Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/6867—Replicase-based amplification, e.g. using Q-beta replicase

Definitions

• the present invention relates to a method and kit for the detection of target polynucleotide sequences making use of terminal transferase and strand displacement techniques employing RNA-dependent RNA polymerases (RdRps) having strand separation activity on double-stranded nucleic acids and being capable of de novo RNA synthesis in the absence of a primer on nucleic acid sequences.
• RdRps RNA-dependent RNA polymerases
• Detection of DNA sequences is basically performed using the TaqMan technology, which is based on the principle of real-time PCR and was developed by Roche Molecular Diagnostics
• the TaqMan technology particularly employs the 5' to 3'-nuclease activity of nucleic acid (in particular DNA) polymerases.
• RNA sequences are so far mostly being performed using RT-PCR, i.e. a reaction including two enzymatic steps, the reverse transcription step followed by the polymerase chain reaction step. Each step involves a different enzyme.
• the RT step involves a reverse transcriptase that transcribes single stranded RNA into a DNA/RNA-duplex. After degradation of the RNA through RNase (H) of the reverse transcriptase, the PCR step leads to an exponential amplification of the reverse transcribed template.
• RNase H
• the PCR reaction occurs in cycles, involving a denaturating heating step, an annealing step of oligonucletide primers to the template, followed by a primer-dependent elongation by the DNA-dependent RNA- polymerase.
• the reverse transcription reaction takes usually 30 to 45 minutes, followed by the PCR reaction which takes about 60 minutes.
• a limiting step as to the sensitivity of the RT-PCR relates to the reverse transcription step that has a variable efficiency ranging from 30 to 70%.
• RNA detection relies so far on two enzymatic steps with a suboptimal efficiency, and taking about 1.5 to 2 hours under thermocycling conditions.
• NASBA see Compton (1991) Nature 350, 91-92
• RNA detection is a further prior art technique for RNA detection which, in comparison to RT-PCR, has the advantage that it can be carried out isothermically, but it also involves a time consuming cycling reaction including reverse transcription and RNA polymerisation steps and, thus requires two enzymes (reverse transcriptase and T7 RNA polymerase).
• Microarrays have also been used for detection of RNA sequences, However, microarray techniques are complex and expensive, in particular as regards equipment for high- throughput routine diagnostics. Microarrays do not allow quantification of the detected RNA.
• RT-PCR is still recognised in routine diagnostics as the "gold standard" for RNA detection.
• WO-A-2007/012329 describes certain RdRps, in particular RdRps of caliciviruses, having a terminal transferase activity.
• WO-A-2007/012329 also discloses that such RdRps are capable of initiating RNA synthesis on polyC templates in the presence of elevated rGTP concentrations.
• WO-A-2010/055134 discloses methods for detection and amplification of RNA sequences making use of strand displacement techniques employing RNA-dependent RNA polymerases having RNA-oligonucleotide duplex separation activity and being capable of de novo RNA synthesis in the absence of a primer, in particular RdRps as described in WO-A- 2007/012329.
• the technical problem underlying the present invention is to provide an improved system for detection of polynucleotide sequences.
• the present invention makes it feasible to detect nucleic acid sequences using one single enzyme, in one step, under isothermal conditions within about 5 to 10 minutes.
• the present invention provides a method for the detection of a target polynucleotide sequence in a sample comprising the steps of: (a) contacting, under hybridisation conditions, single-stranded polynucleotide molecules present in a sample with an oligonucleotide containing a sequence substantially complementary to a sequence of the target polynucleotide and containing a motif of at least 1 C at its 3'-end that does not hybridise with the target polynucleotide to provide a mixture of polynucleotide/oligonucleotide duplexes wherein the
• polynucleotide/oligonucleotide duplexes comprise the target polynucleotide annealed to the olgionucleotide having an unpaired motif of at least 1 C at its 3'-end;
• step (b) incubating the mixture of step (a) with an enzyme having a terminal transferase
• step (c) incubating the reaction product of step (b) with an enzyme having RNA-dependent RNA polymerase (RdRp) and polynucleotide/oligonucleotide duplex separation activities under RNA polymerisation conditions in the absence of a primer and in the presence of elevated rGTP concentrations such that the RdRp activity polymerises an RNA strand complementary to the oligonucleotide beginning with the unpaired 3'-C motif and the polynucleotide/oligonucleotide duplex separation activity releases the target RNA; and
• RdRp RNA-dependent RNA polymerase
• the single-stranded polynucleotide may be provided by any method known in the art such as chemical synthesis, in vitro transcription, preparation of total nucleic acid from cells, tissues or it may be present in other samples such as blood, plasma, liquor etc.
• a double-stranded nucleic acid molecule as starting material may be separated into a single-stranded molecule through heat denaturation.
• nucleic acid molecule to be detected may comprise ribonuclotides as well as deoxynbunucleotides and may thus be DNA, RNA or mixed DNA/RNA.
• the present invention is also directed to a kit for detecting a target polynucleotide sequence in a sample comprising:
• At least one oligonucleotide containing a sequence substantially complementary to a region of the target polynucleotide sequence and having a motif of at least 1 C at its 3'-end which does not hybridise to the target polynucleotide sequence such that at least 1 unpaired C at the 3'-end of the oligonucleotide results when the oligonucleotide is annealed to the target polynucleotide;
• a sample refers to any substance containing or presumed to contain a target nucleic acid such as RNA and includes a sample of tissue or fluid isolated from an individual or individuals or animals or plants or any microorganism including viruses, including but not limited, for example, skin, plasma, serum, spinal fluid, lymph fluid, synovial fluid, urine, tears, blood cells, organs, tumors, and also to samples of in vitro cell culture constituents (including, but not limited to, conditioned medium resulting from the growth of cells in cell culture medium, recombinant cells and cell components).
• the oligonucleotide to be used in the methods claimed in the present invention is not necessarily physically derived from any existing or natural sequence but may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription or a combination thereof.
• An oligonucleotide for use according to the present invention is a single-stranded nucleic acid and maybe DNA or RNA or a sequence consisting of a mixture of both.
• One or more nucleotides in the oligonucleotides useful in the present invention may bear at least one chemical modification.
• the chemical modification may be at the ribose or deoxyribose, base and/or phosphate moiety.
• ribose-modified ribonucleotides are analogues wherein the 2'-OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2 or CN with R being C C 6 alkyl, alkenyl or alkynyl and halo being F, CI, Br or I.
• deoxyribose-modified deoxyribonucleotides are analogues wherein the 2'-H group is replaced by a group selected from OR, R, halo, SH, SR, NH 2 , NHR, NR 2 or CN with R being Ci-C 6 alkyl, alkenyl or alkynyl and halo being F, CI, Br or I.
• nucleotide analogues with a modified ribose or deoxyribose, respectively, at the 2' position include 2 ' -0-methyl-cytidine, 2 ' -amino-2 ' -deoxy-uridine, 2 - azido-2 ' -deoxy-uridine, 2 ' -fluoro-2 ' -deoxy-guanosine and 2'-0-methyl-5-methyl-uridine.
• nuleotides leading to a phosphate backbone modification are phosphothioate analogues.
• the modified nucleotide(s) may also be selected from analogues having a chemical modification at the base moiety.
• Examples of such analogues include 5-aminoallyl-uridine, 6-aza-uridine, 8-aza-adenosine, 5-bromo-uridine, 7-deaza- adenosine, 7-deaza-guanosine, N 6 -methyl-adenosine, 5-methyl- cytidine, pseudo-uridine, and 4-thio-uridine.
