JP2001258569A - Oligonucleotide for detecting vibrio parahaemolyticus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an oligonucleotide specifically binding to a thermostable direct hemolysin-related hemolysin gene (trh1 or trh2) of Vibrio parahaemolyticus or an RNA derived from the gene. SOLUTION: This oligonucletide is used for detecting or amplifying the thermostable direct hemolysin-related hemolysin gene (trh1 or trh2) of the Vibrio parahaemolyticus or the RNA derived from the gene. Furthermore, the oligonucleotide is capable of specifically binding to the trh1 and trh2 or the RNA derived therefrom and comprises at least >=10 contiguous bases in any sequence described in sequence Nos. 1 to 11 in the specification or an oligonucleotide is complementary to the oligonucleotide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an oligonucleotide for detecting Vibrio parahaemolyticus in clinical examination, public health, food examination or food poisoning examination.

[0002]

2. Description of the Related Art Vibrio parenchyma (Vibrio par)
ahaemolyticus) is generally known as a causative agent of infectious food poisoning. More than 95% of Vibrio parahaemolyticus isolated from patients with gastroenteritis are Kanagawa phenomenon-positive bacteria that exhibit hemolytic activity in Wazuma medium , whereas 99% of bacteria isolated from fish and water are Kanagawa phenomenon-negative bacteria. From this, it has been concluded that the pathogenic Vibrio parahaemolyticus and the Kanagawa phenomenon are deeply involved, and thereafter, the Kanagawa phenomenon was caused by the heat-resistant hemotoxin (thermosta) of Vibrio parahaemolyticus.
ble direct hemolysin (TDH)
It has been found that this is a phenomenon that occurs due to the release of TDH out of the cells, and TDH has been attracting attention as a virulence factor of Vibrio parahaemolyticus. More recently, hemolytic toxins having a base sequence similar to TDH and having a partially common antigenicity (thermolytic hemotoxin similar to thermotolerant hemotoxin (TDH-related) have been selected from strains that show pathogenicity despite being a Kanagawa phenomenon-negative strain.
hemolysin (TRH)) was also confirmed.

[0003]

Until now, detection and identification of Vibrio parahaemolyticus has been extremely complicated and time-consuming, such as judging the Kanagawa phenomenon after enrichment culture and separation culture. In recent years, for the detection and identification of Vibrio parahaemolyticus, a hybridization method using a gene probe specific to the sequence in the TDH or TRH gene has been tried. However, it is not possible to obtain sufficient detection sensitivity in food inspection and the like. It was difficult.

As described above, detection and identification of Vibrio parahaemolyticus require complicated operations and a long time, and it is difficult to detect a trace amount of Vibrio parahaemolyticus present in a sample in a short time. Therefore, in the field of food inspection and the like, the emergence of a rapid and highly sensitive detection method is desired. Furthermore, development of an automatic inspection apparatus is desired in order to make inspection easier.

In order to perform detection with high sensitivity, a gene to be detected and identified and RNA derived from the gene are required.
It is preferable to amplify and detect specific sequences in the following (hereinafter, these are referred to as target nucleic acids).

[0006] As an amplification method when the target nucleic acid is DNA, a polymerase chain reaction (PCR) method is known. In this method, a cycle consisting of heat denaturation, primer annealing, and extension is repeated in the presence of a set of primers complementary and homologous to both ends of a specific sequence in the target DNA and a thermotolerant DNA polymerase. Amplifying the specific sequence. However, the PCR method requires a complicated operation of repeatedly raising and lowering the temperature rapidly, which is an obstacle to automation. Further, in order to amplify the specific sequence by the PCR method, an oligonucleotide having high specificity to the specific sequence is required, and in order to perform the detection and identification with high sensitivity, the specificity to the target DNA is required. Are required.

As the amplification method when the target nucleic acid is RNA, the NASBA method of amplifying the specific sequence by the concerted action of reverse transcriptase and RNA polymerase or 3SR
The law is known. In this method, a double-stranded DNA containing a promoter sequence is synthesized with respect to a specific sequence in a target RNA using a primer containing a promoter sequence, a reverse transcriptase, and ribonuclease H, and the double-stranded DNA is synthesized.
Is used as a template to synthesize RNA containing the specific sequence by RNA polymerase, and the RNA is subsequently subjected to a chain reaction as a template for the synthesis of double-stranded DNA containing a promoter sequence. The NASBA method and the 3SR method can perform nucleic acid amplification at a constant temperature, and are considered to be suitable methods for automation. However, since these amplification methods carry out the reaction at a relatively low temperature (for example, 41 ° C.), the target RN
A forms an intramolecular structure, inhibits the binding of the primer,
It is conceivable that the reaction efficiency may be reduced. Therefore, by performing thermal denaturation of the target RNA before the amplification reaction,
An operation for breaking the intramolecular structure of the target RNA and improving the binding efficiency of the primer was required. In order to amplify the specific sequence by the NASBA method or the like, an oligonucleotide having high specificity to the specific sequence is required.
Oligonucleotides with high specificity for NA were required. Furthermore, even when RNA is detected at a low temperature, an oligonucleotide capable of binding to RNA having an intramolecular structure as described above is required.

