U.S. patent application number 11/372586 was filed with the patent office on 2006-10-05 for nucleic acid sequences that can be used as primers and probes in the amplification and detection of all subtypes of hiv-1.
This patent application is currently assigned to bioMerieux SA. Invention is credited to Jaap Goudsmit, Suzanne Jurriaans, Vladimir Vladimirovich Lukashov, Pieter Oudshoorn.
Application Number | 20060223057 11/372586 |
Document ID | / |
Family ID | 8228630 |
Filed Date | 2006-10-05 |
United States Patent
Application |
20060223057 |
Kind Code |
A1 |
Goudsmit; Jaap ; et
al. |
October 5, 2006 |
Nucleic acid sequences that can be used as primers and probes in
the amplification and detection of all subtypes of HIV-1
Abstract
The present invention is related to nucleic acid sequences that
can be used in the field of virus diagnostics, more specifically
the diagnosis of infections with the AIDS causing Human
Immuno-deficiency Virus (HIV). With the present invention
nucleotide sequences are provided that can be used as primers and
probes in the amplification and detection of HIV-1 nucleic acid.
The oligonucleotide sequences provided with the present invention
are located in the LTR part of the HIV viral genome. It has been
found that, by using the sequences of the present invention in
methods for the amplification and detection of nucleic acid a
sensitive and specific detection of HIV-1 can be obtained. The
benefit of the sequences of the present invention primarily resides
in the fact that, with the aid of primers and probes comprising the
sequences according to the invention the nucleic acid of all
presently known subtypes of HIV-1 can be detected with high
accuracy and sensitivity. So far no primer pairs or hybridization
probes have been developed that would allow the detection of such a
broad range of HIV-1 variants. The oligonucleotide sequences
according to the present invention are especially useful in methods
for the amplification of nucleic acid.
Inventors: |
Goudsmit; Jaap; (Amsterdam,
NL) ; Oudshoorn; Pieter; (St. Michielsgestel, NL)
; Jurriaans; Suzanne; (Monnickendam, NL) ;
Lukashov; Vladimir Vladimirovich; (Diemen, NL) |
Correspondence
Address: |
MYERS BIGEL SIBLEY & SAJOVEC
PO BOX 37428
RALEIGH
NC
27627
US
|
Assignee: |
bioMerieux SA
|
Family ID: |
8228630 |
Appl. No.: |
11/372586 |
Filed: |
March 10, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11108223 |
Apr 18, 2005 |
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11372586 |
Mar 10, 2006 |
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09463352 |
Jan 21, 2000 |
6881537 |
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PCT/EP98/04945 |
Aug 5, 1998 |
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11108223 |
Apr 18, 2005 |
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Current U.S.
Class: |
435/5 ; 435/91.2;
536/23.72 |
Current CPC
Class: |
Y10S 435/81 20130101;
C12Q 1/703 20130101; C12Q 1/6848 20130101 |
Class at
Publication: |
435/005 ;
435/006; 435/091.2; 536/023.72 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70; C12Q 1/68 20060101 C12Q001/68; C12P 19/34 20060101
C12P019/34; C07H 21/02 20060101 C07H021/02 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 8, 1997 |
EP |
97202455.8 |
Claims
1-13. (canceled)
14. A pair of oligonucleotide primers, for use as a primer set in
the amplification of a target sequence located within the LTR
region of the genome of HIV-1, said pair consisting of a first
oligonucleotide being 10-50 nucleotides in length and comprising 10
sequential nucleotides of a nucleotide sequence selected from the
group consisting of: TABLE-US-00012 SEQ ID NO:1: G GGC GCC ACT GCT
AGA GA; SEQ ID NO:2: G TTC GGG CGC CAC TGC TAG A; and SEQ ID NO:3:
CGG GCG CCA CTG CTA,
and a second oligonucleotide being 10-50 nucleotides in length and
comprising 10 sequential nucleotides of a nucleotide sequence
selected from the group consisting of: TABLE-US-00013 SEQ ID NO:4:
CTG CTT AAA GCC TCA ATA AA; SEQ ID NO:5: CTC AAT AAA GCT TGC CTT
GA; and SEQ ID NO:12: GAT GCA TGC TCA ATA AAG CTT GCC TTG AGT.
15. A pair of oligonucleotide primers, for use as a primer set in
the amplification of a target sequence located within the LTR
region of the genome of HIV-1, said pair consisting of a first
oligonucleotide, which hybridizes to the target sequence or its
complement, being 10-26 nucleotides in length and comprising 10
sequential nucleotides of a nucleotide sequence selected from the
group consisting of: TABLE-US-00014 SEQ ID NO:1: G GGC GCC ACT GCT
AGA GA; SEQ ID NO:2: G TTC GGG CGC CAC TGC TAG A; and SEQ ID NO:3:
CGG GCG CCA CTG CTA,
and a second oligonucleotide, which hybridizes to the target
sequence or its complement, being 10-26 nucleotides in length and
comprising 10 sequential nucleotides of a nucleotide sequence
selected from the group consisting of: TABLE-US-00015 SEQ ID NO:4:
CTG CTT AAA GCC TCA ATA AA; SEQ ID NO:5: CTC AAT AAA GCT TGC CTT
GA; and SEQ ID NO:12: GAT GCA TGC TCA ATA AAG CTT GCC TTG AGT.
16. The pair of oligonucleotide primers of claim 14, wherein the
first oligonucleotide comprises 10 sequential nucleotides of the
nucleotide sequence of SEQ ID NO: 1: G GGC GCC ACT GCT AGA GA; and
wherein the second oligonucleotide comprises 10 sequential
nucleotides of the nucleotide sequence of SEQ ID NO:5: CTC AAT AAA
GCT TGC CTT GA.
17. The pair of oligonucleotide primers of claim 15, wherein the
first oligonucleotide comprises 10 sequential nucleotides of the
nucleotide sequence of SEQ ID NO: 1: G GGC GCC ACT GCT AGA GA; and
wherein the second oligonucleotide comprises 10 sequential
nucleotides of the nucleotide sequence of SEQ ID NO:5: CTC AAT AAA
GCT TGC CTT GA.
