Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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DETECTION OF HIV-1 BY NUCLEIC ACID AMPLIFICATION
FIELD OF THE INVENTION
This invention relates to diagnostic detection of viral nucleic acids, and
specifically relates to
compositions and assays for detecting HIV-1 sequences using transcription-
mediated nucleic acid
amplification and probe detection of amplified sequences.
BACKGROUND OF THE INVENTION
Human immunodeficiency virus 1 (HIV-1) is the causative agent of acquired
immunodeficiency
syndrome (AIDS) and AIDS related syndrome (ARC). Because the infectious virus
is transmissible in
body fluids, including blood and plasma, it is important to detect infected
body fluids before antibodies to
the virus are detectable or symptoms are evident in the infected individual.
For protection of patients who
might otherwise receive HIV-1 -infected body fluid (e.g., whole blood or
plasma during transfusion), or
products derived from blood or plasma, it is particularly important to detect
the presence of the virus in
the body fluid to prevent its use in such procedures or in products. It is
also important that procedures
and reagents used in detecting HIV-1 be able to detect relatively low numbers
of viral copies which may
be present in an infected individual.
Assays and reagents for detecting HIV-1 have been previously disclosed in, for
example, U.S.
Patent Nos. 5,008,182, 5,594,122, 5,688,637 and 5,843,638; European Patent
Nos. EP 178 978 B1, EP
181,150 B1 and EP 185,444 B1; published European Patent Application Nos. EP
403,333, EP 462,627
and EP 806,484; and PCT No. WO 99/61666.
The present invention includes oligonucleotide sequences used as primers for
amplification and
probes for detection of HIV-1 nucleic acid present in a biological sample,
using an assay that preferably
includes transcription-mediated nucleic acid amplification (e.g., as
previously disclosed by Kacian et al.,
U.S. Pat. Nos. 5,399,491 and 5,554,516). The preferred detection method uses
known homogeneous
detection techniques to detect, in a mixture, a labeled probe that is bound to
an amplified nucleic acid (as
disclosed, for example, in Arnold et al. Clin. Chem. 35:1588-1594 (1989);
Nelson et al., U.S. Pat. No.
5,658,737; and Lizardi et al., U.S. Pat. Nos. 5,118,801 and 5,312,728). The
present invention also
includes nucleic acid oligonucleotide sequences that are useful for capturing
the HIV-1 target using
nucleic acid hybridization techniques that preferably use magnetic particles
in separation of the captured
target (Whitehead et al., U.S. Pat. Nos. 4,554,088 and 4,695,392).
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SUMMARY OF THE INVENTION
Various embodiments of this invention provide a method of detecting HIV-1
RNA in a biological sample, comprising the steps of: contacting the biological
sample with at least one capture oligomer comprising a base sequence that
hybridizes specifically to a target region in LTR or pol sequences of HIV-1
RNA,
thus forming a capture oligomer:HIV-1 RNA complex; separating the capture
oligomer:HIV-1 RNA complex from the biological sample; then amplifying the LTR
or pol sequences, or a cDNA made therefrom, using a nucleic acid polymerase in
vitro and at least two amplification oligomers that bind specifically to LTR
sequences or complementary sequences thereof, and/or at least two
amplification
oligomers that bind specifically to pol sequences or complementary sequences
thereof, to produce an amplified product of the LTR and/or pol sequences; and
detecting the amplified product using a labeled detection probe that
hybridizes
specifically with the amplified product.
Various embodiments of this invention provide an oligomer composition
comprising at least a pair of LTR-specific amplification oligomers selected
from the
group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:30, SEQ ID NO:32,
SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, and SEQ ID
NO:38, and/or a pair of po/-specific amplification oligomers selected from the
group
consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ
ID NO:42, SEQ ID NO:43, and SEQ ID NO:44. The composition may further
comprise at least one LTR-specific capture oligomer and/or at least one po/-
specific
capture oligomer.
Various embodiments of this invention provide a kit comprising a plurality of
oligomers made up of at least a pair of LTR-specific amplification oligomers
selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:30,
SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37,
and SEQ ID NO:38, and/or a pair of po/-specific amplification oligomers
selected
from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ
ID NO:15, SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44. The kit may further
comprise at least one detection probe oligomer.
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Various embodiments of this invention provide a method of detecting HIV-1
RNA in a biological sample, comprising the steps of: contacting the biological
sample
with a capture oligomer comprising a base sequence that hybridizes
specifically to a
target sequence in HIV-1 RNA, thus forming a capture oligomer:HIV-1 RNA
complex;
separating the capture oligomer:HIV-1 RNA complex from the biological sample;
then
amplifying the HIV-1 RNA or a cDNA made therefrom, using a nucleic acid
polymerase
in vitro and at least two amplification oligomers selected from the group
consisting of
SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO:22, SEQ ID
NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID
NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID
NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, and SEQ ID
NO:38 that bind specifically to HIV-1 LTR sequences or complementary sequences
thereof, and at least two amplification oligomers selected from the group
consisting of
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ
ID NO:15, SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44 that bind specifically
to
HIV-1 poi sequences or complementary sequences thereof, to produce an
amplified
product of the HIV-1 RNA; and detecting the amplified product using a labeled
detection probe that hybridizes specifically with the amplified product.
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According to one aspect of the invention, there are provided oligomers
comprising a base
sequence of SEQ ID N0:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,
SEQ ID N0:6,
SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID
NO:21, SEQ ID
NO:42, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:51,
SEQ ID NO:52
or SEQ ID NO:57. One embodiment includes oligomers wherein the base sequence
is that of SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:18 or
SEQ ID NO:45.
Another embodiment includes oligomers further comprising a backbone that
includes at least one 2'-
methoxy RNA group, at least one 2' fluoro-substituted RNA group, at least one
peptide nucleic acid
linkage, at least one phosphorothioate linkage, at least one methylphosphonate
linkage or any
combination thereof. Another embodiment includes oligomers in which the base
sequence comprises the
sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,
SEQ ID NO:6,
SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:45, and
the backbone
comprises at least one 2'-methoxy RNA group.
According to another aspect of the invention, there are oligomers consisting
of a base sequence
of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID
NO:16, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26,
SEQ ID NO:27,
SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID
NO:33, SEQ ID
NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39,
SEQ ID NO:40,
SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:49, SEQ ID
NO:54, SEQ ID
NO:55 or SEQ ID NO:56. In one embodiment, the oligomer has a base sequence of
SEQ ID NO:8, SEQ
ID NO:9, SEQ 1D NO:11, SEQ ID NO:13 or SEQ ID NO:16. In another embodiment,
the base sequence
of the oligomer is joined by a backbone that includes at least one 2'-methoxy
RNA group, at least one 2'
fluoro-substituted RNA group, at least one peptide nucleic acid linkage, at
least one phosphorothioate
linkage, at least one methylphosphonate linkage or any combination thereof.
According to another aspect of the invention, there are provided labeled
oligomers comprising a
base sequence of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:39, SEQ
ID NO:40, SEQ
ID NO:41, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID
NO:50, SEQ ID
NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 or SEQ ID NO:56;
and a
detectable label joined directly or indirectly to the base sequence. In one
embodiment, the detectable
label is a luminescent compound. In another embodiment, the base sequence is
joined by a backbone
comprising at least one 2'-methoxy RNA group. One embodiment is a labeled
oligomer having the base
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sequence of SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18, and the label that is
a chemiluminescent
compound. A preferred embodiment is a labeled oligomer having the base
sequence of SEQ ID NO:16
containing an inosine at residue 7, and an acridinium ester compound as the
label.
According to another aspect of the invention, there is provided a method of
detecting HIV-1 RNA
in a biological sample, comprising the steps of: providing a biological sample
containing HIV-1 RNA;
contacting the biological sample with at least one capture oligomer comprising
a base sequence that
hybridizes specifically to a target region in LTR or pol sequences of HIV-1
RNA, thus forming a capture
oligomer:HIV-1 RNA complex; separating the capture oligomer:HIV-1 RNA complex
from the biological
sample; then amplifying the LTR or pol sequences, or a cDNA made therefrom,
using a nucleic acid
polymerase in vitro to produce an amplified product; and detecting the
amplified product using a labeled
detection probe that hybridizes specifically with the amplified product. In
one embodiment, the contacting
step uses a capture oligomer that further comprises a tail sequence that binds
to a complementary
sequence immobilized on a solid support. In another embodiment, the base
sequence of the capture
oligomer that hybridizes specifically to a target region in LTR or pol
sequences comprises a sequence of
SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:19 or SEQ ID NO:57. In
another embodiment,
the capture oligomer comprises the base sequence of at least one of SEQ ID
NO:2, SEQ ID NO:4, SEQ
ID NO:6, SEQ ID NO:20 or SEQ ID NO:45, or is any combination of oligomers of
SEQ ID NO:2, SEQ ID
NO:4, SEQ ID NO:6, SEQ ID NO:20 or SEQ ID NO:45. In a preferred embodiment,
the capture oligomer
is any combination of at least two oligomers having base sequences selected
from the group of SEQ ID
NO:2, SEQ ID NO:4 and SEQ ID NO:6. In one embodiment, the capture oligomer is
a combination of
oligomers having base sequences of SEQ ID NO:20 and SEQ ID NO:6, or SEQ ID
NO:45 and SEQ ID
NO:6. In another embodiment, the amplifying step uses at least two
amplification oligomers that bind
specifically to LTR or pol sequences or complementary sequences thereof.
Preferably, the amplifying
step uses at least two amplification oligomers for amplifying LTR sequences
selected from the group
consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:21, SEQ ID
NO:22, SEQ ID NO:23,
SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID
NO:29, SEQ ID
NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,
SEQ ID NO:36,
SEQ ID NO:37 and SEQ ID NO:38. Another embodiment uses, in the amplifying
step, at least two
amplification oligomers for amplifying pol sequences selected from the group
consisting of: SEQ ID
NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,
SEQ ID NO:42,
SEQ ID NO:43 and SEQ ID NO:44. In another embodiment, the amplifying step
comprises a
transcription-associated amplification method that includes at least one
promoter-primer comprising a
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promoter sequence that is recognized by an RNA polymerase when the promoter
sequence is double
stranded, wherein the promoter sequence is covalently attached to the 5' end
of a LTR-specific sequence
selected from the group consisting of SEQ ID NO:7, SEQ ID NO:21, SEQ ID NO:23,
SEQ ID NO:25,
SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 and SEQ ID NO:33, or a po/specific
sequence selected
from the group consisting of SEQ ID NO:12 and SEQ ID NO:14; and at least one
primer comprising a
LTR-specific sequence selected from the group consisting of SEQ ID NO:9, SEQ
ID NO:35, SEQ ID
NO:36, SEQ ID NO:37 and SEQ ID NO:38, or a po/-specific sequence selected from
the group consisting
of SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:42, provided that at least one LTR-
specific promoter-
primer is combined with at least one LTR-specific primer for amplifying a LTR
target region, or at least
one po/-specific promoter-primer is combined with at least one po/-specific
primer for amplifying a pol
target region. In one embodiment, the amplifying step comprises a
transcription-associated amplification
method that includes at least one promoter-primer having a LTR-specific
sequence selected from the
group consisting of SEQ ID NO:8, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ
ID NO:28, SEQ
ID NO:30, SEQ ID NO:32 and SEQ ID NO:34, or a po/-specific sequence selected
from the group
consisting of SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:43 and SEQ ID NO:44; and
at least one primer
having a LTR-specific sequence selected from the group consisting of SEQ ID
NO:9, SEQ ID NO:35,
SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, or a po/-specific sequence
selected from the group
consisting of SEQ ID NO:1 0, SEQ ID NO:1 1 and SEQ ID NO:42, provided that at
least one LTR-specific
promoter-primer is combined with at least one LTR-specific primer for
amplifying a LTR target region, or
at least one pol specific promoter-primer is combined with at least one po/-
specific primer for amplifying a
po/ target region. Preferably, the amplifying step uses any of the following
combinations of promoter-
primers and primers: promoter-primers of SEQ ID NO:13 and SEQ ID NO:15, with
primers of SEQ ID
NO:10 and SEQ ID NO:11; promoter-primers of SEQ ID NO:13 and SEQ ID NO:15,
with primers of SEQ
ID NO:42 and SEQ ID NO:11; promoter-primers of SEQ ID NO:43 and SEQ ID NO:15,
with primers of
SEQ ID NO:10 and SEQ ID NO:11; promoter-primers of SEQ ID NO:13 and SEQ ID
NO:44, with primers
of SEQ ID NO:10 and SEQ ID NO:11; promoter-primers of SEQ ID NO:7, SEQ ID
NO:13 and SEQ ID
NO:15, with primers of SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; a promoter-
primer of SEQ ID
NO:8, and a primer of SEQ ID NO:9; a promoter-primer of SEQ ID NO:8, and a
primer of SEQ ID NO:35:
a promoter-primer of SEQ ID NO:8, and a primer of SEQ ID NO:36; a promoter-
primer of SEQ ID NO:30,
and a primer of SEQ ID NO:9; a promoter-primer of SEQ ID NO:30, and a primer
of SEQ ID NO:36; a
promoter-primer of SEQ ID NO:32, and a primer of SEQ ID NO:9; a promoter-
primer of SEQ ID NO:34,
and a primer of SEQ ID NO:36; a promoter-primer of SEQ ID NO:13, and a primer
of SEQ ID NO:10; or a
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promoter-primer of SEQ ID NO:7, and a primer of SEQ ID NO:9. In one
embodiment, the detecting step
uses at least one labeled detection probe having a base sequence selected from
the LTR-specific group
consisting of SEQ ID NO:1 6, SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41, or
the po/-specific
group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:46, SEQ ID NO:47,
SEQ ID NO:48, SEQ
ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID
NO:54, SEQ ID
NO:55 and SEQ ID NO:56, or a combination thereof. In another embodiment, the
detecting step uses a
combination of at least two labeled detection probes having the base sequences
of SEQ ID NO:1 6, SEQ
ID NO:17 or SEQ ID NO:18. Preferably, the labeled detection probe of SEQ ID
NO:16 has an inosine at
position 7. One embodiment, in the detecting step, uses at least one labeled
detection probe having a
base sequence selected from the LTR-specific group consisting of SEQ ID NO:16,
SEQ ID NO:39, SEQ
ID NO:40 and SEQ ID NO:41. Another embodiment, in the detecting step, uses at
least one labeled
detection probe having a base sequence selected from the po/-specific group
consisting of SEQ ID
NO:17, SEQ ID NO:18, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49,
SEQ ID NO:50,
SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 and SEQ
ID NO:56. In
one embodiment, the detecting step uses at least one labeled detection probe
that includes at least one
2'-methoxy backbone linkage. Another embodiment includes the contacting step
that uses capture
oligomers having the sequences of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6;
the amplifying step
that uses promoter-primers having the sequences of SEQ ID NO:8, SEQ ID NO:13
and SEQ ID NO:15
and primers having the sequences of SEQ ID NO:9, SEQ ID NO:10 and SEQ ID
NO:11; and the
detecting step that uses labeled detection probes having the sequences of SEQ
ID NO:16, SEQ ID
NO:17 and SEQ ID NO:18. In one embodiment, the contacting step uses at least
two capture oligomers
that hybridize to different sequences in the target region; the amplifying
step uses at least two different
promoter-primers that hybridize to a first set of sequences within the target
region and at least two
different primers that hybridize to a second set of sequences within the
target region; and the detecting
step uses at least two labeled probes that bind specifically to different
sequences located between the
first set and second set of sequences within the target region. In another
embodiment, the contacting
step uses capture oligomers having the sequences of SEQ ID NO:4 and SEQ ID
NO:6; the amplifying
step uses promoter-primers having the sequences of SEQ ID NO:13 and SEQ ID
NO:15 and primers
having the sequences of SEQ ID NO:10 and SEQ ID NO:11; and the detecting step
uses labeled probes
having the sequences of SEQ ID NO:17 and SEQ ID NO:18. In a preferred
embodiment, the amplifying
step uses at least two promoter-primers that hybridize to a first set of
overlapping sequences within the
target region, at least two primers that hybridize to a second set of
overlapping sequences within the
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target region, or a combination thereof.