Accordingly, the present invention provides a thermotolerant hemotoxin-like hemolytic toxin gene for Vibrio parahaemolyticus (trh1 and trh2),
Another object of the present invention is to provide oligonucleotides useful for specifically amplifying RNAs derived from these genes and for detecting and identifying them with high sensitivity.

[0009]

Means for Solving the Problems To achieve the above object, the invention of claim 1 of the present application is directed to a thermotolerant hemotoxin gene (trh1 and trh1) of Vibrio parahaemolyticus.
2) or an oligonucleotide for detecting or amplifying RNA derived from these genes, wherein trh1
And oligonucleotides consisting of at least 10 consecutive nucleotides or more in any of the sequences shown in SEQ ID NOs: 1 to 11 that are capable of specifically binding to trh2 or RNA derived therefrom, or complementary to these oligonucleotides Is an oligonucleotide.

[0010] In order to achieve the above object, the invention of claim 2 of the present invention is directed to a thermostable hemotoxin-like hemotoxin gene (trh1) of Vibrio parahaemolyticus or R derived from the gene.
An oligonucleotide for detecting or amplifying NA, which is at least 10 consecutive nucleotides in any of the sequences shown in SEQ ID NOs: 12 to 14, which can specifically bind to trh1 or RNA derived from the gene. Oligonucleotides comprising the above or oligonucleotides complementary to those oligonucleotides.

In order to achieve the above object, the invention of claim 3 of the present invention provides a thermostable hemotoxin-like hemolytic toxin gene of Vibrio parahaemolyticus (trh2) or an R gene derived from said gene.
An oligonucleotide for detecting or amplifying NA, wherein at least 10 consecutive nucleotides in any of the sequences shown in SEQ ID NOS: 15 to 17 are capable of binding specifically to trh2 or RNA derived from the gene. Oligonucleotides consisting of bases or more or oligonucleotides complementary to those oligonucleotides.

The invention of claim 4 of the present application is directed to claim 1
An oligonucleotide primer for a DNA extension reaction, comprising the oligonucleotides of items 3 to 3,
The invention of claim 5 of the present application is an oligonucleotide probe in which a part of the oligonucleotide described in claims 1 to 3 is modified or labeled with a detectable label. Hereinafter, the present invention will be described in detail.

The oligonucleotide of the present invention is an oligonucleotide which forms a complementary bond specifically to a region free of intramolecular structure of the target RNA upon amplification of the above-mentioned RNA, and undergoes the above-mentioned heat denaturation. It can specifically bind to target RNA without any problem. As described above, the present invention provides a relatively low temperature and constant temperature (35 ° C. to 50 ° C.).
C., preferably 41.degree. C.).
And / or by providing an oligonucleotide that binds to the intramolecular structure-free region of the RNA derived from trh2, an oligonucleotide for specifically amplifying or detecting trh1 and trh2, specifically only trh1 Oligonucleotides for amplification or detection, etc.
The present invention also provides an oligonucleotide for specifically amplifying or detecting only trh2. More specifically, in the present invention, the target D
Oligonucleotide primers for amplifying NA,
An oligonucleotide primer for amplifying the target RNA or the like by the NASBA method or the like, and an oligonucleotide probe for detecting a target nucleic acid without such amplification or after such amplification are included. The use of the oligonucleotide of the present invention provides a simple, rapid and highly sensitive detection method for food inspection, food poisoning inspection and the like.

SEQ ID NOS: 1 to 11 are trh1 and tr
h2 or an oligonucleotide for detecting or amplifying RNA derived from these genes.
Here, the RNAs derived from these genes include RNAs produced using these genes as templates. These oligonucleotides may be oligonucleotides composed of at least 10 consecutive bases or more of the described base sequences, and may be complementary oligonucleotides.

SEQ ID NOs: 12 to 14 are oligonucleotides for detecting trh1 or RNA derived from the gene, and are useful when amplifying or detecting trh1 separately from trh2. These oligonucleotides may be oligonucleotides composed of at least 10 consecutive bases or more of the described base sequences, and may be complementary oligonucleotides.

SEQ ID NOs: 15 to 17 are oligonucleotides for detecting trh2 or RNA derived from the gene, and are useful when amplifying or detecting trh2 separately from trh1. These oligonucleotides may be oligonucleotides composed of at least 10 consecutive bases or more of the described base sequences, and may be complementary oligonucleotides.

One embodiment of the present invention is an amplification primer comprising the above oligonucleotide. If a nucleic acid amplification reaction is performed using the above oligonucleotide as a primer, only the target nucleic acid can be amplified. Examples of the amplification method include a PCR method, a NASBA method, and a 3SR method. Among them, a constant temperature nucleic acid amplification method such as the NASBA method and the 3SR method is preferable. By detecting the amplification product by various methods, it becomes possible to detect Vibrio parahaemolyticus. In this case, the above-described oligonucleotide other than the oligonucleotide used in the amplification may be used as a probe,
The amplified fragment of the specific sequence may be confirmed by electrophoresis or the like.

