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WO2000075369A1 - Procede pour detecter des produits d'amplification d'acides nucleiques au moyen d'amorces comportant des sequences rapporteuses integrees (pirs) - Google Patents

Procede pour detecter des produits d'amplification d'acides nucleiques au moyen d'amorces comportant des sequences rapporteuses integrees (pirs) Download PDF

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Publication number
WO2000075369A1
WO2000075369A1 PCT/EP2000/005023 EP0005023W WO0075369A1 WO 2000075369 A1 WO2000075369 A1 WO 2000075369A1 EP 0005023 W EP0005023 W EP 0005023W WO 0075369 A1 WO0075369 A1 WO 0075369A1
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Prior art keywords
primers
sequence
pirs
primer
nucleic acid
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PCT/EP2000/005023
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German (de)
English (en)
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Reinald Repp
Wolfgang Rascher
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Reinald Repp
Wolfgang Rascher
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Application filed by Reinald Repp, Wolfgang Rascher filed Critical Reinald Repp
Priority to AU52191/00A priority Critical patent/AU5219100A/en
Publication of WO2000075369A1 publication Critical patent/WO2000075369A1/fr

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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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6823—Release of bound markers

Definitions

  • the present invention relates to methods for the detection of products of a nucleic acid amplification reaction, whereby novel primers with a primer-integrated reporter sequence (PIRS) and probes specific therefor enable highly sensitive quantitative determination of the amplificates.
  • PIRS primer-integrated reporter sequence
  • the invention further relates to reagent kits and nucleic acids which are suitable as PIRS primers.
  • PCR polymerase chain reaction
  • the TaqMan system uses the 5'-nuclease activity of Taq DNA polymerase, using an oligonucleotide which is labeled with a fluorescent molecule at the 5 'end and with a quencher molecule at the 3' end, the Taq- DNA polymerase, through its 5'-exonuclease activity, releases the fluorescence molecule, the signal of which is detected in the ABI-PRISM - Seq uen zd ete kti on syste m.
  • Fluorescent dyes are available in the 6-FAM, TET, JOE, VIC TM and HEX molecules, which can be combined with the quenching molecule TAMRA.
  • so-called “dark quenchers” can be used, which emit in the infrared range, which means that TAMRA can also be used as a reporter dye.
  • the synthesis of TaqMan TM probes is offered by various manufacturers.
  • the second system concerns the Light-Cycler TM system from Boehringer Mannheim / Hoffmann La Röche.
  • the fluorescent dyes are distributed over two different oligonucleotides.
  • the fluorescent dye fluorescein is located at the 3 'end of the first oligonucleotide, while the second oligonucleotide attached downstream of the first oligonucleotide has the fluorescent dye LC Red 640 at its 5' end.
  • the fluorescein is excited by an LED as a light source and emits light that excites the second fluorescence molecule (LC Red 640 or LC Red 705) via fluorescence resonance energy transfer.
  • the light emitted by the second dye is detected via an appropriate filter.
  • the second dye can only be excited if the two dye molecules are in spatial proximity (distance of 1 to 5 nucleotides) due to the addition of the two oligonucleotides to their complementary strand.
  • the principle of the system is based on the secondary excitation of a second fluorescent dye, which is only made possible by the generation of the PCR product.
  • light emitted by the fluorescent dye molecule is absorbed by the quencher molecule, which is why the quencher effect and the emitted light can only be canceled after the hydrolysis of the fluorescent dye molecule by the oligonucleotide by the 5'-3 'exonuclease activity of the Taq DNA polymerase .
  • a third technique for fluorescence-optical detection of the PCR product generation uses so-called “molecular beacons".
  • "Molecular Beacons” are made up of three domains and used as probes.
