WO2000046400A1

WO2000046400A1 - Method for detecting and quantifying analytes in samples using rna replicons

Info

Publication number: WO2000046400A1
Application number: PCT/EP2000/000875
Authority: WO
Inventors: Christof K. Biebricher; Rüdiger Luce; Frank Berendes; Maria Kesseler; Jutta Kalkus; Katja Gellersen; Gerhard Gottschalk
Original assignee: MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.; Clavigen Gmbh
Priority date: 1999-02-03
Filing date: 2000-02-03
Publication date: 2000-08-10
Also published as: EP1147221A1; DE19904285A1

Abstract

The invention relates to a method for the qualitative and/or quantitative detection of an analyte in a sample using an analytical reagent containing a RNA replicon or a DNA sequence coding for such a sequence. According to said method the analyte is detected by amplification of the RNA replicon using a DNA-dependent RNA polymerase and subsequent detection of the amplification products. The invention also relates to a nucleic acid which codes for an analytical reagent provided for in the invention and to a test kit for carrying out the above method.

Description

Method for the detection and quantification of analytes in samples using RNA replicons

description

The invention relates to a method for the qualitative and quantitative detection of an analyte in a sample, the detection being carried out with the aid of a detection reagent specific for the analyte which contains a replicon sequence, the replicon being able to be amplified by a DNA-dependent RNA polymerase. The invention further relates to a nucleic acid which codes for a detection reagent according to the invention, and a test kit for carrying out the method according to the invention.

Known methods for the detection of certain substances are, for example, immunoassays and nucleic acid hybridization assays, which are widely used today in the fields of diagnostics and quality control. These methods are based on the principle that a substance to be detected specifically binds to another substance, i.e. forms a complex with this, with the help of this other substance the presence of the substance to be detected can be detected. Immunoassays utilize the highly specific binding of antibodies to certain antigens, while nucleic acid hybridization assays take advantage of the fact that complementary single strands of nucleic acids also form highly specific stable double strands.

An important factor, especially when detecting pathogenic organisms, is the sensitivity of the assays. For this reason

Methods developed to be able to amplify substances to be detected, for example by amplification. The most important of these procedures PCR (polymerase chain reaction) is in the field of nucleic acid analysis, in which very small concentrations of nucleic acids can be amplified to macroscopically detectable amounts with the help of specific oligonucleotide primer molecules. In this reaction, the double-stranded nucleic acid is melted into single strands by heating, and the two single strands are supplemented to form the double strand by a DNA polymerase and suitable primer molecules at low temperature. This cycle is repeated until the amplification of the desired nucleic acid section is sufficient for detection. This process has already been made automatable through the use of thermostable DNA polymerases.

A number of analogous reactions, e.g. Ligation Chain Reaction (LCR) (Backman and Wang, EP-A-0 320 308; Backman et al., EP-A-0 439 1 82) and Strand Displacement Enhancement (SDA) (Walker et al., Nucleic Acids Res. 20 ( 1 992), 1 691 -1 696) were then developed. However, all of these methods have the disadvantage that they are very complex. The nucleic acids, usually DNA, must be isolated from the biological material with as little loss as possible, and it must be by independent methods, e.g. gel electrophoretic length determination or hybridization of the amplified DNA with a specific oligonucleotide, verified that the correct product was amplified. In addition, there is always a high risk of amplification of non-specific or contaminating sequences, which leads to false positive results.

Isothermal RNA amplification processes are extremely sensitive. In one method, the RNA is transcribed into DNA using reverse transcription. The resulting RNA.DNA hybrid is converted into a DNA double strand by digestion of the RNA strand by RNaseH and reconstruction by DNA polymerase, which in turn enables the formation of further RNA molecules by transcription. Two such procedures were implemented as self-sustaining sequence amplification (3SR) and NASBA (Kwoh et al. (1 981), Proc. Natl. Acad. Sci. USA 86, 1 1 73-1 1 77; Guatelli et al. (1 990), Proc. Natl. Acad. Sci. USA 70, 934-938). However, these are complicated reactions in which side reactions can occur which easily lead to the formation and selection of non-specific sequences (Breaker and Joyce (1 993), Proc. Natl. Acad. Sei USA 91, 6093-6097).

The above-mentioned enzymatic duplication methods are very susceptible to contamination. It is therefore not possible to work with raw material, such as a blood or food sample without extracting the nucleic acid material. Such methods can only be used successfully with complex and low-loss isolation of the smallest amounts of nucleic acids in high purity.

Another method for amplifying RNA is replication by means of RNA-dependent RNA polymerases. The prime example of such a polymerase is the RNA polymerase of bacteriophage Qß, also called Qß replicase. Their function in vivo is to replicate the RNA genome of the Qβ phage. The genomic single-stranded RNA from the Qβ virion, which is referred to as the plus strand, serves as the template. The Qß replicase produces a minus strand RNA copy of the plus strand, which can act as a template just like plus strands. In this way, exponential duplication can be achieved, in contrast to transcription, which leads to linear duplication of the template. For this reason, the Qβ system has already been widely used for the detection of analytes, such as nucleic acids (Chu et al. (1,986), Nucleic Acids Research J_4, 5591 -5603; Lizardi et al. (1,988), Biotechnology 6 , 1 1 97-1 202).

An example of a wild-type Qβ RNA replicon is the "midivariant" or MDV sequence. In a conventional Qβ amplification assay, a Probe with an oligonucleotide sequence that hybridizes specifically with a target nucleic acid and additionally uses a Qß replicon sequence, eg MDV. Theoretically, a single RNA molecule suitable as a template is sufficient to trigger an avalanche of duplication, but these sensitivities could not be achieved in practical experiments. Another major disadvantage is that the reaction is very susceptible to faults.

Another problem is that Qβ replicase must be isolated from E. coli cells infected with Qβ phage. It is inevitable that replicable RNA molecules are also involved. When Qβ replicase is used as an amplification enzyme in a detection method, these RNA molecules - even if they are initially only present in small amounts - compete with the replicable nucleic acid used as a probe. On the one hand, the test sensitivity of the assay is reduced by a high background signal, and on the other hand false positive results are often obtained. Qß replication is also very sequence-specific. In some cases it is possible to insert a target sequence into a replicable RNA molecule (Miele et al. (1 983), J. Mol. Biol. 1 71, 281-285), but this manipulation affects the replication speed. In a rapid evolution, the RNA used is modified or cut out by mutation, and the resulting products are preferably amplified. Verification of the test result by independent methods is therefore essential, but much more difficult than in PCR, since hybridization is made more difficult by the rapid formation of double strands between the two complementary strands formed during replication.

In the prior art, attempts have been made to overcome or at least reduce some of these disadvantages. For example, EP-A-0 454 461 discloses a Qβ replicase detection method in which inhibitory chemicals are used to suppress the replication of wild-type MDV. At the same time a probe is used which is linked to a recombinant MDV sequence which is resistant to the inhibitor.

WO 90/06376 proposes to use Qβ replicase as a primer-dependent RNA-dependent RNA polymerase for the amplification of a target sequence. Two oligonucleotide sequences are used for this. A first contains in the 3'-5 'direction a sequence that hybridizes specifically with the target nucleic acid and a Qß-replicable sequence. This first oligonucleotide sequence acts as a primer for RNA template-directed RNA primer initiated chain extension. The double strand is then separated into single strands and a second oligonucleotide sequence serves as a primer for a second Qβ replicase-controlled elongation along the product of the first elongation. After strand separation, there is a replicable RNA which has Qβ replicon sequences at both ends and the target sequence in the middle. However, the inventors of the present application have now shown that the proposed reaction does not work.

