WO2000075369A1 - Method for the detection of nucleic acid amplification products, using primers containing primer-integrated reporter sequences (pirs) - Google Patents

Abstract

The invention relates to methods for detecting products of a nucleic acid amplification reaction, whereby novel primers containing a primer-integrated reporter sequence and specific probes therefor enable a highly sensitive, quantitative determination of the amplification products. The invention also relates to reagent kits and nucleic acids which are suitable as PIRS primers.

Description

Method for the detection of nucleic acid amplification products using primers with "primer-integrated reporter sequences" (PIRS)

description

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. The invention further relates to reagent kits and nucleic acids which are suitable as PIRS primers.

The polymerase chain reaction (PCR) is a method for the exponential multiplication of specific DNA segments.

To automate the detection of products of a polymerase chain reaction, three methods have been developed so far, which enable a fluorescence-optical detection of the reaction products during the ongoing reaction in quantitative terms and thus the number of molecules of the target sequence that were present in the PCR approach before the start of the reaction help to determine.

The first involves the TaqMan ™ technology from Applied Biosystems / Perkin Elmer, which includes the ABI-PRISM ™ sequence detection system. 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. As Fluorescent dyes are available in the 6-FAM, TET, JOE, VIC ™ and HEX molecules, which can be combined with the quenching molecule TAMRA. In addition, 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 ™ probes is offered by various manufacturers.

The second system concerns the Light-Cycler ™ 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. In the TaqMan ™ system already described, on the other hand, 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. As a result of the synthesis of PCR product, more and more "molecular beacons" with their central domain can bind to the target sequence, as a result of which the reporter dye and quencher are spatially separated from one another Light is detectable in an intensity proportional to the amount of the target sequence. Various reporter dyes are available (e.g. 6-FAM, TAMRA, TET, Cy3, Cy5, Texas Red ™, HEX). DABCYL is generally used as the quencher. 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 ™ 7700, 7200 and 5700 from PE Biosystems, the Rotor-Gene RG2000 ™ Real-Time Cycler from Corbett Research or the FMBIO II ™ Fluorescence Imaging System from Hitachi.

These systems, which are based on detection of the fluorescence signals generated by the PCR reaction, enable highly sensitive determination of molecules of a DNA target sequence and make complex product analysis, such as. B. by gel electrophoretic separation after the actual PCR reaction superfluous. A major disadvantage of the conventional systems is that a specific probe is required for each target sequence to be detected, either one on both Ends fluorescence-labeled oligonucleotide as in the TaqMan ™ system, or two single fluorescence-labeled oligonucleotides as in the LightCyclerTM system or the use of molecular beacons.

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.

Another problem that arises in the polymerase chain reaction is the relatively frequent formation of primer dimers. 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. To solve this problem, 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). These 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. 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. also used tail-specific primers (tags) which can be used for priming after a few amplification cycles, namely after the homo-tail sequence has been inserted into the section to be amplified. 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. In addition, 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 ™ and Light Cycler ™ systems, and other systems fluorescence-optical detection of PCR products to increase the number of target sequences to be detected in parallel.

According to the invention, 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

Use of one or more probes specific therefor, characterized in that a) 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, b) 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.

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. In addition to expanding the number of target sequences that can be detected in parallel in a PCR reaction, 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

Provides reporter sequences ("PIRS primer"). The PIRS primers (with integrated probe binding sequence / reporter sequence) have the following general structure

5'-homo-tail - probe binding sequence / PIRS - specific sequence-3 '

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:

5'- homo-tail - specific sequence - 3 '

However, it is also possible to insert 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.

An essential feature of the invention is that 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 (PIRS) 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.

In a preferred embodiment of the invention, primers are used in which, apart from the homo-tail section, at least one of the thymidine bases has been replaced by uracil. In this case the Amplification reaction mixture added an enzyme which is able to degrade nucleic acids with uracil bases, uracil-N-glycosylase is preferred.

In all areas of the PCR primers used, apart from the "homo-tail" section, one or more thymidine bases are replaced by uracil (Grace et al., 1 998, degradable dUMP outer primers in merged tandem (M / T) -nested PCR : Low and Single target amplification, Analytical Biochemistry 263: 85-92), the generation of primer dimers can be reduced further. 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. In addition, by eliminating the PIRS contained in the multidomain primers, their competition with the fluorescence-labeled reporter probes is eliminated, which contributes to a more efficient signal generation during the PCR.

It is not necessary to replace all of the thymindine bases with uracil in order to implement the stated embodiment. However, it is preferred that the digestion result in primer fragments that are no longer than 8 to 10 nucleotides. In a multiplex PCR according to the invention with PIRS primers, 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. When 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 ™ system. This is illustrated using the examples below.

The use of the 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 ™ and Light Cycler ™ 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. In addition, 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

3) Cytokine responses are appropriate, as illustrated in the examples.

