WO2000068436A1

WO2000068436A1 - Detection system for human immunodeficiency virus based on nucleic acid amplification

Info

Publication number: WO2000068436A1
Application number: PCT/IB1999/000828
Authority: WO
Inventors: Stefan Bosbach; Jürg BÖNI; Jörg SCHÜPBACH
Original assignee: Nationales Zentrum Für Retroviren
Priority date: 1999-05-07
Filing date: 1999-05-07
Publication date: 2000-11-16
Also published as: AU3438999A

Abstract

Sequences allowing the detection and quantification of human immunodeficiency virus-1 (HIV-1) are described. For the detection of the various subtypes A to H (group M) sequences of a specific pol region are preferred whereby a selective group O sequence has been derived from the LTR region. Both types of sequences are either alone or in combination particularly suitable as probes in TaqMan ® analysis.

Description

DETECTION SYSTEM FOR HUMAN IMMUNODEFICIENCY V IRUS BASED ON NUCLEIC ACID AMPLIFICATION

Technical field

The present invention concerns oligonucleo- tides and oligonucleotide systems that are particularly suited for the detection and preferably also quantif ica- tion of infections with human immunodeficiency viruses (HIV) , in particular HIV-1 .

Background Art

The rapid and specific detection of infectious agents such as HIV, in donated blood, is of utmost importance. In order to reduce the diagnostic window period, or for monitoring, sequence-based detection of vi- ral RNA or DNA is increasingly used. So far, however, respective detection and quantification methods for HIN-1 suffer from reduced reliability due to the virus' ability for rapid mutations and existence of a broad sequence variety, as well as undesired interactions such as hairpin and duplex formation, false priming and hybridisation etc. often occurring in the samples during analysis.

It is therefore still very much desired to get tools and methods for the reliable detection and preferably quantification of HIV-1 infection independent of sequence particularities.

Disclosure of Invention

Thus, one object of the present invention was to provide sequences that enable a fast detection and quantification of HIV-1 infections by nucleic acid ampli-

C0ΝFIRMATI0Ν COPY fication technologies (NAT) . The expression NAT comprises both sequence and signal amplification. Such methods are well known by the persons skilled in the art and comprise general PCR method, RT-PCR, Nested-PCR, antigen-capture- PCR, m-situ-PCR and TaqMan ; ligase chain reactions ( CR) , in particular gap- CR, and asymmetric gap-LCR; strand displacement amplification (SDA) ; and transcription-mediated amplification (TMA) or nucleic acid sequence-based amplification (NASBA) ; tyramide based signal amplification and branched DNA signal amplification

(bDNA) . The most preferred method is the so called

® TaqMan -method .

It is general knowledge that primer and probe sequences are best selected in highly conserved and char- acteristic regions. Such regions can be found in the pol and the LTR regions of the HIV-1 genome. It has been found in connection with the present invention that the range from about nucleotide 4920 to about 5090 for pol (numbering referred to HIVHXB2CG; gene bank ace. no. K03455 and M38432, see Table 1, and SEQ. ID. NO. 1) and the range from about nucleotide 600 to about 750 for LTR (numbering referred to HIVANT70C; gene bank ace. no. L20587, see Table 2, and SEQ. ID. NO. 6) are of great importance .

Table 1:

Pol region of HIVHXB2CG (known sequence gene bank accession no. K03455 and M38432) with information on the localization of region of greatest interest (underlined) and preferred probe and primer sequences (separately marked) :

4890 4900 4910 4920 4930 HIVHXB2CG AATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAAATCCACTTTGGA TTAAGTTTTAAAAGCCCAAATAATGTCCCTGTCGTCTTTAGGTGAAACCT

N S K F S G L L Q G Q Q K S T L E> POL POLYPROTEIN (NH2-TERMINUS UNCERTAIN) >

SB5s

TGGA>

HIVHXB2CG TGGA

4940 4950 4960 4970 4980 HIVHXB2CG AAGGACCAGCAAAGCTCCTCTGGAAAGGTGAAGGGGCAGTAGTAATACAA TTCCTGGTCGTTTCGAGGAGACCTTTCCACTTCCCCGTCATCATTATGTT

