U.S. patent application number 10/085056 was filed with the patent office on 2003-01-09 for oligonucleotide and method for detecting verotoxin.
This patent application is currently assigned to TOSOH CORPORATION. Invention is credited to Ishiguro, Takahiko, Maruyama, Takahiro, Taya, Toshiki.
Application Number | 20030008305 10/085056 |
Document ID | / |
Family ID | 18917912 |
Filed Date | 2003-01-09 |
United States Patent
Application |
20030008305 |
Kind Code |
A1 |
Maruyama, Takahiro ; et
al. |
January 9, 2003 |
Oligonucleotide and method for detecting verotoxin
Abstract
An oligonucleotide capable of binding to the intramolecular
structure-free region of Verotoxin type 1 RNA or Verotoxin type 2
RNA at relatively low and constant temperature, and which can be
used in a constant temperature nucleic acid amplification method,
is provided. Also, a simple, speedy and highly sensitive method for
detecting Verotoxin type 1 RNA or Verotoxin type 2 RNA is
provided.
Inventors: |
Maruyama, Takahiro;
(Yokohama-shi, JP) ; Ishiguro, Takahiko;
(Yokohama-shi, JP) ; Taya, Toshiki;
(Sagamihara-shi, JP) |
Correspondence
Address: |
OBLON SPIVAK MCCLELLAND MAIER & NEUSTADT PC
FOURTH FLOOR
1755 JEFFERSON DAVIS HIGHWAY
ARLINGTON
VA
22202
US
|
Assignee: |
TOSOH CORPORATION
4560, Kaisei-cho
Shinnanyo-shi
JP
746-8501
|
Family ID: |
18917912 |
Appl. No.: |
10/085056 |
Filed: |
March 1, 2002 |
Current U.S.
Class: |
435/6.16 ;
435/91.2; 536/23.1 |
Current CPC
Class: |
C12Q 1/6865 20130101;
C12Q 1/6865 20130101; C12Q 1/689 20130101; C12Q 2527/101
20130101 |
Class at
Publication: |
435/6 ; 435/91.2;
536/23.1 |
International
Class: |
C12Q 001/68; C07H
021/02; C12P 019/34 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 2, 2001 |
JP |
2001-58143 |
Claims
What is claimed is:
1. An oligonucleotide for detection or amplification of VT1 RNA,
which oligonucleotide is capable of specifically binding to VT1
RNA, and comprises at least 10 contiguous bases of any of the
sequences listed as SEQ. ID. Nos. 1 to 5.
2. An oligonucleotide for detection or amplification of VT2 RNA,
which oligonucleotide is capable of specifically binding to VT2
RNA, and comprises at least 10 contiguous bases of any of the
sequences listed as SEQ. ID. Nos. 6 to 14.
3. A process of detecting VT1 RNA, wherein a specific sequence of
VT1 RNA present in a sample is used as a template for synthesis of
a cDNA employing an RNA-dependent DNA polymerase, the RNA of the
formed RNA/DNA hybrid is digested by ribonuclease H to produce a
single-stranded DNA, said single-stranded DNA is then used as a
template for production of a double-stranded DNA having a promoter
sequence capable of transcribing RNA comprising said specific
sequence or a sequence complementary to said specific sequence
employing a DNA-dependent DNA polymerase, said double-stranded DNA
produces an RNA transcription product in the presence of an RNA
polymerase, and said RNA transcription product is then used as a
template for cDNA synthesis employing said RNA-dependent DNA
polymerase, the amplification process being characterized by
employing a first oligonucleotide capable of specifically binding
to VT1 RNA and comprising at least 10 contiguous bases of any of
the sequences listed as SEQ. ID. Nos. 1 to 5 and a second
oligonucleotide comprising at least 10 contiguous bases of any of
the sequences listed as SEQ. ID. Nos. 15 to 18, where either said
first or second oligonucleotide includes said RNA polymerase
promoter sequence at the 5' end.
4. A process of detecting VT2 RNA, wherein a specific sequence of
VT2 RNA present in a sample is used as a template for synthesis of
a cDNA employing an RNA-dependent DNA polymerase, the RNA of the
formed RNA/DNA hybrid is digested by ribonuclease H to produce a
single-stranded DNA, said single-stranded DNA is then used as a
template for production of a double-stranded DNA having a promoter
sequence capable of transcribing RNA comprising said specific
sequence or a sequence complementary to said specific sequence
employing a DNA-dependent DNA polymerase, said double-stranded DNA
produces an RNA transcription product in the presence of an RNA
polymerase, and said RNA transcription product is then used as a
template for cDNA synthesis employing said RNA-dependent DNA
polymerase, the amplification process being characterized by
employing a first oligonucleotide capable of specifically binding
to VT2 RNA and comprising at least 10 contiguous bases of any of
the sequences listed as SEQ. ID. Nos. 6 to 14 and a to second
oligonucleotide comprising at least 10 contiguous bases of any of
the sequences listed as SEQ. ID. Nos. 19 to 23, where either said
first or second oligonucleotide includes the RNA polymerase
promoter sequence at the 5' end.
5. The process according to claim 3 or 4, wherein said
amplification process is carried out in the presence of an
oligonucleotide probe capable of specifically binding to the RNA
transcription product resulting from said amplification and labeled
with an intercalator fluorescent pigment, and changes in the
fluorescent properties of the reaction solution is measured, with
the proviso that the labeled oligonucleotide has a sequence
different from those of the first oligonucleotide and the second
oligonucleotide in the sequence.
6. The detection process according to claim 5, characterized in
that said oligonucleotide probe is designed so as to
complementarily bind to at least a portion of the sequence of said
RNA transcription product, and the fluorescent property changes
relative to that of a situation where a complex formation is
absent.
7. The detection process according to claim 5, characterized in
that said oligonucleotide probe for detecting said VT1 mRNA
comprises at least 10 contiguous bases of SEQ. ID. No. 24 or its
complementary sequence.
8. The detection process according to claim 5 for detecting said
VT2 RNA, characterized in that said oligonucleotide probe comprises
at least 10 contiguous bases of SEQ. ID. No. 25 or its
complementary sequence.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to oligonucleotides for use in
detecting Verotoxin (hereafter, abbreviated as "VT") in clinical
examinations, public health examinations, food evaluations and food
poisoning examinations, as well as a detection method using said
oligonucleotides. The oligonucleotide provided by the present
invention can be used as a gene diagnosing reagent for cleaving,
amplifying and detecting RNA or DNA, and is, for example, useful as
a reagent for quantifying or diagnosing VT.
PRIOR ART
[0002] Verotoxin is a potent toxin produced by Verotoxin-producing
Escherichia coli (hereafter, abbreviated as "VTEC"), typically
pathogenic E. coli 0157. Although the primary symptom caused by
infection with VTEC can be food poisoning represented by
hemorrhagic colitis, it is reported that, in some cases, the
symptom will advance to a hemolytic uremic syndrome (HUS) and, at
worst, it will cause death.
[0003] Although VTEC has many various serotypes, which may be 60
types or more, in view of their detection frequencies, it is deemed
that the major serotype is 0157:H7. Further, VT includes VT type 1
which has the same structure as Shiga toxin produced by Shigella
dysenteriae as well as VT type 2 having different physicochemical
and immunological properties.
[0004] In Japan, VTEC mass infection occurs frequently and,
therefore, in order to accomplish early detection and exclusion of
the infectious source, speedy detection is desired. Further, from a
clinical standpoint, since it is demonstrated that dosing with
antibacterial composition comprising antibiotics at an early
condition stage, i.e. within a few days from the onset of the
disease is effective, speedy identification of the bacteria is
becoming important.