18. The pair of oligonucleotide primers of claim 14, 15, 16 or 17,
wherein the first oligonucleotide is additionally provided with a
promoter sequence recognized by a DNA dependent RNA polymerase.
19. The pair of oligonucleotide primers of claim 18, wherein the
promoter sequence is a T7 promoter sequence.
20. The pair of oligonucleotide primers of claim 19, wherein the
first oligonucleotide consists essentially of the nucleotide
sequence of SEQ ID NO:9: aat tct aat acg act cac tat agg gAG AGG
GGC GCC ACT GCT AGA GA and wherein the second oligonucleotide
consists essentially of the nucleotide sequence of SEQ ID NO:5: CTC
AAT AAA GCT TGC CTT GA.
21. A method for the detection of HIV-1 nucleic acid in a sample,
comprising: a) contacting the sample with the pair of
oligonucleotide primers of any of claims 14-20 under conditions
whereby an amplification reaction can occur; and b) detecting the
presence of amplified HIV-1 nucleic acid, thereby detecting HIV-1
nucleic acid in the sample.
22. The method of claim 21, wherein the detection of amplified
nucleic acid is carried out by reacting the sample with one or more
oligonucleotides selected from the group consisting of:
TABLE-US-00016 SEQ ID NO:6: TCT GGT AAC TAG AGA TCC CTC; SEQ ID
NO:7: TAG TGT GTG CCC GTC TGT; and SEQ ID NO:8: AGT GTG TGC CCG TCT
GTT,
under conditions whereby a hybridization complex can form, and
detecting the presence of the hybridization complex, thereby
detecting HIV-1 nucleic acid in the sample.
23. A method for amplifying a target sequence located within the
LTR region of the genome of HIV-1, comprising contacting the sample
with the pair of oligonucleotide primers of any of claims 14-20
under conditions whereby an amplification reaction can occur.
24. The method of claims 21 or 23, wherein the amplification
reaction is a transcription based amplification system
reaction.
25. The method of claim 24, wherein the transcription based
amplification system reaction is a nucleic acid sequence based
amplification reaction.
26. A test kit for the detection of HIV-1 in a sample, comprising:
a) the pair of oligonucleotide primers of any of claims 14-20; b)
one or more detectably labeled oligonucleotides comprising a
nucleic acid sequence substantially complementary to at least part
of a target sequence produced in an amplification reaction
comprising the pair of oligonucleotides of (a); and c) suitable
amplification reagents.
Description
RELATED APPLICATIONS
[0001] This application is a continuation application of, and
claims priority to U.S. application Ser. No. 11/108,233 (pending),
filed Apr. 18, 2005, which is a continuation of U.S. application
Ser. No. 09/463,352, filed Jan. 21, 2000 and issued Apr. 19, 2005
as U.S. Pat. No. 6,881,537, which is a 35 U.S.C. .sctn. 371
National Phase Application of International Application Serial No.
PCT/EP98/04945, filed Aug. 5, 1998 and published in English as PCT
Publication No. WO 99/07898 on Feb. 18, 1999, which claims priority
to European Patent Application No. EP 97202455.8, filed Aug. 8,
1997. Each of these applications is incorporated herein by
reference in its entirety.
FIELD OF INVENTION
[0002] The present invention is related to nucleic acid sequences
that can be used in the field of virus diagnostics, more
specifically the diagnosis of infections with the AIDS causing
Human Immunodeficiency Virus (HIV).
BACKGROUND OF THE INVENTION
[0003] Whereas conventional virus diagnosis has been based
predominantly on the detection of viral antigens or specific
antibodies thereto, in recent years attention has shifted towards
methods for the direct detection of the genome of viruses or
nucleic acid sequences derived thereof, both RNA and DNA. These
methods are usually based on nucleic acid hybridization. Nucleic
acid hybridization is based on the ability of two strands of
nucleic acid containing complementary sequences to anneal to each
other under the appropriate conditions, thus forming a double
stranded structure. When the complementary strand is labeled, the
label can be detected and is indicative for the presence of the
target sequence. Especially in combination with methods for the
amplification of nucleic acid sequences these methods have become
an important tool in viral diagnosis, in particular for the
detection of human immunodeficiency virus (HIV).
[0004] Nucleic acid amplification techniques are especially useful
as an additional technique in cases where serological methods give
doubtful results or in cases where there may be a considerable time
period between infection and the development of antibodies to the
virus. With HIV, seroconversion usually can occur some 36 months
after exposure to the virus. Thus, whereas no antibodies will be
detected with conventional immunoassays, proviral DNA or
circulating viral RNA may already be detectable. Also in monitoring
antiviral therapy, methods based on nucleic acid amplification have
several advantages over serological methods. Especially
quantitative amplification methods provide a powerful tool in
assessing the changes in the amount of virus present before and
during therapy.
[0005] The choice of the oligonucleotides to be used as primers and
probes in the amplification and detection of nucleic acid sequences
is critical for the sensitivity and specificity of the assay. The
sequence to be amplified is usually only present in a sample (for
example a blood sample obtained from a patient suspected of having
a viral infection) in minute amounts. The primers should be
sufficiently complementary to the target sequence to allow
efficient amplification of the viral nucleic acid present in the
sample. If the primers do not anneal properly (due to mispairing of
the bases on the nucleotides in both strands) to the target
sequence, amplification is seriously hampered. This will affect the
sensitivity of the assay and may result in false negative test
results. Due to the heterogeneity of viral genomes false negative
test results may be obtained if the primers and probes are capable
of recognizing sequences present in only part of the variants of
the virus. The HIV virus shows a high heterogeneity. Genetic
variability has been demonstrated amongst isolates from different
continents but also between individuals and between different
stages of the disease. Based on sequence analysis two groups within
HIV-1 have been identified: group M (M for "major"), and group O (O
for "outlier"). Within group M subtypes (A-H), each constituting a
phylogenetic separate set of sequences, have been assigned and
additional ones are being identified. This sequence variation is
not uniformly distributed throughout the genome. The HIV-1 genome,
like all retroviral genomes, roughly consists of the following
regions: The gag gene of the HIV-1 genome is the region encoding
the core proteins of the virus (for example, p24). The env gene
encodes a large precursor protein, gp160, which is processed into
the envelop proteins gp120 and gp41. The pol gene encodes the
polymerase of the virus (reverse transcriptase). The Long Terminal
Repeat regions (LTRs) are the regions on the viral genome that
participate in the integration of the virus with the host cell and
in the regulation of transcription of the viral genes. Some regions
are more prone to sequence variation than others. Especially in the
env domain sequence variation can be as high as 30% between members
of the different subtypes. Ideally, primer selection should be
based on knowledge of interstrain variability in candidate primer
sequences and the consequences of mismatching at primer sites.