According to another aspect of the invention these is provided a kit
comprising a plurality of
oligomers having the sequences of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13,
SEQ ID NO:15, SEQ
ID NO:17 and SEQ ID NO:18, wherein the oligomers having the sequences of SEQ
ID NO:17 and SEQ
ID NO:18 are labeled with a detectable label. In one embodiment, the kit
further includes oligomers
having the sequences of SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:16, wherein the
oligomer of SEQ
ID NO:16 is labeled with a detectable label.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic drawing of HIV-1 RNA (indicated in a 5' to 3'
orientation by the thick
dashed line divided into portions by vertical lines) showing the relative
positions of genes ("gag", "pol",
"env") and untranslated 5' and 3' regions ("R U5" and "U3 R" respectively)
which contain long terminal
repeats ("LTR"); target regions are indicated by the double-dashed lines
labeled "LTR" and "Pol".
FIG. 2 is a schematic drawing of the components of an embodiment of the
present invention for
detecting a'Target Region" of HIV-1 nucleic acid (represented by the thick
vertical line), where the
positions of the following nucleic acids are shown relative to the target
region: a "Capture Oligomer"
refers to the nucleic acid used to hybridize to and capture the target nucleic
acid prior to transcription-
mediated amplification, where "T" refers to a 3' tail sequence used to
hybridize to an immobilized
oligomer having a complementary sequence (not shown); "Non-T7 Primer" and 77
Primer" represent two
amplification oligonucleotides used in transcription-mediated amplification
where "P" indicates the
promoter sequence of the T7 primer; and "Probe" refers to a labeled probe used
to detect the amplified
nucleic acid.
FIG. 3 is a schematic drawing, labeled as in FIG. 2, of the components of
another embodiment of
the present invention for detecting a HIV-1 nucleic acid Target Region using
two capture oligomers, two
non-T7 primers, two T7 primers and two labeled probes.
FIG. 4A is a schematic drawing of a preferred embodiment, as illustrated
generically in FIG. 2, for
detecting an HIV-1 LTR target region in which the capture oligomer is
represented by SEQ ID NO:2, the
non-T7 primer is represented by SEQ ID NO:9, the T7 primer is represented by
SEQ ID NO:8, and the
labeled probe is represented by SEQ ID NO:16.
FIG. 4B is a schematic drawing of a preferred embodiment, as illustrated
generically in FIG. 3, for
detecting an HIV-1 po/target region in which the capture oligomers are
represented by SEQ ID NO:4 and
SEQ ID NO:6, the non-T7 primers are represented by SEQ ID NO:10 and SEQ ID
NO:11, the T7 primers
are represented by SEQ ID NO:13 and SEQ ID NO:15, and the labeled probes are
represented by SEQ
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ID NO:17 and SEQ ID NO:18.
The accompanying drawings illustrate some embodiments of the invention. These
drawings,
together with the description, serve to explain and illustrate the principles
of the invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention includes methods of detecting HIV-1 nucleic acids
present in biological
samples derived from humans, preferably in blood, serum or plasma. The present
invention also
includes compositions which include nucleic acid capture oligomers (or capture
oligonucleotides) used to
specifically capture HIV-1 target sequences present in a biological sample,
nucleic acid amplification
oligomers (or primers) used to specifically amplify selected HIV-1 nucleic
acid sequences and nucleic
acid probe oligomers (probes or labeled probes) for detecting amplified HIV-1
sequences.
The nucleic acid sequences of this invention are useful for capturing,
amplifying and detecting
HIV-1 nucleic acid present in a biological sample such human blood, serum,
plasma or other body fluid
containing HIV-1. The methods of the present invention are valuable for
detecting HIV-1 nucleic acid in a
biological sample, and thus are important for diagnosis of HIV-1 infection and
for screening blood and
blood products that may contain infectious virus, to prevent infecting
individuals through transfusion with
infected blood or plasma. Such screening is also important to prevent HIV-1
contamination in blood-
derived therapeutics.
By "biological sample" is meant any tissue or material derived from a living
or dead human which
may contain the target HIV-1 nucleic acid, including, for example, peripheral
blood or bone marrow,
plasma, serum, cervical swab samples, biopsy tissue including lymph nodes,
respiratory tissue or
exudates, gastrointestinal tissue, urine, feces, semen or other body fluids,
tissues or materials. The
biological sample may be treated to physically or mechanically disrupt tissue
or cell structure, thus
releasing intracellular components into a solution which may contain enzymes,
buffers, salts, detergents
and the like which are used to prepare the biological sample using standard
methods for analysis.
By "nucleic acid" is meant a multimeric compound comprising nucleosides or
nucleoside analogs
which have nitrogenous heterocyclic bases, or base analogs, where the
nucleosides are linked together
by phosphodiester bonds to form a polynucleotide. The term "nucleic acid"
includes conventional RNA
and DNA oligomers and those that include base analogs or substitutions. The
"backbone" of a nucleic
acid may be made up of a variety of known linkages, including one or more of
sugar-phosphodiester
linkages, peptide-nucleic acid bonds (i.e., "peptide nucleic acids" as
described by Hydig-Hielsen et al.,
PCT No. WO 95/32305), phosphorothioate linkages, methylphosphonate linkages or
combinations
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thereof. Sugar moieties of the nucleic acid may be either ribose or
deoxyribose, or similar compounds
having known substitutions, such as, for example, 2' methoxy substitutions and
2' halide substitutions
(e.g., 2'-F). The nitrogenous bases may be conventional bases (A, G, C, T, U),
known analogs thereof
(e.g., inosine; see The Biochemistry of the Nucleic Acids 5-36, Adams et al.,
ed., 11th ed., 1992), known
derivatives of purine or pyrimidine bases (e.g., N4-methyl deoxygaunosine,
deaza- or aza-purines and
deaza- or aza-pyrimidines, pyrimidine bases having substituent groups at the 5
or 6 position, purine
bases having an altered or a replacement substituent at the 2, 6 or 8
positions, 2-amino-6-
methylaminopurine, 06-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines,
4-dimethylhydrazine-
pyrimidines, and 04-alkyl-pyrimidines; see, Cook, PCT No. WO 93/13121) and
"abasic" residues where
the backbone includes no nitrogenous base for one or more residues of the
polymer (see Arnold et al.,
U.S. Pat. No. 5,585,481). A nucleic acid may comprise only conventional
sugars, bases and linkages
found in RNA and DNA, or may include both conventional components and
substitutions (e.g.,
conventional bases linked via a methoxy backbone, or a nucleic acid including
conventional bases and
one or more base analogs).
The backbone composition of an oligomer may affect stability of a
hybridization complex (e.g.,
formed by hybridization of a capture oligomer to a target nucleic acid).
Preferred backbones include
peptide linkages as in peptide nucleic acid, sugar-phosphodiester type
linkages as in RNA and DNA, or
derivatives thereof. Peptide nucleic acids are advantageous for forming a
hybridization complex with
RNA. In some embodiments, the backbone is made up of sugar-phosphodiester type
linkages in which
the sugar group and/or the linkage joining the groups is altered relative to
standard DNA or RNA to
enhance hybridization complex stability. For example, an oligomer having one
or more 2'-methoxy
substituted RNA groups or a 2'-fluoro substituted RNA forms a stable
hybridization complex with a
complementary 2' OH RNA. A linkage joining two sugar groups may affect
hybridization complex
stability by affecting the overall charge or the charge density, or by
affecting steric association (e.g., steric
interactions due to bulky linkages may reduce hybridization complex
stability). Preferred embodiments
include linkages with charged (e.g., phosphorothioates) or neutral (e.g.,
methylphosphonates) groups to
affect complex stability.
By "oligonucleotide" or "oligomer" is meant a nucleic acid having generally
less than 1,000
residues, including polymers falling in a size range having a lower limit of
about 2 to 5 residues and an
upper limit of about 500 to 900 residues. Preferably, oligomers of the present
invention fall in a size
range having a lower limit of about 5 to about 15 residues and an upper limit
of about 50 to 600 residues.
More preferably, oligomers of the present invention fall in a size range
having a lower limit of about 10 to
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WO 01/04361 PCT/US00/18685
about 20 residues and an upper limit of about 22 to 100 residues. Oligomers
may be purified from
naturally occurring sources, but preferably are synthesized using any of a
variety of well known
enzymatic or chemical methods.
By "amplification oligonucleotide" or "amplification oligomer" is meant an
oligonucleotide that
hybridizes to a target nucleic acid, or its complement, and participates in a
nucleic acid amplification
reaction. Examples of amplification oligonucleotides include primers and
promoter-primers. Preferably,
an amplification oligonucleotide contains at least about 10 contiguous bases,
and more preferably at
least about 12 contiguous bases, which are complementary to a region of the
target nucleic acid
sequence (or a complementary strand thereof). The contiguous bases are
preferably at least about 80%,
more preferably at least about 90%, and most preferably greater than 95%
complementary to a region to
which the amplification oligonucleotide binds. An amplification
oligonucleotide is preferably about 10 to
about 60 bases long and optionally may include modified nucleotides or
analogs.
Amplification oligonucleotides or oligomers also may be referred to as
"primers" or "promoter-
primers." A "primer" refers to an optionally modified oligonucleotide which is
capable of hybridizing to a
template nucleic acid and which has a 3' end that can be extended in a known
polymerization reaction.
The 5' region of the primer may be non-complementary to the target nucleic
acid; if the 5' non-
complementary region includes a promoter sequence, it may be referred to as a
"promoter-primer."
Those skilled in the art will appreciate that any oligomer that can function
as a primer (i.e., an
amplification oligonucleotide that hybridizes specifically to a target
sequence and has a 3' end capable of
being extended by a polymerase activity) can be modified to include a 5'
promoter sequence, and thus
could function as a promoter-primer. Similarly, any promoter-primer can be
modified by removal of, or
synthesis without, a promoter sequence and still function as a primer.
By "LTR-specific sequence" is meant any sequence of nucleic acid bases that
hybridizes
specifically to a sequence in an HIV-1 LTR region or its complement under
standard hybridization
conditions; an LTR-specific sequence may further contain or be covalently
linked to nucleic acid bases
that do not hybridize specifically to an LTR sequence or its complement,
provided that such non-LTR-
specific bases do not interfere with hybridization of the LTR-specific
sequence to its target sequence.
Similarly, by "Pol-specific sequence" is meant any sequence of nucleic acid
bases that hybridizes
specifically to a sequence in an HIV-1 pol region or its complement under
standard hybridization
conditions as described herein; a pol-specific sequence may further contain or
be covalently linked to
nucleic acid bases that do not hybridize specifically to a pol sequence or its
complement, provided that
such non-pol-specific bases do not interfere with hybridization of the po/-
specific sequence to its target
sequence.