  • a central sequence section is complementary to a target sequence in the PCR product. 5'- and 3'-wards of it are two mutually complementary sequence sections. Outermost 5'- and 3 ' If the probe is not bound with its central section to the complementary target sequence, the two mutually complementary terminal sections form an intramolecular double strand, resulting in the reporter dye at one end and the quencher at the other end in close proximity, which means that the quencher neutralizes the light signal emitted by the reporter dye.
  • Molecular beacons can be used in various devices that are suitable for the detection of the resulting fluorescence signals, such as. B. the Sequence Detection Systems TM 7700, 7200 and 5700 from PE Biosystems, the Rotor-Gene RG2000 TM Real-Time Cycler from Corbett Research or the FMBIO II TM Fluorescence Imaging System from Hitachi.
  • the systems mentioned above can basically be used for multiplex applications, i.e. several target sequences can be amplified simultaneously.
  • a disadvantage of this type of application in these known methods is, however, that for each target sequence a separate probe or pair of probes is required for detection, which must specifically bind to an internal region of the target sequence resulting from the amplification. If many different target sequences are to be detected side by side, the reaction approach becomes relatively expensive due to the increasing number of different detection probes and the increasing number of probes can impair the RCR amplification.
  • Primer dimers are double-stranded nucleic acid molecules that arise when two primers hybridize directly with each other over a few nucleotides and then elongate along the complementary strand. These products can in turn hybridize with further primers, which leads to a loss of primer material with the formation of the undesired primer dimers.
  • the use of homo-tail sequences at the 5 'ends of the primers has been proposed (Brownie et al. (1 997), Nucleic Acids Research 25, 3235-3241).
  • primers thus contain a section specific to the sequence to be amplified at the 3 'end and a homo-tail section at the 5' end.
  • the homo-tail sequence is a sequence that is identical for the forward and reverse primers used.
  • primer dimers When primer dimers are formed, single strands with complementary 5 'and 3' ends are formed after the melting of the elongated double strands, which hybridize with one another and so-called "panhandles" - Form structures. These structures are no longer available for further hybridizations with primers and prevent the amplification of the primer dimers. Brownie et al.
  • tail-specific primers tags
  • the advantage of the tags is that they are shorter and reduce the formation of primer dimers by several powers of ten.
  • the system was not developed for fluorescence-coupled detection of the resulting PCR products. This would be possible, but again with the disadvantage that a specific probe would be required for each sequence to be amplified.
  • the specific reaction preceding the amplification via the homo-tail sequences is carried out at lower temperatures than the reaction with the tags. In principle, the specificity of the PCR reaction can be increased by choosing the highest possible primer binding temperature, which in turn requires relatively long primers that can still bind at such temperatures.
  • the upper temperature limit of around 74 ° C results from the optimum activity of the polymerase enzyme used. Since this temperature range with optimal specificity is reserved for the reaction with the tags, it follows inevitably that the amplification carried out at a lower temperature via the sequence-specific primers does not take place under optimal specificity conditions. There is a risk that the areas (tails, reporter sequences) which are still present in addition to the primer sections which bind to the target sequences cause unspecific binding.
  • the object on which the present invention is based is therefore to use suitable changes to the primer structures when using nucleic acid amplification techniques, in particular the TaqMan TM and Light Cycler TM systems, and other systems fluorescence-optical detection of PCR products to increase the number of target sequences to be detected in parallel.
  • this object is achieved by a method for the detection of products of a nucleic acid amplification reaction, comprising the steps: i) providing nucleic acids to be amplified, ii) performing an amplification reaction using at least two primers, iii) detecting the amplification products formed in ii) under
