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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/118—Prognosis of disease development

Definitions

• This invention relates to the field of cancer diagnosis, more specifically to the monitoring of disease development during and after treatment.
• Cytostatic or cytotoxic treatment induces remission in the majority of patients with lymphoid malignancies. Nevertheless many of these patients relapse. Apparently the current cytostatic or cytotoxic treatment protocols are not capable of killing all malignant cells in these relapsing patients, although they reached so-called complete remission according to cytomorpho ogical criteria. Since the detection limit of cytomorphoiogical techniques is not lower than 1-5% malignant cells, it is obvious that such techniques can only provi ⁇ e superficial information about the effectiveness of treatment.
• MRD multiple malignart cell between 10 3 to 10 5 normal cells
• ALL acute lymphoblastic leukemia
• MRD information can oe applied to the clinical decision process, e.g. for stratification of treatment protocols.
• Bone marrow (BM) samples were collected at up to 9 time points during and after treatment: at the end of induction treatment, before consolidation treatment, before re-induction treatment, before maintenance treatment, three samples during maintenance treatment, at the end of treatment, and one year after cessation of treatment.
• Classical polymerase chain reaction (PCR) analysis of patient-specific immunoglobulin and T cell receptor gene rearrangements and TALI deletions were used as targets for semi- quantitative estimation of MRD levels: >10 ⁇ 2 , 10 ⁇ 3 , and ⁇ 10 "4 together with radioactive patient-specific junctional region probes.
• MRD negativity at the various follow-up time points was associated with low relapse rates (3- 16%), but five to fourteen-fold higher relapse rates (41- 86%) were found in MRD positive patients.
• the distinct MRD levels appeared to have independent prognostic value (p (trend) ⁇ 0.001) at all time points. Especially at the first two time points three-fold higher relapse rates were found in patients with high tumour loads (>10 ⁇ 2 ) as compared to patients with low tumour loads ( ⁇ 10 ⁇ 4 ) . At later time points (including the end of treatment) also low tumour loads were associated with a high relapse rate. Positivity in CCR patients after treatment was rarely ( ⁇ 1%) observed, even when multiple sensitive PCR analyses were performed.
• MRD techniques need to have at least a sensitivity of 10 ⁇ 4 to allow for earlier identification of patients belonging to high and especially to low risk groups, allowing a clinical decision process that would allow assigning the proper treatment to the proper patient. This would allow to reduce false-negative results and avoid being too late with therapy when a patient with high risk has not yet been detected as such, and would allow reducing false positive results and avoid cumbersome therapy in patients with low risk. Furthermore, more sensitive MRD techniques would allow sampling patients via routine blood sampling, instead of by the painfull or cumbersome bone marrow aspiration currently employed.
• the group of MRD-based high risk patients might benefit from intensive treatment protocols, including stem cell transplantation. Therefore, follow-up samples need to be collected during and after treatment for obtaining insight into the kinetics of tumour reduction and for determining the risk of relapse per patient .
• junctional regions which can be regarded as "fingerprint-like" sequences due to deletion and random insertion of nucleotides during the rearrangement process.
• PCR analysis of junctional regions generally reaches sensitivities of 10 ⁇ 4 .
• PCR analysis of a specific chromosome aberration can use primers at opposite sites of the fusion regions of the breakpoints e.g. TALI deletion, t(14;18), t(ll;14), t(l;14), and t(10;14).
• TALI deletion t(14;18), t(ll;14), t(l;14), and t(10;14.
• a random deletion and insertion of nucleotides may have occurred similar to the junctional regions of Ig and TCR gene rearrangements.
• MRD detection in lymphoid malignancies has prognostic value.
• the prognostic value of MRD detection has especially been studied in patients with non-Hodgkin lymphoma (e.g. before and after bone marrow transplantation) and in patients with acute lymphoblastic leukemia (ALL) . Absence of MRD in ALL patients after induction therapy is suggested to predict good outcome. However approximately half of the ALL patients are still positive at that time. Therefore the level of MRD positivity was evaluated and found to have predictive value. If multiple bone marrow samples are repeatedly analysed during follow-up, steady decrease of MRD levels to negative PCR results is associated with favorable prognosis, whereas persistence of MRD generally leads to clinical relapse.