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By "amplification" is meant any known in vitro procedure for obtaining
multiple copies of a target
nucleic acid sequence or its complement or fragments thereof. Amplification of
"fragments thereof" refers
to production of an amplified nucleic acid containing less than the complete
target region nucleic acid
sequence or its complement. Such fragments may be produced by amplifying a
portion of the target
nucleic acid, for example, by using an amplification oligonucleotide which
hybridizes to, and initiates
polymerization from, an internal position of the target nucleic acid. Known
amplification methods include,
for example, transcription-mediated amplification, replicase-mediated
amplification, polymerase chain
reaction (PCR) amplification, ligase chain reaction (LCR) amplification and
strand-displacement
amplification (SDA). Replicase-mediated amplification uses self-replicating
RNA molecules, and a
replicase such as QB-replicase (e.g., see Kramer et al., U.S. Pat. No.
4,786,600; PCT No. WO
90/14439). PCR amplification is well known and uses a DNA polymerase, primers
and thermal cycling to
synthesize multiple copies of the two complementary strands of DNA (e.g., see
U.S. Pat. Nos. 4,683,195,
4,683,202, and 4,800,159; Methods in Enzymology, 1987, Vol. 155: 335-350). LCR
amplification uses at
least four separate oligonucleotides to amplify a target and its complementary
strand by using multiple
cycles of hybridization, ligation, and denaturation (see EP Pat. App. No. 0
320 308). SDA is a method in
which a primer contains a recognition site for a restriction endonuclease such
that the endonuclease will
nick one strand of a hemimodified DNA duplex that includes the target
sequence, followed by
amplification in a series of primer extension and strand displacement steps
(see Walker et al., Proc. Natl.
Acad. Sci. USA 89:392-396 (1992); and U.S. Pat. No. 5,422,252) Transcription-
associated amplification
is a preferred embodiment of the present invention. It will be apparent to one
skilled in the art, however,
that the amplification oligonucleotides disclosed herein are readily
applicable to other amplification
methods that use primer extension.
By `Transcription-associated amplification" or `Transcription-mediated
amplification" is meant any
type of nucleic acid amplification that uses an RNA polymerase to produce
multiple RNA transcripts from
a nucleic acid template. Transcription-associated amplification generally
employs an RNA polymerase, a
DNA polymerase, deoxyribonucleoside triphosphates, ribonucleoside
triphosphates, and a promoter-
template complementary oligonucleotide, and optionally may include one or more
analogous
oligonucleotides. Variations of transcription-associated amplification are
well known in the art as
disclosed in detail in Burg et al., U.S. Pat. No. 5,437,990; Kacian et al.,
U.S. Pat. Nos. 5,399,491 and
5,554,516; Kacian et al., PCT No. WO 93/22461; Gingeras et al., PCT No. WO
88/01302; Gingeras et
al., PCT No. WO 88/10315; Malek et al., U.S. Pat. No. 5,130,238; Urdea et al.,
U.S. Pat. Nos. 4,868,105
and 5,124,246; McDonough et al., PCT No. WO 94/03472; and Ryder et al., PCT
No. WO 95/03430.
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The methods of Kacian et al. are used in preferred embodiments of the present
invention.
By "probe" is meant a nucleic acid oligomer that hybridizes specifically to a
target sequence in a
nucleic acid, preferably in an amplified nucleic acid, under standard
conditions that promote hybridization,
thereby allowing detection of the target sequence or amplified nucleic acid.
Detection may either be
direct (i.e., resulting from a probe hybridizing directly to the target
sequence or amplified nucleic acid) or
indirect (i.e., resulting from a probe hybridizing to an intermediate
molecular structure that links the probe
to the target sequence or amplified nucleic acid). A probe's "target"
generally refers to a sequence
within (i.e., a subset of) an amplified nucleic acid sequence or target region
which hybridizes specifically
to at least a portion of a probe oligomer using standard hydrogen bonding
(i.e., base pairing). A probe
may comprise target-specific sequences and other sequences that contribute to
three-dimensional
conformation of the probe (e.g., as described in Lizardi et al., U.S. Pat.
Nos. 5,118,801 and 5,312,728).
Sequences that are "sufficiently complementary" allow stable hybridization of
a probe oligomer to a target
sequence even thought it is not completely complementary to the probe's target-
specific sequence.
By "sufficiently complementary" is meant a contiguous nucleic acid base
sequence that is
capable of hybridizing to another base sequence by hydrogen bonding between a
series of
complementary bases. Complementary base sequences may be complementary at each
position in the
base sequence of an oligomer using standard base pairing or may contain one or
more residues that are
not complementary using standard hydrogen bonding (including abasic
"nucleotides"), but in which the
entire complementary base sequence is capable of specifically hybridizing with
another base sequence in
appropriate hybridization conditions. Contiguous bases are preferably at least
about 80%, more
preferably at least about 90%, and most preferably greater than 95%
complementary to a sequence to
which an oligomer is intended to specifically hybridize. To those skilled in
the art, appropriate
hybridization conditions are well known, can be predicted based on base
composition, or can be
determined empirically by using routine testing (e.g., see Sambrook et al.,
Molecular Cloning, A
Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY, 1989) at
1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57 particularly at 9.50-9.51,
11.12-11.13, 11.45-11.47
and 11.55-11.57).
By "capture oligonucleotide" or "capture oligomer" is meant at least one
nucleic acid oligomer that
provides means for specifically joining a target sequence and an immobilized
oligomer due to base pair
hybridization. A capture oligomer preferably includes two binding regions: a
target sequence-binding
region and an immobilized probe-binding region, usually contiguous on the same
oligomer, although the
capture oligomer may include a target sequence-binding region and an
immobilized probe-binding region
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which are present on two different oligomers joined together by one or more
linkers. For example, an
immobilized probe-binding region may be present on a first oligomer, the
target sequence-binding region
may be present on a second oligomer, and the two different oligomers are
joined by hydrogen bonding
with a linker that is a third oligomer containing sequences that hybridize
specifically to the sequences of
the first and second oligomers. Sometimes, a capture oligomer is referred to
as a "capture probe."
By "immobilized probe" or "immobilized nucleic acid" is meant a nucleic acid
that joins, directly or
indirectly, a capture oligomer to an immobilized support. An immobilized probe
is an oligomer joined to a
solid support that facilitates separation of bound target sequence from
unbound material in a sample.
Any known solid support may be used, such as matrices and particles free in
solution, including for
example, nitrocellulose, nylon, glass, polyacrylate, mixed polymers,
polystyrene, silane polypropylene
and metal particles, preferably, magnetically attractable particles. Preferred
supports are monodisperse
magnetic spheres (i.e., uniform in size about 5%), thereby providing
consistent results, to which an
immobilized probe is joined directly (e.g., via covalent linkage, chelation,
or ionic interaction), or indirectly
(e.g., via one or more linkers), where the linkage or interaction is stable
during nucleic acid hybridization
conditions.
By "separating" or "purifying" is meant that one or more components of a
biological sample are
removed from one or more other components of the sample. Sample components
include nucleic acids
in a generally aqueous solution phase which may also include materials such as
proteins, carbohydrates,
lipids and labeled probes. Preferably, this step removes at least about 70%,
more preferably at least
about 90% and, most preferably, at least about 95% of the other sample
components from the desired
component.
By "label" or "detectable label" is meant a molecular moiety or compound that
can be detected or
can lead to a detectable response. A label is joined, directly or indirectly,
to a nucleic acid probe. Direct
labeling can occur through bonds or interactions that link the label to the
probe, including covalent bonds
or non-covalent interactions (e.g., hydrogen bonding, hydrophobic and ionic
interactions) or through
formation of chelates or coordination complexes. Indirect labeling can occur
through use of a bridging
moiety or "linker', such as an antibody or additional oligonucleotide(s),
which is either directly or indirectly
labeled, and which can amplify a detectable signal. A label can be any known
detectable moiety, such
as, for example, a radionuclide, ligand (e.g., biotin, avidin), enzyme or
enzyme substrate, reactive group,
chromophore, such as a dye or particle that imparts a detectable color (e.g.,
latex or metal particles),
luminescent compond (e.g., bioluminescent, phosphorescent or chemiluminescent
labels) and
fluorescent compound.
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Preferably, the label on a labeled probe that is detectable in a homogeneous
assay system, i.e.,
where, in a mixture, bound labeled probe exhibits a detectable change, such as
stability or differential
degradation, compared to unbound labeled probe, without physically removing
hybridized from
unhybridized forms of the label or labeled probe. A "homogeneous detectable
label" refers to a label
whose presence can be detected in a homogeneous fashion as previously
described in detail in Arnold et
al., U.S. Pat. No. 5,283,174; Woodhead et al., U.S. Pat. No. 5,656,207; and
Nelson et al., U.S. Pat. No.
5,658,737. Preferred labels for use in a homogenous assay include
chemiluminescent compounds (e.g.,
see Woodhead et al., U.S. Pat. No. 5,656,207; Nelson et al., U.S. Pat. No.
5,658,737; and Arnold, Jr., et
al., U.S. Pat. No. 5,639,604). Preferred chemiluminescent labels are
acridinium ester ("AE") compounds,
such as standard AE or derivatives thereof (e.g., naphthyl-AE, ortho-AE, 1- or
3-methyl-AE, 2,7-dimethyl-
AE, 4,5-dimethyl-AE, ortho-dibromo-AE, ortho-dimethyl-AE, meta-dimethyl-AE,
ortho-methoxy-AE, ortho-
methoxy(cinnamyl)-AE, ortho-methyl-AE, ortho-fluoro-AE, 1- or 3-methyl-ortho-
fluoro-AE, 1- or 3-methyl-
meta-difluoro-AE, and 2-methyl-AE). Synthesis and methods of attaching labels
to nucleic acids and
detecting labels are well known in the art (e.g., see Sambrook et al.,
Molecular Cloning, A Laboratory
Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Habor, NY,
1989), Chapter 10;
Nelson et al., U.S. Pat. No. 5,658,737; Woodhead et al., U.S. Pat. No.
5,656,207; Hogan et al., U.S. Pat.
No. 5,547,842; Arnold et al., U.S. Pat. No. 5,283,174; Kourilsky et al., U.S.
Pat.. No. 4,581,333; and
Becker et al., European Pat. App. No. 0 747 706).
By "consisting essentially of" is meant that additional component(s),
composition(s) or method
step(s) that do not materially change the basic and novel characteristics of
the present invention may be
included in the compositions or kits or methods of the present invention. Such
characteristics include the
ability to detect HIV-1 nucleic acid in a biological sample such as whole
blood or plasma, at a copy
number of about 100 copies of HIV-1. Any component(s), composition(s), or
method step(s) that have a
material effect on the basic and novel characteristics of the present
invention fall outside of this term.
Unless defined otherwise, all scientific and technical terms used herein have
the same meaning
as commonly understood by those skilled in the relevant art. General
definitions of many of the terms
used herein are provided, for example, in Dictionary of Microbiology and
Molecular Biology, 2nd ed.
(Singleton et al., 1994, John Wiley & Sons, New York, NY) or The Harper
Collins Dictionary of Biology
(Hale & Marham, 1991, Harper Perennial, New York, NY). Unless mentioned
otherwise, the techniques
employed or contemplated herein are standard methodologies well known to one
of ordinary skill in the
art. Examples are included to illustrate some embodiments of the invention.
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The present invention includes compositions (nucleic acid capture oligomers,
amplification
oligomers and probes) and methods for detecting HIV-1 nucleic acid in a human
biological sample. To
design oligomer sequences appropriate for such uses, known HIV-1 DNA
sequences, including subtypes,
partial sequences, or complementary sequences, available from publicly
accessible databases (e.g.,
GenBank) were aligned by matching regions of the same or similar sequences and
compared using well
known molecular biology techniques. The sequences used in the comparisons
included (using their
database designations): HIV3ALTRc, HIVANT70, HIVANT70C, HIVBAL1, HIVBaLTRc,
HIVBbLTRc,
HIVBRUCG, HIVCAM1, HIVCDC41, HIVD31, HIVEaLTRc, HIV1EbLTRc, HIVELI, HIVELICG,
HIVHAN,
HIVHXB2, HIVH9NL43, HIVJ233, HIVJRCSF, HIVJRFL, HIVJH31, HIVKEBO, HIVLAI,
HIVMAL,
HIVMCK1, HIVMN, HIVMNCG, HIVMVP5180, HIVNDK, HIVNL43, HIVNY5, HIVNY5CG,
HIVOYI, HIVRF,
HIVSC, HIVSF162, HIVSF2, HIV-subC, HIVU455, HIVTH475A, HIVYU2, HIVZ2Z6,
HIVISG3X,
HIV1 U12055, HM U21135, HIV1 U23487,HIV1 U26546, HIV1 U26942, HIV1 U34603, HM
U34604,
HIV1U39362, HIV1U3RA, HIV2ALTRc, HIV2BLTRc, HIV2CLTRc, HIV2132, HIV3BLTRc,
HIV4ALTRc,
HIV4BLTRc, HIV4CLTRc, and REHTLV3. The regions of the HIV-1 genome targeted
for detection using
the designed capture oligomers, primers and probes were the LTR and pol
regions as shown
schematically in FIG. 1. Although sequence comparisons may be facilitated by
use of algorithms, those
skilled in the art can readily perform such comparisons manually. Portions of
sequences containing
relatively few sequence variants between the compared sequences were chosen as
a basis for designing
synthetic oligomers suitable for use in capture, amplification and detection
of amplified sequences. Other
considerations in designing oligomers included the relative GC content of the
sequence (ranging from
about 30% to about 55%) and the relative absence of predicted secondary
structure (e.g., hairpin turns
forming intramolecular hybrids) within a sequence, using methods well known in
the art.
