DE4416446A1 - Sequences specific for human DNA - Google Patents

Abstract

Nucleotide sequences which can be used for the species-specific detection of human nucleic acids. The oligonucleotides are derived from parts of the human cytoplasmic beta -actin gene which are not conserved in respect of other human actins or other species. Methods for using the oligonucleotides as internal controls and controls for sample integrity in nucleic acid amplification reactions are also provided.

Description

The present invention relates to oligonucleotides which have species spec hybridize fish with human beta-actin nucleotide sequences. In particular, the invention relates to the use of these Oligonucleotides as internal controls for nucleic acid amplification reactions and as a means of determining intactness of samples for nucleic acid amplification.

Methods for amplifying nucleic acids were found a variety of diagnostic and clinical applications as useful, including the diagnosis of diseases and the Determination of the genotype. Certain instructions for the Am plification of nucleic acids are based on hybridization Sequence specific primer with the target nucleic acid to the Initiate polymerization of the complementary nucleic acid sequence. For example, the polymerase chain reaction (PCR; U.S.-Pa No. 4,683,195, U.S. Patent No. 4,683,202, Mullis et al. 1987th Mtds. Enz. 155: 335-350, Saiki et al. 1988. Science 239: 489-491) two oligonucleotide primers containing the DNA segment to be amplified flank. The primers are attached to their complementary sequences stores and with the help of a thermostable DNA polymerase verlän siege. The primers hybridize with opposite strands of the Target sequence and are oriented so that the DNA synthesis on the  Section between the primers takes place. Repeated cycles Denaturation in the heat, annealing of primers and extension double the amount of segment in each cycle. The strand ver displacement amplification (SDA; G. T. Walker et al. (1992) PNAS 89, 392-396; Walker, T. et al. (1992) Nucleic Acids Res. 20, 1691-1696) uses complementary oligonucleotide primers containing a HincII range to flank the target sequence. After Extension of the primers by DNA polymerase is with HincII Single-strand break introduced. The next round of polymerization begins at the single-strand break and displaces the previously synthesized th strand. The displaced strand then stands for a Hybridisie tion with the appropriate primer and a repetition of the cycle Available. It is with these types of amplification guides difficult to find out if a failure of the amplification of a Target sequence for lack of target sequence or problems with the sample or with reagents, which leads to a while existing target sequence is not amplified, is due. That is, the reliability of nucleic acid amplification Reaction is determined by the quality of the sample (sample suitability) as well as by the quality of the reagents. For nucleic acid Am plication reactions carry both the existing amount of Test sample as well as the suitability of the target nucleic acid sequence Amplification for sample suitability at. It is therefore desirable to have a means to determine if a sample is available for the Amplification is useful or not if the target nucleic acid sequence in particular an internal control which is in place examining sample simultaneously with the amplification reaction could be carried out. For clinical tests on humans would be the appropriate control for the sample suitability of a human Nucleic acid sequence used in every human clinical trial is present and occurs only in human cells. Perkin Elmer Cetus (Norwalk, CT) markets an internal control as Test for sample suitability under the name GENEAMPLIMER beta-globin Primer 1 and primer 2. The primers target the beta globin gene, which is also highly conserved in mammal species, and it is  Not known if the test is specific for human nucleic acid sequences is or not.

Actin is a common protein found in all eukaryotic Cells finds. It practices many in the cells of higher organisms structural and regulatory functions, among others mitosis, motility and structural integrity. Six isoforms of actin have been identified in vertebrates - four muscle types (actin for skeletal muscles, heart muscles, smooth Musculature of the aortic type and smooth muscle of the abdominal type) and two types of non-muscle (cytoplasm-beta and cytoplasmic gamma) Actin). The muscle actins are tissue-specific and on the muscle contraction involved. In contrast, the cytoplasmic actin is found in Virtually all cells, and they are involved in many cell functions involved. Despite the variety of actin cells present the amino acid sequence among the Act in types and among eukaryotic highly conserved. In addition to the rat, the chicken and yeast (Ng et al., 1980. PNAS 77: 3912-3916; Gallwits et al., 1980. PNAS 77: 2546-2550; Nudel et al. 1983. Nucleic Acids Res. 11: 1759-1771; Kost et al. 1983. Nucleic Acids Res. 11: 8287-8301) also sequenced the gene of human cytoplasmic beta-actin and compared with the beta-actin genes of other species (Nakajima Iÿima et al. 1985. PNAS 82: 6133-6137; European Patent Application No. 01 74 608; Ponte et al. 1984. Nucleic Acids Res. 12: 1687 to 1696). Primer for use in the amplification of the whole human beta-actin gene are from Clontech Laboratories, Inc. (Palo Alto, CA) under the name MAPPING Amplimers for Human Beta. Actin.

