EP1187936A2

EP1187936A2 - Automatable rapid test for detecting cancers, on the basis of telomerase(htc) mrna and specific primers and probes

Info

Publication number: EP1187936A2
Application number: EP00920657A
Authority: EP
Inventors: Wolfgang Springer; Gustav Hagen; Maresa Wick; Dmitry Zubov
Original assignee: Bayer AG
Current assignee: Bayer AG
Priority date: 1999-04-15
Filing date: 2000-04-04
Publication date: 2002-03-20
Also published as: DE19916929A1; JP2003519466A; AU4115300A; WO2000063429A2; CA2370305A1; WO2000063429A3; AU774182B2; US6808883B1

Abstract

The invention relates to an automatable rapid test for detecting cancers on the basis of telomerase(hTC) mRNA, starter nucleotides and oligonucleotide probes suitable for said test, a corresponding method of detection and a test kit.

Description

An automated rapid test for the detection of cancer based on telomerase (hTC) mRNA with specific primers and probes

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.

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).

The 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.

Furthermore, 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).

Another important task is performed by telomeres in the DNA replication of eukaryotic cells. In contrast to the circular genomes of prokaryotes the linear chromosomes of the eukaryotes cannot be fully replicated by the DNA polymerase complex. RNA primers 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).

In addition, 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,

1990). Various observations support the hypothesis that a cell determines how often it can divide based on the length of its telomeres (Allsopp et al, 1992).

In summary, the telomeres have central functions in the aging of cells and the stabilization of genetic material and prevention of cancer.

The enzyme telomerase synthesizes the telomeres

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).

Activation of telomerase in human tumors

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).

However, these cells still showed a reduction in 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 ). to

Clarification would require proof of telomerase activity in a single cell.

Interestingly, however, significant telomerase 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).

Detection of telomerase mRNA for cancer diagnosis

Based on these results, a "telomerase hypothesis" was developed that combines the loss of telomer sequences and cell aging with the activity of telomerase and the development of cancer. In long-lived species like humans, the 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.

Reactivation of telomerase is believed to be the main mechanism for tumor cells to stabilize their telomeres.

From these observations and considerations it follows that an increased expression of the telomerase should allow a diagnosis of tumors. Telomerase 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.

113, 237, 1977) or random primed techniques (Feinberg and Vogelstein, Anal. Biochem. 132,6, 1983) 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.

For the specific hybridization between the nucleic acid to be detected and the specific gene probe, 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. With suitable hybridization conditions, 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 (read out) 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.

The 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

87/06270) or HAS technology (EP 427074) can be achieved. With these techniques, up to a 10-fold multiplication of the DNA to be detected can be achieved. The combination of amplification and hybridization makes it possible to detect individual DNA molecules.

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.

Oligonucleotides in purified form with a sequence which are identical or exactly complementary to a 10 to 500 nucleotide long, contiguous sequence of the mRNA of hTC.

Such an oligonucleotide can in particular be an oligodeoxyribonucleotide or an oligoribonucleotide or a peptide nucleotide acid (PNA)

Preferred are 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

The process then comprises the following steps:

A. Detection of the hTC mRNA in cellular samples to obtain a diagnostic value;

B. Comparison of the diagnostic value with standard values for the hTC mRNA in non-neoplastic cells of the same type as the test sample;

C. Diagnostic values that are significantly higher than standard comparative values indicate a neoplastic condition.

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

Cancer. In particular, the test kit contains:

A compilation consisting of a pair of human hTC polynucleotide PCR primers, the primers preferably consisting of sequences which correspond to the sequence of the human hTC mRNA or are complementary to this sequence and / or a compilation which comprises a polynucleotide Contains hybridization probe for the human hTC gene, the probe containing 20-36, for example 30 consecutive nucleotides, which correspond to the sequence of the human hTC mRNA or are complementary to this.

In the case of 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:

Set of a starter oligonucleotide containing the nucleotide sequence according to SEQ ED No 1 and a starter oligonucleotide containing the nucleotide sequence according to SEQ

ID No 2.

Set of a starter oligonucleotide containing the nucleotide sequence according to SEQ ID No 4 and a starter oligonucleotide containing the nucleotide sequence according to SEQ ID No 5. Set of a starter oligonucleotide containing the nucleotide sequence according to SEQ ID No 7 and a starter oligonucleotide containing the nucleotide sequence according to SEQ ID N0 8.

Set of a starter oligonucleotide containing the nucleotide sequence according to SEQ ID

No 9 and a starter oligonucleotide containing the nucleotide sequence according to SEQ ID No 10.