Another embodiment of the present invention is a probe in which a part of the above oligonucleotide is modified or labeled with a detectable label. When the target nucleic acid is to be detected, the oligonucleotide labeled with a detectable labeling substance may be hybridized with a single-stranded target nucleic acid, and the label may be detected with respect to the hybridized probe. The detection of the label may be performed by a method suitable for the labeling substance. For example, when an intercalating fluorescent dye is used for the labeling of the oligonucleotide, the double-stranded nucleic acid comprising the target nucleic acid and the oligonucleotide probe may be interleaved. If a dye or the like having a property of increasing the fluorescence intensity by callation is used, it is possible to easily detect only the hybridized probe without removing the probe not hybridized with the target nucleic acid. When an ordinary fluorescent dye or the like is used as a label, the label may be detected after removing a probe that has not hybridized to the target nucleic acid. For detection, the target nucleic acid in the sample was analyzed by PCR, NAS
It is preferable to amplify to an amount detectable by various nucleic acid amplification methods such as BA method and 3SR method.
Constant temperature nucleic acid amplification methods such as the ASBA method and the 3SR method are most preferred. Here, when a probe labeled with the above nucleotide is coexisted in the reaction solution at the time of amplification, modification such as adding glycolic acid to the 3 ′ end thereof is performed so that the probe does not function as a nucleotide primer. Is particularly preferred.

[0019]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 It was confirmed whether the oligonucleotide of the present invention specifically binds to trh1-RNA at 41 ° C. Note that t
rh1-RNA is a double-stranded D containing the nucleotide sequence of trh1.
RNA synthesized and purified by in vitro transcription using NA as a template.

First, trh1-RNA of Vibrio parahaemolyticus
Base numbers 1 to 610 (RNA base numbers are as described in Nishibuchi et al., "Appl. Environ. Microbiol.,
1992, 58, pp. 2449-2457) as a sample.
After quantification by UV absorption at 60 nm, the RNA diluent (1
0 mM Tris-HCl (pH 8.0), 0.1 mM
EDTA, 0.5 U / μl RNase Inhib
It diluted so that it might become 3.0 * 10 < ^-12 > mol / microliter using (itor).

Next, 14.0 μl of a reaction solution having the following composition was added to a 0.5 ml PCR tube (trade name: Gene A).
mp Thin-Walled Reaction T
ubes, manufactured by PerkinElmer Co., Ltd.).

Composition of reaction solution 20.0 mM Tris-HCl buffer (pH 7.5) 20.0 mM potassium chloride 10.0 mM magnesium chloride 0.1 mM DTT 0.1 mM EDTA 1.3 μM oligonucleotide primer solution 1.0 × 10 ⁻¹² mol standard trh1-RNA sample distilled water for volume adjustment The oligonucleotide primer solution includes the oligonucleotide solution of SEQ ID NO: 1, the oligonucleotide solution of SEQ ID NO: 2, the oligonucleotide solution of SEQ ID NO: 3, the SEQ ID NO: 4, , The oligonucleotide solution of SEQ ID NO: 13, the oligonucleotide solution of SEQ ID NO: 5, the oligonucleotide solution of SEQ ID NO: 5, and the oligonucleotide solution of SEQ ID NO: 14. The oligonucleotide of SEQ ID NO: 1 is tr
The oligonucleotide of SEQ ID NO: 2 is complementary to the 20-mer at positions 31 to 50 of h1-RNA and the 20-mer at positions 67 to 86 of trh1-RNA.
The oligonucleotide of SEQ ID NO: 3 is complementary to the sequence of the 20-mer from position 132 to 151 of trh1-RNA, and the oligonucleotide of SEQ ID NO: 4 is complementary to the sequence of SEQ ID NO: 4.
Is complementary to the 20-mer sequence at positions 229 to 248 of trh1-RNA, and the oligonucleotide of SEQ ID NO: 12 is trh1-RNA.
Complementary to the 20-mer sequence at positions 274 to 293 of the RNA, the oligonucleotide of SEQ ID NO: 5 is the 20-mer at positions 308 to 327 of the trh1-RNA.
and the oligonucleotide of SEQ ID NO: 13 is complementary to the sequence of trh1-RNA 414-43.
The oligonucleotide is complementary to the third 20-mer sequence and the oligonucleotide of SEQ ID NO: 14 is complementary to the 20-mer sequence from trh1-RNA at positions 563 to 582.

Next, the above reaction solution was kept at 41 ° C. for 5 minutes, and 0.1 U of RNase H (manufactured by Takara Shuzo Co., Ltd.) was added (RNase H is a DNA / RNA double-stranded RNase).
A), and then the PCR tube was kept at 41 ° C. for 15 minutes.

To confirm the cleavage fragments after the reaction, a polyacrylamide gel (acrylamide concentration: 6%, urea: 7%)
M) Electrophoresis was performed. Staining after electrophoresis is SYBR
Green II (manufactured by Takara Shuzo Co., Ltd.). When the oligonucleotide binds to a specific site of the target RNA, RNase H causes the DNA / RNA double-stranded R
NA is cleaved and a specific band is observed.