• oligonucleotide may also be denoted as “oligoprobe” and may be a polynucleotide of any length. Since their inexpensiveness and easier handling compared to RNA oligos, DNA oligonucleotides, i.e. oligodeoxyribonucleotides are preferred.
• oligonucleotide furthermore intends a polynucleotide of genomic DNA or RNA, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of the polynucleotide with which it is associated in nature; and/or (2) is linked to a polynucleotide other than that to which it is linked in nature; and (3) is not found in nature.
• the olginoucleotide having a motif of at least 1 C at its 3'-end may be prepared by any method known in the art such as chemical or enzymatic synthesis or a combination thereof.
• the oligonucleotide excluding the motif of at least 1 C at its 3'-end may be prepared by chemical synthesis or enzymatically, and afterwards the C motif is added to the 3'-end by using an enzyme having terminal transferase activity in the presence of rCTP, preferably by an RdRp as defined below.
• oligonucleotide is further preferred to be a rather small polynucleotide, i.e. preferably it has a length of from 5 to 100, more preferred 5 to 20, most preferred 10 to 12 nucleotides, excluding the motif of at least 1 C at its 3'-end.
• an oligonucleotide of the present invention such as the oligonucleotide having a motif of at least 1 C at its 3'-end (“C n oligonucleotide” or “C n oligoprobe” or simply "C n oligo”) may be labeled.
• label refers to any atom or molecule which can be used to provide a detectable (preferably quantifiable) signal, and which can be attached to a nucleic acid, in particular the oligonucleotide.
• Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like.
• Preferred labels according to the present invention are chromophores such as fluorescent labels which may preferably be added to the 5'-end of the oligonucleotide having a motif of at least 1 C at its 3'-end.
• the single-stranded nucleic acid molecule including the target polynucleotide in the sample may as well be of any origin and length.
• Typical examples of single-stranded nucleic acid species to be detected by the methods according to the present invention are viral nucleid acids, prokaryotic nucleic acids such as bacterial RNA or DNA and eukaryotic nucleic acids such as RNA or DNA from animals, humans, fungi and protozoa. This also includes cellular RNAs, e.g. messenger RNA, or extracellular RNA and the like.
• the single-stranded target polynucleotide for example an RNA, may, in certain embodiments, have preferably a length of from 18 to 200, preferably 16 to 40, most preferably 10 to 25 nucleotides.
• miRNA profiling has important implications for cancer aetiology, diagnosis and treatment (see, e.g. Esquela-Kerscher et al. (2006) Nat. Rev. Cancer 6, 259-269; Calin et al.
• Crucial to the present invention is the activity of special RNA-dependent RNA polymerases having strand displacement activity and being furthermore capable of de novo RNA synthesis on polyC containing templates in the absence of any primer.
• Enzymes of this category typically have the feature that de novo RNA synthesis can be accomplished on a single-stranded polynucleotide strand, in case of a single-stranded polynucleotide strand having a polyC stretch elevated rGTP concentrations are usually required.
• Such RNA-dependent RNA polymerases typically show a "right hand conformation" and have a primary sequence comprising a conserved arrangement of the following sequence motives:
• right hand conformation means that the tertiary structure (conformation) of the protein folds like a right hand with finger, palm and thumb, as observed in most template-dependent polymerases.
• the sequence motif "XXDYS” is the so-called A-motif.
• the A-motif is responsible for the discrimination between ribonucleosides and deoxyribonucleosides.
• the motif "GXPSG” is the so-called B-motif.
• the B-motif is conserved within all representatives of the RdRp family of the corresponding polymerases from Calicivirdae.
• the motif "YGDD” (“C-motif ) represents the active site of the enzyme. This motif, in particular the first aspartate residue (in bold, YGDD) plays an important role in the coordination of the metal ions during the Mg 2+ /Mn 2+ - dependent catalysis.
• the motif "XXYGL” is the so-called D-motif.
• the D-motif is a feature of template-dependent polymerases.
• the "XXXXFLXRXX” motif (“E-motif") is a feature of RNA-dependent RNA polymerases which discriminates them from DNA-dependent RNA polymerases.
• RdRps Typical representatives of the above types of RdRps are the corresponding enzymes of the calicivirus family (Caliciviridae).
• the RdRps of the calicivirus family are capable of synthesizing complementary strands using as a template any ssRNA template in vitro, including heterologous viral, eukaryotic and prokaryotic templates.
• the ssRNA template may be positive stranded or negative stranded.
• the RdRp for use in the present invention is capable of synthesizing a complementary strand to the oligonucleotide having a motif of at least 1 C at the 3'-end which has been elongated by the terminal transferase activity employed in step (b) and which oligonucleotide is hybridised with its substantially
• RNA complementary sequence to the ssRNA to be detected.
• de novo synthesis in the absence of a primer in the context of the present invention means that the RdRp is capable of synthesizing a complementary RNA strand on a single-stranded nucleic acid template without requiring a nucleic acid duplex such as RNA (either formed by a separate primer molecule or by back folding of the template) for initiation of polymerisation.
• RNA either formed by a separate primer molecule or by back folding of the template
• the RdRp enzyme recognises the elongated C n 3'-terminal repeat (with n being at least 2 after step (b)) and initiates RNA synthesis at elevated rGTP concentration on the C n repeat.
• the enzyme is polymerising the strand complementary to the oligonucleotide hybridised to the template it reaches the duplex region formed by the target polynucleotide sequence and the sequence of the oligonucleotide substantially complementary to the target polynucleotide.
• the duplex region between target polynucleotide and oligonucleotide is unwinded and the RdRp further polymerises a complementary strand to the oligonucleotide releasing the target polynucleotide.
• the products of this process are the
• oligonucleotide/complementary strand duplex and the released single-stranded nucleic acid molecule.
• One or both products may be detected by procedures known in the art.
• rGTP is added in surplus (preferably, 2x 3x, 4x or 5x more) over at least rATP and rUTP, in certain embodiments also over rCTP, respectively.
• rGTP and rCTP may be present in surplus (for example as outlined above for rGTP) over rATP and rUTP.
• rCTP and rGTP may be present at different concentrations or they may be present at substantially the same concentration.
• rGTP and rCTP are present at substantially equal concentrations.
• RdRp are corresponding enzymes of a human and/or non- human pathogenic calicivirus.
• an RdRp of a norovirus, sapovirus, vesivirus or lagovirus for example the RdRp of the norovirus strain
• HuCV/NL/Dresden174/1997/GE GenBank Acc. No. AY741811
• sapovirus strain pJG-Sap01 GenBank Acc. No. AY694184
• RdRp of the vesivirus strain HuCV/NL/Dresden174/1997/GE (GenBank Acc. No. AY741811) or of the sapovirus strain pJG-Sap01 (GenBank Acc. No. AY694184) or an RdRp of the vesivirus strain
• FCV/Dresden/2006/GE GenBank Acc. No. DQ424892.
• the RdRp is a protein having an amino acid sequence according to SEQ ID NO: 6 (norovirus-RdRp), SEQ ID NO: 7
• RdRp sapovirus-RdRp
• SEQ ID NO: 8 vesivirus-RdRp
• SEQ ID NO: 9 lagovirus-RdRp
• suitable expression vectors and host organisms cf. WO-A- 2007/012329
• the RdRp is expressed with a suitable "tag" (for example GST or (His) 6 -tag) at the N- or C-terminus of the corresponding sequence.
