U.S. patent application number 11/040417 was filed with the patent office on 2006-01-05 for primer and probe design for efficient amplification and detection of hcv 3' non-translating region.
This patent application is currently assigned to bioMerieux, Inc.. Invention is credited to Lawrence Burg, Prakash Purohit, Hwa-Tang Thomas Wang, Brian Washburn.
Application Number | 20060003343 11/040417 |
Document ID | / |
Family ID | 34812082 |
Filed Date | 2006-01-05 |
United States Patent
Application |
20060003343 |
Kind Code |
A1 |
Wang; Hwa-Tang Thomas ; et
al. |
January 5, 2006 |
Primer and probe design for efficient amplification and detection
of HCV 3' non-translating region
Abstract
The present invention provides selected oligonucleotides
corresponding to portions of the 3' non-translating region of
Hepatitis C virus. The invention also includes methods of detecting
and of quantitating HCV nucleic acids in a sample using these
oligonucleotides.
Inventors: |
Wang; Hwa-Tang Thomas;
(Encinitas, CA) ; Washburn; Brian; (Durham,
NC) ; Purohit; Prakash; (Shrewsbury, MA) ;
Burg; Lawrence; (Framingham, MA) |
Correspondence
Address: |
BIOMERIEUX, INC.
PATENT DEPARTMENT
100 RODOLPHE STREET
DURHAM
NC
27712
US
|
Assignee: |
bioMerieux, Inc.
|
Family ID: |
34812082 |
Appl. No.: |
11/040417 |
Filed: |
January 21, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60538815 |
Jan 23, 2004 |
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60538814 |
Jan 23, 2004 |
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60538816 |
Jan 23, 2004 |
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Current U.S.
Class: |
435/5 ;
435/345 |
Current CPC
Class: |
C12Q 1/6851 20130101;
C12Q 1/707 20130101; C12Q 1/6844 20130101; C12Q 1/6851 20130101;
C12Q 1/6844 20130101; C12Q 1/6846 20130101; C12Q 1/6806 20130101;
C12Q 1/6846 20130101; C12Q 2527/101 20130101; C12Q 2527/137
20130101; C12Q 2527/137 20130101; C12Q 2527/125 20130101; C12Q
2527/101 20130101; C12Q 2527/125 20130101; C12Q 2527/113 20130101;
C12Q 2527/101 20130101; C12Q 2527/125 20130101; C12Q 2527/125
20130101; C12Q 2527/125 20130101; C12Q 2527/137 20130101; C12Q
2527/125 20130101; C12Q 2527/125 20130101; C12Q 2527/107 20130101;
C12Q 2527/107 20130101; C12Q 1/686 20130101; C12Q 1/686 20130101;
C12Q 1/6806 20130101; C12Q 1/686 20130101; C12Q 1/6846
20130101 |
Class at
Publication: |
435/006 ;
435/345 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12N 5/06 20060101 C12N005/06; C12N 5/16 20060101
C12N005/16 |
Claims
1. An isolated nucleic acid comprising a nucleotide sequence
selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 11,
SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 27, SEQ ID
NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO:32, SEQ ID NO:24,
SEQ ID NO:41, SEQ ID NO:43, and SEQ ID NO:42.
2. An isolated nucleic acid consisting essentially of a nucleotide
sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID
NO: 11, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 27,
SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO:32, SEQ ID
NO:24, SEQ ID NO:41, SEQ ID NO:43, and SEQ ID NO:42.
3. An isolated nucleic acid consisting essentially of a nucleotide
sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID
NO: 11, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 27,
SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO:32, SEQ ID
NO:24, SEQ ID NO:41, SEQ ID NO:43, and SEQ ID NO:42, wherein said
nucleotide sequence further has from one to four mismatches and
hybridizes under NASBA hybridization conditions, to the 3'NTR of
HCV.
4. An isolated nucleic acid comprising a nucleotide sequence set
forth in SEQ ID NO: 37.
5. An isolated nucleic acid consisting essentially of a nucleotide
sequence set forth in SEQ ID NO:37.
6. An isolated nucleic acid comprising (a) a tag oligonucleotide
having a 5' end and a 3' end, and (b) a primer nucleic acid having
a 5' end and a 3' end and comprising a nucleotide sequence selected
from the group consisting 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:7, and SEQ ID NO:
8, wherein the tag oligonucleotide consists of about 18 to about 23
randomly selected nucleotides heterologous to HCV and is linked at
its 3' end to the 5' end of the primer nucleic acid.
7. The isolated nucleic acid of claim 6, wherein the tag
oligonucleotide comprises the nucleotide sequence set forth in SEQ
ID NO: 9.
8. An isolated nucleic acid comprising (a) a promoter
oligonucleotide that, when in double-stranded form, can function as
a T7 promoter, and (b) a primer nucleic acid having a 5' end and a
3' end and comprising a nucleotide sequence selected from the group
consisting of SEQ ID NO:11, SEQ ID NO: 15, SEQ ID NO:27, SEQ ID
NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, wherein the
promoter oligonucleotide is linked at its 3' end to the 5' end of
the primer nucleic acid.
9. The isolated nucleic acid of claim 8, wherein the promoter
oligonucleotide has the nucleic acid sequence set forth in SEQ ID
NO:38.
10. The isolated nucleic acid of claim 8, wherein the promoter
oligonucleotide has the nucleic acid sequence set forth in SEQ ID
NO:23.
11. A method of assaying for the presence of HCV in a nucleic acid
sample comprising a. Amplifying a selected HCV nucleic acid to form
an HCV amplification product, utilizing as a primer of
amplification a nucleic acid comprising a nucleotide sequence
selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 11,
SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24; SEQ ID
NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO:32,
SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO:43; and b. Detecting
the presence of amplified product.
12. The method of claim 11, wherein the amplification utilizes a
first primer comprising the nucleotide sequence set forth in SEQ ID
NO:11 and a second primer comprising the nucleotide sequence set
forth in SEQ ID NO:8.
13. The method of claim 12, wherein the first primer further
comprises, at its 5' end, a promoter oligonucleotide that, when in
double-stranded form, can function as a T7 promoter.
14. The method of claim 13, wherein the T7 promoter is SEQ ID NO:
38.
15. The method of claim 11, wherein the amplification utilizes a
first primer comprising the nucleotide sequence set forth in SEQ ID
NO: 15 and a second primer comprising the nucleotide sequence set
forth in SEQ ID NO:8.
16. The method of claim 15, wherein the first primer further
comprises, at its 5' end, a promoter oligonucleotide that, when in
double-stranded form, can function as a T7 promoter.
17. The method of claim 11, wherein the amplification utilizes a
first primer and a second primer, said second primer comprising (a)
a tag oligonucleotide having a 5' end and a 3' end, and (b) a
primer nucleic acid having a 5' end and a 3' end and comprising a
nucleotide sequence selected from the group consisting 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:7, and SEQ ID NO: 8, wherein the tag
oligonucleotide consists of about 10 to about 25 randomly selected
nucleotides heterologous to HCV and is linked at its 3' end to the
5' end of the primer nucleic acid.