• MRD data of a large prognostic study of ALL patients show that high tumor loads (>10 3 ) at two successive time points in the early phase of therapy result in a RFS (relapse free survival) of only 25%.
• RFS relapse free survival
• low levels of MRD ( ⁇ 10 ⁇ 4 ) or MRD negativity on both these time points result in RFS of -95%.
• the present invention provides a method for the detection of minimal residual disease by determining the presence of malignant cells in a sample comprising amplification of nucleic acid molecules corresponding to common rearranged gene segments using for example in a PCR a forward and/or reverse primer reactive with said gene segments and further comprising the identification of malignancy-specific nucleic acid molecules found among those rearranged gene segments by hybridisation with a fluorogenic probe specifically and selectively reactive with said malignancy-specific nucleic acid molecules (note that malignancy-specific is in most cases also patient-specific, since a malignancy in general occurs in a patient-specific manner) .
• the current MRD techniques including immunological marker analysis and current PCR techniques have several disadvantages which make a prognostic assessment inaccurate.
• the invention provides a method for the detection of minimal residual disease by determining the presence of malignant cells in a sample by detecting amplified nucleic acid molecules via real-time quantitative nucleic acid amplification (e.g. using the ABI PRISM Sequence Detection System and the Light Cycler system) , which avoid using radio-active isotopes and contamination and have a short processing time, and allow detection of specifically amplified nucleic acid sequences.
• real-time quantitative nucleic acid amplification e.g. using the ABI PRISM Sequence Detection System and the Light Cycler system
• Samples in which a specific nucleic acid molecule needs to be detected are subjected to amplification (for example by real-time quantitative (RQ-) PCR) using a primer pair specific for said molecule. Detection of the thus amplified molecule occurs fluorogenically in real time (i.e. during each amplification cycle) via a fluorogenic probe consisting of one of more oligonucleotides. Only detection in real-time allows reliable and reproducible quantification of MRD. RQ-PCR makes use of data generated in the early productive PCR cycles where the fidelity of the PCR is still high. It enables detection with a high throughput of samples allowing automated testing. Samples are not restricted to bone marrow samples but can also be blood samples or other cell samples. In a preferred embodiment of the invention, a patient- or malignancy-specific fluorogenic probe according to the invention is used.
• TaqMan fluorogenic probe consists of an oligonucleotide to which a reporter dye and a quencher dye are attached. During amplification, the probe anneals to the template molecule somewhere between the location of the forward and reverse primer sites. However, during the amplification process the annealed probe is cleaved by the 5' nuclease activity of the polymerase. This separates the reporter dye from the quencher dye, generating an increase in the reporter dye's fluorescence, which finally allows real time detection of the amplified molecule, with an increase of the fluorescent signal per PCR cycle.
• Another example is based on the proximity of two oligonucleotides, together forming a fluorogenic probe for fluorescence emission.
• the first oligonucleotide is labelled with a donor fluorochrome that is excited by an external light source and emits light that is absorbed by an acceptor fluorochrome present on a second oligonucleotide.
• a fluorogenic probe according to the invention which comprises an oligonucleotide specifically and selectively reacting with a junctional or fusion region which is only found with a specific patient and which is characteristic for his or her malignancy.
• the so-called fluorescence resonance energy transfer (FRET) probe consisting of both nucleotides hybridises to the nucleic acid between the two primers to detect the amount of target, resulting in emission of light by the acceptor fluorochrome.
• FRET fluorescence resonance energy transfer
• Samples that contain the wanted nucleic acid molecules that are reactive with said forward and reverse primer are thus identified by the presence of amplified product which is detected in real time by using said fluorogenic probe.
• Another embodiment of the invention is a method wherein the fluorescence of said probe is detected in real-time, during execution of the amplification, allowing the sensitive and quantitative detection of minimal residual disease to allow for earlier identification of patients belonging to high or low risk groups, allowing a clinical decision process that allows assigning the proper treatment to the proper patient.
• the invention also provides a fluorogenic probe which comprises an oligonucleotide specifically and selectively reacting with a junctional or fusion region which is only found with a specific patient and which is characteristic for his or her malignancy.
• a fluorogenic probe which comprises an oligonucleotide specifically and selectively reacting with a junctional or fusion region which is only found with a specific patient and which is characteristic for his or her malignancy.
• a fluorogenic probe is linked to a reporter dye and to a quencher dye, however in another embodiment, said probe comprises an oligonucleotide- linked donor fluorochrome and acceptor fluorochrome.