The invention further relates to oligonucleotide probes, optionally labeled, containing a nucleotide sequence selected from the group consisting of SEQ ID

No 3, SEQ ID No 6 and SEQ ID No 11.

The invention further relates to a method for the detection of increased telomerase activity, in which

a) amplified in a sample of telomerase (hTC) mRNA using one or more starter oligonucleotides according to claim 1 and

b) evaluates the amplification results.

The invention further relates to a test kit for detecting increased telomerase activity, containing one or more of the starter oligonucleotides.

In the present invention, an automatable genetic test is described 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

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.

By using an RNA detector probe in combination with a DNA / RNA antibody, 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. As a result, 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. With the read out with magnetic beads using the methods described in Examples 5 and 6, 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. In addition, this test is particularly suitable for the cellular analysis of any sample material (eg smears) and for in situ hybridization.

I. 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

Sequence listing sequences 1-11.

The preferred primers were selected from the range

a) which is specific for the telomerase T motif (primer 1 +2 SEQ ID

No 1 + 2)

b) which is specific for the 5 'region (promoter region) (primer 4 + 5 SEQ ID No 4 + 5)

c) which is specific for the 3 'splice area with splice variants (primer 7 + 8 SEQ ID No7 + 8 or 9 + 10)

II. The oligonucleotide probes were prepared by chemical synthesis

III. The mRNA was isolated from clinical samples using special RNA isolation methods.

IV. 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.

VI. The amplification was carried out using known amplification techniques, preferably the RT-PCR amplification method (U.S. Patent 5322770).

VII. The specific amplification product was detected

a) by direct electrophoresis of the amplification product in the agarose sail and staining by intercalating agents such as ethidium bromide

b) by determining the fluorescence during amplification with

Fluorescence nucleotides or fluorescence-labeled primers of labeled amplification product. The amplification product is separated using additional biotin (primer or nucleotide)

c) and preferably by hybridization of the amplification product labeled during the amplification, e.g. digoxigenin d-UTP) with the above-mentioned fluorescein-labeled oligonucleotide probes. The hybridization complex is separated with magnetic particles coated with fluorescein antibodies.

d) 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. 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.

Selection and synthesis of primers

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.

Selection and synthesis of the oligonucleotide probes

The 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 chemical synthesis was carried out using the phosphoramidite method of S.L. Beaucage and M.Caruthers, Tetrahedron Letters, 22, 1859, 1981.

Amplification of m-RNA from hTC telomerase

For the amplification of the mRNA sequence of human telomerase, 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. With the mRNA of neoplastic cells in the agarose gel, they produce a visible amplification product.

In RTPCR amplification, in addition to the 4 dNTPs (deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate and thymidine triphosphate), e.g. 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.

Alternatively, there is the possibility of incorporating fluorescence-labeled nucleoside triphosphates such as fluorescein-dUTP or coumarin-dUTPs into the amplification product and identifying the amplification product with much higher sensitivity than with ethidium bromide staining. When using 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. In this invention, 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. With 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.

Detection of telomerase expression

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

Analytical methods can be determined.

A possible read out method is the staining of the amplification product separated by agarose gel electrophoresis with intercalating agents such as ethidium bromide.

Another possibility is the incorporation of fluorescence-labeled nucleoside triphosphates into the amplification product. This leads to a significant improvement in test sensitivity.

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

Amplicons by hybridization with a fluoresein-labeled capture probe and an additional RNA probe as a detector sample or sole fluorescein-labeled RNA capture and detector sample. 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

Immunol vending machines and successors developed.

example 1

Synthesis of starter oligonucleotides (primers)

The selected starter oligonucleotides (primers) 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:

Detection of the telomerase motif: PCR primer 1: SEQ ID No 1

PCR primer 2 SEQ ID No 2

Detection of the 5 'area PCR-Primer4 + 5 SEQ ID No 4 + 5

Evidence of the 3 'area

PCR Primer 7 + 8 SEQ ID No 7 + 8

PCR primer 9 + 10 SEQ ID No 9 + 10

Example 2

Synthesis and selection of the oligonucleotide probes

The oligonucleotide probes were selected from the nucleotide region which contains the amplified sequence of the different primer sets. The chemical

The selected oligonucleotide probes were synthesized using the phosphoramidite method of S.L. Beaucage and M. Caruthers, Tetrahedron Letters, 22, 1859, 1981.

T-motif area: sample 3 SEQ ID No 3 5 'area: sample 6 SEQ ID No 6

3 'Area: Sample 11 SEQ ID No 11 The capture probe was labeled at the 3 'end using the method of BoUum, The enzymes, Boyer ed, Vol 10, p 145 Academic Press New York. End group labeling was not radioactive with fluorescein dUTP. (Chang, LMS, Bollum TJ, J.Biol. Chem. 246, 909, 1971).