• a histidine tag allows the purification of the protein by affinity chromatography over a nickel or cobalt column in a known fashion.
• RNA polymerases fused to a histidine tag are the proteins comprising (or having) an amino acid sequence according to SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.
• SEQ NO: NO: 10 and SEQ ID NO: 11 correspond to an RNA polymerase of a norovirus having a histidine tag (SEQ ID NO: 10: C-terminal His-tag; SEQ ID NO: 1 1 : N-terminal His-tag).
• SEQ ID NO: 12 and SEQ ID NO: 13 correspond to amino acid sequences of an RNA polymerase of a sapovirus having a histidine tag (SEQ ID NO: 12: C-terminal His-tag; SEQ ID NO: 13: N- terminal His-tag).
• SEQ ID NO: 14 corresponds to the amino acid sequence of an RNA polymerase of a vesirius having a histidine tag (C-terminal).
• SEQ ID NO: 15 corresponds to the amino acid sequence of an RNA polymerase of a lagovirus having a histidine tag (C- terminal).
• the oligonucleotide containing a motif of at least 1 C at its 3'-end used herein is selected to be "substantially" complementary to a region of the target polynucleotide (i. e. the nucleic acid sequence to be detected).
• the oligonucleotide needs not to reflect the exact sequence of the target, but must be sufficiently complementary to at least a region of the target for hybridising selectively to it.
• Non-complementary bases or longer sequences can be interspersed into the oligonucleotide or located at the 5'-end or before the at least 1 C at the 3'-end of the oligonucleotide, provided that it retains sufficient complementarity with the template strand to form a stable duplex therewith.
• the preferably labeled oligonucleotide having a motif of at least 1 C at its 3'-end can hybridise to any region of the target polynucleotide. According to certain embodiments it is preferred that the region of substantial complementarity of the
• oligonucleotide is located near or at the 5'-end of the target polynucleotide. "Near" the 5'-end of the target polynucleotide sequence in this context preferably means that the last nucleotide at the 3'-end of the oligonucledide (excluding the motif of at least 1 C at the 3'- end) hybridises to the second, third, fourth, fifth, sixth, seventh, eighth, ninth or tenth nucleotide of the target single-stranded nucleic acid molecule counted from its 5'-end.
• step (b) of the method according to the present invention there is a motif of at least 2 unpaired C at the 3'-end of the oligonucleotide which also makes sure that the RdRp, preferably an RdRp having the features as defined above, does not use the 3'-end of the oligonucleotide for priming RNA synthesis when the oligonucleotide is hybridised to the target polynucleotide more remote to its 5'-end.
• the RdRp recognises the unpaired C n motif (n being at least 2) at the 3'-end of the oligonucleotide and starts, at eleveated rGTP concentrations, RNA synthesis complementary to this C stretch of the annealed
• the C n motif at the 3'-end of the oligonucleotide as defined above has a length of more than 1 , preferably 2 or 3 C, more preferably 4, 5, 6, 7, 8, 9 or 10 to 15 or even more such as 20 rCs.
• the optimal length of the Cn motif at the 3'-end of the oligonucleotide to be used in step (a) will depend on several parameters such as the region where the oligonucleotide hybridises to the target RNA: in case of a region of complementarity that lies more remote from the 5'-end of the target polynucleotide it is preferred that the the C n motif at the 3'-end of the oligonucleotide is longer, for example it may have at least 3, 4 or 5 Cs, than in those cases where the oligonucleotide anneals at or near the 5'-end of the target single-stranded polynucleotide in order to ensure proper recognition of the enzyme having terminal transferase activity and/or the enzyme in step (c).
• the enzymatic activities according to steps (b) and (c) of the inventive method and according to items (ii) and (iii) of the kit according to the present invention reside in the same protein.
• Particularly preferred examples of this type of enzyme are the RdRps having the above-defined structural features (right hand conformation, motives a. to e.), preferably RdRps of caliciviruses as defined above. Using this type of RdRps it becomes feasible to carry out steps (b) and (c) simultaneously in a single reaction which makes the process according to the invention particularly fast and convenient.
• steps (b) and (c) it is also possible, and preferred, to provide reaction conditions in which steps (a), (b) and (c) are carried out in a single reaction mixture.
• rCTP and rGTP are present at elevated concentrations in comparison to ATP and UTP (see above for particularly preferred values).
• the RdRps having the above structural features recognise the free C motif of the oligonucleotide hybridised to the target polynucleotide and, in the presence of elevated rCTP concentrations, preferably add further rC nucleotides to this C motif.
• the RdRp After having added at least 1 further C, usually 2, 3, 4 or 5 to 10, 20 or even 30 C, depending on the particular reaction conditions (especially the relation of rCTP concentration to rGTP concentration), the RdRp stops transferring further nucleotides to the 3'-end of the oligonucleotide and starts polymerisation of a complementary strand to the free C (oligo C or poly C) stretch previously generated (by incorporating G ribonucleotides). As already outlined above, the RdRp polymerising the RNA strand complementary to the oligonucleotide then will reach the duplex region of the oligonucleotide hybridised to the target polynucleotide. At this time point the strand displacement activity of the RdRp comes into play which leads to the release of the target polynucleotide from the oligonucleotide.
• oligonucleotides and also any single-stranded nucleic acid molecule in the context of the present invention, may be prepared by any suitable methods.
• Methods for preparing oligonucleotides of specific sequence are known in the art, and include, for example, cloning and restriction of appropriate sequences and direct chemical synthesis.
• Chemical synthesis methods may include, for example, the phosphotriester method described by Narang et al. (1979) Method in Enzymology 68:90, the phoshpdiester method disclosed by Brown et al. (1979) Methods in Enzymology 68: 109, the diethylphosphoramidate method disclosed in Beaucage et al. (1981) Tetrahedron Letters 22: 1859, and the solid support method disclosed in US-A-4,458,066.
• oligonucleotide is labeled, as described below, by incorporating moieties detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
• the method of linking or conjugating the label to the oligonucleotide depends, of course, on the type of label(s) used and the position of the label on the oligonucleotide.
• labels that would be appropriate for use in the present invention, as well as methods for their inclusion in the oligonucleotide, are known in the art and include, but are not limited to, enzymes (for example alkaline phosphatase and horseradish peroxidase) and enzyme substrates, radioactive atoms, fluorescent dyes, chromophores, chemiluminescent labels, electrochemiluminescent labels, such as OrigenTM (Igen), ligands having specific binding partners or any other labels that may interact with each other to enhance, alter, or diminish a signal.
• enzymes for example alkaline phosphatase and horseradish peroxidase
• enzyme substrates for example alkaline phosphatase and enzyme substrates
• radioactive atoms for example alkaline phosphatase and horseradish peroxidase
• fluorescent dyes for example alkaline phosphatase and horseradish peroxidase
• chromophores for example alkaline phosphatase and horserad
• 32 P is preferred.
• Methods for introducing 32 P into nucleic acids are known in the art, and include, for example 5'-labeling with a kinase, or random insertion by nick translation. Enzymes are typically detected by their activity.
• "Specific binding partner” refers to a protein capable of binding a ligand molecule with high specificity, as for example in the case of an antigen and a monoclonal antibody specific therefore.
• Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in the art. The above description is not meant to categorize the various labels into distinct classes, as the same label may serve in several different modes.