The results of the electrophoresis are shown in FIG. 1 (FIG. 1 is a photograph (black and white reversal) showing the state of the oligonucleotide). The size of the newly appearing specific band is as shown in FIG. For example, in the experiment using the oligonucleotide of SEQ ID NO: 1, the size of the band when the trh1-RNA was cleaved at the 31st position from the 5 'end, that is, 31 mer and 585 mer are displayed. From this, in the experiments using each of the above oligonucleotides, a specific band was confirmed in each case. Therefore, all of these oligonucleotides were trh1-
It was shown to bind strongly to RNA at 41 ° C.

Example 2 It was confirmed whether the oligonucleotide of the present invention specifically binds to trh2-RNA at 41 ° C. Note that t
rh2-RNA is a double-stranded D containing the nucleotide sequence of trh2.
RNA synthesized and purified by in vitro transcription using NA as a template.

First, trh2-RNA of Vibrio parahaemolyticus
Base numbers 1 to 610 (RNA base numbers are as described in Nishibuchi et al., "Appl. Environ. Microbiol.,
1992, 58, pp. 2449-2457) as a sample.
After quantification by UV absorption at 60 nm, the RNA diluent (1
0 mM Tris-HCl (pH 8.0), 0.1 mM
EDTA, 0.5 U / μl RNase Inhib
It diluted so that it might become 3.0 * 10 < ^-12 > mol / microliter using (itor).

Next, 14.0 μl of a reaction solution having the following composition was added to a 0.5 ml PCR tube (trade name: Gene A).
mp Thin-Walled Reaction T
ubes, manufactured by PerkinElmer Co., Ltd.).

Composition of reaction solution 20.0 mM Tris-HCl buffer (pH 7.5) 20.0 mM potassium chloride 10.0 mM magnesium chloride 0.1 mM DTT 0.1 mM EDTA 1.3 μM oligonucleotide primer solution 1.0 × 10 ⁻¹² mol standard trh2-RNA sample distilled water for volume adjustment The oligonucleotide primer solutions are the oligonucleotide solution of SEQ ID NO: 1, the oligonucleotide solution of SEQ ID NO: 2, the oligonucleotide solution of SEQ ID NO: 3, the SEQ ID NO: The oligonucleotide solution of No. 12, the oligonucleotide solution of SEQ ID NO: 5, the oligonucleotide solution of SEQ ID NO: 13, the oligonucleotide solution of SEQ ID NO: 14, and the oligonucleotide solution of SEQ ID NO: 8 were used. The oligonucleotide of SEQ ID NO: 1 is tr
The oligonucleotide of SEQ ID NO: 2 is complementary to the 20-mer at positions 31 to 50 of h1-RNA and the 20-mer at positions 67 to 86 of trh1-RNA.
The oligonucleotide of SEQ ID NO: 3 is complementary to the 20-mer sequence from the 132nd to the 151st of trh1-RNA, and the oligonucleotide of SEQ ID NO: 1
Oligonucleotide 2 is 274 of trh1-RNA
And the oligonucleotide of SEQ ID NO: 5 is complementary to the 20-mer sequence at position 293 from trh1-R
Complementary to the 20-mer sequence from 308 to 327 of NA, the oligonucleotide of SEQ ID NO: 13 is the 20-mer from 414 to 433 of trh1-RNA.
er, and the oligonucleotide of SEQ ID NO: 14 is trh1-RNA 563-58.
Complementary to the sequence of the second 20 mer, the oligonucleotide of SEQ ID NO: 8
Complementary to the 20-mer sequence at positions 59 to 278.

Next, the above reaction solution was kept at 41 ° C. for 5 minutes, and 0.1 U of RNase H (manufactured by Takara Shuzo Co., Ltd.) was added (RNase H is a DNA / RNA double-stranded RNase).
A), and then the PCR tube was kept at 41 ° C. for 15 minutes.

In order to confirm the fragment after the reaction, a polyacrylamide gel (acrylamide concentration: 6%, urea: 7%) was used.
M) Electrophoresis was performed. Staining after electrophoresis is SYBR
Green II (manufactured by Takara Shuzo Co., Ltd.).
When the oligonucleotide binds to a specific site of the target RNA, RNase H causes the DNA / RNA double-stranded R
NA is cleaved and a specific band is observed.

The results of the electrophoresis are shown in FIG. 2 (FIG. 2 is a photograph (inversion of black and white) showing the state of the oligonucleotide). The size of the newly appeared specific band is as shown in FIG. For example, in the experiment using the oligonucleotide of SEQ ID NO: 2, the size of the band when the trh1-RNA was cleaved at the 67th position from the 5 'end, that is, 67mer and 549mer are displayed.