Although it occurs in all eukaryotic cells, Cytoplas ma-actin has not yet been used as an internal control in nucleic acid Amplification reactions used. That's because the high conserved nature of the actin sequence suggests that amplification primers that represent a general actin target sequence (e.g. Clontech's MAPPING amplimers), with the actin genes of others eukaryotic organisms cross-react, resulting in a sample con  tamers, so that it will be impossible to determine whether the Sample DNA itself is suitable for amplification. This is from of particular concern in clinical nucleic acid amplification assays, where a human sample with eukaryotic pathogens such as Yeast, whose actins have extensive homology, kon could be tampered with. The present invention overcomes this Problem by providing nucleic acid sequences which specific for human cytoplasmic beta-actin and those as Primers can be used to exclude only human cytoplasmic beta Actin target sequences in a human clinical sample specifically to amplify.

The present invention provides nucleic acid sequences which specific for the human beta-actin gene. These sequences can be used to prepare oligonucleotide primers for the species spec fish amplification of human beta-actin target sequences build. Also provided are methods of use the specific human beta-actin nucleic acid sequences as internal Controls and tests for sample suitability in amplification reactions with human samples. The primers can be used in conjunction with any amplification-based diagnostic test for human clinical samples are used on the Extension of primers, including PCR and SDA. In addition, the sequences of the invention show among the human actin gene sequences have little homology with skeletal or Herzmuskelactinen and therefore show no tissue-specific Cross-reaction. When used as a primer, amplify the nucleic acid sequences comprise small portions of the target nucleic acid (about 100 bp) and thus provide for each nucleic acid amplification an effective control amplification.

Figure 1 illustrates the regions for hybridization of the oligonucleotides to the human beta-actin gene. The base fumigation is from Nakajima-Iÿima et al. (see above) taken.

The present invention provides oligonucleotides with nucleic acid sequences that are present in the sequence of the human cytoplasmic contain beta-actin gene and specific for human cells are. These sequences can therefore be used to Presence of human nucleic acid in a sample specific determine. In one embodiment, the oligonucleotides used as probes for the hybridization of target nucleic acids (for example in dot blots, Southern blots or Northern blots), wherein the probes are labeled with direct or indirect detectable are provided gene. The marker can be any in the art for Be used labeling of nucleic acids, and the Labeling can be achieved by chemical conjugation to the finished oligo nucleotide can be attached, or the marker can be during its synthesis into the oligonucleotide (eg nick translation [shift of single-strand breaks] or chemical synthesis). Directly detectable markings for Attachment to the oligonucleotides can be carried out without further chemical or biological reaction manually or by instruments be pointed. These include, but are not limited to, fluorescent compounds, visible dyes and radioisoto pe. Indirect detectable labels require another chemical (eg enzymatic production of a detectable Reaction product) or biological reaction (e.g., binding to a labeled antibody or a specific binding partner). Examples of indirectly detectable markings for attachment at the oligonucleotides are, though not exclusively, Enzymes that react with other reagents to form a colored React (eg horseradish peroxidase or alkaline Phosphatase), biotin, avidin, streptavidin and haptens such as Digoxigenin. Suitable methods for detecting such indirect detectable labels are well known in the art.

In a preferred embodiment, the sequences are described as human-specific primers for nucleic acid amplification used, for. B. for determining the sample suitability or as internal positive control for the amplification reaction. For nucleic acid  re-amplification reactions, the primer can be used to either the amplification of DNA or RNA target sequences prepare. DNA target sequences can be any genomic sequences which are contained in the human beta-actin gene or flank this, or cDNA derived from human beta-actin mRNA reverse transcribed. RNA target sequences are in general mRNAs transcribed from the human beta-actin gene. As further explained below, one of the oligonucleotides derives tide sequences specific for human beta-actin, of the area flanking the gene on the 5 'side. This here term used beta-actin "gene" is intended to refer both to tran scribbled as well as on nontranscribed portions of the gene including flanking sequences on the 5 'and the 3 'side, the regulatory elements and recognition regions included with the expression of the gene and the posttrans criptional modification of mRNA.

