CA2354184A1

CA2354184A1 - Multiplex real-time pcr

Info

Publication number: CA2354184A1
Application number: CA002354184A
Authority: CA
Inventors: Dieter Klein; Walter Gunzburg; Brian Salmons
Original assignee: Individual
Current assignee: Bavarian Nordic AS
Priority date: 1999-01-29
Filing date: 2000-01-28
Publication date: 2000-08-03
Also published as: WO2000044935A2; AU2799400A; EP1147225A1; WO2000044935A3; JP2002539769A; US20030087397A1

Abstract

The present invention relates to a real-time Polymerase Chain Reaction (PCR) method for the detection and quantification of variants of nucleic acid sequences which differ in the probe-bindung site. The method is based on the complete or partial amplification of the same region of the variants and the addition of two or more oligonucleotide probes to the same PCR mixture, each probe being specific for the probe-binding site of at least one variant. The method can be applied e.g. to estimate the viral load in a sample, to differentiate between subgroups, subtypes isolates or clades of a viral species or to estimate the impact of the viral load on tumorigenesis.</SDOAB >

Description

Multiplex Real-Time PCR
The present invention relates to a real-time Polymerase Chain Reaction (PCR) method for the detection and quantification of variants of nucleic acid sequences which differ in the probe-binding site. The method is based on the complete or partial amplification of the same region of the variants and the addition of two or more oligonucleotide probes to the same PCR mixture, each probe being specific for the probe-binding site of at least one variant. The method can be applied e.g. to estimate the viral load in a sample, to differentiate between subgroups, subtypes, isolates or I o Glades of a viral species or to estimate the impact of the viral load on tumorigenesis.
Background of the invention The detection and quantification of nucleic acid sequences is of importance for a wide range of experiments and applications. Several methods for the detection and quantification of nucleic acid sequences have been described previously. Most methods are based on polymerase chain reaction (PCR): The PCR is used to amplify a segment of DNA flanked by stretches of known sequences. Two oligonucleotides binding to these known flanking sequences are used as primers for a series of in vitro reactions that are catalyzed by a DNA polymerase. Typically, these oligonucleotides have different sequences and are complementary to sequences that ( 1 ) lie on opposite strands of the template DNA and (2) flank the segment of DNA that is to be amplified. The template DNA is first denatured by heat in the presence of large molar excess of each of the two oligonucleotides and the four dNTPs. The reaction mixture is then cooled to a temperature that allows the oligonucleotide primers to anneal to their target sequences. Afterwards, the annealed primers are extended by the DNA polymerase. The cycle of denaturation, annealing, and DNA-synthesis is then repeated about 10 to 50 times. Since the products of one cycle are used as a template for the next cycle the amount of the amplified DNA fragment is theoretically doubled with each cycle resulting in a PCR-efficiency of 100%.
The specific amplification of a target sequence is due to the annealing of the primers to a complementary region of the DNA. If the primer differs in its sequence from the sequence of the annealing region of the target DNA, the PCR may fail.
Accordingly, if a target sequence is analyzed that differs between samples in the primer-annealing region, the amplification of the target sequence in some samples will fail or will be less efficient. Therefore, degenerated primers are often used, i.e. primers that have unspecific nucleotide analogous at the positions at which the sequence varies between samples.
If two or more target sequences are amplified simultaneously in the same PCR
reaction, a multiplex PCR is performed. Then, more than one primer pair is added to the PCR mixture and each primer pair allows the specific amplification of one target sequence.
The enzyme used for PCR is specific for DNA. If an RNA template is amplified by PCR, the RNA has first to be transcribed into complementary DNA (cDNA) by the 2o enzyme reverse transcriptase. Afterwards the cDNA is used as a template in a PCR.
Accordingly, the method of the amplification of RNA is called reverse-transcriptase (RT) PCR.
The PCR results in a large copy number of the sequence flanked by the primers.
The large copy number of this sequence allows the detection and quantification of the target sequence after the PCR reaction. The detection of the amplification products is usually performed by gel electrophoresis and staining of the DNA. The intensity of the band after gel electrophoresis also allows to estimate the copy number of the sequence of interest in the original sample mostly by comparison with a standard with a known copy number (Sambrook et al., Molecular Cloning, 2"d edition, cold spring harbor laboratory press 1989, p. l:J. 30).
The conventional PCR is widely used. However, the method has several disadvantages that are mostly linked with the detection of the amplification products by gel electrophoresis. The gel electrophoresis requires additional handling of the sample which is time-consuming and prone to sample mix-ups. In addition, the sensitivity of the detection method is low. Finally, quantification of the copy number of the template sequence requires a standard and is often difficult.
More recently, a new technology for the detection and quantification of target sequences was developed which does not show the disadvantages mentioned above.
The method is called "real-time" PCR. Here, the DNA generated within a PCR is detected on a cycle-by-cycle basis during the PCR reaction. The amount of DNA
increases the faster the more template sequences are present in the original sample.
When enough amplification products are made a threshold is reached at which the PCR products are detected. Hence, amplification and detection are performed simultaneously in the same tube.
Most instruments that are used for a real-time PCR detect an increase of fluorescence of a specific wave length as a result of an increasing amount of PCR product.
For example, the Applied Biosystems Prism 7700 sequence detection system is based on the combination of PCR and hybridization of a fluorogenic, target-specific probe.
The probe is an oligonucleotide with both a reporter and a quencher dye attached at the 5'and 3'end respectively. The fluorescence of the reporter dye is efficiently quenched by the quencher dye as long as both fluorochromes are present in close proximity. If the target sequence is present, the probe anneals between the forward and reverse primers. During PCR amplification and thus elongation of the primers the probe is cleaved by the 5'nuclease activity of the DNA-polymerase. This cleavage of the probe separates the reporter dye from the quencher dye, making reporter dye signal detectable. Additional reporter dye molecules are cleaved from their respective probes with each cycle, effecting a proportional increase in fluorescence intensity of the reporter dye and a decrease of the fluorescence intensity of the quencher dye. An algorithm of the software of the instrument compares the amount of reporter dye emission with the quenching dye emission once every few seconds during the PCR
reaction, generating a normalized reporter signal 0R". The first cycle m which the l o normalized reporter signal is above a defined threshold is defined as the threshold cycle CT. The CT value is proportional to the copy number of the template and used for quantification (Held, et al., Genome Research, 6: 986f~. The real-time PCR
provides greater quantitative precision and dynamic range compared to other quantitative PCR methods, and is easier to handle.
If the template is not DNA but RNA a real-time reverse-transcription (RT) PCR
is performed. As described for the conventional PCR the RNA is first transcribed into cDNA before the actual real-time PCR is performed.
Detailed description of the invention For the determination of the viral load in samples from animals infected with the same type of virus a real-time PCR was performed. After detection and quantification of the viral nucleic acid sequence by real-time PCR the viral load was calculated. Considerable variation was found between the samples. In some samples the calculated copy number of the target sequence was very small or even no target sequence was detected. Unexpectedly, the calculated viral load did not correlate with WO Otl/44935 PCT/EP00/00677 the severity of the disease of those animals the samples were taken from. In order to verify the results of the real-time PCR, a conventional PCR with staining of the amplified DNA after gel electrophoresis was performed. Although this method is much less sensitive as compared to the real-time PCR method, amplification 5 products were surprisingly detected in all analyzed samples, i.e. even for samples in which no viral sequences were detected by real-time PCR, positive results were obtained by conventional PCR.
Further investigations showed that the animals, the samples were taken from, were to infected with different subtypes of the same virus. The subtypes were characterized by variants of the viral nucleic acid sequence. Apparently, some variants of a nucleic acid sequence that were present in specific viral subtypes, were not detected by real-time PCR.
Accordingly, it was an object of the present invention, to provide a real-time PCR
method for the detection and/or quantification of variants of a nucleic acid sequence.
The problem underlying the present invention is solved by the complete or partial amplification of the same region of variants of a nucleic acid sequence comprising 2o nucleotide variations within the probe-binding site and the addition of two or more oligonucleotide probes to the same PCR mixture, each probe being specific for the probe-binding site of at least one variant. Said variants of a nucleic acid sequence are found e.g. in different subtypes of phylogenetically related groups of organisms such as in subtypes of families, genera, and species. The variants analyzed by a method according to the present invention are preferably derived from subtypes of a species such as e.g. Glades, isolates or breeds. The variants of nucleic acid sequences may be identical in 50 to 70%, preferably 70 to 90% and most preferably 90 to 99% of the nucleotides.