  • one of the primers used for the amplification reaction comprises the following elements in the 5'-3 'direction: a 5'-homo-tail sequence, a probe binding sequence which acts as a primer integrated reporter sequence (PIRS), and a sequence specific for the nucleic acid to be amplified
  • another of the primers used for the amplification reaction in the 5'-3 'direction comprises the following elements: a 5'-homo-tail sequence, and one for the one to be amplified
  • Nucleic acid specific sequence, and c) the probes used for detection can interact with the complementary strand of the probe binding sequence of one of the two amplification primers, and the probes used carry detectable labeling groups.
  • PIRS primers By using primers with primer-integrated reporter sequences (PIRS primers), only one probe oligonucleotide sequence per PIRS is necessary in multiplex PCR applications with fluorescence optical product detection. Labeling of each individual specific primer with fluorescent dye molecules is omitted, so that the invention This represents a great advantage over conventional detection methods of products of an amplification reaction, particularly in multiplex PCR.
  • the fluorescent dyes can be assigned not only individual target sequences but a whole group of target sequences, which are then simultaneously detected in multiplex reactions and discriminated from one another on the basis of the characteristic fluorescence can be.
  • the possibility of detecting multiple target sequences with a single probe construct saves costs, since the production of fluorescence-labeled probes is relatively more expensive.
  • the multiplex application which is becoming increasingly important for today's diagnostics, should not be affected.
  • the invention is based on a novel primer design which, in a first aspect of the invention, primers with primer-integrated
  • PIRS primer Provides reporter sequences ("PIRS primer").
  • the PIRS primers (with integrated probe binding sequence / reporter sequence) have the following general structure
  • the second primer with opposite polarity to be used in the PCR does not have to contain PIRS and thus has the following schematic structure:
  • an additional primer-integrated domain between these two domains, which is not the serves fluorescence-based product detection, but enables certain post-PCR product analyzes.
  • This must differ in its sequence from the PIRS domain used for fluorescence-optical PCR product detection and can e.g. B. serve as an attachment site for a sequencing primer with which the nucleic acid sequence of the PCR product is determined after the PCR reaction.
  • both PCR primers have an additionally attached sequence called "5'-homo-tail" at their 5 'end.
  • This "5'-homo-tail” sequence can be chosen freely, but it is preferably also identical in the case of a combination of several primer sets to form a multiplex PCR for all primers and should be somewhat higher in melting temperature than that of the "specific sequence" defining primer section.
  • the underlying principle corresponds to the "HANDS” -PCR technology (Homo-Tag-Assisted-Non-Dimer-System) (Brownie et al., See above; EP-0 731 1 77; EP-0 332 435). By using the "HANDS" -CRC principle, the undesired formation of primer dimers during the PCR reaction is reduced by several orders of magnitude.
  • the primer integrated sequence is a sequence that is specific for the probes used, i.e. that the amplification products can hybridize to the probes used on the basis of these sequences.
  • the specific sequence is specific for the nucleic acid section to be amplified, with which it hybridizes and initiates the elongation from this hybridized section.
  • primers are used in which, apart from the homo-tail section, at least one of the thymidine bases has been replaced by uracil.
  • the Amplification reaction mixture added an enzyme which is able to degrade nucleic acids with uracil bases, uracil-N-glycosylase is preferred.
  • the PCR approach is used after the first three to five cycles during which the sequence-specific 3 'ends of the multi-domain primers (specific sequence + homo-tail + PIRS) attach to their respective target sequences and PCR products with the universal 5 '- "Homo-Tail" are generated, a digestion with the enzyme uracil-N-glycosylase interposed. Removal of the uracil base residues from the DNA chain and subsequent hydrolysis at 95 ° C. destroys all primer domains that are no longer required for the further amplification mediated exclusively via the “homo-tails” and only for the generation of undesired products, such as Primer dimers contribute.