WO 90/14439 discloses an improvement of the already known Qß method, which aims to reduce background signals. Two probes are used as primers, which can initiate target-specific hybridization and a DNA synthesis reaction. This creates a double-stranded DNA, which by transcription can deliver an RNA that contains a replicable sequence. A DNA-dependent RNA polymerase is used for the transcription, and an RNA-dependent RNA polymerase, in particular the Qβ replicase, is used for the subsequent amplification.

Numerous extraction protocols have been developed to isolate nucleic acids in as pure a form as possible. A successful method is the so-called "gene capture" method (Ranki et al. (1 983), Gene 21, 77-

85). By hybridization with one immobilized on a carrier The target sequence is extracted from a sample specifically nucleic acid. The hybridization reaction is not very susceptible to disruption, it takes place even under the conditions under which enzymes denature and lyse cells. Degradation of the nucleic acids by ubiquitous nucleases can thus be prevented by the choice of denaturing conditions. The unbound nucleic acids as well as other components, nucleases and buffer chemicals are removed by extensive washing, and an enzymatic amplification reaction can be connected. Despite the versatile application possibilities of the gene capture method on various raw samples, it has proven to be difficult in practice to develop a reliable implementation protocol.

In the 1970s (Biebricher and Orgel (1 973), Proc. Natl. Acad. Sci. USA 70, 934-938) it was discovered that DNA-dependent RNA polymerases can also replicate RNA molecules. This finding was confirmed by Konarska and Sharp (Cell 57, 423-431, 1 989). Biebricher and Luce (EMBO J. 11, 51 29-51 35, 1 992; Biochemistry 32, 4848-4854, 1 993) were able to demonstrate that an RNA template for the T7 RNA polymerase is single-stranded and a secondary structure in the form of a "Hairpins" (hairpin) has. The replication is independent of the primer. Both the plus strand and the minus strand can serve as a matrix, but the minus strand is preferred, so that more plus strand is formed. Further RNA matrices for the T7 RNA polymerase could later be identified (Biebricher and Luce (1 996), EMBO J., 15, 3458-3465). The plus strand of the replicon has a hairpin secondary structure and preferably the sequence GG on both terms. In particular, three RNA species were identified that are particularly well suited as templates for replication by T7 RNA polymerase: T7rp 1, T7rp2 and T7rp3. Some templates can also be replicated by other RNA polymerases, such as T3 and SP6 polymerase, but not by Qβ replicase. Surprisingly, it was found that the RNA replication by DNA-dependent RNA polymerase can be used as an extremely sensitive and less prone to interference amplification reaction in a detection method for the qualitative and / or quantitative determination of an analyte. A particular advantage is that the 3 'end of the RNA replicon used as a template can be variable, so that - in contrast to the Qβ system - additional sequences can be added without difficulty.

The invention thus relates to a method for the qualitative and / or quantitative detection of an analyte in a sample, using a detection reagent which has an RNA replicon or a DNA sequence coding for such a sequence, and the analyte by amplifying the Detects RNA replicons by means of a DNA-dependent RNA polymerase and subsequent detection of the amplification products.

The sample is a material that is to be tested for the presence of one or more specific analytes. In general, the sample is a biological material or a material containing biological substances. The sample can be a liquid, e.g. Drinking water or body fluids or extracts from tissues or cells. However, the method is also suitable for examining external contamination of solids.

Molecules which are able to form complexes with other molecules are suitable as analytes. Biomolecules present in one of the above-mentioned samples, such as proteins or nucleic acids, are preferably detected. The analyte is particularly preferably a nucleic acid. The detection of nucleic acids is particularly important for the determination of contamination by microorganisms. The detection reagent is a molecule that enables the specific detection of the analyte. This detection can be carried out by binding the detection reagent directly to the analyte or indirectly, for example by competition with the analyte or by binding to a further analyte-binding substance, for example a nucleic acid sequence complementary to the analyte. The detection reagent contains at least one identifier domain for the detection of the analyte and an amplifier or reporter domain, which consists of the replicon or the DNA coding for the replicon. The identifier and amplifier domains are preferably covalently coupled to one another.

In a preferred embodiment, the detection reagent is a single-stranded nucleic acid molecule whose identifier domain hybridizes with a nucleic acid to be detected or a sequence complementary thereto and whose amplifier domain contains an RNA replicon or a DNA coding therefor. The identifier domain may optionally contain a modified nucleic acid, e.g. a peptide nucleic acid (PNA), or a nucleic acid with modified nucleotides, in particular those which are more stable against enzymatic degradation than natural nucleic acid building blocks.

In the sense of this invention, however, the detection reagent can also contain identifier domains which do not consist of nucleic acid, but instead, for example, polypeptides, such as antibodies or antibody fragments or peptides, which are specific for specific antibodies. Thus, the detection reagents according to the invention can be used not only for nucleic acid hybridization methods, but also for other types of detection methods, e.g. be used for immunochemical methods for the detection of antigens or antibodies.

The amplifier domain is an RNA replicon accepted as a template by a DNA-dependent RNA polymerase or a DNA coding therefor. Such RNA replicons have been described previously. The plus strand of the replicon preferably has the base sequence pppGpG at its 5 'end and the base sequence GG (OH) at its 3' end. The sequence is at least partially palindromic, so that the replicon can form a hairpin-shaped secondary structure. The majority of the replicon is preferably base-paired, only the middle nucleotides and the terminal nucleotides being unpaired. The inner loop preferably consists of 4 to 5 bases on each strand, each with the base sequence CC in the middle in the plus strand and the base sequence GG in the minus strand. The halves of the replicon are themselves partially palindromic, so that an alternative secondary structure is possible, but is less stable. The RNA amplifier domain can also contain modified nucleotide building blocks (e.g. 2'-0-alkyl, 2'-0-alkenyl, 2'-0-alkynyl, 2'-amino, optionally substituted, 2'- Halogen, 2'-thio-nucleosides and the like), provided that this does not impair replication by a DNA-dependent RNA polymerase.

On the one hand, the amplifier domain can be present in the detection reagent in an already replicable form, e.g. as an RNA replicon. On the other hand, the amplifier domain can also only be formed during the detection reaction, e.g. by transcription of a DNA fragment and / or by ligation of partial sequences.

Any type of DNA-dependent RNA polymerase capable of amplifying an RNA replicon can be used for the method according to the invention. Bacterial RNA polymerases and in particular RNA polymerases from bacteriophages, for example from “T-odd” E. coli phages such as T1, T3, T5 or T7 or phages related to them such as SP6, are suitable. The polymerases of T3 and T7 are particularly preferred. Most preferred is T3 RNA polymerase. In a first embodiment, the detection reagent consists of a replicon which is preferably linked at its 3 'end to an identifier domain which binds directly to the analyte to be detected. The identifier sequence preferably contains an oligonucleotide sequence which hybridizes specifically with the analyte or a sequence complementary thereto. The identifier is preferably capable of hybridizing with a sequence section which is strictly specific to the analyte, for example with a sequence section which is species-specific or individual-specific for an organism to be detected. The identifier domain is particularly preferably specific for a single bacterial species or subspecies. Suitable identifier sequences are those that can hybridize with ribosomal DNA or RNA, such as the 16S rDNA / RNA or the 23S rDNA / RNA.

The amplifier domain contained in the detection reagent can have any sequence which can be amplified in the form of an RNA by a DNA-dependent RNA polymerase. The replicon sequences T7rp1, T7rp2 and T7rp3 (SEQ ID NO. 1-3) have proven to be particularly suitable. The replicon sequence is preferably 50 to 200 bases in length.