The nucleic acid sequence of the different subtypes may differ not only in the probe-binding site but also in the primer-binding site. In this case, primers may not anneal to the primer-binding site, resulting in PCR failure. Hence, according to a preferred embodiment of the invention, more than one primer pair is added to the reaction mixture, wherein each primer specifically anneals to the nucleic acid sequence of at least one subtype (multiplex real-time PCR).
The primers and probes used for the method according to the present invention 1o should be at least 60 to 80%, preferably 80, to 90%, and most preferably 90 to 100%
homologous to the nucleic acid sequence of at least one variant of the nucleic acid sequence.
Within the completely or partially amplified region more than one probe-binding site may be included. If in this case the complete region is amplified the amplification products have more than one probe-binding site and more than one probe may anneal to the amplification product. This may e.g. cause interactions between the reporter and quencher dyes of the different annealed probes influencing the quantification.
Hence, according to a preferred embodiment of the invention, two or more parts of 2o the region of each variant are amplified, each part of the region comprising only one probe-binding site.
When two or more parts of the region of the nucleic acid sequence are amplified, primer pairs and probes may be chosen to be specific for one variant. In this case, the fluorescence signal of a specific probe may be characteristic for a specific variant.
The probes are labeled at the 3-prime end with a quencher and at the 5-prime end with a reporter dye. According to a preferred embodiment of the invention, different probes are labeled with the same quencher dye but with different reporter dyes. In that case, the different amplification products can be distinguished. Any reporter dye can be attached to the probe. However, preferably FAMTM or VICTM is used as a reporter dye.
The differentiation between amplification products using different reporter dyes may be applied for the classification of subtypes. First the inventors tried to classify the subtypes by monoplex real-time PCRs. In this case, only one primer pair and one probe is added to the reaction mixture, wherein the primer-pair and probe are chosen to to be specific for one subtype. Then, PCR products should only be detected if this specific subtype is present in the sample. However, non-specific amplification and/or detection was observed in some cases, resulting in wrong classification of the subtype. Then the inventors used a multiplex real-time PCR according to the present invention for the classification. They added several of the subtype-specific primers 1 s and probes to a PCR mixture, wherein the different probes were labeled with different reporter dyes. In this case, a specific subtype is identified, when the fluorescence signal of the respective probe is detected. Using this multiplex real-time PCR method, all subtypes could be classified correctly. Hence, according to a preferred embodiment of the present invention, a multiplex real-time PCR with 2o subtype-specific primers and probes is performed for the classification of subtypes, wherein the probes are labeled with different reporter dyes.
The present invention may be used to study viral diseases such as diseases caused by lentiviruses. Lentiviruses are associated with immunodeficiency and malignacies. The 25 mechanisms involved in tumorigenesis are still not fully understood, but it is suspected that a correlation between tumorigenesis and the viral load exists.
Cats infected with Feline Immunodeficiency Virus (FIV) represent a model for the role of the viral load in the pathogenesis of tumors, since cats infected with FIV
develop quite often tumors, especially lymphomas. Accordingly, in a preferred embodiment of the present invention, the real-time PCR is especially used for the detection and/or quantification of nucleic acid sequences of different subtypes of lentiviruses, especially of FIV.
Since lentiviruses are retroviruses, the nucleic acid sequence of the genome present in the viral particles consists of RNA. According to the life cycle of a retrovirus, the RNA genome is transcribed into DNA after the infection of a host cell. Then, the transcribed, retroviral DNA may be integrated into the genome of the host cell, t 0 forming the so-called provirus. If the already integrated viral genome shall be analyzed and accordingly amplified, no reverse transciption is necessary before the real-time PCR is performed. However, if the genome of the viral particles is analyzed, a reverse-transcription (RT) real-time PCR is performed.
FIV isolates comprising known and unknown variants of the viral nucleic acid sequence have been analyzed by FIV specific real-time PCR. Accordingly, the present invention also provides oligonucleotide probes as well as primer pairs for the detection and/or quantif cation of variants of nucleic acid sequences derived from FIV. Preferably probes and primer pairs according to Seq. ID No. 1 to 21 are provided. The primers and probe according to Seq. ID No. I to 3 are especially used for the detection and/or quantification of Glade A of FIV, whereas oligonucleotides according to Seq. ID No. 7 to 9 are especifically used for the detection and/or quantification of Glade B of FIV. Preferably, both sets of oligonucleotides are used simultaneously in a multiplex real-time PCR. Accordingly, with the two sets of oligonucleotides a method is provided which enables to distinguish between Glade A
and B of FIV in a sample. The probes according to Seq. ID No. 3, 6 or 9 may be combined with primers different from those according to Seq. ID No. 1, 2, 4, 5, 7 or 8, especially when FIV samples are analyzed that comprise unknown FIV
isolates. In one embodiment of the invention, the forward primer according to Seq. ID No. 1 is replaced by a primer according to Seq. ID No. 12. The probe according to Seq.
ID
No. 6 may be used in combination with the forward primers according to Seq. ID
No.
4, 14 or 15 and the reverse primers according to Seq. ID No. 5 or 13. The probe according to Seq. ID No. 9 may be used in combination with the forward primer according to Seq. ID No. 7, 20 or 21 and the reverse primer according to Seq.
ID No.
8. Furthermore, a set of oligonucleotides is provided comprising the probe according to Seq. ID No. 18, the forward primer according to Seq. ID No. 16, and the reverse primer according to Seq. ID No. 17 or 19.
The primers and probes with a sequence according to Seq. ID No. 1 to 21 as well as primers and probes with a homology of at least 70% to the sequences according to Seq. ID No. 1 to 21 may be used in general to amplify sequences specific for FIV.
Additionally, the primer pairs can also be applied without the probes when a conventional PCR is performed instead of a real-time PCR.
In summary, the present invention provides a highly reliable and reproducible method for the detection and quantification of variable nucleic acid sequences.
Brief description of the tables Table 1: The PCR-efficiencies of four different FIV isolates, and the corresponding sequences of the PCR-products are listed. The sequence given in this table is always from the same strand, despite the fact that the probe and the reverse primer bind to the complementary strand compared to the forward primer. The exact sequences (5'-3' orientation) of the primers and probe are described above.

