WO2012069484A2

WO2012069484A2 - Method for qualitatively and quantitatively detecting specific nucleic acid sequences in real time

Info

Publication number: WO2012069484A2
Application number: PCT/EP2011/070699
Authority: WO
Inventors: Elmara Graser; Timo Hillebrand
Original assignee: Aj Innuscreen Gmbh
Priority date: 2010-11-22
Filing date: 2011-11-22
Publication date: 2012-05-31
Also published as: WO2012069484A3; DE102010052524A1

Abstract

The method and test kit are designed to qualitatively and quantitatively detect nucleic acid sequences in real time. The invention relates to the use of a DNA probe marked only with a fluorochrome (fluorophor), wherein the base marked with the fluorophor is always present in the vicinity of a guanine base and thus quenches the fluorophor without the use of a quenching dye. A mixture of duplexes is generated under hybridization conditions by means of primers, wherein the duplexes comprise the target nucleic acids deposited on the marked oligonucleotide (probe). By adding a polymerase having exonuclease activity, cutting the deposited marked oligonucleotide (probe) between the fluorophor marking and the guanine base, and thus terminating the quenching, a measurable fluorescence signal is generated.

Description

METHOD FOR THE QUALITATIVE AND QUANTITATIVE DETECTION OF SPECIFIC NUCLEIC ACID SEQUENCES IN REAL TIME

description

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. In a particularly efficient embodiment of the present invention, 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.

State of the art

Genetic diagnostics has become an indispensable part of modern medical laboratory diagnostics, forensic diagnostics, veterinary laboratory diagnostics or food and environmental diagnostics.

Genetic diagnostics have been revolutionized with the invention of PCR technology, which allows to specifically multiply any nucleic acid sequence.

However, in addition to its clear advantages (such as sensitivity, specificity), 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. Here, 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. By using two hybridizing probes in addition to the primers used, the specificity of this detection system is extremely high.

Another real-time PCR application for the detection of specific nucleic acid targets can be performed with so-called. 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 During the elongation phase, the polymerase hits the probe and hydrolyzes it polymerase to hydrolyze an oligonucleotide (or probe) during strand synthesis as 5 ^λ -3 ^λ exonuclease activity Not all polymerases have a 5 ^λ -3 ^λ 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 ™ and the like). However, a clear differentiation between specific amplification event or artifact is absolutely necessary. To achieve this, one uses a so-called melting point analysis at the end of the actual PCR reaction. By means of real-time PCR applications, it is also possible to carry out a quantification of the targets to be detected.

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.

It is known that the base guanine acts as a fluorescence acceptor and that the fluorescence of some fluorophores is quenched as a result of the interaction with guanine (Cooper et al. (1990) "Analysis of fluorescence energy-transfer in duplex and branched DNA"). Lee et al. (1994) "A fluorometric assay for DNA cleavage reaction characterized by BamHI restriction endonuclease." Anal. Biochem. 220: 377-383., Horn T et al. 1997) "Nucleic Acids Res. 25: 4842-4849.)." "Chemical synthesis and characterization of branched oligodeoxyribonucleotides (bDNA) for use as signal amplifiers in nucleic acid quantification assays."

Maruyama et al. describe in "Mutation detection in DNA oligonucleotides based on a guanine quenching method coupled with enzymatic digestion of single-stranded DNA" Biotechnology Letters (2005) 27: 1349-1354, the determination of G> T mutations by guanine quenching. Guanine quenching enables DNA hybridization to be observed and point mutations to be determined. The resulting duplex is cleaved by T7 endonuclease.

More detailed investigations on guanine quenching are by Torimura et al. in Analytical Sciences (2001) Vol. 17, p. 155ff.

So far, in no real-time PCR method guanine quenching using a polymerase with 5 ^λ -3 ^λ exonuclease activity has been described.

Object of the invention

The invention had the object to simplify a method for the qualitative and quantitative detection of specific nucleic acid sequences. Solution of the task

The object has been solved according to the features of the claims.

