EP1187936A2 - Test rapide automatisable pour la detection de cancers a base d'arnm de telomerase(htc), ainsi qu'amorces et sondes specifiques - Google Patents

Test rapide automatisable pour la detection de cancers a base d'arnm de telomerase(htc), ainsi qu'amorces et sondes specifiques

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Publication number
EP1187936A2
EP1187936A2 EP00920657A EP00920657A EP1187936A2 EP 1187936 A2 EP1187936 A2 EP 1187936A2 EP 00920657 A EP00920657 A EP 00920657A EP 00920657 A EP00920657 A EP 00920657A EP 1187936 A2 EP1187936 A2 EP 1187936A2
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European Patent Office
Prior art keywords
seq
telomerase
htc
mrna
starter
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EP00920657A
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German (de)
English (en)
Inventor
Wolfgang Springer
Gustav Hagen
Maresa Wick
Dmitry Zubov
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Bayer AG
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Bayer AG
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the invention relates to an automated rapid test for the detection of cancer on the basis of telomerase (hTC) mRNA, suitable start nucleotides and oligonucleotide probes for this test as well as a corresponding detection method and a test kit.
  • hTC telomerase
  • telomeres The genetic material of eukaryotic cells is distributed on linear chromosomes.
  • the ends of the genes are derived from the Greek words telos (end) and meros (part, segment) as telomeres.
  • Most telomeres consist of repetitions of short sequences, which are mainly composed of thymine and guanine (Zakian, 1995).
  • the telomeric sequences of related organisms are often similar and even conserved between more distant species. It is remarkable that in all vertebrates examined so far, the telomeres are built up from the sequence TTAGGG (Meyne et al, 1989).
  • telomeres perform various important functions. They prevent the fusion of chromosomes (McClintock, 1941) and thus the development of dicentric inheritance. Such chromosomes with two centromeres can lead to the development of cancer by loss of heterozygosity or doubling or loss of genes.
  • telomeres serve to distinguish intact hereditary systems from damaged ones. For example, yeast cells stopped dividing when they contained a chromosome without telomer (Sandell and Zakian, 1993).
  • telomeres are required to initiate DNA replication. After cleavage of the RNA primer, extension of the Okazaki fragments and subsequent ligation, the newly synthesized DNA strand lacks the 5 'end, because there the RNA primer cannot be replaced by DNA. Without special protective mechanisms, the chromosomes would shrink with every cell division ("end-replication problem"; Harley et al, 1990). The non-coding telomer sequences presumably represent a buffer zone to prevent the loss of genes (Sandeil and Zakian, 1993).
  • telomeres also play an important role in regulating cellular aging (Olovnikov, 1973). Human somatic cells show a limited replication capacity in culture; after a certain time they become senese. In this state, the cells no longer divide even after stimulation with growth factors, but do not die, but remain metabolically active (Goldstein,
  • telomeres have central functions in the aging of cells and the stabilization of genetic material and prevention of cancer.
  • telomeres synthesize the telomeres
  • telomere As described above, organisms with linear chromosomes can only partially replicate their genome without a special protective mechanism. Most eukaryotes use a special enzyme, telomerase, to regenerate the telomer sequences. Telomerase is constitutively expressed in the unicellular organisms examined so far. In contrast, telomerase activity was only cells and tumor cells were measured, whereas neighboring somatic tissue contained no telomerase (Kim et al, 1994).
  • telomerase activity was originally only detectable in human germline cells, but not in normal somatic cells (Hastie et al, 1990; Kim et al, 1994). After developing a more sensitive detection method (Kim et al, 1994), low telomerase activity was also detected in hematopoietic cells (Broccoli et al, 1995; Counter et al, 1995; Hiyama et al, 1995).
  • telomeres Vaziri et al, 1994; Counter et al, 1995. It has not yet been clarified whether the amount of enzyme in these cells is not sufficient to compensate for telomer loss or whether the measured telomerase activity stems from a subpopulation, e.g. incompletely differentiated CD34 + 38 + precursor cells (Hiyama et al, 1995 ).
  • Clarification would require proof of telomerase activity in a single cell.
  • telomere activity was detected in a large number of tumor tissues tested to date (1734/2031, 85%; Shay, 1997), while no activity was found in normal somatic tissue (1/196, ⁇ 1%, Shay, 1997).