Table 2: A comparison of the results from the real-time PCR and the sequence of the probe-binding site of PCR-products derived from four field isolates. The nucleotide sequence in the table is complementary to the sequence of the probe used. (nd) not determined.

Table 3: Results of the amplification of 30 unknown FIV isolates by the two monoplex real-time PCR assays l O1 Op and 1372p or by one multiplex real-time PCR
composed of the lOlOv- and the 1372p-assay. The subtype of the isolate was identified according to the results of the real-time PCR: (+) a PCR-product was detected with this assay; (-) no PCR-product was detected using this assay;
(*) The subtype can only be determined by a multiplex real-time PCR.
Summary of the invention ~5 A real-time Polymerase Chain Reaction (PCR) method for the detection and/or quantification of variants of a nucleic acid sequence, wherein the same region of said variants is completely or partially to be amplified, each variant differing in one or more nucleotides within the probe-binding site, said method comprising addition of 2o two or more oligonucleotide probes to the same PCR mixture, each probe being specific for the probe-binding site of at least one variant.
The real-time PCR method as above, wherein said variants of the nucleic acid sequence differ in one or more nucleotides within the primer-binding sites and wherein more than one primer pair is added to the reaction mixture each primer 25 specifically annealing to the primer-binding site of at least one subtype The real-time PCR method as above, wherein two or more parts of the region of each variant are amplified, each part of the region comprising only one probe-binding site.
The real-time PCR method as above, wherein the different probes are labeled with different fluorescent reporter dyes.
The real-time PCR method as above, wherein the probes are labeled with FAMTM
or VICTM
The real-time PCR method as above for the detection and/or quantification of variants of the nucleic acid sequence of a virus.
The real-time PCR method as above for the detection and/or quantification of l0 variants of the nucleic acid sequence of a retrovirus.
The real-time PCR method as above for the detection and/or quantification of variants of the nucleic acid sequence of a lentivirus.
The real-time PCR method as above for the detection and/or quantification of variants of the nucleic acid sequence of a Feline Immunodeficiency Virus (FIV).
~ 5 The real-time PCR method as above, wherein the probes according to Seq. ID
No. 3 and 6 are added to the reaction mixture.
The real-time PCR method as above, wherein the probes according to Seq. ID No.

and 9 are added to the reaction mixture.
The real-time PCR method as above, wherein the forward primer according to Seq.
20 ID No. 1 and/or 12 and the reverse primer according to Seq. ID No. 2 are added to the reaction mixture.

The real-time PCR method as above, wherein the forward primer according to Seq.
ID No. 4, 14, and/or 15 and the reverse primer according to Seq. ID No. 5 and/or 13 are added to the reaction mixture.
The real-time PCR method as above, wherein the forward primer according to Seq.
ID No. 7, 20, and/or 21 and the reverse primer according to Seq. ID No. 8 are added to the reaction mixture.
The real-time PCR method as above, wherein the forward primer according to Seq.
ID No. 16 and the reverse primer according to Seq. ID No. 17 or 19 are added to the reaction mixture.
i o The real-time PCR method as above, wherein the above listed primers and probes are added to the reaction mixture.
The real-time PCR method as above, wherein said PCR is a reverse-transcription (RT) PCR.
The real-time PCR method as above, wherein said variants of nucleic acid sequences are nucleic acid sequences derived from subtypes, isolates, Glades or any other subgroup of a species.
Use of the real-time PCR method, as above, for the determination of the overall viral load in a sample comprising variants of a viral nucleic acid sequence.
The use of the real-time PCR method, as above, wherein the variants are derived from nucleic acid sequences derived from subtypes, isolates, Glades or any other subgroup of a viral species.
The use of the real-time PCR method as above for the investigation of the impact of the viral load on tumorigenesis.

