U.S. patent application number 12/644982 was filed with the patent office on 2010-09-02 for method and rapid test for detection of specific nucleic acid sequences.
This patent application is currently assigned to AJ INNUSCREEN GmbH. Invention is credited to Elmara Graser, Timo Hillebrand.
Application Number | 20100221718 12/644982 |
Document ID | / |
Family ID | 40030866 |
Filed Date | 2010-09-02 |
United States Patent
Application |
20100221718 |
Kind Code |
A1 |
Hillebrand; Timo ; et
al. |
September 2, 2010 |
METHOD AND RAPID TEST FOR DETECTION OF SPECIFIC NUCLEIC ACID
SEQUENCES
Abstract
A universally usable method for specific detection of target
nucleic acid sequences, which method can be performed very rapidly
and also simply and furthermore which does not need any expensive
instrumental systems. The method is intended to be suitable as a
molecular genetic rapid test and to respect the requirements of
diagnostic specificity assurance. In this regard it is important
that only one specific amplification product be detected and that
amplification artifacts can be unambiguously discriminated. A
nucleic acid amplification kit suitable for performing this
method.
Inventors: |
Hillebrand; Timo; (Hoenow,
DE) ; Graser; Elmara; (Berlin, DE) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND MAIER & NEUSTADT, L.L.P.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
AJ INNUSCREEN GmbH
Berlin
DE
|
Family ID: |
40030866 |
Appl. No.: |
12/644982 |
Filed: |
December 22, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/EP08/57857 |
Jun 20, 2008 |
|
|
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12644982 |
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Current U.S.
Class: |
435/6.1 ;
435/6.18 |
Current CPC
Class: |
C12Q 1/689 20130101;
C12Q 2600/158 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 22, 2007 |
DE |
10 2007 029 772.8 |
Claims
1. A method for assaying at least one specific nucleic acid
sequence (target sequence) comprising: amplifying a nucleic acid
sequence to be assayed with at least one primer, if necessary,
followed by strand separation, and hybridizing with at least one
probe completely or partly complementary to the target sequence,
and detecting the hybridization reaction; wherein a) the
amplification, and if necessary, the strand separation, and the
hybridization take place in one reaction vessel, and b) at least
one primer is labeled with a molecule, and c) the hybridization
probe is provided with a label and it hybridizes to the strand of
the target sequence that contains the labeled primer and d) the
detection of the hybridization reaction takes place on a solid
phase outside the reaction vessel mentioned under a) and e) the
solid phase contains a binding site for the label either of the
primer or of the probe and thereby the hybridization product is
thereby bound to the solid phase and f) the detection of the
hybridization reaction takes place on a solid phase outside the
reaction vessel mentioned under a) by the fact that the solid phase
has a binding site that permits binding with the label of the
primer or with the label of the hybridization probe, whereby the
hybridization product is bound to the solid phase and detection of
the bound hybridization product takes place by direct or indirect
detection of the label that is still free or the label of the
primer or the label of the hybridization probe enters into binding
with a detection molecule and then the free label enters into
binding with a binding site of the solid phase, whereby the
hybridization product is bound to the solid phase and detection of
the hybridization product bound to the solid phase takes place via
the detection molecule.
2. The method according to claim 1, wherein the visualization or
the measurement of the PCR hybridization result takes place by
means of an optical device.
3. The method according to claim 1, wherein in that the
hybridization probe is protected against the 5'.fwdarw.3'
polymerase activity.
4. The method according to claim 3, wherein the hybridization probe
is protected against the 5'.fwdarw.3' polymerase activity by
labeling or by phosphorylation.
5. The method according to claim 1, wherein the at least one primer
is labeled with biotin.
6. The method according to claim 1, wherein the hybridization probe
is labeled with FITC (fluorescein isothiocyanate).
7. The method according to claim 1, wherein there is used as the
solid phase a test strip that contains a streptavidin site for
coupling the biotin-labeled label and an FITC binding site for
functional control of the test strip.
8. The method according to claim 7, wherein the PCR mixture
(amplification mixture) is mixed with a running buffer and applied
on the test strip.
9. The method according to claim 7, wherein gold particles coated
with anti-FITC antibodies are located in the lower zone of the test
strip, where the sample is applied, and in that the streptavidin
binding site is located further along the test strip.
10. The method according to claim 1, wherein an asymmetric PCR is
performed instead of the standard PCR reaction.
11. The method according to claim 1, wherein a reverse
transcription takes place in the case of RNA assay before
amplification.
12. A test kit for performing the method according to claim 1,
comprising: a reaction vessel for performing the amplification, the
strand separation and the hybridization with the probe, at least
one primer labeled with a molecule, at least one probe that is
completely or partly complementary to the target sequence, that is
protected against the 5'.fwdarw.3' polymerase activity and/or is
provided with a label, and that hybridizes to the strand of the
target sequence that contains the labeled primer, at least one
solid phase, which contains a binding site for the label either of
the primer or of the probe, and/or PCR reagents known in
themselves, such as PCR buffers, polymerases, dNTPs and if
necessary further additives as well as at least one running agent
for detection of hybridization.