18. The method of claim 11, wherein the amplification utilizes a
first primer and a second primer, said first primer comprising (a)
a promoter oligonucleotide that, when in double-stranded form, can
function as a T7 promoter, and (b) a primer nucleic acid having a
5' end and a 3' end and comprising a nucleotide sequence selected
from the group consisting of SEQ ID NO:11, SEQ ID NO:15, SEQ ID
NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32,
wherein the promoter oligonucleotide is linked at its 3' end to the
5' end of the primer nucleic acid.
19. The method of claim 11, wherein the detecting is performed by
hybridizing to the amplification product a detectable probe.
20. The method of claim 19, wherein the detectable probe comprises
a nucleic acid comprising a nucleotide sequence selected from the
group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24,
SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43.
21. The method of claim 11, wherein the detecting is performed by
capturing the amplification product on an immobilised capture
probe.
22. The method of claim 21, wherein the capture probe comprises a
nucleic acid comprising a nucleotide sequence selected from the
group consisting of SEQ ID NO: 19.
23. The method of claim 11, wherein the amplification is performed
by isothermal amplification or thermocyclic amplification.
24. A method of assaying for the presence of HCV in a nucleic acid
sample comprising a. Amplifying a target nucleic acid comprising a
selected portion of the 3'NTR of HCV and b. Detecting the presence
of amplified product utilizing a probe comprising a nucleotide
sequence selected from the group consisting of SEQ ID NO: 21, SEQ
ID NO: 22; SEQ ID NO:19, SEQ ID NO: 24, SEQ ID NO: 41, SEQ ID NO:
42, and SEQ ID NO: 43.
25. A method of detecting the presence of HCV nucleic acid in a
sample comprising hybridizing with the sample, under selective
hybridization conditions, a nucleic acid comprising a nucleotide
sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID
NO: 11, SEQ ID NO: 15, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30,
SEQ ID NO:31, and SEQ ID NO:32.
26. A method of quantifying the amount of HCV nucleic acid in a
sample comprising a. Amplifying selected HCV nucleic acid to form
an HCV amplification product, utilizing as a primer of
amplification a nucleic acid comprising a nucleotide sequence
selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 11,
SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24; SEQ ID
NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO:32,
SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO:43, and b. Quantifying
the amount of amplified product.
27. The method of claim 26, wherein the amplification utilizes a
first primer comprising the nucleotide sequence selected from the
group consisting of SEQ ID NO:11 and SEQ ID NO: 15 and a second
primer comprising the nucleotide sequence set forth in SEQ ID NO:8.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 60/538,815 filed on Jan. 23, 2004, U.S.
Provisional Patent Application No. 60/538,814 filed on Jan. 23,
2004, and U.S. Provisional Patent Application No. 60/538,816 filed
on Jan. 23, 2004.
FIELD OF THE INVENTION
[0002] The present invention relates to oligonucleotides that bind
to the 3' non-translating region (3'NTR) of Hepatitis C Virus
(HCV), useful for the detection and/or quantification of HCV. Also
provided are methods of nucleic acid assays useful in the
detection, capture and amplification of HCV utilizing the 3'NTR as
the target region.
BACKGROUND OF THE INVENTION
[0003] After the development of diagnostic tests for hepatitis A
and hepatitis B viruses in the 1970s, an additional parenterally
transmitted agent responsible for the majority of
transfusion-associated non-A, non-B hepatitis cases was recognized.
The identification of this agent turned out to be very difficult
(Bartenschlager and Lohmann, J. Gen. Virol. 81: 1631-1648 (2000).
With the advent of recombinant DNA, the genome of the virus that
was termed hepatitis C virus (HCV) was cloned (Choo et al., Science
244: 359-362 (1989)). Its RNA has been difficult to study because
biological materials are scarce and RNA replication is of low
efficiency (Shi and Lai, Cell. Mol. Life Sci. 58: 1276-1295
(2001)).
[0004] Many general techniques are known for studying nucleic
acids. When necessary, enzymatic amplification of nucleic acid
sequences will enhance the ability to detect a desired nucleic acid
sequence. Generally, the currently known amplification schemes can
be broadly grouped into two classes based on whether the enzymatic
amplification reactions are driven by continuous cycling of the
temperature between the denaturation temperature, the primer
annealing temperature, and the amplicon (product of enzymatic
amplification of nucleic acid) synthesis temperature, or whether
the temperature is kept constant throughout the enzymatic
amplification process (isothermal amplification). Typical cycling
nucleic acid amplification technologies (thermocycling) are
polymerase chain reaction (PCR), and ligase chain reaction (LCR).
Specific protocols for such reactions are discussed in, for
example, Short Protocols in Molecular Biology, 2.sup.nd Edition, A
Compendium of Methods from Current Protocols in Molecular Biology,
(Eds. Ausubel et al., John Wiley & Sons, New York, 1992)
chapter 15. Reactions which are isothermal include:
transcription-mediated amplification (TMA), nucleic acid
sequence-based amplification (NASBA), and strand displacement
amplification (SDA).
[0005] U.S. Pat. Nos. 4,683,195 (Mullis); 4,965,188 (Mullis); and
4,683,202 (Mullis) describe a polymerase chain reaction (PCR)
utilizes DNA polymerase, complementary primer molecules and
repeated cycles of thermal reactions to exponentially replicate
target nucleic acid molecules. Isothermal target amplification
methods include transcription-based amplification methods, in which
an RNA polymerase promoter sequence is incorporated into primer
extension products at an early stage of the amplification (WO
89/01050), and further target sequence, or target complementary
sequence, is amplified by transcription steps and digestion of an
RNA strand in a DNA/RNA hybrid intermediate product. See, for
example, U.S. Pat. Nos. 5,169,766 and 4,786,600. These methods
include transcription mediated amplification (TMA), self-sustained
sequence replication (3SR), Nucleic Acid Sequence Based
Amplification (NASBA), and variations there of. See, for example,
Guatelli et al. Proc. Natl. Acad. Sci. U.S.A. 87:1874-1878 (1990);
U.S. Pat. Nos. 5,766,849 5,399,491; 5,480,784; 5,766,849; 5,466,586
(NASBA); 5,409,818(NASBA); 5,554,517(NASBA); 6,063,603(NASBA);
5,130,238 (NASBA); and 5,654,142 (TMA); and 5,130,238 (Malek et
al.); 5,409,818 (Davey et al.); 5,654,142 (Kievits); and 6,312,928
(Van Gemen et al.) (nucleic acid sequence-based amplification
(NASBA) techniques). U.S. Pat. No. 5,792,607 (Backman) describes
amplification methods referred to as ligase chain reactions (LCR).
U.S. Pat. Nos. 5,744,311 (Fraiser); 5,648,211 (Fraiser) and
5,631,147 (Lohman), describe isothermal amplification systems based
on strand displacement amplification (SDA). Other approaches
include Q.beta. replicase, strand displacement assay (SDA),
transcription mediated iso CR cycling probe technology, nucleic
acid sequence-based amplification (NASBA) and cascade rolling
circle amplification (CRCA). Additional U.S. Patent documents which
describe nucleic acid amplification include U.S. Pat. Nos.