The amplified target sequence can be separated by separation of the nucleases acids of the sample by size using gel electrophoresis and observation of a band of a predetermined size corresponds to be demonstrated. Alternatively, the am Plested target sequence detected by means of oligonucleotides which are as described above with a detectable mark are provided. In a first embodiment of the proof is the amplified target sequence with the labeled oligonucleotide hybridized, which is either one of the for the Amplification used human-specific primer or a Oligonucleotide which is linked to an internal sequence of the amplifi annealed target nucleic acid hybridizes. Alternatively, the Human-specific primers are self-labeled and amplified be used in which case the label in the amplified target sequence is incorporated. After hybridization tion of the probe or the incorporation of the labeled primer is the Labeling as described above using in the art proven methods.  

To determine if an area of the human cytoplasm-beta Actin gene exists, which is sufficient of other Actinen distinguishes it as a human-specific nucleic acid target sequence to be able to use or not, the entire amino acid was sequence of human beta-actin with that of other human Actine and compared to other types of Actinas. The ideal sequence for use as a control target sequence, a distinction would be made between cytoplasmic beta-actin and tissue-specific Actinen (from heart, smooth and Skeletal musculature) as well as actinas of other species, especially potent eukaryotic human pathogen (eg dysentery and yeast) differentiate. The first step was the FINDSEQ program of IntelliGenetics (Mountain View, CA) used to be in the after all Databases available to search for actin sequences, with only the human sequences were retained. The selected ones Sequences included cytoplasmic beta-actin, the actin mRNA smooth muscle, alpha-actin mRNA, skeletal alpha actin, Fibroblast actin, non-muscle gamma-actin, alpha-actin of smooth muscle, aortic-type smooth muscle actin, Cardiac alpha actin, cytoplasmic gamma actin, cytoskeletal gamma actin, Cardiac muscle actin, skeletal muscle actin and the beta-actin pseudogene. Similarly, non-human actin sequences were also selected and loaded on disk, including cytoplasmic actin Rat, cytoplasmic actin of mouse, actin of dysentery Entamoeba histolytica, actin of Saccharomyces cerevisiae, actin of phyto phora megasperma, Actin of Trypanosoma brucei, Actin of Physarum polycephalum, Act in of Tetrahymena thermophila and cytoplasmic beta-actin of the mouse.

Then, selected amino acid sequences of actins were used of the GENEALIGN program of IntelliGenetics. human Actin sequences were compared, and human beta-actin became compared with the acts of other species. In addition, the Nucleic acid sequences of human beta-actin and human genes Compared yeast actins separately. A very strong sequence Mood has been under the human actin sequences at the level the amino acids found, with the greatest variability in the  amino-terminal ends of the sequences. A comparison of the human cytoplasmic beta-actin with the actin of other species also showed areas of potential sequence divergence at the amino-terminal ends, and approximately at residues 292-300 and 313-320. A comparison of the gene sequences of yeast actin and the human beta-actin showed little agreement at the 5 'end of the expressed region of the gene and in the untranslated 5 ' Range, but the 3 'end of the exon showed a strong sequence homo logy.

Although the most divergent portion of the sequence was found at the 5 'end of the coding region, this region has a high GC content as well as a significant amount of secondary structure that would interfere with nucleic acid amplification reactions. Moreover, an HincII site was found in this region which would prevent SDA amplification. Therefore, nucleotide sequences of 24 base pairs each were analyzed in the 100 bp regions on both sides of the HincII site (but without overlapping) using the OLIGO program (© 1989 Wojciech Rychlik) for a minimal secondary structure and similar melting temperatures. The following primer pairs of 24 base pairs each were selected. They hybridize to the untranslated region on the 5 'side and the first exon, as shown in Fig. 1:

As shown in Figure 1, SEQ ID NO: 1 hybridizes to the 5 'untranslated region. SEQ ID NO: 2 and SEQ ID NO: 3 hybridize with two regions in the first exon. SEQ ID NO: 4 hybridizes to the C-terminal part of the first exon and is extended slightly into the first intron. When used as primers in nucleic acid amplification reactions, SEQ ID NO: 1 and SEQ ID NO: 2 sequence a portion from the 5 'untranslated region (-81) to the first exon (+27). SEQ ID NO: 3 and SEQ ID NO: 4 amplify a portion located primarily within the first exon (+31 to +126). The numbering of the nucleotides is based on Nakajima-Iÿima et al. (see above). The portion of the cytoplasmic beta-actin gene covered by these four oligonucleotides can either be amplified as part of the genomic DNA, or the portion can e.g. B. restrictively using DdeI or PleI and amplified as a separate fragment.