R TT S K A P L E R * R G S S N T> _POL POLYPROTEIN (NH2-TERMINUS UNCERTAIN) _

1. SBP3as 30 20 10 <AAGGTGAAGGGGCAGTAGTAATACAA

HIVHXB2CG AAGGTGAAGGGGCAGTAGTAATACAA 3. SB5s 10 AAGGACCAGCAAAGO

HIVHXB2CG AlAlGlGlAlClClAlGlClAlAlAlGlCl

4990 5000 5010 5020 5030

HIVHXB2CG GATAATAGTGACATAAAAGTAGTGCCAAGAAGAAAAGCAAAGATCATTAG CTATTATCACTGTATTTTCATCACGGTTCTTCTTTTCGTTTCTAGTAATC

R H K SS SS AA KK KK KK SS KK DD H *> .POL POLYPROTEIN (NH2-TERMINUS UNCERTAIN).

1. SBP3as

<GATAA ill! HI HXB2CG GATAA

2. SB6as 20 10 <TAAAAGTAGTGCCAAGAAGAAAAG HIVHXB2CG TAAAAGTAGTGCCAAGAAGAAAAG 5040 5050 5060 5070 5080

HIVHXB2CG GGATTATGGAAAACAGATGGCAGGTGATGATTGTGTGGCAAGTAGACAGG CCTAATACCTTTTGTCTACCGTCCACTACTAACACACCGTTCATCTGTCC

G L W K T D G R * * L C G K * G> POL POLYPROTEIN (NH2-TERMINUS UNCERTAIN) >

5090 5100 5110

HIVHXB2CG ATGAGGATTAGAACATGGAAAAGTT

TACTCCTAATCTTGTACCTTTTCAA

* G L> >

Table 2 :

LTR region of HIVANT70C (known sequence gene bank accession no. L20587) with information on the localization of region of greatest interest (underlined) and preferred probe and primer sequences (separately marked) :

600 610 620 630 640 HIVANT70C TCATCTGTTCAACCCTGGTGTCTAGAGATCCCTCAGATCACTTAGACTGA AGTAGACAAGTTGGGACCACAGATCTCTAGGGAGTCTAGTGAATCTGACT LTR; 5' LTR >

1. SBls 10 20 CTGGTGTCTAGAGATCCCTCAGATO

HIVANT70C CTGGTGTCTAGAGATCCCTCAGATC

650 660 670 680 690 HIVANT70C AGCAGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAGTGAAAGT TCGTCTTTTAGAGATCGTCACCGCGGGCTTGTCCCTGAACTTCACTTTCA LTR; 5' LTR >

SBPls 10 20 TCTCTAGCAGTGGCGCCCGAACA>

HIVANT70C TCTCTAGCAGTGGCGCCCGAACA

700 710 720 730 740 HIVANT70C GAAACCAGGGAAGAAAACCTCCGACGCAACGGGCTCGGCTTAGCGGAGTG CTTTGGTCCCTTCTTTTGGAGGCTGCGTTGCCCGAGCCGAATCGCCTCAC

3. SB2as 10 <GCAACGGGCTCGGCTTA HIVANT70C GCAACGGGCTCGGCTTA

750

HIVANT70C CACCCG GTGGGC

The most interesting sequence to be used for the detection of a broad variety of HIV-1 virus subtypes (detection sequence) in the pol region comprises a sequence within or corresponding to nucleotides 4953 to 4992 with a length of 20 to 40 bp (base pairs) , preferably a length of 25 to 40 bp, much preferably a length of 31 bp. The sequence chosen within said region is also referred to as hybridizing sequence or sequence with a specific hybridizing length. The whole detection sequence can be longer, i.e. it may comprise at least one non hybridizing partial sequence. The hybridizing sequence can be the specified sequence or a partial sequence within the specified range or such sequence comprising universal bases as substitute bases. At places where mismatches are expected due to nucleotide mutations, the detection sequence can comprise any replacement base which allows hydrogen-bounding with the mutated nucleotide. All these sequences are also referred to as sequences derived from the specific region. Such sequences can be obtained by generally known methods, in particular by synthesis.