  • Various studies also showed that in senescent cells transformed with viral oncoproteins, the telomeres continued to shrink and telomerase could only be discovered in the subpopulation that survived the growth crisis (Counter et al, 1992). The telomeres were also stable in these immortalized cells (Counter et al, 1992). Similar findings from studies in mice (Blasco et al, 1996) support the assumption that reactivation of telomerase is a late event in tumorigenesis. Information on telomerase and in particular on human catalytic telomerase subunit and its sequence are contained in WO 98/14592 (Geron Corp.) and WO 98/59040 (Bayer AG).
  • telomerase mRNA for cancer diagnosis
  • telomerase hypothesis combines the loss of telomer sequences and cell aging with the activity of telomerase and the development of cancer.
  • shrinking of telomeres can be seen as a mechanism for tumor suppression. Differentiated cells that do not contain telomerase stop their cell division at a certain length of the telomeres. If such a cell mutates, it can only develop into a tumor if the cell can extend its telomeres. Otherwise, the cell would continue to lose telomere sequences until its chromosomes become unstable and it eventually perishes.
  • telomere Reactivation of telomerase is believed to be the main mechanism for tumor cells to stabilize their telomeres.
  • telomere activity was detected in almost all tumor tissues tested so far, so that a genetic test could be used to diagnose all types of cancer. This genetic test is particularly suitable for monitoring the course of cancer, but can also be used as a prognostic test or for early diagnosis of certain cancers
  • Gene probe diagnostics in particular in conjunction with amplification techniques, is a fast, specific and highly sensitive method that enables early detection of specific genes, gene fragments or individual mutations at the DNA / RNA level.
  • the technique can be carried out directly in the test material. It is based on the DNA / RNA hybridization technique, ie the specific one in vitro binding of complementary single-stranded nucleic acid to form Watson-Crick base pairs.
  • the DNA / DNA or DNA RNA double strands formed are also referred to as DNA hybrids.
  • Complementary sequence-specific gene probes are used to detect the specific DNA or RNA by means of the hybridization reaction. These gene probes are short, chemically synthesized oligonucleotide probes with a length of 10-200 nucleotides.
  • the gene probes can be photochemically (N. Dattagupta, PMMRae, ED Huguenel, E. Carlson, A. Lyga, JSShapiro, JPAlbarella, Analytical Biochem. 177,85,1989) or enzymatically by nick translation (Rigby, P.WJ. et al, J. Mol. Biol.
  • radioactive or non-radioactive label can be provided with a radioactive or non-radioactive label. Suitable for this are labels with 32 TPs or non-radioactive labels with digoxigenin-dUTP, biotin-dUTP or direct labeling with enzymes such as alk. Phosphatase or Horseradish Peroxidase.
  • the nucleic acids are first separated into single strands by denaturation (heat or alkali treatment) and then specifically hybridized with one another under stringent conditions which are achieved by temperature, ionic strength of the buffers and organic solvents.
  • the gene probe only binds to complementary sequences of the DNA or RNA to be detected.
  • This hybridization reaction can be carried out in various test formats, for example as solid-phase hybridization to a carrier such as, for example, nitrocellulose-coupled target DNA or gene probe, or as a liquid hybridization.
  • the evaluation takes place via the labeling of the gene probe with a reporter molecule as listed above or, as in the reversed phase hybridization system shown here, via the target DNA which is labeled with digoxigenin-dUTP during the amplification and the gene probe which is used for binding on magnetic particles is labeled with fluorescein.
  • the hybridization complex of target DNA and labeled gene probe is removed quantified by unbound gene probe via the reporter molecule used.
  • This read out can take place directly with fluorescence labeling or radioactive labeling or indirectly through enzyme tests and immunological methods with antibody conjugates, the enzymes such as the alk. Contain phosphatase and then allow a color reaction or chiluminescence reaction.
  • test sensitivity with gene probe diagnostics is in the range of 10 ⁇ to 10 "copies based on the detection of single genes.
  • An increase in test sensitivity can be achieved by combining it with DNA or RNA amplification techniques such as PCR (EP 200362) .LCR (EP 320308), NASBA (EP 329822), Qß (PCT
  • the invention relates to primers and probes (probes) for the amplification and detection of the mRNA of the human catalytically active telomerase subunit (hTC).
  • the human catalytic telomerase subunit (hTC) is described in WO 98/59040, to which express reference is made.
  • Such an oligonucleotide can in particular be an oligodeoxyribonucleotide or an oligoribonucleotide or a peptide nucleotide acid (PNA)
  • PNA peptide nucleotide acid
  • oligonucleotides which hybridize specifically with the hTC mRNA of the telomerase from the T-motif area, 5 'area and 3' area A DNA sequence or a degenerate variation of this sequence which encodes the protein hTC or a fragment of this protein, or DNA sequence which hybridizes with the DNA sequence under standard hybridization conditions.