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

Auxiliaries.

In addition, 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 ™, 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.

In a preferred embodiment, a 50 μl reaction mixture produced using a reagent kit according to the invention contains: 2 μl of the sample to be examined

5 μl 1 0 x PCR buffer

4 μ \ 25 mM MgCl ₂

2 μ \ dNTP mix (each dATP, dCTP, dGTP and TTP)

5 μ "homo-tail" primer (500 nM final concentration) 0.15 vl thermostable DNA polymerase

0.5 μ \ PIRS primer and back primer (10 nM final concentration) 5 μ \ probe oligonucleotide A with fluorescent dye 1 (200 nM final concentration)

5 μ \ probe oligonucleotide B with fluorescent dye 2 (200 nM final concentration) sterile aqua bidest. ad 50 μl.

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.

Oligonucleotide primers of the type described above (PIRS primers) are a further subject of the invention.

The following PIRS primers according to the invention are the basis of the method according to the invention in the embodiment of the claimed

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. 5: HSV2 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 5: * TELfwinREHHANHNNO +

SEQ ID No. 1 6: * ETOrvinKasuHANHNNO +

SEQ ID No. 1 7: * ABLrvinBeideHanNPY + -U

SEQ ID No. 1 8: * TELfwinREHHANHNNO + -U SEQ ID No. 1 9: * ETOrvinKasuHANHNNO + -U

The drawing consists of FIGS. 1 and 2, which show the result of carrying out example 1.

1 is the result of a multiplex PCR using PIRS primers with the TaqMan ™ method. The result is shown as it is obtained from the ABI-PRISM ™ Sequence Detection System. The number of completed PCR cycles (0 - 35) is shown on the X axis. The Y-axis indicates the fluorescence intensities measured for their 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. Only the FAM signals are visible in Figure 1. The samples are designated as follows: A4 = m-BRC (baddy), A5 = M-BCR (baddy), A6 = TEL / AML

(Goody), A7 = AML / ETO (Goody); B4 - B7 = four identical negative controls (mRNA of the HL60 cell line without translocation); C4 = water control of the PCR (without template).

Fig. 2 is the result of a multiplex PCR using PIRS primers with the TaqMan ™ method. The result is shown as it is obtained from the ABI-PRISM ™ 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:

A4 = m-BRC (Baddy), A5 = M-BCR (Baddy), A6 = TEL / AML (Goody), A7 = AML / ETO (Goody); B4 - B7 = four identical negative controls (mRNA of the HL60 cell line without translocation); C4

= PCR water control (without template).

EXAMPLES 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 ™ probe was labeled 5'-VIC / TAMRA-3 '. The 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 from leukemia cell lines, which each have one of the chromosomal translocations to be detected, was used as the starting material, or a cell line (HL60), which has no such genetic modification, as a negative control. 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. To increase the sensitivity, 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 ™ 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. In the first run of the nested PCR, the target sequences are pre-amplified. As a rule, 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. In principle, it is not necessary to carry out a nested PCR in the proposed method using PIRS primers. It should only be demonstrated that the method can also be used in a nested PCR. 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.

External primer for upstream run of a nested PCR: "Baddies":

Baddy 1 and 2 t (9; 22) (mBCR positive cell line: SD 1; MBCR positive cell line:

K562) → m-BCRupmRNA

5'-CAGCTCCAATGAGAACCTCACCTCCAGCG

→ M-BCRupmRNA 5'-AGAGAAGAGGGCGAACAAGGGCAGCAAGG

→ ABLdomRNA 5'-CTCAGCGGATACTCAGCGGCATTGCGG

"Goodies":

Goody 1 t (1 2; 21) (positive cell line used: REH)

→ TELupmRNA 5'-GGCACTCCGTGGATTTCAAACAG

→ AMLdomRNA 5'-AACGCCTCGCTCATCTTGCCTGG

Goody 2 t (8; 21) (positive cell line used: Kasumi)

→ AMLI upmRNA 5'-TTCACAAACCCACCGCAAGTCG

→ ETOdomRNA 5'-TGAACTGGTTCTTGGAGCTCCTTG

Primer sequences:

5'-homo-tail = 5'-GCGTACTAGCGTACCACGTGTCGACT

between homo-tail (HANDS day) and probe binding site (PIRS) two additional bases: GA FAM probe HNNO * (for goodies): 5 '- (FAM) -TCCTGCTGCGATGCAATGCTTTTC- (TAMRA)

VIC probe NPY-Rec * (for baddies): 5 '- {VIC) -TGTGGTAGCATTTGCAGTCTGCTGGC- (TAMRA)

* The designations "HNNO" and "NPY-Rec" represent internal laboratory abbreviations for the identification of the different probe sequences.