4,876,187; 5,030,557; 5,399,491; 5,485,184; 5,554,517; 5,437,990;
5,399,491 and 5,554,516.
[0006] Nucleic acid hybridization techniques have been described
for example, in Sambrook et al. Molecular Cloning A Laboratory
Manual, 2nd Ed. Cold Spring Lab. Press, December 1989; U.S. Pat.
Nos. 4,563,419 (Ranki) and 4,851,330 (Kohne) and in Dunn, et al.,
Cell 12, pp. 23-26 (1978) among many other publications.
[0007] Detection methods utilizing nucleic acids are also known.
Nucleic acids are often labeled for various detection purposes. For
example, methods described in U.S. Pat. Nos. 4,486,539 (Kourlisky);
4,411,955 (Ward); 4,882,269 (Schneider) and 4,213,893 (Carrico),
illustrate preparation of labeled detection probes for detecting
specific nucleic acid sequences. Furthermore, before or after
exposing an extracted nucleic acid to a probe, the target nucleic
acid can be immobilized by target-capture means, either directly or
indirectly, using a "capture probe" bound to a substrate, such as a
magnetic bead. Examples of target-capture methodologies are
described by Ranki et al., U.S. Pat. No. 4,486,539, and Stabinsky,
U.S. Pat. No. 4,751,177. Further uses of probes have been
described, for example, in U.S. Pat. Nos. 5,210,015; 5,487,972;
5,804,375; 5,994,076.
[0008] HCV has been classified as the sole member of a distinct
genus called Hepacivirus in the family Flaviviridae HCV is an
enveloped particle harboring a plus-strand RNA with a length of
approximately 9600 nucleotides. The genome carries a single long
open reading frame (ORF) encoding a polyprotein that is
proteolytically cleaved into a set of distinct products. An
approximately 340 nucleotide-long 5' non-translated region (NTR)
functions as an internal ribosome entry site (IRES) for translation
of the HCV ORF (Tsukiyama-Kohara et al., J. Virol. 66: 1476-1483
(1992); Wang et al., J. Virol. 67: 3338-3344 (1993)). The 3'NTR was
more recently studied (Kolykhalov et al., J. Virol. 70: 3363-3371
(1996); Tanaka et al., Biochem. Biophys. Res. Comm. 215: 744-749
(1995), J. Virol. 70: 3307-3312 (1996); Yamada et al., Virology
223: 255-261 (1996)). The HCV 3'nontranslated region has been found
to include four elements (positive sense, 5' to 3'): (i) a short
sequence with significant variability among genotypes, (ii) a
homopolymeric poly(U) tract, (iii) a polypyrimidine stretch
consisting of mainly U with interspersed C residues, (iv) a novel
sequence of 98 bases (the X-region). This latter nucleotide
sequence is not present in human genomic DNA and is highly
conserved among HCV genotypes (Kolykhalov et al. (1996); Pavio and
Lai, J. Biosci. 28(3): 287-304 (2003)). Most HCV infections
persist, leading in about 50% of all cases to chronic hepatitis,
which can develop into chronic active hepatitis, liver cirrhosis
and hepatocellular carcinoma. Furthermore, HCV is distributed
worldwide, with the number of infected individuals being estimated
to be .about.170 million. (Bartenschlager and Lohmann (2000). Thus,
a definitive diagnostic test is needed to identify infected
individuals. Furthermore, HCV genotyping in patients is essential
for diagnostic and epidemiological studies, as well as in studies
of the natural history and treatment of HCV.
[0009] Detection of HCV RNA is the diagnostic test utilized for
acute and chronic HCV infection. However, it is often complicated
by the low levels of HCV replication or small numbers of infected
cells in hepatitis C patients (Shi and Lai (2000)). Most
qualitative and quantitative diagnostic tests are RTPCR aiming at
the 5'-UTR, the most conserved region of the HCV genome. However,
the sensitivity of this method has been reported to be accompanied
by problems of false priming, presumably in areas of RNA secondary
structure (Shi and Lai (2000)). This has been partially addressed
by the use of tagged primers or a thermostable reverse
transcriptase (Lanford, Virology 202: 606-614 (1994)). Since the
core and NS5B genes are relatively conserved and do not contain
extensive secondary structures, they have also been employed in
various detection methods.
[0010] The X region of the 3'-UTR is another highly conserved
region in the HCV genome, but it is highly structured. The 3' HCV
NTR is considered a difficult amplification target for at least two
reasons: it forms highly stable secondary structures; and the NTR
is very small. It has not been used in the detection of HCV RNA
due, at least in part, to its lack of practical advantages over the
well-established tests based on the 5'-UTR sequence (Shi and Lai
(2000)).
[0011] Thus there is a clear need for primer and probe designs that
provide more useful and reliable results in detection of the
presence of HCV. Specifically, there is a need for primers and
probes that are aimed at overcoming the complications of 3' NTR
secondary structure, type specificity and palindrome sequences that
could affect the hybridization process and providing improved means
for the detection, capture, and/or amplification of HCV nucleic
acids. The present invention provides oligonucleotides that address
these needs to help solve such issues.
SUMMARY OF THE INVENTION
[0012] The present invention provides an isolated Hepatitis C
virus-derived nucleic acid comprising a nucleotide sequence
selected from the group consisting of specific nucleic acid
sequences corresponding to a portion of the 3'NTR of HCV. Such
oligonucleotides are useful in detecting the presence of HCV
nucleic acids. Specifically, the present invention provides an
isolated nucleic acid comprising a nucleotide sequence selected
from the group consisting of SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID
NO: 15, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24; SEQ ID NO: 27,
SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO:32, SEQ ID
NO: 41, SEQ ID NO: 42, and SEQ ID NO:43.
[0013] The present invention further provides an isolated nucleic
acid comprising (a) a tag oligonucleotide having a 5' end and a 3'
end, and (b) a primer nucleic acid having a 5' end and a 3' end and
comprising a nucleotide sequence corresponding to a portion of the
3'NTR of HCV, wherein the tag oligonucleotide consists of about 18
to about 23 randomly selected nucleotides heterologous to HCV and
is linked at its 3' end to the 5' end of the primer nucleic acid.
More specifically, the present invention further provides an
isolated nucleic acid comprising (a) a tag oligonucleotide having a
5' end and a 3' end, and (b) a primer nucleic acid having a 5' end
and a 3' end and comprising a nucleotide sequence selected from the
group consisting 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:7, and SEQ ID NO: 8,
wherein the tag oligonucleotide consists of about 18 to about 23
randomly selected nucleotides heterologous to HCV and is linked at
its 3' end to the 5' end of the primer nucleic acid.