• 125 l may serve as a radioactive label or as an electron-dense reagent.
• HRP may serve as enzyme or as antigen for a monoclonal antibody.
• one may combine various labels for a desired effect. For example, one might label a probe with biotin, and detect the presence of the oligonucleotide with avidin labeled with 125 l, or with an anti- biotin monoclonal antibody labeled with HRP.
• Other permutations and possibilities will be readily apparent for the skilled person and considered as equivalents within the scope of the present invention.
• Fluorophores for use as labels in constructing labeled oligonucleotides of use according to the present invention are preferred and include rhodamine and derivatives, such as Texas Red, 5-carboxytetramethyl rhodamine (5-TAMRA), 6-carboxytetramethyl rhodamine (6- TAMRA) and their respective succinimidyl esters, fluorescein and derivatives, such as 5- bromomethyl fluorescein, 5-carboxy fluorescein (5-FAM), 6-carboxy fluorescein (6-FAM), and their respective succinimidyl esters, Lucifer Yellow, IAEDANS, 7-Me 2 -N-coumarin-4-acetate, 7-OH-4-CH 3 -coumarin-3-acetate, 7-NH 2 -4-CH 3 -coumann-3-acetate (AMCA),
• rhodamine and derivatives such as Texas Red, 5-carboxytetramethyl rhodamine (5-TAMRA),
• monobromobimane pyrene trisulfonates, such as Cascade Blue, and monobromotrimethyl- ammoniobimane. It is further preferred, that, if fluorescence is used to detect the released
• the single-stranded nucleic acid molecule is provided with a molecule quenching the fluorescence of the fluorescent label of the oligonucleotide when it is hybridised to the target polynucleotide.
• the oligonucleotide may be labeled with a fluorescein derivative, such as 5- or 6-FAM, preferably at the 5'-end thereof, and the single-stranded nucleic acid molecule is provided with a quencher for the fluorescein label, for example 5-TAMRA or 6-TAMRA.
• the oligonucleotide comprising the (fluorescent) label preferably at its 5'-end, hybridises to the target sequence at or near its 5'- end which carries the quencher molecule.
• the quencher/donor may be generally selected as FRET (fluorescence resonance energy transfer) pairs such as in the case of FAM/TAMRA.
• FRET fluorescence resonance energy transfer
• the fluorescence donor-quencher pair can be provided in the form of two oligonucleotides (which may each be of RNA or DNA type or mixed DNA/RNA) substantially complementary (with respect to the meaning of "substantially complementary” it is referred to the corresponding section above) to the target polynucleotide but hybridising thereto at different, non-overlapping regions of said target polynucleotide, and whereby the regions of complementarity to the target/template polynucleotide are selected such that, when the oligonucleotides are hybridized to the target polynucleotide, the fluorescence by the labeled oligonucleotide is quenched by the second oligonucleotide carrying the quencher, in particular by FRET between the fluorescence donor and quencher.
• two oligonucleotides which may each be of RNA or DNA type or mixed DNA/RNA
• the oligonucleotide having a motif of at least 1 C at its 3'-end carries, for example near or at its 5'-end the quenching moiety, and the second oligonucleotide carries a fluorophore quenched by the quenching moiety when both oligonucleotides are hybridised to the target polynucleotide.
• the RdRp When the RdRp according to the present invention initiates de novo RNA synthesis and synthesises an RNA strand complementary to the C n oligonucleotide, it displaces the target polynucleotide from the C n oligonucleotide whereas the donor oligonucleotide (the
• the quencher oligo is the oligonucleotide having the C motif at its 3'-end which carries the quenching moiety at its 5'-end and hybridizes to or near the 5'-end of the target/template polynucleotide.
• the donor oligo has the fluorescence donor at its 5'-end and hybridizes to or near the 3'end of the target/template polynucleotide.
• quencher and donor moieties can be present at either probe.
• a quencher moiety could be present at the 3'- end of one oligo hybridizing to or near the 5'-end of the target/template and the donor group could be bound to the 5'-end of the other oligo (i.e. the C n oligonucleotide) hybridizing near or to the 3'-end of the target/template polynucleotide, and vice versa. In the latter scenario, it would be possible to use hybridizing regions which are farther remote from one another. With regard to preferred lengths and other characteristics of the quencher oliconucleotide (i.e.
• the second oligonucleotide present in step (a) of the above defined method, or present as a further component of the inventive kit) it is referred to the above sections in connection with the Cn oligonucleotide.
• useful donor/quencher pairs in the context of the present invention have already been described above.
• Particularly preferred examples for providing donor/quencher pairs in the form of two oligonucleotides are a 5- or 6-FAM labeled oligonucleotide (donor) and a 5- or 6-TAMRA-coupled oligonucleotide (quencher).
• the above-described preferred embodiment of the inventive polynucleotide detection method employing a combination of two oligonucleotides (one carrying a quencher moiety and the other carrying the fluorescence donor moiety) has particular advantages: (i) Through the use of two oligonucleotides having non-overlapping regions of complementarity to the target, the polynucleotide detection is highly specific, since false positive signals are very unlikely because two regions of the target sequence are probed, (ii) By optimisation of the length of the oligoprobes per se and of the length of complementary sequences in the oligoprobes it is possible to create customized pairs for specific targets: e.g.
• the second oligonucleotide (which does not carry an unpaired C at its 3'-end) has a region of complementary to the target polynucleotide that ensures very tight and specific binding to the target, whereas, in comparison thereto, the C n oligoprobe may be selected such that its complementary region to the target is shorter which provides for a very sensitive detection signal, since the (elongated) C n oligoprobe is released in very short time by the
• the present invention provides a polynucleotide detection process, in particular when using the above-described pair of oligoprobes (C n oligoprobe and second oligoprobe) that is, compared to prior art methods, in particular RT-PCR, fast (specific signal generation takes only minutes), highly specific (two non-overlapping regions of complementarity are probed) and very sensitive (down to femtogramm amounts of the target polynucleotide).
• hybridizing at the 5'-end or “hybridizing at the 3'-end” does not necessarily mean that the Cn oligonucleotide or second oligonucleotide hybridises exactly to the 5'-end or 3'-end, respectively, of the target sequence. Rather, these terms also include that the
• oligonucleotide may hybridize near (such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides away from) the 5'-end or 3'-end, respectively, which means that there may be additional nucleotides within the target sequence between its very 5'-end or 3'-end, respectively, of the sequence hybridizing with the respective oligonucleotide.
• the labels for use in the present invention may be attached to the oligonucleotide directly or indirectly by a variety of techniques. Depending on the precise type of label used, the label can be located at the 5'- or the 3'-end of the oligonucleotide (with respect to the C n oligonucleotide this means excluding the at least 1 C), located internally in the
• oligonucleotide or attached to spacer arms of various sizes and compositions to facilitate signal interactions Using commercially available phosphoramidite reagents, one can produce oligomers containing functional groups (e.g., thiols or primary amines) at either the 5'- or the 3'-terminus via an appropriately protected phosphoramidite, and can label them using protocols described in, for example, PCR Protocols: A Guide to Methods and
• a 5'-phosphate group can be introduced as a radioisotope by using polynucleotide kinase and gamma- 32 P-ATP to provide a reporter group.
• Biotin can be added to the 5'-end by reacting an aminothymidine residue, or a 6-amino hexyl residue, introduced during synthesis, with an N-hydroxysuccinimide ester of biotin.