Surprisingly, it is evident in a test setup that a real-time measurement of target nucleic acids is also possible if one does not use a double-dye probe system as described in US Pat. No. 5,538,848. It was found that a FRET effect can be used when using a fluorochrome-labeled probe only. According to the invention, a variant is currently being used which actually represents a limitation in the case of TaqMan probes and is never used there. This concerns the positioning of the reporter dye in close proximity to a guanine base.

Such an arrangement shows exactly the effect that has a double-dye probe system to the principle. As long as the probe is intact, the fluorescence released is small, since almost all the energy produced by the excitation of the reporter is absorbed and reshaped by the proximity of the quencher. This phenomenon is now fulfilled by the guanine base, ie the guanine base located on the probe fulfills the function of the quencher of a double-dye probe system according to the invention. 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.

For each probe set, a so-called C _T value is determined, as in other real-time technologies. The intersection between the fluorescence of the sample and the threshold (background fluorescence) projected onto the abscissa is called the cycle threshold (C _T ) and represents the lowest measurable positive value of the real-time PCR. Thus, the C _T value gives a number of cycles at. It is directly related to the initial amount of DNA used. If the C _T value is low, the amount of DNA used is large. If the C _T value is high, the initial amount of DNA is small. The C _T value is therefore the basis for the Quantification of a reaction. In addition, a simple qualitative statement about the presence of a specific target nucleic acid is possible with this evaluation method. In this way, 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 (real-time PCR devices) 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. Using a polymerase with 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. 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. The term "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. As markers, all fluorophores can be used, which can be quenched with the base guanine. Such 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.

Surprisingly, the method according to the invention discloses an additional and considerable advantage in a further specific embodiment. Thus, 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". When both probes were labeled with the same dye, 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. Thus, 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. This is especially important when specific target sequences often vary due to mutations. With a second probe, a larger sequence range can thus be covered specifically for the detection reaction. In such a case, for example, 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.

The invention is illustrated below with reference to embodiments. The embodiments do not represent a limitation of the method according to the invention.

embodiments Embodiment 1

Comparison of fluorescence of one or two guanine-quenched probes with TaqMan detection

In the approach, three different approaches are tested against each other. The target nucleic acid (HilA gene from Salmonella sp.) Was amplified in all three batches with the same primers:

PCR primer:

Primer 1 (5'-GAG AGA AGC GGG TTG GTG TTC ACT C -3 ')

Primer 2 (5'-CCG GGC AGA TGA TAC CCG ATG -3 ')

In approach 1 a quango quenched probe was used. The probe is labeled at the 5 'end with the fluorescent FAM ((3', 6'-dipivaloylfluoresceinyl) -6-carboxamidohexyl) -10- (2-cyanoethyl) - (N, N-diisopropyl) -phosphoramidite) and am 3'-end protected with a phosphate group against the polymerase activity (Figure 1).

Probe 1: (5'-FAM-TGC ACC AGG AAA GCA TTA AGT-Pho -3 ')

In approach 2, two quenched quench probes were used, which bind to the opposite strands of the target nucleic acid (Figure 1).

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.

In approach 3, a BHQl (Black Hole Quencher-1) quenched TaqMan probe is used:

Probe 3: (5'-FAM-TGC ACC AGG AAA GCA TTA AGT-BHQ1 -3 ')

Reaction batch (amplification / hybridization)

Per sample: sense primer (50 pmol / μΐ) 0, 1 μΐ

antisense primer (50 pmol / μΐ) 0, 1 μΐ

Probe (25 pmol / μΐ) per 0.1 μΐ

dNTP mix (12.5 mM) 0.3 μΐ

10X PCR Buffer (MgCl ₂ included) 1, 5 μΐ

Taq DNA polymerase 0.75 U

PCR grade H ₂ 0 add 15 μΐ

For testing, a negative sample (containing only PCR chemicals and H ₂ 0) and a positive sample (including additionally S. enterica DNA (1.5 μΐ, total DNA concentration about 50 ng / μΐ)).

The real-time PCR was carried out in the Q-Tower (Analytik Jena) using the Rapid-Cycler technology:

Amplification / hybridization conditions

Step 1: Denaturation 95 ° C 120 "

Step 2: Amplification 50 Cycles

95 ° C 4 "

50 ° C 40 "

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.