[0014] The present invention additionally provides a method of
assaying for the presence of HCV in a nucleic acid sample
comprising (a) amplifying a selected HCV nucleic acid to form an
HCV amplification product, utilizing as a primer of amplification a
nucleic acid comprising a nucleotide sequence selected from the
group consisting of specific nucleic acid sequences corresponding
to a portion of the 3'NTR of HCV, and (b) detecting the presence of
amplified product. Specifically, the present invention additionally
provides a method of assaying for the presence of HCV in a nucleic
acid sample comprising (a) amplifying a selected HCV nucleic acid
to form an HCV amplification product, utilizing as a primer of
amplification a nucleic acid comprising a nucleotide sequence
selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 11,
SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24; SEQ ID
NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO:32,
SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO:43, and (b) detecting
the presence of amplified product.
[0015] The instant invention further provides a method of
quantifying the amount of HCV nucleic acid in a sample comprising
(a) amplifying selected HCV nucleic acid to form an HCV
amplification product, utilizing as a primer of amplification a
nucleic acid comprising a nucleotide sequence selected from the
group consisting of specific nucleic acid sequences corresponding
to a portion of the 3'NTR of HCV, and (b) quantifying the amount of
amplified product.
[0016] The instant invention further specifically provides a method
of quantifying the amount of HCV nucleic acid in a sample
comprising (a) amplifying selected HCV nucleic acid to form an HCV
amplification product, utilizing as a primer of amplification a
nucleic acid comprising a nucleotide sequence selected from the
group consisting of SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 15, SEQ
ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24; SEQ ID NO: 27, SEQ ID NO:
29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO:32, SEQ ID NO: 41, SEQ
ID NO: 42, and SEQ ID NO:43, and (b) quantifying the amount of
amplified product.
[0017] Additionally provided is a method of assaying for the
presence of HCV in a nucleic acid sample comprising (a) amplifying
a target nucleic acid comprising a selected portion of the 3'NTR of
HCV and (b) detecting the presence of amplified product utilizing a
probe comprising a nucleotide sequence selected from the group
consisting of SEQ ID NO: 21, SEQ ID NO: 22; SEQ ID NO:19, SEQ ID
NO: 24, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 44.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 provides a schematic of the 3' NTR of HCV (98
sequences), showing the general region of hybridization of P1
primers, P2 primers and detection probes.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The 3' HCV NTR is considered a difficult amplification
target for at least two reasons. First, it forms highly stable
secondary structures. The intramolecular pairing (including
potential double-stranded (stem) and palindrome regions) is
expected to cause difficulties for primer binding, primer
extension, and probe binding. Secondly, the NTR is very small (98
nt), resulting in unusually limited primer and probe choices, and
an amplicon of sub-optimal length (amplicons are ideally >100
nt). The inability to reduce secondary structure by raising the
temperature (as in PCR) makes it particularly problematic for
isothermal amplification methods such as TMA and NASBA. The present
invention provides primers and probes that help overcome these
challenges.
[0020] The 3' NTR of Hepatitis C virus has the following sequence:
TABLE-US-00001 Genotype 1:
GGUGGCUCCAUCUUAGCCCUAGUCACGGCUAGCUGUGAAAGGUCCGUGAGCCGCAUGACUG (SEQ
ID NO: 25) CAGAGAGUGCUGAUACUGGCCUCUCUGCAGAUCAUGU Genotype 2:
GGUGGCUCCAUCUUAGCCCUAGUCACGGCUAGCUGUGAAAGGUCCGUGAGCCGCAUGACUG (SEQ
ID NO: 26) CAGAGAGUGCCGUAACUGGUCUCUCUGCAGAUCAUGU
[0021] The most common sequence differences between Genotype 1 and
Genotype 2 are indicated by underlining. Although the region is
highly conserved, additional nucleotide differences may also be
present in the individual isolates.
[0022] As stated above, the present invention provides
oligonucleotides, derived from the 3' NTR of Hepatitis C virus,
found to be particularly useful in assays to detect the presence,
or quantify the amount of, HCV nucleic acids in selected
samples.
[0023] Throughout this application, nucleic acid sequences may have
descriptors that include an "nt" range of numbers. Such descriptor
indicates where, within the 98 nucleotides of the 3'HCV NTR, the
sequence corresponds, the first of the 98 nucleotides, reading in a
5'-3' direction along the viral genome, having the number "1". For
example, the following oligonucleotide "1206 (nt5-24)" indicates
that the sequence of the oligonucleotide named 1206 has a nucleic
acid sequence that corresponds to nucleotides 5-24 of the 3'NTR.
Additionally, it is noted that references to sequences that include
thymidine can be readily adapted to utilize uridine in substitution
for thymidine, where useful for the particular assay. Furthermore,
nucleotides may be modified by addition of chemical groups, or
substitution of individual residues by analogues (e.g.,
2'-O-methoxy versions). Additional such modified nucleotides are
known in the art; some examples include hydroxymethyl nucleotides,
methylated nucleotides, fluorinated nucleotides, alpha this
phosphate nucleotides, amine-modified nucleotides, methoxy
nucleotides, carboxymethyl nucleotides, thio nucleotides, inosine,
dihydrouridine, psuedouridine, wybutosine, queuosine, C7dGTP.
Additional modified nucleotides are found in U.S. Pat. Nos
5,405,950 and 5,633,364 (both, Mock and Lovern).
[0024] The invention particularly provides an isolated Hepatitis C
virus-derived nucleic acid comprising a nucleotide sequence
selected from the group consisting of specific nucleic acid
sequences corresponding to a portion of the 3'NTR of HCV.
Specifically, the present invention provides an isolated nucleic
acid comprising a nucleotide sequence selected from the group
consisting of SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID
NO: 21, SEQ ID NO: 22, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 30,
SEQ ID NO: 31, and SEQ ID NO:32. It also provides an isolated
nucleic acid consisting essentially of a nucleotide sequence
selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 11,
SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 27, SEQ ID
NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO:32. Given the
localization of useful target regions for hybridization of the
primers and probes to the HCV 3' NTR, slight modifications,
primarily mismatches from the provided sequences, are contemplated
as being useful, provided they still hybridize to the target region
of HCV under appropriate hybridization conditions. The nucleotide
sequence of the oligonucleotide thus can further have from one to
four mismatches from the recited nucleotide sequence, wherein the
oligonucleotide hybridizes, under NASBA hybridization conditions,
to the 3'NTR of HCV. NASBA conditions are known in the art and can
include, for example, parameters creating stringency provided by
about 41.degree. C. and about 70 mM KCl (see. e.g., U.S. Pat. Nos.
5,466,586; 5,409,818; 5,554,517; 6,063,603; and 5,130,238; Deiman
B, van Aarle P, Sillekens P. Characteristics and applications of
nucleic acid sequence-based amplification (NASBA).
[0025] Mol Biotechnol. 2002 February; 20(2):163-79.). Further, such
sequences can have from one to four mismatches from HCV 3'' NTR
genomic sequence, wherein the oligonucleotide hybridizes, under
NASBA hybridization conditions, to the 3'NTR of HCV. Examples of
mismatches are exemplified herein.