[0005] Means which had been used for examining VT include detection
of 0157 antigen. However, it is known that some Salmonella and
Citrobacter strains show cross-antigenecity with 0157 antigen, and
it is reported that this detection method sometimes provides false
positive results. In addition, mass infections caused by serotypes
other than 0157 have been reported, and therefore it is required to
carry out tests using antisera against various serotypes.
[0006] Recently, a method of selectively detecting
Verotoxin-producing bacteria comprising construction of
oligonucleotides that selectively hybridize against a VTEC gene,
and use of these oligonucleotides in a gene-amplification process
(PCR process) as primers, has been proposed. However, since
identification of the amplified DNA fragment is carried out with
agarose electrophoresis, there remains a problem in view of the
lack of speeds.
[0007] Contrary to the other types of food poisoning, VT results in
a great deal of harm with smaller amounts of contaminating bacteria
and, therefore, the food examination field, and the like, desire a
more rapid and highly sensitive detection method. However, previous
methods carry problems regarding their speed and simplicity. In
addition, in order to simplify the an examination, an examining
instrument which carries out the detection automatically is
desired.
[0008] It is known that when the target nucleic acid is RNA,
Reverse Transcription-Polymerase Chain Reaction (RT-PCR) can be
used. This method involves synthesizing a cDNA from the target RNA
in a reverse transcription step, and then amplifying a specific
sequence of said cDNA by repetition of a cycle comprising heat
denaturation, primer annealing and extension reactions, in the
presence of a pair of primers complementarily and homologous to
both ends of said specific sequence (the antisense primer may be
the same as the one used in reverse transcription step) as well as
a thermostable DNA polymerase. However, RT-PCR method requires a
two-step operation (a reverse transcription step and a PCR step) as
well as an operation involving repetition of rapidly increasing and
decreasing the temperature, which prevent its automation.
[0009] As amplification methods in cases where the target nucleic
acid is RNA, in addition to the above, NASBA and 3SR method are
known, whereby the specific sequence is amplified by the concerted
action of a reverse transcriptase and an RNA polymerase. In these
methods, the following procedures are carried out: using the target
RNA as a template, a double-stranded DNA including a promoter
sequence is synthesized with a primer containing the promoter
sequence, reverse transcriptase and Ribonuclease H; this
double-stranded DNA is used as a template in synthesizing an RNA
containing the specific sequence with an RNA polymerase and,
subsequently, this RNA provides a template in a chain reaction for
synthesizing a double-stranded DNA containing the promoter
sequence.
[0010] NASBA, 3SR, and the like, allow amplification at a constant
temperature and are considered suitable for automation.
[0011] Because amplification methods such as NASBA and 3SR methods
involve relatively low temperature reactions (41.degree. C., for
example), however, the target RNA may form an intramolecular
structure that inhibits binding of the primer, which may reduce the
reaction efficiency. Therefore, they require subjecting the target
RNA to heat denaturation prior to the amplification reaction so as
to destroy the intramolecular structure thereof and thus to improve
the primer binding efficiency. As a result, the simplicity and
speed of the methods are impaired.
[0012] Thus, an object of the present invention is to provide an
oligonucleotide capable of complementarily binding to an
intramolecular structure-free region of the target RNA, the binding
of which against the target RNA would not be inhibited even when
being manipulated at relatively low temperature (for example,
between 35 and 50.degree. C., preferably, about 41.degree. C.),
whereby its reaction efficiency would not be impaired. In
particular, an object of the present invention is to provide an
oligonucleotide capable of binding to the intramolecular
structure-free region of VT1 RNA or VT2 RNA at relatively low
temperature, or to provide an oligonucleotide primer which can be
used in a nucleic acid amplification method so as to detect VT1 RNA
or VT2 RNA, and also to provide simple, speedy and highly sensitive
detecting method using such an oligonucleotide.
[0013] The invention according to claim 1 and intended to
accomplish the objects relates to an oligonucleotide for detection
or amplification of VT1 RNA, which oligonucleotide is capable of
specifically binding to VT1 RNA, and comprises at least 10
contiguous bases of any of the sequences listed as SEQ. ID. Nos. 1
to 5.
[0014] Moreover, the invention according to claim 2 and intended to
accomplish the objects relates to an oligonucleotide for detection
or amplification of VT2 RNA, which oligonucleotide is capable of
specifically binding to VT2 RNA, and comprises at least 10
contiguous bases of any of the sequences listed as SEQ. ID. Nos. 6
to 14.
[0015] Furthermore, the invention according to claim 3 and intended
to accomplish the objects relates to a process of detecting VT1
RNA, wherein a specific sequence of VT1 RNA present in a sample is
used as a template for synthesis of a cDNA employing an
RNA-dependent DNA polymerase, the RNA of the formed RNA/DNA hybrid
is digested by ribonuclease H to produce a single-stranded DNA, the
single-stranded DNA is then used as a template for production of a
double-stranded DNA having a promoter sequence capable of
transcribing RNA comprising the specific sequence or the sequence
complementary to the specific sequence employing a DNA-dependent
DNA polymerase, the double-stranded DNA produces an RNA
transcription product in the presence of an RNA polymerase, and the
RNA transcription product is then used as a template for cDNA
synthesis employing the RNA-dependent DNA polymerase, the
amplification process being characterized by employing a first
oligonucleotide capable of specifically binding to VT1 RNA and
comprising at least 10 contiguous bases of any of the sequences
listed as SEQ. ID. Nos. 1 to 5 and a second oligonucleotide
comprising at least 10 contiguous bases of any of the sequences
listed as SEQ. ID. Nos. 15 to 18, where either the first or second
oligonucleotide includes the RNA polymerase promoter sequence at
the 5 end.
[0016] Still furthermore, the invention according to claim 4 and
intended to accomplish the objects relates to a process of
detecting VT2 RNA, wherein a specific sequence of VT2 RNA present
in a sample is used as a template for synthesis of a cDNA employing
an RNA-dependent DNA polymerase, the RNA of the formed RNA/DNA
hybrid is digested by ribonuclease H to produce a single-stranded
DNA, the single-stranded DNA is then used as a template for
production of a double-stranded DNA having a promoter sequence
capable of transcribing RNA comprising the specific sequence or the
sequence complementary to the specific sequence employing a
DNA-dependent DNA polymerase, the double-stranded DNA produces an
RNA transcription product in the presence of an RNA polymerase, and
the RNA transcription product is then used as a template for cDNA
synthesis employing the RNA-dependent DNA polymerase, the
amplification process being characterized by employing a first
oligonucleotide capable of specifically binding to VT2 RNA, and
comprising at least 10 contiguous bases of any of the sequences
listed as SEQ. ID. Nos. 6 to 14 and a second oligonucleotide
comprising at least 10 contiguous bases of any of the sequences
listed as SEQ. ID. Nos. 19 to 23, where either the first or second
oligonucleotide includes the RNA polymerase promoter sequence at
the 5' end.