  • a recombinant polynucleotide probe that contains a DNA sequence or a degenerate variation of that sequence that hybridizes hTC or a fragment of hTC
  • the invention further relates to a method for the detection of a neoplastic disease of a patient, in particular a method for determining the presence of the hTC protein in a cell or cellular sample, which is based on the amplification of an hTC polynucleotide or hybridization of an hTC polynucleotide, primers or an hTC complementary sequence with an hTC polynucleotide.
  • the process then comprises the following steps:
  • the invention further relates to a test kit for the detection of hTC mRNA in cellular samples and body fluids based on the above test principle.
  • the test kit is preferably used for the diagnosis of
  • test kit contains:
  • oligo- or polynucleic acids functional equivalents should be understood to mean those compounds which differ in the nucleotide sequence but code for the same protein. This is e.g. attributed to the degenerate genetic code.
  • the invention relates in particular to starter oligonucleotides comprising a nucleotide sequence selected from the group consisting of SEQ ID No 1, SEQ ID No 2, SEQ ID No 4, SEQ ID No 5, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9 and SEQ DD No 10.
  • the starter oligonucleotides are preferably used in suitable pairs, in the following sets:
  • the invention further relates to oligonucleotide probes, optionally labeled, containing a nucleotide sequence selected from the group consisting of SEQ ID NO: 1
  • the invention further relates to a method for the detection of increased telomerase activity, in which
  • telomerase (hTC) mRNA a sample of telomerase (hTC) mRNA using one or more starter oligonucleotides according to claim 1 and
  • the invention further relates to a test kit for detecting increased telomerase activity, containing one or more of the starter oligonucleotides.
  • an automatable genetic test for the detection of cancer diseases based on hTC-specific mRNA.
  • the previously described in situ tests based on the RNA component of telomerase had the disadvantage that no tumor-specific relevance was recognizable.
  • TRAP tests (Kim et al, Science 266, 2011-2015, 1994) had shown that telomerase activity was increased in various malignant tumors. The test specificity and sensitivity has so far been unsatisfactory for this test and not relevant for prognostic or diagnostic use (eg bladder cancer).
  • the advantage of the described invention is that the use of special primers optimized with regard to length and sequence and a readout that can be automated automatically enables a direct measurement of the amount of the telomerase amplicon formed via a chemiluminescence test or colorimetric test, and that Amplicon serves as a direct measure of telomerase expression or telomerase activity. Since the hTC telomerase obviously represents the speed-limiting step in the catalytic activity of the telomerase, this test provides a direct correlation between tumor tissue and telomerase activity at the nucleic acid level. So could in different
  • telomeres Tumors of the stomach, intestines, lungs, breast, ovary, prostate as well as melanomas and osteosarcomas, strongly increased telomerase values can be detected, whereas in normal tissues such as lungs, brain, kidneys, intestines and blood, only small signals were found.
  • the signal strength of the amplicon could be increased by a factor of 10 and the sensitivity of the test compared to conventional DNA tests by a factor of 10-100.
  • the amount of test material required was greatly reduced and the reliability of the test result was significantly improved by the significantly higher signals, even with low test material. Thanks to the already developed automation of the process, a large number of samples (> 100) can be read out within 20 minutes.
  • the present invention describes specific primers and oligonucleotide probes and their use for the rapid detection of telomerase expression based on hTC mRNA.
  • an automatic implementation is possible, for example on the Immuno I, Bayer Diagnostics, Tarrytown.
  • the test can also be carried out with the described primers and probes in Taqman or Lightcycler.
  • this test is particularly suitable for the cellular analysis of any sample material (eg smears) and for in situ hybridization.
  • the primers were prepared from the gene sequence of the telomerase gene by chemical synthesis.
  • the invention relates to primers and probes with a length of 15 to 40 (e.g. 15 to 30) nucleotides from the T motif area, 5 'area upstream from the start codon and 3' area of a splicing variant according to the im
  • the preferred primers were selected from the range
  • oligonucleotide probes were prepared by chemical synthesis
  • the mRNA was isolated from clinical samples using special RNA isolation methods.
  • the amplification of parts of the hTC mRNA was carried out with the specific primers from the T motif region, promoter region or splicing variant region.