13. The test kit according to claim 12, wherein the reaction vessel
contains the primer, the probe and the PCR reagents known in
themselves in solid form.
14. The method of claim 1, which is a qualitative method for
detecting the target nucleic acid.
15. The method according to claim 1 that is a rapid test in which
detection takes less than one hour.
16. The method according to claim 1 that is a rapid test that
comprises multiplex detection, wherein several primers and probes
labeled by either identical or different molecules are
employed.
17. The method according to claim 1, wherein the target nucleic
acid is from a virus or a bacterium.
18. A method for food diagnosis, environmental analysis, or
hospital hygiene comprising the method of claim 1.
19. The method according to claim 1, wherein the target nucleic
acid is from Salmonella, Listeria, E. coli, Campylobacter,
Shigella, Enterobacter, MRSA microbes or Legionella.
20. The method according to claim 1, wherein the target nucleic
acid is from Borrelia, Rickettsia, Erlichia, Babesia, or another
tick-born pathogen.
21. The method according to claim 1, wherein said target nucleic
acid comprises a SNP, mutation or methylated sequence motif.
22. A method for detecting a target nucleic acid comprising:
conducting a polymerase chain reaction (PCR) on a sample suspected
of containing the target nucleic acid in a PCR reaction mixture
comprising: two PCR primers suitable for amplifying the target
nucleic acid, one of which is labeled at its 5' end with a labeling
molecule, and a hybridization probe which is able to hybridize to
the strand of the target nucleic acid containing the sequence of
primer labeled at its 5' end, but which has been chemically
modified so that it is not elongated by a 5'.fwdarw.3' polymerase
used for the PCR; denaturing the PCR reaction mixture at a
temperature sufficient to separate the strands of a PCR
amplification product generated by the PCR, cooling the denatured
PCR reaction mixture to a hybridization temperature of the
hybridization probe for a time and under conditions sufficient for
the probe to bind to the complementary strand of the nucleic acid
amplification product, and contacting the resulting mixture with a
solid phase support comprising a nucleic acid complementary to the
PCR probe; wherein the occurrence of, or amount of, binding to the
solid phase support compared to a control indicates the presence of
the target nucleic acid in the sample.
23. The method of claim 22, wherein contacting the resulting
mixture with a solid phase support comprising a nucleic acid
complementary to the PCR probe is conducted outside of a reaction
vessel used to perform amplification and hybridization.
24. The method of claim 23, wherein said solid phase is a
lateral-flow test strip.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of PCT/EP2008/057857,
filed Jun. 20, 2008, and claims priority to Germany 10 2007 029
772.8, filed Jun. 22, 2007, both of which are hereby incorporated
by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to a method and a test kit for
detection of specific nucleic acid sequences with the steps of
amplification, hybridization by means of probes, and detection of
the hybridization event; wherein the detection of the hybridization
event takes place on a solid phase outside the reaction vessel for
amplification/hybridization.
[0004] 2. Description of the Related Art
[0005] Genetic diagnostics has become an indispensable tool of
modern medical laboratory diagnostics, forensic diagnostics,
veterinary medical laboratory diagnostics or food and environmental
diagnostics.
[0006] Genetic diagnostics was revolutionized with the invention of
PCR technology, with which it is possible to amplify any arbitrary
nucleic acid sequence specifically.
[0007] The use of PCR covers a diversity of methods, which in
combination with the PCR technology additionally permit specific
detection of completed amplification. Especially the requirements
of an exact genetic diagnosis must make use of methods that ensure
that a generated amplification product also corresponds to the
target sequence that is specifically to be detected. The widespread
use of visualization of a PCR product by means of gel
electrophoresis is not sufficient for this purpose.
[0008] One possibility for detection of specific nucleic acid
sequences in a way that in principle can be achieved very rapidly
and without great experimental time and effort is what are known as
real-time PCR methods. In this case the amplification reaction is
directly coupled with the actual detection reaction.
[0009] A widely used method for detection of specific nucleic acids
is light cycler technology (Roche). For this purpose Roche has
developed special hybridization probes, consisting of two different
oligonucleotides, each labeled with only one fluorochrome. The
acceptor is located at the 3'-end of the one probe and the other
oligonucleotide has a donor at the 5'-end. The probes are chosen
such that they both bind to the same DNA strand, the distance
between acceptor and donor being permitted to be at most 1 to 5
nucleotides, so that what is known as the FRET effect can occur.