An oligonucleotide probe for use in a real-time PCR method selected from the group of probes comprising (a) the nucleic acid sequences according to Seq. ID No. 3 and/or Seq. ID No. 6 and/or Seq. ID No. 9 and/or Seq. ID No. 18, s (b) their complementary strands, and/or (c) nucleic acid sequences with a homology of at least about 70% to the nucleic acid sequences according to Seq. ID No. 3 and/or Seq. ID No. 6 and/or Seq. ID No. 9 and/or Seq. ID No. 18.
A primer for use in a PCR method selected from the group of primers comprising l0 (a) a primer according to Seq. ID No. 1, 2, 4, 5, 7 or 8 or 10 to 17 or 19 to 21, (b) a primer complementary to one of said sequences, and/or (c) a primer with a homology of at least about 70% to the nucleic acid sequences of one of said primers.
A set of primers selected from the group of primer sets comprising 15 (a) the primers according to Seq. ID No. 1 and/or 12 and according to Seq.
ID No. 2, (b) primers with a nucleic acid sequence complementary to one or more of the primers according to (a), and/or (c) a primer with a nucleic acid sequence with a homology of at least about 70% to the primers according to (a).
20 A set of primers selected from the group of sets of primers comprising (a) the primers according to Seq. ID No. 4, 14, and/or 15 and according to Seq. ID
No. 5 and/or 13, (b) primers with a nucleic acid sequence complementary to one or more of the primers according to (a), and/or 25 (c) a primer with a nucleic acid sequence with a homology of at least about 70% to the primers according to (a).

A set of primers selected from the group of sets of primers comprising (a) the primers according to Seq. ID No. 7, 20, and/or 21 and according to Seq. ID
No. 8, (b) primers with a nucleic acid sequence complementary to one or more of the primers according to (a), and/or (c) a primer with a nucleic acid sequence with a homology of at least about 70% to the primers according to (a).
A set of primers selected from the group of sets of primers comprising (a) the primers according to Seq. ID 16 and according to Seq. ID No. 17 and/or 19, (b) primers with a nucleic acid sequence complementary to one or more of the primers according to (a), and/or (c) a primer with a nucleic acid sequence with a homology of at least about 70% to the primers according to (a).
A set of oligonucleotides for use in a real-time PCR method, comprising a primer set ~ 5 selected from the group of primer sets as above and a probe selected from the group of probes as above.
The set of oligonucleotides as above for use in the method as above.
2o Example: Detection of Provirai DNA
The following example will further illustrate the present invention. It will be well understood by a person skilled in the art that the provided example in no way may be interpreted in a way that limits the applicability of the technology provided by 25 the present invention to this example.

Recently, a method based on the ABI 7700 system (Perkin Elmer, Foster City, California) was established and validated for the quantification of FIV
proviral and viral loads (Leutenegger et al. J Virol Methods 1999, 78(1):10.5-11 ~. In this method, the S'nuclease activity of the Taq-polymerase allows the cleavage of a labeled probe 5 and the subsequent liberation of a reporter fluorescent dye which can be excited with an argon laser and leads to the emission of light. The amount of emitted fluorescence, which is proportional to the amount of DNA produced during the PCR, is measured at regular intervals during the PCR and allows the monitoring of the PCR in a real-time manner. The method has been shown to be very useful in viral load 1o determinations if just one isolate is used (e.g. in challenge experiments for vaccination trials where the isolate is known and optimized primers and probes could be used).
However, as soon as viral loads from different isolates should be compared the equality of the PCR efficiency for the different isolates must be ensured. In the following example, the influence of mutations in the primer- and probe-binding site 15 on the PCR-efficiency and the subsequent estimation of this influence on the viral load determination is analyzed. A real-time PCR system was established that allows the estimation of viral loads in patients, infected with different isolates as a basis for the determination of the impact of the viral load on tumorigenesis.
1 Conditions and parameters of the real-time PCR for the analysis of FIV
sequences First the conditions for the amplification of FIV sequences by real-time PCR
were established and evaluated. Primers and probes for the amplification and detection of the gag gene of FIV were designed. The linear range within which the copy number of a template can be quantified was calculated using a plasmid with a corresponding region of a FIV isolate. PCR-efficiencies were calculated for this plasmid and for a genomic standard.
1.1 Real-time PCR primers, probes, and cycling conditions For the real-time PCR the subsequently described primer and probe sequences were designed using the Primer Express software (Perkin Elmer, Foster City, California).
All oligonucleotides were purified by high-performance liquid chromatography and purchased from Perkin Elmer (Weiterstadt, Germany).
to Three different real-time PCR assays (FIV 101 Op/v, FIV 1416p, and FIV 1372p) were developed. For the FIV1010p-assay, the PCR primers used were FIV0771f (5'-AGA
ACC TGG TGA TAT ACC AGA GAC-3') and FIV1081r (5'-TTG GGT CAA
GTG CTA CAT ATT G-3'). The primers were designed to be 100% homologous to ~ 5 the sequence of the Glade A FIV isolates which comprise among other strains the isolates Petaluma (Genebank accession number M25381 ), San Diego PPR (M36968), Zurichl (X57002), and Utrecht113 (X68019). It was also considered that for an efficient amplification the size of the amplified fragment should be smaller than 350 by and, if possible, smaller than 100 bp.
For a monoplex PCR, the probe was labeled at the 5'end with the fluorochrome FAM (6-carboxy-fluorescein) which serves as a reporter fluorochrome and at the 3'end with the fluorochrome TAMRA (6-carboxy-tetramethyl-rhodamine) which functions as a quencher. In a multiplex PCR the same probe was labelled with the reporter fluorochrome VIC to distinguish between the signal of the different PCR
systems.