The following section lists the exact sequences of the PCR primers used: (5'-homo-tail = italic; probe binding site (PIRS) = printed in bold; specific sequence = underlined).

"Baddies":

m-BCR internal hands sense:

5 '- G C G TA C TA G C G TA C C A C G T G T C G A C T

CCAATGAGAACCTCACCTCCAGCG

M-BCR internal Hands sense: δ '- G C G TA C TA G C G TA C C A C G T G T C G A C T AGAGGGCGAACAAGGGCAGCAAGG

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):

5 '- G C G TA C TA G C G TA C CA C G TG TC GA C T- G A - TGTGGTAGCATTTGCAGTCTGCTGGC-GCGGATACTCAGCGGCATTGCGG

'Goodies':

TEL intern Hands HNNO sense (SEQ ID No.2):

5 '- GCG TA C TA GCG TA C CA CG TG TC GA C T- GA TCCTGCTGCGATGCAATGCTTTTC-CGGCACTCCGTGGATTTCAAACAG AML1 internal hands antisense

5 '- G C G TA C TA G C G TA C C A C G T G T C G A C T -

AACGCCTCGCTCATCTTGCCTGG

AML1 internal Hands sense δ '- G C G TA C TA G C G TA C C A C G T G T C G A C T

CTTCACAAACCCACCGCAAGTCG

ETO internal Hands HNNO antisense (SEQ ID No.3):

5 '- G C G TA C TA G C G TA C CA C G TG TC GA C T- G A

TCCTGCTGCGATGCAATGCTTTTC-TGAACTGGTTCTTGGAGCTCCTTG

PCR conditions for this approach:

A 50 μl reaction mixture contains the following components:

5μl 10x PCR buffer μl 25 nM MgCI ₂ 2 μl dNTP-Mix (dATP, dCTP, dGTP and TTP)

5 μl "homo-tail" primer (500 nM final concentration)

5μl FAM probe HNNO (200 nM final concentration)

5 μl VIC probe NPY-Rec (200 nM final concentration)

0.25 μl UNG (substance for eliminating contamination) 0.125 μl AmpliTaq Gold Polymerase

0.5 ul of each specific primer (PIRS primer and respective

Counter primer, 10 nM final concentration)

2 μl of the sample to be examined sterile Aqua bidest. ad 50 μl

PCR cycler conditions:

50 ° C - 2 min (contamination inactivation by UNG)

95 ° C - 10 min (denaturation and activation of the AmpliTaq Gold

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

The results of the application example are shown in FIGS. 1 and 2.

It can be seen that only the "baddies" have a positive VIC signal, while only the "goodies" have a positive FAM signal. All other samples are negative. There are therefore no false positive results.

Example 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. On the basis of identical PIRS, the corresponding groups of primers can each be assigned to a disease in question. To clarify the cause in children with a lung disease, 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. In patients with clinical suspicion of meningitis or encephalitis, 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. One detects both types of the herpes simplex virus. (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 (meningococci, hemophilus influenza and pneumococci) 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. The species specificity is achieved solely by the sense primers. For this reason, the primer must already carry the "HANDS sequence" 5 'from the target sequence-specific section in the first run. If the primer had not yet the "HANDS sequence" in the first run, then primers with and primers without "HANDS sequence" would compete with each other in the second run, which was due to the low concentration of the target sequence-specific primers in the decisive second run of the nested PCR could lead to an inefficient incorporation of the "HANDS sequence", with the consequence of a significantly impaired sensitivity. Primer sequences:

Except for the primer "Eubac-exdo" or "Eubac-Hands-antisense", as mentioned above, all primers used in the first run of the nested PCR correspond in structure to normal PCR primers without any additional sequences. They are all marked with an arrow (→) in front of the abbreviation.

External primer for upstream run of a nested PCR:

"Bacteria (Eubacteriae)":

→ Eubac-exup 5'-AACT (C / A) CGTGCCAGCAGCCGCGGTAA

Eubac-exdo = Eubac-Hands-antisense:

5 '- 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

"Herpes virus":

→ Herpes exup 5'-AGCGAATGCGATGAATTTCGATT

→ Herpes exdo 5'-GAGGCGGAGCTTCACCAGGC

Primer sequences:

5'-homo-tail = 5'-GCGTACTAGCGTACCACGTGTCGACT

Two additional bases between homo-tail (HANDS day) and probe binding site: 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)

* The designations "HNNO" and "NPY-Rec" represent internal laboratory abbreviations for the identification of the different probe sequences.

The following section lists the exact sequences of the PCR primers used: (5'-homo-tail = italics, probe binding site (PIRS) = printed in bold; specific sequence = underlined).