From the above results, in the experiments using the oligonucleotides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 5, specific bands could be confirmed. Therefore, the oligonucleotides of these sequences were used for trh of Vibrio parahaemolyticus.
It was shown that it bound strongly to 2-RNA at 41 ° C.
On the other hand, the oligonucleotides of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14 did not bind to trh2-RNA. The oligonucleotide of SEQ ID NO: 8 is trh2
-Used as a control sequence that binds strongly to RNA at 41 ° C. Together with the results of Example 1, the oligonucleotides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 5 strongly bind to both trh1-RNA and trh2-RNA at 41 ° C. Indicated. on the other hand,
It was shown that the oligonucleotides of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14 strongly bind only to trh1-RNA at 41 ° C.

Example 3 It was confirmed whether the oligonucleotide of the present invention specifically binds to trh2-RNA at 41 ° C.

First, a standard RNA (616mer) containing base numbers 1 to 610 of trh2-RNA was used as a sample.
After quantification by ultraviolet absorption at 260 nm, an RNA diluent (10 mM Tris-HCl (pH 8.0), 0.1
mM EDTA, 0.5 U / μl RNase Inh
3.0 × 10 ⁻¹² mol / μl
And diluted.

Next, 14.0 μl of a reaction solution having the following composition was added to a 0.5 ml PCR tube (trade name: Gene A).
mp Thin-Walled Reaction T
ubes, manufactured by PerkinElmer Co., Ltd.).

Composition of reaction solution 20.0 mM Tris-HCl buffer (pH 7.5) 20.0 mM potassium chloride 10.0 mM magnesium chloride 0.1 mM DTT 0.1 mM EDTA 1.3 μM oligonucleotide primer solution 1.0 × 10 ^-12 mol standard trh2-RNA sample distilled water for volume adjustment The oligonucleotide primer solutions include the oligonucleotide solution of SEQ ID NO: 6, the oligonucleotide solution of SEQ ID NO: 15, the oligonucleotide solution of SEQ ID NO: 7, the SEQ ID NO: The oligonucleotide solution of No. 8, the oligonucleotide solution of SEQ ID NO: 16, the oligonucleotide solution of SEQ ID NO: 17, the oligonucleotide solution of SEQ ID NO: 9, the oligonucleotide solution of SEQ ID NO: 10, and the oligonucleotide solution of SEQ ID NO: 11 were used. In addition, the oligonucleotide of SEQ ID NO: 6 is trh2-R
Complementary to the 20-mer sequence at positions 55 to 74 of NA, the oligonucleotide of SEQ ID NO: 15
The oligonucleotide of SEQ ID NO: 7 is complementary to the 20-mer sequence at positions 84 to 103 of rh2-RNA,
The oligonucleotide of SEQ ID NO: 8 is complementary to the sequence of
The oligonucleotide of SEQ ID NO: 16, which is complementary to the sequence of the 20-mer at position 78, is trh2-RNA
And the oligonucleotide of SEQ ID NO: 17 is complementary to the 20-mer sequence of positions 322 to 341 of
The oligonucleotide of SEQ ID NO: 9 is complementary to the 20-mer sequence of positions 380 to 399 of rh2-RNA, and the oligonucleotide of SEQ ID NO: 9 is complementary to the sequence of 20-mer of positions 407 to 426 of trh2-RNA. 10
Is complementary to the 20-mer sequence at positions 456 to 475 of trh2-RNA, and the oligonucleotide of SEQ ID NO: 11 is trh2-RNA.
Complementary to the 20-mer sequence at positions 540 to 459 of RNA.

Next, the above reaction solution was kept at 41 ° C. for 5 minutes, and 0.1 U of RNase H (manufactured by Takara Shuzo Co., Ltd.) was added (RNase H is a DNA / RNA double-stranded RNase).
A), and then the PCR tube was kept at 41 ° C. for 15 minutes.

To confirm the cleavage fragments after the reaction, a polyacrylamide gel (acrylamide concentration: 6%, urea: 7%) was used.
M) Electrophoresis was performed. Staining after electrophoresis is SYBR
Green II (manufactured by Takara Shuzo Co., Ltd.).
When the oligonucleotide binds to a specific site of the target RNA, RNase H causes the DNA / RNA double-stranded R
NA is cleaved and a specific band is observed.

The results of the electrophoresis are shown in FIG. 3 (FIG. 3 is a photograph (inversion of black and white) showing the state of the oligonucleotide). The size of the newly appeared specific band is as shown in FIG. For example, in the experiment using the oligonucleotide of SEQ ID NO: 6, the size of the band when trh2-RNA was cleaved at the 55th position from the 5 'end, that is, 55mer and 561mer are displayed.

From the above, specific bands were confirmed in the experiments using each of the above oligonucleotides, indicating that all of these oligonucleotides strongly bind to trh2-RNA of Vibrio parahaemolyticus at 41 ° C. Was done.

Example 4 It was confirmed whether the oligonucleotide of the present invention specifically binds to trh1-RNA at 41 ° C.

First, a standard RNA (616 mer) containing base numbers 1 to 610 of trh1-RNA was used as a sample.
After quantification by ultraviolet absorption at 260 nm, an RNA diluent (10 mM Tris-HCl (pH 8.0), 0.1
mM EDTA, 0.5 U / μl RNase Inh
3.0 × 10 ⁻¹² mol / μl
And diluted.