  • N For the detection of the amplicons, a capture probe is used which hybridizes with the amplified nucleotide region.
  • the amplification was carried out using known amplification techniques, preferably the RT-PCR amplification method (U.S. Patent 5322770).
  • Fluorescence nucleotides or fluorescence-labeled primers of labeled amplification product Fluorescence nucleotides or fluorescence-labeled primers of labeled amplification product.
  • the amplification product is separated using additional biotin (primer or nucleotide)
  • amplification product labeled during the amplification e.g. digoxigenin d-UTP
  • the hybridization complex is separated with magnetic particles coated with fluorescein antibodies.
  • the evaluation of the hybridization complex formed as a measure of the telomerase expression and thus the telomerase activity is carried out by a chemiluminescence test with antidigoxigenin antibodies which are coupled with alkali phosphatase and which react with the digoxigenin built into the amplicon.
  • the test is carried out with one batch of test tissue mRNA and one batch of normal control tissue mRNA.
  • normal telomerase gene expression With normal telomerase gene expression, the amplification with the specific primers gives only little amplicon and thus also only a low chemiluminescence signal. A lot of amplicon and thus a strong chemiluminescence signal is produced in neoplastic tissue.
  • the great advantage of this test is that it can be decided immediately after the amplification whether there is an increased telomerase mRNA level, the test can be fully automated and can be carried out very quickly and with little effort and expense due to only a single amplification step.
  • the primer sets specific for the amplification products were selected from areas of the telomerase gene which are specific for the hTC telomerase gene and which do not result in homology with other RT motifs or other RT sequences. Primers with the sequence SEQ ID No 1, 2, 4, 5, 7, 8, 9, 10 were synthesized, which give specific amplification products. Suitable primers preferably have a length of 15 to 25 base pairs, particularly preferably 17 to 22 base pairs.
  • the selected primers were chemically synthesized using the phosphoramidite method of S.L. Beaucage and M.Carathers, Tetrahedron Letters, 22, 1859, 1981.
  • oligonucleotide probes specific for the amplification products of the primer sets were selected from regions of the telomerase gene which are specific for the hTC telomerase gene and which do not result in homology and hybridization with other RT motifs or other RT sequences. 30-36 mers were synthesized with the sequence SEQ ID No 3, 6, 11, which are specific for the amplification products.
  • Suitable probes preferably have a length of 20 to 36 base pairs, particularly preferably 25 to 36, very particularly preferably 30 to 36 base pairs.
  • Suitable probes can also have a solution of 25 to 30 base pairs.
  • the above-mentioned primers were each used as primer sets (Primer 1 + 2), (Primer4 + 5) or (Primer 7 + 8, 9 + 10), for the specific RTA amplification of the human telomerase m-RNA used.
  • primer sets (Primer 1 + 2), (Primer4 + 5) or (Primer 7 + 8, 9 + 10)
  • dNTPs deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate and thymidine triphosphate
  • Digoxigenin-dUTP can be incorporated into the amplification product. This allows the amplification product to be combined with an antidigoxigenin antibody, e.g. alk. Phosphatase coupled can be evaluated via a chemiluminescence test with AMPPD as a substrate or a dye test with pNPP.
  • fluorescence-labeled nucleoside triphosphates such as fluorescein-dUTP or coumarin-dUTPs
  • biotinylated primers it is possible to separate the fluorescence-labeled, biotinylated amplification product via streptavidin-coated magnetic particles and to determine them quantitatively in the fluorescence photometer.
  • a DNA capture probe and a digoxigenin-labeled amplification product are preferably used. set.
  • a sample in the form of a fluorescein-labeled RNA sample can also be used, which serves as a capture and detector sample.
  • This genetic test using a DNA / RNA antibody significantly better sensitivities are achieved than with the previously usual genetic tests for other targets and therefore very little starting material is required to carry out the test.
  • the level of telomerase expression can be amplified by any of the primer sets described in the invention directly after amplification of part of the htCmRNA
  • a possible read out method is the staining of the amplification product separated by agarose gel electrophoresis with intercalating agents such as ethidium bromide.
  • a further possibility is the use of fluorescence-labeled primers for the amplification or the combination of biotinylated primers with fluorescence nucleotides, so that a terminally biotinylated, fluorescence-labeled amplification product is formed which can be bound and separated to magnetic particles coupled to streptavidin and the fluorescence can be determined semiquantitatively .
