JP2002281972A - Oligonucleotide for detecting salmonella and method for detecting salmonella - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an oligonucleotide, or the like, capable of complementarily binding to an intramolecular structure-free region of Salmonella toxin gene mRNA. SOLUTION: This invention provides oligonucleotides for detecting Salmonella toxin genes invA mRNA and stn mRNA, which oligonucleotides specifically bind to invA mRNA or stn mRNA at a relatively low constant temperature (for example, 41 deg.C), and a process for amplifying Salmonella toxin gene invA mRNA or stn mRNA and a method for detecting the above genes by using the oligonucleotides are provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to the detection of the mRNA of the toxin gene invA or stn (hereinafter sometimes referred to as target RNA in the present specification) of Salmonella spp., Which is generally known as a causative organism of food poisoning. And a detection method using the same.

[0002]

2. Description of the Related Art In clinical tests, public health, food tests, and food poisoning tests, detection and identification of Salmonella spp. Have conventionally been carried out by directly applying food or patient feces to a culture medium or culturing the culture using an enrichment medium. After culturing, a method of culturing in a separation medium is used.

[0003] Such a culturing method requires a culturing time of 18 hours or more, and thus lacks rapidity. In recent years, in order to realize rapid detection, detection methods based on gene amplification methods such as PCR have been developed.However, when DNA is to be detected, DNA of dead bacteria contained in sterilized foods and the like can be detected. It may be amplified and determined to be positive. In addition, in the detection by electrophoresis after amplification, which is generally performed in a PCR method, a negative sample may be contaminated by scattering of an amplification product, and a false positive may occur.

Since RNA is rarely present in dead bacteria, it is used to convert RNA into DN by reverse transcription.
A, and then perform PCR (RT-PC
R method) has also been used to detect RNA.
However, the originally existing DNA is also amplified together with the RNA, so that the dead bacterial DNA is amplified as described above, and may be determined to be positive. In order to avoid this, an operation for removing the originally existing DNA is required, and the operation is eventually complicated and lacks in speed.

[0005] The NASBA method and the 3SR method for amplifying a specific sequence of RNA by reverse transcriptase and RNA polymerase are known (for example, the NASBA method is described in Patent No. 265).
No. 0159, the 3SR method is disclosed in European Patent No. 3739.
No. 60). This method uses a specific sequence as a template, a primer containing a promoter sequence, a reverse transcriptase,
And ribonuclease H to synthesize a double-stranded DNA containing a promoter sequence, use the double-stranded DNA as a template to synthesize RNA containing a specific sequence by RNA polymerase, and the RNA continues to have the same promoter sequence as described above. It performs a chain reaction that serves as a template for double-stranded DNA synthesis. The NASBA method, the 3SR method, and the like are considered to be suitable methods for automation because they can amplify only a specific sequence at a constant temperature and can amplify at a constant temperature.

[0006]

In the above-described RNA amplification methods such as the NASBA method and the 3SR method, the amplification reaction is performed at a relatively low temperature (for example, 41 ° C.). May inhibit the binding of, and reduce the reaction efficiency. Therefore, prior to the amplification reaction, an operation for heat denaturing the RNA (for example, heat denaturation at 65 ° C.) to break its internal structure and improve the binding efficiency of the primer is required, resulting in a simple operation There is a problem that performance and speed are impaired. Further, if the electrophoresis method is used for detection after the amplification reaction, there is a problem that the problem of false positive due to scattering of the amplification product as described above cannot be avoided.

Accordingly, an object of the present invention is to provide an oligonucleotide capable of complementary binding to a region free of intramolecular structure of Salmonella toxin gene mRNA. That is, the present invention provides an oligonucleotide for amplifying and detecting Salmonella toxin gene mRNA at a relatively low temperature (35 ° C. to 50 ° C.) and capable of binding to its intramolecular structure-free region, and using such an oligonucleotide. And amplifying a specific sequence of a target RNA to provide a simple, rapid and highly sensitive detection method for clinical tests, food tests, food poisoning tests, and the like.

[0008]

Means for Solving the Problems The invention of claim 1 made to achieve the above object is an oligonucleotide for detecting salmonella toxin gene invA mRNA, which is specific for salmonella gene invA mRNA. And an oligonucleotide consisting of at least 10 consecutive nucleotides in any of the sequences shown in SEQ ID NOS: 1 to 12 that can bind to

In order to achieve the above object, the invention of claim 2 of the present invention provides a salmonella toxin gene stnm
An oligonucleotide for detecting RNA,
An oligonucleotide comprising at least 10 consecutive nucleotides or more in any of the sequences shown in SEQ ID NOS: 13 to 18, which can specifically bind to the Salmonella toxin gene stn mRNA.