Based on these analyses, the capture oligomer, amplification oligomers and
probe sequences of
SEQ ID NO:1 to SEQ ID NO:45 were designed. Generally, for capture oligomer
sequences, the
sequence that specifically binds to HIV-1 sequence is shown without a 3' tail
(SEQ ID NO:1, 3, 5, and
19), and with a 3' tail sequence (SEQ ID NO:2, 4, 6, and 20). Generally, for
sequences of T7 promoter
primers, which include a T7 promoter sequence, the primer sequences are shown
without the T7
promoter sequence (SEQ ID NO:7, 12, 14, 21, 23, 25, 27, 29, 31, and 33) and
with a 5' T7 promoter
sequence (SEQ ID NO:8, 13, 15, 22, 24, 26, 28, 30, 32, and 34). Although some
T7 promoter primer
sequences are shown only with a T7 promoter sequence (SEQ ID NO:43 and SEQ ID
NO:44), those
skilled in the art will appreciate that the primer sequence specific for HIV-
1, with or without the T7
promoter sequence, may be useful as a primer under some amplification
conditions.
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Preferred capture oligomers include a sequence that binds specifically to an
HIV-1 sequence in
the LTR region or pol region (i.e., an HIV-1 -binding sequence) which is
covalently attached to a tail
sequence. Any backbone to link the base sequence of a capture oligomer may be
used, and in preferred
embodiments the capture oligomer includes at least one methoxy linkage in the
backbone. The tail
sequence, which is preferably at the 3' end of a capture oligomer, is used to
hybridize to a
complementary base sequence to provide a means of capturing the hybridized
target HIV-1 nucleic acid
from the other components in the biological sample. Any base sequence that
hybridizes to a
complementary base sequence may be used in a tail sequence, which preferably
has a sequence length
of about 5 to 50 nucleotide residues. Preferred tail sequences are
substantially homopolymeric,
containing about 10 to about 40 nucleotide residues. For example, a tail may
comprise about (A)10 to
about (A)40 residues. Preferably, the tail of a capture oligomer includes
about 14 to about 30 residues
and is substantially homopolymeric in sequence. Preferred tail sequences
include sequences of
TTT(A)14 to TTT(A)30 and (A)14 to (A)30=
Preferred methods of the present invention are described and are illustrated
by the examples.
FIG. 2 illustrates one system for detecting a target region (i.e., a selected
portion) of the HIV-1 genome
(shown by the thick solid horizontal line). This system includes four
oligomers (shown by the shorter solid
lines): one capture oligomer which includes a sequence that hybridizes
specifically to an HIV-1 sequence
in the target region and a tail (`T") that hybridizes to complementary
sequence immobilized on a solid
support to capture the target region present in a biological sample; one T7
primer which includes a
sequence that hybridizes specifically to an HIV-1 sequence in the target
region and a T7 promoter
sequence ("P") which, when double-stranded, serves as a functional promoter
for T7 RNA polymerase;
one non-T7 primer which includes a sequence that hybridizes specifically to a
first strand cDNA made
from the target region sequence using the T7 primer; and one labeled probe
which includes a sequence
that hybridizes specifically to a portion of the target region that has been
amplified using the two primers.
Using the components illustrated in FIG. 2, an assay to detect HIV-1 sequences
in a biological
sample includes the steps of capturing the target nucleic acid using the
capture oligomer, amplifying the
captured target region using at least two primers, preferably by using a
transcription-associated
amplification reaction, and detecting the amplified nucleic acid by
hybridizing the labeled probe to a
sequence contained in the amplified nucleic acid and detecting a signal
resulting from the bound labeled
probe.
The capturing step preferably uses a capture oligomer as illustrated in FIG.
2, where under
hybridizing conditions one portion of the capture oligomer specifically
hybridizes to a sequence in the
CA 02374385 2001-11-19
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target nucleic acid and a tail portion serves as one portion of a binding
pair, such as a ligand (e.g., a
biotin-avidin binding pair) that allows the target region to be separated from
other components of the
sample. Preferably, the tail portion of the capture oligomer is a sequence
that hybridizes to a
complementary sequence that is immobilized by being bound to a solid support
particle. Preferably, first,
the capture oligomer and the target nucleic acid are in solution to utilize
solution phase hybridization
kinetics. Hybridization produces a capture oligomer:target nucleic acid
complex which can be bound to
an immobilized probe by hybridization of the tail portion of the capture
oligomer with a complementary
immobilized sequence. Thus, a complex comprising a target nucleic acid,
capture oligomer and
immobilized probe is formed under hybridization conditions. Preferably, the
immobilized probe is a
repetitious sequence, and more preferably a homopolymeric sequence (e.g., poly-
A, poly-T, poly-C or
poly-G), which is complementary to the tail sequence and attached to a solid
support. For example, if the
tail portion of the capture oligomer contains a poly-A sequence, then the
immobilized probe would
contain a poly-T sequence, although any combination of complementary sequences
may be used. The
capture oligomer may also contain "spacer" residues, which are one or more
bases located between the
base sequence that hybridizes to the target and the base sequence of the tail
that hybridizes to the
immobilized probe. For example, a tail portion consisting of TTT(A)16 that
hybridizes to a poly-dT
immobilized probe contains a spacer consisting of TTT separating the base
sequence that hybridizes to
the target sequence and the (A)16 sequence that hybridizes to the immobilized
probe. Any solid support
may be used such as matrices and particles free in solution (e.g.,
nitrocellulose, nylon, glass,
polyacrylate, mixed polymers, polystyrene, silane polypropylene and,
preferably, magnetically attractable
particles). Methods of attaching an immobilized probe to the solid support are
well known. The support
is preferably a particle which can be retrieved from solution using standard
methods (e.g., centrifugation,
magnetic attraction of magnetic particles, and the like). Preferred supports
are paramagnetic
monodisperse particles (i.e., uniform in size about 5%).
Retrieving the target nucleic acid:capture oligomer:immobilized probe complex
concentrates the
target nucleic acid (relative to its concentration in the biological sample)
and purifies the target nucleic
acid from amplification inhibitors which may be present in the biological
sample. The captured target
nucleic acid may be washed one or more times, further purifying the target
(e.g., by resuspending the
particles with the attached target nucleic acid:capture oligomer:immobilized
probe complex in a washing
solution and then retrieving the particles with the attached complex from the
washing solution as
described above). In a preferred embodiment, the capturing step takes place by
sequentially hybridizing
the capture oligomer with the target nucleic acid and then adjusting the
hybridization conditions to allow
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hybridization of the tail portion of the capture oligomer with an immobilized
complementary sequence
(e.g., as described in PCT No. WO 98/50583). When the capturing step, and any
optional washing steps
included in it, have been completed, the target nucleic acid is amplified,
preferably without releasing it
from the capture oligomer.
Amplifying the captured target region using the two primers can be
accomplished using a variety
of known nucleic acid amplification reactions, but preferably uses a
transcription-associated amplification
reaction. In such an embodiment, many strands of nucleic acid are produced
from a single copy of target
nucleic acid, thus permitting detection of the target by using detectable
probes bound to the amplified
sequences. Transcription-associated amplification has been described in detail
elsewhere (Kacian et al.,
U.S. Pat. Nos. 5,399,491 and 5,554,516) and described briefly below.
Preferably, transcription-
associated amplification uses two types of primers (one referred to as a
promoter-primer because it
contains a promoter sequence, labeled "P" in FIG. 2, for an RNA polymerase),
enzymes (a reverse
transcriptase and an RNA polymerase), and substrates (deoxyribonucleoside
triphosphates,
ribonucleoside triphosphates) with appropriate salts and buffers in solution
to produce multiple RNA
transcripts from a nucleic acid template. Briefly, in the first step, a
promoter-primer hybridizes specifically
to a target sequence and reverse transcriptase creates a first strand cDNA by
extension from the 3' end
of the promoter-primer. The cDNA becomes available for hybridization with the
second primer by using
well known techniques, such as, by denaturing the duplex or using RNase H
activity, which is preferably
supplied by the reverse transcriptase, to degrade the RNA in a DNA:RNA duplex.
A second primer then
binds to the cDNA and a new strand of DNA is synthesized from the end of the
second primer using
reverse transcriptase, to create a double-stranded DNA having a functional
promoter sequence at one
end. RNA polymerase binds to the double-stranded promoter sequence and
transcription produces
multiple transcripts or "amplicons." These amplicons then are used in the
transcription-associated
amplification process, each serving as a template for a new round of
replication as described above, thus
generating large amounts of single-stranded amplified nucleic acid (about 100
to about 3,000 RNA
transcripts synthesized from a single template). Preferably, amplification
uses substantially constant
reaction conditions (e.g., a single temperature).
A promoter-primer oligonucleotide contains a 5' sequence promoter sequence
that, when double-
stranded, serves as a functional promoter for an RNA polymerase, and a 3'
sequence region that
hybridizes specifically to a sequence in the target region. A preferred
promoter-primer includes a
promoter sequence specific for a T7 RNA polymerase, and such a promoter-primer
may be referred to as
a "T7 primer." The second primer that does not include a promoter sequence is
often referred to as a
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"non-T7 primer" to distinguish it from the T7 primer.
Referring to FIG. 2, during transcription-mediated amplification, the captured
target nucleic acid
is hybridized to a first primer shown as a T7 primer. Using reverse
transcriptase, cDNA is synthesized
from the T7 primer using the target RNA as the template. The second primer,
shown as a non-T7 primer,
hybridizes to the cDNA strand and reverse transcriptase forms a DNA duplex,
thus forming a double-
stranded functional T7 promoter. Then, T7 RNA polymerase generates multiple
RNA transcripts by
using the functional T7 promoter. By repeating these hybridization and
polymerization steps following the
initial cDNA synthesis step, additional RNA transcripts are produced thus
amplifying target region-specific
nucleic acid sequences.
The detecting step uses at least one labeled probe that binds specifically to
the amplified nucleic
acid (e.g., the RNA transcripts or amplicons resulting from transcription-
mediated amplification).
Preferably, the probe is labeled with a detectable label that produces a
signal that can be detected
without purifying the bound probe from unbound probe (i.e., a homogeneous
detection system). More
preferably, the probe is labeled with an acridinium ester compound from which
a chemiluminescent signal
is produced and detected, as described in detail previously (Arnold et al.,
U.S. Pat. No. 5,283,174 and
U.S. Pat. No. 5,656,744; Nelson et al., U.S. Pat. No. 5,658,737).
As illustrated in FIG. 3, transcription-associated amplification reactions may
use multiple
promoter-primers and primers for a target region. Referring to FIG. 3, two
capture oligomers are used to
capture target region nucleic acids. Then, during transcription-associated
amplification, multiple T7
primers and non-T7 primers are used as described above. During the detection
step, multiple labeled
probes that bind specifically to the transcripts are used to detect the
amplicons. For the purposes of
illustration, FIG. 3 shows two different forms of each of the above
components, although more than two
of each component may be used in an assay reaction.
FIG. 3 illustrates two different capture oligomers used in the capture step,
each hybridizing
specifically to a different sequence of the target region. Preferably, one
capture oligomer hybridizes to a
5' portion and a second capture oligomer hybridizes to a 3' portion of the
target region. In embodiments
which use multiple capture oligomers in the capture step, preferably both
capture oligomers have
substantially the same tail sequence to allow hybridization of both capture
oligomers to the same
immobilized probe species as described above. For example, the tail of two
different capture oligomers
may be poly-T sequences and the immobilized sequences are then complementary
poly-A oligomers.
Referring to FIG. 3, following target capture, a portion of the target region
is amplified, preferably
using a transcription-associated amplification method that employs at least
two T7 primers and at least
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two non-T7 primers. Although only two primers are needed for transcription-
associated amplification (a
T7 promoter primer and a non-T7 primer), the use of two or more different
primers of each type has been
found to enhance amplification of some target sequences. Surprisingly, even if
two primers bind
specifically to target sequences that overlap (as illustrated in FIG. 3),
enhanced amplification was
observed. That is, by providing primers that specifically bind to overlapping
target sequences, enhanced
amplification of the target region was observed, rather than inhibited
amplification as might be expected,
for example, if the primers compete for hybridization to the target sequence.
An additional unexpected
benefit was that, for some targets (e.g., HIV-1 Group 0 pofj, the combination
of at least two amplification
oligomers that bind to overlapping target sequences allowed more efficient
amplification of the target
than was achieved using a system as illustrated in FIG. 2, which uses one
primer and one promoter-
primer.
Again referring to FIG. 3, following amplification of the target sequences,
two or more different
labeled probes that specifically hybridize to amplified sequences are used in
the detecting step of the
assay. As discussed above, any detectable label may be used and different
labels may be used for the
different probes. For example, one probe may be radiolabled another probe
fluorescently labeled, thus
providing different distinguishable signals. Preferably, the two or more
labeled probes are each labeled
with a label that allows for signal detection in a homogeneous system where
unbound probe is
distinguished from bound probe without requiring physical separation. In a
preferred embodiment, the
two or more labeled probes are labeled with a luminescent label, and more
preferably the luminescent
label is an AE compound that is detected as a chemiluminescent signal in a
homogenous detection
assay using well known procedures.