The respective detection sequence thus comprises a sequence selected within or corresponding to the following sequence (SEQ. ID. NO. 2)

TATTATCTTGTATTACTACTGCCCCTTCACCTTTCCAGAG or the respective complementary sequence, including specific G-U base pairing.

The most interesting sequence to be used for the detection of HIV-1 viruses of the 0 group is in the LTR region. Said sequence comprises a sequence within or is the sequence from nucleotides 653 to 680 and has a hybridizing length (see above) of 17 to 28 bp, preferably a hybridizing length of 23 bp. The respective sequence is preferably selected within the following sequence (SEQ. ID. NO. 7) AAATCTCTAGCAGTGGCGCCCGAACAGG whereby the sequence CGAACA is preferably present, or the respective complementary sequence, including specific G-U base pairing.

These detection sequences can - dependent on the detection method to be used - act as primer sequences or probe sequences . In the scope of the present invention, primer and probe are distinguished in that sequences with the predominant purpose to start and maintain amplification are called primers, sequences with the predominant purpose to indicate hybridization due to spe- cific labeling are called probes.

It has surprisingly been found that the reliability of primer and/or probe involving methods can further be enhanced if the primer (s) /probe (s) , in particular the detection sequences, are selected thus that they meet or are optimized, respectively, with regard to specific requirements . Said requirements are :

- G and C content

- no duplex formation between primer (s) and/or probe(s), - no hairpin formation within the primer (s) and/or probe(s),

- no false priming/hybridization sites for selected primer (s) /probe (s) ,

- no hybridization/priming with itself for the selected primer (s) /probe (s) ,

and, if primer (s) and as detection sequence acting probe (s) are simultaneously present,

- a T_m of the probe (s) that is about 9 to

12 °C, preferably about 10 - 11 °C higher than that of the primer (s) .

In the scope of the further description of the invention and the claims, wherever reference is made to sequences or sets of sequences it shall be understood that such references concern the complementary sequences as well.

The preferred detection sequence in the pol region comprises the sequence TTATCTTGTATTACTACTGCCCCTTCACCTT (nucleotides

4960 to 4990, SEQ. ID. NO. 3) .

A preferred method for detecting and quanti- fymg HIV-1 infection is the so called TaqMan ® method largely described in the literature. Said method com- prises

- a sense primer

- an antisense primer

- a probe (detection sequence, sense or antisense) comprising a labelling system allowing distinc- tion between unbound probe and degraded probe.

A preferred labeling system is a detectable reporter covalently linked to the probe, the detectable signal of said reporter being quenched by a quencher also covalently linked to the probe, so that the detectable signal is only detected if the reporter and the quencher are separated. Such separation is obtained if a probe is first annealed to the sequence of interest and then degraded due to the PCR reaction starting from a primer also present. It is of course also possible and it is a further object of the present invention to use different sets of sequences together, e.g. the set of the pol range and the set of the LTR range, or one or both of those in combination with at least one other set. It is still another object of the present invention to provide a kit for such analysis such kit comprising probes and primers enabling the detection of at least one sequence of interest. Modes for Carrying out the Invention

As already mentioned above, the present invention in one aspect concerns detection sequences, i.e. nucleotide sequences that are especially suitable to detect and quantify HIV-1 infections. Such nucleotide sequences comprise a sequence hybridizing to a particularly well conserved, characteristic region of human immunodeficiency virus-1 (HIV-1) . Dependent on the analytical method chosen, the length of the detection sequence may vary within certain limits. For the preferred, quite generally applicable pol sequence derived detection sequences, the hybridizing part should not be shorter than 20 bp and not longer than 40 bp, and it has preferably a length of about 31 bp. It is of course possible to add non- hybridizing, or non-matching, respectively, nucleotides as far as they do neither affect the desired hybridization nor the nucleic acid sequence synthesis such as PCR. Preferred detection sequences of the present invention are uninterrupted partial sequences of the nucleotide sequence (SEQ. ID. NO. 2)