• Labels at the 3'-terminus may employ polynucleotide terminal transferase to add the desired moiety, such as for example, cordycepin 35 S-dATP, and biotinylated dUTP.
• Oligonucleotide derivatives are also available as labels.
• etheno-dA and etheno- A are known fluorescent adenine nucleotides that can be incorporated into an
• oligonucleotide etheno-dC or 2-amino purine deoxyriboside is another analogue that could be used in oligonucleotide synthesis.
• the oligonucleotides containing such nucleotide derivatives may be hydrolyzed to release much more strongly fluorescent mononucleotides as the RdRp unwinds the duplex formed between the target single- stranded nucleic acid molecule and the labeled C n oligonucleotide.
• RNA polymerization conditions means the conditions, in particular relating to buffer, temperature, salt and metal ion (if applicable), that allow the RdRp to synthesize an RNA strand complementary to a template strand in the absence of a primer.
• buffer, salt, metal ion, reducing agent (if applicable) and other conditions of RdRps are known to the skilled person.
• RdRps of caliciviruses it is referred to WO-A-2007/012329.
• the concentration of the ribonucleoside triphosphates is preferably in the range of from 0.1 mM to 1 mM, for example 0.4 ⁇ / ⁇ .
• the concentration of the RdRp may be for example 1 ⁇ to 6 ⁇ .
• Typical buffer conditions are 10 to 80 mM, more preferred 20 to 50 mM HEPES pH 8.0, 1 to 200 mM, for example 5 to 150 mM, particularly preferred 80 to 120 mM, most preferably 100 mM magnesium acetate, magnesium chloride, manganese acetate or manganese chloride and 1 to 4 mM of a reducing agent, for example DTT.
• substantially the same conditions apply except that only one ribonucleotide is present, i.e. rCTP for providing the additional rC residues at the 3'-end of the C n oligonucleotide.
• rCTP and rGTP are present in higher concentrations, preferably 2x, 3x, 4x, 5x, 6x or even more, in comparison to rATP and UTP.
• Especially preferred conditions are 0.4 mM of each of rATP and rUTP, and 2 mM of each of rCTP and rGTP.
• the method according to the present invention may be stopped by introducing a stop solution into the reaction mixture.
• a typical stop solution contains 2 to 10 mM, preferably 4 to 8 mM ammonium acetate and 50 to 200 mM, for example 100 mM EDTA.
• a thermal cycler such as the
• the detection or verification of the C n oligonucleotide/complementary strand duplex and/or the single-stranded nucleic acid molecule displaced by the action of the RdRp may be accomplished by a variety of methods and may be dependent on the source of the label or labels employed.
• One convenient embodiment of the invention is to subject the reaction products, including the released preferably labeled C n oligonucleotide/complementary strand duplex, to size analysis. Methods for determining the size of the labeled nucleic acid fragments are known in the art, and include, for example, gel electrophoresis (using e.g. polyacrylamide), sedimentation ingredients, gel exclusion chromatography and
• kits in particular diagnostic ktis.
• Kits according to the invention include the target specific Cn oligonucleotide, terminal transferase and RdRp activities.
• the C n oligonucleotide is preferably labeled as described above or may comprise a quenching moiety as outlined above.
• the kit may further contain a second oligonucleotide as described above comprising a quenching moiety, preferably at its 5'- or 3'-end, or label, for example a fluorescent label as described in detail above.
• the second oligonucleotide is preferably blocked at its 3'-end, in particular in order to prevent usage of the second oligonucleotide as a primer for RNA polymerisation by the RdRp activity.
• the RdRp for use in the present invention will usually not use such (second) oligonucleotide as a primer for RNA
• the second oligonucleotde according to the invention is to be regarded as a non-priming oligonucleotide for the RdRp.
• the kit may also contain other suitably packaged reagents and materials needed for carrying out the methods according to the present invention, for example, buffers, ribonucleotides (rATP, rGTP, rCTP, rUTP) and, optionally, a stop solution (preferably a stop solution as defined above), all solutions are more preferred in the form of 5x or 10x stock solutions, as well as instructions for conducting the methods.
• kits according to the invention contain rATP and rUTP in one stock solution (for example 5x or 10x concentrated) and rCTP and rGTP together in a different stock solution or rCTP and rGTP each as a single stock solution (preferably concentrated as outlined with respect to rUTP and rATP).
• the polynucleotide detection method according to the present invention shows excellent sensitivity. Especially using quencher/donor oligo pairs as described in detail herein and below, sensitivities of more than 1 , 2 or even 3 orders of magnitude higher compared to prior art polynucleotide detection methods are feasible such that the detection of very small amounts of target polynucleotides such as RNA in the range of femtogram and below becomes possible.
• the polynucleotide detection method according to the invention also allows the quantification of the target polynucleotide in a sample by using one or more standards containing a known amount of the target polynucleotide and comparing the measured signal in the sample with that of the standard(s).
• the present invention is also directed to a method of quantifying the amount/concentration of a target polynucleotide in a sample comprising (1) measuring the signal detected by carrying out the method of the invention as described herein using one or more samples containing a known amount/concentration of the target polynucleotide; (2) measuring the signal detected by carrying out the method of the invention using the sample of target polynucleotide to be quantified; and (3) comparing the signal obtainged in step (2) with the signal(s) obtained in step (1). It is clear for the skilled person that step (2) could also be carried out before step (1).
• the method according to the invention can be used to detect nucleic acids of microorganisms (pathogenic or non-pathogenic) or viral nucleic acids such as RNA or DNA of pathogenic or non-pathogenic viruses. Since the present method is capable to detect femtogramm amounts of a target polynucleotide within minutes, it would be possible to detect viral infections at an early stage and to have a diagnosis of virus infection within an extremely short time.
• Another field of application is detection of cellular nucleic acid species such as microRNAs and disrupted microRNAs.
• Fig. 1 is a schematic illustration of a preferred embodiment of the polynucleotide detection method according to the present invention.
• step (a) target single-stranded nucleic acid molecule and two DNA oligonucleotides (one carrying 5 to 10 rC residues at the 3'-end and a quencher (Q) at the 5'-end, the second carrying a fluorescence donor (D) at the 5'-end) complementary to different regions of the target polynucleotide: the oligonucleotide carrying the quencher and having 5 to 10 rC residues at its 3'-end hybridises near or at the 5'-end of the target polynucleotide whereas the second oligo (carrying the fluorescence donor at the 5'- end) hybridises near or at the 3'-end of the target polynucleotide.
• the distance of the hybridisation regions of the oligos is selected such that the quen
• step (b) RdRp having terminal transferase activity is added (together with rNTPs (here 1x rATP and rUTP and 5x rGTP and 5x rCTP) and buffer).
• rNTPs here 1x rATP and rUTP and 5x rGTP and 5x rCTP
• the RdRp elongates the rC stretch at the 3'-end of the quencher oligonucleotide by several further rC residues resulting in an (unpaired) oligoC or polyC sequence at the 3'-end of the quencher oligonucleotide.
• the RdRp initiates RNA polymerisation de novo on the oligoC/polyC sequence of the quencher oligonucleotide and polymerises an RNA strand complementary to the quencher oligonucleotide.
• the RdRp reaches the duplex region between the quencher oligonucleotide and the target polynucleotide.
• the RdRp further polymerises the complementary strand to the quencher oligonucleotide which leads to displacement of the target polynucleotide from the quencher oligonucleotide by the strand displacement activity of the RdRp.