  • the most sensitive and preferred method is the described method of hybridizing the amplification products with the described oligonucleotide probe. If, for example, digoxigenin-dUTP is incorporated during the amplification and use of a biotinylated or fluorescent oligonucleotide probe, the Separate the hybridization complex of streptavidin / fluorescein antibody-coated magnetic particles and when using antidigoxigenin antibodies that are mixed with alk. Phosphatase are coupled, with AMPPD or CSPD as substrate semi-quantitatively via chemiluminescence.
  • An alternative method is the amplification without any incorporation of marker molecules and the detection of the
  • the detection of the hybridized amplicon takes place with a DNA / RNA antibody. This read out results in a high sensitivity and was specially developed for the automated process in
  • the selected starter oligonucleotides were chemically synthesized using the phosphoramidite method of S.L. Beaucage and M.Camthers, Tetrahedron Letters, 22, 1859, 1981.
  • the following nucleotide sequences were synthesized:
  • PCR primer 1 SEQ ID No 1
  • the oligonucleotide probes were selected from the nucleotide region which contains the amplified sequence of the different primer sets.
  • the selected oligonucleotide probes were synthesized using the phosphoramidite method of S.L. Beaucage and M. Caruthers, Tetrahedron Letters, 22, 1859, 1981.
  • reaction buffer potassium cocodylate 1 mol / 1; Tris / HCl 125 mmol / l; bovine serum albumin 1.25 mg / ml; pH 6.6; 25 ° C
  • oligonucleotide 25 units of terminals Transferase, C0CI2 2.5 mmol / 1 and 1 ml fluorescein-d-UTP (1 mmol / L) are calibrated after 60 minutes at 37 ° C approx. 50% 3 'end label.
  • the mRNA or total RNA was diluted and used in the concentrations 100 ng, 50 ng and 25 ng (10 ⁇ l).
  • the prepared mixtures were mixed with the prepared mixes (50 ⁇ l total volume / tube). For checking on
  • ethidium bromide was used as the intercalating agent after the amplification.
  • Biotin-dUTP or digoxigenin-dUTP can also be used and alk coupled with antibodies. Phosphatase a dye read out can be performed. Correspondingly fluorescent-labeled primers can also be used with lower sensitivity.
  • the amplification product was applied to a 0.8% agarose gel and electrophoresed at 100 mA for 30 minutes.
  • the fluorescence signals were evaluated directly under a UV transilluminator.
  • PCR Polymerase Chain Reaction
  • LCR LCR
  • gene probe technology a significant increase in sensitivity compared to conventional gene probe read-out methods is achieved.
  • the liquid hybridization tests were carried out with 100 ng digoxigenized amplicon and fluorescent capture probe according to Example 3 in a volume of 50 ⁇ l.
  • the blocking reaction and antibody reaction for the detection of hybridization via chemiluminescence was then carried out.
  • the beads loaded with DNA were added 1x with 500 ⁇ l buffer 2 (0.1 M maleic acid; 0.15 M NaCl pH 7.5; 1% blocking reagent (Boehringer)). After 5 minutes of incubation at room temperature, the mixture was separated, pipetted off and 250 ⁇ l of antibody conjugate solution (AK 1: 2500 in buffer 2) were added and incubated for 10 minutes at room temperature, then separated, pipetted off and treated with 500 ⁇ l of washing buffer 2 ⁇ 30 seconds, 1 x 2 minutes with weak movement. It was then incubated with detection solution with AMPPD 1: 100 in buffer 3 for 10 minutes at 37 ° C. in a water bath, then the chemiluminescence was measured in the luminescence photometer at 477 nm (Lumacounter from Lumac).
  • PCR polymerase chain reaction
  • LCR LCR
  • gene probe technology results in a significant increase in sensitivity compared to conventional gene probe read-out methods.
  • the liquid hybridization tests were carried out with 100 ng fluorescein-labeled RNA probe and amplified DNA according to Example 3 in a volume of 50 ⁇ l.
  • the coupled hybridization complex was separated with the beads, the residual liquid was pipetted off and washed once with buffer B (0.1 SSC; 0.1% SDS) once.
  • the blocking reaction and antibody reaction for the detection of hybridization via chemiluminescence was then carried out.
  • the loaded beads were added 1x with 500 ⁇ l buffer 2 (0.1 M maleic acid; 0.15 M NaCl pH 7.5; 1% blocking reagent (Boehringer)). After 5 minutes of incubation at room temperature, the mixture was separated, pipetted off and 250 ⁇ l of antibody conjugate solution
  • Lumac Lumac measured.