The graph comparisons and Ct values demonstrate that the guanine-quenched probe exhibits comparable fluorescence release during amplification with the Taqman probe. However, the two simultaneously probes function synergistically, showing higher signal strength and Ct compared to the two single-probe approaches.

Embodiment 2

Determination of the Detection Limits of the Three Probe Systems Described in Application Example 1

In the approach, two different approaches are tested against each other. The target nucleic acid (HilA gene of Salmonella sp.) Is amplified in both approaches with the same primers: PCR primer:

Primer 1 (5'-GAG AGA AGC GGG TTG GTG TTC ACT C -3 ')

Primer 2 (5'-CCG GGC AGA TGA TAC CCG ATG -3 ')

In approach 1, two queuing quenched probes were used, which bind to the opposite strands of the target nucleic acid Figure 1:

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 ') In 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 ')

Reaction batch (amplification / hybridization)

Per sample:

sense primer (50 pmol / μΐ) 0, 1 μΐ

antisense primer (50 pmol / μΐ) 0, 1 μΐ

Probe (25 pmol / μΐ) per 0.1 μΐ

dNTP mix (12.5 mM) 0.3 μΐ

10X PCR Buffer (MgCl ₂ included) 1.5 μΐ Taq DNA polymerase 0.75 U

PCR grade H ₂ 0 add 15 μΐ

For testing, a negative sample (containing only PCR chemicals and H ₂ O) and a dilution series of positive samples (containing additionally S. enterica DNA: about 50 ng / μΐ, 0.5 ng / μΐ, 0.05 ng / μΐ and 0.005 ng / μΐ sample DNA).

Amplification / hybridization conditions

Step 1: Denaturation 95 ° C 120 "

Step 2: Amplification 50 Cycles

95 ° C 4 '

50 ° C 40 '

The fluorescence released was measured in real time during the annealing phase of each cycle and is shown in FIG.

Explanation to FIG. 3:

Sample 1: inventive method with two guaningequentschten probes; 50 ng / μΐ DNA Sample 2: method according to the invention with two guanine-quenched probes; 0.5 ng / μΐ DNA Sample 3: method according to the invention with two guanine-quenched probes; 0.05 ng / μΐ DNA Sample 4: Method according to the invention with two guanine-quenched probes; 0.005 ng / μΐ

DNA

Sample 5: Taqman's method; 50 ng / μΐ DNA

Sample 6: Taqman method; 0.5 ng / μΐ DNA

Sample 7: Taqman method; 0.05 ng / μΐ DNA

Sample 8: Taqman's method; 0.005 ng / μΐ DNA

NK: Negative controls of the respective approaches Sample DNA concentration Ct value

Approach 1 50 ng / μΐ 22.9

Batch 2 0.5 ng / μΐ 30.3

Batch 3 0.05 ng / μΐ 32.8

Approach 4 0.005 ng / μΐ 36.0

Approach 1-4 NK - no Ct

Batch 5 50 ng / μΐ 30.4

Batch 6 0.5 ng / μΐ 37, 1

Batch 7 0.05 ng / μΐ 41.9

Batch 8 0.005 ng / μΐ no Ct

Approach 5-8 NK - no Ct

The comparison of the Kurvenverl on and the Ct values clearly demonstrate that the synergistic effect of the two simultaneously used probes not only leads to an increased signal strength, but also to a much more sensitive detection of the target nucleic acid compared to a Taqman assay ,

Example 3

Use of a system of two guanine-quenched probes residing on the same DNA strand compared to TaqMan probe-based detection.

In the approach, two different approaches are tested against each other. 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) is amplified in both approaches with the same primers:

PCR primer:

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 ')

Figures 4 and 5 show the bonding scheme of the quenched probe.

In approach B, a BHQ1 (Black Hole Quencher-1) quenched TaqMan probe was used (FIG. 5).