Although FIG. 2 illustrates an embodiment in which one of each component is
used and FIG. 3
illustrates an embodiment in which two of each components is used, those
skilled in the art will
appreciate that different variations that combine features as illustrated in
FIGS. 2 and 3 may be used.
For example, an assay may use one capture oligomer to capture the target
region, then may use two
promoter-primers and two second primers during amplification, and finally may
detect amplified products
using one probe that binds specifically to all amplicons. In another example,
multiple capture oligomers
may be used to capture a target region nucleic acid, followed by transcription-
mediated amplification
using multiple promoter-primers but a single second primer sequence, followed
by detection using one
labeled probe. That is, different combinations of components of the basic
assay shown in FIG. 2 may be
used, so long as at least one of each component is present in the assay.
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Examples of specific embodiments for detecting target regions in the HIV-1 LTR
and pol
sequences are illustrated in FIGS. 4A and 4B. As shown in FIG. 4A, for
detecting HIV-1 LTR sequences,
the capture oligomer includes the sequence of SEQ ID NO:2 , the non-T7 primer
includes the sequence
of SEQ ID NO:9, the T7 primer includes the sequence of SEQ ID NO:8, and the
labeled probe includes
the sequence of SEQ ID NO:16, where the relative positions of these sequences
are illustrated by the
short horizontal lines above and below the long vertical line representing the
LTR target region.
Preferably, the labeled probe includes an AE-derived label as shown by the
short vertical line joined to
the horizontal line labeled SEQ ID NO:16. As shown in FIG. 4B, for detecting
HIV-1 pol sequences, two
of each type of oligomer are included in the assay, where the relative
positions of these sequences are
illustrated by the short horizontal lines above and below the long vertical
line representing the pol target
region. That is, two capture oligomers are used, the first having the sequence
of SEQ ID NO:4 and the
second having the sequence of SEQ ID NO:6; two non-T7 primers are used, the
first having the
sequence of SEQ ID NO:10 and the second having the sequence of SEQ ID NO:11;
two T7 primers are
used, the first having the sequence of SEQ ID NO:13 and the second having the
sequence of SEQ ID
NO:15; and two labeled probes are used, the first including the sequence of
SEQ ID NO:17 and the
second including the sequence of SEQ ID NO:18. Preferably, the labeled probe
includes an AE-derived
label as shown by the short vertical lines joined to the horizontal lines
labeled SEQ ID NO:17 and SEQ ID
NO:18.
For the methods described above, capture oligomers, amplification oligomers
and labeled probes
having specific sequences have been identified as useful for detecting HIV-1
target sequences that are
localized to the LTR and pol regions of the HIV-1 genome. These specific
sequences may include
contiguous nucleic acid bases as occur in naturally occurring nucleic acids
(A, T, G or C), analogs (e.g.,
inosine) or synthetic purine and pyrimidine derivatives, such as, e.g., P or K
bases (Lin & Brown, 1989,
Nucl. Acids Res. 17:10373-83; Lin & Brown, 1992, Nucl. Acids Res. 20: 5149-
52), or combinations
thereof in a single contiguous sequence. Preferred capture oligomers for the
LTR target region include
the HIV-1-binding sequences of SEQ ID NO:1 and SEQ ID NO:19, which are
attached to any moiety that
can serve as a binding partner (i.e., ligands) for linking the target region
sequences to a retrievable solid
phase. Preferably, the ligand is a 3' tail sequence that hybridizes to an
immobilized complementary
oligomer. Preferred LTR-specific capture oligomers that include a tail
sequence are shown by the
sequences of SEQ ID NO:2, SEQ ID NO:20 and SEQ ID NO:45. Preferred capture
oligomers for the pol
target region include the HIV-1 -binding sequences of SEQ ID NO:3 and SEQ ID
NO:5, attached to a tail
sequence that hybridizes to an immobilized complementary oligomer. Preferred
capture oligomers for
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the po/target region that include tail sequences include the sequences of SEQ
ID NO:4 and SEQ ID
NO:6.
Preferred amplification oligomer sequences for amplifying sequences in the LTR
region include
SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO:22, SEQ ID
NO:23, SEQ ID
NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,
SEQ ID NO:30,
SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID
NO:36, SEQ ID
NO:37 and SEQ ID NO:38. Primer sequences for amplification of HIV-1 LTR
sequences that are
preferred T7 promoter primers include SEQ ID NO:8, SEQ ID NO:22, SEQ ID NO:24,
SEQ ID NO:26,
SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32 and SEQ ID NO:34. Preferred non-T7
primer sequences
for amplification of HIV-1 LTR sequences include SEQ ID NO:9, SEQ ID NO:21,
SEQ ID NO:23, SEQ ID
NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35,
SEQ ID NO:36,
SEQ ID NO:37 and SEQ ID NO:38. Preferred combinations of primers for
amplifying the HIV-1 LTR
region in a transcription-mediated amplification reaction include at least one
T7 promoter primer that
includes the sequence of SEQ ID NO:8, SEQ ID NO:22, SEQ ID NO:24, SEQ ID
NO:26, SEQ ID NO:28,
SEQ ID NO:30, SEQ ID NO:32 or SEQ ID NO:34; and at least one non-T7 primer
that includes the
sequence of SEQ ID NO:9, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 or SEQ ID
NO:38.
Preferred amplification oligomer sequences for amplifying sequences in the pol
region include
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID
NO:15, SEQ ID
NO:42, SEQ ID NO:43 and SEQ ID NO:44. Primer sequences for amplification of
HIV-1 po/sequences
that are preferred T7 promoter primers include SEQ ID NO:13, SEQ ID NO:15, SEQ
ID NO:43 and SEQ
ID NO:44. Preferred non-T7 primer sequences for amplification of HIV-1
po/target sequences include
SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:42. Preferred combinations of
primers for amplifying
the HIV-1 po/target region in a transcription-mediated amplification method
include at least one T7
promoter primer that includes the sequence of SEQ ID NO:13, SEQ ID NO:15, SEQ
ID NO:43 or SEQ ID
NO:44; and at least one non-T7 primer that includes the sequence of SEQ ID
NO:10, SEQ ID NO:1 1 or
SEQ ID NO:42.
Preferred probes for hybridizing to amplified HIV-1 LTR sequences to provide
detectable signals
include the sequences of: SEQ ID NO:16, wherein the "N" residue may be any
base (A, T, G, C) or
inosine or analogs thereof (e.g., 5-nitro-indole or a 2'-methoxy base), and
preferably is inosine; SEQ ID
3 o NO:39, wherein the "N" residues may be any base or inosine or analogs
thereof; SEQ ID NO:40, wherein
the "R" residue may be either A or G, and preferably is A; and SEQ ID NO:41.
In one embodiment, the
labeled probe sequence for detecting amplified LTR sequences has a methoxy
backbone or at least one
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methoxy linkage in the nucleic acid backbone. Preferably the label in a
labeled probe is a
chemiluminescent AE compound (e.g., 2-methyl-AE) which is attached to the
probe sequence via a linker
substantially as described in Arnold et al., U.S. Pat. No. 5,585,481; and
Arnold et al., U.S. Pat. No.
5,639,604, particularly as described at column 10, line 6 to column 11, line
3, and in Example 8. That is,
preferred labeling positions are a central region of the probe and near a
region of All base pairs, at a 3'
or 5' terminus of the probe, or at or near a mismatch site with a known
sequence that is not the desired
target sequence. For example, using a probe having the sequence of SEQ ID
NO:17, an AE label is
preferably attached to a 3' or 5' terminus or at positions between adjacent
residues from residues 5 to 14
or from residues 16 to 19, such as, for examples, between residues 6 and 7, or
between residues 7 and
8, or between residues 10 and 11.
Individual embodiments of probes having the sequences of SEQ ID NO:16, SEQ ID
NO:39, SEQ
ID NO:40 and SEQ ID NO:41 were tested for binding to HIV-1 LTR sequences of
subtype B, subtype G
(having a base substitution in the LTR relative to subtype B) and Group 0. For
example, when a probe
having SEQ ID NO:16 includes inosine at position 7, the Tm for both subtype B
and Group 0 were
essentially the same (72-73 C) and was higher for subtype G (about 80 C),
whereas when 5-nitro-indole
was used at position 7, the Tm for all three tested strains was about the same
(70-73 C). Similar tests
were performed using the different embodiments of SEQ ID N0:39, and from all
of the tests the following
conclusions were drawn: substitution of one 2'-methoxyribose base for a
deoxyribose base generally
lowered the Tm about 2 C; inosine substituted for a base complementary to a
residue that varied
between target sequences aided hybridization to the target; and substitution
of 5-nitro-indole for a base
may increase hybridization kinetics.
Preferred probes for hybridizing to amplified HIV-1 po/target sequences to
provide detectable
signals include the sequences of SEQ ID NO:17, SEQ ID NO:18, SEQ ID N0:46, SEQ
ID N0:47, SEQ
ID N0:48, SEQ ID N0:49, SEQ ID N0:50, SEQ ID NO:51, SEQ ID N0:52, SEQ ID
N0:53, SEQ ID
N0:54, SEQ ID N0:55 and SEQ ID N0:56, and more preferably include SEQ ID NO:17
and SEQ ID
N0:18. In one embodiment, the labeled probe sequence for detecting amplified
HIV-1 po/sequences
has a methoxy backbone. A preferred label is a chemiluminescent AE label, more
preferably 2-methyl-
AE, which is attached to the probe sequence substantially as described above.
The general principles of the present invention may be more fully appreciated
by reference to the
following non-limiting examples.
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Example 1
Detection of HIV-1, Subtype B (HIV-1, I llb) Pol Target Sequences Using
SEQ ID NO:10 or SEQ ID NO:42 in Amplification Oligomer Mixture
A sample of human plasma containing a known amount of HIV-1 subtype B (100
copies of HIV-1
Illb per reaction tube) was mixed with an equal volume of a lysis buffer ( 790
mM HEPES, 230 mM
succinic acid, 10% (w/v) lithium lauryl sulfate, 680 mM lithium hydroxide
monohydrate). To capture the
HIV-1 pol target RNA, the mixture also contained about 1.75 pmols each of
oligomers of SEQ ID NO:4
and SEQ ID NO:6, and about 100 p g of immobilized poly-dT14 probe attached to
paramagnetic particles
(0.7-1.05 particles, Seradyn, Indianapolis, IN). Probes were attached to the
particles using
carbodiimide chemistry as previously described (Lund, et al., 1988, Nuc. Acids
Res.16:10861-10880).
The mixture was heated at 55 C to 60 C for about 15 to 30 min and then cooled
to room temperature to
allow hybridization. Then a magnetic field was applied to attract the magnetic
particles with the complex
containing immobilized probe HIV-1, capture oligomer and HIV-1 RNA to a
location on the reaction
container (substantially as described in Wang, U.S. Pat. No. 4,895,650). The
particles were then washed
twice with 1 ml of a washing buffer (10 mM HEPES, 6.5 mM NaOH, 1 mM EDTA, 0.3%
(v/v) ethanol,
0.02% (w/v) methyl-paraben, 0.01% (w/v) propyl-paraben, 150 mM NaCl, 0.1%
(w/v) sodium lauryl
sulfate) by resuspending the particles in the buffer and then repeating the
magnetic separation step.
Washed particles were then suspended in 75 p I of a nucleic acid amplification
reagent solution for
transcription-associated amplification using methods substantially as
described previously (Kacian et al.,
U.S. Pat. Nos. 5,399,491 and 5,554,516).
Briefly, the washed particles with the attached complexes were mixed with 7.5
pmol each of
amplification oligonucleotides that were either (1) SEQ ID NO:10, SEQ ID NO:1
1, SEQ ID NO:13 and
SEQ ID NO:15, or (2) SEQ ID NO:42, SEQ ID NO:11, SEQ ID NO:13 and SEQ ID
NO:15, in a reaction
mixture (40 mM Trizma base, pH 7.5, 17.5 mM KCI, 20 mM MgCl21 5%
polyvinylpyrrolidone (PVP), 1 mM
each dNTP, 4 mM each rNTP), covered with a layer (200 p I) of inert oil to
prevent evaporation, and
incubated at 60 C for 10-15 min, and then at 41.5- 42 C for 5 min. Enzymes
(about 750 Units of
reverse transcriptase and about 2,000 of Units T7 RNA polymerase per reaction)
were added, mixed,
and the target HIV-1 nucleic acid was amplified at 41.5-42 C for 2 hr.
Amplified HIV-1 target sequences were detected using an AE-labeled probe (SEQ
ID NO:17)
which was detected by chemiluminescence and expressed in relative light units
(RLU) substantially as
described previously (U.S. Pat. No. 5,658,737 at column 25, lines 27-46;
Nelson et al., 1996, Biochem.
35:8429-8438 at 8432). For each assay condition, negative controls consisted
of all of the same
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reagents but substituting an equal volume of plasma that contained no HIV-1.
The detected RLU of
these assays for eleven HIV-1 positive ("HIV-1 +") samples and eight HIV-1
negative samples for each
set of amplification oligonucleotides are shown in Table 1.