The fluorescence is measured during the annealing step, in which
only light of this wavelength is detectable as long as both probes
are bound to the DNA. In this system the melting point of both
probes should be identical. Because of the use of two hybridizing
probes in addition to the primers used, the specificity of this
detection system is extremely high.
[0010] A further real-time PCR application for detection of
specific nucleic acid targets can be performed with what are known
as double-dye probes, which are disclosed in U.S. Pat. Nos.
5,210,015 and 5,487,972 (TaqMan probes), both of which are
incorporated by reference. Double-dye probes carry two
fluorochromes on one probe. The reporter dye is located in this
case at the 5'-end and the quencher dye at the 3'-end. In addition,
a phosphate group is also located at the 3'-end of the probe if
necessary, 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 transformed by
virtue of the spatial proximity of the quencher. The emitted light
of the reporter dye is "quenched", or in other words extinguished.
This FRET effect is preserved even after the probe has bonded to
the complementary DNA strand. During the elongation phase, 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). This principle was first described for the Taq
polymerase. The principle is known as the TaqMan principle. After
probe hydrolysis, the reporter dye is no longer located in spatial
proximity to the quencher. The emitted fluorescence is now no
longer transformed and this fluorescence increase is measured.
[0011] 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 Green.TM. and the like). After being excited by high-energy UV
light, these dyes emit light in the lower-energy visible wavelength
region (fluorescence). If the dye is present as a free dye in the
reaction mixture, the emission is very weak. Only by intercalation
of the dye, whereby it fits into the small furrows of double-strand
DNA molecules, is the light emission greatly intensified. The dyes
are inexpensive and universally usable, since in principle any PCR
reaction can be followed in real time with them. In addition, they
have high signal strength, since every DNA molecule binds several
dye molecules. From the advantages, however, there also results an
extreme disadvantage for application: In principle it is not
possible by means of intercalating dyes to distinguish between
correct product and amplification artifacts (such as primer dimers
or defective products). While primer dimers and other artifacts are
being formed, they naturally also bind intercalating dyes and thus
lead to an unspecific increase in fluorescence even in negative
samples. However, a clear differentiation between specific
amplification event or artifact is absolutely necessary. In order
to achieve this in any case, what is known as a melting-point
analysis is performed at the end of the actual PCR reaction. For
this purpose the reaction mixture is heated in steps of 1 degree
from 50.degree. C. to 90.degree. C. The fluorescence is measured
continuously during this process. The point at which double-strand
DNA melts is characterized by a decrease (peak) of the fluorescence
of the intercalating dye, since the intercalating dye dissociates
from the single-strand DNA. When the PCR is optimally adjusted, a
melting-point peak that tapers sharply is to be expected. This
melting point represents the specific product to be expected.
Products of different sizes and products of other sequences have
different melting points.
[0012] When the fluorescence is plotted graphically against
temperature, the fluorescence decrease of the two products can be
perceived as two separate melting points. Thus this system gains
specificity and makes it possible to distinguish a specific
amplification product from artifacts. In this way it is possible to
distinguish even between homozygotes (single peak) and
heterozygotes (two peaks).
[0013] Furthermore, it is also possible to achieve quantitation of
the targets to be detected by means of REAL-time PCR
applications.
[0014] As already explained, the described methods fulfill the need
for specific detection of an amplification product.
[0015] A great disadvantage, however, is that they are implemented
on very expensive instrumental platforms, which have to unite the
process of amplification and that of subsequent optical detection,
in a manner corresponding to the problem, in one hardware solution.
Furthermore, many of these described detection methods are still
based on real-time tracking of the amplification process. On the
basis of this strategy, even workup of the measured fluorescence
values takes place in the course of the amplification reaction. It
is clear to those skilled in the art that, in this connection, an
enormously large body of analysis algorithms must also be
integrated into real-time systems. Ultimately this explains the
high financial expenditure that must be invested for the use of
real-time PCR systems, Also ultimately, the operation of such
instrumental systems requires a high degree of expertise.
[0016] Besides the described diagnostic detections based on
REAL-time PCR, however, alternative variants for specific detection
of nucleic acids also exist.
[0017] An example of less expensive methods for detection of
nucleic acids in this connection is PCR-ELISA. In this method, the
DNA sequence to be examined is amplified and the generated DNA
fragment is then covalently immobilized on a solid phase (such as
microtiter plates or strips), denatured to a single strand and
hybridized with a sequence-specific probe. Successful binding of
the probe can be visualized with an antibody-mediated color
reaction. Another variant is based on immobilizing the probes on a
solid phase, denaturing the PCR product and then bringing it into
contact with the immobilized probe. Detection of a completed
hybridization event takes place by analogy with the first variant
of the method.
[0018] In principle, PCR-ELISA methods are easy to perform, but
they comprise multiple procedural steps. Besides the time needed to
perform the PCR, therefore, several hours of working time are also
needed to perform the subsequent detection method. Such a method
usually needs 8 hours and therefore is also not suitable as a rapid
test.