Probe 2: (5'-FAM-CCA TGG CAC CAC CAG CAT CT - BHQ1 -3 ') Reaction batch (amplification / hybridization)

Per sample:

sense primer (50 pmol / μΐ) 0, 1 μΐ

antisense primer (50 pmol / μΐ) 0, 1 μΐ

Probe (25 pmol / μΐ) per 0.1 μΐ

dNTP mix (12.5 mM) 0.3 μΐ

10X PCR Buffer (MgCl ₂ included) 1, 5 μΐ

Taq DNA polymerase 0.75 U

PCR grade H ₂ 0 add 15 μΐ

For testing a negative sample (containing only PCR chemicals and H ₂ 0) and the synthetic standard (1.5 μΐ, total DNA concentration about 50 ng / μΐ DNA) was used by each approach.

Amplification / hybridization conditions

Step 1: Denaturation 95 ° C 120 "

Step 2 Amplification 50 cycles

95 ° C 4 "

53 ° C 40 " The fluorescence released was measured in real time during the anealing phase of each cycle (Figure 6).

Explanation to FIG. 6:

A: batch with the probe 1;

B: approach with TaqMan probe

NK. The negative controls of the respective approaches

The comparisons of the curve and the Ct values demonstrate that in this case the signal strength of the TaqMan probe is higher, but the sensitivity of the reaction with respect to the CT values is again better in the method according to the invention. By using two queued probes placed one behind the other, much earlier Ct signals are obtained. This property of the probe system can thus lead to a shortening of the transit time of a PCR detection.

Claims

claims

A method of qualitatively or quantitatively detecting specific target nucleic acid sequences, characterized by the steps of: a) a nucleic acid-containing probe is provided with at least one fluorophore-labeled oligonucleotide (probe) containing a sequence complementary to a target nucleic acid sequence strand , wherein the fluorophore label is in proximity to a guanine base on the same strand of the labeled oligonucleotide (probe) and thus quenches the fluorophore without the use of a quencher dye to produce a mixture of primers under hybridization conditions Producing duplexes, wherein the duplexes comprise the target nucleic acid attached to the labeled oligonucleotide (probe), and

b) Addition of a polymerase with exonuclease activity, which cuts the annealed labeled oligonucleotide (probe) between the fluorophore label and the guanine base, thus abolishing quenching and producing a measurable fluorescence signal.

2. The method according to claim 1, characterized in that the guanine base is one to four bases removed from the base with the fluorophore label, preferably located directly next to the base with the fluorophore label on the same strand.

3. The method according to claim 1 or 2, characterized in that a polymerase with 5 ^' - 3 ^' -Exonukleaseaktivität is used.

4. The method according to any one of claims 1 to 3, characterized in that as

Fluorophores are used which emit light of a wavelength of 300 to 700 nm, preferably of the wavelengths 400 to 500 nm.

5. The method according to claim 4, characterized in that are used as fluorophores FAM, FITC, BODIPY FL, TAMRA or CCP dyes.

6. The method according to any one of claims 1 to 5, characterized in that the pH is between 6.5 and 9.5, preferably between 7.3 and 8.5.

7. The method according to any one of claims 1 to 6, characterized in that two or more labeled probes in which the fluorophore label is in spatial proximity to a guanine base on the same strand of the labeled oligonucleotide (probe) and so on Using a quencher dye substance - the

Fluorophore quenches, is used.

8. The method according to claim 8, characterized in that a probe to the "plus strand" and the second probe to the "minus strand" of the target nucleic acid or both probes hybridize to the same strand.

9. test kit for carrying out the method according to one of claims 1 to 8,

full:

at least one fluorophore-labeled oligonucleotide (probe) containing, on the one hand, a sequence that is complementary to a target nucleic acid sequence strand, wherein the fluorophore label is in proximity to a guanine base on the same strand of the labeled oligonucleotide (probe) and, on the other hand, has a nuclease interface between the base with the fluorophore label and the guanine base

a polymerase with exonuclease activity that cuts the oligonucleotide (probe) between the fluorophore label and the guanine base

at least one primer

> per se known PCR buffers and dNTPs.

10. Use of the method according to any one of claims 1 to 8 or the test kit according to claim 9 for the qualitative or quantitative detection of specific nucleic acid sequences in real time.