WO2012069484A2 - Procédé de détection qualitative et quantitative de séquences d'acides nucléiques spécifiques en temps réel - Google Patents

Procédé de détection qualitative et quantitative de séquences d'acides nucléiques spécifiques en temps réel Download PDF

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Publication number
WO2012069484A2
WO2012069484A2 PCT/EP2011/070699 EP2011070699W WO2012069484A2 WO 2012069484 A2 WO2012069484 A2 WO 2012069484A2 EP 2011070699 W EP2011070699 W EP 2011070699W WO 2012069484 A2 WO2012069484 A2 WO 2012069484A2
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Prior art keywords
probe
nucleic acid
fluorophore
base
strand
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PCT/EP2011/070699
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German (de)
English (en)
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WO2012069484A3 (fr
Inventor
Elmara Graser
Timo Hillebrand
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Aj Innuscreen Gmbh
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Publication of WO2012069484A2 publication Critical patent/WO2012069484A2/fr
Publication of WO2012069484A3 publication Critical patent/WO2012069484A3/fr

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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

Definitions

  • the presented method is used for the qualitative and quantitative detection of nucleic acid sequences in real time and thus the molecular diagnostics.
  • the basis of the novel process is the use of a DNA probe labeled only with a fluorochrome (fluorophore), wherein the fluorophore-labeled base is always in close proximity to a guanine base.
  • fluorochrome fluorophore
  • two probes are used for a qualitative or quantitative real-time measurement of a specific target nucleic acid. As a result, a significant increase in the detection sensitivity and signal strength can be generated.
  • Genetic diagnostics has become an indispensable part of modern medical laboratory diagnostics, forensic diagnostics, veterinary laboratory diagnostics or food and environmental diagnostics.
  • this technology also has its disadvantages.
  • To carry out the amplification reaction expensive equipment is needed, which can ensure the rapid change in temperature; the results of the amplification or the probe hybridization coupled with the amplification must also be evaluated by means of expensive and expensive laboratory technology.
  • a "simple" visualization by gel electrophoresis is sometimes insufficient because of its inaccuracy.
  • a widely used method for detecting specific nucleic acids is, for example, the light cycler technology (Roche).
  • the Roche company developed special hybridization probes consisting of two different oligonucleotides, each labeled with only one fluorochrome. At the 3-end of one probe is the acceptor, the other oligonucleotide is provided at the 5-end with a donor.
  • the probes are chosen so that they both bind to the same DNA strand, with the distance between acceptor and donor may only be a maximum of 1 to 5 nucleotides, so that it can come to the so-called. FRET effect.
  • the measurement of the fluorescence takes place during the annealing step, whereby only light of this wavelength is detectable, as long as both probes are bound to the DNA and are in close proximity.
  • the melting point of both probes should be identical in this system.
  • Double Dye probes which are disclosed in the patent US 5210015 and US 5487972 (TaqMan probes). Double Dye Probes carry two fluorochromes on a probe. The reporter dye is here at the 5 - end, the quencher color fabric at the 3 - end. In addition, there may still be a phosphate group on the 3-end of the probe, so that the probe can not function as a primer during elongation. As long as the probe is intact, the intensity of light released is small, since almost all of the light energy produced by the reporter's excitation is absorbed and transformed by the quencher due to its proximity.
  • the emitted light from the reporter dye is "quenched", ie quenched This FRET effect is also retained after the probe has bound to the complementary DNA strand
  • the polymerase hits the probe and hydrolyzes it polymerase to hydrolyze an oligonucleotide (or probe) during strand synthesis as 5 ⁇ -3 ⁇ exonuclease activity