[0017] The invention according to claim 5 relates to the process
according to claim 3 or 4, wherein said amplification is carried
out in the presence of an oligonucleotide probe capable of
specifically binding to the RNA transcription product resulting
from the amplification and labeled with an intercalator fluorescent
pigment, and measuring changes in the fluorescent properties of the
reaction solution. The invention according to claim 6 relates to
the process according to claim 5, characterized in that the
oligonucleotide probe is designed so as to complementarily bind to
at least a portion of the sequence of the RNA transcription
product, and the fluorescent property changes relative to that of a
situation where a complex formation is absent. The invention
according to claim 7 relates to the process according to claim 5
for detecting VT1 RNA, characterized in that the oligonucleotide
probe comprises at least 10 contiguous bases of SEQ. ID. No. 24 or
its complementary sequence. The invention according to claim 8
relates to the process according to claim 5 for detecting VT2 RNA,
characterized in that the oligonucleotide probe comprises at least
10 contiguous bases of SEQ. ID. No. 25 or its complementary
sequence. The present invention will be explained below.
[0018] First, the present invention provides an oligonucleotide
useful in detecting VT1 RNA, which oligonucleotide is capable of
specifically binding to VT1 RNA, and comprises at least 10
contiguous bases of any of the sequence listed as SEQ. ID. Nos. 1
to 5. This oligonucleotide is capable of binding to VT1 RNA at
relatively low and constant temperature (35 to 50.degree. C.,
preferably, about 41.degree. C.).
[0019] The RNA detecting process involving the step of amplifying
VT1 RNA in a sample provided by the present invention includes PCR
method, NASBA method, 3SR method, or the like. However, it is
preferred that the nucleic acid amplification is a one which can be
conducted under constant temperature, such as NASBA or 3SR method
in which specific sequence within VT1 RNA is amplified with the
concerted action of reverse transcriptase and RNA polymerase.
[0020] For example, in the NASBA method, a specific sequence of VT1
RNA present in a sample is used as a template for synthesis of a
cDNA employing an RNA-dependent DNA polymerase, the RNA of the
RNA/DNA hybrid is digested by ribonuclease H to produce a
single-stranded DNA, the single-stranded DNA is then used as a
template for production of a double-stranded DNA having a promoter
sequence capable of transcribing RNA comprising the specific
sequence or the sequence complementary to the specific sequence
employing a DNA-dependent DNA polymerase, the double-stranded DNA
produces an RNA transcription product in the presence of an RNA
polymerase, and the RNA transcription product is then used as a
template for cDNA synthesis employing the RNA-dependent DNA
polymerase. The process of the present invention is characterized
by employing a first oligonucleotide capable of specifically
binding to VT1 RNA and comprising at least 10 contiguous bases of
any of the sequences listed as SEQ. ID. Nos. 1 to 5 and a second
oligonucleotide comprising at least 10 contiguous bases of any of
the sequences listed as SEQ. ID. Nos. 15 to 18 and having a
sequence homologous to a portion of the VT1 RNA sequence to be
amplified, where either the first or second oligonucleotide
includes the RNA polymerase promoter sequence at the 5' end.
[0021] Although the RNA-dependent DNA polymerase, the DNA-dependent
DNA polymerase and the ribonuclease H are not critical, AMV reverse
transcriptase that has all of these types of activity is most
preferably used. Moreover, although the RNA polymerase is not
critical, T7 phage RNA polymerase or SP6 phage RNA polymerase is
preferably used.
[0022] In the above amplification process, an oligonucleotide that
is complementary to the region adjacent to and overlapping with the
5' end region of the specific sequence (bases 1 to 10) of VT1 RNA
sequence is added, and the VT1 RNA is cleaved (with ribonuclease H)
at the 5' end region of the specific sequence to provide the
initial template for nucleic acid amplification, thereby allowing
amplification of VT1 RNA even when the specific sequence is not
positioned at the 5' end. The oligonucleotide used for this
cleaving may, for example, be any of those of SEQ. ID. Nos. 1 to 5,
provided that it differs from the one used as the first
oligonucleotide in the amplification process. In addition, the
oligonucleotide for cleaving is preferably chemically modified (for
example, aminated) at the 3' hydroxyl group in order to prevent an
extension reaction from the 3' end.
[0023] Although the RNA transcription product obtained by the above
nucleic acid amplification can be detected by a known method, per
se, preferably, it is detected by carrying out the above
amplification process in the presence of an oligonucleotide probe
labeled with an intercalator fluorescent pigment, and measuring
changes in the fluorescent properties of the reaction solution.
Examples of the oligonucleotide probe include one in which the
intercalator fluorescent pigment is bonded to a phosphorus atom in
the oligonucleotide through a linker. The probe is characterized in
that when it forms a double-stranded chain with the target nucleic
acid (complementary nucleic acid), separation analysis is not
required because the intercalator portion intercalates into the
double-stranded chain portion to vary the fluorescent
characteristics (Ishiguro, T. et al. (1996), Nucleic Acids Res. 24
(24) 4992-4997).
[0024] The probe sequence is not critical so long as it has a
sequence complementary to at least a portion of the RNA
transcription product. However, the probe sequence is preferably
one comprising at least 10 contiguous bases of the sequence listed
as SEQ. ID. No. 24. Moreover, chemical modification (for example,
glycolic acid addition) at the 3' end hydroxyl group of the probe
is preferred in order to inhibit an extension reaction in which the
probe acts as a primer.
[0025] It becomes possible to amplify and detect RNA comprising the
same sequence as the specific sequence of VT1 RNA in a single tube
at a constant temperature and in a single step by carrying out the
amplification process in the presence of the probe, as explained
above, and, thus, the amplification process is easily
automated.
[0026] Next, the present invention provides an oligonucleotide
useful in detecting VT2 RNA, which oligonucleotide is capable of
specifically binding to VT2 RNA, and comprises at least 10
contiguous bases of any of the sequence listed as SEQ. ID. Nos. 6
to 14. This oligonucleotide is capable of binding to VT2 RNA at
relatively low and constant temperature (35 to 50.degree. C.,
preferably, about 41.degree. C.).
[0027] The RNA detecting process involving the step of amplifying
VT2 RNA in a sample provided by the present invention includes PCR
method, NASBA method, 3SR method, or the like. However, it is
preferred that the nucleic acid amplification is a one which can be
conducted under constant temperature, such as NASBA or 3SR method
in which specific sequence within VT2 RNA is amplified with the
concerted action of reverse transcriptase and RNA polymerase.
[0028] For example, in the NASBA method, a specific sequence of VT2
RNA present in a sample is used as a template for synthesis of a
cDNA employing an RNA-dependent DNA polymerase, the RNA of the
RNA/DNA hybrid is digested by ribonuclease H to produce a
single-stranded DNA, the single-stranded DNA is then used as a
template for production of a double-stranded DNA having a promoter
sequence capable of transcribing RNA comprising the specific
sequence or the sequence complementary to the specific sequence
employing a DNA-dependent DNA polymerase, the double-stranded DNA
produces an RNA transcription product in the presence of an RNA
polymerase, and the RNA transcription product is then used as a
template for cDNA synthesis employing the RNA-dependent DNA
polymerase. The process of the present invention is characterized
by employing a first oligonucleotide capable of specifically
binding to VT2 RNA and comprising at least 10 contiguous bases of
any of the sequences listed as SEQ. ID. Nos. 6 to 14 and a second
oligonucleotide comprising at least 10 contiguous bases of any of
the sequences listed as SEQ. ID. Nos. 19 to 23 and having a
sequence homologous to a portion of the VT2 RNA sequence to be
amplified, where either the first or second oligonucleotide
includes the RNA polymerase promoter sequence at the 5' end.
[0029] Although the RNk-dependent DNA polymerase, the DNA-dependent
DNA polymerase and the ribonuclease H are not critical, AMV reverse
transcriptase that has all of these types of activity is most
preferably used. Moreover, although the RNA polymerase is not
critical, T7 phage RNA polymerase or SP6 phage RNA polymerase is
preferably used.