• Another embodiment of the invention is a method wherein said fluorogenic probe is reactive with a junctional region or fusion region of a malignancy-specific rearranged gene segments of Ig or TCR gene rearrangements or chromosome aberrations.
• a probe provided by the invention and/or a method provided by the invention is specifically used to detect and quantify the level of MRD, for example by using the ABI PRISM sequence detection system or the Light Cycler.
• the invention also provides said probes in the context of a diagnostic assay, comprising necessary means and methods to use a method provided by the invention.
• Said diagnostic assays or kit may also comprise primers, enzymes, buffers or other components that are necessary for the amplification of the rearranged gene segments.
• MRD analysis can predict outcome by determining the reduction of the leukemic burden during the first months of therapy.
• Methods that allow sensitive MRD detection are (i) flow cytometric detection of leukemia-specific immunophenotypes, (ii) polymerase chain reaction (PCR) amplification of leukemia specific chromosomal aberrations, and (iii) PCR amplification of clonogeneic rearrangements of immunoglobulin (Ig) and T- cell receptor (TCR) genes.
• the last method has the broadest applicability in ALL. Using PCR techniques it is possible to detect one leukemic cell in a background of approximately 10 normal cells. This is about 100 to 10,000 times more sensitive than obtained with morphology.
• Immature B and T lymphocytes rearrange the V, D, and J gene segments of their Ig and TCR genes in order to achieve antigen diversity.
• a molecular fingerprint is provided by the deletion and insertion of random nucleotides between the joined gene segments, the so- called junctional regions.
• junctional regions of Ig/TCR gene rearrangements can be regarded as leukemia specific DNA fingerprints.
• oligoclonality of Ig/TCR gene rearrangements at diagnosis may occur, since these rearrangements are not linked to the oncogenic process.
• a patient specific PCR primer or probe is usually designed to the sequence of the junctional region in order to detect the leukemia within the background of normal cells that may have similar gene rearrangements but different junctional regions.
• the usage of a patient-specific junctional region probe has shown to be highly effective in the detection of MRD.
• the PCR product is blotted after gel-electrophoresis or directly spotted on a nitrocellulose membrane, the so-called dot-blot method.
• the PCR product is not fixed but hybridized as free DNA in liquid hybridization.
• the probe and restriction enzyme combinations used for Southern blot analysis comprised: for the IGH locus the IGHJH6 probe in Bglll and BamHI/Jfindlll digests; for the TCRD locus the TCRDJ1 probe in BcoRI and Bglll digests; for the TCRG locus the J ⁇ l .3 and J ⁇ 2.1 probes in an BcoRI digest and the JI .2 probe in a Bglll digest; for the IGK locus the
• MNC Mononuclear cells
• PB peripheral blood
• BM samples peripheral blood (PB) or BM samples at diagnosis by ficoll density centrifugation. MNC samples were frozen and stored in liquid nitrogen. Good quality medium molecular weight DNA was isolated from MNC samples using the QIAamp kit (Qiagen Inc, Chatsworth, CA) (Verhagen et al., manuscript in preparation).
• Sequences of the junctional regions were obtained by direct sequence analysis of the Ig/TCR gene rearrangements with the dye terminator ready reaction cycle sequencing kit on an ABI PRISM 377 automated sequencer of PE Biosystems.
• the template DNA used in the sequence reaction was either the PCR product or a homo- tor hetero-) duplex band excised and eluted from a polyacrylamide gel in case of a bi-allelic gene rearrangement.
• patient-specific oligonucleotides were developed complementary to the sequence of the junctional region (Table 1) . Oligonucleotides that were likely to form of secondary structures were avoided.
• Fluorochrome labeled TaqMan probes were designed with the Primer Express software (PE Biosystems) .
• the TaqMan probe did not start with a G and contained more C s than G' s according to the guidelines of PE Biosystems.
• the melting temperature (Tm) was around 68 °C, 8-9°C above the Tm of the matching primers, to ensure proper hybridization to the target sequence.
• FAM was chosen as reporter dye at the 5' end of the TaqMan probe and TAMRA as the quencher dye at the 3' end.
• the forward primer 5' -GTACTTAAGATACTTGCACCATCAGAGA-3' and the reverse primer 5' -GAAGCTGCTTGCTGTGTTTGTC-3' were chosen and give a PCR product of 190 base pairs.