"Herpes viruses":

Herpes virus type 1:

HSV1-intem-Hands-HNNO-sense (SEQ ID No.4): '-GCGTACTAGCGTACCACGTGTCGACT-GA TCCTGCTGCGATGCAATGCTTTTC-GTCCTCCGCGTCGGGTCGGGCC

HSV1-internal-hands-antisense:

5'-GCG74C74GCG74CCqCG7G7CG ^ C7-TCGCACTACTGCCAGGGGCAG

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

HSV2-internal-hands-antisense:

5 ^, -GCG7 C74GCG74CC .CG7G7CG / 4C7-TCGCACTACTGCCAGGGCCGC

"Bacteria":

Neisseria 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

Haemophilus influenzae:

Hae-intern-Hands-NPY-Rec-sense (SEQ ID No.7):

5 '- G C G TA C TA G C G TA C CA C G TG TC GA C T- G A

TGTGGTAGCATTTGCAGTCTGCTGGC-CCTAAGAAGAGCTCAGAG

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 μl reaction mixture contains the following components:

5μl 10x PCR buffer

4 µl 25 mM MgCl ₂

2 μl dNTP mix (dATP, dCTP, dGTP and TTP)

5 μl "Homo-Tail" primer (500 nM final concentration) 5 μl FAM probe HNNO (200 nM final concentration)

5 μl VLC probe NPY-Rec (200 nM final concentration)

0.25 μl UNG (substance to eliminate contamination)

0.125 ul AmpliTaq Gold Polymerase 0.5 μl of each specific primer (PIRS primer and respective counterprimer, 1 0 nM final concentration) 2 μl of the sample to be examined sterile aqua bidist. ad 50 μl

PCR cycler conditions:

50 ° C - 2 min (contamination inactivation by UNG) 95 ° C - 1 0 min (denaturation and activation of the AmpliTaq Gold Polymerase = Hot Start) after 5 cycles to 95 ° C - 1 5 s → 55 ° C - 1 min → 72 ° C - 1 min after that 35 cycles of 95 ° C - 1 5 s → 68 ° C - 1 min → 72 ° C - 1 min

Example 4

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. Many infectious agents or allergens polarize to one of the two cytokine patterns T _H1 and T _H2 , which can lead to the protection of the organism from the corresponding agent or to an immunopathological process (see work by Lanzavecchia, Identifying strategies for immune intervention, Science Vol. 260 : 937-944, 1,993).

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.

As with the method described above for the detection of prognostically relevant chromosomal translocations in leukemia cells, a nested PCR is carried out to increase the sensitivity.

All primers used in the first run of the nested PCR correspond in structure to normal PCR primers without any additional sequences. They are all marked with an arrow (- *) in front of the abbreviation.

External primer for upstream run of a nested PCR:

- Lm— - Interleukin-2:

→ IL-2exup

5'-CAAGAATCCCAAACTCACCAGG

→ IL-2-exdo 5'-CCATCTGTTCAGAAATTCTACAATG

Interferon gamma:

→ IF-G-exup

5'-CCAAAAGAGTGTGGAGACCATCAAGG

→ IF-G-exdo 5'-CCATTACTGGGATGCTCTTCG

"TH ₂ _ Interleukin-4:

→ IL-4-exup 5'-GAACAGCCTCACAGAGCAGAAG

→ IL-4-exdo 5'-CGTTTCAGGAATCGGATCAG

Interleukin-5:

→ IL-5-exup

5'-TCAGGGAATAGGCACACTGGAG

→ IL-5-exdo 5'-ACTCGGTGTTCATTACACCAAG

Interleukin-1 0:

→ IL-1 0-exup

5'-CCAAGACCCAGACATCAAGG

→ IL-1 0-exdo 5'-ACCCTGATGTCTCAGTTTCG

Primer sequences:

5'-homo-tail =

5'-GCGTACTAGCGTACCACGTGTCGACT

Two additional bases between homo-tail (HANDS day) and probe binding site: GA

FAM probe HNNO * (for T _H1 ): 5 '- (FAM) -TCCTGCTGCGATGCAATGCTTTTC- (TAMRA)

VlC probe NPY-Rec * for (T _H2 ): 5 '- (VIC) -TGTGGTAGCATTTGCAGTCTGCTGGC- (TAMRA) * The designations "HNNO" and "NPY-Rec" represent internal laboratory abbreviations for the identification of the different probe sequences.