[0030] In the above amplification process, an oligonucleotide that
is complementary to the region adjacent to and overlapping with the
5' end region of the specific sequence (bases 1 to 10) of VT2 RNA
sequence is added, and the VT2 RNA is cleaved (with ribonuclease H)
at the 5' end region of the specific sequence to give the initial
template for nucleic acid amplification, thereby allowing
amplification of VT2 RNA even when the specific sequence is not
positioned at the 5' end. The oligonucleotide used for this
cleaving may, for example, be any of those of SEQ. ID. Nos. 6 to
14, provided that it differs from the one used as the first
oligonucleotide in the amplification process. In addition, the
oligonucleotide for cleaving is preferably chemically modified (for
example, aminated) at the 3' hydroxyl group in order to prevent an
extension reaction from the 3' end.
[0031] Although the RNA transcription product obtained by the above
nucleic acid amplification can be detected by a method known per
se, preferably, it is detected by carrying out the above
amplification process in the presence of an oligonucleotide probe
labeled with an intercalator fluorescent pigment, and measuring
changes in the fluorescent properties of the reaction solution.
Examples of the oligonucleotide probe include one in which the
intercalator fluorescent pigment is bonded to a phosphorus atom in
the oligonucleotide through a linker. The probe is characterized in
that when it forms a double-stranded chain with the target nucleic
acid (complementary nucleic acid), separation analysis is not
required because the intercalator portion intercalates into the
double-stranded chain portion to vary the fluorescent
characteristics (Ishiguro, T. et al. (1996), Nucleic Acids Res. 24
(24) 4992-4997).
[0032] The probe sequence is not critical so long as it has a
sequence complementary to at least a portion of the RNA
transcription product. However, the probe sequence is preferably
one comprising at least 10 contiguous bases of the sequence listed
as SEQ. ID. No. 25. Moreover, chemical modification (for example,
glycolic acid addition) at the 3' end hydroxyl group of the probe
is preferred in order to inhibit an extension reaction based on the
probe used as a primer.
[0033] It becomes possible to amplify and detect RNA comprising the
same sequence as the specific sequence of VT2 RNA in a single tube
at a constant temperature and in a single step by carrying out the
amplification process in the presence of the probe, as explained
above, and, thus, the amplification process is easily
automated.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 is a urea modified 6% polyacrylamide electrophoresis
diagram for samples obtained by performing cleaving experiments on
VT1 RNA standard at 41.degree. C., using Oligos 1 to 6 and
AMV-Reverse Transcriptase (black and white inverted). The lanes
without any indications are unrelated to the present invention.
[0035] FIG. 2 is a urea modified 6% polyacrylamide electrophoresis
diagram for samples detained by performing cleaving experiments on
VT2 RNA standard at 41.degree. C., using Oligos 7 to 15 and
AMV-Reverse Transcriptase (black and white inverted). The lanes
without any indications are unrelated to the present invention.
[0036] FIG. 3 is a 4% agarose gel electrophoresis diagram for RNA
amplification reactions of VT1 RNA standard performed as described
in Example 3 using oligonucleotide probe combinations (a) to (c)
shown in Table 3 (black and white inverted), with an initial RNA
amount of 10.sup.4 copies/30 .mu.l and 10.sup.3 copies/30 .mu.l.
Lane 1 is the result for combination (a) with an initial RNA amount
of 10.sup.4 copies/30 .mu.l; lanes 2 and 3 are for combination (a)
with an initial RNA amount of 10.sup.3 copies/30 .mu.l; lane 4 is
for combination (a) using only the diluent instead of RNA samples
(control); lane 5 is the result for combination (b) with an initial
RNA amount of 10.sup.4 copies/30 .mu.l; lanes 6 and 7 are for
combination (b) with an initial RNA amount of 10.sup.3 copies/30
.mu.l; lane 8 is for combination (b) using only the diluent instead
of RNA samples (control); lane 9 is the result for combination (c)
with an initial RNA amount of 10.sup.4 copies/30 .mu.l; lanes 10
and 11 are for combination (c) with an initial RNA amount of
10.sup.3 copies/30 .mu.l and lane 12 is for combination (c) using
only the diluent instead of RNA samples (control). Specific bands
were confirmed in every combination.
[0037] FIG. 4 is a 4% agarose gel electrophoresis diagram for RNA
amplification reactions of VT1 RNA standard performed as described
in Example 3 using oligonucleotide probe combinations (d) to (f)
shown in Table 3 (black and white inverted), with an initial RNA
amount of 10.sup.4 copies/30 .mu.l and 10.sup.3 copies/30 .mu.l.
Lane 1 is the result for combination (d) with an initial RNA amount
of 10.sup.4 copies/30 .mu.l; lanes 2 and 3 are for combination (d)
with an initial RNA amount of 10.sup.3 copies/30 .mu.l; lane 4 is
for combination (d) using only the diluent instead of RNA samples
(control); lane 5 is the result for combination (e) with an initial
RNA amount of 10.sup.4 copies/30 .mu.l; lanes 6 and 7 are for
combination (e) with an initial RNA amount of 10.sup.3 copies/30
.mu.l; lane 8 is for combination (e) using only the diluent instead
of RNA samples (control); lane 9 is the result for combination (f)
with an initial RNA amount of 10.sup.4 copies/30 .mu.l; lanes 10
and 11 are for combination (f) with an initial RNA amount of
10.sup.3 copies/30 .mu.l; and lane 12 is for combination (f) using
only the diluent instead of RNA samples (control). Specific bands
were confirmed in every combination.
[0038] FIG. 5 is a 4% agarose gel electrophoresis diagram for RNA
amplification reactions of VT2 RNA standard performed as described
in Example 4 using oligonucleotide probe combinations (g) to (i)
shown in Table 4 (black and white inverted), with an initial RNA
amount of 10.sup.4 copies/30 .mu.l and 10.sup.3 copies/30 .mu.l.
Lane 1 is the result for combination (g) with an initial RNA amount
of 10.sup.4 copies/30 .mu.l lanes 2 and 3 are for combination (g)
with an initial RNA amount of 10.sup.3 copies/30 .mu.l lane 4 is
for combination (g) using only the diluent instead of RNA samples
(control); lane 5 is the result for combination (h) with an initial
RNA amount of 10.sup.4 copies/30 .mu.l; lanes 6 and 7 are for
combination (h) with an initial RNA amount of 10.sup.3 copies/30
.mu.l; lane 8 is for combination (h) using only the diluent instead
of RNA samples (control); lane 9 is the result for combination (i)
with an initial RNA amount of 10.sup.4 copies/30 .mu.l; lanes 10
and 11 are for combination (i) with an initial RNA amount of
10.sup.3 copies/30 .mu.l and lane 12 is for combination (i) using
only the diluent instead of RNA samples (control). Specific bands
were confirmed in every combination.
[0039] FIG. 6 is a 4% agarose gel electrophoresis diagram for RNA
amplification reactions of VT2 RNA standard performed as described
in Example 4 using oligonucleotide probe combinations (j) to (1)
shown in Table 4 (black and white inverted), with an initial RNA
amount of 10.sup.4 copies/30 .mu.l and 10.sup.3 copies/30 .mu.l.