Next, 14.0 μl of a reaction solution having the following composition was added to a 0.5 ml PCR tube (trade name: Gene A).
mp Thin-Walled Reaction T
ubes, manufactured by PerkinElmer Co., Ltd.).

Composition of reaction solution 20.0 mM Tris-HCl buffer (pH 7.5) 20.0 mM potassium chloride 10.0 mM magnesium chloride 0.1 mM DTT 0.1 mM EDTA 1.3 μM oligonucleotide primer solution 1.0 × 10 ^-12 mol standard trh2-RNA sample distilled water for volume adjustment The oligonucleotide primer solutions include the oligonucleotide solution of SEQ ID NO: 6, the oligonucleotide solution of SEQ ID NO: 15, the oligonucleotide solution of SEQ ID NO: 7, the SEQ ID NO: The oligonucleotide solution of SEQ ID NO: 8, the oligonucleotide solution of SEQ ID NO: 16, the oligonucleotide solution of SEQ ID NO: 9, and the oligonucleotide solution of SEQ ID NO: 3 were used.

Next, the above reaction solution was kept at 41 ° C. for 5 minutes, and 0.1 U of RNase H (manufactured by Takara Shuzo Co., Ltd.) was added (RNase H is a DNA / RNA double-stranded RNase).
A), and then the PCR tube was kept at 41 ° C. for 15 minutes.

To confirm the cleavage fragments after the reaction, a polyacrylamide gel (acrylamide concentration: 6%, urea: 7%) was used.
M) Electrophoresis was performed. Staining after electrophoresis is SYBR
Green II (manufactured by Takara Shuzo Co., Ltd.).
When the oligonucleotide binds to a specific site of the target RNA, RNase H causes the DNA / RNA double-stranded R
NA is cleaved and a specific band is observed.

The results of the electrophoresis are shown in FIG. 4 (FIG. 4 is a photograph (black and white reversal) showing the state of the oligonucleotide). The size of the newly appeared specific band is as shown in FIG. For example, in the experiment using the oligonucleotide of SEQ ID NO: 6, the size of the band when trh2-RNA was cleaved at the 55th position from the 5 'end, that is, 55mer and 561mer are displayed.

From the above results, in the experiments using the oligonucleotides of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, specific bands could be confirmed in all cases.
It was shown that it bound strongly to 1-RNA at 41 ° C.
On the other hand, the oligonucleotides of SEQ ID NO: 15 and SEQ ID NO: 16 did not bind to trh1-RNA. In addition, the oligonucleotide of SEQ ID NO: 3 has 41
It was used as a control sequence that binds strongly at ° C. In combination with the results of Example 3, the oligonucleotides of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9
It was shown that both -RNA and trh2-RNA strongly bind at 41 ° C. On the other hand, the oligonucleotides of SEQ ID NO: 15 and SEQ ID NO: 16
It was shown to bind strongly only to 2-RNA.

Example 5 It was confirmed whether the oligonucleotide of the present invention specifically binds to trh1-RNA or trh2-RNA at 41 ° C.

First, trh1-RNA or trh2-
Standard RNAs containing base numbers 1 to 610 of RNA (61
6mer) as a sample, and after quantification by ultraviolet absorption at 260 nm, an RNA diluent (10 mM Tris-HCl)
(PH 8.0), 0.1 mM EDTA, 0.5 U / μl
3.0 × using RNase Inhibitor
It was diluted to 10 ⁻¹² mol / μl.

Next, 14.0 μl of a reaction solution having the following composition was added to a 0.5 ml PCR tube (trade name: Gene A).
mp Thin-Walled Reaction T
ubes, manufactured by PerkinElmer Co., Ltd.).

Composition of reaction solution 20.0 mM Tris-HCl buffer (pH 7.5) 20.0 mM potassium chloride 10.0 mM magnesium chloride 0.1 mM DTT 0.1 mM EDTA 1.3 μM oligonucleotide primer solution 1.0 × 10 ⁻¹² mol standard trh2-RNA sample distilled water for volume adjustment The oligonucleotide primer solution includes the oligonucleotide solution of SEQ ID NO: 17, the oligonucleotide solution of SEQ ID NO: 10, the oligonucleotide solution of SEQ ID NO: 11, and the sequence The oligonucleotide solution of No. 4 was used.

Next, the above reaction solution was kept at 41 ° C. for 5 minutes, and 0.1 U of RNase H (manufactured by Takara Shuzo Co., Ltd.) was added (RNase H is a DNA / RNA double-stranded RNase).
A), and then the PCR tube was kept at 41 ° C. for 15 minutes.

To confirm the cleavage fragments after the reaction, a polyacrylamide gel (acrylamide concentration: 6%, urea: 7%) was used.
M) Electrophoresis was performed. Staining after electrophoresis is SYBR
Green II (manufactured by Takara Shuzo Co., Ltd.).
When the oligonucleotide binds to a specific site of the target RNA, RNase H causes the DNA / RNA double-stranded R
NA is cleaved and a specific band is observed.