The following section lists the exact sequences of the PCR primers used: (5'-homo-tail = italic; probe binding site (PIRS) = printed in bold; specific sequence = underlined)

- Ü-U - Interleukin-2:

IL-2-internal-hands-ENT-sense (SEQ ID No.9): δ'-GCGTACTAGCGTACCACGTGTCGACT-

GA-TCCTGCTGCGATGCAATGCTTTTC-CCCAAGAAGGCCACAGAACTG

IL-2 internal hands antisense: δ '- G C G TA C TA G C G TA C C A C G T G T C G A C T

TTGATATTGCTGATTAAGTCCCTG

Interferon gamma:

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: δ'-GCGTACTA GCGTACCA CGTGTCGA C7-AAATTCAAATATTGCAGGCAGG

- 'TH2— "

Interleukin-4:

IL-4-internal-Hands-NPY-Rec-sense (SEQ ID No.11): δ '- G C G TA C TA G C G TA C CA C G TG TC GA C T- G A

T G T G G T A G C A T T T G C A G T C T G C T G G C

CTGCCTCCAAGAACACAACTGAGAAG

IL-4-internal-hands-antisense: 5 ^, -GCG74C74GCG74CC ^ CG7G7CG .C7-GAGTGTCCTTCTCATGGTGG Interleukin-5:

IL-5-internal-Hands-NPY-Rec-sense (SEQ ID No. 12):

5 '- G C G T, C 7. G C G 7. C C. C G 7G 7C G. C 7- G A -

TGTGGTAGCATTTGCAGTCTGCTGGC-ACTCGGTGTTCATTACACCAAG

IL-5 internal hands antisense: δ'-GCGTACTA GCGTA CCA CGTGTCGA C7-TTGCAGGTAGTCTAGGAATTGG

Interleukin-10: IL-10-internal-Hands-NPY-Rec-sense (SEQ ID No. 13):

5 '- GCG 7 1 C 7 1 6 C 6 7 ^" /. CC ^ C 6 Γ G 7 ^' C 6 ^ C 7- GA - TGTGGTAGCATTTGCAGTCTGCTGGC-AAGACCCTCAGGCTGAGGCTACG

IL-10 internal hands antisense: δ'-GCGTA CTA GCGTA CCA CGTGTCGA C7-CTTTGTAGATGCCTTTCTCTTGG

PCR conditions:

A 50 μl reaction mixture contains the following components:

5μl 10x PCR buffer

4 µl 25 mM MgCl ₂

2 μl dNTP mix (dATP, dCTP, dGTP and TTP) 5 μl "homo-trail" primer (500 nM final concentration)

5 μl HNNO FAM probe (200 nM final concentration)

5 μl VLC probe NPY-Rec (200 nM final concentration)

0.25 μl UNG (substance for eliminating contamination)

0.125μl AmpliTaq Gold Polymerase 0.5μl of each specific primer (PIRS primer and respective

Counter primer, 10 nM final concentration)

2 μl of the sample to be examined sterile Aqua bidest. ad 50 μl PCR cycler conditions:

50 ° C - 2 min (contamination inactivation by UNG) 95 ° C - 1 0 min (denaturation and activation of the AmpliTaq Gold Polymerase = Hot Start) then 5 cycles at 95 ° C - 1 5 s → 60 ° C - 1 min → 72 ° C - 1 min after that 35 cycles of 95 ° C - 1 5 s → 68 ° C - 1 min → 72 ° C - 1 min

Example 5:

Example 5 serves the same goal as Example 1. The same leukemia cell lines were used as starting material. In contrast to Example 1, a modified primer / probe combination was chosen which allows amplification at higher temperatures.

External primer for upstream run of a nested PCR (1st run is not listed in the PCR protocol):

"Baddies":

Bady 1 and 2 t (9; 22) (mBCR positive cell line: SD 1; MBCR positive cell line: K562)

m-BCRfwexSD 1

* → 5'-GCTATACCCCGGACTGCAGCTCCAATGAG

* → M-BCRfwexK562

5'-AGAGGCTGAAGAAGAAGCTGTCGGAGCAGG

* → ABLrvexBoth 5'-GAGTGTTTCTCCAGACTGTTGACTGGCGTGATG

"Goodies":

Goody 1 t (1 2; 21) (positive cell line used: REH)

* → TELfwexREH

5'-TCTCCGAGGACGGGCTGCATAGGGAAGG * → AML1rvexREH 5'-GCCACCACCTTGAAAGCGATGGGCAGG

Goody 2 t (8; 21) (positive cell line used: Kasumi)

* → AML1 fwexKasumi 5'-TACGCACTGGCGCTGCAACAAGACCCTG

* → ETOrvexKasumi 5'- GCCCATTGCTGAAGCCATTGGGTGGTGAG

Primer sequences and PCR cycler conditions that were used in the decisive PCR run according to the TaqMan ™ method using "PIRS":

5'-Homo-Tail = δ'-gCg TACTAgCgTA CCA Cg Tg TCgACT

FAM probe HNNOplus * (for goodies):

5 '- (FAM) -CCCTCCTgCTgCgATgCAATgCTTTTCCgC- (TAMRA)

VlC probe NPYRECplus * (for badies): 5 '- (VIC) -CCCgTgTggTAgCATTTgCAgTCTgCTggC- (TAMRA)

# The names "HNNOplus" and "NPYRECplus" represent internal laboratory abbreviations for identifying the different probe sequences.