Lane 1 is the result for combination (j) with an initial RNA amount
of 10.sup.4 copies/30 .mu.l; lanes 2 and 3 are for combination (j)
with an initial RNA amount of 10.sup.3 copies/30 .mu.l; lane 4 is
for combination (j) using only the diluent instead of RNA samples
(control); lane 5 is the result for combination (k) with an initial
RNA amount of 10.sup.4 copies/30 .mu.l; lanes 6 and 7 are for
combination (k) with an initial RNA amount of 10.sup.3 copies/30
.mu.l; lane 8 is for combination (k) using only the diluent instead
of RNA samples (control); lane 9 is the result for combination (1)
with an initial RNA amount of 10.sup.4 copies/30 .mu.l; lanes 10
and 11 are for combination (1) with an initial RNA amount of
10.sup.3 copies/30 .mu.l; and lane 12 is for combination (1) using
only the diluent instead of RNA samples (control). Specific bands
were confirmed in every combination.
[0040] FIG. 7 is a 4% agarose gel electrophoresis diagram for RNA
amplification reactions of VT2 RNA standard performed as described
in Example 4 using oligonucleotide probe combination (m) shown in
Table 4 (black and white inverted), with an initial RNA amount of
10.sup.4 copies/30 .mu.l and 10.sup.3 copies/30 .mu.l. Lane 1 is
the result with an initial RNA amount of 10.sup.4 copies/30 .mu.l;
lanes 2 and 3 are the results with an initial RNA amount of
10.sup.3 copies/30 .mu.l and lane 4 is the result obtained by using
only the diluent instead of RNA samples (control). Specific bands
were confirmed in every combination.
[0041] FIG. 8 shows the results obtained in Example 5 for samples
prepared from the VT2 RNA standard with an initial RNA amount of
from 10.sup.4 copies/30 .mu.l to 10.sup.5 copies/30 .mu.l. Panel
(a) is a fluorescence profile exhibiting the fluorescence increase
ratio that increases with the reaction time-course formation of
RNA. Panel (b) is a calibration curve exhibiting the relationship
between the logarithm of the initial RNA amount and the detection
time (time at which the relative fluorescence reaches 1.2).
.quadrature. shows the result for 10.sup.5 copies, .largecircle. is
for 10.sup.4 copies, .DELTA. is for 10.sup.3 copies, .diamond. is
for 10.sup.2 copies, + is for 10 copies and .times. is for control.
It was demonstrated that RNA with initial copies of 10.sup.1
copies/30 .mu.l can be detected by a reaction for about 20 minutes,
and that there is a correlation between the initial RNA amount and
the detection time.
EXAMPLES
[0042] The present invention will now be explained in greater
detail by way of examples, with the understanding that the
invention is not limited by these examples.
Example 1
[0043] (1) An oligonucleotide which specifically binds to VT1 RNA
at 41.degree. C. was selected. A standard RNA comprising a region
of base Nos. 228 to 1558 of the VT1 RNA base sequence (Calderwood,
S. B. et al., Proc. Natl. Acad. Sci. U.S.A., 84, 4364-4368 (1987),
U.S. GenBank Registered No. M16625) was quantified by ultraviolet
absorption at 260 nm, and then diluted to a concentration of 1.33
pmol/.mu.l with an RNA diluent (10 mM Tris-HCl (pH 8.0)), 0.1 mM
EDTA, 1 mM DTT, 0.5 U/.mu.l RNase Inhibitor).
[0044] (2) 14.0 .mu.l of a reaction solution having the following
composition was dispended into 0.5 ml volume PCR tubes (Gene Amp
Thin-Walled Reaction Tube.TM., Perkin-Elmer Co. Ltd.)
[0045] Reaction Solution Composition
[0046] 60.0 mM Tris-HCl buffer (pH 8.6)
[0047] 90.0 mM potassium chloride
[0048] 13.0 mM magnesium chloride
[0049] 1.0 mM DTT
[0050] 80.0 nM standard RNA
[0051] 0.8 .mu.M oligonucleotide (one of the oligonucleotides shown
below).
[0052] Oligo-1: SEQ. ID. No. 1;
[0053] Oligo-2: SEQ. ID. No. 2;
[0054] Oligo-3: SEQ. ID. No. 26;
[0055] Oligo-4: SEQ. ID. No. 3;
[0056] Oligo-5: SEQ. ID. No. 4;
[0057] Oligo-6: SEQ. ID. No. 5
[0058] Distilled Water for Adjusting Volume
[0059] (3) The reaction solutions were then incubated at 41.degree.
C. for 5 minutes, and then 1 .mu.l of 8.0 U/.mu.l AMV-Reverse
Transcriptase (Takara Shuzo Co. Ltd.; an enzyme which cleaves RNA
of a double stranded-DNA/RNA) was added thereto.
[0060] (4) Subsequently, the PCR tubes were incubated at 41.degree.
C. for 10 minutes. Modified-urea polyacrylamide gel (acrylamide
concentration: 6%; urea: 7M) electrophoresis was conducted to
confirm the cleaved fragments after the reaction. Dyeing following
the electrophoresis was carried out with SYBR Green II.TM. (Takara
Shuzo Co. Ltd.). Upon binding of the oligonucleotide to the
specific site of the target RNA, RNA of the double stranded DNA/RNA
is cleaved by the ribonuclease H activity of AMV-Reverse
Transcriptase and, thereby, a characteristic band can be
observed.
[0061] (5) The results of the electrophoresis are shown in FIG. 1
(black and white inverted). If the oligonucleotide binds
specifically to the standard RNA, the standard RNA will be cleaved
at this region, yielding a decomposition product having a
characteristic chain length. Table 1 shows the positions of the
standard RNA where each oligonucleotide had specifically bind and
the expected band lengths of the fragments. Cleavages at the
expected positions were confirmed with oligos 1 to 6. These
indicated that these oligonucleotides bind strongly to the VT1 RNA
under a constant temperature of 41.degree. C.
1TABLE 1 Expected band Length Oligo Position.sup.1) (base) Oligo -1
425 425, 912 Oligo -2 555 555, 782 Oligo -3 710 710, 627 Oligo -4
890 890, 447 Oligo -5 980 980, 357 Oligo -6 1031 1031, 306
.sup.1)The position designates the 5' end number of the
oligonucleotide which binds to the VT1 RNA standard (1337
base).
Example 2
[0062] (1) An oligonucleotide which specifically binds to VT2 RNA
at 41.degree. C. was selected. A standard RNA comprising a region
of base Nos. 81 to 1437 of the VT2 RNA base sequence (Schmitt, C.
K. et al., Infect. Immun, 59, 1065-1073 (1991), US GenBank
Registered No. X07865) was quantified by ultraviolet absorption at
260 nm, and then diluted to a concentration of 1.75 pmol/.mu.l with
an RNA diluent (10 mM Tris-HCl (pH 8.0)), 0.1 mM EDTA, 1 mM DTT,
0.5 U/.mu.l RNase Inhibitor).
[0063] (2) 14.0 .mu.l of a reaction solution having the following
composition was dispended into 0.5 ml volume PCR tubes (Gene Amp
Thin-Walled Reaction Tube.TM., Perkin-Elmer Co. Ltd.)
[0064] Reaction Solution Composition
[0065] 60.0 mM Tris-HCl buffer (pH 8.6)
[0066] 90.0 mM potassium chloride
[0067] 13.0 mM magnesium chloride
[0068] 1.0 mM DTT
[0069] 80.0 nM standard RNA
[0070] 0.8 .mu.M oligonucleotide (one of the oligonucleotides shown
below).