[0019] Furthermore, some items of equipment are also needed, such
as a temperature-control station, what is known as a washer, or
even a measuring instrument for detection of the hybridization
signal. Furthermore, other special instruments or special
consumable materials may also be necessary.
[0020] Further simple methods for detection of amplification
products are based on amplification of the target sequences and
subsequent hybridization of amplification products on a membrane.
These methods also have several variants known to those skilled in
the art. Once again, however, these methods are also laborious to
perform, need a large number of procedural steps to be performed
and therefore are not suitable as rapid tests. This then also
applies to the use of biochip strategies, which use hybridization
of PCR products with hybridization probes for detection of the
specificity. These methods also are laborious and associated with
very expensive instrumental platforms.
[0021] A distinct reduction of working steps is disclosed in Korean
Patent 1020060099022A (Method and kit for rapid and accurate
detection and analysis of nucleotide sequence with naked eye by
using membrane lateral flow analysis).
[0022] In this case what is known as a lateral flow method is used
to detect nucleic acids. This method also makes use of the
technology of hybridization of nucleic acids on a solid phase.
Advantageously, a lateral flow method has a small, handy test
format (strip test).
[0023] In contrast to the above patent specification, a very fast
detection method, which also makes use of detection of
amplification products by means of a test strip and is commercially
available, is in turn based on a completely different principle. In
this case the PCR reaction is performed with a biotinylated primer
and a non-biotinylated primer. After the PCR has been performed,
there is obtained a PCR product that is therefore biotin-labeled at
one end. Detection is achieved using a test strip (for example of
the Millenia Co.), which contains two separate binding sites: a
streptavidin site for coupling the biotin-labeled DNA strand and an
FITC binding site for functional control of the test strip.
[0024] Detection of the PCR product is achieved by denaturing the
PCR mixture on completion of the PCR and hybridizing it with a
probe complementary to the biotin-labeled DNA strand. The probe is
FITC-labeled.
[0025] For detection, the PCR mixture is mixed with a running
buffer and applied on the test strip. According to the description
of the test, the biotinylated DNA strand binds to the streptavidin
binding site of the strip. Detection takes place via the FITC
labeling of the probe hybridized with the DNA strand. A typical
signal in the form of a strip is developed. This signal is supposed
to be the specific detection of the amplification product. However,
the method does not combine hybridization of the probe with the PCR
process but instead performs the latter process as a separate
procedural step. However, the method suffers from a fundamental and
dramatic error source.
[0026] Detection of the target nucleic acid to be detected is not
specific. The reason is that artifacts such as primer dimers are
formed during PCR and naturally also bind specifically to the
streptavidin binding sites of the test strip, and so they can cause
a positive reaction just as does a specific PCR product.
[0027] International Document WO 2004/092342 A2 describes the
technology of the lateral-flow assay, which is incorporated by
reference. As examples of application to molecular biology, there
are used already known and in some cases commercially available
technologies, which are adapted to the lateral-flow assay of that
invention. In Example 1 of WO 2004/092342 A2, one of the RT
reactions and subsequent amplification is performed with two
labeled primers. This method may lead to false-positive results due
to primer-dimer formation and mispriming. The second option (FIG.
20 d-e) represents a subsequent hybridization with two labeled
probes. The problem of primer-dimer formation and mispriming is not
acknowledged in that publication.
[0028] The important problem of false-positive results due to
primer-dimer formation was correctly recognized in the publication
of Kozwich, et al. (Development of a novel, rapid integrated
Cryptosporidium parvum detection assay. Appl. Environ. Microbiol.
(2000) 66 (7) 2711 7, page 2712, right column, 2.sup.nd par., FIG.
3), incorporated by reference. The solution of the problem (nested
PCR with the labeled and non-labeled primers) differs in principle
from the solution of the present invention, for which protection is
applied for herewith (one labeled primer and one labeled probe).
The solution proposed in the publication of Kozwich, et al.
excludes the formation of primer dimers only as a matter of
probability but not of principle. The mispriming that occurs so
often is also not completely excluded as an error source in the
solution proposed by Kozwich, et al.
[0029] All of the described alternative methods for detection of
nucleic acid sequences without REAL-time PCR technologies therefore
also have a substantial common feature, regardless of the
considerable manual working effort that is still necessary. The
necessary hybridization reaction between PCR product and specific
probe always takes place outside the PCR process. This feature is
at the base of all of these methods. A major advantage of REAL-time
PCR technologies, however, is precisely that the processes of
amplification and specific hybridization take place in one reaction
vessel, and so the processes of amplification and hybridization are
not disconnected. Furthermore, amplification artifacts frequently
lead to a false-positive signal in these cases.