The oligonucleotide sequences were also bacteriel selected len and invertebrate sequences in the NIH gene bank, by the clinical samples could be contaminated. There were no sequence homologies found. The actin sequences of the invention therefore do not hybridize with other actin sequences or with Nucleotide sequences of bacterial or invertebrate pathogens; you are therefore particularly advantageous as internal controls or tests for the sample suitability of human clinical samples.

Because the oligonucleotide pairs are linked by a HincII (HindII) restriction may be a control amplification tion reaction, in which SEQ ID NO: 1 and SEQ ID NO: 4 be used. The amplification product of these two primers has a length of 195 bp and was intended by HincII (HindII) in two Fragments are cut at about 100 bp each. On similar control test could be other restriction enzymes which intersect within this 195 bp fragment, for example Example: AluI, AcyI, Asu, AvaI, BbvII, BcefI, BglI, BsePI, EcoRI and HaeIII.

The following experimental examples are illustrative specified specific embodiments of the invention and are not to be considered as limiting the invention, as defined in the appended claims.

example 1

SEQ ID NO: 1 and SEQ ID NO: 2 were used as primers for amplification tion of a target sequence at the 5 'end of the human cytoplasmic beta Actin gene tested. The PCR reaction mixture consisted of 1 × PCR Buffer (10 mM Tris, pH 8.8, 50 mM KCl and 1.5 mM MgCl₂), 5 mM dNTP, 4.5 mM MgCl₂ and 10 mM 2-mercaptoethanol. PCR was performed with 5 μl of AMPLITAQ Taq polymerase in 495 μl of PCR reaction mixture were used. Fifty μl of this mixture was used for each test with 50 μl of the DNA sample and 30 μM each of the 5 'and 3' Primers used. The thermocycles were carried out as they by Clontech for their MAPPING human beta-actin amplimers be proposed, d. H. 94 ° C for 1 minute, 61-62 ° C for 1 minute and 72 ° C for 1 minute for 30 cycles. On the last cycle followed by incubation at 72 ° C for 7 minutes. The samples were then kept at 4 ° C until analysis.

The DNA samples tested for amplification were extracted from human isolated cervix, bronchial lavages, penis and nasopharynx and diluted 1: 5 in sterile Tris-EDTA buffer. Several samples out each source was combined to a final volume of 3.5-4.5 ml poured. In each case 1/10 of the volume was 1% sodium decyl sulfate (SDS), 0.1 M dithiothreitol (DTT). Samples were mixed on a vortex mixer and 250 μl of PREPAGENE Beads were added. The volume was made up with binding buffer 15 ml, and isolation of the DNA was performed according to the instructions carried out by the manufacturer. In short, the samples were Shaken for 30 minutes at room temperature, centrifuged for 5 minutes, and the supernatant liquid was sucked off and discarded. The Sludge was washed in 5 ml of binding buffer and again as previously centrifuged. The sediment was twice in binding buffer  The DNA was washed by adding 0.5 ml twice Elution buffer dissolved at 50 ° C from the beads. Elek Trophoresis on a 1% agarose gel in TAE buffer revealed that DNA from all samples with the following approximate concentration had been extracted:

source concentration bronchial 0.158 μg / ml nasopharyngeal 0.172 μg / ml penis 0.145 μg / ml cervix 0.162 μg / ml

This DNA isolated from these samples was left untreated, with Proteinase K treated and boiled or boiled only boiled. The negative control was Tris-EDTA buffer, and the positive one Control was 2 μl of amplified beta-actin from Clontech. Everyone Assay consisted of 50 μl of DNA dilution and 50 μl of PCR mix, the containing primer and Taq polymerase. The results of Amplification reactions were performed on 10% polyacrylamide gels analyzed stained with ethidium bromide.

The human DNA samples showed an amplified target sequence with the expected size, which confirmed that the beta-actin gene specifically amplified in DNA from a variety of human tissues is zable. The positive control showed a smaller at plied segment, probably because of the missing 5'- untranslated region in the Clontech amplification product. In the negative control there was no visible amplification product. These results show that SEQ ID NO: 1 and SEQ ID NO: 2 specifically with human cytoplasmic beta -actin nucleic acid hybridize and are able to act as primers for a human to serve specific nucleic acid amplification reaction. It was a slight improvement in the efficiency of amplification  detected when the sample was cooked or with proteinase K was treated and cooked.