In addition to the detection reagent, a capture reagent can also be used, in particular if the method according to the invention is carried out as a heterogeneous test using a solid carrier. The capture reagent contains a capture domain, which is preferably used for binding the analyte and particularly preferably for immobilizing the analyte on the solid phase. In contrast to the identifier domain of the detection reagent, the capture domain of the capture reagent need not be strictly analyte-specific. If the capture domain of the capture reagent is a nucleic acid, it can react, for example, in a highly conserved, non-species-specific sequence section of the organism to be detected. Of course, a strictly analyte-specific catcher domain can also be used. In a preferred embodiment, the capture reagent is immobilized on a support or contains an immobilization group. It is particularly preferred to use a specific binding pair for the immobilization, one partner of which is coupled to the capture reagent as an immobilization group and the other partner of which is located on the carrier. Such binding pairs suitable for immobilization on carrier materials are known, for example biotin-streptavidin, biotin-avidin or oligo (C) -oligo (G) can be used.

The detection of the amplification products can be done in any way, preferably using labeling substances, which can be incorporated into the amplification products, for example in the form of labeled nucleoside triphosphates. Alternatively, the amplification products can also be detected by nucleic acid-binding labeling substances. Fluorescent or luminescent labeling substances are preferably used. Intercalating fluorescent substances, for example ethidium bromide, propidium iodide, acridine orange, thiazole orange and their derivatives ToProl and YoProl (Molecular Probes, Eugene, OR), are particularly suitable. The analyte can be quantified using labeling substances and, if necessary, an internal standard. For example, the amount of analyte in the sample can be determined by measuring the time it takes to obtain linear RNA growth. Conventional fluorescence measuring devices, as are known for the analysis of microtiter plates, can be used for the detection. The method according to the invention allows quantification with a sensitivity of up to 100 nucleic acid molecules in the sample (cf. Fig. 7). If the ribosomal RNA is detected as an analyte, it is therefore possible to use the method according to the invention to detect a single bacterium in the sample, because bacteria that grow exponentially contain up to 10000 ribosomes per cell. For the detection of bacteria, the ribosomal RNA is preferably extracted and separated from other cell components. The conditions are favorably chosen so that RNases are inhibited. A lysis buffer containing a detergent, a reducing agent, for example a thiol reagent and 0.1-1.5 M salt, for example LiCl, is particularly suitable. A particularly suitable lysis buffer contains 1% sodium dodecyl sulfate / 1% β-mercaptoethanol / 0.2 M LiCl / 10 mM TES (pH 6.5). It is also preferred to immobilize the ribosomal RNA on a support with the aid of a capture reagent.

A first embodiment of the method according to the invention is shown in FIG. 1. The analyte (1) is a nucleic acid, e.g. a ribosomal RNA. The detection reagent (2) consists of an RNA replicon (3) which is linked at its 3 'end to an identifier sequence (4). The identifier sequence (4) hybridizes with a section of the analyte (1). The analyte (1) is immobilized on a support (7) by a capture reagent (5) which has a sequence which can hybridize specifically with the analyte (6). The capture reagent (5) is bound to the carrier via a specific binding pair (8a / 8b).

The sample containing the analyte is brought into contact with the capture reagent and the detection reagent, the order of addition of the two reagents being irrelevant. The nucleotide sequence in the capture reagent is preferably chosen so that it can be used to recognize the ribosomal RNA of any species of bacteria, and is therefore preferably a highly conserved sequence. The capture reagent can, for example, be immobilized on a microtiter plate, for example NucleoLink ™ (Nunc AS, Roskilde, Denmark). As shown, the capture reagent can also be directly covalently bound to the support via a specific binding pair, for example through the 5'-phosphate group or through 5'- or 3'-amino linker groups of the oligonucleotide. Nucleotides coupled via amino linkers are preferred here because they are stable against hydrolysis are . In addition to the RNA replicon, the detection reagent contains a species-specific or isolate-specific identifier sequence which binds directly to the analyte, but this sequence is different from that of the oligonucleotide of the capture reagent. For this embodiment, the RNA replicon is particularly preferably at the 5 'end and the identifier sequence at the 3' end. After the analyte has been brought into contact with the capture reagent and the detection reagent, a washing procedure is carried out. Then the DNA-dependent RNA polymerase is added and the RNA replicon is amplified. While the amplifiers and / or amplifiers are in use, the amplification products can be detected by adding a marker substance.

In a further embodiment, the sensitivity of the method can be increased in that the RNA replicon can be formed in situ and only in the presence of the analyte to be detected. This embodiment comprises the steps:

(i) contacting the sample with a capture reagent comprising one

Nucleic acid domain that hybridize with a nucleic acid analyte and its 3 'end as a primer for an enzymatic

Elongation can serve, and with a blocker reagent, which at a certain point downstream of the capture reagent to the

Can bind analytes and is capable of an enzymatic

Stop elongation, (ii) enzymatically elongating the capture reagent using the

Analytes as a template, one complementary to the analyte

Nucleic acid strand is formed up to the binding site of the

Blocker reagent on the analyte is sufficient,

(iii) separating the analyte and contacting the complementary strand with a nucleic acid detection reagent comprising one

Amplifier domain, which encodes an RNA replicon

Contains sequence operatively linked to a promoter, and an identifier domain which can hybridize with the 3 'end of the complementary strand formed in step (ii),

(iv) further enzymatically elongating the one formed in step (ii)

Complementary strand using the detection reagent as a template, with a nucleic acid double strand from the

N u c h w e is rea g e n z u n d e i n e m d a z u ko m p le mentä re

Nucleic acid strand is formed

(v) transcribing the double strand to form an RNA replicon, (vi) amplifying the transcribed RNA replicon by a DNA-dependent RNA polymerase and

(vii) detecting the amplification product.

In this embodiment, the analyte is a nucleic acid, which is preferably present as a single-stranded DNA or RNA. The capture reagent preferably has a nucleic acid domain which can hybridize specifically with the analyte - preferably with a highly conserved sequence section - for example with many and preferably all species of eubacteria. The 3 'end of this nucleic acid domain can act as a primer for an enzymatic elongation. The capture reagent is preferably immobilized on a solid support or contains a group capable of immobilization. After the analyte has bound to the capture domain of the capture reagent, an enzymatic elongation takes place at its free 3 'end, for example with the aid of a reverse transcriptase or a DNA polymerase, a nucleic acid strand complementary to the analyte, in particular a DNA strand, being formed. It is essential in this reaction that a defined 3 'end is formed on the newly formed DNA strand. It is therefore preferred to add a blocker reagent before the initiation of the elongation, which also binds specifically to the analyte and is able to stop the elongation of a polymerase at a defined point, so that a defined 3 'end is formed on the newly formed complementary strand. Such a blocker reagent can for example, an oligonucleotide sequence which hybridizes with the analyte at a defined location downstream from the location at which the capture reagent hybridizes. The blocker reagent is preferably such that it does not have a free 3 'end suitable as an elongation primer. Since most RNA-dependent DNA polymerases do not have 3 '→ 5' exonuclease activity, the presence of a double-stranded region stops elongation at this point. The blocker reagent can optionally contain modified nucleotide building blocks, for example from PNA, or also be a polypeptide which is able to bind to the analyte in a sequence-specific manner and to stop the elongation.

In step (iii) of the method, the analyte used as the template for the elongation in step (ii) is separated off. This can be done in several ways, e.g. by denaturing the hybrid between analyte and capture reagent by means of heating and / or alkaline treatment. If the analyte is an RNA, the separation can also be accomplished by degrading the analyte using chemical or enzymatic methods, e.g. using an RNase, for example RNase H. After the analyte has been separated off, a single-stranded nucleic acid molecule is formed which has a sequence section complementary to the analyte and a defined 3 'end.