The following section lists the exact sequences of the PCR primers used: (5'-homo-tail = italic; probe binding site = printed in bold; specific sequence = underlined)

"Badies":

* mBCRfwinSD1HAN: b '- G C G TA C TA G C G TA C CA C G T G T C G A C T- T - CCCTCGCAGAACTCGCAACAGTCCTTCG

^ MBCRfwinK562HAN: '- g C g TACTA g C g TACCAC g T g TC g ACT - TGCAGAGTGGAGGGAGAACATCCGGGAG

* ABLrvinBeideHanNPY + (SEQ ID No. 14) 5 '- g C g TA C TA g C g TA C C A C g Tg T C g A C T- T - C C C G T g T g g T A g C A T T T g C A g T C T g C T g g C - T -

GTGCAACGAAAAGGTTGGGGTCATTTTCACTGG

"Goodies":

* TELfwinREHHANHNNO + (SEQ ID No. 15)

5 '- g C g TA C TA g C g TA C C A C g Tg T C g A C T- T -

C C C T C C T g C T g C g A T g C A A T g C T T T T C C G C - T - GCCTGAAGAGCACGCCATGCCCATTGG

^* AML1rvinHANREH

5 '- g C g TA C TA g C g TA C C A C g T g T C g A C T -

CGGCAACGCCTCGCTCATCTTGCCTG

* AML1fwinHANKasumi

S '- g C g TA C TA g C g TA C C A C g T g T C g A C T -

GCTGAGCTGAGAAATGCTACCGCAGCCATG * ETOrvinKasuHANHNNO + (SEQ ID No.16)

5 '- g C g TA C TA g C g TA C C A C g Tg T C g A C T- T - C C C T C C T g C T g C g A T g C A A T g C T T T T C C G C - T -

GGTGGCATTGTTGGAGGAGTCAGCCTAGATTG

PCR conditions for this approach:

A 50 μl reaction mixture contains the following components:

5μl 10x PCR buffer 4μl 25mM MgCl ₂

2 μl dNTP mix (ATP, CTP, GTP and TTP)

5 μl "homo-tail" primer (500 nM final concentration)

5 μl HNNO FAM probe (400 nM final concentration)

5μl VlC probe NPY-Rec (400 nM final concentration) 0.25 μl UNG (substance to eliminate contamination)

0.125 ul AmpliTaq Gold Polymerase 0.5 ul of each specific primer (SIRS-modified primer and respective

Counter primer, 1 0 nM final concentration) 2 μl of the sample to be examined sterile aqua bidist. ad 50 μl

PCR cycler conditions:

50 ° C - 2 min (contamination inactivation by UNG) 95 ° C - 1 0 min (denaturation and activation of the AmpliTaq Gold Polymerase = Hot Start) then 40 cycles at 95 ° C 1 5 s → 70 ° C - 1 min

Example 6

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). In this way, 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. This results in a partial elimination of any primer dimers that may have formed. In addition, 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":

Bady 1 and 2 t (9; 22) (mBCR positive cell line: SD 1; MBCR positive cell line: K562)

m-BCRfwexSD 1 -U

* → 5'-GCUAUACCCCGGACUGCAGCUCCAAUGAG

* → M-BCRfwexK562-U 5'-AGAGGCUGAAGAAGAAGCUGUCGGAGCAGG

* → ABLrvexBeide-U 5'-GAGUGUUUCUCCAGACUGUUGACUGGCGUGAUG

"Goodies":

Goody 1 t (1 2; 21) (positive cell line used: REH)

^* → TELfwexREH-U 5'-UCUCCGAGGACGGGCUGCAUAGGGAAGG

* → AML1 rvexREH-U 5'-GCCACCACCUUGAAAGCGAUGGGCAGG

Goody 2 t (8; 21) (positive cell line used: Kasumi)

^* → AML1 fwexKasumi-U 5'-UACGCACUGGCGCUGCAACAAGACCCUG

* → ETOrvexKasumi-U

5'- GCCCAUUGCUGAAGCCAUUGGGUGGUGAG Primer sequences and PCR Cclerbedinqunqen that were used in the decisive PCR run according to the TaqMan ™ method using "PIRS":

5'-homo-tail = δ'-gCgTACTAgCgTACCACgTgTCgACT ("Ts" as "Us" within primers with multiple domains)

an additional base between homo-tail (HANDS tag) and probe binding site: "U"

FAM probe HNNOplus * (for goodies): 5 '- (FAM) -CCCTCCTgCTgCgATgCAATgCTTTTCCgC- (TAMRA)

VlC probe NPYRECplus * (for badies):

5 '- (VIC) -CCCgTgTggTAgCATTTgCAgTCTgCTggC- (TAMRA)

# The names "HNNOplus" and "NPYRECplus" represent internal laboratory abbreviations for identifying the different probe sequences.