  • any primer dimers that may have already formed are largely destroyed, since the primer portions contained in them are broken down with the uracil residues.
  • their competition with the fluorescence-labeled reporter probes is eliminated, which contributes to a more efficient signal generation during the PCR.
  • the digestion result in primer fragments that are no longer than 8 to 10 nucleotides.
  • the above-described methods can be used to combine a plurality of primer sets which, owing to different “specific sequences” at the 3 ′ end of the first type of primer, allow the detection of several target sequences in a single reaction mixture.
  • PCR products are formed, a sequence is formed which is complementary to the probe binding site / reporter sequence and with which the probe oligonucleotides must hybridize.
  • Different groups of target sequences can be distinguished by using several probes with appropriate fluorescent dyes, for example using the TaqMan TM system. This is illustrated using the examples below.
  • PIRS primers according to the invention in combination with the measures described to avoid the undesired formation of primer dimers (HANDS-PCR) allows the fluorescence-optical detection systems described above, such as e.g. TaqMan TM and Light Cycler TM with a single probe or a pair of probes, which is labeled with one of the available fluorescent dyes, to detect a large number of different target sequences in one reaction batch.
  • all available fluorescent dyes can be used in parallel with the help of specific probes. It is thus possible to assign different groups of target sequences to a probe or an associated fluorescent color. The target sequences of the individual groups can be detected in parallel in a single PCR reaction and their group membership can be identified on the basis of the assigned color of the probe used, as a result of which a very high degree of automation of the PCR diagnosis is achieved.
  • Another object of the invention is a reagent kit which contains the PIRS primers according to the invention and for use in the diagnosis of 1) leukaemias, 2) meningitis or encephalitis and
  • the reagent kit for the detection of nucleic acid amplification products comprises: a) at least one nucleic acid which is used as a primer and which comprises the following elements in the 5'-3 'direction: a 5'-homo-tail sequence, a probe binding sequence which acts as a primer -integrated reporter sequence, and a sequence specific for the nucleic acid to be amplified, b) at least one nucleic acid which is used as a primer and which comprises the following elements in the 5'-3 'direction: a 5'-homo-tail Sequence, and a sequence specific for the nucleic acid to be amplified, c) at least one probe which can be detected on the basis of labeling groups and which is specific for the complementary strand of the nucleic acid according to a), d) deoxyribonucleotides, e) one for extending the nucleic acid Primer according to a) and b) suitable enzyme, and f) a suitable buffer and optionally further
  • the kit can comprise nucleic acids with the homo-tail sequence, or it can comprise primers in which at least one thymidine base is replaced by uracil, in which case the kit furthermore contains the enzyme uracil-N-glycosylase.
  • the enzyme suitable for the extension is preferably a thermostable DNA polymerase which has a 5'-3 'exonuclease activity.
  • the probe preferably carries the dye molecules 6-FAM, TET, JOE, HEX, VIC TM, fluorescein, LCRed 640 or LCRed 705 at its 5 'end and the quencher molecule TAMRA or DABCYL at its 3' end.
  • the probe is particularly preferably a “molecular beacon”.
  • the kit may also include three different probes that interact with the complementary strand of the probe binding sequence, with the first of the three probes carrying the fluorescein dye molecule at its 3 'end, and the LC Red 640 dye molecule at its 5' end and the third probe carries the dye molecule LC Red 705 at its 5 'end.
  • the kit preferably contains primers which hybridize with m-BCR-cDNA or / and M-BCR-cDNA or / and TEL / AML-cDNA or / and AML / ETO-cDNA.
  • the primers and PIRS primers particularly preferably hybridize with genomic herpesvirus DNA or / and with genomic DNA coding for the 1 6S rRNA from Haemophilus influenzae or / and Neisseria meningitidis or / and Streptococcus pneumoniae, even more preferably with interleukin-2 cDNA or / and interleukin-4 cDNA or / and interleukin-5 cDNA or / and interleukin-10 cDNA or / and interferon-cDNA.