The probe was designed, based on several criteria: (i) 8-10°C higher melting temperature than the primers, (ii) no G's at the 5' end of the probe, (iii) no stretches of identical nucleotides longer than four, especially not of G's, (iv) lack of self annealing, (v) lack of predicted dimer formation with corresponding primers.
Furthermore, the probe is blocked at the 3' end to prevent elongation during the amplification. The probe was at least 80%, but preferably 95 to 100%
homologous to the sequence of different FIV isolates. The probe used to establish standard assay conditions was FIV l O l Op/v (5'-FAM/VIC-TAT GCC TGT GGA GGG CCT TCC
T-TAMRA-3' ).
Io Under the same considerations as described for the FIV 101 Op-assay the two other assays were designed. The FIV1416p-assay was specific for the Glade A FIV
isolate Glasgow 8 (Hosie, M.J., Jarrett, O. AIDS 1990, ;I, 21.5-220) and several FIV
isolates from southern Germany or Austria. The system consisted of the primers FIV
1360f (5'-GCA GAA GCA AGA TTT GCA CCA-3') and FIV1437r (5'-TAT GGC GGC
CAA TTT TCC T-3'), and the probe FIV1416p (5'-FAM-TGC CTC AAG ATA
CCA TGC TGT ACA CTG CA-TAMRA-3'). The FIV1372p-assay was designed to be 100% homologous to Glade B FIV isolates Italy-M2 (Y13867), Italy-M3 (Y13866), Italy-M8 (Z96111), Amori-1 (D37823), Amori-2 (D37824), Sendai-2 (D37821), Yokohama (D37819), and a local, subtype B-like isolate. The system consisted of the primers FIV 1280f (5'-ATC CTC CTG ATG GGC CTA GAC-3') and FIV 1426r (S'-ACT TTC CTA ATG CTT CAA GGT ACC A-3') and the probe FIV 1372p (5'-TTT GCA CCA GCC AGA ATG CAG TGT AG-3').
The target sequence was amplified in a 251 reaction volume using the following PCR-conditions: 10 mM Tris (pH 8.3), 50 mM KC1, 3 mM MgClz, 200 nM dATP, dCTP, dGTP, 400 nM dUTP, 300 nM of each primer, 200 nM of the fluorogenic probe, and 2.5 units of Taq DNA polymerase were used. After the initial denaturation (2 min at 95°C), amplification was performed in 45 cycles each comprising 15 sec at 95°C and 60 sec at 60°C. For a multiplex PCR the same reaction conditions were applied.
1.2 Preparation of DNA
A Construct of the FIV Zurich 2 isolate (plasmid, pBSCompZ2 [Allenspach, et al., Schweiz Arch Tierheilkd 1996, 138, 87-92]) was used as a control to determine the 1 o linear range of the real-time PCR. The plasmid was propagated in E. coli cells and extracted using the Qiagen Plasmid Kit according to manufacturer's instructions (Qiagen, Hilden, Germany). The copy number of the plasmid was estimated from the absorbtion at 260nm. A set of tenfold dilutions was performed in PCR grade water containing calf thymus DNA as a carrier in a concentration of 30 ~g/ml.
A genomic DNA standard was developed that mimics the in vivo situation of a provirus integrated into genomic DNA of a cell. The DNA from CrFK cells [Crandell, et al., In Vitro 1973, 9, 176-185] stably infected with different FIV isolates (Petaluma [Pedersen, et al., Science 1987, 23~, 790-793], Glasgow 8 [Hosie &
Jarrett, 2o Aids 1990, 4, 215-220], Amsterdam 6 [Siebelink, et al., Vet Immunol Immunopathol 1995, ;t6, 61-69], Utrecht 113 [Verschoor, et al., J Clin Microbiol 1993, 3I, 2355] was extracted using the QIAamp Kit according to manufacturer's instructions (Qiagen, Hilden Germany). The DNA concentration was estimated by OD
measurement at 260 nm and a tenfold dilution series was performed in PCR grade water containing 30 ~g cellular DNA per ml.

When the proviral load was studied DNA was extracted from peripheral blood leucocytes. Otherwise, it was proceeded as described above for the DNA-extraction from infected CrFK cells.
1.3 Calculation of the template copy number An algorithm of the Sequence Detection Software compares the amount of reporter dye emission (R) with the quenching dye emission (Q) once every few seconds during the PCR amplification, generating a normalized reporter signal OR".
This value o represents the fluorescence signal of the reporter dye divided by the fluorescence signal of the quencher signal minus the baseline signal established in the first few cycles of the PCR when cleaved probe is generally not detectable. The OR"
values are plotted as a function of the PCR cycle. The first cycle which is above a defined threshold (normally ten times the standard deviation of the background fluorescence) ~ 5 is defined as the threshold cycle CT. Within a certain range of template concentrations the C-~ value is proportional to the template copy number present at the beginning of the reaction and reflects the first opportunity for quantification of the template (Held, et al., 1996, Genome Research, 6, p. 986ff).
20 1.4 Accuracy of the quantification of control DNA
A dilution of the plasmid pBSCompZ2 was obtained as described above and the concentration was estimated in three independent real-time PCRs using the FIV 1 O1 Op-assay for each of the diluted samples. The calculation of the initial copy 25 number using DR" is highly reproducible as it was shown by small standard deviations of the CT values, ranging from 0.13 to 2.99%. With a decreasing number of template copies the number of cycles increased and larger standard deviations are obtained. Hence, the accuracy of the measurement deteriorates.
Using the PCR conditions described above a linear relationship between CT and the 5 standard template concentration was achieved for nine log units (S x 10° to 5 x 109 copies). The coefficient of correlation, that is defined as the percentage of standard deviation of the threshold cycle numbers, was 0.9977. This high coefficient of correlation is a prerequisite for the calculation of the PCR-efficiency. The result confirms that the method is highly accurate over a wide range of template 10 concentrations.
2.4 Calculation of the PCR-efficiency 15 The PCR-efficiency can be used to evaluate the PCR-conditions. If the PCR-efficiency was 100% the concentration of the target sequence should be doubled every cycle. However, usually the PCR-efficiency (E) is less than 100% and the amount of PCR-product (Y) amplified from an initial template copy number Z
after n cycles can be calculated according to the following equation Y=Z x (1+E)" or after 20 logarithmic transformation as log Y = log Z + n x log (1 + E). The PCR-efficiency E
can be calculated from the slope of a standard curve where the CT-value is plotted against the logarithm of the copy number of a dilution serie. Now the PCR-efficiency can be described as E=10-~~S -1 with s representing the slope of the straight line.
A set of tenfold dilutions of the FIV plasmid was prepared as described above.
A
real-time PCR using the FIV 1 O1 Op-assay was performed for each dilution and a standard curve was obtained. A PCR-efficiency of 0.9815 was calculated from the slope of that standard curve.