The detection reagent is a single-stranded nucleic acid, for example a DNA, which contains an identifier domain which can hybridize with the 3'-terminal region of the analyte complementary strand elongated in step (ii). Furthermore, the capture reagent contains, as an amplifier domain, a DNA sequence coding for an RNA replicon in operative association with a promoter recognized by a DNA-dependent RNA polymerase. Preferably, the identifier domain is downstream of the amplifier domain. The identifier domain hybridizes to the 3 'end of the strand synthesized in step (ii) and can then act as a primer for a second serve enzymatic elongation reaction. This elongation is preferably carried out using a DNA polymerase, so that a DNA double strand is formed. By adding a DNA-dependent RNA polymerase, this DNA double strand can be transcribed, resulting in RNA molecules which have an RNA replicon at their 5 'end. This RNA replicon can then be amplified using the DNA-dependent RNA polymerase, the presence of additional nucleotides at the 3 'end of the replicon of the originally transcribed RNA not being a disadvantage. These additional sequences are not also amplified.

A schematic representation of this process variant is shown in FIG. 2. The replicon (3) is formed from the detection reagent (1 2) by transcription in situ. As in FIG. 1, the analyte (1) is bound to the carrier (7) via the capture domain (6) of a capture reagent (5) and a binding pair (8a / 8b). A blocker reagent (9) is located upstream from the point at which the capture reagent hybridizes with the analyte. This serves to stop the enzymatic elongation when the blocker reagent is reached, so that a complementary strand 1) reaching up to this point is synthesized.

In the next step, the analyte is broken down by RNAse-H digestion and the newly synthesized complementary strand is exposed. The detection reagent (1 2) can in turn hybridize with this complementary strand, which has a promoter (1 3), a sequence coding for a replicon (1 4) and an identifier sequence (1 5) in the 5'-3 'direction. Then another elongation is carried out with a DNA polymerase. The newly formed double strand is then transcribed using an RNA polymerase to provide a replicable RNA (17). This can be replicated with a DNA-dependent RNA polymerase to RNA replicons (3). The present invention furthermore relates to a nucleic acid, preferably in the form of a DNA, which comprises the following elements in the 5'-3 'direction: a promoter, one for an RNA replicon, preferably selected from T7rp 1, T7rp2 and T7rp3, coding sequence and an identifier sequence, wherein the nucleic acid can deliver an RNA replicon by transcription, the 3 'end of which has the identifier sequence. The promoter is preferably a promoter for a DNA-dependent RNA polymerase, preferably from bacteriophages T7, T3 or SP6, so that in the second variant of the method according to the invention the same RNA polymerase can be used for the transcription step and the amplification step.

Yet another object of the invention is a nucleic acid comprising an RNA replicon, the 3 'end of which has an identifier domain. The identifier domain can be defined as above and the RNA replicon can optionally contain modified nucleotide building blocks.

Yet another object of the invention is an assay kit that can be used for the method according to the invention. It comprises a detection reagent, optionally a capture reagent and a blocker reagent, a DNA-dependent RNA polymerase and optionally further polymerases (such as, for example, the reverse transcriptase or DNA polymerase required for the second embodiment), and also customary excipients, auxiliaries and additives.

The invention is further illustrated by the following figures and examples:

Figure 1 shows the schematic representation of a first preferred embodiment of the method according to the invention. Figure 2: shows the schematic representation of a second preferred

Embodiment of the method according to the invention.

FIG. 3A: shows a nucleic acid which has a T3 promoter, a T7rp1 replicon and a Bpm I recognition site in the 5'-3 'direction

CTGGAG (Bpm I rec) and a 1 6 nucleotides cleavage site (Bpml) has.

Figure 3B: shows the insertion of heterologous sequences, e.g. an identifier sequence for 23 S rRNA from E. coli in the Bpml

Gap.

Figure 4: shows the sequences and secondary structures of the plus and

Minus strands of the RNA replicon T7rp1.

FIG. 5: shows the production of 5'-terminal fusions of the 5'-

Partial strands of T7rp1. The transcription begins with GGG. The identifier sequence is inserted into the asymmetric BstEII restriction site. The partial strand of T7rp1 (minus strand) starts with CCAAAA. The termination of the

Transcription is achieved by cleavage with the restriction enzyme Eco57l, the recognition sequence of which (GTCAAG) is indicated.

FIG. 6: shows the production of 3'-terminal fusions of the 3'-

Partial strands of T7rp1. An oligonucleotide with a catcher sequence (complementary to a sequence of the 16 S rRNA conserved in bacteria) is ligated into the Bpml cleavage site. The termination of the transcription is forced by cleavage of the plasmid with SacI. This creates a

Poly (C) end that can be used to immobilize onto a poly (G) coated support. The partial molecules of T7rp1 shown in FIGS. 5 and 6, which are produced by transcription, give T7rp1 which is capable of growth after ligation.

Figure 7: shows amplification profiles of different dilutions of a

Probe in the presence of the YoPro dye, recorded with a microtiter plate fluorimeter.

SEQ ID NO. 1 shows the plus strand of the RNA replicon T7rp1.

SEQ ID NO.2 shows the plus strand of the RNA replicon T7rp2.

SEQ ID NO.3 shows the plus strand of the RNA replicon T7rp3.

SEQ ID NO.4 shows the probe CB111 (23S rRNA E. coli 1179-1160).

SEQ ID NO.5 shows the probe CB112 (23S rRNA E. coli 1160-1181).

SEQ ID NO.6 shows the probe CB117 (23S rRNA E.coli 1160-1115).

SEQ ID NO.7 shows the probe CB118 (23S rRNA E.coli 1950-1920).

SEQ ID NO.8 shows the probe CB178 (poly G probe).

SEQ ID NO.9 shows the probe CB174 (1-27: 16S rRNA E.coli 469-

443).

SEQ ID NO. 10 shows the probe CB176 (1-24: 23S rRNA E.coli 1946-

1921).

SEQ ID NO. 11 shows the probe CB182 (3-18: T3 promoter; 16-99

T7rp1; 99-124: 16S rRNA E. coli 226-250). SEQ ID NO. 12 shows the probe CB188 (12-36: 16S rRNA E. coli 361-

347).

SEQ ID NO. 13 shows the probe CB184 (1-26: 16S rRNA E. coli 224-199).

Examples

1. Synthesis of oligonucleotides

Oligoribonucleotides and oligodeoxyribonucleotides were made by a synthesizer or purchased commercially. Oligonucleotides with chain lengths of more than 40 building blocks were additionally purified by means of gel electrophoresis. Oligonucleotides used for ligation were phosphorylated at the 5 'end by polynucleotide kinase and ATP.

Modified oligonucleotides with biotin groups at the 5 'and 3' end and with an amino group at the 5 'and 3' end were also produced.

2. Provision of DNA-dependent RNA polymerases

2.1 Purification of the RNA polymerases

E.coli strains which overexpress the DNA-dependent RNA polymerases of bacteriophages T7, T3 or SP6 were used as starting materials (Davanloo et al. (1984), Proc. Natl. Acad. Sci. USA 81, 2035-2039; Morris et al. (1986), Gene 41, 193-200 and Jorgensen et al. (1991), J. Biol. Chem. 266, 645-651). The bacteria were cultivated in 20 l of complete medium + 100 / yg / ml ampicillin at 37 ° C. After reaching an optical density A600 of 0.5, they were induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside and cultured for a further 4 h. Then the cells were harvested by centrifugation.