Table 1
RLU Detected for Combinations of Amplification Oligonucleotides
Sample SEQ ID NO:10, SEQ ID NO:42,
SEQ ID NO:11, SEQ ID NO:11,
SEQ ID NO:13 and SEQ ID NO:13 and
SEQ ID NO:15 SEQ ID NO:15
HIV-1 + 2.11 x 106 1.90 x 106
HIV-1 + 1.69 x 106 1.88 x 106
HIV-1 + 1.67x106 1.86x106
HIV-1 + 1.63 x 106 1.78 x 106
HIV-1+ 1.47 x 106 1.74 x 106
HIV-1 + 1.47 x 106 1.73 x 106
HIV-1 + 1.42 x 106 1.71 x 106
HIV-1 + 1.24 x 106 1.64 x 106
HIV-1 + 1.18x106 1.54x106
HIV-1 + 1.17x 106 1.44 x 106
HIV-1 + 1.13x106 1.22x106
Average HIV-1 + 1.47 x 106 1.68 x 106
HIV-1 negative 3.79 x 103 5.02 x 103
HIV-1 negative 3.57 x 103 4.95 x 103
HIV-1 negative 3.71 x 103 3.39 x 103
HIV-1 negative 2.73 x 103 3.00 x 103
HIV-1 negative 3.82 x 103 4.27 x 103
HIV-1 negative 4.67 x 103 4.49 x 103
HIV-1 negative 9.31 x 104 4.66 x 103
HIV-1 negative 3.81 x 103 3.91 x 103
Average HIV-1 negative 1.49 x 104 4.21 x 103
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These results show that HIV-1 can be readily detected in a biological sample
using the
compositions and methods of the present invention. Moreover, the two different
combinations of
amplification oligomers were able to similarly amplify the captured po/target
sequence to produce
detectable amplified products. The next example shows that a different
subtype, HIV-1 subtype C, can
also be detected using the same combinations of capture oligomers,
amplification oligomers and labeled
probe.
Example 2
Detection of HIV-1, Subtype C, Po/Target Sequences in Biological Samples
Samples of normal uninfected blood plasma were mixed with known amounts of HIV-
1 subtype C
RNA to produce samples containing about 100 copies of target nucleic acid per
reaction. Negative
controls were plasma samples without HIV-1 RNA that were treated identically
in the assay. The
samples were subjected to target capture, transcription-mediated amplification
and labeled probe
detection substantially as described in Example 1, and the results of these
assays are shown in Table 2.
Table 2
RLU Detected for Amplification Oligonucleotides:
Sample SEQ ID NO:10, SEQ ID NO:42,
SEQ ID NO:11, SEQ ID NO:11,
SEQ ID NO:13 and SEQ ID NO:13 and
SEQ ID NO:15 SEQ ID NO:15
HIV-1 subtype C + 4.49 x 105 6.63 x 105
HIV-1 subtype C + 4.12 x 105 5.79 x 105
HIV-1 subtype C + 4.07 x 105 4.61 x 105
HIV-1 subtype C + 3.42 x 105 2.94 x 105
HIV-1 subtype C + 3.42 x 105 2.72 x 105
Average subtype C + 3.90 x 105 4.54 x 105
HIV-1 neg. 3.58 x 104 3.07 x 103
HIV-1 neg. 3.06 x 103 2.85 x 103
HIV-1 neg. 2.60 x 103 1.97 x 103
HIV-1 neg. 1.56 x 104 7.26 x 103
Average of HIV-1 neg. 1.42 x 104 3.79 x 103
Although the detected signals in these assays were somewhat lower than those
of Example 1,
these results show that the assay conditions also allow detection of HIV-1
subtype C nucleic acid in a
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biological sample.
The next example shows detection of HIV-1 target which is present in a
biological sample at
between 600 and 60 copies per ml.
Example 3
Detection of Varying Copy Numbers of HIV-1 Subtype B (HIV-1 Illb) Pol Target
Sequences
In this example, plasma samples containing a known number of copies of HIV-1
Illb (600, 200 or
60 copies/ml) were assayed using the procedures substantially as described in
Example 1, but using
different combinations of amplification oligomers. In these assays, the
transcription-mediated
amplification was performed using either (1) SEQ ID NO:10, SEQ ID NO:11, SEQ
ID NO:13 and SEQ ID
NO:15, or (2) SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:43 and SEQ ID NO:15.
Following the capture
and amplification steps, the RLU signals shown in Table 3 were detected. Table
3 presents the average
(mean) RLU and the standard deviation obtained from ten reactions for each of
the different copy number
samples (copy number = 600, 200 or 60 per ml) tested, or from five reactions
for the negative controls
(copy number = 0 per ml).
Table 3
Average RLU Detected for Amplification Oligonucleotides:
HIV-1 Copies/ml of Sample SEQ ID NO:10, SEQ ID N0:10,
SEQ ID N0:11, SEQ ID N0:11,
SEQ ID NO:13 and SEQ ID NO:43 and
SEQ ID N0:15 SEQ ID NO:15
600 373,464 164,269 93,207 70,948
200 74,695 113,555 49,448 49,938
60 22,838 17,487 7,249 8,876
0 1,861 174 1,996 180
In these assays, the combination of capture oligomer, amplification oligomers
and detection
labeled probe that included the amplification promoter-primer having the
sequence of SEQ ID NO:13
provided higher detectable signals than the combination that included SEQ ID
NO:43, but both
combinations were able to detect the presence of at least 200 or more copies
of HIV-1 per ml of sample.
Moreover, the combination that included the primer of SEQ ID NO:13 was
generally able to detect as low
as 60 copies of HIV-1 per ml.
These assays were also performed in separate tests on samples containing 500
copies of HIV-1
IIIb per reaction. In these assays, the combination of capture oligomers,
amplification oligomers and
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detection labeled probe that included the amplification promoter-primer having
the sequence of SEQ ID
NO:13 provided an average RLU signal for ten samples of 2.03 x 106 7.03 x
104 compared to an
average RLU signal of 1,624 174 for five negative controls. The combination
of capture oligomers,
amplification oligomers and detection labeled probe that included the
amplification promoter-primer
having the sequence of SEQ ID NO:43 provided an average RLU signal for ten
samples of 1.30 x 106
4.52 x 105 compared to an average RLU signal of 1,693 196 for four negative
controls.
Other combinations of primers can also be used in a similar assay to detect
HIV-1 in a biological
sample as shown in the next example.
Example 4
Detection of Varying Copy Numbers of HIV-1 Po/Target Sequences Using an
Alternative Promoter-
Primer Oligonucleotide
In additional sets of experiments, plasma samples containing 200, 100 or 20
copies of HIV-1 Illb
per reaction were tested using a similar combination of capture oligomers,
amplification oligomers and
detection labeled probe as one embodiment described in Example 3, but using a
promoter-primer having
the sequence of SEQ ID NO:44 in place of the primer having the sequence of SEQ
ID NO:15 in the
amplification reaction mixture. That is, target capture, amplification and
detection steps were performed
substantially as described in Example 1, but the amplification
oligonucleotides used in the amplification
reactions included those having the sequences of SEQ ID NO:10, SEQ ID NO:11,
SEQ ID NO:13 and
SEQ ID NO:44.
The results of these assays are shown in Table 4, in which the average (mean)
detected RLU
are shown for each of the sets of assays.
Table 4
Detection of Different Numbers of HIV-1 Target in a Sample
Experiment No. HIV-1 Copies Per Mean RLU Samples
1 200 1.59 x 106 5
1 20 1.25 x 105 5
3 3
2 200 1.10 x 106 5
2 20 1.79 x 105 5
3
3 100 2.54 x 106 5
3 20 2.49 x 106 5
3 0 (background) 2.94 x 103 2
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These results show that the assay can detect as few as 20 copies of HIV-1
target nucleic acid in
a biological sample.
The following examples show that a similar assay using capture oligomers,
amplification
oligonucleotides and labeled probes specific for the LTR target region of HIV-
1 can also be used to
detect HIV-1 target nucleic acid in a biological sample.
Example 5
Detection of HIV-1 LTR Sequences
This assay used different capture oligomers to capture the target HIV-1 RNA,
which was present
in the sample at known numbers of copies, and then amplify the HIV-1 sequences
using amplification
oligomers (SEQ ID NO: 8 and SEQ ID NO:9) specific for the LTR region, as
diagrammed in FIG. 1. The
amplified sequences were then detected using a labeled probe that specifically
bound to LTR sequences
(SEQ ID NO:41). The HIV-1 target RNA was present in tested samples made of 500
l human plasma
containing 1,000, 200 or 50 copies of HIV-1, and compared to negative samples
of plasma containing no
HIV-1 RNA which were tested under the same conditions. For each set of
conditions, six samples were
tested.
The protocol used was substantially as described in Example 1 with the
following changes.
Target capture was performed using capture oligomers having SEQ ID NO: 2 (LTR
specific, 2
pmol/reaction), SEQ ID NO: 20 (LTR specific, 2 pmol/reaction) or a mixture of
SEQ IN NO:4 and SEQ ID
NO:6 (both pol specific, 1.75 pmol each/reaction) and using oligo-dT14
oligonucleotides attached to
magnetic particles substantially as described in Example 1. The mixture of
sample, capture oligomers
and immobilized probes on magnetic particles was heated to 60 C for 20 min,
cooled to room
temperature for 20 min and then the magnetic particles and their attached
complexes were separated
and washed twice. Following target capture and washing, amplification was
carried out substantially as
described in Example 1, using 75 /,cl of amplification reagents without
enzymes and containing
amplification oligonucleotides having SEQ ID NO: 8 and SEQ ID NO:9, which was
overlaid with 100 Rl of
oil, heated at 65 C for 10 min and then incubated at 41.5-42 C for 5 min. The
enzymes were added (25
Rl) and the amplification reaction was incubated 1 hr at 42 C. Then 100 ,al of
probe reagent containing
0.1 pmol/reaction of oligomer having SEQ ID NO:41 was added and the mixture
was heated at 60 C for
15 min, followed by addition of 300 RJ of selection reagent, incubation at 60
C for 10 min, cooling to
room temperature and detection of relative light units ("RLU" for 2 sec) in a
luminometer (e.g., LEADER,
Gen-Probe, Inc., San Diego, CA) (substantially as described in U.S. Pat. No.
5,658,737 at column 25,
lines 27-46; and Nelson et al., Biochem. 35:8429-8438(1996) at page 8432).
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The results of these assays are shown in Table 5, reporting the average RLU
(mean standard
deviation) for each of the test conditions of six samples each. The detected
RLU are reported for each
group containing different numbers of HIV-1 target (1,000, 200 or 50 copies)
compared to 0 copies in the
negative ("Neg.") controls.
Table 5
LTR Amplification and Detection
Capture 1,000 Copies 200 Copies 50 Copies 0 Copies
Oligomers HIV-1 HIV-1 HIV-1 (Negative)
SEQ ID NO:2 1.67x106 2.47x105 7.27x104 3.38x103
4.07x105 2.38x105 5.93x104 4.02 x 102
SEQIDNO:20 2.15x105 4.18x104 1.04x104 5.02x104
2.24 x 105 4.30 x 104 1.00 x 104 1.25 x 103
SEQIDNO:4+ 1.40x106 4.75x105 5.15x104 2.92x103
SEQ ID NO:6 3.52 x 105 2.49 x 105 4.51 x 104 5.34 x 102
The results in Table 5 show that the target nucleic acid can be captured by
oligomers specific for
the region to be amplified (e.g., LTR, rows 2 and 3) or by capture oligomers
specific for another region
(e.g., Pol, row 4). The results also show that different capture oligomers for
the LTR region (SEQ ID
NO:2 and SEQ ID NO:20) were both effective in generating positive signals from
HIV-1-containing
biological samples compared to HIV-1 -negative samples.
The next example shows different variations of capture oligomers used to
detect HIV-1 subtype
B and Group 0 sequences.
Example 6
Detection of LTR Regions of HIV-1 Subtype B and Group 0
The assays in this example were performed substantially as described in
Example 5, except that
another capture oligomer was used that is specific for the HIV-1 LTR region,
having a sequence (SEQ ID
N0:45) that varies from that of SEQ ID N0:2 by one base (A instead of T at
position 9). The HIV-1 target
nucleic acid in different plasma samples was either Subtype B (1,000 or 100
copies per assay) or Group
0 (MVP5180 virion at 1,000 or 100 copies per assay).
The samples (six per assay condition) were treated, substantially as described
in Example 5,
with capture oligomers having sequences of SEQ ID N0:2, SEQ ID N0:45 or the
mixture of SEQ ID
N0:4 and SEQ ID N0:6. Amplification was done substantially as described in
Example 6, and the
signals resulting from binding of an acridinium-labeled probe having the
sequence of SEQ ID N0:16 to
the amplified sequences were detected as described in Example 5. The average
RLU (mean standard
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deviation) for the six samples for each of the assay conditions are shown in
Table 6.