[0010] In order to achieve the above object, the invention of claim 3 of the present invention provides a method using a specific sequence of the Salmonella gene invA mRNA present in a sample as a template.
CDNA is synthesized by dependent DNA polymerase, RNA in the RNA / DNA hybrid is degraded by ribonuclease H to generate single-stranded DNA, and the single-stranded DNA is used as a template to perform DNA-dependent DNA polymerase. 2 having a promoter sequence capable of transcribing an RNA consisting of a specific sequence or a sequence complementary to the specific sequence
Producing a single-stranded DNA, and the double-stranded DNA producing an RNA transcript in the presence of the RNA polymerase;
In any one of the sequences shown in SEQ ID NOs: 1 to 12, which can specifically bind to the Salmonella gene invA mRNA in the RNA amplification step in which the transcript subsequently serves as a template for cDNA synthesis by the RNA-dependent DNA polymerase. A first oligonucleotide consisting of at least 10 consecutive nucleotides or more;
And the amplified Salmonella gene invA consisting of at least 10 consecutive nucleotides or more in any of the sequences shown in
A second oligonucleotide having a sequence homologous to a part of the mRNA sequence (here, either one of the first and second oligonucleotides contains an RNA polymerase promoter sequence on its 5 'side). This is the step of amplifying the Salmonella gene invA mRNA.

[0011] Further, the invention of claim 4 of the present invention made to achieve the above object provides a method of synthesizing cDNA by RNA-dependent DNA polymerase using a specific sequence of the salmonella gene stn mRNA present in a sample as a template, The RNA in the RNA / DNA hybrid is degraded by Nuclease H to produce single-stranded DNA,
A double-stranded DNA having a promoter sequence capable of transcribing RNA comprising the specific sequence or a sequence complementary to the specific sequence is generated by DNA-dependent DNA polymerase using NA as a template, and the double-stranded DNA is A sequence capable of specifically binding to the Salmonella gene stn mRNA in an RNA amplification step in which an RNA transcript is generated in the presence of polymerase and the RNA transcript subsequently serves as a template for cDNA synthesis by the RNA-dependent DNA polymerase. A first oligonucleotide consisting of at least 10 consecutive nucleotides or more in any of the sequences shown in Nos. 13 to 18 and a first oligonucleotide consisting of at least 10 consecutive nucleotides or more in any of the sequences shown in SEQ ID Nos. 24 to 27 , Amplified Salmonella gene stnmR
A second oligonucleotide having a sequence homologous to a part of the NA sequence (here, one of the first and second oligonucleotides contains a promoter sequence of RNA polymerase on its 5 'side). This is the step of amplifying the Salmonella gene stn mRNA.

The invention of claim 5 of the present application is directed to the amplification step according to claim 3 or 4, wherein the amplification step is capable of specifically binding to an RNA transcript generated by amplification, and is labeled with an intercalating fluorescent dye. A detection method characterized in that the method is carried out in the presence of a labeled oligonucleotide probe, and the change in the fluorescence property of the reaction solution is measured (wherein the labeled oligonucleotide is the first oligonucleotide and the second oligonucleotide). Is a different array). The invention according to claim 6 of the present application is directed to the invention according to claim 5, wherein the probe is designed so as to complementarily bind to at least a part of the sequence of the RNA transcript, and is compared with a case where no complex is formed. It is characterized in that the fluorescence characteristics change. The invention of claim 7 of the present application is directed to the invention of claim 6, wherein the invA mRNA detection probe comprises at least 10 or more consecutive nucleotides in the sequence shown in SEQ ID NO: 28 or its complementary sequence. The invention of claim 8 of the present application is directed to the invention of claim 6, wherein the stn mRNA detection probe comprises at least 10 consecutive nucleotides or more in the sequence shown in SEQ ID NO: 29 or its complementary sequence. . Less than,
The present invention will be described in detail.

The salmonella toxin gene invA of the present invention
at least 10 contiguous sequences of any of the sequences set forth in SEQ ID NOs: 1-12 that are capable of specifically binding to mRNA.
Oligonucleotides consisting of bases or more, and oligonucleotides consisting of at least continuous 10 bases or more of any of the sequences shown in SEQ ID NOs: 13 to 18, which can specifically bind to Salmonella toxin gene stn mRNA, are relatively Low temperature and constant temperature (35 ℃ -50 ℃)
Thus, they have the characteristic that they can specifically bind to portions of the target RNA that do not take a three-dimensional structure. As a result, the oligonucleotide of the present invention is useful as a primer or the like for performing a nucleic acid amplification step such as a PCR method, a NASBA method, or a 3SR method on a target RNA. Since the oligonucleotide of the present invention can specifically bind to the target RNA at a relatively low temperature and a constant temperature (35 ° C. to 50 ° C.), the amplification step is performed in the above temperature range and at a constant temperature (eg, 41 ° C.). Can be used in the target RNA amplification step by the NASBA method or the 3SR method.
The effect of eliminating the need for heat denaturation of A is achieved.