BRIEF SUMMARY OF THE INVENTION
[0030] An objective of the present invention was to provide a
universally usable method for specific detection of target nucleic
acid sequences, which method can be performed very rapidly and also
simply and furthermore which does not need any expensive
instrumental systems. The method is intended to be suitable as a
molecular genetic rapid test and to respect the requirements of
diagnostic specificity assurance. In this regard it is important
that only one specific amplification product be detected and that
amplification artifacts can be unambiguously discriminated.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1 shows the amplification event/the hybridization
reaction as detected by means of gel-electrophoretic separation of
the amplification/hybridization mixture. Lane 1: DNA ladder; lane
2: positive control from mixture 1; lane 3: negative control from
mixture 1; lane 4: positive control from mixture 2; lane 5:
negative control from mixture 2.
[0032] FIG. 2 shows the detection of the specific hybridization
event on a lateral-flow test strip. Strip 1: positive control from
mixture 1; strip 2: negative control from mixture 1; strip 3:
positive control from mixture 2; strip 4: negative control from
mixture 2.
[0033] FIG. 3 shows that after completion of the coupled
amplification/hybridization method, the specific detection of the
exciting nucleic acid can be visualized by means of gel
electrophoresis. Lane 1: DNA ladder; lane 2: negative sample; lane
3: positive sample; lane 4: negative sample; lane 5: positive
sample; lane 6: negative sample; lane 7: positive sample; lane 8:
PCR blank control.
[0034] FIG. 4 shows that after completion of the coupled
amplification/hybridization method, the specific detection of the
exciting nucleic acid can be visualized by means of a lateral-flow
test strip. Strip 1: negative sample; strip 2: positive sample;
strip 3: negative sample; strip 4: positive sample; strip 5:
negative sample; strip 6: positive sample; strip 8: PCR blank
control.
DETAILED DESCRIPTION OF THE INVENTION
[0035] This object and others were achieved as described below.
Conventional PCR procedures, including amplification and
hybridization steps are well-known and are incorporated by
reference to the publications described above. The significant
conceptual and technical problems inherent to conventional methods,
such as those described above, were solved by the inventors as
described below.
[0036] Herein the present invention solves the existing problem in
the most ideal way. Furthermore, the inventive method for the first
time combines the amplification reaction and specific probe
hybridization in one and the same reaction vessel and is
nevertheless able to dispense completely with the extremely
expensive instrumental systems of REAL-time PCR.
[0037] The inventive method for detection of specific nucleic acid
sequences is based on a probe hybridization integrated into the
PCR, followed by simple detection of the specific hybridization
event. This detection takes place outside the PCR reaction vessel.
Preferably there is used, for example, a lateral-flow technology
(detection strips). Thus the test procedure now needs nothing more
than one PCR apparatus and one test strip and can be performed
simply, extremely rapidly and without problems, even by unskilled
personnel. In a preferred alternative embodiment, the rapid-cycler
technology (patent) is used. The combination of rapid PCR and
detection strips makes it possible to perform the test for
detection of a specific nucleic acid in not even one hour and to do
so for extremely low test costs.
[0038] This inventive method is based on the following steps:
A. Preparation of a PCR Reaction Mixture Comprising:
[0039] 1. two PCR primers, one of the primers being labeled at the
5'-end with a labeling molecule (such as biotin) [0040] 2. a
specific hybridization probe (also provided with labeling; for
example FITC), which is able to hybridize to the strand of the
target sequence containing the labeled primer [0041] 3. PCR
reagents known in themselves: PCR buffers, polymerases, dNTPs and
if necessary further additives. B. Performance of the Amplification
Process with Integrated Probe Hybridization
[0042] The amplification reaction takes place under standard
conditions. The actual amplification reaction is followed by a
denaturing step at a temperature of >90.degree. C. for thermal
separation of the strands of the amplification product generated
during the PCR. After denaturing, the PCR reaction mixture is
cooled to the hybridization temperature of the probe. During this
step the hybridization probe binds specifically to the
complementary DNA strand of the amplification product. This strand
then contains the biotin labeling, which was incorporated by the
biotin-labeled primer into the PCR product.
C. Detection of the Hybridization Event
[0043] Detection of the specific hybridization event takes place
via specific coupling of the biotinylated DNA strand to a solid
phase and specific detection of the label of the hybridization
probe, which is hybridized to the sequence of the biotinylated DNA
strand complementary to the probe. In a preferred variant,
commercially available lateral-flow test strips (for example, from
Millenia) are used for detection. As already explained, the test
strip contains two separate binding sites: a streptavidin site for
coupling the biotin-labeled label and an FITC binding site for
functional control of the test strip. For detection, the PCR
mixture is mixed with a running buffer and applied on the test
strips. The following binding events may occur. [0044] 1. In the
lower zone of the test strip, where the sample is applied, all
FITC-labeled nucleic acids (non-hybridized FITC-labeled
hybridization probe or hybridization product between biotin-labeled
DNA strand and FITC-labeled hybridization probe) bind to gold
particles, which are coated with anti-FITC antibodies. [0045] 2.