  • a 50 ⁇ l reaction mixture produced using a reagent kit according to the invention contains: 2 ⁇ l of the sample to be examined
  • thermostable DNA polymerase 5 ⁇ "homo-tail" primer (500 nM final concentration) 0.15 vl thermostable DNA polymerase
  • the reagent kit according to the invention comprises at least one nucleic acid for use as a primer in nucleic acid amplification reactions, characterized in that it comprises the following elements in the 5'-3 'direction: a 5'-homo-tail sequence, a probe binding sequence which acts as a primer Integrated reporter sequence (PIRS) and whose complementary strand can interact with at least one probe with detectable marker groups, and a sequence specific for the nucleic acid to be amplified.
  • PIRS primer Integrated reporter sequence
  • Oligonucleotide primers of the type described above are a further subject of the invention.
  • Reagent kits the implementation of which is described in Examples 1, 3, 4, 5 and 6. Their sequences are shown in the sequence listing.
  • SEQ ID No. 1 ABL internal Hands NPY antisense
  • SEQ ID No. 2 TEL internal Hands ENT sense
  • SEQ ID No. 3 ETO internal hands ENT antisense
  • SEQ ID No. 4 HSV1 internal hands ENT sense
  • SEQ ID No. 6 Men intern Hands NPY Rec sense
  • SEQ ID No. 7 Hae intern Hands NPY Rec sense
  • SEQ ID No. 8 Pneu internal Hands NPY Rec sense
  • SEQ ID No. 9 IL-2 internal hands HNNP sense
  • SEQ ID No. 1 0 IF-g internal hands ENT sense
  • SEQ ID No. 1 1 IL-4 internal hands NPY Rec sense
  • SEQ ID No. 1 2 IL-5 internal hands NPY Rec sense
  • SEQ ID No. 1 3 IL-1 0 internal Hands NPY Rec sense.
  • SEQ ID No. 1 4 * ABLrvinBeideHanNPY +
  • SEQ ID No. 1 8 * TELfwinREHHANHNNO + -U
  • SEQ ID No. 1 9 * ETOrvinKasuHANHNNO + -U
  • FIGS. 1 and 2 show the result of carrying out example 1.
  • Fig. 2 is the result of a multiplex PCR using PIRS primers with the TaqMan TM method. The result is shown as it is obtained from the ABI-PRISM TM Sequence Detection System. On the X axis is the number of completed PCR cycles (0 - 35) is shown. The Y axis indicates the associated fluorescence intensities measured in the respective cycles. The black line parallel to the X axis indicates the limit below which the fluorescence signal is referred to as negative (threshold). It is determined mathematically by the device. In Figure 2 only the VIC signals are visible. The samples are labeled as follows:
  • Example 1 Using PIRS primers, a method was developed which, with a single PCR reaction, makes it possible to differentiate between several genetic markers in leukemia cells which are associated with a particularly good or bad prognosis of the disease. The results of such a test can be used directly to determine the therapy.
  • Two splicing variants of the BCR / ABL rearrangement (referred to as m-BCR and M-BCR), which corresponds cytogenetically to the translocation t (9; 22), were also included as markers with poor prognosis (hereinafter referred to as "baddies") known as the Philadelphia chromosome.
  • the corresponding TaqMan TM probe was labeled 5'-VIC / TAMRA-3 '.
  • chromosomal translocation t (1 2; 21) and t (8; 21) were included as a chromosomal translocation with a good prognosis in childhood leukemia.
  • the resulting chimeric mRNAs are referred to as "TEL / AML” or "AML / ETO" fusion genes.
  • the corresponding probe was labeled 5'-FAM / TAMRA-3 '.
  • RNA Since chimeric mRNA molecules are to be detected (these result from a fusion of two different gene fragments, which was caused by the chromosomal translocation), the RNA must be rewritten into cDNA (complementary DNA) according to conventional methods before the actual PCR.
  • cDNA complementary DNA
  • a normal PCR run was carried out before the actual PCR reaction was connected under fluorescence-optical product detection.
  • the decisive PCR reaction in the TaqMan TM procedure was carried out as the second run of a nested PCR.
  • the nested PCR (originally described by Chamberlain et al.) Is a known and widely used method for increasing the PCR sensitivity and specificity.
  • the target sequences are pre-amplified.
  • a microiter of the reaction product of the first run is transferred as starting material for the second run of the nested PCR.
  • the primers used in the second run bind within the pre-amplified DNA sections.
  • All primers used in the first run of the nested PCR correspond to their structure according to normal PCR primers without any additional sequences. They are all marked with an arrow ( ⁇ ) in front of the abbreviation.