The results of the electrophoresis are shown in FIG. 5 (FIG. 5 is a photograph (black and white reversal) showing the state of the oligonucleotide). The size of the newly appearing specific band is as shown in FIG. For example, in an experiment using the oligonucleotide of SEQ ID NO: 10, the size of the band when trh2-RNA was cleaved at position 456 from the 5 ′ end,
That is, 456mer and 160mer are displayed.

From this, SEQ ID NO: 10 and SEQ ID NO: 11
Since specific bands were confirmed in all experiments using the oligonucleotides of the above, oligonucleotides of these sequences were added to trh1-RNA of Vibrio parahaemolyticus at 41 ° C.
Indicated that they were strongly bound. On the other hand, SEQ ID NO: 17
Did not bind to trh1-RNA. Together with the results of Example 3, it was shown that the oligonucleotides of SEQ ID NO: 10 and SEQ ID NO: 11 strongly bind to both trh1-RNA and trh2-RNA at 41 ° C. On the other hand, it was shown that the oligonucleotide of SEQ ID NO: 17 strongly binds only to trh2-RNA at 41 ° C. In addition, in the experiment using SEQ ID NO: 4, a specific band was confirmed, indicating that the oligonucleotide of SEQ ID NO: 4 strongly binds to trh2-RNA at 41 ° C. Together with the results of Example 1, it was shown that the oligonucleotide of SEQ ID NO: 4 strongly binds to both trh1-RNA and trh2-RNA at 41 ° C.

[0059]

As described above, the oligonucleotide of the present invention has a relatively low temperature and a constant temperature (35 ° C. to 50 ° C.) in which RNA forms an intramolecular structure and may inhibit the binding of primers and probes. C., preferably 41 ° C.) under conditions of trh1 and / or trh of Vibrio parahaemolyticus.
2 is an oligonucleotide that complementarily binds to RNA derived from 2. Therefore, it is possible to specifically bind the oligonucleotide without thermally denaturing the target RNA. As described above, the oligonucleotide of the present invention can be used for the heat-resistant hemotoxin-like hemotoxin gene (t
oligonucleotides for amplifying or detecting RNA derived from rh1 or trh2), ie, RN
It is useful as an oligonucleotide primer or an oligonucleotide probe used in the amplification method of A.

In addition to the above, it is clear that the oligonucleotide of the present invention is useful for amplifying or detecting trh1 or trh2. Oligonucleotides complementary to the specific oligonucleotides described above are also useful for amplifying double-stranded DNA by PCR or detecting cDNA obtained by reverse transcription of RNA.

The oligonucleotide of the present invention is not limited to the specifically described 20-mer oligonucleotide, but includes oligonucleotides consisting of at least 10 consecutive bases or more in these sequences. This is 10 me to ensure sufficient specificity of the primer or probe to the target nucleic acid.
It is clear from the fact that a base sequence of about r is sufficient.

[0062]

[Sequence list]

SEQUENCE LISTING <110> TOSOH Corporation <120> Oligonucleotide for detecting Vibrio parahaemolyticus <130> PA211-0104 <160> 17 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220><223> oligonucleotide <400> 1 ttttagtttc ataattaatc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220><trh1 and trh2 or oligonucleotides capable of specifically binding to RNA derived therefrom 223> oligonucleotides capable of specifically binding to trh1 and trh2, or RNA derived therefrom <400> 2 tatcgaagcc aatagcaaac 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220><223> oligonucleotide <400> 3 tccgaacctg gagaaggaaa 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220><trh1 and trh2, or oligonucleotides capable of specifically binding to RNA derived therefrom 223> trh1 and trh2 or RN derived therefrom Oligonucleotide <400> 4 tcgttttatg tttcggtttg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220><223> trh1 and trh2 derived from these Oligonucleotides capable of specifically binding to RNA <400> 5 ttaccgttat ataggcgctt 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220><223> trh1 and trh2 or derived from these Oligonucleotides capable of specifically binding to RNA <400> 6 tagcaaactg aatgcaaagt 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220><223> trh1 and trh2, or derived from these Oligonucleotides that can specifically bind to RNA <400> 7 cggaaccagg agaaggaaaa 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220><223> trh1 and trh2, or derived from these Specific binding to RNA Ligonucleotide <400> 8 cgattgaccg tatacatctt 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220><223> Specific binding to trh1 and trh2, or RNA derived therefrom Oligonucleotide <400> 9 cttggtttag gcttgttttc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220><223> Specific binding to trh1 and trh2, or RNA derived therefrom Oligonucleotide <400> 10 ggtattgatt cttcgctatc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220><223> Specific binding to trh1 and trh2, or RNA derived therefrom Oligonucleotide <400> 11 ataacaaaca tatgtccatt 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220><223> Oligonucleotide that can specifically bind to trh1 or RNA derived from the gene <400> 12 tgaagtcgtg aaaatagatt 2 0 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220><223> Oligonucleotide that can specifically bind to trh1 or RNA derived from the gene <400> 13 gaatagttct gatttaggct 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220><223> Oligonucleotide capable of specifically binding to trh1 or RNA derived from the gene <400> 14 atgatgattt attggaaata 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220><223> Oligonucleotide that can specifically bind to trh2 or RNA derived from the gene <400> 15 gatttagata ttgaaaatat 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220><223> Oligonucleotide that can specifically bind to trh2 or RNA derived from the gene <400> 16 gtgaccattg atgttgactg 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220><223> trh2 or RNA derived from the gene Oligonucleotides that can be differentially bound <400> 17 ttgtgaagac cgtagaagta 20