The streptavidin binding site is located further along the test
strip. The following nucleic acids are able to bind to this binding
site: 1. the biotin-labeled primer, 2. the biotin-labeled DNA
strands and 3. the products of hybridization between biotin-labeled
DNA strand and FITC-labeled hybridization probe. [0046] However, a
detection signal is able to be visible only when the specific
hybridization product between biotin-labeled DNA strand and
FITC-labeled hybridization probe exists, since only this product is
also coupled to the detection system (FITC/anti-FITC gold
particles). [0047] 3. Further along the test strip, there then bind
excess gold particles coated with anti-FITC antibodies, which serve
as control of the functional capability of the test strip.
[0048] After the described method was performed (Practical example
1), it was possible to achieve detection of an amplification
product without problems. However, it was found that the negative
control conducted in parallel may also cause a strong positive test
signal on the test strip. The following circumstance was discovered
as the cause of the false-positive result. During the PCR, the
FITC-labeled hybridization probe is also able to function as a
primer. Thereby a shortened amplification product is formed and is
therefore detected just as accurately as the specific amplification
product would be. Such a result is not problematic in principle,
since naturally it would also be specific. However, the problem is
that amplification artifacts naturally are also formed when the
hybridization probe acts as a primer. These primer dimers, which
are so often formed, then lead on the test strip to a
false-positive signal, since they bind specifically to the
streptavidin site and are detected via the incorporated FITC label.
This experimental result therefore shows that, in the described
form, the detection probe cannot be integrated in the PCR mixture
and thus the coupling of amplification and specific hybridization
in one reaction vessel cannot function.
[0049] This may explain why a detection system of this type has not
existed heretofore.
[0050] The inventive method surprisingly solves the problem by
modifying the hybridization probe chemically such that it is no
longer able to function as primer in the process of amplification,
and so elongation by the polymerase is no longer possible. This is
achieved by blocking the probe against the 5'.fwdarw.3' polymerase
activity, preferably by phosphorylation of the last nucleotide of
the probe. The process is further intensified by the fact that the
melting temperature of the probe lies well below the temperatures
at which the PCR takes place. By use of a modified probe it was
possible to eliminate the described problem completely (see
Practical example 1).
[0051] A further increase in efficiency of the test method can be
achieved by modifying not only the described denaturing step after
completion of the amplification reaction but also the PCR protocol.
Thus, an increase of detection efficiency (higher signal strength
on the test strip) is achieved by performing an asymmetric PCR
(instead of the standard PCR reaction).
[0052] In summary, an extremely simple detection method is now
available by virtue of the inventive method. The inventive
integration of a hybridization probe into the PCR adds the
certainty that the amplified fragment actually contains the target
sequence. Thereby the false-positive results caused by mispriming
are excluded. The use of the chemically modified probe (preferably
phosphorylation of the last nucleotide of the probe) prevents
elongation of the probe by 5'.fwdarw.3' polymerase activity, thus
preventing the probe from functioning as a primer and generating
unspecific PCR artifacts (primer dimers) that would be detected as
false-positive signals.
[0053] In contrast to REAL-time PCR methods, the specific detection
signal is not detected by means of fluorescence released by the
probe hydrolysis caused by the Taq polymerase (EP 0972848 A2).
Nevertheless, the advantage of real-time technologies is used, in
that the PCR and hybridization take place in one reaction vessel,
albeit not by quenching and exonuclease activity. The inventive
method is also distinguished from that of a patent (EP 0826066 B1),
which also represents a combination of PCR and hybridization. In
this method also, a fluorescence signal mediated by FRET effect is
again detected. This occurs during the amplification process by
hybridization of a probe having a lower annealing temperature than
does the primer. In this case, release of the fluorescence does not
take place by hydrolysis of the probe as a result of exonuclease
activity of the polymerase, but by the fact that the secondary
structure of the probe becomes decomposed during hybridization, and
so the fluorescence is less quenched. In this connection only
enzymes having no exonuclease activity (such as Klenow fragment or
T4 or T7 polymerases) can be used for amplification.
[0054] The fluorescence is always measured at the probe
hybridization temperature. Thus, this method always needs extremely
expensive real-time PCR instruments. As examples for final
detection, the inventive method uses strips (lateral-flow formats)
or other solid phases, which are easy to handle and which are
capable of binding the DNA strand of the PCR product to be
detected. The label of the probe is then detected by means of
technologies known to those skilled in the art.
[0055] By means of the inventive method, an extremely simple, rapid
and universal method is available for the first time for specific
detection of an amplification event, and from the instrumentation
viewpoint it needs only one PCR instrument. The combination of PCR
and probe hybridization in one reaction vessel means that detection
is now achieved merely by bringing the PCR reaction mixture into
contact with the test strips. Thus the inventive method represents
a test format that in principle can also be achieved under field
conditions.