  • 5'-homo-tail 5'-GCGTACTAGCGTACCACGTGTCGACT
  • VIC probe NPY-Rec * (for baddies): 5 '- ⁇ VIC) -TGTGGTAGCATTTGCAGTCTGCTGGC- (TAMRA)
  • ABL-internal Hands NPY antisense (SEQ ID No. 1): (This is the counterprimer for both primers mentioned above, both for M-BCR and for mBCR):
  • a 50 ⁇ l reaction mixture contains the following components:
  • Polymerase hot start) then 5 cycles at 95 ° C - 1 5 s ⁇ 60 ° C - 1 min ⁇ 72 ° C - 1 min then 35 cycles at 95 ° C - 1 5 s ⁇ 68 ° C - 1 min ⁇ 72 ° C - 1 min
  • FIGS. 1 and 2 The results of the application example are shown in FIGS. 1 and 2.
  • a multiplex PCR with PIRS primers can be used to detect several genetically heterogeneous diseases at the same time.
  • a probe color is reserved for each disease to be considered.
  • the individual PCR primers receive the specificity for a single point mutation due to different 3 'ends.
  • the corresponding groups of primers can each be assigned to a disease in question.
  • a fluorescent color could be reserved for the detection of various point mutations in the CFTR gene in order to detect cystic fibrosis.
  • a second PIRS could be installed in a PCR primer set in order to detect mutations in the PIZ gene which are present in the alpha-1 antitrypsin deficiency in the same PCR reaction.
  • Example 3 A multiplex PCR with PIRS primers can also be used to detect different groups of infectious agents in a single PCR approach. This can be particularly helpful for clinical questions if the individual groups require a different therapeutic approach.
  • both bacterial and viral diseases can be detected in a single multiplex PCR. Becomes a probe color for all bacterial - and a different probe color for all If the viral pathogen is reserved, the necessary therapy can be derived immediately from the PCR result: broad-spectrum antibiotics (e.g. cefotaxime) for bacterial pathogens or antivirals (e.g. acyclovir) for herpes virus infections. Only two primer sets are used in the first run of the nested PCR.
  • broad-spectrum antibiotics e.g. cefotaxime
  • antivirals e.g. acyclovir
  • the primer sequences are taken from the following work: Casas et al, Detection of both Herpes simplex and Varicella -Zoster Viruses in cerebrospinal fluid from patients with encephalitits, J. Med. Virol. Vol. 50: 82-92, 1 996).
  • the second binds to highly conserved bacterial DNA regions that code for sections of the 1 6S rRNA of bacteria (Eubacteriae). It can be used to detect and amplify the corresponding genome areas of a large number of bacteria (see Radström et al., J. Clin. Microbiology, Vol. 32: 2738-2744, 1 994).
  • the delimitation of the clinically relevant pathogenic bacteria takes place only in the second run of the nested PCR using specific primer sets with PIRS primers on a fluorescence-optical system for the detection of the PCR products.
  • the bacteria-specific primer sequences are the work of Radström et al. taken (see above); the herpes virus-specific primer sequences come from the work of Casas et al. (see above).
  • Another special feature is the execution of the bacteria-specific primer set as a "semi-nested PCR”.
  • the universal antisense primer for the detection of bacteria is also used in the second run as a counterprimer to the bacterial species-specific PIRS primers.
  • Eubac-exdo Eubac-Hands-antisense:
  • 5'-homo-tail 5'-GCGTACTAGCGTACCACGTGTCGACT
  • GA FAM probe HNNO * for the two types of herpes virus: 5 '- (FAM) -TCCTGCTGCGATGCAATGCTTTTC- (TAMRA)
  • VLC probe NPY-Rec * (for the three pathogenic bacteria mentioned above) 5 '- (VIC) -TGTGGTAGCATTTGCAGTCTGCTGGC- (TAMRA)
  • Herpes virus type 1 Herpes virus type 1:
  • HSV1-intem-Hands-HNNO-sense SEQ ID No.4: '-GCGTACTAGCGTACCACGTGTCGACT-GA TCCTGCTGCGATGCAATGCTTTTC-GTCCTCCGCGTCGGGTCGGGCC
  • Herpesvirus type 2 HSV2-intem-hands-HNNO-sense (SEQ ID No.5): '- G C G TA C TA G C G TA C CA C G TG TC GA C T- G A - TCCTGCTGCGATGCAATGCTTTTC-GTCCTCCGCGTGGGCCCGGAG
  • Neisseria meningitidis meningitidis (meningococcal):
  • Men-intern-Hands-NPY-Rec-sense (SEQ ID No.6): 5 '- GCG TA C TA GCG TA C CA CG TG TC GA C T- GA - TGTGGTAGCATTTGCAGTCTGCTGGC-TGTTGGGCAACCTGATTG
  • Streptococcus pneumoniae (pneumococci):
  • Pneu-intern-Hands-NPY-Rec-sense SEQ ID No.8: '- G C G TA C TA G C G TA C CA C G TG TC GA C T- G A TGTGGTAGCATTTGCAGTCTGCTGGC-GTACAACGAGTCGCAAGC
  • Eubac-Hands-antisense '- G C G TA C TA G C G TA C C A C G T G T C G A C T AAGGAGGTGATCCA (G / A) CCGCA (G / C) (G / C) TTC