[0071] Oligo-7: SEQ. ID. No. 6;
[0072] Oligo-8: SEQ. ID. No. 7;
[0073] Oligo-9: SEQ. ID. No. 8;
[0074] Oligo-10,: SEQ. ID. No. 9;
[0075] Oligo-11: SEQ. ID. No. 10;
[0076] Oligo-12: SEQ. ID. No. 11;
[0077] Oligo-13: SEQ. ID. No. 12;
[0078] Oligo-14: SEQ. ID. No. 13;
[0079] Oligo-15: SEQ. ID. No. 14;
[0080] Distilled water for adjusting volume
[0081] (3) The reaction solutions were then incubated at 41.degree.
C. for 5 minutes, and then 1 .mu.l of 8.0 U/.mu.l AMV-Reverse
Transcriptase (Takara Shuzo Co. Ltd.; an enzyme which cleaves RNA
of a double stranded-DNA/RNA) was added thereto.
[0082] (4) Subsequently, the PCR tubes were incubated at 41.degree.
C. for 10 minutes. Modified-urea polyacrylamide gel (acrylamide
concentration: 6%; urea: 7M) electrophoresis was conducted to
confirm the cleaved fragments after the reaction. Dyeing following
the electrophoresis was carried out with SYBR Green II.TM. (Takara
Shuzo Co. Ltd.). Upon binding of the oligonucleotide to the
specific site of the target RNA, RNA of the double stranded DNA/RNA
is cleaved by the ribonuclease H activity of AMV-Reverse
Transcriptase and, thereby, a characteristic band can be
observed.
[0083] (5) The results of the electrophoresis are shown in FIG. 2
(black and white inverted). If the oligonucleotide binds
specifically to the standard RNA, the standard RNA will be cleaved
at this region, yielding a decomposition product having a
characteristic chain length. Table 2 shows the positions of the
standard RNA where each oligonucleotide had specifically bound and
the expected band lengths of the fragments. Cleavages at the
expected positions were confirmed with Oligos 7 to 15. These
indicated that these oligonucleotides bind strongly to the VT2 RNA
under a constant temperature of 41.degree. C.
2TABLE 2 Expected band Length Oligo Position.sup.1) (base) Oligo -7
102 102, 1259 Oligo -8 260 260, 1101 Oligo -9 365 365, 996 Oligo
-10 436 436, 925 Oligo -11 675 675, 686 Oligo -12 723 723, 638
Oligo -13 787 787, 574 Oligo -14 848 848, 513 Oligo -15 986 986,
375 .sup.1)The position designates the 5' end number of the
oligonucleotide which binds to the VT2 RNA standard (1361
base).
Example 3
[0084] RNA amplification reactions were carried out using the
oligonucleotides which specifically bind to VT1 RNA.
[0085] (1) As described in example 1, VT1 standard RNA was diluted
to 10.sup.4 copies/2.5 .mu.l and 10.sup.3 copies/2.5 .mu.l with an
RNA diluent (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5 U/.mu.l RNase
Inhibitor (Takara Shuzo Co. Ltd.), 5 mM DTT). In the control test
sections (negative), only the diluent was used.
[0086] (2) 23.3 .mu.l of a solution having the following
composition was dispended into 0.5 ml volume PCR tubes (Gene Amp
Thin-Walled Reaction Tuber, Perkin-Elmer Co. Ltd.), followed by
addition of 2.5 .mu.l of the above RNA sample.
[0087] Reaction Solution Composition (each concentration represents
a concentration in a final reaction solution volume of 30
.mu.l)
[0088] 60 mM Tris-HCl buffer (pH 8.6)
[0089] 17 mM magnesium chloride
[0090] 90 mM potassium chloride
[0091] 39 U RNase Inhibitor
[0092] 1 mM DTT
[0093] 0.25 .mu.l of each dATP, dCTP, dGTP, dTTP
[0094] 3.6 mM ITP
[0095] 3.0 .mu.l of each ATP, CTP, GTP, UTP
[0096] 0.16 .mu.M first oligonucleotide
[0097] 1.0 .mu.M second oligonucleotide
[0098] 1.0 .mu.M third oligonucleotide
[0099] 13% DMSO
[0100] Distilled Water for Adjusting Volume
[0101] (3) RNA amplification reactions were carried out using the
oligonucleotide sequences listed in Table 3, as the first, second
and third oligonucleotides. Solutions were prepared so that the
combinations of the first, second and third oligonucleotides would
be those as listed in Table 3.
[0102] (4) After incubating the above reaction solutions for 5
minutes at 41.degree. C., 4.2 .mu.l of an enzyme solution having
the following composition was added.
[0103] Composition of Enzyme Solution (each figure represents the
amount in a final reaction solution volume of 30 .mu.l)
[0104] 1.7% sorbitol
[0105] 3 .mu.g bovine serum albumin
[0106] 142 U T7 RNA polymerase (Gibco)
[0107] 8 U AMV-Reverse Transcriptase (Takara Shuzo Co. Ltd.)
[0108] Distilled Water for Adjusting Volume
[0109] (5) Subsequently, the PCR tubes were incubated at 41.degree.
C. for 30 minutes. In order to identify the RNA amplified portion
after the reaction, agarose gel (agarose concentration 4%)
electrophoresis was performed. Dyeing following the electrophoresis
was performed with SYBR Green II (Takara Shuzo Co. Ltd.). When an
oligonucleotide probe binds to the specific portion of the target
RNA, the RNA portion between the second and third oligonucleotide
is amplified and, thereby, a characteristic band could be
observed.
[0110] The results of the electrophoresis are shown in FIGS. 3 and
4 (black and white inverted). The lengths of the specific bands
amplified in this reaction are shown in Table 3. Since specific
bands were confirmed in any of the combinations shown in Table 3,
it was demonstrated that these oligonucleotides are effective in
detecting VT1 RNA.
3TABLE 3 Amplification 1st Oligo- 2nd Oligo- 3rd Oligo- Product
nucleotide nucleotide nucleotide Length Combination Probe Probe
Probe (Base) (a) 5S 5F 6R 141 (b) 6S 6F 7R 166 (c) 6S 6F 8R 346 (d)
7S 7F 8R 191 (e) 7S 7F 9R 281 (f) 8S 8F 9R 101
[0111] Table 3 shows the combinations of first, second and third
oligonucleotides used in this example, as well as the chain lengths
of the amplified specific bands resulted from the RNA amplification
reaction using these combinations. The 3' end hydroxyl group of
each first oligonucleotide base sequence was aminated. In each
second oligonucleotide base sequence, the region of the 1st "A" to
the 22nd "A" from the 5' end corresponds to the T7 promoter region,
and the subsequent region from the 23rd "G" to the 28th "A"
corresponds to the enhancer sequence.
[0112] First Oligonucleotide
[0113] 5S (SEQ. ID. No. 27)
[0114] 6S (SEQ. ID. No. 28)
[0115] 7S (SEQ. ID. No. 29)
[0116] 8S (SEQ. ID. No. 30)
[0117] Second Oligonucleotide
[0118] 5F (SEQ. ID. No. 36)
[0119] 6F (SEQ. ID. No. 37)
[0120] 7F (SEQ. ID. No. 38)
[0121] 8F (SEQ. ID. No. 39)
[0122] Third Oligonucleotide
[0123] 6R (SEQ. ID. No. 2)
[0124] 7R (SEQ. ID. No. 26)
[0125] 8R (SEQ. ID. No. 3)
[0126] 9R (SEQ. ID. No. 4)
Example 4
[0127] RNA amplification reactions were carried out using the
oligonucleotides which specifically bind to VT2 RNA.