TATTATCTTGTATTACTACTGCCCCTTCACCTTTCCAGAG. Comprising in connection with the sequences of the present invention means that they hybridize over at least the mentioned length (at most three, usually two mismatches) and may or may not have further nucleotides (non-matching or non-hybridizing, respectively) added. Respective sequences of the LTR region are uninterrupted partial sequences of the following sequence (SEQ. ID. NO. 7)

AAATCTCTAGCAGTGGCGCCCGAACAGG with a hybridizing length of 17 to 28 bp, preferably with a length of about 23 bp. It is furthermore preferred if the detection sequence comprises the sequence CGAACA. Preferred detection sequences of the present invention are furthermore optimized with regard to the following characteristics:

- G and C content, - no duplex formation between primer (s) and/or probe(s),

- no hairpin formation within the primer (s) and/or probe(s),

- no false priming/hybridization sites for selected primer (s) /probe (s) ,

- no priming/hybridization with itself for the selected primer (s) /probe (s) , whereby the G and C content is no criterium in the specific LTR region and would be at most about 45%, preferably less 40% in the pol region.

It has to be understood that the optimization has always to be performed for the lowest temperature reasonably applied for a specific analytical method, i.e. the most critical temperature for nucleotide interac- tions. Said temperature usually is about 37 to 40 °C.

Much preferred sequences being optimized with regard to said further characteristics are in the pol region

TTATCTTGTATTACTACTGCCCCTTCACC T (SEQ. ID. NO. 3) and in the LTR region

TCTCTAGCAGTGGCGCCCGAACA (SEQ. ID. NO. 8) .

In a further aspect of the present invention, such detection sequences are not used either the one or the other, but in combination. The combined use is possible either simultaneously or one after the other. The present invention therefore also encompasses a set of detection sequences that comprises at least one of the sequences derived from the pol region and at least one of the sequences derived from the LTR region. Alternatively, it is of course also possible to use a sequence derived from the pol region and/or a sequence derived from the LTR region in combination with other detection sequences . However, since the sequences known so far are less sensitive, such combination is not preferred at present.

In a further aspect, the present invention also provides an analytical system of primers/probe (s) comprising as at least one probe a sequence as defined above as detection sequence and having a T_m of the probe that is 9 to 12°C, preferably about 10 - 11 °C higher than the one of the primers. For such systems, the com- plementary strand of the detection sequence is less preferred.

A preferred analytical system or set comprises at least one analytical system of forward and reverse primer and probe selected from the group consisting of the pol region derived system.

Although the primers in such a system comprising primers and probe add to the sensitivity, selectivity and reliability of the system, they are not referred to as detection sequences within the scope of the present invention.

It has been found that preferred regions and length for primers to be used in combination with the inventive detection sequences are: in the LTR region primer length in bp range in bp range optimal sense 19 - 33 25 600 - 650 antisense 15 - 32 17 710 - 750 in the pol region primer length in bp range in bp range optimal antisense 19 - 30 24 4995 - 5090 sense 15 - 26 19 4920 - 4955

The distance between the primer and the 5 ' - end of the probe for the pol system is from 1 bp to 15 bp, preferably 8 bp, in the LTR system from 1 to 20 bp, whereby 20 bp are preferred.

Much preferred analytical systems or sets of analytical systems comprise the pol region derived se- quences primer (sense) TGGAAAGGACCAGCAAAGC

(SEQ. ID. NO. 4) primer (antisense) CCTTTCTTCTTGGCACTACTTTTA (SEQ. ID. NO. 5) probe (antisense) TTATCTTGTATTACTACTGCCCCTTCACCTT

(SEQ. ID. NO. 3)

and/or the LTR region derived sequences

primer (sense) CTGGTGTCTAGAGATCCCTCAGATC

(SEQ. ID. NO. 9) primer (antisense) TAAGCCGAGCCCGTTGC

(SEQ. ID. NO. 10) probe (sense) TCTCTAGCAGTGGCGCCCGAACA (SEQ. ID. NO. 8) .