[0026] Oligonucleotides of the present invention can be utilized,
for example, as primers and/or as probes for the detection of HCV
nucleic acids in a sample. Throughout this application, a
particular oligonucleotide may be exemplified in use as a
particular type (e.g. P1-type (linked to a sequence that provides a
promoter region when in double-stranded form) or P2 type (used
alone or linked to a tag oligonucleotide)) primer or as a probe;
however, such use should not limit the use(s) for which the
oligonucleotide may be useful. For example, a primer exemplified as
a P1 primer may be useful as a P2-type primer. Additionally, an
oligonucleotide exemplified for use as a probe may be useful as the
base HCV-hybridizing sequence for other styles of probes (e.g.,
having different labels or capture oligonucleotides or other
structures for functioning of that probe type).
[0027] The present invention provides amplicon length modulation
through the use of the attachment of non-related sequence at the 5'
end of primers. Such non-related sequences are particularly useful
for non-promoter-linked primers, such as P2 primers herein. With
this invention, our results demonstrate that one can, given the
teachings herein, modulate amplicon length and modulate its
secondary structure for highly efficient probe-based detection.
[0028] Thus, the present invention further provides an isolated
nucleic acid comprising (a) a tag oligonucleotide having a 5' end
and a 3' end, and (b) a primer nucleic acid having a 5' end and a
3' end and comprising a nucleotide sequence corresponding to a
portion of the 3'NTR of HCV, wherein the tag oligonucleotide
consists of about 18 to about 23 randomly selected nucleotides
heterologous to HCV and is linked at its 3' end to the 5' end of
the primer nucleic acid. More specifically, the present invention
further provides an isolated nucleic acid comprising (a) a tag
oligonucleotide having a 5' end and a 3' end, and (b) a primer
nucleic acid having a 5' end and a 3' end and comprising a
nucleotide sequence selected from the group consisting 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:7, and SEQ ID NO: 8, wherein the tag
oligonucleotide consists of about 18 to about 23 randomly selected
nucleotides heterologous to HCV and is linked at its 3' end to the
5' end of the primer nucleic acid.
[0029] In a preferred embodiment, the tag oligonucleotide (or
"tail") is a sequence heterologous to the target nucleic acid, and,
more preferably, heterologous to any region of HCV. The tag
oligonucleotide can comprise any selected heterologous sequence of
about 18-23 nucleotides. An approximately 20-mer oligonucleotide
"tail" can readily be designed by one skilled in the art, given
these teachings, to create additional detection tails useful with
(non-promoter-carrying) primers to increase detectable amplicons
from an amplification reaction. The tail is preferably comprised of
sequences heterologous to the target nucleic acid. The
oligonucleotide tail can be from about 18 to about 23 nucleotides
in length, is preferably about 19-22 nucleotide in length, more
preferably about 20-21 nucleotides in length, and most preferably
20 nucleotides in length. One example of an oligonucleotide tail is
the "ECL (electrochemiluminescence) tail," set forth in SEQ ID
NO:9.
[0030] In a specifically preferred embodiment, the tag
oligonucleotide comprises an ECL tag; more particularly, the
nucleotide sequence set forth in SEQ ID NO: 9. In a preferred
embodiment, the oligonucleotide comprises a tag oligonucleotide
linked at its 3' end to the 5' end of a primer having a nucleic
acid sequence set forth in SEQ ID NO: 8. Thus, the present
invention provides a preferred isolated nucleic acid comprising a
nucleotide sequence set forth in SEQ ID NO: 37, which consists of
an ECL tag linked at its 3' end to the 5' end of the primer (herein
referred to as "1259") having a nucleotide sequence set forth in
SEQ ID NO:8.
[0031] The present invention further provides an isolated nucleic
acid comprising (a) a promoter oligonucleotide that, when in
double-stranded form, can function as a T7 promoter, and (b) a
primer nucleic acid having a 5' end and a 3' end and comprising a
nucleotide sequence comprising a nucleotide sequence corresponding
to a portion of the 3'NTR of HCV. More specifically, the present
invention provides an isolated nucleic acid comprising (a) a
promoter oligonucleotide that, when in double-stranded form, can
function as a T7 promoter, and (b) a primer nucleic acid having a
5' end and a 3' end and comprising a nucleotide sequence selected
from the group consisting of SEQ ID NO:11, SEQ ID NO:15, SEQ ID
NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32,
wherein the promoter oligonucleotide is linked at its 3' end to the
5' end of the primer nucleic acid.
[0032] The nucleic acid sequence of the T7 promoter is well-known
to persons skilled in the art, and though a particular sequence is
exemplified herein, functional equivalents having slight variations
may be designed. In a preferred embodiment, the sequence of the T7
promoter is that set forth in SEQ ID NO:38. A further example is
provided by SEQ ID NO: 23. An oligonucleotide having T7 promoter
sequences is useful as a primer, and, when utilized as such, is
herein referred to as a "P1 primer," P1-type primer,"
"promoter-oligonucleotide," or simply as "P1." In a
transcription-based amplification reaction (e.g., NASBA, TMA), as
is known in the art, these T7 promoter sequences have a function in
the amplification reaction, priming the transcription of RNA from
the target template, in this case, HCV. The nucleotide sequences of
primers exemplified herein as of the "P1" type are typically listed
without the T7 promoter sequences, but in the experiments, such T7
promoter sequences are present.
[0033] Additionally provided is a method of assaying for the
presence of HCV in a nucleic acid sample comprising (a) amplifying
a target nucleic acid comprising a selected portion of the 3'NTR of
HCV and (b) detecting the presence of amplified product utilizing a
probe comprising a nucleotide sequence selected from the group
consisting of SEQ ID NO: 21, SEQ ID NO: 22; SEQ ID NO:19, SEQ ID
NO: 24, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 44. In such a
method, any useful HCV primer(s) can be utilized; examples are
provided herein. Assay conditions are known to those of skill in
the are and exemplified herein.
[0034] Oligonucleotides of the present invention can have various
uses. They can, for example, be utilized as probes in capture and
detection reactions, and as primers and/or probes in various
amplification reactions. Provided herein are primers and probes
utilized for 3' HCV amplification and exemplified by TMA-and NASBA.
Both of these methods use isothermal amplification to produce RNA
amplicons through the combined use of reverse transcriptase and T7
polymerase. Because of these similarities, the primers and probes
are expected to share some performance similarity in the two
systems, and experiments (see Examples) support this.
[0035] The present invention additionally provides a method of
assaying for the presence of HCV in a nucleic acid sample
comprising (a) amplifying a selected HCV nucleic acid to form an
HCV amplification product, utilizing as a primer of amplification a
nucleic acid comprising a nucleotide sequence selected from the
group consisting of specific nucleic acid sequences corresponding
to a portion of the 3'NTR of HCV, and (b) detecting the presence of
amplified product. Specifically, invention provides a method of
assaying for the presence of HCV in a nucleic acid sample
comprising (a) amplifying a selected HCV nucleic acid to form an
HCV amplification product, utilizing as a primer of amplification a
nucleic acid comprising a nucleotide sequence selected from the
group consisting of SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 15, SEQ
ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID
NO:32, and (b) detecting the presence of amplified product.