[0056] The inventive method will be explained hereinafter on the
basis of practical examples, but the practical examples are not to
be construed as any restriction of the method.
PRACTICAL EXAMPLES
Example 1
Detection of Listeria monocytogenes DNA by Means of the
Hybridization Method Integrated into the PCR and of Lateral Flow
Detection. Comparison of an Unphosphorylated and a Phosphorylated
Probe
[0057] Two types of labeled probes were tested against one another
in the mixture. The first probe is FITC-labeled at the 5'-end, and
the second probe is also singly phosphorylated at its 3'-end. The
3'-phosphorylation of the probe prevents it from being elongated by
the Taq polymerase.
Mixture 1 (unphosphorylated hybridization probe) PCR
primer/probe
TABLE-US-00001 L. monocytogenes sense primer (SEQ ID NO: 1) (5'-CGC
AAC AAA CTG AAG CAA AGG-3') L. monocytogenes antisense primer (SEQ
ID NO: 2) (5'-BIOTIN-TCC GCG TGT TTC TTT TCG AT-3') L.
monocytogenes probe (SEQ ID NO: 3) (5'-FITC-CCA TGG CAC CAC CAG CAT
CT-3')
Reaction mixture (amplification/hybridization) Per sample:
TABLE-US-00002 sense primer (50 pmol/.mu.L) 0.1 .mu.L antisense
primer (50 pmol/.mu.L) 0.1 .mu.L probe (25 pmol/.mu.L) 0.1 .mu.L
dNTP Mix (12.5 mM) 0.3 .mu.L 10X PCR buffer (MgCl.sub.2 included)
1.5 .mu.L Taq-DNA polymerase 0.75 U PCR-grade H.sub.2O add 15
.mu.L
Mixture 2 (phosphorylated hybridization probe) PCR primer/probe
TABLE-US-00003 L. monocytogenes sense primer (SEQ ID NO: 1) (5'-CGC
AAC AAA CTG AAG CAA AGG-3') L. monocytogenes antisense primer (SEQ
ID NO: 2) (5'-BIOTIN-TCC GCG TGT TTC TTT TCG AT-3') L.
monocytogenes probe (SEQ ID NO: 4) (5'-FITC-ATG CAT CTG CAT TCA
ATA-Pho-3')
Reaction mixture (amplification/hybridization) Per mixture:
TABLE-US-00004 sense primer (50 pmol/.mu.L) 0.1 .mu.L antisense
primer (50 pmol/.mu.L) 0.1 .mu.L probe (25 pmol/.mu.L) 0.1 .mu.L
dNTP Mix (12.5 mM) 0.3 .mu.L 10X PCR buffer (MgCl.sub.2 included)
1.5 .mu.L Taq-DNA polymerase 0.75 U PCR-grade H.sub.2O add 15
.mu.l
[0058] For testing, one negative sample (containing only PCR
chemicals and H.sub.2O) and one positive sample-containing
additionally L. monocytogenes DNA (1.5 .mu.L, total DIN
concentration approximately 50 ng/.mu.L)--from each mixture were
used.
[0059] The PCR was performed in the SpeedCycler (Analytik Jena),
using the rapid-cycler technology:
Amplification/hybridization conditions
TABLE-US-00005 Step 1: Denaturing 95.degree. C. 120 minutes Step 2:
Amplification 37 cycles 95.degree. C. 4 minutes 55.degree. C. 4
minutes 72.degree. C. 20 minutes Step 3: Denaturing 95.degree. C.
300 minutes Step 4: Hybridization 15.degree. C. 600 minutes
[0060] The amplification event/the hybridization reaction was
detected by means of gel-electrophoretic separation of the
amplification/hybridization mixture (FIG. 1) as well as by means of
lateral-flow test strips (GeneLine HybriDetect; Millenia Biotec
GmbH; FIG. 2).
[0061] Comparison of the two figures demonstrates the disadvantages
of the probe not protected from polymerase activity (mixture 1) and
thus the unsuitability of the lateral-flow method in the case of
probes without polymerization blocking. On the gel photograph, the
negative control does not contain any specific DNA bands, whereas
the test strip exhibits a strongly positive signal caused by doubly
labeled primer dimers.
[0062] In contrast, the amplification mixture/hybridization mixture
with the hybridization probe phosphorylated at the 3'-end exhibits
only one positive signal, for the positive control, on the test
strip. Thus, the result on the test strip correlates unambiguously
with the gel photograph.
Explanation of FIG. 1:
[0063] Lane 1: DNA ladder; lane 2: positive control from mixture 1;
lane 3: negative control from mixture 1; lane 4: positive control
from mixture 2; lane 5: negative control from mixture 2.