The purification was carried out essentially according to the Davanloo et al. (1 984), supra. The bacteria were lysed by treatment with lysozyme and then with sodium deoxycholate. The crude extract was homogenized, the nucleic acids removed by Polymin P precipitation and the enzyme collected by ammonium sulfate precipitation. The purification to homogeneity was carried out by cation exchange chromatography on S-Sepharose FF (Pharmacia), filtration by TSK CM and anion exchange chromatography on TSK DEAE (Merck, Darmstadt). The purified enzyme was concentrated by ammonium sulfate precipitation and stored at a concentration of 2 mg / ml in 50 mM Tris HCl pH 7.7, 1 mM dithiothreithol, 50% glycerol at -80 ° C. The enzyme preparation remained active for a period of three years. Thinner enzyme preparations stored at -30 ° C lost their replication activity within a few weeks, surprisingly their transcription activity was far less affected.

2.2 Characterization of the RNA polymerase

The transcription activity was determined by incubating 100 nM RNA polymerase (T3, T7, SP6) for 30 min at 37 ° C. in 0.05 M Tris HCl pH 7.5, 10 mM MgCl ₂ , 1 mM dithioerythrithol (DTE) , 100 mg / ml (30 nM) pBluescript SK DNA (Stratagene), 0.5 mM ATP, CTP, GTP and UTP determined. One unit is defined as the incorporation of 1 nmol NTP in RNA in 30 min. 1 mg T3 RNA polymerase had an activity of 472,000 units under these conditions. Replication activity was determined by incubating 1 μM RNA polymerase (T3, T7, SP6) in 0.05 M Tris HCl buffer, pH 7.5, 10 mM MgCl ₂ , 1 mM DTE, 1 mM and ATP, CTP, GTP and UTP and 1 amol to 1 μmol RNA template measured for 1 80 min 37 ° C.

The amount of material generated was determined by

(1) measuring the incorporation of radioactively labeled nucleotides,

(2) separation of the products by gel electrophoresis and staining and

(3) addition of RNA intercalating fluorescent dyes, e.g. Add 2 μM YoPro 1 to the replication buffer.

The latter method allows real-time measurements to be carried out.

By choosing suitable conditions, the activity of the RNA polymerase could be directed towards transcription or replication. When an excess of DNA was added, RNA replication was suppressed. On the other hand, when the enzyme concentration is high, the autocatalytically amplified RNA replicons outgrow the only linearly growing transcripts.

The replication of a replicon by RNA polymerase produced an exactly reproducible RNA concentration profile. At the beginning, the RNA template is usually in large deficit to the RNA polymerase. The newly synthesized strand and the reactivated template find new enzyme molecules to start new rounds of replication. There is therefore an exponential increase in the RNA concentration. Finally, the RNA strands have increased to such an extent that all enzyme molecules are saturated with the template. The further increase is limited by the enzyme concentration and a linear increase in the RNA concentration is observed. With further duplication, the replication speed decreases due to product inhibition. In the case of very small RNA concentrations, the initial exponential amplification initially remains invisible in the profile until a sufficiently large amount of RNA has been synthesized, the signal of which is easily detectable. The replication profiles (see Fig. 7) thus show a start-up phase, the length of the start-up time being an exact measure of the initial concentration of the RNA molecules in the sample. Start-up time and logarithm of the initial concentration are linearly correlated with one another, so that the measurable RNA concentration range spans about eight orders of magnitude.

The RNA species T7rp1 is amplified by T7 or T3 RNA polymerase in a concentration of 1 μM in the presence of 1 mM triphosphates by exactly 1 dB / min, so that when the RNA is diluted by a factor of 100 the curve changes Moved 20 minutes on the time axis, with 8 orders of magnitude by 80 minutes. As can be seen from FIG. 7, a replication cascade can be triggered by approximately at least 100 RNA molecules.

3. Manipulation of the replicon sequence

Recombinant RNA was generated by manipulation of DNA and subsequent transcription. For this purpose, the cDNA of the replicon T7rp 1 was placed in the cloning vector pUC1 8 behind a T3 promoter sequence (Biebricher and Luce (1 996), supra), so that the transcription begins exactly at the 5 'terminus of the replicon. A restriction site was used to cut the DNA sequence at the 3 'terminus of the replicon. In order to be independent of sequence restrictions at the 3 'end of the replicon, a restriction enzyme of type IV, Bpml, was chosen, the recognition site of which is located 1 4 nucleotides downstream of the interface (FIG. 3). A second Bpml restriction site in the cloning vector pUC 1 8 was eliminated by directed mutation, so that this site occurs only once. The cut plasmid could be produced by in vitro transcription with T3 RNA polymerase RNA, which is known in its replication properties as proven indistinguishable from replicon RNA synthesized by replication.

Since transcription and replication use the same enzyme and the same substrates, both steps can be carried out in one pot under the replication conditions. First, the plasmid generates RNA by transcription, which is then amplified by replication. After a short time, the transcription is surpassed by the replication and no longer plays a role. In this way, the DNA template can be used directly for replicon amplification. Precise measurements showed that in this system profiles are obtained as in the case of RNA amplification, but that the sensitivity can be increased to the value of 1 DNA molecule per sample.

Further restriction sites lie behind the Bpm1 site of the cloning vector. When the vector is linearized at these restriction sites, an RNA extended by sequence parts of the cloning vector is generated by transcription. Gel electrophoresis demonstrated that the resulting transcripts were actually longer by the calculated number of nucleotides. These extended transcripts are also accepted as replication matrices, however, only the replicon sequences themselves were detectable as synthesis products. The additionally attached nucleotides were thus ignored. Serial dilution made it possible to create replication profiles that were congruent with those of non-elongated replicons.

The Bpm1 restriction site located at the 3 'terminus of the replicon is very well suited for inserting any sequences, for example analyte-specific identifier sequences. For example, sequences were chosen which were complementary to species-specific sections of the rRNA sequence from E. coli. As an example in Figure 3B is the ligation of the oligonucleotides CB1 1 1 / CB1 1 2 (SEQ ID NO. 4 and 5) shown, which correspond to positions 1 1 60 to 1 1 80 of the 23S RNA from E. coli. In the resulting construct, the Bpml site has slipped to the end of the ligated sequence and can therefore be used to ligate further sequences. In this way, a complex sequence can be built up from short sequence sections.

The RNA extended by the identifier was generated by transcription and examined for its replication properties. As in the previous experiment, the identifier was ignored during amplification and only the replicon sequence was amplified.

4. Hybridization of the identifier sequence with rRNA from E. coli

The hybridization conditions of the DNA probe CB1 1 2 and the corresponding identifier-reporter RNA probe were examined by labeling both with P ³² at the 5 'end. For this purpose, 4 pmol probe and 4 pmol rRNA were mixed in 5 μl SSC buffer and the hybridization was detected by comigration in the gel electrophoresis. It was found that after heating to 65 ° C for 2 min, followed by slow cooling to 37 ° C within 60 min, both the DNA probe and the RNA probe were quantitatively hybridized with 23S rRNA. Under the appropriate conditions, other probe sequences tested also hybridized with 23S or 16S rRNA. Even at higher dilution, the hybridization in solution was complete after about 30 minutes.

5. Lysis of bacteria and release of the rRNA

A large number of methods for extracting RNA from biological materials are available on the market. They are based on extraction by guanidinium rhodanide or phenol. In the case of smaller sample quantities, however, these extraction methods are often lossy and not very reproducible. Hybridization directly under lysis conditions could be achieved using a lysis hybridization buffer consisting of 1% sodium dodecyl sulfate, 1% mercaptoethanol, 0.2 M LiCl, 0.01 M piperazine-1,4- to 2-ethanesulfonic acid, pH 6.5. Under these conditions, the rRNA could be released quantitatively after 30 minutes of treatment at 60 ° C. and hybridized with the probes. Titration with the probes gave values of 20,000 1 6S and 23S rRNA molecules for an E. coli cell cultured in the full medium. Degradation of the probe or rRNA was not observed, ie RNases are inactivated under these conditions. An investigation with other bacterial species showed that these conditions can be transferred well to other Gram-negative bacteria. For Gram-positive cells, pretreatment with lysozyme or lysostaphin is required before adding the lysis buffer.