In addition, the PCR-efficiency of the genomic standard obtained from transduced CrFK cells as described above was calculated. A real-time PCR was performed and PCR-efficiencies were calculated. The PCR-efficiencies varied between 0.9742 and 0.8711. The best PCR-efficiency (0.9742 for CrFK cells infected with FIV
Petaluma) is almost as good as the PCR-efficiency of the plasmid dilution series.
For all constructs the correlation coefficient was larger than 0.99 which demonstrates that a comparison of the PCR-efficiencies is possible. The results show that the chosen PCR conditions are suitable for an efficient amplification of the FIV
fragment.
2.5 Influence of mismatches on the PCR-efficiency The viral load can only be compared between different samples if the PCR-efficiencies for the different samples are similar, i.e. when the reaction conditions are suitable for the amplification of a specific template. In vaccination trails where an organism is challenged with a specific viral subtype the PCR conditions can be optimised for this specific viral subtype allowing the comparison of data between 2o patients infected with the same isolate. The same accuracy needs to be achieved in naturally infected patients, where a swarm of viruses differing in their nucleotide sequence can be present. The FIV model was chosen in order to investigate the influence of mismatches on the PCR-efficiency.
2.5.1 Sequence analysis of a conserved region of the FIV genome The complete gag gene (1.6 kb) of the characterised isolates of FIV (Petaluma, Glasgow 8, Amsterdam 6 and Utrecht 113) were amplified and sequenced using the primers FIV566f (5'-ACC TTC AAG CCA GGA GAT TC-3') and FIV2167r (5'-CCT CCT CCT ACT CCA ATC AT-3'). Additionally, a 311 by region of the FIVgag gene of some of the unclassified isolates (Munich 3, 4, 6 and 7) was amplified and sequenced with the same primers as for the FIV 1010p-assay. The used primers were FIV0771f (5'-AGA ACC TGG TGA TAT ACC AGA GAC-3') and FIV 1081 r (5'-TTG GGT CAA GTG CTA CAT ATT G-3').
A conventional PCR was performed on a 9600 thermal cycler (Perkin Elmer, Foster City, California). PCR reactions contained 10 mM Tris (pH 8.3), 50 mM KCI, 3 io mM MgCl2, 200 nM dATP, dCTP, dGTP, dTTP, 300 nM of each primer and 2.5 U
of Taq DNA polymerase. Amplification was performed with 1 cycle of 3 min at 95°C, 60 sec at 51°C and 3 min at 72°C, followed by 39 cycles of 15 sec at 94°C, 40 sec at 51 °C and 90 sec at 72°C. PCR-products were separated on a 0.8 % agarose gel and visualized after ethidium bromide staining with the Eagle Eye system (Stratagene, Heidelberg, Germany). The appropriate bands were isolated and DNA was purified using the QIAamp gel extraction Kit (Qiagen, Hilden Germany) according to the manufacturer's instructions. Approximately 20 - 50 ng PCR-product were used in the subsequent sequencing reaction mixture containing 4 pl BigDye premix (Perkin Elmer, Foster City, California), 4 pmol of the primer FIV0771f, and water in a total 2o volume of 10 ~.1. The cycling was performed on a 9600 thermocycler (Perkin Elmer, Foster City, California) with the following programme: 30 sec at 96°C, 10 sec at 50°C, 4 min at 60°C for 30 cycles. The sequencing reaction was purified according to the manufacturer's instructions. The sequence analysis was performed with an ABI
310 Genetic Analyzer (Perkin Elmer, Foster City, California).
2.5.2 Comparison of sequence data and real-time PCR-efficiency for standard isolates Four isolates were sequenced and, in parallel, amplified by real-time PCR
using the FIV l Ol Op-assay as described above. The sequence data and the corresponding PCR-efficiencies are listed in Table 1. In the region of the forward primer (FIV0771fj no mutation was found in any of the four isolates. In the region where the probe FIV 1 O 1 Op anneals only one mutation was found. The T to C change at by 1020 was found when the sequence of the Amsterdam 6 isolate [Siebelink, et al., yet Immunol Immunopathol 1995, .16, 61-69] was compared with the sequence of FIV Petaluma (Genebank accession number M25381). Interestingly, the nucleotide sequence of this isolate was still detected but with the lowest PCR-efficiency (0,8711 }
compared to 1 o all other isolates. In the region of the reverse primer FIV 1081 r one point mutation was found. In the Glasgow 8 isolate [Hosie, & Jarrett, Aids 1990, ~, 21 S-220]
an A
to T change at position 1071 was detected, which was associated with the second lowest PCR-efficiency of 0.9284. In summary, in the four standard isolates two point mutations were found, which reduced the PCR-efficiency.
2.5.3 Comparison of sequence data and PCR-efficiencies for field isolates The real-time PCR system was analysed for FIV isolates of ten naturally infected cats. The cats were selected from southern Germany and Austria. This region has 2o previously been shown to contain a heterogenous FIV population. In that region isolates from three different subtypes and from several other genetic outliers have been found [Bachmann, M. H., et al., J Virol 1997, 71, pp. 4241ff].
Five out of ten tested cats infected with unknown FIV isolates were positive in the real-time PCR assay (Table 2). However, two of the five cats (Munich 3 and 4) that were negative in the real-time PCR, were positive when the PCR-product was analysed by agarose gel electrophoresis. Sequencing showed that the sequence of the two FIV isolates Munich 3 and 4 differed from the previously published sequences by three and four mutations in the probe-binding site respectively. The mismatches are located in a part of the probe which is initially not displaced by the Taq polymerase and which is responsible for the binding of the probe before the probe is cleaved by the 5' nuclease activity of the Taq-polymerase. The failed amplification can either be explained by the lack of binding or by the displacement of the probe before appropriate cleavage.
In contrast, no mutations were found in the probe-binding site of the two isolates, which were positive in the conventional and in the real-time PCR (Table 2).
For these to two samples the curve of the real-time PCR showed an exponential increase of the fluorescence signal similar to the one which was seen for the plasmid standard indicating a high PCR-efficiency. In conclusion, variation in the probe-binding site result in apparently reduced PCR-efficiency or even PCR-failure.
6 Detection of different FIV Glades by multiplex real-time PCR
The proviral load was studied according to the invention for samples of cats infected with FIV. It was shown that using more than one primer pair and probe allows the 2o detection of a larger number of viral strains and to differentiate between subtypes.
FIV isolates of an unknown subtype were analysed using the above described conditions. Three monoplex real-time PCRs were performed: The assays FIV l O1 Ov and FIV1416p that are specific for Glade A FIV isolates and the FIV1372p-assay that is specific for the Glade B subtype. Two multiplex PCRs were performed: The FIV 1 O 1 Ov- and the FIV 1416p- assay were used in a multiplex real-time PCR
to detect Glade A FIV isolates. The FIV l O l Ov- and the FIV 1372p-assay were used to detect Glade A and Glade B FIV isolates in a multiplex setup. The use of different reporter dyes (VICTM in the FIV1010v-assay and FAMTM in the FIV1372p and in the FIV 1416p-assay) allowed to distinguish between the signal of the two PCR-systems in the multiplex setup.
5 The results of the FIV 1010p, FIV 1416p or the FIV 1372p monoplex real-time PCR
are compared with the multiplex real-time PCR FIV l O l Ov/FIV 1416p and FIV1010v/FIV1372p. The results of the FIV1010p and FIV1372p assay are summarized in Table 3. For more than one third of the samples that were analyzed by one monoplex real-time PCR no signal was detected. Surprisingly, combining the t0 FIV 101 Ov and the FIV 1372p assay none of 30 samples was negative. Hence, the use of more than one primer pair and more than one probe enabled the detection of all samples which were not detected by one monoplex real-time PCR.
The inventors used the results to group the viruses of the different samples into ~5 Glades. In 6 out off 30 samples the results of two monoplex real-time PCRs were inconclusive. In contrast, the results of one multiplex real-time PCR allowed the grouping into Glade A or B for all samples. In conclusion, the present invention allows to detect sequences of different viral Glades and also to group the viruses according to their sequence into the different Glades.
The final goal however will be a system that allows the detection and quantification of a broad range of different isolates with similar PCR efficiencies. Based on this optimized system the comparison of viral loads from cats infected with different FIV
isolates can be performed much more precisely. Such an optimized system provides the tool for the investigation of the impact of the viral load on the development of cancers in lentiviral infection, as well as providing the basis for the investigation of the efficiency of therapeutic agents tested in naturally infected cats.
Furthermore, real-time PCR strategies can be designed to detect mutations in oncogenes present in WO 00!44935 PCT/EP00/0067?