  • Not all polymerases have a 5 ⁇ -3 ⁇ exonuclease activity (Taq and Tth polymerase)
  • the principle is called the TaqMan principle, and after probe hydrolysis, the reporter dye is no longer in close proximity to the quencher, and the emitted fluorescence is no longer transformed, and this increase in fluorescence is measured.
  • Another possibility for the specific detection of amplification products by means of real-time PCR technology is the use of intercalating dyes (ethidium bromide, Hoechst 33258, Yo-Pro-1 or SYBR Green TM and the like).
  • intercalating dyes ethidium bromide, Hoechst 33258, Yo-Pro-1 or SYBR Green TM and the like.
  • a clear differentiation between specific amplification event or artifact is absolutely necessary.
  • one uses a so-called melting point analysis at the end of the actual PCR reaction.
  • real-time PCR applications it is also possible to carry out a quantification of the targets to be detected.
  • TaqMan assay As already mentioned, the use of the so-called. TaqMan assay is state of the art. This elegant method of real-time measurement of target nucleic acids is used worldwide for qualitative and quantitative molecular diagnostics. However, the probes to be used are expensive because of the required two markings.
  • the invention had the object to simplify a method for the qualitative and quantitative detection of specific nucleic acid sequences.
  • the emitted light of the reporter colorant substance is "quenched", ie erased, and this FRET effect is also retained in the method according to the invention after the probe has bound to the complementary strand of DNA, All subsequent steps of the reaction then proceed exactly as in the case of By using polymerases, preferably with S ' -S ' exonuclease activity, the probe is hydrolyzed during the elongation phase, with the fluorochrome of the probe no longer being in spatial proximity to the quenching guanine base.
  • the emitted fluorescence is no longer reshaped and the increase in fluorescence is measured.
  • C T cycle threshold
  • a probe labeled only with a dye in the embodiment of the probe design according to the invention can enable real-time detection of nucleic acids at a lower cost.
  • the device systems available for such a technology can be used without changes. This also applies to the evaluation algorithms.
  • the process according to the invention can be carried out on all these systems.
  • the implementation of the method according to the invention differs from the known Taq-Man method in that in the probe the base labeled with the fluorophore is always in close proximity to a guanine base - that is what in the Taq Man method just should be excluded.
  • Spatial proximity in the sense of the invention means that the guanine base is one to four bases apart from the base with the fluorophore on the same strand. For best results, place the guanine base next to the base with the fluorophore on the same strand.
  • the probe is hydrolyzed during the elongation phase, with the fluorochrome of the probe no longer being in spatial proximity to the quenching guanine base.
  • Corresponding polymerases with exonuclease activity are known to the person skilled in the art.
  • the probe used must be complementary to the sequence to be determined.
  • complementary is identical to the term “complementary” of the patents US 5210015 and US 5487972.
  • a complement of a nucleic acid sequence is when aligned with the nucleic acid sequence such that the 5 'end of one sequence is paired with the 3' end of the other, in the "antiparallel association".
  • Complementarity exists when stable duplex molecules are formed. They may contain mismatched base pairs or unpaired bases. A person skilled in the art is familiar with the term "complementary".
  • the key idea of the invention is the exploitation of an undesirable effect in the Taq-Man process, namely guanine quenching, which dispenses with quencher color substances.
  • markers all fluorophores can be used, which can be quenched with the base guanine.
  • fluorophores are state of the art. Examples include FAM, FITC, BODIPY FL, TAMRA or the CCP dyes. Investigations have shown that such fluorophores can be used which emit light of the wavelengths 300 to 700 nm, preferably of the wavelengths 400 to 500 nm.