• RNA template mir375 (5 ' - UUUGUUCGUUCGGCUCGCGUGA-3 ' ; SEQ ID NO: 16) was diluted in 1 : 10 steps yielding a standard curve ranging from 25 ng/ ⁇ (standard no. 1) to 2.5 fg/ ⁇ (standard no. 10).
• standard no. 1 25 ng/ ⁇
• standard no. 3 0.25 ng/ ⁇
• standard no. 6 25 pg/ ⁇
• the oligonucleotide DS16 (5 ' -CGAACGAACAAACCCCC-3 ' ; SEQ ID NO: 17) labeled at its 5 ' end with TAMRA and the oligonucleotide DS10 (5 ' - TCACGCGAGC-3 ' ; SEQ ID NO: 18) labeled at its 5 ' end with FAM were added to the reaction to a final concentration of 1 ⁇ each.
• the mix was incubated at 65°C for 30 min followed by cooling on ice for 15 min.
• the following reagents were added to the reaction: 5 ⁇ buffer B (Hepes 250 mM, MnCI2 25 mM, DTT 5 mM, pH 7.6), 0.4 mM ATP and UTP, 2 mM CTP and GTP, 8 ⁇ of sapovirus RdRp, and RNase-DNase-free water to a total volume of 25 ⁇ .
• RNA template mir375 (5 ' - UUUGUUCGUUCGGCUCGCGUGA-3 ' ; SEQ ID NO: 16), the RNA template ssR35 (5 ' -UAAGCACGAAGCUCAGAGUCCCCC-3 ' ; SEQ ID NO: 19) and the RNA template ssR25 (5 ' -GCUGAUGCCGUCAAGUUUA-3 ' ; SEQ ID NO: 20) were used at a concentration of 25 ng/ ⁇ .
• each template was incubated with 1 ⁇ of buffer A (50 mM, Tri-Hcl, 100 mM NaCI, 5 mM EDTA, pH 8.0) and 3 ⁇ of RNase-DNase-free water at 95°C for 5 min.
• buffer A 50 mM, Tri-Hcl, 100 mM NaCI, 5 mM EDTA, pH 8.0
• RNase-DNase-free water 3 ⁇ of RNase-DNase-free water at 95°C for 5 min.
• the oligonucleotide DS16 (5 ' -CGAACGAACAAACCCCC-3 ' ; SEQ ID NO: 17
• TAMRA oligonucleotide DS10
• FAM oligonucleotide DS10
• oligonucleotide probes used are both specific for mir375 but not for ssR35 or ssR25.
• the mix was incubated at 65°C for 30 min followed by cooling on ice for 15 min.
• the following reagents were added to the reaction: 5 ⁇ buffer B (Hepes 250 mM, MnCI2 25 mM, DTT 5 mM, pH 7.6), 0.4 mM ATP and UTP, 2 mM CTP and GTP, 8 ⁇ of sapovirus RdRp, and RNase-DNase-free water to a total volume of 25 ⁇ .
• the reaction mix was incubated in a LightCycler (Roche, Basel, Switzerland) and the fluorescence measured by F1/F2 channel mode. A total of 150 cycles of 15 seconds each were performed at 30 °C, and measurements performed at each cycle. Quantification of the emitted signal allowed the
• Ct cycle threshold
• RNA mir375 was diluted in 1 : 10 steps yielding a standard curve ranging from 25 ng/ ⁇ (standard no. 1) to 2.5 fg/ ⁇ (standard no. 10).
• ssR35 (5 ' - UAAGCACGAAGCUCAGAGUCCCCC-3 ' ; SEQ ID NO: 19) and ssR25 (5 ' - GCUGAUGCCGUCAAGUUUA-3 ' ; SEQ ID NO: 20) were used at a concentration of 25 ng/ ⁇ .
• ssR35 5 ' - UAAGCACGAAGCUCAGAGUCCCCC-3 ' ; SEQ ID NO: 19
• ssR25 (5 ' - GCUGAUGCCGUCAAGUUUA-3 ' ; SEQ ID NO: 20) were used at a concentration of 25 ng/ ⁇ .
• standard no. 1 25 ng/ ⁇
• standard no. 3 (0.25 ng/ ⁇
• each template ssR35 or ssR25 were incubated each with 1 ⁇ of Buffer A (50 mM, Tri- Hcl, 100 mM NaCI, 5 mM EDTA, pH 8.0) and 3 ⁇ of RNase-DNase-free water at 95°C for 5 min.
• Buffer A 50 mM, Tri- Hcl, 100 mM NaCI, 5 mM EDTA, pH 8.0
• the oligonucleotide DS17 (5 ' -CGAACGAACAAACCCCCCCC- 3 ' ; SEQ ID NO: 21) labeled at its 5 ' end with TAMRA and the oligonucleotide DS10 (5 ' -TCACGCGAGC-3 ' ; SEQ ID NO: 18) labeled at its 5 ' end by FAM were added to the reaction at a final concentration of 1 ⁇ , as well as 5 ⁇ buffer B (Hepes 250 mM, MnCI2 25 mM, DTT 5 mM, pH 7.6), 0.4 mM ATP and UTP, 2 mM CTP and GTP, 8 ⁇ of sapovirus NS7-RdRp, and RNase-DNase-free water to a total volume of 25 ⁇ .
• the reaction mix was incubated in a LightCycler (Roche, Basel,
• Ct cycle threshold
• No Ct was detected for ssR35 or ssR25.
• RNAse-DNase-free water was used as negative control, instead of the template. The noise band is shown.
• RNA template mir375 (5 ' - UUUGUUCGUUCGGCUCGCGUGA-3 ' s ; SEQ ID NO: 16) was diluted in 1 : 10 steps yielding a standard curve ranging from 25 ng/ ⁇ (standard no. 1) to 2.5 fg/ ⁇ (standard no. 10).
• RNA template ssR35 (5 ' - UAAGCACGAAGCUCAGAGUCCCCC-3 ' ; SEQ ID NO: 19) and the RNA template ssR25 (5 ' -GCUGAUGCCGUCAAGUUUA-3 ' ; SEQ ID NO: 20) were used at a concentration of 25 ng/ ⁇ .
• One microliter of each template corresponding to standard no. 1 (25 ng/ ⁇ ), standard no. 2 (2.5 ng/ ⁇ ), and standard no.
• each template ssR35 or ssR25 were incubated each with 1 ⁇ of buffer A (50 mM, Tri-Hcl, 100 mM NaCI, 5 mM EDTA, pH 8.0) and 3 ⁇ of RNase-DNase-free water at 95°C for 5 min.
• buffer A 50 mM, Tri-Hcl, 100 mM NaCI, 5 mM EDTA, pH 8.0
• the oligonucleotide DS18 (5 ' - ACGAACAAACCCCCCCCCC-3 ' ; SEQ ID NO: 22) labeled at its 5 ' end with TAMRA and the oligonucleotide DS10 (5 ' -TCACGCGAGC-3 ' ; SEQ ID NO: 18) labeled at its 5 ' end with FAM were added to the reaction at a final concentration of 1 ⁇ , as well as 5 ⁇ buffer B (Hepes 250 mM, MnCI2 25 mM, DTT 5 mM, pH 7.6), 0.4 mM ATP and UTP, 2 mM CTP and GTP, 8 ⁇ of sapovirus NS7-RdRp, and RNase-DNase-free water to a total volume of 25 ⁇ .