6. Capture of analyte sequences via covalently immobilized oligonucleotides

Hybridization with immobilized or immobilizable oligonucleotides was used to capture ribosomal RNA. These oligonucleotides were complementary to sequence regions of the 16S or 23S rRNA which are conserved for all eubacteria (Amann et al. (1,995), Microbiol. Rev. 59, 143-1 69). The usability of these sequences was confirmed by dot blot hybridization with different strains of eubacteria.

Various immobilization techniques have been investigated. In principle, the immobilization could be carried out directly by hybridization with already bound oligonucleotides. Alternatively, the hybridization was carried out in solution with a capture reagent which was provided with an immobilizable group and bound to the surface in a subsequent reaction. For the direct method, the oligonucleotides were covalently linked to the surface. The two commercial microtiter plate preparations CovaLink and NucleoLink (Nalge-NUNC, Roskilde, Denmark) were used for this. The coupling to the surface was carried out according to the manufacturer's protocols with 5'-phosphorylated and with a 5'-amino group provided oligonucleotides with the coupling agent 1-ethyl-3- (3-d-imethylamino propyl) carbodimide . Up to 10 pmol oligonucleotides per well could be immobilized in a microtiter plate. This amount is sufficient for the immobilization of analyte nucleic acids.

The hybridization of dissolved nucleic acids (e.g. ribosomal RNA) to the immobilized oligonucleotides can be improved by (i) a hybridization time of at least 6, preferably at least 1 2 h (e.g. overnight);

(ii) Additional unpaired nucleotides at the 5 'end of the oligonucleotide.

Sometimes nucleic acids are also bound non-specifically to the surface. These non-specifically bound nucleic acids can largely be removed by washing with water and in particular with lysis buffer.

An alternative method for immobilizing oligonucleotides is indirect immobilization, in which not the capture reagent itself is covalently bound to the support, but two substances are selected that quickly and specifically form a stable complex. These types of immobilization are described in the following examples.

7. Biotin streptavidin immobilization

Magnetic beads (Dynabeads M-280) or microtiter plates were used as streptavidin-coated supports. As capture reagents 5'- or 3'-biotinylated oligonucleotide probes were used. These probes were hybridized in solution to complementary analyte nucleic acids and then immobilized via the streptavidin-biotin interaction.

This immobilization was demonstrated using labeled oligonucleotides. 2.9 μg (2.8 pmol) of 23 S rRNA were bound per 0.5 mg of Dynabeads.

A 5 '- [ ³² P] -labeled RNA probe (fusion of T7rp 1 with identifier for E. coli 23S rRNA) was produced to test the overall reaction. 10 pmol rRNA, 10 pmol scavenger (3'-biotinylated probes CB1 1 7 and CB1 1 8 corresponding to SEQ ID NO. 6 and 7) and 5 pmol labeled RNA probe were incubated together in 25 μl lysis buffer under the hybridization conditions mentioned above and mixed with 50μl Dynabeads (0.5 mg). Negative controls were performed with no catcher or RNA. After washing the Dynabeads, the bound amount of labeled probe was determined by measuring the immobilized radioactivity. In the negative controls, immobilized radioactivity was no longer measurable after washing. In the overall reaction, however, 1.4 pmol probe was bound to the Dynabeads.

The Dynabead samples were then suspended in 50 μl 0.1 X SSC buffer (SSC buffer: 0.1 5 M NaCl, 0.01 5 M Na citrate buffer pH 7.0), heated to 80 ° C. and the Supernatant removed. 1 μl of this supernatant was mixed with 1 00 μl replication buffer and 1 00 pmol T3 RNA polymerase and incubated at 37 ° C. Empty samples without additional matrices were also routinely checked. After 30, 60, 1 00, 1 20 and 1 80 min incubation, 10 μl aliquots were removed in each case and separated by gel electrophoresis. While the blank samples showed no RNA replication, RNA replication was found in all other samples. Due to the apparently non-quantitative removal of the probe during washing, a low background signal, which could be distinguished from positive samples, was also found in control samples to which no rRNA had been added.

8. Po.y (C): Poly (G) immobilization

An oligonucleotide probe CB1 78 (SEQ ID NO. 8) provided with a poly (G) tail was covalently bound to a nucleolink microtiter plate (1.2 pmol oligonucleotide per well) according to the method described above. The bound oligonucleotide showed rapid and quantitative hybridization with poly (C) capture probes such as CB1 74 and CB1 76 (SEQ ID NO. 9 and 10).

1 0 ⁵ E. coli cells were incubated with 0.1 pmol capture reagent CB1 76 and probe in 100 μl lysis buffer at 60 ° C. for 10 minutes and then transferred to the microtiter plate coated and washed with the poly (G) oligonucleotide CB178. An approach in which the bacterial solution was omitted served as a control. After binding for 30 min at 30 ° C was washed with lysis buffer and then with water. Then replication buffer and T3 RNA polymerase were added. The appearance of macroscopic amounts of RNA was monitored by gel electrophoresis. A quantitative estimate of the amount of bacteria used was perfectly possible down to about 1 00 bacteria.

9th generation of replicons during the reaction

A reduction in the test background can be achieved if the replicon in the test mixture is only generated in situ by the presence of the analyte. 9.1 Ligation of partial sequences of the replicon

A replication-capable molecule was obtained from non-replicating part sequences of a replicon by ligation, in that both parts were sterically arranged by hybridization to two adjacent sequence regions in such a way that the ligation after hybridization proceeds several orders of magnitude faster than without. Plasmid constructs were generated by cloning, in which corresponding partial sequences of T7rp1 can be generated by transcription (see FIGS. 5 and 6). The fusion with the hybridizing regions - for the 5 'terminal partial fragment at the 5' end, for the 3 'terminal fragment at the 3' end - was carried out according to Figures 5 and 6. It could be shown that the 3'-terminal section was not replicable at all and that the 5'-terminal section was only converted into a molecule capable of repetition after a long time. The 5'-terminus of the 3'-terminal section was converted into the 5'-monophosphate form suitable for ligation by dephosphorylation with alkaline phosphatase and subsequent phosphorylation with ATP and polynucleotide kinase.

At 37 ° C, the sections showed partial formation of a double strand even without hybridization to the analytes. The sections could be ligated in a form hybridized to the analyte by DNA ligase.

9.2 Elongation of the hybridized sequence

Another strategy for producing a molecule capable of repetition assumes that the analyte after hybridization can serve as a template for an instructed synthesis of complementary sequences.

This takes advantage of the fact that a DNA single or double strand with the sequence of an RNA replicon is completely incapable of replication. A single strand is also unsuitable for RNA synthesis by means of transcription, at least the promoter sequence must be in double-stranded form in order to enable transcription.

The DNA probe CB1 82 (SEQ ID NO. 1 1) was therefore synthesized, which contains a promoter followed by the replicon sequence, to the 3 'end of which the analyte identifier sequence (in positive polarity) is added. This probe was not replicable. By adding a DNA oligonucleotide and DNA polymerase from E.coli complementary to the 3 'end of the probe, a DNA double strand was formed, which gave a quantifiable amplification signal by means of transcription / replication by T3 RNA polymerase.