Table 6
LTR HIV-1 Detection of Subtype B and Group 0 Viral RNA
Target & Capture Oligomer Capture Oligomer Capture Oligomers
Amount SEQ ID NO:2 SEQ ID NO:45 SEQ ID NO:4 +
in Sample (T at residue 9) (A at residue 9) SEQ ID NO:6
Subtype B 5.49 x 106 6.24 x 106 5.41 x 106
1000 copies 2.21 x 106 1.44 x 105 2.65 x 106
Subtype B 1.87 x 106 2.54 x 106 3.40 x 106
100 copies 2.42 x 106 1.90 x 106 2.09 x 106
Subtype B 3.31 x 103 3.14 x 103 3.23 x 103
0copies 6.72x102 5.14x102 1.10x102
Group0 4.13x106 4.13x106 3.65x106
1000 copies 1.12 x 106 1.13 x 106 1.33 x 106
Group 0 3.29 x 105 6.78 x 105 8.65 x 105
100 copies 3.69 x 105 7.19 x 105 1.38 x 106
Group 0 3.24 x 103 2.98 x 103 3.14 x 103
0 copies 4.99 x 102 7.53 x 102 2.13 x 102
These results show that modified capture oligomers such as that of SEQ ID
N0:45 are effective
in an assay that amplifies and detects the captured HIV-1 target, including
Group 0 HIV-1 target
sequences. In additional assays, capture oligomers that were a mixture of
oligomers having sequences
of SEQ ID N0:2 and SEQ ID N0:45 (i.e., synthetic oligomers in which position 9
was either A or T) were
also effective in assays to detect HIV-1, including Group 0. This example also
shows that capture of the
target HIV-1 nucleic acid may be performed using capture oligomers specific
for one region (e.g., pofj
and then amplified and detected with oligonucleotide primers and probe that
are specific for another
region (e.g., LTR).
The next example shows that different combinations of promoter-primers and non-
T7 primers
can be used to amplify HIV-1 LTR target sequences to produce detectable
amplification products from a
biological sample containing HIV-1.
Example 7
Comparison of HIV-1 LTR Amplification Using Different Combinations of Primers
HIV-1 RNA purified from high titer tissue culture specimens was mixed with
amplification
reagents at known numbers of copies of HIV-1 for testing different
combinations of amplification
oligonucleotides in assays that used the amplification and detection steps
substantially as described in
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Example 5. The biological samples contained 10,000, 1,000, 250 or 100 copies
of HIV-1 per reaction,
and were compared to negative controls (human plasma containing no HIV-1 RNA).
These samples
were amplified in seven replicate tests for each pair of amplification
oligomers tested (about 1.5 to 2.0
pmol of each primer per assay). The combinations of primers used were: a T7
promoter-primer having
the sequence of SEQ ID NO:8 combined with a non-T7 primer having the sequence
of SEQ ID NO:9,
SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:38; and a T7 promoter-
primer having the
sequence of SEQ ID NO:30 combined with a non-T7 primer having the sequence of
SEQ ID NO:9, SEQ
ID NO:35, SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:38. For each of these ten
combinations, the
biological samples that contained 100 or more copies of target HIV-1 produced
a positive detectable
signal in the assay. That is, amplified sequences were produced and then
detected using a labeled LTR-
specific probe (SEQ ID NO:16 or SEQ ID NO:41), producing a signal of 105 to
106 RLU (mean of seven
samples per assay condition), compared to the negative controls that produced
less than 103 RLU
(mean) under the same conditions. For comparison, the HIV-1 -positive samples
were also amplified
using a combination of po/-specific amplification oligomers (SEQ ID NO:10 and
SEQ ID NO:13), which
also produced a detectable average RLU signal of 105 to 106 for all of the HIV-
1 -positive samples.
In additional assays, greater dilutions of the HIV-1 target nucleic acid were
amplified using the
SEQ ID NO:8 promoter-primer combined with a primer having the sequence of SEQ
ID NO:9, SEQ ID
NO:35, SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:38. In these tests, the HIV-1
virion RNA was
present at 100, 50, 30 or 10 copies per assay. Amplified sequences were
detected at an average of 105
to 106 RLU when at least 30 copies of HIV-1 virion RNA were present in the
sample. At 10 copies of HIV-
1 per sample, the detected RLU indicating amplified LTR sequences (104 to 105
average) were at least
10-fold higher than those of the negative controls (averaging less than 103
RLU).
In separate assays, using HIV-1 target nucleic acid present at 100, 50, 30 or
10 copies per
sample, the LTR target sequences were amplified using a promoter primer having
SEQ ID NO:32 and a
non-T7 primer having the sequence of SEQ ID NO:9, SEQ ID NO:35, SEQ ID NO:36,
SEQ ID NO:37 or
SEQ ID NO:38. In these assays, the combinations of a primer having SEQ ID
NO:32 and a primer
having SEQ ID NO:9, SEQ ID NO:35 or SEQ ID NO:36 produced about 104 to 105 RLU
(average) when
50 to 100 copies of target were present, whereas the combinations of a primer
having SEQ ID NO:32
and a primer having SEQ ID NO:37 or SEQ ID NO:38 produced about 103 to 104 RLU
(average) when 50
to 100 copies of target were present, all compared to negative controls of
less than 400 RLU.
HIV-1 target nucleic acid present at 100, 50, 30 or 10 copies per sample were
also amplified
using combinations of a promoter-primer having the sequence of SEQ ID NO:34
and the non-T7 primers
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tested above (SEQ ID NO:9, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 or SEQ ID
NO:38). In these
assays, the combinations of a primer having SEQ ID NO:34 and a primer having
SEQ ID NO:9 or SEQ ID
NO:36 produced about 105 RLU (average) when 50 to 100 copies of target were
present, whereas the
combinations of a primer having SEQ ID NO:34 and a primer having SEQ ID NO:35,
SEQ ID NO:37 or
SEQ ID NO:38 produced about 103 to 104 RLU (average) when 50 to 100 copies of
target were present,
all compared to negative controls of less than 103 RLU.
Based on these results and additional tests, preferred combinations of
amplification
oligonucleotides for amplifying HIV-1 LTR sequences include: SEQ ID NO:8 and
SEQ ID NO:35; SEQ ID
NO:8 and SEQ ID NO:9; SEQ ID NO:8 and SEQ ID NO:36; SEQ ID NO:30 and SEQ ID
NO:9; SEQ ID
NO:30 and SEQ ID NO:36; SEQ ID NO:32 and SEQ ID NO:9; and SEQ ID NO:34 and SEQ
ID NO:36.
The next example shows that both po/-specific and LTR-specific assays can
detect HIV-1
subtype B and Group 0 nucleic acids in biological samples.
Example 8
Assay for HIV-1 Subtype B and Group 0 Nucleic Acids
In these tests, LTR target sequences were amplified and detected for different
HIV-1 isolates.
Plasma samples containing known numbers of copies of HIV-1 were prepared using
subtype B (1,000,
100 or 30 copies of virion RNA per assay) or two Group 0 isolates (CA9 and
MVP5180, purified
individually from high titer tissue culture and used at dilutions equivalent
to about 1,000, 100 or 30 copies
per assay). The samples were subjected to target capture using the methods
substantially as described
in Example 5, using capture oligomers having sequences of SEQ ID N0:2 (LTR-
specific), and SEQ ID
N0:4 and SEQ ID NO:6 (both p0/specific). Then the captured target was
amplified using substantially
the procedures described in Example 5, using a LTR-specific promoter-primer
having the sequence of
SEQ ID N0:8 and a LTR-specific primer having the sequence of SEQ ID N0:9. The
amplified sequences
were detected using an AE-labeled probe (0.1 pmol/ reaction) having the
sequence of SEQ ID N0:16 as
described above. The average RLU detected (mean standard deviation) for five
samples for each set
of conditions are presented in Table 7.
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Table 7
Amplified and Detected (RLU) HIV-1 LTR Sequences of Subtype B and Group 0
Target Amount Subtype B Group 0 Group 0
Strain MVP5180 Strain CA9
1,000 copies 7.72 x 106 8.00 x 106 7.04 x 106
7.10 x 105 4.56 x 105 1.76 x 106
100 copies 4.84 x 106 7.45 x 106 5.91 x 105
2.14x106 5.21 x105 1.23x106
30 copies 3.81 x 105 1.13 x 106 5.52 x 104
3.56 x 105 1.56 x 106 6.96 x 104
0 copies 7.15 x 103 6.98 x 103 5.80 x 103
7.38 x 102 3.71x103 1.12x103
These results show that the assay can detect both subtype B and Group 0 HIV-1
and can detect
different Group 0 strains using HIV-1 LTR-specific primers and probe.
A similar assay was used to detect both LTR and po/ sequences in the same
amplification
mixture. In this assay, the same target HIV-1 nucleic acids were used at the
same concentrations and
target capture was performed using a mixture of capture oligomers that
included the LTR-specific
oligomer having SEQ ID N0:2 and two pol-specific oligomers having SEQ ID N0:4
and SEQ ID N0:6.
During amplification as described above, a combination of primers was used
that included LTR-specific
primers having the sequences of SEQ ID N0:8 and SEQ ID NO:9, and pol specific
primers having the
sequences of SEQ ID NO:10, SEQ ID NO:11, SEQ ID N0:13 and SEQ ID NO:15. This
multiplex
amplification reaction includes multiple primers for different targets in the
same amplification reagent to
simultaneously amplify the target nucleic acids. Following amplification, the
amplified sequences were
detected independently using LTR-specific AE-labeled probe having SEQ ID
N0:16, or poi -specific AE-
labeled probe having SEQ ID N0:17, using 0.1 pmol//.cl of each in the standard
protocol as described in
Example 1. The results of these tests are shown in Table 8, showing the
average RLU (mean standard
deviation) for five samples for each set of assay conditions.
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Table 8
Detection of HIV-1 LTR and Pol Sequences Following Multiplex Amplification
Subtype/Group (Strain), LTR Detection Pol Detection
Target Amount or Dilution Factor
Subtype B 7.78 x 106 4.10 x 106
1,000 copies 4.74 x 105 9.81 x 104
Subtype B 1.57 x 106 2.99 x 106
100 copies 1.36 x 106 1.65 x 106
Subtype B 3.20 x 105 3.09 x 106
30 copies 2.70 x 105 1.07 x 106
Subtype B 3.71 x 103 1.19 x 103
0 copies (negative control) 4.01 x 102 2.81 x 102
Group 0 (Strain MVP5180) 7.92 x 106 1.79 x 106
1,000 copies 3.07 x 105 3.34 x 105
Group 0 (Strain MVP5180) 3.64 x 106 2.16 x 105
100 copies 1.79 x 106 2.49 x 105
Group 0 (Strain MVP5180) 2.28 x 105 3.22 x 104
30 copies 2.16 x 105 4.90 x 104
Group 0 5.06 x 103 1.64 x 103
0 copies (negative control) 3.13 x 102 9.52 x 102
Group 0 (Strain CA9) 1.94 x 106 1.38 x 106
1,000 copies 4.12 x 105 1.21 x 106
Group 0 (Strain CA9) 3.67 x 105 3.10 x 10'+-
100 copies 6.25 x 105 6.16 x 105
Group 0 (Strain CA9) 2.20 x 105 1.27 x 103
copies 4.58 x 105 39
Group 0 3.64 x 103 1.24 x 103
0 copies (negative control) 3.20 x 102 75
These results show that amplified sequences specific for both the HIV-1 LTR
and po/ regions
were produced during the multiplex amplification and the amplified sequences
were detected using LTR-
specific or po/-specific probes, respectively. In similar experiments, a
combination of labeled probes
having sequences of SEQ ID N0:16, SEQ ID N0:17 and SEQ ID N0:18 were used to
successfully detect
the amplified HIV-1 sequences of both subtype B and Group 0 isolates. In
separate experiments, similar
effective detection was accomplished using a labeled probe having SEQ ID N0:16
with an inosine at
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position 7.
Similar results were obtained in separate experiments in which the amplified
target region was
HIV-1 pol sequences from subtype B and Group 0, probed with a 2-methyl-AE
labeled probe having
SEQ ID NO:17. In these experiments, the target was initially present at 1,000,
100, 50, 20 or 10 copies
per reaction, compared to a negative control containing no target. Five
replicate assays were performed
for each reaction condition. For HIV-1 subtype B, the mean RLU detected was in
a range of 9.20 x 105 to
4.44 x 105 for reactions containing target, compared to a mean RLU of 1.97 x
103 for the negative
controls. For HIV-1 Group 0, the mean RLU detected was in a range of 3.53 x
105 to 1.69 x 104 for
reactions containing 1,000 to 50 copies of target, with variable results
obtained for tubes containing 20 or
10 copies of target, compared to a mean RLU of 2.60 x 103 for the negative
controls.
These results all show that different groups of HIV-1 can be detected using
target capture and
amplification of sequences using the combinations of capture probes, primers
and detection probes
described herein. Use of additional combinations are described in Examples 9
and 10.
Example 9
Assay for HIV-1 Pol Target Nucleic Acids in HIV-1 Subtype B and Group 0
In a series of tests, HIV-1 of subtype B and Group 0 were assayed
substantially as described in
the previous examples, using different probes. The samples were subjected to
target capture using the
methods substantially as described in Example 1, using po/-specific capture
oligomers having sequences
of SEQ ID N0:4 and SEQ ID N0:6. Then the captured HIV-1 target was amplified
using substantially the
procedures described in Example 1, using a combination of promoter-primers
having the sequences of
SEQ ID NO:13 and SEQ ID NO:15 and a combination of primers having the
sequences of SEQ ID N0:10
and SEQ ID NO:1 1. These experiments compared detection with different HIV-1 -
specific probes. The
reactions were performed using HIV-1 subtype B at 104 copies/ml and Group 0 at
106 copies/ml.