[0036] Nucleic acid hybridization techniques and conditions are
known to the skilled artisan and have been described for example,
in Sambrook et al. Molecular Cloning A Laboratory Manual, 2nd Ed.
Cold Spring Lab. Press, December 1989; U.S. Pat. Nos. 4,563,419
(Ranki) and 4,851,330 (Kohne) and in Dunn, et al., Cell 12, pp.
23-26 (1978) among many other publications. Various modifications
to the hybridization reactions are known in the art including
in-solution hybridization or hybridization to capture probes on a
solid support in one or more reaction steps.
[0037] Detection methods utilizing nucleic acids are also known.
Nucleic acids can be labeled for use in detection. For example,
detectable labels have been conjugated, directly or indirectly
through linker arms on either the base, sugar or phosphate moiety
of one or more specific oligonucleotides (see, e.g., U.S. Pat. Nos.
4,486,539 (Kourlisky); 4,411,955 (Ward); 4,882,269 (Schneider) and
4,213,893 (Carrico)). Labels known in the art include
fluorochromes, radioisotopes, dyes, enzymes such as alkaline
phosphatase, and luminescent or chemiluminescent molecules.
Detectably labeled probes may, for example, be used to bind to
amplified nucleic acid reaction products or amplicons during or
after an amplification reaction. Particularly useful probes are
those that can detect the target under the preferred conditions for
the assay and type of probe utilized; typical hybridization
conditions can be stringent hybridization conditions such as taught
by Sambrook, et al.
[0038] This invention involves the primer and probe designs for
efficient capture, amplification and detection of HCV 3' end
98-base non-translated region. The primer designs can be applied
for TMA, NASBA, SDA, PCR and other amplification methods. The
present oligonucleotides are useful in the present detection and
quantification assays. Amplification can be performed by
thermocycling methods or isothermal methods. The present
oligonucleotides are particularly useful, and preferably used in,
transcription based amplification methods, for example, NASBA and
TMA. Transcription based amplification methods often utilize single
stranded RNA as the input material, although single or double
stranded DNA can likewise be used as input material. When a
transcription based amplification method is practiced on a sample
with single stranded RNA (of the "plus" sense) with additional
sequences on both the 3'-end and the 5' end of the target sequence,
a pair of oligonucleotides that is conveniently used with the
methods can include (1) a first oligonucleotide (often referred to
as a "promoter-oligonucleotide", or "P1" primer) that is capable of
hybridizing to the 3-end of the target sequence, which
oligonucleotide has the sequence of a promoter (preferably the T7
promoter) attached to its 5' end (the hybridizing part of this
oligonucleotide has the opposite polarity as the plus RNA used as
input material); and (2) a second oligonucleotide ("primer") which
comprises the 3' end of the target sequence (this oligonucleotide
has the same polarity as the plus RNA).
[0039] When such a pair of oligonucleotides, together with all
enzymes having the appropriate activities, and a sufficient supply
of the necessary ribonucleotides and deoxyribonucleotides are put
together in one reaction mixture and are kept under the appropriate
conditions (that is, under the appropriate buffer conditions and at
the appropriate temperature) (such conditions being known in the
art) for a sufficient period of time an isothermal continuous
amplification reaction will start.
[0040] For use in non-transcription-based methods, the first primer
can be an oligonucleotide provided here and can lack a T7 promoter
sequence. For any amplification method, the present
olgionucleotides can provide probes for detection of amplicons. The
probe designs provided herein are for target capture (sample
preparation), amplicon capture, and amplicon detection, which
methods are know in the art. Probe designs for different detection
methods: HPA, TAQman, molecular Beacons and Sandwich hybridization
have also been discovered. These designs demonstrate the efficient
capture and detection by hybridization to amplicons
EXAMPLES
[0041] Within this application and particularly within the
Examples, the primers utilized may be referred to as P1 primers or
P2 primers. This terminology simply indicates that, within a NASBA
or TMA reaction in these examples, the P1 primer is the primer
having a T7 promoter sequence attached to its 5' end (promoter
primer); the P2 primer does not. It is not limiting
terminology.
TMA Amplification and Detection of 3' HCV
[0042] To evaluate the usefulness of various primers in HCV
amplification, standard VIDAS Probe D2 qHCV assay conditions were
used as follows. For lysis, 0.5 ml sample (such as EDTA-plasma, or
in vitro RNA transcript diluted in base matrix) was mixed with 0.4
ml urea-based lysis buffer, incubated 67.5.degree. C. (20 min) and
cooled to room temp (20 min). During these incubations the released
nucleic acid was captured by capB probe (SEQ ID NO: 19), which was
linked to magnetic beads by a polyA tail. Complexes were washed and
resuspended in RAR buffer (see Table 1). RNA target was then
denatured at 65.degree. C., then amplified by TMA (standard
conditions; McDonough et al., Nucleic acid amplification
technologies 1998:113-123 BioTechniques Books Natick, Mass., Lee H
Morse S Olsvik .0. eds.) at 42.degree. C. with primers 1259 (SEQ ID
NO: 8) and 1236 (SEQ ID NO: 11). Specifically, the RAR-resuspended
purified target was then added to VIDAS Probe strips (containing
all additional reagents required for amplification and detection,
such as enzymes, probes, wash solutions, and detection substrate).
The amplification was carried out in bioMerieux AmpStations, and
then transferred to VIDAS instruments, which carried out
sequence-specific capture of the amplicons, washing, and detection
with a fluorescent-conjugate probe specific for the 3' end. The SPR
Probe 1247 (SEQ ID NO: 22) captures the amplicon on the VIDAS SPR.
The AKP Probe (1246) (SEQ ID NO: 21) was conjugated to alkaline
phosphatase and binds to the purified amplicon, providing detection
via fluorescent AKP substrate. TABLE-US-00002 TABLE 1 RAR Buffer
Tween 80 12.5% Tris pH 7.9 95 mM EDTA 0.375 MM KCl 50 MM ZnAc
0.0765 MM MgCl 20.75 MM Glycerol 10% DMSO 5% ATP 4 MM CTP 3 MM GTP
6 MM UTP 2 MM d-ATPs 1 MM d-CTPs 1 MM d-GTPs 1 MM d-TTPs 1 MM Na
Azide 0.06% P1 Primer 40 NM
Additional Primer Sequences Used for TMA Amplification
[0043] Several P1 (T7 promoter) (SEQ ID NOs: 10-18) and P2
(reverse) (SEQ ID NOs: 1-8) primers (Table 3) were evaluated for 3'
HCV amplification in the VIDAS Probe assay as described above.
