sec; 72° C./10 sec)
  • the amplification event/the hybridization reaction in Batch 1 was detected by means of an end-point measurement of the fluorescence by means of SpeedScan (Analytik Jena AG; FIG. 4 ). At the same time, the change of fluorescence intensity of the sample was observed in real time (real-time PCR) during the entire reaction ( FIG. 5 ).
  • the measured data of the SpeedScan instrument were subjected to a Pos/Neg determination according to the formula described hereinabove (see Example 1). In Batch 2, the fluorescence release was measured conventionally by means of real-time PCR.
  • FIG. 1 shows a diagram of the process flow.
  • FIG. 2 shows a fluorescence measurement after the end of the amplification/hybridization reaction.
  • Fields B3-B5 are POS samples
  • B6-B8 are NEG samples
  • B9-B11 are NTC samples (measurement of the fluorescence by means of SpeedScan (Analytik Jena AG)).
  • FIG. 3 shows a real-time fluorescence measurement during the entire amplification/hybridization reaction.
  • Curves 1-3 are POS samples
  • 4-6 are NEG samples
  • 7-9 are NTC samples (real-time measurement for tracking of the detection reaction).
  • FIG. 4 shows a fluorescence measurement after the end of the amplification/hybridization reaction.
  • FIG. 5 shows a real-time fluorescence measurement during the entire amplification/hybridization reaction.
  • Curves 1-4 are NTC samples; samples 5-14 are concentrated as follows: 5, 6-100000 particles/sample; 7, 8-5000 particles/sample; 9, 10-500 particles/sample; sample 11, 12-50 particles/sample; sample 13, 14-5 particles/sample.
  • FIG. 6 shows a real-time fluorescence measurement during a real-time reaction with a conventional TaqMan probe.
  • Curves 6, 7 are NTC samples; samples 1-5 are concentrated as follows: 1-100000 particles/sample; 2-5000 particles/sample; 3-500 particles/sample; sample 4-50 particles/sample; sample 5-5 particles/sample.

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US11209368B2 (en) 2010-04-08 2021-12-28 Ist Innuscreen Gmbh Method for detecting specific nucleic acid sequences

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ES2911458T3 (es) 2022-05-19
DE102010003781B4 (de) 2012-08-16
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US11209368B2 (en) 2021-12-28
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