The following section lists the exact sequences of the PCR primers used: (5'-homo-tail = italic; probe binding site = printed in bold; specific sequence = underlined)

"Badies":

* mBCRfwinSD1HAN-U: b '- G C G UA C UA G C G UA C CA C G U G U C GA C U- ü

CCCUCGCAGAACUCGCAACAGUCCUUCG

* MBCRfwinK562HAN-U: 5 '- g C g UACUA g C g UACCAC g U g UC g ACU - UGCAGAGUGGAGGGAGAACAUCCGGGAG

* ABLrvinBeideHanNPY + -U (SEQ ID No. 17) b '- g C g UA C UA g C g UA C CA C g Ug UC g ACU - U - CCCGU g U gg UA g CAUUU g CA g UCU g CU gg C - U - GUGCAACGAAAAGGUUGGGGUCAUUUUCACUGG

"Goodies":

* TELfwinREHHANHNNO + -U (SEQ ID No. 18) b '- g C g UA C UA g C g UA C CA C g Ug U C g A C U - C C C C U C C U g C U g C g A U g C A A U g C U U U U C C G C - U GCCUGAAGAGCACGCCAUGCCC

* AML1rvinHANREH-U b '- g C g U A C U A g C g U A C C A C g U g U C g A C U

CGGCAACGCCUCGCUCAUCUUGCCUG

* AML1 fwinHANKasumi-U b '- g C g U A C U A g C g U A C C A C g U g U C g A C U

GCUGAGCUGAGAAAUGCUACCGCAGCCAUG

* ETOrvinKasuHANHNNO + -U (SEQ ID No. 19) b '- g C g UA C UA g C g UA C CA C g Ug U C g A C U - C C C U C C U g C U g C g A U g C A A U g C U U U U C C G C - U

GGUGGCAUUGUUGGAGGAGUCAGCCUAGAUUG

PCR conditions for this approach: A 50 μl reaction mixture contains the following components:

3, 5 μl 1 0x PCR buffer

4 ul 25mM MgCl ₂

2 μl dNTP mix (ATP, CTP, GTP and TTP)

0.1.25 ul AmpliTaq Gold Polymerase

0.5 ul of each specific primer (SIRS-modified primer and respective

Counter primer, 1 0 nM final concentration) 2 μl of the sample to be examined sterile aqua bidist. ad 35 μl

PCR cycler conditions: pre-amplification:

95 ° C - 1 0 min (denaturation and activation of the AmpliTaq Gold Polymerase = Hot Start) then 5 cycles at 95 ° C - 1 s → 70 ° C - 1 min 99 ° C - 1 0 min (destruction of the Taq polymerase )

Add 2 units of uracil-N-glycosylase (UNG) + 0.15 μl AmpliTaq Gold + 5 μl "Homo-Tail" primer (500 nM final concentration) + 5 μl FAM probe HNNOplus (400 nM final concentration) +

5 ul VLC probe NPYRECplus (400 nM final concentration) + 1.5 ul PCR buffer 10x

50 ° C - 1 hour (UNG digestion)

then 95 ° C - 10 min (activation of the newly added polymerase and

Hydrolysis of the abasic nucleotides)