• IGH-1 primers forward, 5' -CACGGCTGTGTATTACTGTGCAA-3' and reverse, 5'- GGTCGAACCAGTACCCAATAGC-3' .
• IGH-2 primers forward, 5'- GAGGACACGGCTGTGTATTACTGT-3' and reverse, 5'- ACCTGAAGAGACGGTGACCAT-3' .
• IGH-3 primers forward, 5'- GAGGACACGGCTGTGTATTACTGT-3' and reverse, 5'-
• IGK primers forward for IGFC-1, 5'-AGCAGGGTGGAGGCTGA-3' , for IGK-2 5'-
• TCRG primers ( TCRG-1 and -2): forward, 5' -GCATGAGGAGGAGCTGGA-3' and reverse, 5'- GGAAATGTTGTATTCTTCCGATACTTAC-3' .
• ICRD-1 primers forward, 5' -GTACTTAAGATACTTGCACCATCAGAGA-3' and reverse, 5'-GAAGCTGCTTGCTGTGTTTGTC-3' .
• TCRD-2 primers forward, 5'-GCAAAGAACCTGGCTGTACTTAAG-3' and reverse, 5'- GTTTTTGTACAGGTCTCTGTAGGTTTTGTA-3' .
• the TaqMan TM PCR core reagent kit was used (PE Biosystems) .
• Reaction mixtures of 50 ⁇ l contained the RQ-PCR buffer with the ROX dye as the passive reference, 5 mM MgC12, dNTP's: 0.3 mM dATP, 0.3 mM dCTP, 0.3 mM dGTP, and 0.6 mM dUTP, 50-900 mM primers, 1.25 U AmpliTaq GoldTM (PE Biosystems), 1 U uracil-N- glucosidase (UNG) and 50-1000 ng of DNA.
• PE Biosystems Reaction mixtures of 50 ⁇ l contained the RQ-PCR buffer with the ROX dye as the passive reference, 5 mM MgC12, dNTP's: 0.3 mM dATP, 0.3 mM dCTP, 0.3 mM dGTP, and 0.6 mM dUTP, 50-900 mM
• the two-step amplification protocol consisted of a 2 minutes incubation step at 50°C (digestion of PCR product contaminants by UNG) , 10 minutes at 95°C (inactivation of UNG, denaturation of target DNA, and activation of AmpliTaq Gold TM) , followed by target amplification via
• PRISM 7700 Sequence Detection System containing a 96 well thermal cycler (PE Biosystems).
• the TaqMan probe first hybridizes to the DNA target, followed by primer annealing. With the TaqMan probe still intact, the emission of the reporter dye is quenched, but during the extension phase of the reaction the TaqMan probe is cleaved by the exonuclease activity of the Taq polymerase. Subsequently, a fluorescent reporter signal is generated per cycle, which is proportional to PCR product accumulation.
• the fluorescence intensity is normalized using the passive reference ROX present in the buffer solution. Normalization corrects for fluorescence fluctuations which are PCR independent.
• a real time amplification plot is generated using the normalized reporter signal (Rn) .
• the PCR product yield or ⁇ Rn is defined as the Rn minus the baseline signal established in the first few cycles of the PCR and is at least ten times the standard deviation of the noise.
• the cycle threshold (CT) is the PCR cycle at which a statistically significant increase in ⁇ Rn is first detected.
• DNA from the sample at diagnosis was diluted in 10-fold steps into DNA from normal mononuclear cells (MNC) , down to 10 " ⁇ .
• MNC mononuclear cells
• the normal MNC DNA consisted of equivalent mixtures from ten different healthy donors.
• the dilution series was subjected to (RQ)PCR analysis together with appropriate positive and negative controls.
• the furthest dilution of diagnosis DNA that gave a radioactive or fluorescent signal, in the absence of a signal from the polyclonal control (MNC DNA) was defined as the sensitivity threshold of the PCR-target .
• the sensitivity threshold based on the theoretical calculations, can be 10 " (-8 copies of the target gene) or 10 (-0.8 copies of the target gene).
• the primers used for the PCR analysis of the Ig/TCR gene rearrangements were described previously.