Such analytical systems or sets of sequences or systems of course can also be present in the form of kits comprising separable specific primers and probes, enzyme reaction buffer, MgCl2 stock solution, nucleotide master mix, Taq polymerase, reverse transcriptase, RNase inhibitor, positive and negative control RNA, internal run control and quantitative HIV-1 RNA standard.

The present invention also provides for a method for detecting and quantifying HIV-1. Said method comprises at least one PCR reaction involving as primer or probe one of the detection sequences described above, or a system of nucleotide sequences comprising such a detection sequence together with suitable primers . Respec- tive methods are known to the skilled person and a selection has already been mentioned above . A much preferred method is the so called

® TaqMan method that uses a probe comprising a reporter dye and a quencher dye bound to different nucleotides of the probe such that during removal of the hybridized probe said probe is cleaved such that the reporter dye and the quencher dye are separated. Due to said separation, the reporter signal becomes detectable because of the "absence" of the quencher dye.

® The TaqMan method thus involves at least one analytical system comprising sense and antisense primers and a probe labeled with a system of reporter dye and quencher dye bound to different nucleotides of the probe. A sample to be analyzed is treated with the probe and primers under conditions first allowing hybridization of said primers and probe to a target of interest, whereupon the probe is removed due to a nucleic acid sequence synthesis starting from the corresponding primer. Due to the fact that the probe is not only separated from the target during the sequence synthesis but also cleaved, the se- quence synthesis leads to a separation of the reporter dye and the quencher dye and thus to a detectable reporter signal.

® A preferred TaqMan method is based on the intrinsic 5 ' —> 3' exonuclease activity of preferred Taq, Tth or Tfl DNA polymerases which cleaves fluorescein (FAM; reporter dye) labelled and rhodamine (TAMRA; quencher dye) labelled probes hybridized to amplicons of the pol gene or, respectively, to the LTR gene. Probe cleavage generates specific fluorescent signals whose in- tensity is real-time quantified during amplification by laser-based fluorometry using a suitable detector such as e.g. the ABI PRISM 7700 Sequence Detector (PE Applied

Biosystems) .

® In order to get a good working TaqMan analy- sis, the distance between forward primer and probe has to be at least 1, usually about 4 nucleotides. The upper limit for the distance as well as the preferred distance are dependent from the system and have already been mentioned above. The TaqMan ® method of the present invention thus provides a very suitable 5' nuclease PCR assay to rapidly detect and quantify HIV-1 RNA in plasma samples.

® Preferred sequences for the TaqMan method are in the pol region: primer (sense) TGGAAAGGACCAGCAAAGC (SEQ. ID. NO. 4) primer (anti-sense) CCTTTCTTCTTGGCACTACTTTTA

(SEQ. ID. NO. 5) probe (anti-sense) TTATCTTGTATTACTACTGCCCCTTCACCTT

(SEQ. ID. NO. 3)

in the LTR region: primer (sense) CTGGTGTCTAGAGATCCCTCAGATC

(SEQ. ID. NO. 9) primer (anti-sense) TAAGCCGAGCCCGTTGC (SEQ. ID. NO. 10) probe (sense) TCTCTAGCAGTGGCGCCCGAACA

(SEQ. ID. NO. 8) .

Using the above described TaqMan ® method, with the disclosed primers and probes it was possible to reliably detect 10 copies of purified viral full-length RNA or plas id DNA with a dynamic range of 5-6 logs of HIV-1 subtype A, B, C, D, E, F, G, H, i.e. group M, (all with the pol selective sequences) and group 0 (with the LTR selective sequences) .

The presently described PCR system tolerates up to 3, usually 2 mismatches between the template and the 31-mer (pol) and 23-mer (LTR) probes or the flanking primers, respectively. Best results with the above mentioned systems were found with the AMV/Tfl DNA Pol enzyme combination. Even low copy numbers of a variety of HIV-1 subtypes in plasma specimens are quantitatively and reproducibly de-

® . . . tectable . A TaqMan assay of the present invention is specific and sensitive for B- and non-B HIV-1 subtypes, eliminates the need for laborious post PCR processing and permits testing of 96 samples within 2.5 hours. This makes it ideal for routine diagnostic use.