biopsy material, where tumor and normal cells may be present. In such cases, real-time PCR permits a quantification of the number of tumor cells present.

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Table 3: Amplification of 25 unknown FTV isolates by three monoplex or two multiplex real-time PCRs P C R-assay FIV isolate lOlOp 1372 p lOlOv/1372p subtype Munich 11 r - +/- A

Munich 14 - + -/+ g Munich 1$ - + -/+ B

Munich 20 - + _/+ g 'Munich 27 _ + _i+ B

Munich 29 - + _/+ g Munich 31 + ~/- .

Munich 32 + - +/- A

Munich 35 + - +/- A

Munich 36 + - +/- A

Munich 38 + -/+ B

Munich 39 - + _/+ B

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Munich 41 + - +/- A

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Munich 44 + _ +/_ A

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Amsterdam 6 ' -~ +/- A

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Austria TE* + + -/+ B

Austria ~S , + + /rt B
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SEQUENCE LISTING
<110> Bavarian Nordic Research Institute A/S
<120> Multiplex Real-Time PCR
<130> Sequence listing for BN29PCT
<140>
<141>
<150> PA 1999 00114 <151> 1999-01-29 <160> 21 <170> PatentIn Ver. 2.1 <210> 1 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer for the amplification of a FIV sequence (FIV0771f) <400> 1 agaacctggt gatataccag agac 24 <210> 2 <211> 22 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer for the amplification of a FIV sequence (FIV1081r) <400> 2 ttgggtcaag tgctacatat tg 22 <210> 3 <211> 22 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Probe for the detection of a FIV sequence (FIV1010p) <400> 3 tatgcctgtg gagggccttc ct 22 <210> 4 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer for the amplification of a FIV sequence (FIV1360f) <400> 4 gcagaagcaa gatttgcacc a 21 <210> 5 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer for the amplification of a FIV sequence (FIV1437r) <400> 5 tatggcggcc aattttcct 1g <210> 6 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Probe for the detection of a FIV sequence (FIV1416p) <400> 6 tgcctcaaga taccatgctc tacactgca 29 <210> 7 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer for.the amplification of a FIV sequence (FIV1280f) <400> 7 atcctcctga tgggcctaga c <210> 8 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer for the amplification of a FIV sequence (FIV1426r) <400> 8 actttcctaa tgcttcaagg tacca 25 <210> 9 <211> 26 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Probe for the detection of a FIV sequence (FIV1372p) <400> 9 tttgcaccag ccagaatgca gtgtag 26 <210> 10 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer for the amplification of a FIV sequence (FIV566f) <400> 10 accttcaagc caggagattc 20 <210> 11 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer for the amplification of a FIV sequence (FIV2167r) <400> 11 cctcctccta ctccaatcat 20 <210> 12 <212> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer for the amplification of a FIV sequence (FIV0754f) <400> 12 aatgtatcta caggacgaga acct 24 <210> 13 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer for the amplification of a FIV sequence (FIV1437rb) <400> 13 tatggctgcc aactttcct 19 <210> 14 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer for the amplification of a FIV sequence (FIV1366fa) <400> 14 gcaagatttg caccagctag g 21 <210> 15 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer for the amplification of a FIV sequence (FIV1366fb) <400> 15 gcaagatttg caccagccag a 21 <210> 16 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer for the amplification of a FIV sequence (FIV1182f) <400> 16 atggccacat taataatggc 20 <210> 17 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer for the amplification of a FIV sequence (FIV1307r) <400> 17 ggtaatggtc taggaccatc a <210> 18 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Probe for the amplification of a FIV sequence (FIV1212v) <400> 18 tcatccaata tttctttatc tgcagcgca 29 <210> 19 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer for the amplification of a FIV sequence (FIV1307z) <400> 19 ggtaatggtc tgggagcatc a 21 <210> 20 <211> 27 <212> DNA
<2I3> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer for the amplification of a FIV sequence (FIV1332f) <400> 20 gggaatagga ttaactcaag aacaaca 27 <210> 21 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer for the amplification of a FIV sequence (FIV1324f) <400> 21 gagattatgg gaataggatt aactcaaga 29