[0064] FIG. 2 shows the detection of the specific hybridization
event on a lateral-flow test strip.
Explanation of FIG. 2:
[0065] Strip 1: positive control from mixture 1; strip 2: negative
control from mixture 1; strip 3: positive control from mixture 2;
strip 4: negative control from mixture 2.
Example 2
Performance of the Method by Means of Asymmetric PCR and Check of
Specificity of the Test on the Basis of Testing of Positive and
Negative Starting Samples
[0066] The inventive method was used as an example for detection of
Rickettsia DNA isolated from tick tissue. The specificity of the
method was determined by means of parallel tests on
Rickettsia-negative DNA samples, also isolated from tick
tissue.
PCR primer probe:
TABLE-US-00006 Rickettsia sense primer: (SEQ ID NO: 5) 5'-GGG ACC
TGC TCA CGG CGG-3' Rickettsia antisense primer: (SEQ ID NO: 6)
5'-Biotin-TCT ATT GCT ATT TGT AAG AGC GGA TTG-3' Rickettsia probe:
(SEQ ID NO: 7) 5'-FITC- CAA AGA AGT ATT AAA GGA ACT C-Pho-3'
Reaction mixture (amplification/hybridization) Per sample:
TABLE-US-00007 sense primer (50 pmol/.mu.L) 0.05 .mu.L antisense
primer (50 pmol/.mu.L) 0.1 .mu.L probe (25 pmol/.mu.L) 0.1 .mu.L
dNTP Mix (12.5 mM) 0.3 .mu.L 10X PCR buffer (MgCl.sub.2 included)
1.5 .mu.L Taq-DNA polymerase 0.75 U DNA (positive or negative) 1.5
.mu.L (approx. 50 ng) PCR-grade H.sub.2O add 15 .mu.l
[0067] The PCR was performed in the SpeedCycler (Analytik Jena),
using the rapid-cycler technology:
Amplification/Hybridization Conditions
TABLE-US-00008 [0068] Step 1: Denaturing 95.degree. C. 120 minutes
Step 2: Amplification 37 cycles 95.degree. C. 4 minutes 55.degree.
C. 4 minutes 72.degree. C. 20 minutes Step 3: Denaturing 95.degree.
C. 300 minutes Step 4: Hybridization 45.degree. C. 600 minutes
[0069] After completion of the coupled amplification/hybridization
method, the specific detection of the exciting nucleic acid was
again visualized by means of a lateral-flow test strip (FIG. 4) as
well as by means of gel electrophoresis (FIG. 3). The results show
impressively the specific detection of the target nucleic acid to
be detected. The entire process needed approximately 50
minutes.
Explanation of FIG. 3:
[0070] Lane 1: DNA ladder; lane 2: negative sample; lane 3:
positive sample; lane 4: negative sample; lane 5: positive sample;
lane 6: negative sample; lane 7: positive sample; lane 8: PCR blank
control.
[0071] FIG. 4 shows the detection of the specific hybridization
events on a lateral-flow test strip.
Explanation of FIG. 4:
[0072] Strip 1: negative sample; strip 2: positive sample; strip 3:
negative sample; strip 4: positive sample; strip 5: negative
sample; strip 6: positive sample; strip 8: PCR blank control.
[0073] Various modifications and variations of the described
nucleic acid products, compositions and methods as well as the
concept of the invention will be apparent to those skilled in the
art without departing from the scope and spirit of the invention.
Although the invention has been described in connection with
specific preferred embodiments, it should be understood that the
invention as claimed is not intended to be limited to such specific
embodiments. Various modifications of the described modes for
carrying out the invention which are obvious to those skilled in
the molecular biological, chemical, medical, biological,
pharmacological arts or related fields are intended to be within
the scope of the following claims.
[0074] Each document, patent application, or patent publication
cited by or referred to in this disclosure is incorporated by
reference in its entirety, especially of the material disclosed in
the same paragraph or section surrounding the citation. Any patent
document to which this application claims priority is also
incorporated by reference in its entirety.
Sequence CWU 1
1
7121DNAListeria monocytogenes 1cgcaacaaac tgaagcaaag g
21220DNAListeria monocytogenesmodified_base(1)..(1)5'-biotin-T
2tccgcgtgtt tcttttcgat 20320DNAListeria
monocytogenesmodified_base(1)..(1)5'-FITC-C 3ccatggcacc accagcatct
20418DNAListeria monocytogenesmodified_base(1)..(1)5'-FITC-a
4atgcatctgc attcaata 18518DNARickettsia sp. 5gggacctgct cacggcgg
18627DNARickettsia sp.modified_base(1)..(1)5'-biotin-t 6tctattgcta
tttgtaagag cggattg 27722DNARickettsia
sp.modified_base(1)..(1)5'-FITC-c 7caaagaagta ttaaaggaac tc 22
* * * * *