  • the process can be carried out automatically on the Immunol or subsequent devices.
  • 2xl0 7 cells (total) were centrifuged for 5 min at 2500 ⁇ m. Then the supernatant was poured off and dried overhead. The pellet was resuspended in 800 ⁇ L Lysis Buffer OL1 and incubated on ice for 3 min. 400 ⁇ L of the pellet lysed in OL1 were applied to 2 homogenizing columns. The mixture was then centrifuged for 1 min at 13000 ⁇ m and the column was discarded.
  • reaction tube was discarded and the column was placed on a new reaction tube containing 2 ⁇ L RNAsin.
  • 125 ⁇ L of elution buffer OEB (70 ° C.) was added to the column, mixed with the pellet and eluted for 5 min at 13000 ⁇ m. The two eluates were combined and the OD measured at 260 nm.
  • telomerase mRNA in clinical sample material (normal / neoplastic tissue)
  • the mRNA was isolated from the clinical sample material according to the method described in Example 7.
  • the RNA lysate was then amplified with the aid of suitable amplification methods as described in Example 5 with specific oligonucleotide primers.
  • the amplified nucleic acid was then with the
  • Oligonucleotide probes which are described in the sequence listing and the specific hybridization complex which forms under stringent conditions were separated using magnetic particles from Dynal and, as in Example 6 or preferably 7, determined quantitatively by chemiluminescence readout.
  • RNA or mRNA The tumor material of various origins listed in Table 1 was used to isolate total RNA or mRNA.
  • Cell cultures such as HeLa cells or Hek cells served as further positive controls.
  • Normal tissues from the lungs, brain, kidneys, intestines and blood (leukocytes) served as negative controls.
  • RNA was worked up using an RT-PCR as endpoint PCR on a normal thermal cycler or as kinetic PCR on the Lightcycler or the Perkin-Elmer-Taqman.
  • Primer sets and probes of the new telomerase assay have high signals that are comparable were with the signals in the HeLa and Hek cells.
  • the normal tissue on the other hand, gave only very low background signals even in the highest RNA concentration.
  • telomerase activity was also successful in the urine of patients with bladder tumors or in the sputum of patients with lung tumors.
  • various body fluids such as urine, sputum and blood are also suitable as starting materials for testing for increased telomerase activity.
  • the amplification was carried out via one- or two-stage RT-PCR and the detection via chemiluminescence test, fluorescence or colorimetric read out.
  • Sequences Telomerase T-motif sequences from
  • Probe Telo Tmotiv SeqlDNo 3 5 'TCCgTgACATAAAAgAAAgACCTgAgCAgCTCgA3' Probe
  • Rev5180 SEQIDNo 5 5 'TagTggCTgCgCAgCAgggA3'
  • Probe5100 SEQIDNo 6 5 'AagCCCTggCACCggTCACCCCCgCgATgCCgCgCg3 ⁇

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Abstract

L'invention concerne un test rapide automatisable pour la détection de cancers à base d'ARNm de télomérase(hTC), des nucléotides amorces appropriés et des sondes oligonucléotidiques pour ce test. L'invention concerne également un procédé de détection correspondant et un nécessaire de test.
EP00920657A 1999-04-15 2000-04-04 Test rapide automatisable pour la detection de cancers a base d'arnm de telomerase(htc), ainsi qu'amorces et sondes specifiques Withdrawn EP1187936A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19916929A DE19916929A1 (de) 1999-04-15 1999-04-15 Ein automatisierbarer Schnelltest zum Nachweis von Krebserkrankungen auf der Basis von Telomerase(hTC) mRNA mit spezifischen Primern und Sonden
DE19916929 1999-04-15
PCT/EP2000/002980 WO2000063429A2 (fr) 1999-04-15 2000-04-04 Test rapide automatisable pour la detection de cancers a base d'arnm de telomerase(htc), ainsi qu'amorces et sondes specifiques

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US (1) US6808883B1 (fr)
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AU (1) AU774182B2 (fr)
CA (1) CA2370305A1 (fr)
DE (1) DE19916929A1 (fr)
WO (1) WO2000063429A2 (fr)

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AU774182B2 (en) 2004-06-17
DE19916929A1 (de) 2000-10-19
WO2000063429A3 (fr) 2002-01-03
US6808883B1 (en) 2004-10-26
AU4115300A (en) 2000-11-02
WO2000063429A2 (fr) 2000-10-26
JP2003519466A (ja) 2003-06-24
CA2370305A1 (fr) 2000-10-26

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