McCutchan et al, J. AIDS, 4, 1241-1250, 1991, used PCR to make a
genetic comparison of different HIV-1 isolates. Using anchored PCR
(varying sense primers were used with a constant antisense primer.
primers were chosen from relatively conserved regions in gag, env
and LTR), the effect of primer mispairing on the amount of PCR
product obtained was also investigated. Mispairing at the 3' end of
the primer decreased the amount of product sometimes more then
100-fold.
[0006] The detection of all presently known subtypes of HIV-1 is of
extreme importance, especially with regard to patient management,
security of blood and blood products and clinical and
epidemiological studies. Current assays for the amplification and
subsequent detection of HIV-1 derived nucleic acid sequences are
usually based on amplification of sequences in the gag region of
the viral genome. These assays have been developed for subtype B,
which is the major subtype in European countries and the United
States. However, the presence of other subtypes, which were
geographically confined before, is increasing due to frequent
travel between these countries and, for example, African countries.
Sensitive assays are therefore needed that are capable of detecting
as much variants of the HIV-1 virus as possible (preferably
all).
SUMMARY OF THE INVENTION
[0007] Research aimed at identifying suitable primer sets for the
reliable amplification of HIV-1 derived nucleic acid sequences has
been ongoing for the past years. Engelbrecht et al., J. Virol.
Meth., 55, 391400, 1995, describe a study aimed at the development
of a specific and sensitive PCR protocol using env, gag and LTR
primer pairs to detect subtypes present in the Western Cape, South
Africa. Twenty four strains of which it was known that they
belonged to subtypes B, C and D were analyzed. It was found that
the performance of the primer pairs was greatly dependent on the
optimization of the reaction conditions for the different primer
pairs. Only when less stringent conditions were used (for example,
with the LTR primer pair an increased cycle time and lower
annealing temperatures were required) these particular strains of
HIV could be detected with sufficient sensitivity and
reproducibility with all primer pairs.
DETAILED DESCRIPTION OF THE INVENTION
[0008] Zazzi et al., J. Med. Virol., 38, 172-174, 1992, developed a
two-step PCR reaction (using nested primers) for the detection of
HIV-1 DNA in clinical samples. The primers used for amplification
were derived from the gag gene and the LTR region. The patients
tested in this study were all from neighbouring areas, which makes
it likely that they represent only a limited number of different
viral strains.
[0009] A quantitative PCR method using LTR derived nested primers
was described by Vener et al. in BioTechniques, 21, 248255, 1996.
This procedure was only tested on HIV-1.sub.MN infected peripheral
blood mononuclear cells (PBMC). Thus nothing can be said about the
suitability of the primers used for detecting different subtypes of
the virus.
[0010] With the present invention nucleotide sequences are provided
that can be used as primers and probes in the amplification and
detection of HIV-1 nucleic acid. The oligonucleotide sequences
provided with the present invention are located in the LTR part of
the HIV viral genome. It has been found that, by using the
sequences of the present invention in methods for the amplification
and detection of nucleic acid a sensitive and specific detection of
HIV-1 can be obtained. The benefit of the sequences of the present
invention primarily resides in the fact that, with the aid of
primers and probes comprising the sequences according to the
invention the nucleic acid of all presently known subtypes of HIV-1
can be detected with high accuracy and sensitivity. So far no
primer pairs or hybridization probes have been developed that would
allow the detection of such a broad range of HIV-1 variants.
[0011] The oligonucleotide sequences according to the present
invention are especially useful in methods for the amplification of
nucleic acid.
[0012] Various techniques for amplifying nucleic acid are known in
the art. One example of a technique for the amplification of a DNA
target segment is the so-called "polymerase chain reaction" (PCR).
With the PCR technique the copy number of a particular target
segment is increased exponentially with a number of cycles. A pair
of primers is used and in each cycle a DNA primer is annealed to
the 3' side of each of the two strands of the double stranded
DNA-target sequence. The primers are extended with a DNA polymerase
in the presence of the various mononucleotides to generate double
stranded DNA again. The strands of the double stranded DNA are
separated from each other by thermal denaturation and each strand
serves as a template for primer annealing and subsequent elongation
in a following cycle. The PCR method has been described in Saiki et
al., Science 230, 135, 1985 and in European Patents no. EP 200362
and EP 201184.
[0013] Another technique for the amplification of nucleic acid is
the so-called transcription based amplification system (TAS). The
TAS method is described in International Patent Appl. no. WO
88/10315. Transcription based amplification techniques usually
comprise treating target nucleic acid with two oligonucleotides one
of which comprises a promoter sequence, to generate a template
including a functional promoter. Multiple copies of RNA are
transcribed from said template and can serve as a basis for further
amplification.
[0014] An isothermal continuous transcription based amplification
method is the so-called NASBA process ("NASBA") as described in
European Patent no. EP 329822. NASBA includes the use of T7 RNA
polymerase to transcribe multiple copies of RNA from a template
including a T7 promoter. Other transcription based amplification
techniques are described in EP 408295. EP 408295 is primarily
concerned with a two-enzyme transcription based amplification
method. Transcription based amplification methods, such as the
NASBA method as described in EP 329822, are usually employed with a
set of oligonucleotides, one of which is provided with a promoter
sequence that is recognized by a DNA dependent RNA polymerase such
as, for example, T7 polymerase. Several modifications of
transcription based techniques are known in the art. These
modifications comprise, for example, the use of blocked
oligonucleotides (that may be provided with a promoter sequence).