Among these, the 1236 (SEQ ID NO: 11)/1259 (SEQ ID NO: 8) primer
pair was optimal, and derivatives of these (e.g., SEQ ID NO: 27
derived from 1236, SEQ ID NO: 37 derived from 1259) were also
tested by NASBA (described below). Primer 1279 (SEQ ID NO: 15) also
worked well for TMA and NASBA. TABLE-US-00003 TABLE 2 Primer
sequences P2 Primers 1206 (SEQ ID NO: 1) GCTCCATCTTAGCCCTAGTC 1242
(SEQ ID NO: 2) TCTTAGCCCTAGTCA 1243 (SEQ ID NO: 3) TTAGCCCTAGTCACG
1244 (SEQ ID NO: 4) AGCCCTAGTCACGGC 1245 (SEQ ID NO: 5)
CCCTAGTCACGGCTA 1257 (SEQ ID NO: 6) AGCCCTAGTCACGGCTAG 1258 (SEQ ID
NO: 7) AGCCCTAGTCACGGCTAGC 1259 (SEQ ID NO: 8) CCCTAGTCACGGCTAGC P1
primers 1211 (SEQ ID NO: 10) AGGCCAGTATCAGCACTCTC 1236 (SEQ ID NO:
11) AGGCCAGTAACGGCACTCTCTGC 1237 (SEQ ID NO: 12)
AGGCCAGTATCGGCACTCTCTG 1238 (SEQ ID NO: 13) AGGCCAGTAACGGCACTCUCTGC
1278 (SEQ ID NO: 14) CAGTATCGGCACTCTCTGCAGT 1279 (SEQ ID NO: 15)
CCAGTATCGGCACTCTCTGCAG 1280 (SEQ ID NO: 16) AGGCCAGTATCGGCACTCTCTGC
1281 (SEQ ID NO: 17) AGTATCGGCACTCTCTGCAGT 1282 (SEQ ID NO: 18)
CAGTAACGGCACTCTCTGCAGT
NASBA Amplification of 3' HCV
[0044] The 1236/1259 (P1/P2) primer pair, which performed optimally
in the TMA assay, was analyzed for use in NASBA amplification.
Additional primers providing a larger predicted amplicon were also
tested, for example primer P1: 1211 (nt83-64) (SEQ ID NO: 10) and
primer P2:VP1206 (nt5-24) (SEQ ID NO: 1).
[0045] For detection of amplicons produced by NASBA, probes were
designed to bind to a region corresponding to a combination of the
VIDAS Probe amplicon capture probe and amplicon detection probe
sites from the previous example. Probes were based upon an HCV base
sequence of either TGAAAGGTCCGTGAGCCGC (nt 36-54) (SEQ ID NO: 43)
or TGTGAAAGGTCCGTGAGCCGC (nt34-54) (SEQ ID NO: 44). These probes
were tested as capture probes (EG6, EG7) in the ECL assay (see
below) or as the amplicon-binding ("loop") portion of molecular
beacons (MB140 (EG18) (SEQ ID NO: 24); MB102 (Q1) (SEQ ID NO:
41)
[0046] ECL (electrochemiluminescence) is a NASBA (bioMerieux, Inc.,
Durham, N.C.) endpoint detection system that detects amplicons
after sequence-specific capture. The ECL system utilizes an ECL
detection tag ("ECL tail") linked to the 5' end of a P2 primer.
Utilizing an ECL detection tag (SEQ ID NO: 9) with above-described
primers and capture probe EG7, amplification was considerable and
better with the 1259/1236 pair than 1206/1211 (Table 3).
TABLE-US-00004 TABLE 3 Performance of various ECL-tagged P1 and P2
primers (normalized fluorescence values) HCV dilution P2, P1 1 10x
100x 1000x 10000x neg 1259ECL, 1236 2.8E+07 3.3E+06 3.3E+04 9.9E+02
1.2E+02 1.0E+00 1206ECL, 1211 8.6E+02 2.9E+03 1.0E+00 1.0E+00
1.0E+00 1.0E+00
[0047] Similarly, in the absence of ECL tags the 1236 and 1259
primers performed better than 1206 and 1211 in NASBA reactions. As
shown in table 4, the 1236/1259 pair performed well. TABLE-US-00005
TABLE 4 Performance of various P1 and P2 primers (normalized
fluorescence values). P1 1211 1211 1236 1236 Prim- er: P2 1206 1259
1206 1259 Prim- er: De- -0.0227978 0.0323256 0.0336202 0.7147294
tec- tion:
ECL detection is presumed to be relatively insensitive to secondary
structure, suggesting that the difference in sensitivity is more
likely due to increased amplification than increased detection.
Modified P2 Primer Designs
[0048] In the above assays, it was also found that addition of the
20-nucleotide universal ECL tag to the P2 primer 1259 increased
performance. Approximately a 10-fold increase in sensitivity was
observed when using the ECL tail on the 1259 P2 primer, as compared
to the P2 primer without the ECL tail (as shown in table 5, tagged
detection of 10.times.-diluted sample is roughly equivalent to
untagged detection of 1.times. sample). TABLE-US-00006 TABLE 5
Comparison of ECL-tagged vs. untagged P2 primers (P1 used is 1236,
probe is EG18, fluorescence values are normalized): HCV dilution: 1
10x neg ECL tag no yes no yes no yes Detection 0.80 3.58 0.13 0.97
0.09 0.09
[0049] ECL assays using an HCV-specific probe (i.e., not
recognizing the ECL tag) also demonstrated improved performance
with the tag, suggesting that this increase in performance was
likely due to increased amplification (Table 6). TABLE-US-00007
TABLE 6 Comparison of ECL-tagged 1259 vs. untagged, in ECL assay
(P1 used is 1236, capture probe is EG7, fluorescence values are
normalized): HCV dilution P2 1 10x 100x 1000x neg ECL-tagged 1259
3.2E+07 4.4E+06 2.0E+05 3.1E+03 1 1259 2.1E+06 6.9E+05 2.1E+04
1.3E+01 1
One likely explanation for the effect of the tag is that the HCV
amplicon is very small (70 nt without tag, increased to 90 with
tag), and small amplicons may be relatively inefficient in NASBA.
Six P2 primers were designed to test whether additional extensions
(of 10 or 20 additional nucleotides) to the ECL tag (either
homologous to the target or heterologous) could improve
amplification. None of the additional heterologous sequences
increased performance, and additional homologous sequence decreased
performance. Additional P1 Primers for NASBA
[0050] Another set of P1 (promoter) primers was tested for function
in NASBA, as listed in Table 7. This set of primers spans, and
moves in the 3' direction in one-nucleotide increments, from the
position of primer 1236 to (at the 3' end of the final primer
P1.89) the position 3 bases from the probe-binding site. Of this
set of primers, the best functioning (comparably to primer 1236) to
produce detectable signal were P1.63, P1.82, P1.83, P1.84, and
P1.85. The P2 used for these tests was EG4 and probe was EG18
(fluorescence values were normalized). TABLE-US-00008 TABLE 7 P1.63
(SEQ ID NO: 27) AGACCAGTTACGGCACTCTCTGC P1.81 (SEQ ID NO: 28)
GACCAGTTACGGCACTCTCTGCA P1.82 (SEQ ID NO: 29)
ACCAGTTACGGCACTCTGTGCAG P1.83 (SEQ ID NO: 30)
CCAGTTACGGCACTCTCTGCAGT P1.84 (SEQ ID NO: 31)
CAGTTACGGCACTCTCTGCAGTC P1.85 (SEQ ID NO: 32)
AGTTACGGCACTCTCTGCAGTCA P1.86 (SEQ ID NO: 33)
GTTACGGCACTCTCTGCAGTCAT P1.87 (SEQ ID NO: 34)
TTACGGCACTCTCTGCAGTCATG P1.88 (SEQ ID NO: 35)
TACGGCACTCTCTGCAGTCATGC P1.89 (SEQ ID NO: 36)
ACGGCACTCTCTGCAGTCATGCG
[0051] TABLE-US-00009 TABLE 8 Amplification with various P1 primers
Primer: P1.63 P1.81 P1.82 P1.83 P1.84 P1.85 P1.86 P1.87 P1.88 P1.89
Signal: 5.250 3.184 4.886 5.099 5.239 3.746 2.668 1.995 0.025
0.142
[0052] Throughout this application, various publications are
referenced. The disclosures of each of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this invention pertains.