Similar tests with SEQ ID NO: 3 and SEQ ID NO: 4 as primers showed also a specific priming and a specific amplification tion of a human beta-actin gene segment with the prediction Size in DNA samples taken from a variety of tissues had been isolated.

Example 2

To confirm the target sequence specificity of the primers of the invention SEQ ID NO: 3 and SEQ ID NO: 4 were used as primers in Amplifizie tion reactions containing DNA consisting of several human and non-human species. The amplification reactions were carried out essentially as in Example 1, wherein to 1 ml PCR reaction mixture 10 ul Taq polymerase, 20 μl SEQ ID NO: 3 (final concentration 0.22 μM) and 30 μl of SEQ ID NO: 4 (final concentration 0.21 μM) were added. Fifty μl of this preparation became 50 μl of each of the following Given DNA samples:

DNA source Concentration (per 50 μl) Mycobacterium tuberculosis | 525 ng Candida albicans 875 ng HeLa cells 500 ng Rhodococcus rhodocrous 402ng McCoy cells 568 ng Escherichia coli 500 ng Clostridium perfringens 500 ng Micrococcus lysodeikticus 500 ng dog 2 520 ng cat 2 070 ng chicken 2,170 ng pig 1 700 ng rat 2 390 ng Human (source: blood) 1 550 ng

The PCR reactions were overlaid with mineral oil and thermocycled as follows: 1) 94 °, 3 min (melting), 2 ) 94 °, 1 min (melting) / 70 °, 1 min (annealing) / 72 °, 1 min (Extension) during 30 cycles and 3) 720, 7 min (final extension). Ten μl of each amplified reaction mixture was run on a 10% polyacrylamide gel at 100 V in Tris-borate-EDTA buffer. The gels were stained with ethidium bromide, washed in water and photographed under ultraviolet irradiation. The length of the specifically amplified human beta-actin target sequence was predicted to be 97 base pairs. This band was seen only in the amplification reactions containing human DNA (HeLa cells and human blood DNA), confirming that the primers under these thermocycle conditions are specific for human DNA.

Non-human DNA samples showed an amplified product a size of less than 50 bp, indicating nonspecific Primer dimerization or nonspecific amplification seemed to lead. This lack of specificity can be an artifact of a PCR "cold start" due to the fact that so many  Samples were taken simultaneously. This hypothesis was supported by further experiments involving fewer samples were prepared faster, as previously thermocycled. The non-specific 50 bp product was present in non-human mammalian DNA samples (cat, rat, dog, chicken) that way were prepared, not seen. The specificity for humans was amplified by the specific 97 bp amplification product was present in human DNA samples, again confirmed.

To rule out the possibility that the specificity of Am plifizierungsreaktion on the inhibition of the PCR reaction by derived from non-human DNA, samples containing mammalian DNA at lower temperatures to reduce stringency amplified again. Mammalian specimens were raised because of the increased similarity the presence of the beta-actin Gene sequences chosen. With reduced stringency you would in this Samples expect generation of a 97 bp target sequence, as Confirmation that PCR is not inhibited. PCR was as before carried out with a lowering of the attachment temperature according to the following thermocyclic protocol: 1) 94 °, 3 min, 2) 94 °, 1 min / 50 °, 1 min / 72 °, 1 min during 30 cycles and 3) 720, 7 min. All non-human species except the dog (cat, rat, pig, Chicken) showed an amplified target sequence of 97 bp, which confirms that the amplification of beta-actin is not due to nonhuman DNA is inhibited. At an attachment temperature of 70 °, PCR amplification is therefore specific to human beta-actin DNA.  