Claims

1. A real-time Polymerase Chain Reaction (PCR) method for the detection and/or quantification of variants of a nucleic acid sequence, wherein the same region of said variants is completely and/or partially to be amplified, each variant differing in one or more nucleotides within the probe-binding site, said method comprising addition of two or more oligonucleotide probes to the same PCR mixture, each probe being specific for the probe binding site of at least one variant.

2. The real-time PCR method according to claim 1, wherein said variants of the nucleic acid sequence differ in one or more nucleotides within the primer binding sites and wherein more than one primer pair is added to the reaction mixture each primer specifically annealing to the primer binding site of at least one subtype

3. The real-time PCR method according to claim 1 or 2, wherein two or more parts of the region are amplified, each part of the region comprising only one probe binding site.

4. The real-time PCR method according to any of the preceding clams 1 to 3, wherein the different probes are labeled with different fluorescent reporter dyes.

5. The real-time PCR method according to claim 4, wherein the probes are labeled with FAM TM or VIC TM.

6. The real-time PCR method according to any of the preceding clams 1 to 5 for the detection and/or quantification of variants of the nucleic acid sequence of a virus.

7. The real-time PCR method according to claim 6 for the detection and/or quantification of variants of the nucleic acid sequence of a retrovirus.

8. The real-time PCR method according to claim 7 for the detection and/or quantification of variants of the nucleic acid sequence of a lentivirus.

9. The real-time PCR method according to claim 8 for the detection and/or quantification of variants of the nucleic acid sequence of a Feline Immunodeficiency Virus (FIV).

10. The real-time PCR method according to claim 9, wherein the probes according to Seq. ID No. 3 and 6 are added to the reaction mixture.

11. The real-time PCR method according to claim 9, wherein the probes according to Seq. ID No. 3 and 9 are added to the reaction mixture.

12. The real-time PCR method according to claim 9, wherein the forward primer according to Seq. ID No. 1 and/or 12 and the reverse primer according to Seq.
ID
No. 2 are added to the reaction mixture.

13. The real-time PCR method according to claim 9, wherein the forward primer according to Seq. ID No. 4, 14, and/or 15 and the reverse primer according to Seq.
ID No. 5 and/or 13 are added to the reaction mixture.

14. The real-time PCR method according to claim 9, wherein the forward primer according to Seq. ID No. 7, 20, and/or 21 and the reverse primer according to Seq.
ID No. 8 are added to the reaction mixture.

15. The real-time PCR method according to claim 9, wherein the forward primer according to Seq. ID No. 16 and the reverse primer according to Seq. ID No. 17 and/or 19 are added to the reaction mixture.

16. The real-time PCR method according to claim 9, wherein the primers according to claim 12 and/or 13 and the probes according to claim 10 are added to the reaction mixture.

17. The real-time PCR method according to claim 9, wherein the primers according to claim 12 and/or 14 and the probes according to claim 11 are added to the reaction mixture.

18. The real-time PCR method according to any of the preceding claims 1 to 17, wherein said PCR is a reverse-transcription (RT) PCR.

19. The real-time PCR method according to any of the preceding claims 1 to 18, wherein said variants of nucleic acid sequences are nucleic acid sequences derived from subtypes, isolates, clades or any other subgroup of a species.

20. Use of the real-time PCR method according to any of the preceding claims 6 to 19 for the determination of the overall viral load in a sample comprising variants of a viral nucleic acid sequence .

21. The use of the real-time PCR method according to claim 20, wherein the variants are derived from nucleic acid sequences derived from subtypes, isolates, clades or any other subgroup of a viral species.

22. The use of the real-time PCR method according to claims 20 or 21 for the investigation of the impact of the viral load on tumorigenesis.

23. An oligonucleotide probe for use in a real-time PCR method according to claim 9 selected from the group of probes comprising (a) the nucleic acid sequences according to Seq. ID No. 6 and/or Seq. ID No. 9 and/or Seq. ID No. 18, and/ or (b) their complementary strands.

24. A primer for use in a PCR method according to claim 9 selected from the group of primers comprising a primer according to Seq. ID No. 4, 5, 7 or 8 or 10 to 17 or 19 to 21.

25. A set of primers for use in a PCR method according to claim 9 selected from the group of primer sets comprising the primers according to Seq. ID No. 12 and according to Seq. ID No. 2.

26. A set of primers for use in a PCR method according to claim 9 selected from the group of sets of primers comprising the primers according to Seq. ID No. 4, 14, and/or 15 and according to Seq. ID
No. 5 and/or 13.

27. A set of primers for use in a PCR method according to claim 9 selected from the group of sets of primers comprising the primers according to Seq. ID No. 7, 20, and/or 21 and according to Seq. ID
No. 8.

28. A set of primers for use in a PCR method according to claim 9 selected from the group of sets of primers comprising the primers according to Seq. ID 16 and according to Seq. ID No. 17 and/or 19.

29. A set of oligonucleotides for use in a real-time PCR method according to claim 9, comprising a primer set selected from the group of primer sets according to any of the preceding claims 25 to 28 and a probe selected from the group of probes according to claim 23.