[0053] Thus, while there have been described what are presently
believed to be the preferred embodiments of the present invention,
those skilled in the art will realize that other and further
embodiments can be made without departing from the spirit and scope
of the invention, and it is intended to include all such further
modifications and changes as come within the true scope of the
invention.
Sequence CWU 1
1
43 1 20 DNA Hepatitis C virus misc_feature (1)..(20) "1206" 1
gctccatctt agccctagtc 20 2 15 DNA Hepatitis C virus misc_feature
(1)..(15) "1242" 2 tcttagccct agtca 15 3 15 DNA Hepatitis C virus
misc_feature (1)..(15) "1243" 3 ttagccctag tcacg 15 4 15 DNA
Hepatitis C virus misc_feature (1)..(15) "1244" 4 agccctagtc acggc
15 5 15 DNA Hepatitis C virus misc_feature (1)..(15) "1245" 5
ccctagtcac ggcta 15 6 18 DNA Hepatitis C virus misc_feature
(1)..(18) "1257" 6 agccctagtc acggctag 18 7 19 DNA Hepatitis C
virus misc_feature (1)..(19) "1258" 7 agccctagtc acggctagc 19 8 17
DNA Hepatitis C virus misc_feature (1)..(17) "1259" 8 ccctagtcac
ggctagc 17 9 20 DNA Artificial oligonucleotide 9 gatgcaaggt
cgcatatgag 20 10 20 DNA Hepatitis C virus misc_feature (1)..(20)
"1211" 10 aggccagtat cagcactctc 20 11 23 DNA Hepatitis C virus
misc_feature (1)..(23) "1236" 11 aggccagtaa cggcactctc tgc 23 12 22
DNA Hepatitis C virus misc_feature (1)..(22) "1237" 12 aggccagtat
cggcactctc tg 22 13 23 DNA Hepatitis C virus misc_feature (1)..(23)
"1238" 13 aggccagtaa cggcactcuc tgc 23 14 22 DNA Hepatitis C virus
misc_feature (1)..(22) "1278" 14 cagtatcggc actctctgca gt 22 15 22
DNA Hepatitis C virus misc_feature (1)..(22) "1279" 15 ccagtatcgg
cactctctgc ag 22 16 23 DNA Hepatitis C virus misc_feature (1)..(23)
"1280" 16 aggccagtat cggcactctc tgc 23 17 21 DNA Hepatitis C virus
misc_feature (1)..(21) "1281" 17 agtatcggca ctctctgcag t 21 18 22
DNA Hepatitis C virus misc_feature (1)..(22) "1282" 18 cagtaacggc
actctctgca gt 22 19 19 DNA Hepatitis C virus misc_feature (1)..(19)
"capB" 19 gggctaagat ggagccacc 19 20 16 DNA Hepatitis C virus
misc_feature (1)..(16) "1289" 20 acatgatctg cagaga 16 21 14 DNA
Hepatitis C virus misc_feature (1)..(14) "1246" 21 ctgtgaaagg tccg
14 22 16 DNA Hepatitis C virus misc_feature (1)..(16) "1247" 22
tgagccgcat gactgc 16 23 24 DNA Bacteriophage T7 misc_feature
(1)..(24) T7 promoter 23 aatttaatac gactcactat aggg 24 24 31 DNA
Artificial Synthetic construct 24 cgatgctgaa aggtccgtga gccgcgcatc
g 31 25 98 RNA Hepatitis C virus Type I 3'UTR (1)..(98) 25
gguggcucca ucuuagcccu agucacggcu agcugugaaa gguccgugag ccgcaugacu
60 gcagagagug cugauacugg ccucucugca gaucaugu 98 26 98 RNA Hepatitis
C virus Type II 3'UTR (1)..(98) variation (72)..(72) Sequence
variation from Hepatitis C virus Type I 26 gguggcucca ucuuagcccu
agucacggcu agcugugaaa gguccgugag ccgcaugacu 60 gcagagagug
ccguaacugg ucucucugca gaucaugu 98 27 23 DNA Hepatitis C virus
misc_feature (1)..(23) "P1.63" 27 agaccagtta cggcactctc tgc 23 28
23 DNA Hepatitis C virus misc_feature (1)..(23) "P1.81" 28
gaccagttac ggcactctct gca 23 29 23 DNA Hepatitis C virus
misc_feature (1)..(23) "P1.82" 29 accagttacg gcactctctg cag 23 30
23 DNA Hepatitis C virus misc_feature (1)..(23) "P1.83" 30
ccagttacgg cactctctgc agt 23 31 23 DNA Hepatitis C virus
misc_feature (1)..(23) "P1.84" 31 cagttacggc actctctgca gtc 23 32
23 DNA Hepatitis C virus misc_feature (1)..(23) "P1.85" 32
agttacggca ctctctgcag tca 23 33 23 DNA Hepatitis C virus
misc_feature (1)..(23) "P1.86" 33 gttacggcac tctctgcagt cat 23 34
23 DNA Hepatitis C virus misc_feature (1)..(23) "P1.87" 34
ttacggcact ctctgcagtc atg 23 35 23 DNA Hepatitis C virus
misc_feature (1)..(23) "P1.88" 35 tacggcactc tctgcagtca tgc 23 36
23 DNA Hepatitis C virus misc_feature (1)..(23) "P1.89" 36
acggcactct ctgcagtcat gcg 23 37 37 DNA Artificial synthetic
construct 37 gatgcaaggt cgcatatgag ccctagtcac ggctagc 37 38 25 DNA
Bacteriophage T7 misc_feature (1)..(25) T7 promoter 38 aattctaata
cgactcacta taggg 25 39 35 DNA Artificial Synthetic construct 39
gcactcgtgt gaaaggtccg tgagccgccg agtgc 35 40 35 DNA Artificial
Synthetic construct 40 cgactcgtgt gaaaggtccg tgagccgccg agtcg 35 41
33 DNA Artificial synthetic construct 41 gcatgctgtg aaaggtccgt
gagccgcgca tgc 33 42 21 DNA Hepatitis C virus misc_feature
(1)..(21) "EG6" 42 tgtgaaaggt ccgtgagccg c 21 43 19 DNA Hepatitis C
virus misc_feature (1)..(19) "EG7" 43 tgaaaggtcc gtgagccgc 19
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