In a 50 ml batch with 10 ml reaction buffer (potassium cocodylate 1 mol / 1; Tris / HCl 125 mmol / l; bovine serum albumin 1.25 mg / ml; pH 6.6; 25 ° C) 1-2 mg 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.

Example 3

Telomerase-specific amplification using the RT-PCR method

(Titan One Tube RT-PCR system)

The mRNA or total RNA was diluted and used in the concentrations 100 ng, 50 ng and 25 ng (10 μl). The prepared mixtures were mixed with the prepared mixes (50 μl total volume / tube). For checking on

DNA contamination, the enzyme mix was replaced by Taq polymerase.

Mix l lμl PCR-Nucleotide-Mix (lOmM) 200ng forward Primer (0.4-1 μm)

200ng reverse primer (0.4-1 μm)

2.5μl l00mM DTT

0.2μl RNAsin (20 units)

2μl MgCl ₂ (25mM) l, 5μl Dig dUTP l: 10verd (25nM) bidest to 15μl Wed 2nd

10μl 5x RT-PCR buffer l, 5μl enzyme mix, bidest to 25 μl

Prepare PCR tubes with dilutions of RNA (10 μl). Mix with 25μl Mix 2 + 15μl Mix 1.

PCR prof for T motif: 20 '58 ° C / 2' 94 ° C // 30 "94 ° C _ 1 '54 ° C_1' 68 ° C 30 cycles

T 68 ° C / 4 ° C BoehringeπOrder no. 1855476

Example 4

Direct evaluation of the amplification product

For direct evaluation of the DNA amplification product, 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. Example 5

Gene probe test of RT amplification products

By combining suitable target amplification methods like that

Polymerase Chain Reaction (PCR) (EP200362), LCR (EP320308) and 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 μl.

After heating to 100 ° C. for 10 minutes and then cooling to 0 ° C., 50 μl 2x hybridization mix (50 ml 20XSSC, 1 g blocking reagent

(Boehringer), 2 ml of 10% lauroyl sarcosine, 200 ml of 20% SDS ad 100 ml bidest H2O) and hybridized for 5-10 minutes at 37 ° C oligonucleotide probe). The magnetic beads were pretreated with 1 × hybridization mix and, after separation using a magnet, the liquid was pipetted off, the hybridization mixture and 100 μl 1 × hybridization mix were added and incubated for 5-10 minutes at room temperature with gentle agitation. 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).

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 μl 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 μl 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 μl 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).

Example 6

Gene probe test of DNA amplification products with DNA / RNA antibody read out

The combination of suitable target amplification methods such as polymerase chain reaction (PCR) (EP200362), LCR (EP320308) and 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 μl.

After heating for 10 minutes at 100 ° C and then cooling to 0 ° C, 50 μl 2x hybridization mix (50 ml 20XSSC, 1 g blocking reagent (Boehringer), 2 ml 10% lauroylsarcosine, 200 ml 20% SDS ad 100 ml bidist H ₂ O) added and hybridized oligonucleotide probe for 5-10 minutes at 37 ° C). The magnetic beads were pretreated with 1 × hybridization mix and, after separation using a magnet, the liquid was pipetted off, the hybridization mixture and 100 μl 1 × hybridization mix were added and incubated for 5-10 minutes at room temperature with gentle agitation. 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 μl 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 μl of antibody conjugate solution

Add (AK 1: 2500 in buffer 2) and incubate for 10 minutes at room temperature, then separate, pipette off and treat with 500 μl washing buffer 2x30 seconds, 1x2 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 in the luminescence photometer at 477 nm (Lumacounter from

Lumac) measured. The process can be carried out automatically on the Immunol or subsequent devices.

Example 7

mRNA isolation with the Qiagen Oligotex Direct mRNA Micro Kit

2xl0 ⁷ cells (total) were centrifuged for 5 min at 2500 φm. Then the supernatant was poured off and dried overhead. The pellet was resuspended in 800 μL Lysis Buffer OL1 and incubated on ice for 3 min. 400 μL of the pellet lysed in OL1 were applied to 2 homogenizing columns. The mixture was then centrifuged for 1 min at 13000 φm and the column was discarded. After adding 800 μL Dilution Buffer ODB and mixing, the mixture was centrifuged for 5 min at 13000 φm and then the supernatant was transferred to autoclaved 2 mL reaction vessels, 30-50 μL Oligotex suspension added (the suspension is preheated at 37 ° C for 10 min and shaken , then placed on ice, then shaken at RT for 10 min After 5 minutes of centrifugation at 13000 φm, the supernatant is discarded and the pellet is resuspended in 350 μL Wash Buffer OW1, then centrifuged for 5 min at 13000 φm, the supernatant is discarded and Washed twice more with Wash Buffer OW2 After the second time, the suspension is added to the column and centrifuged. The reaction tube was discarded and the column was placed on a new reaction tube containing 2 µL RNAsin. 125 μL of elution buffer OEB (70 ° C.) was added to the column, mixed with the pellet and eluted for 5 min at 13000 φm. The two eluates were combined and the OD measured at 260 nm.