• 1 ⁇ g DNA of diluted diagnosis material was used and 30 pmol of each primer in reaction mixtures of 100 ⁇ l containing 1 unit AmpliTaq Gold TM was used (PE
• the cycling protocol consisted of 3 minutes of initial denaturation at 92°C, followed by 40 cycles of 45 seconds at 92°C, 90 seconds at 60°C, 2 minutes at 72°C, and a final extension phase of 10 minutes at 72°C.
• Rearrangements of the IGH gene locus were amplified in 50 ⁇ l reactions containing, 1 ⁇ g DNA, 30 pmol of each primer, 2 units Taq polymerase, 2 mM MgCl2, and 200 ⁇ M dNTP.
• the cycling protocol consisted of 7 minutes of initial denaturation at 95°C, followed by 30 cycles of 30 seconds at 95°C and 45 seconds at 55°C, and a final extension phase of 7 minutes at 72°C.
• PCR products were examined after gel electrophoresis in 1% agarose and or 6-10% polyacrylamide gels and ethidium bromide staining.
• PCR product Five ⁇ l of PCR product was hybridized with approximately 1 ng of the radioactively labeled probe in 2x SSC buffer for 15 minutes at 60°C after denaturation for 10 minutes at 95°C. Subsequently, the mixtures were size separated by electrophoresis through a 10% polyacrylamide gel. Radioactive signals were evaluated by autoradiography after drying of the gels.
• the threshold cycle is that cycle where the fluorescence emitted during the amplification of the target molecule rises above a certain threshold.
• Ig/TCR gene targets (3 IGH, 2 TCRD, 2 TCRG, and 2 IGK) of three precursor-B-ALL were examined for their sensitivity. For all gene rearrangements it was possible to develop primer/probe pairs that resulted in successful amplification and real time detection upon RQ-PCR analysis. For 4 out of 9 PCR targets it was necessary to design the TaqMan probe complementary to the reverse strand of the junctional region, due to the high extent of G' s (Table 2). The initial primer pair to amplify TCRG-1 and -2 PCR targets resulted upon RQ-PCR analysis in low sensitivities (10 ⁇ 2 ), which was unexpected because both gene rearrangements used the rarely used V ⁇ 7 gene segment.
• the sensitivities of RQ-PCR analysis varied between 10 "2 and 10 "4 (Table 3) .
• the sensitivities obtained by RQ- PCR analysis were most similar to that of the dot-blot method (Table 3, Figure 3) .
• RQ-PCR analysis using a Vklll-Kde gene rearrangement was performed in triplicate, and diagnosis dilutions and follow-up samples were analyzed in parallel.
• diagnosis and follow-up samples were checked for the amount and integrity of DNA by performing a albumin RQ-PCR. Quantities were determined using a standard curve of MNC DNA diluted in milli-Q. By dividing the diagnosis quantity by the follow-up quantity a ratio is established that can be used to correct the MRD level generated by the leukemia specific RQ-PCR.
• the aim of this study was to test the value of RQ- PCR analysis using the TaqManTM technology for sensitive and quantitative detection of MRD in follow-up samples using rearranged Ig and TCR genes as PCR targets.
• the majority of PCR targets tested (7 out of 10) were derived from patient 5199.
• PCR targets in patient 5199 might be due to oligoclonality at diagnosis. This was not evident from Southern blot analysis, except for the three rearrangements of albeit equal intensity in the TCRG gene locus. This may be explained by an extra chromosome 7 or by two clonal populations of similar size.
• the sensitivities obtained with the RQ-PCR technique were without further optimization similar to those obtained with the dot-blot method.
• the liquid hybridization with radioactively labeled probes appeared to be most sensitive. In principle, it should be possible to reach even higher sensitivities with RQ-PCR since hybridization with the TaqMan probe is also a liquid hybridization, unless the total detection system based on fluorescence, is less sensitive as compared to radiography.
• one point mutation appeared to be sufficient for allelic discrimination by the TaqMan probe.
• the Tm of the TaqMan probe was in this case 5 to 6°C above that of the corresponding primers.
• This adaptation may also be necessary to increase the specificity for RQ-PCR analysis of the TCRD-2 in this study, which lacked randomly inserted nucleotides. This would mean that for this target a new TaqMan probe should be developed.
• An alternative approach to RQ-PCR analysis of Ig/TCR gene rearrangements might be to use a TaqMan probe positioned at germline sequences (V, D, or J gene segments) in combination with one or two patient specific junctional region primers.