These oligos are blocked so as to inhibit an extension reaction
proceeding therefrom (U.S. Pat. No. 5,554,516). One or more
"promoter-primers" (oligonucleotides provided with a promoter
sequence) may be used in transcription based amplification
techniques, optionally combined with the use of one or more
oligonucleotides that are not provided with a promoter sequence.
For RNA amplification, a transcription based amplification
technique, is a preferred technology. Amplification using PCR can
also be based on an RNA template. The actual PCR needs to be
preceded by a reverse transcription step to copy the RNA into DNA
(RT-PCR). However, if RT-PCR is used for the detection of viral
transcripts differentiation of mRNA- and DNA-derived PCR products
is necessary. DNAse treatment prior to RT-PCR can be employed
(Bitsch, A. et al., J. Infect. Dis 167, 740-743, 1993; Meyer, T. et
al., Mol. Cell Probes. 8, 261-271., 1994), but sometimes fails to
remove contaminating DNA sufficiently (Bitsch, A. et al.,
1993).
[0015] In contrast to RT-PCR, NASBA, which is based on RNA
transcription by T7 RNA polymerase (Kievits et al., 1991; Compton,
1991), does not need differentiation between RNA- and DNA-derived
amplification products since it uses RNA as its principal target.
NASBA enables specific amplification of RNA targets even in a
background of DNA.
[0016] The use of the oligonucleotides according to the invention
is not limited to any particular amplification technique or any
particular modification thereof. It is evident that the
oligonucleotides according to the invention find their use in many
different nucleic acid amplification techniques and various methods
for detecting the presence of (amplified) nucleic acid of HIV. The
oligonucleotides of the present invention can likewise be used in
quantitative amplification methods. An example if such quantitative
method is described in EP 525882.
[0017] The term "oligonucleotide" as used herein refers to a
molecule comprised of two or more deoxyribonucleotides or
ribonucleotides. Such oligonucleotides may be used as primers and
probes.
[0018] Of course, based on the sequences of the oligonucleotides of
the present invention, analogues of oligonucleotides can also be
prepared. Such analogues may constitute alternative structures such
as "PNA" (molecules with a peptide-like backbone instead of the
phosphate sugar backbone of normal nucleic acid) or the like. It is
evident that these alternative structures, representing the
sequences of the present invention are likewise part of the present
invention.
[0019] The term "primer" as used herein refers to an
oligonucleotide either naturally occurring (e.g. as a restriction
fragment) or produced synthetically, which is capable of acting as
a point of initiation of synthesis of a primer extension product
which is complementary to a nucleic acid strand (template or target
sequence) when placed under suitable conditions (e.g. buffer, salt,
temperature and pH) in the presence of nucleotides and an agent for
nucleic acid polymerization, such as DNA dependent or RNA dependent
polymerase. A primer must be sufficiently long to prime the
synthesis of extension products in the presence of an agent for
polymerization. A typical primer contains at least about 10
nucleotides in length of a sequence substantially complementary or
homologous to the target sequence, but somewhat longer primers are
preferred. Usually primers contain about 15-26 nucleotides but
longer primers may also be employed, especially when the primers
contain additional sequences such as a promoter sequence for a
particular polymerase.
[0020] Normally a set of primers will consist of at least two
primers, one `upstream` and one `downstream` primer which together
define the amplificate (the sequence that will be amplified using
said primers);
[0021] Primarily for the use in transcription based amplification
techniques, the oligonucleotides according to the invention may
also be linked to a promoter sequence. The term "promoter sequence"
defines a region of a nucleic acid sequence that is specifically
recognized by an RNA polymerase that binds to a recognized sequence
and initiates the process of transcription by which an RNA
transcript is produced. In principle any promoter sequence may be
employed for which there is a known and available polymerase that
is capable of recognizing the initiation sequence. Known and useful
promoters are those that are recognized by certain bacteriophage
RNA polymerases such as bacteriophage T3, T7 or SP6.
Oligonucleotides linked to a promoter sequence are commonly
referred to as "promoter primers". Their function as a primer, e.g.
the starting point for an elongation reaction, however, may be
blocked, as already mentioned above, or absent in some embodiments
of transcription based amplification reactions.
[0022] An oligonucleotide according to the present invention is
substantially complementary to a sequence of the LTR region of a
nucleic acid sequence of a HIV genome, said oligonucleotide being
1050 nucleotides in length and comprising, at least a fragment of
10 nucleotides, of a sequence selected from the group consisting
of: TABLE-US-00001 SEQ ID 1: G GGC GCC ACT GCT AGA GA SEQ ID 2: G
TTC GGG CGC CAC TGC TAG A SEQ ID 3: CGGGCGCCACTGCTA SEQ ID 4: CTG
CTT AAA GCC TCA ATA AA SEQ ID 5: CTC AAT AAA GCT TGC CTT GA SEQ ID
6: TCT GGT AAC TAG AGA TCC CTC SEQ ID 7: TAG TGT GTG CCC GTC TGT
SEQ ID 8: AGT GTG TGC CCG TCT GTT SEQ ID 12: GAT GCA TGC TCA ATA
AAG CTT GCC TTG AGT
or the complementary sequence thereof.
[0023] It is understood that oligonucleotides consisting of the
sequences of the present invention may contain minor deletions,
additions and/or substitutions of nucleic acid bases, to the extent
that such alterations do not negatively affect the yield or product
obtained to a significant degree. Where oligonucleotides according
to the present invention are used as probes, the alterations should
not result in lowering the hybridization efficiency of the probe.
For example, in case of transcription based amplification
techniques, wherein one or more of the primers may be provided with
a promoter sequence, the introduction of a purine-rich (=G or A)
hybridizing sequence, just after the promoter sequence may have
positive effects on the transcription (when there are C's and T's
abortive transcription may occur). If no such sequence is available
in the target nucleic acid a purine-rich sequence can be inserted
in the oligonucleotide just following the last three G residues of
the promoter sequence.