35 cycles at 95 ° C 1 5 s → 70 ° C - 1 min

Claims

 Expectations A method for the detection of products of a nucleic acid amplification reaction, comprising the steps ) Providing nucleic acids to be amplified, i) performing an amplification reaction under Use of at least two primers, iii) detecting those formed in ii) Amplification products using one or more probes specific thereto, characterized in that a) one of the primers used for the amplification reaction in the 5'-3 'direction comprises the following elements: a 5'-homo-tail sequence, a probe binding sequence which acts as primer -integrated reporter sequence (PIRS), and a sequence specific for the nucleic acid to be amplified, b) another of the primers used for the amplification reaction in the 5'-3 'direction comprises the following elements: a 5'-homo-tail sequence , and a sequence specific for the nucleic acid to be amplified, 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 marker groups. Method according to claim 1, dadu rc hgekennzeichn et that additionally nucleic acids with the homo-tail sequence are used in a molar excess to the primers. 3. The method of claim 1, d a d u r c h g e k e n n z e i c h n et that at least one of the thymidine bases is replaced by uracil in the primers and the enzyme uracil-N-glycosyiase is added to the reaction mixture. 4. The method according to any one of claims 1 to 3, d a d u rc h g e k e n n z e i c h n et that the probes used carry fluorescent-optically detectable marker groups. 5. The method according to claim 4, dadu rc hgekennzeichn et that the probes used are labeled at their 5 'ends with fluorescent dye molecules and at their 3' ends with quencher molecules, the 3 'ends being modified such that an extension by Polymerases is not possible. 6. The method according to any one of claims 4 or 5, that the fluorescent dye molecules are 6-FAM, TET, JOE, HEX, VIC ™, fluorescein, LCRed 640 or LCRed 705 and that the quencher molecule is TAMRA or DABCYL. 7. The method according to any one of the preceding claims, dadurchgekennzeichn et that a "molecular beacon" is used as a probe. 8. The method according to any one of the preceding claims, d a d u r c h g e k e n n z e i c h n e t that two different probes interact with the probe binding sequence. 9. The method according to claim 8, dadu rc hge ke nnzeichn et that one of the two probes at its 3 'end carries a first fluorescent dye and the other of the two probes at its 5' end carries a second fluorescent dye and light emitted by the the first fluorescent dye is absorbed and remitted, the second fluorescent dye, which is spatially close to the first dye after the binding of the two probes to the complementary strand of the probe binding sequence, is again absorbed and remitted. 10. The method of claim 9, d a d u r c h g e k e n n z e i c h n e t that the first fluorescent dye is fluorescein and the second fluorescent dye is LC Red 640 or LC Red 705. 11. The method according to any one of the preceding claims, d a d u r c h g e k e n n ze i c h n e t that the amplification reaction is a multiplex PCR. 12. 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 is referred to as a primer-integrated reporter sequence (PIRS) is and with its complementary strand at least one probe with detectable Label groups can interact, and a sequence specific for the nucleic acid to be amplified. 13. A reagent kit for the detection of nucleic acid amplification products, comprising: 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 is referred to as a primer-integrated reporter sequence, and a specific one for the nucleic acid to be amplified Sequence, 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) an enzyme suitable for extending the nucleic acid primers according to a) and b), and f) one suitable buffers and optionally other auxiliary substances. 14. Kit according to claim 13, so that it additionally comprises nucleic acids with the homo-tail sequence. 15. Kit according to claim 13, characterized in that: that in the primers at least one of the thymidine bases is replaced by uracil and the kit further comprises the enzyme uracil-N-glycosylase. 16. Kit according to any one of claims 13 to 15, that the enzyme is a thermostable DNA polymerase, which is a 5'-3'-exonuclease activity. 17. Kit according to one of claims 13 to 16, dadu rc hgekennzeichn et that the probe as detectable marker groups at its 5 'end, the dye molecules 6-FAM, TET, JOE, HEX, VIC ™, fluorescein, LCRed 640 or LCRed 705 and carries the quenching molecule TAMRA or DABCYL at its 3 'end. 18. Kit according to one of claims 13 to 17, d a d u rc h g e k e n n z e i c h n et that the probe is a "molecular beacon". 19. Kit according to one of claims 13 to 18, characterized in that three different probes interact with the complementary strand of the probe binding sequence and that the first of the three probes carries the dye molecule fluorescein at its 3 'end, the second of the three probes carries its 5' -End carries the LC Red 640 dye molecule and the third probe carries the LC Red 705 dye molecule at its 5 'end. 20. Kit according to one of the preceding claims, characterized in that that the primers and PIRS primers used for the amplification reaction hybridize with m-BCR-cDNA or / and M-BCR-cDNA or / and TEL / AML-cDNA or / and AML / ETO-cDNA. 21. Kit according to one of claims 13 to 19, characterized in that the primers and PIRS primers used for the amplification reaction with genomic herpes virus DNA and / or with genomic DNA coding for the 16S rRNA from Haemophilus influenzae or / and Neisseria meningitidis or / and Streptococcus pneumoniae hybridize. 22. Kit according to one of claims 13 and 19, d a d u r c h g e k e n n z e i c h n et that the primers used for the amplification reaction and Hybridize PIRS primers with interleukin-2 cDNA or / and interleukin-4 cDNA or / and interleukin-5 cDNA or / and interleukin-10 cDNA or / and interferon cDNA. 23. Nucleic acids for use as PIRS primers in a kit according to claim 20, with which in SEQ ID No. 1, No.2 and No.3 reproduced sequences. 24. Nucleic acids for use as PIRS primers in a kit according to claim 21, with the in SEQ ID No. 5, No. 6, No. 7 and No. 8 reproduced sequences. 25. Nucleic acids for use as PIRS primers in a kit according to claim 22, with the in SEQ ID No.9, No. 10, No. 11, No. 12 and No. 13 reproduced sequences. 26. Nucleic acids for use as PIRS primers in a kit according to claim 20, with the in SEQ ID No. 1 4, No. 1 5 and No. 1 6 reproduced sequences. 27. Nucleic acids for use as PIRS primers in a kit according to claim 20, with the in SEQ ID No. 1 7, No. 1 8 and No. 1 9 reproduced sequences.