  • the pH during the PCR reaction should be between 6.5 and 9.5, preferably between 7.3 and 8.5.
  • the method according to the invention discloses an additional and considerable advantage in a further specific embodiment.
  • the use of two probes in a reaction approach results in much higher diagnostic sensitivity.
  • One probe binds to the "plus-strand” and the second probe to the "minus-strand".
  • this embodiment results in significantly higher fluorescence as well as earlier C T values in the real-time measurement. This observation is shown in the embodiment.
  • a much higher diagnostic sensitivity can be achieved than with a single TaqMan probe.
  • Such a strategy of using two probes according to the invention allows a further significant advantage. By using a second probe, it becomes possible to introduce a second detection sequence region into the detection reaction.
  • the second probe may also be labeled with a different dye than the first probe. This allows both probes to be measured and detected independently of each other.
  • the placement of multiple probes may also be done on the same strand of the target nucleic acid.
  • Embodiment 1 Embodiment 1
  • the target nucleic acid (HilA gene from Salmonella sp.) was amplified in all three batches with the same primers:
  • Primer 1 (5'-GAG AGA AGC GGG TTG GTG TTC ACT C -3 ')
  • Primer 2 (5'-CCG GGC AGA TGA TAC CCG ATG -3 ')
  • Probe 1 (5'-FAM-TGC ACC AGG AAA GCA TTA AGT-Pho -3 ')
  • Probe 1 (5'-FAM-TGC ACC AGG AAA GCA TTA AGT-Pho -3 ')
  • Probe 2 (5'-FAM-TGG TGC AGC AGG TGA TAA CCT TT -Pho -3 ')
  • FIG. 1 shows the binding scheme of the two probes.
  • Probe 3 (5'-FAM-TGC ACC AGG AAA GCA TTA AGT-BHQ1 -3 ')
  • antisense primer 50 pmol / ⁇
  • a negative sample containing only PCR chemicals and H 2 0
  • a positive sample including additionally S. enterica DNA (1.5 ⁇ , total DNA concentration about 50 ng / ⁇ )).
  • Step 1 Denaturation 95 ° C 120 "
  • Step 2 Amplification 50 Cycles
  • the fluorescence released was measured in real time during the annealing phase of each cycle ( Figure 2).
  • Figure 2 shows the approach 1 with the probe 1, the approach 2 with the probes 1 and 2, approach 3 with TaqMan sample and the negative controls (NK) of the respective approaches.
  • Primer 1 (5'-GAG AGA AGC GGG TTG GTG TTC ACT C -3 ')
  • Primer 2 (5'-CCG GGC AGA TGA TAC CCG ATG -3 ')
  • Probe 1 (5'-FAM-TGC ACC AGG AAA GCA TTA AGT-Pho -3 ')
  • Probe 2 (5'-FAM-TGG TGC AGC AGG TGA TAA CCT TT -Pho -3 ')
  • approach 2 a BHQ1 (Black Hole Quencher-1) quenched Taqman probe was used:
  • Probe 3 (5'-FAM-TGC ACC AGG AAA GCA TTA AGT-BHQ1 -3 ')
  • antisense primer 50 pmol / ⁇
  • a negative sample containing only PCR chemicals and H 2 O
  • a dilution series of positive samples containing additionally S. enterica DNA: about 50 ng / ⁇ , 0.5 ng / ⁇ , 0.05 ng / ⁇ and 0.005 ng / ⁇ sample DNA.
  • Step 1 Denaturation 95 ° C 120 "
  • Step 2 Amplification 50 Cycles
  • the fluorescence released was measured in real time during the annealing phase of each cycle and is shown in FIG.
  • Sample 1 inventive method with two guaningequentschten probes; 50 ng / ⁇ DNA
  • Sample 2 method according to the invention with two guanine-quenched probes; 0.5 ng / ⁇ DNA Sample 3: method according to the invention with two guanine-quenched probes; 0.05 ng / ⁇ DNA Sample 4: Method according to the invention with two guanine-quenched probes; 0.005 ng / ⁇
  • Sample 7 Taqman method; 0.05 ng / ⁇ DNA
  • Sample 8 Taqman's method; 0.005 ng / ⁇ DNA
  • the target nucleic acid an artificially synthesized fragment having the primer and probe sequences for the detection of Listeria monocytogenes and a repeating nonsense sequence for the binding of a quanine quenched probe
  • the target nucleic acid is amplified in both approaches with the same primers:
  • Primer 1 (5'-CGC AAC AAA CTG AAG CAA AGG -3 ')
  • Primer 2 (5'-TCC GCG TGT TTC TTT TCG AT-3')
  • Approach A used guanine-quenched probes which bind twice to the same strand of the target nucleic acid (FIG. 4).
  • Probe 1 (5'-FAM-TGT GTT CGT GTC ATC TAG GA -Pho -3 ')
  • FIGS 4 and 5 show the bonding scheme of the quenched probe.
  • Probe 2 (5'-FAM-CCA TGG CAC CAC CAG CAT CT - BHQ1 -3 ') Reaction batch (amplification / hybridization)
  • antisense primer 50 pmol / ⁇
  • Step 1 Denaturation 95 ° C 120 "