[0024] The sequences of the present invention are reflected as DNA
sequences. The RNA equivalents of these sequences are likewise part
of the present invention.
[0025] Preferred oligonucleotides according to the invention are
oligonucleotides consisting essentially of a sequence selected from
the group consisting of: TABLE-US-00002 SEQ ID 1: G GGC GCC ACT GCT
AGA GA SEQ ID 2: G TTC GGG CGC CAC TGC TAG A SEQ ID 3:
CGGGCGCCACTGCTA SEQ ID 4: CTG CTT AAA GCC TCA ATA AA SEQ ID 5: CTC
AAT AAA GCT TGC CTT GA SEQ ID 6: TCT GGT AAC TAG AGA TCC CTC SEQ ID
7: TAG TGT GTG CCC GTC TGT. SEQ ID 8: AGT GTG TGC CCG TCT GTT. SEQ
ID 9: aat tct aat acg act cac tat agg gAG AGG GGC GCC ACT GCT AGA
GA SEQ ID 10: aat tct aat acg act cac tat agg gAG AGG TTC GGG CGC
CAC TGC TAG A SEQ ID 11: aat tct aat acg act cac tat agg
gCGGGCGCCACTGCTA SEQ ID 12: GAT GCA TGC TCA ATA AAG CTT GCC TTG
AGT
[0026] SEQ ID 9-11 actually comprise the sequence as reflected by
SEQ ID 1-3. In SEQ ID 9-11, the sequences of SEQ ID 1-3 are
operably linked to a promoter sequence (the T7 promoter sequence).
This makes the sequences especially suitable for use as upstream
primer in a transcription based amplification technique such as
NASBA.
[0027] A preferred embodiment of the present invention is a
combination of two oligonucleotides according to the invention, for
use as a set in nucleic acid amplification.
[0028] Such a pair of oligonucleotides, for use as a set in the
amplification of a target sequence located within the LTR region of
the genome of HIV-1, consists of a first oligonucleotide being
10-50 nucleotides in length and comprising, at least a fragment of
10 nucleotides, of a sequence selected from the group consisting
of: TABLE-US-00003 SEQ ID 1: G GGC GCC ACT GCT AGA GA SEQ ID 2: G
TTC GGG CGC CAC TGC TAG A SEQ ID 3: CGGGCGCCACTGCTA
[0029] and a second oligonucleotide being 10-50 nucleotides in
length and comprising, at least a fragment of 10 nucleotides, of a
sequence selected from the group consisting of: TABLE-US-00004 SEQ
ID 4: CTG CTT AAA GCC TCA ATA AA SEQ ID 5: CTC AAT AAA GCT TGC CTT
GA. SEQ ID 12: GAT GCA TGC TCA ATA AAG CTT GCC TTG AGT
[0030] One of the oligonucleotides may serve as an "upstream
oligonucleotide", i.e., an upstream-primer, while the second
oligonucleotide serves as a "downstream oligonucleotide", i.e.
downstream primer, in the amplification reaction. The location on
the HIV-genome (or the sequence complementary thereto) to which
both oligonucleotides comprised in such a pair according to the
invention can anneal, will together define the sequence of the
nucleic acid that is amplified. The amplified sequence is located
between the "primer-binding sites" within the LTR region of the
HIV-genome. It has been found that, by using a pair of
oligonucleotides according to the invention in an amplification
reaction, accurate and reliable amplification of nucleic acid
derived from all presently know sub-types of HIV can be
achieved.
[0031] A most preferred pair of oligonucleotides according to the
invention will consist of a first primer comprising the sequence of
SEQ ID NO 1 and a second primer with the sequence of SEQ ID NO 5.
For use in a transcription based amplification method, the
oligonucleotide with SEQ ID NO 9 is preferred, in combination with
an oligonucleotide with the sequence of SEQ ID NO 5.
[0032] Part of the oligonucleotides according to the invention are
particularly suitable for use as a probe in the detection of
nucleic acid amplified with a pair of oligonucleotides according to
the invention. When used as a probe, said oligonucleotides may be
provided with a detectable label. Oligonucleotides according to the
invention that are especially suitable as a probe consist
essentially of the sequence TABLE-US-00005 SEQ ID 6: TCT GGT AAC
TAG AGA TCC CTC SEQ ID 7: TAG TGT GTG CCC GTC TGT or SEQ ID 8: AGT
GTG TGC CCG TCT GTT
provided with a detectable label. A most preferred oligonucleotide
in this respect is an oligonucleotide with a sequence as depicted
in SEQ ID 6.
[0033] Various labeling moieties are known in the art. Said moiety
may, for example, either be a radioactive compound, a detectable
enzyme (e.g. horse radish peroxidase (HRP)), a hapten like biotin,
or any other moiety capable of generating a detectable signal such
as a calorimetric, fluorescent, chemiluminescent or
electrochemiluminescent signal.
[0034] Hybrids between oligonucleotides according to the invention
and (amplified) target nucleic acid may also be detected by other
methods known to those skilled in the art.
[0035] Evidently methods for amplification of nucleic acid, like
the ones that have been mentioned above, using the oligonucleotides
according to the present invention are also part of the
invention.
[0036] The present invention further provides test kits for the
amplification and detection of HIV nucleic acid. The use of said
test-kits enables accurate and sensitive screening of samples
suspected of containing HIV derived nucleic acid. Such test-kits
may contain a pair of oligonucleotides according to the invention
and optionally also an oligonucleotide according to the invention
that can be used as a probe for the detection of the amplified
material. Furthermore the test-kit may contain suitable
amplification reagents. These reagents are for example the suitable
enzymes for carrying out the amplification reaction. A kit, adapted
for use with NASBA, for example may contain suitable amounts of
reverse transcriptase, RNase H and T7 RNA polymerase. Said enzymes
may be present in the kit in a buffered solution but can likewise
be provided as a lyophilized composition, for example, a
lyophilized spherical particle. Such lyophilized particles have
been disclosed in PCT appl. no. EP95/01268. The kit may further be
furnished with buffer compositions, suitable for carrying out an
amplification reaction. Said buffers may be optimized for the
particular amplification technique for which the kit is intended as
well as for use with the particular oligonucleotides that are
provided with the kit. In transcription based amplification
techniques, such as NASBA, said buffers may contain, for example,
DMSO, which enhances the amplification reaction (as is disclosed in
PCT appl. no. US90/04733).