  • PCR reaction conditions (In comparison to the PCR conditions described in the previous application example, there are no differences except for an annealing temperature reduced from 60 to 55 ° C in the first five cycles of multiplex PCR with PIRS primers).
  • a 50 ⁇ l reaction mixture contains the following components:
  • a multiplex PCR with PIRS primers can also use the ability to quantitatively determine the amount of PCR product that the fluorescence-optical detection systems described above possess.
  • the respective total amount of several groups of nucleic acid target sequences can thus be determined with a single PCR reaction. It is thus possible to assign different groups of cytokine mRNAs to a probe in order to then determine with a single PCR reaction whether, for example, a T H1 or T H2 cytokine response predominates in a specific sample.
  • a PIRS was used to detect the cytokines interleukin-2 and interferon gamma.
  • the corresponding probe carries the fluorescence signal FAM.
  • the intensity of the FAM signal is thus proportional to the sum of the two target sequences interleukin-2 and interferon-gamma as markers of a T H1 response.
  • the second PIRS generates the binding site for a VIC marked probe.
  • the PIRS-PCR primer sets serve to detect interleukin-4, interleukin-5 and interleukin-10 as representatives of a T H2 response.
  • a nested PCR is carried out to increase the sensitivity.
  • Interleukin-1 0
  • FAM probe HNNO * (for T H1 ): 5 '- (FAM) -TCCTGCTGCGATGCAATGCTTTTC- (TAMRA)
  • HNNO HNNO
  • NPY-Rec internal laboratory abbreviations for the identification of the different probe sequences.
  • IL-2-internal-hands-ENT-sense SEQ ID No.9: ⁇ '-GCGTACTAGCGTACCACGTGTCGACT-
  • IF-g-intern-Hands-HNNO-sense (SEQ ID No. 10): 5 '- G C G TA C TA G C G TA C CA C G TG TC GA C T- G A TCCTGCTGCGATGCAATGCTTTTC-CTGTCGCCAGCAGCTAAAACAGG
  • IF-g-internal-hands-antisense ⁇ '-GCGTACTA GCGTACCA CGTGTCGA C7-AAATTCAAATATTGCAGGCAGG
  • IL-4-internal-Hands-NPY-Rec-sense SEQ ID No.11: ⁇ '- G C G TA C TA G C G TA C CA C G TG TC GA C T- G A
  • IL-4-internal-hands-antisense 5 , -GCG74C74GCG74CC ⁇ CG7G7CG .C7-GAGTGTCCTTCTCATGGTGG Interleukin-5:
  • IL-5 internal hands antisense ⁇ '-GCGTACTA GCGTA CCA CGTGTCGA C7-TTGCAGGTAGTCTAGGAATTGG
  • Interleukin-10 IL-10-internal-Hands-NPY-Rec-sense (SEQ ID No. 13):
  • IL-10 internal hands antisense ⁇ '-GCGTA CTA GCGTA CCA CGTGTCGA C7-CTTTGTAGATGCCTTTCTCTTGG
  • a 50 ⁇ l reaction mixture contains the following components:
  • Example 5 serves the same goal as Example 1.
  • the same leukemia cell lines were used as starting material.
  • a modified primer / probe combination was chosen which allows amplification at higher temperatures.
  • 5'-Homo-Tail ⁇ '-gCg TACTAgCgTA CCA Cg Tg TCgACT
  • VlC probe NPYRECplus * (for badies): 5 '- (VIC) -CCCgTgTggTAgCATTTgCAgTCTgCTggC- (TAMRA)
  • MBCRfwinK562HAN '- g C g TACTA g C g TACCAC g T g TC g ACT - TGCAGAGTGGAGGGAACATCCGGGAG
  • a 50 ⁇ l reaction mixture contains the following components:
  • VlC probe NPY-Rec 400 nM final concentration
  • UNG substance to eliminate contamination
  • Example 6 serves the same goal as Example 1.
  • the same leukemia cell lines were used as the starting material.
  • the primer sequences from Example 5 were used, but with the decisive modification that Ts (deoxythymidine residues) were replaced by Us (deoxyuracil residues).
  • all primer oligonucleotides that are no longer required can be digested with the aid of the enzyme uracil-N-glycosylase before the amplification with the help of the homo-tail sequences and the simultaneous generation of fluorescence signals.
  • the competition between the primers with multiple domains, which also carry a PIRS domain, and the fluorescence-labeled probes is removed. Competition with the homo-tail primers is also abolished. Such a competition would reduce the PCR efficiency especially if a large number of target sequences are to be detected simultaneously and thus a large number of different primers with several domains have to be used in one reaction mixture.
  • Primer sequences for the first run of a nested PCR (first run is not listed in the PCR protocol): "Baddies":
  • 5'-homo-tail ⁇ '-gCgTACTAgCgTACCACgTgTCgACT ("Ts" as "Us” within primers with multiple domains)
  • FAM probe HNNOplus * (for thoughts): 5 '- (FAM) -CCCTCCTgCTgCgATgCAATgCTTTTCCgC- (TAMRA)
  • VIC VIC
  • TAMRA TAMRA
  • a 50 ⁇ l reaction mixture contains the following components:

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Abstract

L'invention concerne un procédé pour détecter des produits d'une réaction d'amplification d'acides nucléiques. De nouvelles amorces comportant une séquence rapporteuse intégrée (PIRS) et des sondes spécifiques permettent une détermination qualitative ultrasensible des produits d'amplification. L'invention concerne en outre des kits de réactifs et des acides nucléiques appropriés pour servir d'amorces PIRS.
PCT/EP2000/005023 1999-06-02 2000-05-31 Procede pour detecter des produits d'amplification d'acides nucleiques au moyen d'amorces comportant des sequences rapporteuses integrees (pirs) WO2000075369A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU52191/00A AU5219100A (en) 1999-06-02 2000-05-31 Method for the detection of nucleic acid amplification products, using primers containing primer-integrated reporter sequences (pirs)

Applications Claiming Priority (2)

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DE19925448.6 1999-06-02
DE19925448A DE19925448A1 (de) 1999-06-02 1999-06-02 Multiplex-PCR mit fluoreszenzoptischer Produktdetektion durch den Einsatz von Primern mit "primerintegrierten Reportersequenzen" (PIRS)

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WO2000075369A1 true WO2000075369A1 (fr) 2000-12-14

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PCT/EP2000/005023 WO2000075369A1 (fr) 1999-06-02 2000-05-31 Procede pour detecter des produits d'amplification d'acides nucleiques au moyen d'amorces comportant des sequences rapporteuses integrees (pirs)

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AU (1) AU5219100A (fr)
DE (1) DE19925448A1 (fr)
WO (1) WO2000075369A1 (fr)

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WO2003060159A2 (fr) * 2002-01-15 2003-07-24 Matforsk Techniques d'amplification d'acide nucleique
CN107385080A (zh) * 2017-08-31 2017-11-24 南京美宁康诚生物科技有限公司 一种儿童白血病相关融合基因多重pcr检测试剂盒及方法
CN110904187A (zh) * 2019-11-05 2020-03-24 翌圣生物科技（上海）有限公司 一种Taq酶活性测定方法

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003060159A2 (fr) * 2002-01-15 2003-07-24 Matforsk Techniques d'amplification d'acide nucleique
WO2003060159A3 (fr) * 2002-01-15 2004-01-22 Matforsk Techniques d'amplification d'acide nucleique
CN107385080A (zh) * 2017-08-31 2017-11-24 南京美宁康诚生物科技有限公司 一种儿童白血病相关融合基因多重pcr检测试剂盒及方法
CN110904187A (zh) * 2019-11-05 2020-03-24 翌圣生物科技（上海）有限公司 一种Taq酶活性测定方法

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AU5219100A (en) 2000-12-28

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