The invention is now further described by means of examples.

EXAMPLES

Example 1: PCR Primers, fluorogenic probes, target sequences and oligo design

Two pairs of probe/flanking primers in specific and highly conserved pol respective LTR regions of HIV-1 were individually designed after multiple sequence alignment of all HIV-1 subtypes (Online HIV-Sequence Database from the Los Alamos National Laboratory) using the OLIGO 5.0 primer analysis software, extended by the further criteria of the present invention.

The primer pairs and probes (Table 1) were selected under consideration of the rules that no intrinsic loop, di er formation, internal structural instabilities (ΔG) or false priming sites should impair specific template-hybridization and of the optimal melting temperature (range 54°C-68°C) . Moreover, probe self-complementary or probe complementary to the flanking primers; 5 or more identical nucleotides, esp. Gs in a row; no G at the probes ' s 5 'end; and a melting temperature not more than 7°C above annealing temperature, were avoided. Because all major subtypes (and group 0) of

HIV-1 should be detectable, probe and primers were de- signed to have a high cross-reactivity with all known HIV-1 strains.

Table 3 :

Pol resp . LTR primers and probes

*dual-labeled probe; as = antisense , s = sense

Subsequent synthesis and HPLC-grade purification of primers and fluorogenic probes (5 '-labeled with reporter FAM ( 6-carboxyfluorescein) and 3 '-labeled with quencher TAMRA (6-carboxy-tetramethyl-rhodamine) was done by PERKIN ELMER, Germany, MICROSYNTH, Switzerland and EUROGENTEC, Belgium, respectively. The TaqMan probes contained a 3 ' -blocking phosphate group, to prevent probe extension during PCR.

Example 2: Real-Time RT-PCR Assay and data analysis

RNA extracted from plasma, serum or cell cul- ture supernatants was reverse transcribed and amplified in a one-tube reaction (0.05 - 0.1 ml duplicates with 0.001 - 0.025 ml diluted templates; 45 - 50 cycles) using the Access RT-PCR System (PROMEGA) in accordance with manufacturer's instructions, slightly modified:

The 0.05 ml mixture for one PCR reaction contained, except template, 5 U of AMV reverse transcrip- tase, 5 U of Tfl polymerase, 4.5 mM MgS0₄, 0.2 mM each dNTP Mix, 400 nM of up- and downstream primers, 400-800 nM of fluorogenic probe (for primers and probes see Ex. 1), 0.007 ml optimized single-buffer 5x (Access RT-PCR System), 0.0015 ml, including the passive reference dye ROX, PCR buffer A lOx; pH 8.3 (PERKIN ELMER), 40 U recom- binant RNasin Ribonuclease Inhibitor, all in nuclease- free water .

One-step reverse transcription and amplification were carried out in a single thin-wall tube in an ABI PRISM 7700 Sequence Detector based on the 5' nuclease assay. Fluorescence intensity at 518 nm is monitored continuously real time during DNA amplification (96-well plate format) , and the data are captured onto a Macintosh computer. Run profile analysis was done by the Sequence Detection System Software (SDS Vers . 1.6.3).

The following temperature profile was used: 45 min at 48°C reverse transcription; a de- naturation and AMV RT inactivation step of 2 min at 94°C; annealing at 58-62°C for 1 min with no further extension step; strand denaturation at 94°C for 15-30 s.

Because an international WHO HIV-1 Standard is still not available, two different in-house HIV-1 subtype B RNA Standards were used (38E06 - 3.8, resp. 1.5E02 - 1 copy per reaction) . Specificity of amplifications was verified by

2 % agarose gel electrophoresis and ethidium bromide staining. Results :

Independent of virus source (plasma, serum, cell culture supernatants) , it was possible to reproduci- bly (i.e. > 10 independent amplification experiments) detect 1-10 copies of purified HIV-1 RNA (specified subtypes A, B, C, D, E, F, G, H and 0) , with a dynamic range of 6 logs per 0.050 ml RT-PCR reaction.