• 5 ⁇ buffer B Hepes 250 mM, MnCI2 25 mM, DTT 5 mM, pH 7.6
• 0.4 mM ATP and UTP 2
• the reaction mix was incubated in a LightCycler (Roche, Basel, Switzerland) and the fluorescence measured by F1/F2-Channel mode. A total of 150 cycles of 15 seconds each were performed at 30 °C and measurements were performed for each cycle. Quantification of the signal emitted allowed the determination of the cycle threshold (Ct) for each reaction.
• RNA mir375 Quantitative detection of mir375 by strand displacement after isothermal nucleotide transfer using a sapovirus RdRp.
• the target RNA mir375 (5 ' - UUUGUUCGUUCGGCUCGCGUGA-3 ' ; SEQ ID NO: 16) was diluted in 1 : 10 steps yielding a standard curve ranging from 25 ng/ ⁇ (standard no. 1) to 2.5 fg/ ⁇
• RNA template ssR35 (5 ' - UAAGCACGAAGCUCAGAGUCCCCC-3 ' ; SEQ ID NO: 19) was used at a concentration of 25 ng/ ⁇ .
• RNA template ssR35 5 ' - UAAGCACGAAGCUCAGAGUCCCCC-3 ' ; SEQ ID NO: 19
• the labeled oligonucleotide DS19 bears a 2 ' -0-methyl-cytidine at positionsl , 5 and 9 of the sequence (from 5' to 3'). Both probes were added to the reaction at a final concentration of 1 ⁇ , as well as 5 ⁇ buffer B (Hepes 250 mM, MnCI2 25 mM, DTT 5 mM, pH 7.6), 0.4 mM ATP and UTP, 2 mM CTP and GTP, 8 ⁇ of sapovirus RdRp, and RNase- DNase-free water to a total volume of 25 ⁇ .
• the oligonucleotide DS16 (5 ' - CGAACGAACAAACCCCC-3 ' ; SEQ ID NO: 17) labeled at its 5 ' end with TAMRA and the oligonucleotide DS10 (5 ' -TCACGCGAGC-3 ' ; SEQ ID NO: 18) labeled at its 5 ' end with FAM were added to the reaction at a final concentration of 1 ⁇ .
• the mix was incubated at 65°C for 30 min followed by cooling on ice for 15 min.
• the following reagents were added to the reaction: 5 ⁇ buffer B (Hepes 250 mM, MnCI2 25 mM, DTT 5 mM, pH 7.6), 0.4 mM ATP and UTP, 2 mM CTP and GTP, 8 ⁇ of vesivirus or norovirus RdRp, and RNase-DNase-free water to a total volume of 25 ⁇ .
• the reaction mix was incubated in a LightCycler (Roche, Basel, Switzerland) and the fluorescence measured by F1/F2 channel mode. A total of 150 cycles of 15 seconds each were performed at 30 °C, and measurements were carried out at each cycle.
• RNA template mir375 (5 ' - UUUGUUCGUUCGGCUCGCGUGA-3 ' ; SEQ ID NO: 16
• RNA template ssR35 (5 ' -UAAGCACGAAGCUCAGAGUCCCCC-3 ' ; SEQ ID NO: 19)
• RNA template ssR25 (5 ' -GCUGAUGCCGUCAAGUUUA-3 ' ; SEQ ID NO: 20) were used at a concentration of 25 ng/ ⁇ .
• each template was incubated with 1 ⁇ of buffer A (50 mM, Tri-Hcl, 100 mM NaCI, 5 mM EDTA, pH 8.0) and 3 ⁇ of RNase-DNase-free water at 95°C for 5 min.
• buffer A 50 mM, Tri-Hcl, 100 mM NaCI, 5 mM EDTA, pH 8.0
• RNase-DNase-free water 3 ⁇ of RNase-DNase-free water at 95°C for 5 min.
• the oligonucleotide DS16 (5 ' -CGAACGAACAAACCCCC-3 ' ; SEQ ID NO: 17
• TAMRA oligonucleotide DS10
• FAM oligonucleotide DS10
• the mix was incubated at 65°C for 30 min followed by cooling on ice for 15 min.
• the following reagents were added to the reaction: 5 ⁇ buffer B (Hepes 250 mM, MnCI2 25 mM, DTT 5 mM, pH 7.6), 0.4 mM ATP and UTP, 2 mM CTP and GTP, 8 ⁇ of the vesivirus RdRp, and RNase-DNase-free water to a total volume of 25 ⁇ .
• the reaction mix was incubated in a LightCycler (Roche, Basel, Switzerland) and the fluorescence measured by F1/F2-Channel mode.
• the DNA-oligonucleotide DS16 (5 ' - CGAACGAACAAACCCCC-3 ' ; SEQ ID NO: 17) labeled at its 5 ' end with TAMRA was incubated at a concentration of 15 ⁇ with 1 ⁇ buffer B (Hepes 250 mM, MnCI2 25 mM, DTT 5 mM, pH 7.6), 2 mM CTP, 8 ⁇ of sapovirus, norovirus, or vesivirus RdRp, and RNase-DNase-free water to a total volume of 7 ⁇ . The reaction mix was incubated at 30 °C for 30 min.
• the reaction was then lodaed on a 20% native polyacrymalide gel and the terminal transferase activity evidenced by electrophoresis after ethtidium bromide staining and UV transillumination.
• the product of the terminal transferase activity of the RdRps on the template in the presence of rCTP is indicated.
• the DNA-oligonucleotide DS17 (5 ' - CGAACGAACAAACCCCCCCC-3 ' ; SEQ ID NO: 21) labeled at its 5 ' end with TAMRA was incubated at a concentration of 15 ⁇ with 1 ⁇ buffer B (Hepes 250 mM, MnCI2 25 mM, DTT 5 mM, pH 7.6), 2 mM CTP, 8 ⁇ of sapovirus, norovirus, or vesivirus NS7-RdRp, and RNase-DNase-free water to a total volume of 7 ⁇ . The reaction mix was incubated at 30 °C for 30 min.
• the reaction was then loaded on a 20% native polyacrymalide gel and the terminal transferase activity evidenced by electrophoresis after ethtidium bromide staining and UV transillumination.
• the product of the terminal transferase activity of the RdRps on the template in the presence of rCTP is indicated.
• miR375-RNA 5 ' - UUUGUUCGUUCGGCUCGCGUGA-3 ' ; SEQ ID NO: 16
• buffer A 50 mM, Tri-Hcl, 100 mM NaCI, 5 mM EDTA, pH 8.0
• RNase- DNase-free water 3 ⁇ of RNase- DNase-free water at 95 °C for 5 min.
• the oligonucleotide DS16 (5 ' - CGAACGAACAAACCCCC-3 ' ; SEQ ID NO: 17) labeled at its 5 ' end with TAMRA was added to the reaction (final volume 5 ⁇ ) at a final concentration of 15 ⁇ together with 1 ⁇ buffer A.
• the mix was incubated at 65 °C for 30 min followed by cooling on ice for 15 min. Hybridisation of DS16 to mir375 yielded the mir375/DS16 hybrid.
• the following reagents were then added to the reaction: 4 ⁇ buffer B (Hepes 250 mM, MnCI2 25 mM, DTT 5 mM, pH 7.6), 2 mM CTP, 8 ⁇ of sapovirus RRp, norovirus RdRp or vesivirus RdRp, and RNase-DNase-free water to a total volume of 20 ⁇ .