[0037] Furthermore the kit may be provided with an internal control
as a check on the amplification procedure and to prevent the
occurrence of false negative test results due to failures in the
amplification procedure. The use of internal controls in
transcription based amplification techniques is described in PCT
appl. no. EP 93/02248. An optimal control sequence is selected in
such a way that it will not compete with the target nucleic acid in
the amplification reaction. Kits may also contain reagents for the
isolation of nucleic acid from biological specimen prior to
amplification. A suitable method for the isolation of nucleic acid
is disclosed in EP389063.
BRIEF DESCRIPTION OF THE FIGURES
[0038] FIG. 1:
[0039] Amplification products after blotting and detection by
enhanced chemiluminiscence obtained after amplification in presence
(panel A) or absence (panel B) of HIV-1 RNA using the following
primer pairs. Lane 1: SEQ ID 9-SEQ ID 10, lane 2; SEQ ID 9-SEQ ID
5, lane 3: SEQ ID 10-SEQ ID 4, lane 4: SEQ ID 10-SEQ ID 5, lane 5:
SEQ ID 3-SEQ ID 12.
EXAMPLES
Example 1
Amplification and Detection of HIV-1 Genomic RNA
[0040] The following procedures were applied to amplify and detect
HIV-1 genomic RNA from samples as described in the following
examples.
Sample Preparation and Nucleic Acid Isolation.
[0041] The isolation of the nucleic acids was performed according
to Boom et al., 1990, Journal of Clinical Microbiology 28, 495-503
and European patent no. EP0389063. In short: A volume of 200 .mu.l
of pooled plasma's from healthy donors (negative for HBsAg,
anti-HIV and anti-HCV) was added to 900 .mu.l of lysis buffer (47
mM Tris-HCl pH 7.2, 20 mM EDTA, 1.2% Triton X-100, 4.7 M guanidine
thiocyanate (GuSCN, Fluka, Buchs, Switzerland)). After vortexing
and centrifugation (30 seconds, 13,000 rpm) a dilution series of
HIV-1 RNA subtype B standard, characterized and described by Layne
et al., 1992, Virology 189, 695-714 or HIV-1 positive specimens,
was added. Upon addition of 50 .mu.l activated silicon (0.4 g/ml
suspension in 0.1 N HCl), the suspension was incubated for 10
minutes at room temperature with regular vortexing. After
centrifugation (30-60 seconds, 13,000 rpm) the silicon pellet was
washed twice with 1 ml of wash buffer (5.25 M GuSCN, 50 mM Tris-HCl
pH 6.4), followed by two 70% ethanol washes and one acetone wash
step. Subsequently, the silicon pellets were dried during ten
minutes in a 56.degree. C. heating block. The nucleic acids were
eluted from the silicon by adding 50 .mu.l elution buffer (1.0 mM
Tris-HCl, pH 8.5) and incubation at 56.degree. C. for 10 minutes.
Subsequently, 5 .mu.l of the eluate was taken of the pellet for
further use in the amplification reaction. The remaining eluate was
stored at -70.degree. C.
NASBA Amplification.
[0042] Amplifications were carried out in a reaction volume of 20
.mu.l which is composed of 10 .mu.l primer mixture, 5 .mu.l
(isolated) nucleic acids and 5 .mu.l enzyme mixture. The primer
mixture was made by reconstitution of a lyophilized accusphere into
50 .mu.l accusphere diluent, 51.6 .mu.l water, 8.4 .mu.l 2 M KCl
and 5 .mu.l of each primer (10 .mu.M). The mixture was thoroughly
vortexed before use. From this primer mixture 10 .mu.l was added to
5 .mu.l of the (isolated) HIV-1 RNA (standard). This mixture was
incubated for 5 minutes at 65.degree. C. and subsequently incubated
at 41.degree. C. for another 5 minutes. After these incubations 5
.mu.l enzyme mixture was added and incubated for 5 minutes at
41.degree. C. The tubes were transferred to the detection area and
incubated for 90 minutes at 41.degree. C. After the amplification
reaction the tubes were stored at -20.degree. C. until further use.
Each amplification reaction contained the following reagents:
[0043] 40 mM Tris-HCl, pH 8.5 [0044] 12 mM MgCl.sub.2 [0045] 70 mM
KCl [0046] 15% v/v dimethyl sulfoxide [0047] 5 mM dithiotreitol
[0048] 1 mM of each deoxynucleoside triphosphate [0049] 2 mM of the
nucleosides rATP, rCTP, rUTP [0050] 1.5 mM rGTP [0051] 0.5 mM ITP
[0052] 0.105 .mu.g/.mu.l BSA [0053] 0.08 units RNaseH [0054] 32
units T.sub.7 RNA polymerase [0055] 6.4 units avian myeloblastosis
virus reverse transcriptase (Seikagaku, USA) [0056] 0.2 .mu.M of
each primer [0057] 375 mM sorbitol [0058] 45.6 mM sucrose [0059]
28.5 mM mannitol [0060] 0.13 mM dextran T40 Detection of the
Amplified Products. A. Gel Electrophoresis.