Following amplification and before detection, the amplification reactions for
each set of assays were
pooled and then diluted as indicated in Table 9. At limiting dilutions, the
RLU detected was equivalent to
background (data not shown). For each assay reported in Table 9, ten
individual detection assays were
performed and the mean number of RLU detected standard deviation are
presented.
The amplified po/ sequences were detected as described earlier using 2-methyl-
AE labeled
probes (0.1 pmol/ reaction) having the sequence of SEQ ID N0:18 or SEQ ID
N0:46, using different lots
of the probe having SEQ ID N0:46, labeled between residues 10 and 11 (Lot 1)
or residues 7 and 8 (Lot
2).
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Table 9
Comparison of Pol-specific Probes for Detecting HIV-1 Subtype B and Group 0
Target, Dilution Factor SEQ ID NO:46 SEQ ID NO:46 SEQ ID N0:18
Assay Number Probe (Lot 1) Probe (Lot 2) Probe
Subtype B, 10"' Dilution 1.05 x 105 9.83 x 104 1.00 x 105
Assay l 2.66 x 103 2.90 x 103 2.20 x 103
Subtype B, 10"' Dilution 9.62 x 104 9.57 x 104 9.91 x 104
Assay 2 4.88 x 103 4.40 x 103 2.54 x 103
Group 0, 10-' Dilution 8.66 x 105 7.11 x 105 6.61 x 105
Assay 3 1.69 x 104 1.43 x 104 1.51 x 104
Group 0, 10-2 Dilution 3.41 x 105 2.53 x 105 4.77 x 105
Assay 4 1.15 x 104 1.08 x 104 1.32 x 104
The results of these experiments show that different labeled probes can
effectively detect both
HIV-1 subtype B and Group 0 amplified target sequences. In separate
experiments using similar
experimental protocols, probes having SEQ ID N0:46 labeled with 2-methyl-AE
between residues 6 and
7, 7 and 8, or 10 and 11 were all effective in detecting HIV-1 subtype B and
Group 0 amplified target
sequences. These experiments show that a variety of different labeled probes
may be used in these
detection assays for detecting different groups of HIV-1.
Example 10
Assay for HIV-1 Po/Target Nucleic Acids in HIV-1 Subtype B and Group 0
In experiments similar to those described in Example 9, HIV-1 target sequences
from subtype B
and Group 0 were amplified and detected using probes having sequences of SEQ
ID NO:50 or SEQ ID
N0:53 and compared to results with the previously tested SEQ ID N0:17 probe.
In these experiments,
capture of the target RNA was not included and the sample tubes were prepared
containing 104 copies of
either HIV-1 subtype B or HIV-1 Group 0 (five tubes for each type, with a
negative control containing no
HIV-1 RNA) in an amplification reaction mixture containing 40 mM Trizma base
(pH 7.5), 17.5 mM KCI,
20 mM MgCl2, 5% PVP, 1 mM each dNTP and 4 mM each rNTP. Transcription-mediated
amplification
was performed substantially as described in Example 1, except that incubation
temperatures were 65 C
for 10 min, and then 42 C for 10 min before addition of enzymes, followed by
amplification for 1 hr using
primers having the sequences of SEQ ID N0:10, SEQ ID N0:11, SEQ ID N0:13 and
SEQ ID NO:15.
Following amplification, the products were pooled to minimize variation
between different amplification
tubes, making separate pools for the different HIV-1 strains. The pooled
amplification products were
used without dilution (i.e., about 104 copies of target in the sample) or
serially diluted into 2X
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Hybridization Buffer (see Example 1) as shown in Table 10, to provide the
equivalent of 103, 102, 10 or 1
copies of target in the sample. The tubes were then used for hybridization at
60 C for 15 min with AE-
labeled probes having SEQ ID NO: 50, SEQ ID NO:53 or SEQ ID NO:17. Following
treatment of the
hybridization tubes at 60 C for 10 min with the selection reagent to
selectively inactivate unbound probe,
chemiluminescence was detected as described above. Mean RLU for each of the
hybridization reactions
are shown in Table 10.
Table 10
Detected RLU for Amplified HIV-1 Pol Sequences with Different Probes
Target, Copies per SEQ ID NO:50 SEQ ID NO:53 SEQ ID NO:17
Sample
Subtype B, 104 2.14 x 105 4.39 x 105 3.71 x 105
Subtype B, 103 8.11 x 104 2.40 x 105 2.21 x 105
Subtype B, 102 6.84 x 104 2.15 x 105 2.01 x 105
Subtype B, 10 6.45 x 104 2.24 x 105 1.40 x 105
Subtype B, 1 2.37 x 104 8.51 x 104 2.31 x 104
Group 0, 104 7.50 x 104 4.23 x 105 2.89 x 105
Group 0,103 2.74x104 2.16x105 1.71x105
Group 0,102 1.43x104 1.26x105 5.15x104
Group 0, 10 7.20 x 103 2.93 x 104 7.65 x 103
Group 0, 1 5.90 x 103 5.78 x 103 not determined
As in Example 9 and by comparison with those results, the results of Table 10
show that different
groups of HIV-1 can be detected using different pol specific probes. In these
and additional experiments,
probes having SEQ ID NO:53 were shown to be effective in detecting HIV-1 pol
amplified sequences
when labeled at a variety of positions in the probe (e.g., between residues 5
and 6, or residues 6 and 7,
or residues 13 and 14). Similarly, probes having SEQ ID NO:17 were effective
in detecting HIV-1
amplified sequences when labeled at a variety of positions (e.g., between
residues 6 and 7, or residues 7
and 8, or residues 10 and 11). Thus, different probes exemplified by these
experimental results are
effective for detecting amplified HIV-1 sequences.
Although the present invention has been described in the context of particular
examples and
preferred embodiments, it will be understood that the invention is not limited
to such embodiments.
Instead, the scope of the present invention shall be measured by the claims
that follow.
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SEQUENCE LISTING
<110> GEN-PROBE INCORPORATED
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<141> 2000-07-07
<150> 60/143,072
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<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
amplification oligomer for HIV-1 pol sequence
<400> 14
gtttgtatgt ctgttgctat tat 23
<210> 15
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
amplification oligomer with 5' promoter sequence,
for HIV-1 pol sequence
3
CA 02374385 2001-11-19
WO 01/04361 PCT/US00/18685
<400> 15
aatttaatac gactcactat agggagagtt tgtatgtctg ttgctattat 50
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
oligomer probe
<220>
<221> modified-base
<222> (7)
<223> any base, including inosine
<400> 16
ctggtancta gagatccctc 20
<210> 17
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
oligomer probe
<400> 17
ccacaatttt aaaagaaaag gg 22
<210> 18
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
oligomer probe
<220>
<221> modified-base
<222> (8)
<223> inosine
<400> 18
ggattggngg gtacagt 17
<210> 19
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
capture oligomer
<400> 19
acaaccatcc aaargtcagt gg 22
<210> 20
<211> 52
<212> DNA
<213> Artificial Sequence
4
CA 02374385 2001-11-19
WO 01/04361 PCT/US00/18685
<220>
<223> Description of Artificial Sequence: synthetic
capture oligomer with 3' tail sequence
<400> 20
acaaccatcc aaargtcagt ggaaaaaaaa aaaaaaaaaa aaaaaaaaaa as 52
<210> 21
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
amplification oligomer for HIV-1 LTR region
<400> 21
cctgttcggg cgccactgc 19
<210> 22
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
amplification oligomer with 5' promoter sequence,
for HIV-1 LTR region
<400> 22
ttaatacgac tcactatagg gagacctgtt cgggcgccac tgc 43
<210> 23
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
amplification oligomer for HIV-1 LTR region
<400> 23
ggcgccactg ctagagattt t 21
<210> 24
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
amplification oligomer with 5' promoter sequence,
for HIV-1 LTR region
<400> 24
aatttaatac gactcactat agggagaggc gccactgcta gagatttt 48
<210> 25
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
amplification oligomer for HIV-1 LTR region
<400> 25
CA 02374385 2001-11-19
WO 01/04361 PCT/US00/18685
cgggcgccac tgctagagat tttc 24
<210> 26
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
amplification oligomer with 5' promoter sequence,
for HIV-1 LTR region
<400> 26
aatttaatac gactcactat agggagacgg gcgccactgc tagagatttt c 51
<210> 27
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
amplification oligomer for HIV-1 LTR region
<400> 27
gggcgccact gctagagatt ttc 23
<210> 28
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
amplification oligomer with 5' promoter sequence,
for HIV-1 LTR region
<400> 28
aatttaatac gactcactat agggagaggc gccactgcta gagattttc 49
<210> 29
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
amplification oligomer for HIV-1 LTR region
<400> 29
gttcgggcgc cactgctaga g 21
<210> 30
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
amplification oligomer with 5' promoter sequence,
for HIV-1 LTR region
<400> 30
aatttaatac gactcactat agggagagtt cgggcgccac tgctagag 48
<210> 31
<211> 21
6
CA 02374385 2001-11-19
WO 01/04361 PCT/US00/18685
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
amplification oligomer for HIV-1 LTR region
<400> 31
ctgttcgggc gccactgcta g 21
<210> 32
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
amplification oligomer with 5' promoter sequence,
for HIV-1 LTR region
<400> 32
atttaatacg actcactata gggagactgt tcgggcgcca ctgctag 47
<210> 33
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
amplification oligomer for HIV-1 LTR region
<400> 33
gcaagccgag tcctgcgtcg agag 24
<210> 34
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
amplification oligomer with 5' promoter sequence,
for HIV-1 LTR region
<400> 34
aatttaatac gactcactat agggagagca agccgagtcc tgcgtcgaga g 51
<210> 35
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial sequence: synthetic
amplification oligomer for HIV-1 LTR region
<400> 35
gcttaagcct caataaagct tgcctt 26
<210> 36
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
7
CA 02374385 2001-11-19
WO 01/04361 PCTIUSOO/18685
amplification oligomer for HIV-1 LTR region
<400> 36
gcctcaataa agcttgcctt gag 23
<210> 37
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
amplification oligomer for HIV-1 LTR region
<400> 37
gcttaagcct caataaagct tgc 23
<210> 38
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
amplification oligomer for HIV-1 LTR region
<400> 38
ctgcttaagc ctcaataaag c 21
<210> 39
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
oligomer probe
<220>
<221> modified-base
<222> (6)..(7)
<223> any base, including inosine
<400> 39
ctggtnncta gagatccctc 20
<210> 40
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
oligomer probe
<400> 40
ggtarctaga gatccctcag 20
<210> 41
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
oligomer probe
8
CA 02374385 2001-11-19
WO 01/04361 PCT/US00/18685
<400> 41
gactctggta actagagatc 20
<210> 42
<211> 16
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
amplification oligomer for HIV-1 pol sequence
<400> 42
acagcagtac aaatgg 16
<210> 43
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
amplification oligomer with 5' promoter sequence,
for HIV-1 pol sequence
<400> 43
aatttaatac gactcactat agggagagta tgtctgttgc tattatgtct a 51
<210> 44
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial sequence: synthetic
amplification oligomer with 5' promoter sequence,
for HIV-1 pol sequence
<400> 44
aatttaatac gactcactat agggagaagt ttgtatgtct gttgctatta t 51
<210> 45
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
capture oligomer with 3' tail sequence
<400> 45
actgacgcac tcgcacccat ctttaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 54
<210> 46
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
oligomer probe
<220>
<221> modified-base
<222> (26)
<223> any base, including inosine
9
CA 02374385 2001-11-19
WO 01/04361 PCTIUSOO/18685
<220>
<221> modified-base
<222> (29)
<223> any base, including inosine
<400> 46
tcatucacaa ttttaaaaga aaaggnggna 30
<210> 47
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
oligomer probe
<400> 47
ccacaatttt aaaagaaaag gggggattgg 30
<210> 48
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
oligomer probe
<400> 48
gggtacagtg caggggaaag aatagtagac 30
<210> 49
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
oligomer probe
<400> 49
gggtacagtg caggggaaag as 22
<210> 50
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
oligomer probe
<220>
<221> modified-base
<222> (9)
<223> any base, including inosine
<400> 50
agaaaaggng ggattggg 18
<210> 51
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
CA 02374385 2001-11-19
WO 01/04361 PCT/US00/18685
<223> Description of Artificial Sequence: synthetic
oligomer probe
<220>
<221> modified-base
<222> (4)
<223> any base, including inosine
<220>
<221> modified-base
<222> (13)
<223> any base, including inosine
<400> 51
aggngggatt ggngggtaca 20
<210> 52
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
oligomer probe
<220>
<221> modified-base
<222> (3)
<223> any base, including inosine
<220>
<221> modified-base
<222> (11)
<223> any base, including inosine
<400> 52
ggnggattgg ngggtacagt 20
<210> 53
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
oligomer probe
<220>
<221> modified-base
<222> (9)
<223> any base, including inosine
<400> 53
gggattggng ggtacagtg 19
<210> 54
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
oligomer probe
<400> 54
caggggaaag aatagtagac 20
11
CA 02374385 2001-11-19
WO 01/04361 PCT/US00/18685
<210> 55
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
oligomer probe
<400> 55
caggggaaag aatagta 17
<210> 56
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
oligomer probe
<400> 56
ggggaaagaa tagtagac 18
<210> 57
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
capture oligomer
<400> 57
actgacgcac tcgcacccat ct 22
12