Example 8

Detection of 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.

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. To

The 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.

The result is summarized in Table 1. All tumor tissues resulted in the

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.

A positive detection of telomerase activity was also successful in the urine of patients with bladder tumors or in the sputum of patients with lung tumors. In addition to biopsy material, various body fluids such as urine, sputum and blood are also suitable as starting materials for testing for increased telomerase activity.

Table 1

Carcinomas and control tissues:

Controls:

Cell lines (positive controls)

Heia mRNA and Hek 293 mRNA and Total RNA Pos.

Normal tissue: (negative controls)

Lung Total RNA / mRNA Neg.

Him Total RNA Neg.

Kidney Total RNA Neg.

Gut Total RNA Neg.

Blood Total RNA / mRNA Neg.

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

HTC. PD 1> 4014 Primer: TeloTmotiv SeQIDNo 1 5 'AgCgTgCgggACTgCgCT3' Forwardprimer (1592)

Telo Tmotiv SEQIDNo 2 5'ACCCTCTTCAAgTgCTgT3 'reverse primer

(1842) Probe: Telo Tmotiv SeqlDNo 3 5 'TCCgTgACATAAAAgAAAgACCTgAgCAgCTCgA3' Probe

(1749-1712) reverse sequence

Telomerase 5 'area sequences

SAC85 .MPD 1> 8377 Printer:

For5050 SEQIDNo 4 5 * TcgCggCgCgAgTTTCAggCA3 '

Rev5180 SEQIDNo 5 5 'TagTggCTgCgCAgCAgggA3'

Probe:

Probe5100 SEQIDNo 6 5 'AagCCCTggCACCggTCACCCCCgCgATgCCgCgCg3 ^■

Telomerase 3 'area

Primer:

Tforl Intronl4 SEQIDNo 7 5 'TgCCTgCTggTgTTAgTgTgT 3' Pos 1490

Tfor2 Intronl4 SEQIDNo 9 5 'AAACCCAggCCAAgggCTTA3' Item 1790

Trevl Exon 16 SEQIDNo 8 5 'AgggTCTCCACAACACAgA3' Item 2041

Trev2 Exon 16 SEQIDNo 10 5 'TTCTCAgggTCTCCACAACA3'

Pos 2046

Sond:

T3 'Probe Pos SEQIDNo 11 5' TCTCAggAgCAgAggCCgCgTATCACCACgACAgA3 '

1870

Claims

Patent claims

1. Starter oligonucleotides containing 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 ID No 10th

2. Set of a starter oligonucleotide containing the nucleotide sequence according to SEQ ID No 1 and a starter oligonucleotide containing the nucleotide sequence according to SEQ ID No 2.

3. Set of a starter oligonucleotide containing the nucleotide sequence according to SEQ ID No 4 and a starter oligonucleotide containing the nucleotide sequence according to SEQ ID No 5.

4. Set of a starter oligonucleotide containing the nucleotide sequence according to

SEQ ID No 7 and a starter oligonucleotide containing the nucleotide sequence according to SEQ ID No 8.

5. Set of a starter oligonucleotide containing the nucleotide sequence according to SEQ ID No 9 and a starter oligonucleotide containing SEQ ID No 10.

6. Oligonucleotide probes, optionally marked, containing a nucleotide sequence selected from the group consisting of SEQ ID No 3, SEQ ID No 6 and SEQ ID No 11.

7. A method for detecting increased telomerase activity, in which

a) amplified in a sample of telomerase (hTC) mRNA using one or more starter oligonucleotides according to spoke 1 and

b) evaluates the amplification results.

8. The method according to spoke 7, in which a suitable oligonucleotide probe according to spoke 6 is used for the specific hybridization and detection of the telomerase (hTC) amplicon.

9. Test kit for detecting increased telomerase activity, containing one or more of the starter oligonucleotides according to claim 1.

10. Test kit according to spoke 9, which further contains one or more of the oligonucleotide probes according to spoke 6.