[0061] The presence of amplified products was analyzed using an
agarose gel (100 ml of 2% Pronarose and 0.5 .mu.g/ml ethidium
bromide) and 1 * TAE (40 mM Trisacetate, 1 mM EDTA pH 8.0) running
buffer are used. Electrophoresis was carried out at 100 volts for
approximately 30 minutes. The ethidium bromide-stained bands of the
amplified products were visualized using UV irradiation. The blot
was hybridized with biotin probe (3 .mu.M) in hybridization mix
(750 mM NaCl, 75 mM sodium citrate, 20 mM
Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 (pH 6.7), 10* Denhardts) by
incubating the blot for 4 hours at 50.degree. C. After
hybridization the blot was washed two times for 5 minutes at
50.degree. C. with 450 mM NaCl, 45 mM sodium citrate pH 6.4 (2*
SSPE) and 1% sodium dodecyl sulphate (SDS) solution and one time
for 10 minutes with 20 mM Na.sub.2HPO.sub.4 pH 7.4, 360 mM NaCl, 2
mM EDTA and 0.1% SDS at room temperature. Subsequently, the blot
was incubated for 30 minutes with 2 .mu.l streptavidin/horse radish
peroxidase solution (500 U/ml from the enhanced chemiluminescence
detection kit of Amersham Life Science) in 10 ml 50 mM
Na.sub.2HPO.sub.4, 900 mM NaCl, 5 mM EDTA, pH 7.4 and 0.5% SDS.
Following washes respectively two times for 5 minutes in 2*SSPE,
0.1% SDS and once for 10 minutes in 2*SSPE at room temperature the
blot was dried between tissues, developed and exposed to a film
according to the Amersham kit protocol.
B. Electrochemiluminescence (ECL) Probe Hybridization.
[0062] The amplification products were diluted two times in
detection diluent (1.0 mM Tris/HCl, pH 8.5 and 0.2 g/l
2-methylisothiazolone HCl). Subsequently, 5 .mu.l of the diluted
amplification product was incubated for 30 minutes at 41.degree. C.
with 0.084 .mu.M of the HIV-1 specific biotin probe bound to 5
.mu.g streptavidin-coated magnetic beads (mean size 2.8
.mu.m.+-.0.2 .mu.m, Dynal, Great Neck, N.Y., USA) and 2*10.sup.11
molecules of an ECL (Tris[2,2-bipyridine]ruthenium[II]
complex)-labeled probe, in a total volume of 25 .mu.l 750 mM NaCl,
75 mM sodium citrate, pH 6.4 (5*SSC). As negative controls,
detection diluent was also incubated with the bead-probe and
ECL-probe mixtures. During incubation, tubes were agitated every 10
minutes to keep the beads in suspension. Subsequently, 300 .mu.l of
assay buffer solution (100 mM tripropylamine, pH 7.5) was added and
the tubes were placed in an ECL detection instrument (NASBA
QR-system from Organon Teknika BV) for reading the emitted ECL
signals.
Example 2
Amplification and Detection of HIV-1 Genomic RNA
[0063] The following primer pairs were tested on 10.sup.4 copies
(as determined by spectrophotometry at 260 nm) of the HIV-1 RNA
standard. The RNA was added directly into the amplification. The
analysis of the amplified products was performed by gel
electophoresis as described in example 1. The results are shown in
the FIG. 1.
[0064] All primer pairs and probes of the present invention were
able to amplify and detect HIV-1 RNA from subtype B to a similar
extent. Analytical sensitivity is shown for two primer pairs (SEQ
ID 9/SEQ ID 5 and SEQ ID 11/SEQ ID 5) using a dilution series of
the HIV-1 RNA standard which has an initial concentration of
5.5*10.sup.9 copies/ml. The detection was done with probes having
the sequences as depicted in SEQ ID 7 and SEQ ID 6. The
amplification and detection was carried out as described in example
1. Table 1 shows the results obtained with both primer pairs. \
TABLE-US-00006 TABLE 1 HIV-1 RNA SEQ ID 3-SEQ ID 5 SEQ ID 9-SEQ ID
5 copies No. detected % detected No. detected % detected 200 4:4
100% 4:4 100% 100 8:8 100% 8:8 100% 50 8:8 100% 8:8 100% 25 7:8
87.5% 7:8 87.5% 12 6:8 75% 4:8 50% 6 2:8 25% 4:7 57% 3 0:8 0% 2:7
28.5% 0 0:8 0% 0:7 0%
[0065] Both primer pairs have approximately the same sensitivity
for the HIV-1 RNA standard respectively a detection rate of 70% for
the primer pair SEQ ID 9/SEQ ID 5 and a detection rate of 63% for
the primer pair SEQ ID 3/SEQ ID 5.
Example 3
Amplification and Detection of HIV-1 RNA in Presence of an Internal
Control
[0066] In this example the primer pair SEQ ID 9/SEQ ID 5 and probe
with the sequence of SEQ ID 7 was tested on dilution series of the
HIV-1 RNA standard in presence of an internal control (ic-) RNA.
The ic-RNA is an in vitro transcript which contains part of the LTR
sequence of HIV-1 HXB-2 and 10.sup.4 copies (as determined by
spectrophotometry at 260 nm) are added prior to nucleic acids
isolation. The isolation, amplification and ECL detection was
performed as described in example 1. As a comparison, separate
amplification and detection was performed using a primer pair
located in the HIV-1 gag region (P1/P2) previously described by Van
Gemen et al, 1993, Journal of Virological Methods 43, 177-188. The
probes used to detect the amplification products generated by the
gag based primer pair were: TABLE-US-00007 Biotin probe:
5'-TGTTAAAAGAGACCATCAATGAGGA ECL probe:
5'-GAATGGGATAGAGTGCATCCAGTG.
[0067] Results are shown in Table 2. TABLE-US-00008 TABLE 2 HIV-1
RNA LTR SEQ ID 9-SEQ ID 5 GAG P1/P2 copies/200 .mu.l No. detected %
detected No. detected % detected 320 9:10 90% nt* nt* 160 5:10 50%
8:10 80% 80 5:10 50% 5:10 50% 40 3:8 38% 4:10 40% 20 3:10 32% 1:10
10% 10 1:10 10% 2:10 20% 0 0:5 0% 1:5 20%
[0068] Both the LTR primer pair of this example and the gag primer
pair were able to detect a similar amount of HIV-1 RNA in an assay
controlled by an in vitro produced RNA which was added prior to the
nucleic acids isolation.