• RQ-PCR may also be helpful in the identification of suitable PCR targets at diagnosis.
• RQ-PCR can replace Southern blot analysis in the future, which is still considered as the golden standard for MRD target identification but is laborious, time-consuming and requires large amounts of good quality DNA.
• RQ-PCR is applicable for MRD analysis via detection of clone- specific Ig/TCR gene rearrangements.
• RQ-PCR offers many advantages over currently used techniques.
• the dot-blot and liquid hybridization are dependent of using radioactive isotopes and require individual optimization of the hybridization or extra washing steps.
• RQ-PCR analysis is simple and fast; data can be acquired as soon as the PCR is completed without any post-PCR handling, i.e. within 3 hours, instead of 5 days generally required for conventional methods.
• V ⁇ , V ⁇ , and V ⁇ expression excess of l O- ' to 10 3 all TCR f T-cell malignancies Occurrence of normal T-cells will influence the detection TCR-V gene expression (double or limit triple IF staining in T-cell populations Oligoclonal T-cell subsets might occur in the elderly or specific T-cell subsets)
• junctional regions have to be sequenced in order to design junctional region-specific probes for each individual patient
• Oligoclonality and clonal evolution at Ig or TcR gene level may cause false-negative results
• Chromosome aberrations with well- 10 to 10 6 25% to 40% of B NHL Fusion region oligonucleotide probes are useful for defined breakpoints at DNA level identification of PCR products from different patients
• Copelan EA McGuire EA. The biology and treatment of acute lymphoblastic leukemia in adults. Blood 1995;85:1151-1168.
• Vervoordeldonk SF Merle PA, Behrendt H, Steenbergen EJ, Van Leeuwen EF, Van den Berg H, Von dem Borne AE, Van der Schoot CE, Slaper-Cortenbach IC. Triple im unofluorescence staining for prediction of relapse in childhood precursor B acute lymphoblastic leukaemia. Br J Haema tol 1996;92:922-928.
• Van Dongen JJM Wolvers-Tettero ILM. Analysis of immunoglobulin and T cell receptor genes. Part I: Basic and technical aspects. Clin Chim Acta 1991;198:1-91.
• Kitchingman GR Immunoglobulin heavy chain gene VH-D junctional diversity at diagnosis in patients with acute lymphoblastic leukemia. Blood 1993;81:775-782. Ghali DW, Panzer S, Fischer S, Argyriou-Tirita A, Haas OA, Kovar H, Gadner H, Panzer-Grumayer ER. Heterogeneity of the T-cell receptor delta gene indicating subclone formation in acute precursor B-cell leukemias. Blood 1995;85:2795-2801.
• FIG. 2 An RQ-PCR sensitivity experiment of a precursor-B- ALL patient (2145) was performed using an IGH gene rearrangement (VH3-JH5b; OGH-1) .
• A The real time amplification plots of the diagnosis dilutions for one series of experiments.
• B The standard curve shows the linear correlation between the cycle threshold (CT) and the initial amount of DNA (tumor load) of all four experiments. With this IGH gene rearrangement a sensitivity of 10 -4 was reached in
• FIG. 3 The three MRD detection methods for an IGK gene rearrangement of precursor-B-ALL 5199 (JGK-2) .
• JGK-2 precursor-B-ALL 5199
• A Schematic diagram of the V ⁇ II-Kde PCR target with a patient specific junctional region of a total of 7 nucleotides deleted and 9 nucleotides randomly inserted. Given are the sequences and relative positions of the primers used for RQ-PCR and conventional MRD methods, as well as the patient-specific junctional region probes.
• B Result of the dot-blot hybridization with the radioactively labeled junctional region probe after PCR amplification of the V ⁇ II-Kde gene rearrangements in a diagnosis dilution series. With this technique a sensitivity of 10 -3 was obtained.
• D RQ-PCR analysis of the V ⁇ II-Kde PCR target with a different primer set and a fluorogeneic TaqMan probe. The experiment was performed in triplicate on the serial diagnosis dilution. Real-time information of PCR product accumulation is given at the left. The standard curve at the right illustrates the linear correlation between the cycle threshold and the initial amount of DNA. With RQ-PCR analysis a sensitivity of 10 ⁇ 4 was reached in 1 out of 3 experiments .

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