The absolute copy numbers per reaction were determined by Amplicor testing (ROCHE Kit v.1.5) . After amplification, each Taqman reaction was checked in direct comparison to a positive and a negative control on a 2% agarose gel for the prescence of the specific 91 bp-band (corresp. to the pol region) resp. the specific 127 bp- band (corresp. to the LTR region) . PCR products were also confirmed by DNA sequencing. In addition, it was found that the described Taqman PCR system tolerates up to 3 mismatches between the HIV-1 template and the 31-mer pol probe resp. the flanking primers.

Example 3: Full-length HIV-1 DNA plasmids , HIV-1 PBMC gDNA, or other HIV-1 DNA prepat ions

Amplification of DNA templates was done under the conditions described in Example 2 without the reverse transcription step, AMV RT and RNasin.

Results:

It was possible to reliably detect purified HIV-1 PBMC-derived genomic DNA, provirus DNA and purified full-length plasmid DNA in the minimum range of 10-100 copies per 0.050 ml reaction (dynamic range of 5 logs), depending on subtype (A, B, C, E, F, H) and/or plasmid type (pBT-1, pLTR, pTZ18, pCRII, pCR2.1). Moreover, it was also possible to detect, with the same specifity and sensitivity HIV-1 DNA from differnt preparations (e.g. Ficoll-, Trizol-, Phenol/Chloroform- , Silica—based- purified DNA) . Pol (91bp) specifity was checked together with a negative and a positive contol on a 2% agarose gel . DNA concentration per reaction was determined photometrically at 260 n .

While there are shown and described presently preferred embodiments of the invention, it is to be distinctly understood that the invention is not limited thereto but may be otherwise variously embodied and practiced within the scope of the following claims.

Claims

1. A detection sequence comprising a nucleotide sequence hybridizing to a particularly well con- served, characteristic region of human immunodeficiency virus-1 (HIV-1) , having a hybridizing length of 20 to 40 bp, said sequence being a uninterrupted partial sequence derived from the following sequence

TATTATCTTGTATTACTACTGCCCCTTCACCTTTCCAGAG (SEQ. ID. NO. 2) .

2. The nucleotide sequence of claim 1 that is optimized with regard the following characteristics:

- G and C content

- no duplex formation between primer (s) and/or probe(s),

- no hairpin formation within the primer (s) and/or probe (s),

- no false priming/hybridization sites for selected primer (s) /probe (s) , - no priming with itself.

3. The nucleotide sequence of claim 1 or 2 that comprises the sequence

TTATCTTGTATTACTACTGCCCCTTCACCTT (SEQ. ID. NO. 3) .

4. The nucleotide sequence of claim 3 that is the sequence

TTATCTTGTATTACTACTGCCCCTTCACCTT (SEQ. ID. NO. 3) .

5. An analytical system selected with the se- quence of Table 1 (SEQ. ID.NO. 1) at least comprising a sense primer, an antisense primer and a probe (sense or antisense) , wherein said probe comprises a sequence according to one of claims 1 to 4 and has a T_m of 9 to 12°C, preferably about 10 - 11 °C higher, than the one of the primers.

6. The analytical system of claim 5 wherein the sequences are selected as follows - antisense primer with a length from 19 to 30 bp, preferably 24 bp, selected in the range from 4995 to 5090 bp,

- sense primer with a length from 15 to 26 bp, preferably 19 bp, selected in the range from 4920 to 4955 bp,

- probe with a length from 20 to 40 bp, preferably 31 bp, selected in the range from 4953 to 4992 bp, whereby the distance between antisense primer and the 5 '-end of the antisense probe is from 1 to 15 bp, preferably 8 bp .

7. The analytical system of claim 6 that comprises the following sequences primer (sense) TGGAAAGGACCAGCAAAGC (SEQ. ID. NO. 4) primer (antisense) CCTTTCTTCTTGGCACTACTTTTA

(SEQ. ID. NO. 5) probe (antisense) TTATCTTGTATTACTACTGCCCCTTCACCTT (SEQ. ID. NO. 3)

8. A nucleotide sequence comprising as detection sequence a sequence hybridizing to a particularly well conserved, characteristic region of human immunodeficiency virus-1 (HIV-1) , having a hybridizing length of 17 to 32 bp, said sequence being a uninterrupted partial sequence derived from the following sequence

AAATCTCTAGCAGTGGCGCCCGAACAGG (SEQ. ID. NO. 7), in particular a sequence comprising the partial sequence CGAACA.