Example 4
Detection of HIV-1 RNA in HIV-1 Positive Samples
[0069] In this example, 40 HIV-1 positive samples from various
geographical locations were analyzed for presence of HIV-1 RNA. All
samples were isolated by the nucleic acids isolation method
described in example 1. Amplification and detection were carried
out as described in example 1 using primer pair SEQ ID 9/SEQ ID 5
and probe with the sequence of SEQ ID 7 or probe with the sequence
of SEQ ID 8.
[0070] For comparison, separate amplification and detection was
carried out using the gag primer pair and probes as described in
example 3. Presence of amplified products was detected by ECL probe
hybridization according to the method of example 1. Result are
shown in Table 3. TABLE-US-00009 TABLE 3 Primer pair ECL Probe No.
Detected % Detected DEQ ID 9/SEQ ID 5 SEQ ID 7 40/40 100% (LTR) SEQ
ID 9/SEQ ID 5 SEQ 8 10/10 100% (LTR) P1/P2 )gag) 29/40 72.5%
[0071] Both primer probe combinations derived from the LTR region
of HIV-1 described in this example were able to detect HIV-1 from
all the samples tested. In contrast, the primer probe combination
of the gag assay failed to detect HIV-1 genomic RNA from a great
number of samples.
Example 5
Amplification and Detection of HIV-1 RNA with Defined Subtypes
[0072] In this example, 33 samples harbouring HIV-1 RNA of known
envelope-based subtypes were tested. The samples originate from
subtyped viruses propagated in cell cultures. Samples of cell
culture supernatants from both variants of group M subtypes A
through H and variants of group O were spiked to HIV-1 negative
human plasma and treated according to the method from example 1.
Amplification and detection was performed essentially as example 4.
Results are shown table 4 as the number of samples of each type
from which HIV-1 RNA was detected out of the total number of each
type tested. TABLE-US-00010 TABLE 4 HIV-1 Types Primer pair A B C D
E F G H O Total SEQ ID 9/SEQ ID 5 5:5 2:2 2:2 5:5 7:7 1:1 1:1 1:1
9:9 33:33 (LTR) 4:5 2:2 2:2 5:5 6:7 1:1 1:1 1:1 0:9 22:33
[0073] HIV-1 RNA was detected from all 33 HIV-1 samples using LTR
primer pair SEQ ID 9/SEQ ID 5 and probe with sequence of SEQ ID 7.
In contrast, the assay using the gag primers probes combination as
described in example 3 failed to detect subtype A and subtype E
each from one of the samples and all samples containing HIV-1 RNA
from group O members.
Example 6
Amplification and Detection of HIV-1 Clinical Samples
[0074] In this example, 7 samples obtained from seropositive
patients originating from Africa, South America and Asia were
assayed for the presence of HIV-1 genomic RNA. Despite the fact
that blood samples from these patients are both anti-p24 antibody
(Abbott Laboratories, Abbott Park, Ill.) and western blot
(Genelabs) positive, all specimens were scored negative by the
NASBA HIV-1 RNA QL assay (Organon Teknika BV) which uses the gag
primer pair and probe combination from example 3 and only a single
sample (R9612222) was detected and quantitated (29.500 RNA
copies/ml) by the Quantiplex HIV-1 RNA 2.0 (Chiron) assay using a
sample volume of 50 .mu.l. In contrast, using an equal sample
volume the primer pair SEQ ID 9/SEQ ID 5 and the probe with
sequence of SEQ ID 7 detected four out of seven samples (Table 5).
TABLE-US-00011 TABLE 5 SEQ ID 9/SEQ ID 5 Sample code Country (LTR)
R9610155 Thailand positive R9612222 Ghana positive R9700062 Brazil
negative R9612218 Zaire negative R9611710 Liberia positive R9610718
Antilles positive R9607884 Rwanda negative
[0075] The samples missed by the LTR primer probe combination are
not detected due to a low viral load below the detection level
since by using a sample volume of one ml the Quantiplex HIV-1 RNA
2.0 (Chiron) assay quantitated HIV-1 RNA levels of 30 or below 30
HIV-1 RNA copies per 50 .mu.l of these samples.
Sequence CWU 1
1
12 1 18 DNA Artificial Sequence Description of Artificial Sequence
synthetic oligonucleotide 1 gggcgccact gctagaga 18 2 20 DNA
Artificial Sequence Description of Artificial Sequence synthetic
oligonucleotide 2 gttcgggcgc cactgctaga 20 3 15 DNA Artificial
Sequence Description of Artificial Sequence synthetic
oligonucleotide 3 cgggcgccac tgcta 15 4 20 DNA Artificial Sequence
Description of Artificial Sequence synthetic oligonucleotide 4
ctgcttaaag cctcaataaa 20 5 20 DNA Artificial Sequence Description
of Artificial Sequence synthetic oligonucleotide 5 ctcaataaag
cttgccttga 20 6 21 DNA Artificial Sequence Description of
Artificial Sequence synthetic oligonucleotide 6 tctggtaact
agagatccct c 21 7 18 DNA Artificial Sequence Description of
Artificial Sequence synthetic oligonucleotide 7 tagtgtgtgc ccgtctgt
18 8 18 DNA Artificial Sequence Description of Artificial Sequence
synthetic oligonucleotide 8 agtgtgtgcc cgtctgtt 18 9 47 DNA
Artificial Sequence Description of Artificial Sequence synthetic
oligonucleotide 9 aattctaata cgactcacta tagggagagg ggcgccactg
ctagaga 47 10 49 DNA Artificial Sequence Description of Artificial
Sequence synthetic oligonucleotide 10 aattctaata cgactcacta
tagggagagg ttcgggcgcc actgctaga 49 11 40 DNA Artificial Sequence
Description of Artificial Sequence synthetic oligonucleotide 11
aattctaata cgactcacta tagggcgggc gccactgcta 40 12 30 DNA Artificial
Sequence Description of Artificial Sequence synthetic
oligonucleotide 12 gatgcatgct caataaagct tgccttgagt 30
* * * * *