[0128] (1) As described in example 2, VT2 standard RNA was diluted
to 10.sup.4 copies/2.5 .mu.l and 10.sup.3 copies/2.5 .mu.l with an
RNA diluent (10 mM, Tris-HCl (pH 8.0), 1 mM EDTA, 0.5 U/.mu.l RNase
Inhibitor (Takara Shuzo Co. Ltd.), 5 mM DTT). In the control test
sections (negative), only the diluent was used.
[0129] (2) 23.3 .mu.l of a solution having the following
composition was dispended into 0.5 ml volume PCR tubes (Gene Amp
Thin-Walled Reaction Tube.TM., Perkin-Elmer Co. Ltd.), followed by
addition of 2.5 .mu.l of the above RNA sample.
[0130] Reaction Solution Composition (each concentration represents
a concentration in a final reaction solution volume of 30
.mu.l)
[0131] 60 mM Tris-HCl buffer (pH 8.6)
[0132] 17 mM magnesium chloride
[0133] 90 mM potassium chloride
[0134] 39 U RNase Inhibitor
[0135] 1 mM DTT
[0136] 0.25 .mu.l of each dATP, dCTP, dGTP, dTTP
[0137] 3.6 mM ITP
[0138] 3.0 .mu.l of each ATP, CTP, GTP, UTP
[0139] 0.16 .mu.M first oligonucleotide
[0140] 1.0 .mu.M second oligonucleotide
[0141] 1.0 .mu.M third oligonucleotide
[0142] 13% DMSO
[0143] Distilled Water for Adjusting Volume
[0144] (3) RNA amplification reactions were carried out using the
oligonucleotide sequences listed in Table 4, as the first, second
and third oligonucleotides. Solutions were prepared so that the
combinations of the first, second and third oligonucleotides would
be those as listed in Table 4.
[0145] (4) After incubating the above reaction solutions for 5
minutes at 41.degree. C., 4.2 .mu.l of an enzyme solution having
the following composition was added.
[0146] Composition of Enzyme Solution (each figure represents the
amount in a final reaction solution volume of 30 .mu.l)
[0147] 1.7% sorbitol
[0148] 3 .mu.g bovine serum albumin
[0149] 142 U T7 RNA polymerase (Gibco)
[0150] 8 U AMV-Reverse Transcriptase (Takara Shuzo Co. Ltd.)
[0151] Distilled Water for Adjusting Volume
[0152] (5) Subsequently, the PCR tubes were incubated at 41.degree.
C. for 30 minutes. In order to identify the RNA amplified portion
after the reaction, agarose gel (agarose concentration 4%)
electrophoresis was performed. Dyeing following the electrophoresis
was performed with SYBR Green II (Takara Shuzo Co. Ltd.). When an
oligonucleotide probe binds to the specific portion of the target
RNA, the RNA portion between the second and third oligonucleotide
is amplified, thereby a characteristic band could be observed.
[0153] The results of the electrophoresis are shown in FIGS. 5 to 7
(black and white inverted). The lengths of the specific bands
amplified in this reaction are shown in Table 4. Since specific
bands were confirmed in any of the combinations shown in Table 4,
it was demonstrated that these oligonucleotides are effective in
detecting VT1 RNA.
4TABLE 4 Amplification 1st Oligo- 2nd Oligo- 3rd Oligo- Product
nucleotide nucleotide nucleotide Length Combination Probe Probe
Probe (Base) (g) B2S B2F B4R 274 (h) B3S B3F B4R 116 (i) B3S B3F
B5R 187 (j) B4S B4F B7R 321 (k) B5S B5F B7R 250 (l) B5S B5F B8R 298
(m) B7S B7F B9R 123
[0154] Table 4 shows the combinations of first, second and third
oligonucleotides used in this example, as well as the chain lengths
of the amplified specific bands resulted from the RNA amplification
reaction using these combinations. The 3' end hydroxyl group of
each first oligonucleotide base sequence was aminated. In each
second oligonucleotide base sequence, the region of the 1st "A" to
the 22nd "A" from the 5' end corresponds to the T7 promoter region,
and the subsequent region from the 23rd "G" to the 28th "A"
corresponds to the enhancer sequence.
[0155] First Oligonucleotide
[0156] B2S (SEQ. ID. No. 31)
[0157] B3S (SEQ. ID. No. 32)
[0158] B4S (SEQ. ID. No. 33)
[0159] B5S (SEQ. ID. No. 34)
[0160] B7S (SEQ. ID. No . 35)
[0161] Second Oligonucleotide
[0162] B2F (SEQ. ID. No. 40)
[0163] B3F (SEQ. ID. No. 41)
[0164] B4F (SEQ. ID. No. 42)
[0165] B5F (SEQ. ID. No. 43)
[0166] B7F (SEQ. ID. No. 44)
[0167] Third Oligonucleotide
[0168] B4R (SEQ. ID. No. 8)
[0169] B5R (SEQ. ID. No. 9)
[0170] B7R (SEQ. ID. No. 10)
[0171] B8R (SEQ. ID. No. 11)
[0172] B9R (SEQ. ID. No. 12)
Example 5
[0173] Combinations of oligonucleotide primers according to the
present invention were used for specific detection of different
initial copy numbers of the target VT2 RNA.
[0174] (1) As described in example 2, VT2 standard RNA was diluted
with an RNA diluent (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5
U/.mu.l RNase Inhibitor (Takara Shuzo Co. Ltd.), 5 mM DTT) to
concentrations ranging from 10.sup.5 copies/2.5 .mu.l to 10.sup.1
copies/2.5 .mu.l. In the control testing sections, only the diluent
was used (Negative).
[0175] (2) 23.3 .mu.l of a reaction solution having the composition
shown below was dispended into 0.5 ml volume PCR tubes (Gene Amp
Thin-walled Reaction Tube.TM., Perkin-Elmer) followed by addition
of 2.5 .mu.l of the above RNA sample.
[0176] Reaction Solution Composition (each concentration represents
that in a final reaction solution of 30 .mu.l)
[0177] 60 mM Tris-HCl buffer (pH 8.6)
[0178] 17 mM magnesium chloride
[0179] 150 mM potassium chloride
[0180] 39 U RNase Inhibitor
[0181] 1 mM DTT
[0182] 0.25 mM each of dATP, dCTP, dGTP and dTTP
[0183] 3.6 mM ITP
[0184] 3.0 mM each of ATP, CTP, GTP and UTP
[0185] 0.16 .mu.M first oligonucleotide (5S shown in Table 4,
[0186] wherein its 3' end is aminated)
[0187] 1.0 .mu.M second oligonucleotide (5F shown in Table 4)
[0188] 1.0 .mu.M third oligonucleotide (7R shown in Table 4)
[0189] 25 nM intercalator fluorescent pigment-labeled
oligonucleotide (SEQ. ID. No. 25, labeled with an intercalator
fluorescent pigment at the phosphorous atom between the 12th "T"
and the 13th "A" from the 5' end, and modified with a glycol group
at its 3' end hydroxyl)
[0190] 13% DMSO
[0191] Distilled Water for Adjusting Volume
[0192] (3) After incubating the above reaction solution for 5
minutes at 41.degree. C., 4.2 .mu.l of an enzyme solution having
the following composition and pre-incubated for 2 minutes at
41.degree. C. was added.
[0193] Enzyme Solution Composition (each concentration represents
that in a final reaction solution of 30 .mu.l)
[0194] 1.7% sorbitol
[0195] 3 .mu.g bovine serum albumin
[0196] 142 U T7 RNA polymerase (Gibco)
[0197] 8 U AMV-Reverse Transcriptase (Takara Shuzo Co. Ltd.)