• the reaction mix was incubated at 30 °C for 30 min.
• the reaction was then loaded on a 20% native polyacrymalide gel and the terminal transferase activity evidenced by electrophoresis after ethidium bromide staining and UV transillumination.
• the product of the terminal transferase activity of the RdRps on the mir375/DS16 hybrid template in the presence of rCTP is indicated.
• the following reagents were then added to the reaction: 4 ⁇ buffer B (Hepes 250 mM, MnCI2 25 mM, DTT 5 mM, pH 7.6), 2 mM CTP, 8 ⁇ of sapovirus RdRp, norovirus RdRp or vesivirus RdRp, and RNase-DNase-free water to a total volume of 20 ⁇ .
• the reaction mix was incubated at 30 °C for 30 min.
• the reaction was then loaded on a 20% native polyacrymalide gel and the terminal transferase activity evidenced by electrophoresis after ethidium bromide staining and UV transillumination.
• the product of the terminal transferase activity of the RdRps on the mir375/DS17 hybrid template in the presence of rCTP is indicated.
• the RNA template mir375 (5 ' -UUUGUUCGUUCGGCUCGCGUGA-3 ' ; SEQ ID NO: 16) was spiked in serum samples from a healthy volunteer at two concentrations: 80 ⁇ g/ml serum and 80 ng/ml serum. Serum was extracted using the RNEasy-Kit (Qiagen) according to the instructions of the manufacturer, yielding a concentration of 125 ng/ ⁇ and 125 pg/ ⁇ respectively.
• the oligonucleotide DS18 (5 ' - ACGAACAAACCCCCCCCCC-3 ' ; SEQ ID NO: 22) labeled at its 5 ' end with TAMRA and the oligonucleotide DS10 l5 ' -TCACGCGAGC-3 ' ; SEQ ID NO: 18) labeled at its 5 ' end with FAM were added to the reaction at a final concentration of 1 ⁇ .
• the mix was incubated at 65 °C for 30 min followed by cooling on ice for 15 min.
• the following reagents were added to the reaction: 5 ⁇ buffer B (Hepes 250 mM, MnCI2 25 mM, DTT 5 mM, pH 7.6), 0.4 mM ATP and UTP, 2 mM CTP and GTP, 8 ⁇ of sapovirus RdRp, and RNase-DNase-free water to a total volume of 25 ⁇ .
• the reaction mix was incubated in a LightCycler (Roche, Basel, Switzerland) and the fluorescence measured in F1/F2 channel mode. A total of 150 cycles of 15 seconds each were performed at 30 °C, and measurements were carried out at cycle.
• Quantification of the emitted signal allowed the determination of the cycle threshold (Ct) for each template, yielding a Ct of 16.2 for the sample with a mir375 concentration of 80 ⁇ g/ml serum and a Ct of 20.01 for the sample with a mir375 concentration of 80 ng/ml serum.
• the mir375 standard no. 1 yielded a Ct of 16.9.
• RNase-DNase-free water was used as negative control, instead of the template. The noise band is shown.
• RNA template mir191-RNA (5 ' - CAACGGAAUCCCAAAAGCAGCU-3 ' ) or the DNA template mir191-DNA (5 ' - CAACGGAATCCCAAAAGCAGCT-3 ' ) were used at a concentration of 25 ng/ ⁇ .
• One microliter of each template 25 ng/ ⁇ were incubated each with 1 ⁇ of Buffer A (50 mM, Tri-Hcl, 100 mM NaCI, 5 mM EDTA, pH 8.0) and 3 ⁇ of RNase-DNase- free water at 95°C for 5 min.
• Buffer A 50 mM, Tri-Hcl, 100 mM NaCI, 5 mM EDTA, pH 8.0
• the oligonucleotide Probe DS41 labeled at its 5 ' -end by TAMRA (5 ' -TAM RA-TGCTTTTG-3 ' ) and the oligonucleotide Probe DS38 labeled at its 5 ' - End by FAM (5 ' -FAM-ATTCCGTTCCCCCCC-3 ' ) were added to the reaction at a final concentration of 1 ⁇ , as well as 5 ⁇ Buffer B (Hepes 250 mM, MnCI2 25 mM, DTT 5 mM, pH 7.6), 0.4 mM ATP and UTP, 2 mM CTP and GTP, 8 ⁇ of sapovirus NS7-RdRp, and RNase-DNase-free water to a total volume of 25 ⁇ .
• Buffer B Hepes 250 mM, MnCI2 25 mM, DTT 5 mM, pH 7.6
• Buffer B Hepes 250 mM, MnCI2
• the reaction mix was incubated in a LightCycler and the fluorescence measured by F1/F2-Channel mode. A total of 30 cycles of 60 seconds each were performed at 30°C, and measurements were done for each cycle. Quantification of the signal emitted allowed the determination of the cycle threshold (Ct) for each of standards. RNAse-DNase-free water was used as negative control, instead of the template. The noise band is shown.
• Example 1 Specific detection of target RNA by sapovirus RdRp using C n oligoprobe carrying quencher and second oligoprobe carrying fluorescence donor
• Fig. 2B demonstrates that a quantification of the template is feasible from 2.5 fg onwards.
• the detection of mir375 using the oligonucleotides DS16 (Cn oligoprobe carrying quencher) and DS10 (second oligo carrying fluorescence donor) is specific for mir375, since no fluorescence is detectable with unrelated RNA sequences.
• the detection is also specific using a C n oligoprobe having a longer C n motif (oligonucleotide DS17; 10 C) compared to oligonucleotide DS16 (5 C).
• detection is still specific using a C n oligoprobe that has a shorter region of complementarity to the target (oligonucleotide DS18) compared to oligonucleotide DS16.
• Example 2 Specific detection of target RNA using chemically modified C n oligoprobe carrying quencher and second oligoprobe carrying fluorescence donor
• C n oligoprobe having chemical modifications (in this case a 2'-0- methyl cytidine in positions 1 , 5 and 9 of the oligonucleotide, counted from the 5'-end) that provide for a stronger annealing to the target RNA and therefore should enable higher detection specificity.
• Example 3 Specific detection of target RNA using norovirus and vesivirus RdRps
• RNA detection as demonstrated before for sapovirus RdRp is also reliable and specific when using other RdRps such as norovirus RdRp or vesivirus RdRp having the same structural features as the saporvirus RdRp.
• Example 4 Terminal transferase activity of calicivirus RdRps on DNA oligonucleotides
• oligondeoxyribonucleotides DS16 (Fig. 9) and DS17 (Fig. 10) in the presence of the corresponding ribonucleoside triphosphate in the presence of the corresponding ribonucleoside triphosphate.
• Example 5 Terminal transferase activity of calicivirus RdRps on DNA oligonucleotides hybridised to complementary target RNA sequence As outlined above in the legends to Figs. 11 and 12, RdRps of sapovirus, norovirus and vesivirus transfer ribonucleotides (in the present Example rCs) to the 3'-end of
• Example 6 Detection of target RNA in human serum by sapovirus RdRp using C n oligoprobe carrying quencher and second oligoprobe carrying fluorescence donor
• the method of the present invention was successfully used to detect mir375 RNA admixed with human serum.
• Example 7 Specific detection of target RNA and target DNA by sapovirus RdRp using C n oligoprobe carrying quencher and second oligoprobe carrying fluorescence donor
• mir191-RNA and mir191 DNA can be carried out using the method of the present invention.

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