SEQUENCE LISTING (1) GENERAL INFORMATION

• (i) REGISTERS:
  (A) NAME: Becton Dickinson and Company
  (B) ROAD: One Becton Drive
  (C) LOCATION: Franklin Lakes
  (D) BUNDESLAND: NJ
  (E) COUNTRY: US
  (F) POSTCODE: 07417
• (ii) REGISTRATION TITLE: sequences specific to human DNA
• (iii) NUMBER OF SEQUENCES: 4
• (iv) COMPUTER-READABLE FORM:
  (A) DATA CARRIER: floppy disk
  (B) COMPUTER: IBM PC compatible
  (C) OPERATING SYSTEM: PC-DOS / Ms-Dos
  (D) SOFTWARE: Patent Release # 1.0, Version # 1.25 (EPA)

(2) INFORMATION FOR SEQ ID NO: 1

• (i) SEQUENCE CHARACTERISTICS:
  (A) LENGTH: 24 base pairs
  (B) ART: nucleic acid
  (C) STRUCTURE: Single
  (D) TOPOLOGY: linear
• (ii) ART OF MOLECULAR: DNA (genomic)
• (iii) HYPOTHETIC: NO
• (iii) ANTISENSE: NO
• (vi) ORIGINAL ORIGIN:
  (A) ORGANISM: Homo sapiens
• (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TAAGGACTCG GCGCGCCGGA AGTG 24

(2) INFORMATION ABOUT SEQ ID NO: 2

• (i) SEQUENCE CHARACTERISTICS:
  (A) LENGTH: 24 base pairs
  (B) ART: nucleic acid
  (C) STRUCTURE: Single
  (D) TOPOLOGY: linear
• (ii) ART OF MOLECULAR: DNA (genomic)
• (iii) HYPOTHETIC: NO
• (iii) ANTISENSE: NO
• (vi) ORIGINAL ORIGIN:
  (A) ORGANISM: Homo sapiens
• (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GACGACGAGC GCGGCGATAT CATC 24

(2) INFORMATION FOR SEQ ID NO: 3

• (i) SEQUENCE CHARACTERISTICS:
  (A) LENGTH: 24 base pairs
  (B) ART: nucleic acid
  (C) STRUCTURE: Single
  (D) TOPOLOGY: linear
• (ii) ART OF MOLECULAR: DNA (genomic)
• (iii) HYPOTHETIC: NO
• (iii) ANTISENSE: NO
• (vi) ORIGINAL ORIGIN:
  (A) ORGANISM: Homo sapiens
• (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GACAACGGCT CCGGCATGTG CAAG 24

(2) INFORMATION FOR SEQ ID NO: 4

• (i) SEQUENCE CHARACTERISTICS:
  (A) LENGTH: 24 base pairs
  (B) ART: nucleic acid
  (C) STRUCTURE: Single
  (D) TOPOLOGY: linear
• (ii) ART OF MOLECULAR: DNA (genomic)
• (iii) HYPOTHETIC: NO
• (iii) ANTISENSE: NO
• (vi) ORIGINAL ORIGIN:
  (A) ORGANISM: Homo sapiens
• (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 TACCTGGTGC CTGGGGCGCC CCAC 24

Claims (10)

1. Oligonucleotide having the sequence SEQ ID NO: 1. 2. Oligonucleotide having the sequence SEQ ID NO: 2. 3. Oligonucleotide having the sequence SEQ ID NO: 3. 4. Oligonucleotide having the sequence SEQ ID NO: 4. A method of specifically amplifying a human target nucleic acid in a nucleic acid-containing sample, comprising the steps of:

• a) hybridizing a pair of oligonucleotides selected from the group consisting of I) SEQ ID NO: 1 and SEQ ID NO: 2 and II) SEQ ID NO: 3 and SEQ ID NO: 4, with the sample nucleic acid under conditions such that the oligonucleotide pair hybridizes specifically with the target nucleic acid, if present, and;
• b) amplifying the target nucleic acid.

6. The method according to claim 5, further comprising the step of Detection of the amplified target nucleic acid comprises. 7. The method according to claim 6, wherein the amplified Zielse is detected by means of an oligonucleotide, the is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and mixtures thereof, wherein the oligonucleotide has a detectable label is provided.   8. A method of detecting human nucleic acid in a nucleic acid-containing sample, comprising the steps of:

• a) hybridizing an oligonucleotide provided with a detectable label with the sample nucleic acid under conditions such that the oligonucleotide hybridizes specifically with the human nucleic acid, if present, wherein the oligonucleotide has a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and mixtures thereof, and;
• b) detection of the human nucleic acid by detection of the tag of the hybridized oligonucleotide.

The method of claim 8, wherein the label is selected is from the group consisting of fluorescent compounds, Dyes, enzymes and radioisotopes exists. 10. The method according to claim 8, wherein this is a method for Detection of human cytoplasmic beta-actin nucleic acid is.