[0198] Distilled Water for Adjusting Volume
[0199] (4) The PCR tube was then incubated at 41.degree. C. using a
direct-measuring fluorescence spectrophotometer equipped with a
temperature-controller, and the reaction solution was periodically
measured at an excitation wavelength of 470 nm and a fluorescent
wavelength of 510 nm.
[0200] FIG. 8 (A) shows the time-course changes in the fluorescence
increase ratio (fluorescence intensity at predetermined
time/background fluorescence intensity) of the sample, where enzyme
was added at 0 minutes. FIG. 8 (B) shows the relationship between
the logarithm of the initial RNA amount and the rise time (time at
which the relative fluorescence reaches the negative sample's
average value plus 3 standard deviations; i.e., the time to reach a
ratio of 1.2). The initial RNA amount was between 10.sup.1
copies/test and 10.sup.5 copies/test.
[0201] FIG. 8 shows that 10.sup.1 copies were detected at
approximately 20 minutes. A fluorescent profile and calibration
curve depending on the initial concentration of the labeled RNA
were obtained, indicating that it is possible to quantify the VT2
RNA present in unknown samples. This demonstrated that speedy,
highly sensitive detection of VT2 RNA is possible using this
method.
[0202] As explained above, the oligonucleotide provided by the
present invention complementarily binds to the intramolecular
structure-free region of VT1 RNA or VT2 RNA. By using this
oligonucleotide, it is possible to detect an RNA by a process
carried out under a relatively low and constant temperature,
without the need of an operation which destroys the intramolecular
structure of an RNA by heat-degradation so as to improve the primer
binding efficiency. As a result, by use of the oligonucleotide
according to the present invention, it would be possible to provide
an RNA detection method which is speedy, simple, and even suitable
for automation.
[0203] It will be appreciated by those skilled in the art that
while the invention has been described above in connection with
particular embodiments and examples, the invention is not
necessarily so limited and that numerous other embodiments,
examples, uses, modifications and departures from the embodiments,
examples and use may be made without departing from the inventive
scope of this application.
Sequence CWU 1
1
44 1 20 DNA Artificial Sequence Synthetic DNA 1 aaaaaacatt
atttgtcctg 20 2 20 DNA Artificial Sequence Synthetic DNA 2
tggcgattta tctgcatccc 20 3 20 DNA Artificial Sequence Synthetic DNA
3 gatgatgaca attcagtatt 20 4 20 DNA Artificial Sequence Synthetic
DNA 4 ttttattgtg cgtaatccca 20 5 20 DNA Artificial Sequence
Synthetic DNA 5 taatagttct gcgcatcaga 20 6 20 DNA Artificial
Sequence Synthetic DNA 6 tatacaggtg ttccttttgg 20 7 20 DNA
Artificial Sequence Synthetic DNA 7 tatatgttca agaggggtcg 20 8 20
DNA Artificial Sequence Synthetic DNA 8 atggtcaaaa cgcgcctgat 20 9
20 DNA Artificial Sequence Synthetic DNA 9 tagaaagtat ttgttgccgt 20
10 20 DNA Artificial Sequence Synthetic DNA 10 gtaaggcttc
tgctgtgaca 20 11 20 DNA Artificial Sequence Synthetic DNA 11
cagtttcaga cagtgcctga 20 12 20 DNA Artificial Sequence Synthetic
DNA 12 ttgctgattc gcccccagtt 20 13 20 DNA Artificial Sequence
Synthetic DNA 13 attattaaag gatattctcc 20 14 20 DNA Artificial
Sequence Synthetic DNA 14 attgtttatt tttataacag 20 15 25 DNA
Artificial Sequence Synthetic DNA 15 tttttatcgc tttgctgatt tttca 25
16 25 DNA Artificial Sequence Synthetic DNA 16 cgccattcgt
tgactacttc ttatc 25 17 25 DNA Artificial Sequence Synthetic DNA 17
tgatctcagt gggcgttctt atgta 25 18 25 DNA Artificial Sequence
Synthetic DNA 18 tcatcatgca tcgcgagttg ccaga 25 19 25 DNA
Artificial Sequence Synthetic DNA 19 gtatatgaag tgtatattat ttaaa 25
20 25 DNA Artificial Sequence Synthetic DNA 20 atatatctca
ggggaccaca tcggt 25 21 25 DNA Artificial Sequence Synthetic DNA 21
accatcttcg tctgattatt gagca 25 22 25 DNA Artificial Sequence
Synthetic DNA 22 ttctaccgtt tttcagattt tacac 25 23 25 DNA
Artificial Sequence Synthetic DNA 23 cttacgcttc aggcagatac agaga 25
24 20 DNA Artificial Sequence Synthetic DNA 24 tgtaacgtgg
tatagctact 20 25 20 DNA Artificial Sequence Synthetic DNA 25
ttaacgccag atatgatgaa 20 26 20 DNA Artificial Sequence Synthetic
DNA 26 gatcatccag tgttgtacga 20 27 39 DNA Artificial Sequence
Synthetic DNA 27 aaaaaacatt atttgtcctg ttaacaaatc ctgtcacat 39 28
39 DNA Artificial Sequence Synthetic DNA 28 tggcgattta tctgcatccc
cgtacgactg atccctgca 39 29 39 DNA Artificial Sequence Synthetic DNA
29 gatcatccag tgttgtacga aatcccctct gtatttgcc 39 30 39 DNA
Artificial Sequence Synthetic DNA 30 gatgatgaca attcagtatt
aatgccacgc ttcccagaa 39 31 39 DNA Artificial Sequence Synthetic DNA
31 tatacaggtg ttccttttgg ctgaagtaat cagcaccag 39 32 39 DNA
Artificial Sequence Synthetic DNA 32 tatatgttca agaggggtcg
atatctctgt ccgtatact 39 33 39 DNA Artificial Sequence Synthetic DNA
33 atggtcaaaa cgcgcctgat agacatcaag ccctcgtat 39 34 39 DNA
Artificial Sequence Synthetic DNA 34 tagaaagtat ttgttgccgt
attaacgaac ccggccaca 39 35 39 DNA Artificial Sequence Synthetic DNA
35 gtaaggcttc tgctgtgaca gtgacaaaac gcagaactg 39 36 53 DNA
Artificial Sequence Synthetic DNA 36 aattctaata cgactcacta
tagggagatt tttatcgctt tgctgatttt tca 53 37 53 DNA Artificial
Sequence Synthetic DNA 37 aattctaata cgactcacta tagggagacg
ccattcgttg actacttctt atc 53 38 53 DNA Artificial Sequence
Synthetic DNA 38 aattctaata cgactcacta tagggagatg atctcagtgg
gcgttcttat gta 53 39 53 DNA Artificial Sequence Synthetic DNA 39
aattctaata cgactcacta tagggagatc atcatgcatc gcgagttgcc aga 53 40 53
DNA Artificial Sequence Synthetic DNA 40 aattctaata cgactcacta
tagggagagt atatgaagtg tatattattt aaa 53 41 53 DNA Artificial
Sequence Synthetic DNA 41 aattctaata cgactcacta tagggagaat
atatctcagg ggaccacatc ggt 53 42 53 DNA Artificial Sequence
Synthetic DNA 42 aattctaata cgactcacta tagggagaac catcttcgtc
tgattattga gca 53 43 53 DNA Artificial Sequence Synthetic DNA 43
aattctaata cgactcacta tagggagatt ctaccgtttt tcagatttta cac 53 44 53
DNA Artificial Sequence Synthetic DNA 44 aattctaata cgactcacta
tagggagact tacgcttcag gcagatacag aga 53
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