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the results obtained by using oligonucleotides designed for the intramolecular structure-free region of trh1-RNA.
It is a 6% PAGE electrophoresis photograph of the sample after performing the binding experiment to trh1-RNA at ° C. In the figure, lanes 1 and 11 are RNA markers (0.1 to 1.0).
K), lanes 2 to 9 correspond to SEQ ID NO: 1,
Lanes 10 and 11 relate to the oligonucleotide solutions of 2, 3, 4, 12, 5, 13 and 14;
-Standard of RNA (616mer).

FIG. 2 shows the results obtained by using oligonucleotides designed for the intramolecular structure-free region of trh1-RNA by using 41 oligonucleotides.
It is a 6% PAGE electrophoresis photograph of the sample after performing the binding experiment to trh2-RNA at ° C. In the figure, lanes 1 and 11 are RNA markers (0.1 to 1.0).
K), lanes 2 to 9 correspond to SEQ ID NO: 1,
Lanes 10 and 11 relate to oligonucleotide solutions of 2, 3, 12, 5, 13, 14 and 8, and lane 10 is trh2
-Standard of RNA (616mer).

FIG. 3 shows the results obtained by using oligonucleotides designed for the intramolecular structure-free region of trh2-RNA.
It is a 6% PAGE electrophoresis photograph of the sample after performing the binding experiment to trh2-RNA at ° C. In the figure, lanes 1 and 12 show RNA markers (0.1 to 1.0).
K), lanes 2 to 10 correspond to SEQ ID NO: 6,
Lanes 11 and 16 relate to oligonucleotide solutions of 15, 7, 8, 16, 17, 9, 10 and 11, and lane 11 is a sample of trh2-RNA (616mer).

FIG. 4 shows the results obtained by using oligonucleotides designed for the intramolecular structure-free region of trh2-RNA by using 41 oligonucleotides.
It is a 6% PAGE electrophoresis photograph of the sample after performing the binding experiment to trh1-RNA at ° C. In the figure, lanes 1 and 10 are RNA markers (0.1 to 1.0).
K), lanes 2 to 8 correspond to SEQ ID NOs: 6, 1
Lanes 9 are for the oligonucleotide solutions of 5, 7, 8, 16, 9 and 3;
(616mer).

FIG. 5: t at 41 ° C. using an oligonucleotide designed for the intramolecular structure-free region of trh2-RNA
6 of the sample after performing the binding experiment to rh1-RNA
% PAGE electrophoresis results in lanes 2 to 4, tr
trh2-RN at 41 ° C. using an oligonucleotide designed for the intramolecular structure-free region of h1-RNA
The result of electrophoresis of 6% PAGE of the sample after the binding experiment to A is shown in lane 6. In the figure, lanes 1 and 8 are RNA markers (0.1 to 1.0K), and lanes 2 to 6 are SEQ ID NOs: 17, 10, and 11, respectively.
Lane 4 is a sample of trh1-RNA (616mer).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) C12R 1:63) C12R 1:63)

Claims (5)

[Claims] 1. An oligonucleotide for detecting or amplifying thermotolerant hemotoxin-like hemotoxin genes (trh1 and trh2) of Vibrio parahaemolyticus, or RNA derived from these genes, comprising trh1 and trh2, or trh1 or trh2. An oligonucleotide consisting of at least 10 consecutive nucleotides or more in any of the sequences shown in SEQ ID NOS: 1 to 11 and capable of specifically binding to the RNA derived therefrom, or an oligonucleotide complementary to these oligonucleotides. 2. An oligonucleotide for detecting or amplifying a thermostable hemotoxin gene (trh1) of Vibrio parahaemolyticus or an RNA derived from said gene,
an oligonucleotide consisting of at least 10 consecutive nucleotides or more in any of the sequences shown in SEQ ID NOS: 12 to 14, which is capable of specifically binding to trh1 or RNA derived from the gene, or which is complementary to the oligonucleotide. Some oligonucleotides. 3. An oligonucleotide for detecting or amplifying a thermotolerant hemotoxin-like hemotoxin gene (trh2) of Vibrio parahaemolyticus or RNA derived from said gene,
Oligonucleotides consisting of at least 10 consecutive nucleotides or more in any of the sequences shown in SEQ ID NOS: 15 to 17, which are capable of binding specifically to trh2 or RNA derived from the gene, or complementary to those oligonucleotides At the oligonucleotide. 4. An oligonucleotide primer for a DNA elongation reaction, comprising the oligonucleotide according to any one of claims 1 to 3. 5. An oligonucleotide probe, wherein a part of the oligonucleotide according to claim 1 is modified or labeled with a detectable labeling substance.