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  • Chemical Kinetics & Catalysis (AREA)
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  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé et un kit d'essai servant à la détection qualitative et quantitative de séquences d'acides nucléiques en temps réel. L'invention a pour objet l'utilisation d'une sonde ADN marquée uniquement par un fluorochrome (fluorophore), la base marquée par le fluorophore se trouvant toujours à proximité spatiale d'une base guanine et ainsi - sans utilisation d'un colorant désactiveur - désactivant le fluorophore. Au moyen d'amorces, un mélange de duplexes est produit dans des conditions d'hybridation, les duplexes comprenant l'acide nucléique cible, hybridé à l'oligonucléotide marqué (sonde). Par l'ajout d'une polymérase présentant une activité exonucléase, qui coupe l'oligonucléotide marqué (sonde) hybridé entre le marquage au fluorophore et la base guanine et par conséquent casse la désactivation, un signal de fluorescence mesurable est créé.
PCT/EP2011/070699 2010-11-22 2011-11-22 Procédé de détection qualitative et quantitative de séquences d'acides nucléiques spécifiques en temps réel WO2012069484A2 (fr)

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DE201010052524 DE102010052524A1 (de) 2010-11-22 2010-11-22 Verfahren zum qualitativen und quantitativen Nachweis von spezifischen Nukleinsäuresequenzen in Echtzeit
DE102010052524.3 2010-11-22

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020025387A1 (fr) * 2018-08-03 2020-02-06 Robert Bosch Gmbh Procédé de réalisation d'une pcr en temps réel
US20220033883A1 (en) * 2013-08-12 2022-02-03 Bio-Rad Laboratories, Inc. Amplification reporter with base-pairing oligomers

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112852927A (zh) * 2021-02-02 2021-05-28 中国科学院合肥物质科学研究院 一种基于荧光自抑制探针的等温扩增系统及方法

Citations (2)

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US5210015A (en) 1990-08-06 1993-05-11 Hoffman-La Roche Inc. Homogeneous assay system using the nuclease activity of a nucleic acid polymerase
US5538848A (en) 1994-11-16 1996-07-23 Applied Biosystems Division, Perkin-Elmer Corp. Method for detecting nucleic acid amplification using self-quenching fluorescence probe

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DE102007013099A1 (de) * 2007-03-14 2008-09-18 Aj Innuscreen Gmbh Verfahren und Testkit zum schnellen Nachweis spezifischer Nukleinsäuresequenzen, insbesondere zum Nachweis von Mutationen oder SNP's

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US5210015A (en) 1990-08-06 1993-05-11 Hoffman-La Roche Inc. Homogeneous assay system using the nuclease activity of a nucleic acid polymerase
US5487972A (en) 1990-08-06 1996-01-30 Hoffmann-La Roche Inc. Nucleic acid detection by the 5'-3'exonuclease activity of polymerases acting on adjacently hybridized oligonucleotides
US5538848A (en) 1994-11-16 1996-07-23 Applied Biosystems Division, Perkin-Elmer Corp. Method for detecting nucleic acid amplification using self-quenching fluorescence probe

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HORN T ET AL.: "Chemical synthesis and characterization of branched oligodeoxyribonucleotides (bDNA) for use as signal amplifiers in nucleic acid quantification assays", NUCLEIC ACIDS RES., vol. 25, 1997, pages 4842 - 4849, XP002260392, DOI: doi:10.1093/nar/25.23.4842
LEE ET AL.: "A fluorometric assay for DNA cleavage reactions characterized with BamHI restriction endonuclease", ANAL. BIOCHEM., vol. 220, 1994, pages 377 - 383, XP024763207, DOI: doi:10.1006/abio.1994.1353
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TORIMURA ET AL., ANALYTICAL SCIENCES, vol. 17, 2001, pages 155FF

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220033883A1 (en) * 2013-08-12 2022-02-03 Bio-Rad Laboratories, Inc. Amplification reporter with base-pairing oligomers
WO2020025387A1 (fr) * 2018-08-03 2020-02-06 Robert Bosch Gmbh Procédé de réalisation d'une pcr en temps réel

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WO2012069484A3 (fr) 2012-09-27

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