If rRNA with capture nucleotide CB1 88 (SEQ ID NO. 1 2) was used as a primer, a transcript of approximately 350 nucleotides in length could be generated using reverse transcriptase with a yield of 80%.

The efficiency of the double-strand synthesis was improved by adding the blocker reagent CB1 84 (SEQ ID NO. 1 3), which blocked the further elongation of CB1 88 beyond the hybridization sequence. Even better results were achieved by using the modified oligonucleotide CB1 90 (corresponding to CB1 84 with a 3'-terminal amino group), since this oligonucleotide itself could not act as a primer. In good yield, transcripts were produced which, after destruction of the DNA: RNA hybrids, were suitable for priming the DNA polymerase reaction of CB1 82 by heating.

Bacterial rRNA was immobilized with 3'-coupled oligo (dG, dl). It could be shown that the overall reaction works as shown in Figure 2 and that background reactions are much slower. 10. Quantify the response

Easy automation is decisive for the usability of a detection method for routine applications. Methods that require sampling during the reaction and complex analysis methods are too expensive and prone to failure.

In the process according to the invention, intercalating fluorescent dyes can be used for this purpose, the fluorescence intensity of which is greatly increased by intercalation into an RNA or DNA structure. Figure 7 shows semilogarithmic growth profiles of an RNA probe against 1 6S rRNA from E. coli under standard replication conditions in the presence of 1 0 ^"7 M of the fluorescent dye YoPro 1 (Molecular Probes, Yuichin, OR). The Y axis shows the relative fluorescence (without RNA = 1), the X-axis the measuring points (every 5 min) The amplification profiles of various dilutions of the probe are recorded with a commercially available microtiter plate fluorometer, which records the excitation and emission light through the bottom plate of the microtiter plate, and an opening of the reaction chamber is shown was not necessary so that contamination can be avoided.

In the absence of the template, the fluorescence of an incubated amplification mixture increases only very slowly and almost linearly. The much faster and almost exponentially following matrix-dependent amplification can easily be distinguished from this non-specific reaction. The figure shows that quantification up to a concentration of 1 0 ² strands of probe is possible. 1 1. Gene capture on modified polypropylene surfaces

The surfaces described above, Nucleolink and Covalink, had disadvantages that make them seem unsuitable for gene capture; the main disadvantages are the steric hindrance on the surface and the too high non-specific binding of RNA. Decisive improvements were brought about by amino-modified supports, in particular those supports on whose surface amino groups are bound via a swing arm (spacer), preferably with a chain length of 5 to 30, in particular 10 to 25, atoms. Examples of such supports are aminated microtiter plates and strips made of polypropylene (AIMS, Braunschweig). These plates, with an average of 2 mmol amine bound to a swing arm, are largely inert to chemical reactions. We modified the surface to add even longer swing arms.

For this purpose, 50 mM acryloyl chloride and 20 mM N-ethyl-diisopropylamine in ethylene chloride were introduced into the wells and incubated for 2 h at room temperature; after washing with ethylene chloride, 50 M 4-7-1 0-trioxa-tridecane-1, 1 3-diamine in DMF was incubated at 50 ° C. for 1 6 h. The plates can be stored after extensive washing with DMF, ethylene chloride and drying. The activation was carried out by 50 mM dimethyl suberimidate in saturated sodium bicarbonate solution for 1 h at room temperature. After the wells had been sprayed out with water, the oligonucleotides modified at the 5 'or 3' end with Aminolink were coupled in 0.05 M N-imidazole buffer (pH 7.0) at 50 ° C. for 1 -3 h. The binding capacity was determined with increasing amounts (5-1 50 pmol) of 5 '- [ ³ P] labeled CB1 89. At these concentrations, the oligonucleotide was bound virtually quantitatively. About 10% could be detached again with lysis buffer containing 1 mg / ml yeast RNA and 0.1 mg serum albumin. The stability against hydrolysis was excellent: after 7 d incubation with lysis buffer, no measurable amounts of RNA were released. Double-stranded PCR products and oligonucleotides could also be bound to the surface. For this purpose, CB228 and CB229 were hybridized and filled up to the double strand (65 bp) with DNA polymerase I (Klenow fragment). When 30 pmol of the double strand was offered, 1 7 pmol was bound. If less was offered, the percentage commitment was higher, with a larger offer the bound quantity could not be increased further. The non-covalently bound strand could be removed by washing with 0.05 M NaOH; the covalent bond was not measurably broken in this treatment. The binding capacities for PCR products (200 bp) were lower and reached 5 pmol.

The filling reaction could also be carried out on the surface: 100 pmol of CB228 was bound to the modified polypropylene plates described above, CB229 and polymerase solution and Klenow fragment were added and the double-strand formation was measured by immobilizing [ ³² P] - dAMP. 2 pmol could be filled up to the double strand. The result shows that some of the oligonucleotides are accessible to macromolecules and that surface enzymatic reactions are possible.

The non-specific adhesion of the detection probe was measured on plates coated with 1 20 pmol CB1 80. 100 μl lysis buffer and 10 ⁸ strands of probe T7rp1: Ec1 8a were added in duplicate samples and incubated for 30 min at room temperature. The liquid was removed and the wells were washed twice with 400 μl lysis buffer, twice with 2 × SSC + 0.1% SDS, twice with SSC + 0.1% SDS, twice with water and twice with amplification buffer. The wells were then filled with 100 μl amplification mix and T3 RNA polymerase was added. 2 blank samples (no matrix) and 2 blank samples (10 ⁸ strands of matrix added) were run into further wells of the plate. The course of the amplification was monitored in the Fluoroscan Ascent (Labsystems, Frankfurt a. M.) In the presence of the fluorescent dye YoPro-1 (Molecular Probes). The kinetics were recorded every 5 minutes and stopped after 40 measuring points. The maximum rise in the curve was selected to determine the response times. The response times were: Blank test: 83 min correspond to 1 0 ³ strands; Measurement sample: 1 45 min corresponds to 1 0 ³ strands; Blank sample: no signal. When the experiment was repeated, 1 0 ⁴ , 1 0 ⁶ and 1 0 ⁸ strands of the probe were added, washed as above and compared with blank and blank samples. Again, the non-specific binding values for the washed samples were 10 ³ strands and were therefore less than the number of rRNA molecules in bacteria.

The microtiter plates, coated with 40 pmol CB1 80, were used to determine the binding of 1 6S rRNA. For this purpose, 2.5 × 1 0 ⁹ E. coli bacteria were dissolved in 1 ml of lysis buffer and 200 μl of this solution (corresponding to about 1 6 pmol rRNA) and 20 pmol [ ³² P] -labelled CB1 53 probe (identifier sequence) were added. The mixture was incubated at 65 ° C. for 5 minutes and then the temperature was reduced to 40 ° C. in the course of 2 hours. After washing twice with washing buffer (10 mM PIPES pH 6.5, 0.2 M LiCl, 0.25% Tween 20), the immobilized radioactivity was measured. 50 to 60 fmol of immobilized 16 rRNA were measured reproducibly.

The measurement was repeated with decreasing amounts of E. coli bacteria, but with a constant 20 pmol CB1 53. 50 fmol rRNA were immobilized in 1 2.5 x 10 ⁷ bacteria, 58 fmol 1 6S rRNA in 5 x 1 0 ⁶ bacteria. In the absence of bacteria, CB1 53 was not immobilized. This result shows that the rRNA was largely quantitatively bound. The upper threshold value of 60 fmol can be easily explained by the space requirement of the 1 6S rRNA: the 30 mm ² surface of the wells would only be able to hold about 300 fmol rRNA in a densely packed monomolecular layer. With large excesses of rRNA, the bound rRNA amount decreased, since fragments of the rRNA hybridized more quickly and therefore competed with the intact rRNA. 12. Kit composition and reaction protocols

1 2.1 First embodiment

Required equipment: A temperature-controlled heating block for microtiter plates, microtiter plate fluorometers.