9. The nucleotide sequence of claim 8 that is optimized with regard to the following characteristics:

- no duplex formation between primer (s) and/or probe (s),

- no hairpin formation within the primer (s) and/or probe(s), - no false priming/hybridization sites for selected primer (s) /probe (s) ,

- no priming with itself.

10. The nucleotide sequence of claim 8 or 9 that comprises the sequence

TCTCTAGCAGTGGCGCCCGAACA (SEQ. ID. NO. 8) .

11. The nucleotide sequence of claim 10 that is the sequence

TCTCTAGCAGTGGCGCCCGAACA (SEQ. ID. NO. 8) .

12. An analytical system selected within the sequence of Table 2 (SEQ. ID.NO. 6) at least comprising a sense primer, a antisense primer and a probe (sense or antisense) , wherein said probe comprises a sequence according to one of claims 1 to 4 and has a T_m that is about 10 - 11 °C higher than the one of the primers.

13. The analytical system of claim 12 wherein the sequences are selected as follows - sense primer with a length from 19 to 33 bp, preferably 25 bp, selected in the range from 600 to 650 bp,

- antisense primer with a length from 15 to 32 bp, preferably 17 bp, selected in the range from 710 to 750 bp,

- probe with a length from 17 to 32 bp, preferably 23 bp, selected in the range from 651 to 690 bp, whereby the distance between sense primer and the 5 ' -end of the sense probe is from 1 tu 20 bp, pref- erably 20 bp.

14. The analytical system of claim 13 that comprises the following sequences primer (sense) CTGGTGTCTAGAGATCCCTCAGATC (SEQ. ID. NO. 9) primer (anti-sense) TAAGCCGAGCCCGTTGC

(SEQ. ID. NO. 10) probe (sense) TCTCTAGCAGTGGCGCCCGAACA (SEQ. ID. NO. 8)

15. A set of nucleotide sequences that comprises at least one of the sequences of any one of claims 1 to 7 and at least one of the sequences of any one of claims 8 to 14.

16. The set of claim 15 that comprises the following sequences primer (sense) TGGAAAGGACCAGCAAAGC

(SEQ. ID. NO. 4) primer (antisense) CCTTTCTTCTTGGCACTACTTTTA

(SEQ. ID. NO. 5) probe (antisense) TTATCTTGTATTACTACTGCCCCTTCACCTT (SEQ. ID. NO. 3) and primer (sense) CTGGTGTCTAGAGATCCCTCAGATC

(SEQ. ID. NO. 9) primer (antisense) TAAGCCGAGCCCGTTGC (SEQ. ID. NO. 10) probe (sense) TCTCTAGCAGTGGCGCCCGAACA (SEQ. ID. NO. 8) .

17. A method for detecting and quantifying HIV-1 in a sample by nucleic acid amplification technol- ogy that comprises at least one detection sequence selected from the sequences of any one of claims 1 to 4 or at least one detection sequence selected from the sequences of any one of claims 8 to 11 or at least one set of claim 15.

18. A method for detecting and quantifying

HIV-1 in a sample by nucleic acid amplification technology that comprises at least one analytical system of one of claims 5 to 7 or at least one analytical system of one of claims 12 to 14 or at least one set of claim 16.

19. The method of claim 18 wherein the probe comprises a reporter dye and a quencher dye bound to different nucleotides of the probe, and wherein the sample is treated with the probe and primers under conditions first allowing hybridization of the probe and the primers with a target of interest followed by removal of the probe due to nucleic acid sequence synthesis, whereby the removal of the probe leads to a cleavage of the probe thus that the reporter dye and the quencher dye are separated.

20. The method of claim 19 wherein the nucleic acid sequence synthesis is a polymerase chain reac- tion.