Kit components: lysis buffer and replication buffer, T3, T7 or SP6 RNA polymerase, probes and scavengers, 1 2 aliquots each for 8 samples, 1 2 microtiter strips coupled with immobilizer. The probe constructs are described above, as are the catchers; they have to be adapted to the respective application. The catcher can be present as a mixture with the probe or directly immobilized on the microtiter plate.

Procedure: Depending on the mass, the specimen is mixed with an equal volume of lysis buffer in 1.5 ml reaction vessels and brought to 60 ° C. Solid samples are mixed with small plastic disposable pistils with the lysis buffer. The sample is roughly clarified by centrifugation. 1 50 μl of this sample are mixed with 2 μl probe-catcher solution and incubated at 55 ° C. for 30 min. The solution is placed in microtiter plates which have been rinsed freshly with lysis buffer and incubated for a further 15 minutes at 50 ° C. for immobilization. The microtiter plates should be gently agitated for better mixing. The solutions are discarded and the strips are washed thoroughly with lysis buffer and water. 1 aliquot of replication buffer and 1 aliquot of T3 RNA polymerase are mixed and 100 μl of this mixture are pipetted into each well. The fluorescence of the samples is recorded and evaluated by the fluorometer. A blank sample should be run as a control per row to detect any contamination. 1 2.2 Second embodiment

Kit components: lysis buffer and replication buffer, T3, T7 or SP6 RNA polymerase-DNA polymerase mixture, mixture of scavenger and blocking oligonucleotide as above. The retrotranscription buffer and reverse transcriptase are also required. The replication buffer additionally contains 0.1 mM dATP, dCTP, dGTP and dTTP, the RNA polymerase solution additionally contains DNA polymerase from E. coli.

Procedure: Depending on the mass, the specimen is mixed with an equal volume of lysis buffer in 1.5 ml reaction vessels and brought to 60 ° C. Solid samples are mixed with small plastic disposable pistils with the lysis buffer. The sample is roughly clarified by centrifugation. 1 50 μl of this sample are mixed with indirect immobilization with 2 μl capture solution and incubated for 30 min at 55 ° C; this step is not necessary if the catcher is immobilized directly. The solution is placed in microtiter plates which have been rinsed freshly with lysis buffer and incubated for 40 min in the case of direct immobilization and for 5 min at 50 ° C. in the case of indirect immobilization. The solutions are discarded and the strips are washed thoroughly with lysis buffer and water. Retrotranscriptase buffer and reverse transcriptase are mixed and 100 μl of this mixture are pipetted into each well. After 40 minutes at 45 ° C, rinse with warm water. Replication buffer, detection reagent, RNA polymerase and DNA polymerase are mixed and 100 μl of this mixture are pipetted into each well. The fluorescence of the samples is recorded and evaluated by the fluorometer, the time of the macroscopically visible amplification being used to quantify the sample.

Claims

Expectations

1 . Method for the qualitative and / or quantitative detection of an analyte in a sample, characterized in that a detection reagent is used which has an RNA replicon or a DNA sequence coding for such a sequence, and in that the analyte is amplified by the RNA Replicons using a DNA-dependent RNA polymerase and subsequent

Detection of the amplification products.

2. The method according to claim 1, characterized in that the detection reagent is a specific for the analyte

Identifier domain.

3. The method according to claim 2, characterized in that the identifier domain is a peptide or polypeptide or

Represents nucleic acid.

4. The method according to claim 2 or 3, characterized in that the identifier domain is able to directly to the

Bind analytes.

5. The method according to claim 2 or 3, characterized in that the identifier domain represents a nucleic acid which in the

Is able to hybridize with the complementary sequence of a nucleic acid analyte.

6. The method according to any one of claims 1 to 5, characterized in that additionally a capture reagent is used which contains a capture domain which is bindable with the analyte.

7. The method according to claim 1 or 2, characterized in that the catcher domain is a nucleic acid which is capable of hybridizing with a nucleic acid analyte.

8. The method according to any one of claims 6 to 7, characterized in that the capture reagent is immobilized on a support or contains a group capable of immobilization.

9. The method according to any one of claims 6 to 8, characterized in that the capture reagent comprises a partner of a binding pair, the other partner is coupled to the carrier.

1 0. The method according to claim 9, characterized in that the binding pair is selected from biotin streptavidin, biotin avidin and oligo (C) oligo (G).

1 1. Method according to one of the preceding claims for determining a nucleic acid analyte, characterized in that it comprises the following steps: (i) bringing a sample containing the analyte into contact with a capture reagent comprising a nucleic acid domain which hybridize with the analyte and its 3 'end as a primer for enzymatic elongation, and with a blocker reagent which can bind to the analyte at a specific location downstream of the capture reagent and is able to stop enzymatic elongation, (ii) enzymatically elongating the capture reagent using the analyte as a template , wherein a nucleic acid strand complementary to the analyte is formed, which extends to the binding site of the blocker reagent on the analyte, (iii) separating the analyte and bringing the complementary strand into contact with the detection reagent, comprising an amplifier domain which operationally encodes an RNA replicon coding sequence Contains linkage with a promoter and an identifier domain which can hybridize with the 3 'end of the complementary strand formed in step (ii),

(iv) enzymatically elongating the complementary strand formed in step (ii) using the nucleotide reagent as a template, a nucleic acid duplex being formed from the detection reagent and a complementary nucleic acid strand,

(v) Transcribing the DNA duplex to form a

RNA replicons, (vi) amplifying the transcribed RNA replicon by a DNA-dependent RNA polymerase and (vii) detecting the amplification product.

12. The method according to any one of the preceding claims, characterized in that the replicon or the DNA sequence coding therefor is selected from the sequences T7rp1, T7rp2 and T7rp3.

3. The method according to any one of the preceding claims, characterized in that T7 polymerase, T3 polymerase or SP6 polymerase is used as the DNA-dependent RNA polymerase.

4. The method according to any one of the preceding claims, characterized in that the detection of the amplification products is carried out with the aid of a labeling substance.

5. The method according to any one of the preceding claims, characterized in that the marking substance from nucleic acid-intercalating fluorescent substances, preferably from ethidium bromide, propidium iodide, acridine orange, thiazole orange and its

Derivatives YoPro l and ToPro l is selected.

6. The method according to any one of the preceding claims, characterized in that the detection using a carrier with a through

Amino groups modified surface takes place.

7. Nucleic acid, characterized in that it comprises the following elements in the 5'-3 'direction: a promoter, a sequence coding for an RNA replicon, preferably selected from T7rp 1, T7rp2 and T7rp3, and an identifier domain.

1 8. Nucleic acid according to claim 1 7, characterized in that the promoter is selected from the promoter for T7 or T3 RNA polymerase.

1 9. Nucleic acid, characterized in that it comprises the following elements in the 5'-3 'direction: an RNA replicon sequence and an identifier domain.

20. Nucleic acid according to one of claims 1 7 to 1 9, characterized in that it contains modified nucleotide building blocks.

21. Assay kit comprising a nucleic acid according to claim 1 7, 1 8, 1 9 or 20, and optionally a carrier, a capture reagent, a DNA-dependent RNA polymerase and other polymerases, and also conventional carriers, auxiliaries and additives.

22. Use of a DNA-dependent RNA polymerase for the detection of an analyte by amplification of an RNA replicon and detection of the amplification products.