The present invention also provides a nucleic acid amplification step for amplifying a specific sequence of a target RNA, and a method for detecting an RNA transcript generated by the nucleic acid amplification step. For example, in the amplification step in the NASBA method, using a specific sequence of RNA present in a sample as a template, DNA is synthesized by RNA-dependent DNA polymerase, and RNA-DNA hybrid RNA is synthesized by ribonuclease H.
To produce a single-stranded DNA, and using the single-stranded DNA as a template, an RN comprising the specific sequence or a sequence complementary to the specific sequence by DNA-dependent DNA polymerase.
Double-stranded DNA having a promoter sequence capable of transcribing A
And the double-stranded DNA produces an RNA transcript in the presence of the RNA polymerase, and the RNA transcript is subsequently c-linked by the RNA-dependent DNA polymerase.
Although it is a step that serves as a template for DNA synthesis, in the step of amplifying the Salmonella toxin gene invA mRNA provided by the present invention, at least one of the sequences shown in SEQ ID NOS: 1 to 12 that can specifically bind to the mRNA is used. A first oligonucleotide consisting of at least 10 contiguous bases and a portion of the mRNA sequence to be amplified consisting of at least 10 contiguous bases of any of the sequences shown in SEQ ID NOs: 19 to 23, It is characterized in that a second oligonucleotide having a sequence (here, either the first or the second oligonucleotide contains a promoter sequence of RNA polymerase on the 5 ′ side thereof) is used.

[0015] In the step of amplifying the salmonella toxin gene stn mRNA provided by the present invention, if at least 10 consecutive nucleotides of any of the sequences shown in SEQ ID NOS: 13 to 18 that can specifically bind to the mRNA are used. A first oligonucleotide consisting of SEQ ID NOS: 24 to 2
A second oligonucleotide having at least 10 consecutive bases or more of any of the sequences shown in 7 and having a sequence homologous to a part of the mRNA sequence to be amplified (here, the first or second oligonucleotide) One of them is
(Including a promoter sequence of RNA polymerase on its 5 ′ side).

In the above-described amplification step of the present invention,
The RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and ribonuclease H to be used are not particularly limited.
Although various kinds of enzymes may be used, it is particularly preferable to use AMV reverse transcriptase having all of the aforementioned enzyme activities. In the amplification step of the present invention, the RNA polymerase used is not particularly limited, but it is preferable to use T7 phage RNA polymerase or SP6 phage RNA polymerase.

In the above amplification step, an oligonucleotide complementary to a region overlapping (1 to 10 bases) and adjacent to the 5 'terminal region of each specific sequence in the target RNA sequence is added, and ribonuclease is added. By cutting the target RNA at the 5 'end region of the specific sequence by H activity and using it as a template for the initial stage of nucleic acid amplification, even when the specific sequence is not located at the 5' end, it can also be amplified. . For this cleavage, for example, the Salmonella toxin gene invA mRN
In the case of A, the oligonucleotides of SEQ ID NOs: 1 to 12,
In the case of the Salmonella toxin gene stn mRNA, oligonucleotides of SEQ ID NOs: 13 to 18 (however, oligonucleotides other than those used as the first oligonucleotide in the amplification step) may be used. It is preferable that the 3'-hydroxyl group of the oligonucleotide for cleavage is chemically modified (for example, aminated) in order to suppress the extension reaction from the 3'-terminal.

The detection method provided by the present invention is characterized in that the amplification step as described above is performed in the presence of an oligonucleotide probe labeled with an intercalating fluorescent dye, and the change in the fluorescence characteristics of the reaction solution is measured. And Examples of the oligonucleotide probe include those in which an intercalating fluorescent dye is bonded to phosphorus in an oligonucleotide via a linker. This probe has the characteristic that when it forms a double strand with the amplification product, the intercalator portion intercalates into the double-stranded portion and the fluorescence characteristics change, so that separation analysis is not necessary (Ishiguro, T. et al., Supra). (1996) Nuc
leic Acids Res. 24 (24) 4992
-4997).

The sequence of the probe is not particularly limited as long as it has a sequence complementary to at least a part of the amplified product. In the case of the Salmonella toxin gene invA mRNA, at least the sequence shown in SEQ ID NO: 28 Done 1
A sequence consisting of 0 bases is preferable, and in the case of Salmonella toxin gene stn mRNA, a sequence consisting of at least 10 consecutive bases of the sequence shown in SEQ ID NO: 29 is preferable.
In addition, in order to suppress the extension reaction using the probe as a primer, the hydroxyl group at the 3 ′ end of the oligonucleotide probe is preferably chemically modified (for example, glycolic acid is added).

By performing the amplification step in the presence of the above-described probe, the Salmonella toxin genes invA and s
It becomes possible to amplify and detect RNA consisting of a specific sequence in the tn mRNA or an RNA consisting of a sequence complementary to the specific sequence in one tube at a constant temperature and one step, and automation becomes easy. .

[0021]

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 [0022] For Salmonella toxin gene invA mRNA,
Oligonucleotides that specifically bind at 1 ° C were selected. (1) Nucleotide sequence of Salmonella toxin gene invA (Ga
lan, J .; E. FIG. Et al. Bacteriol. , 17
4, 4338-4349 (1992), GenBa, USA
NK accession number M90846) base numbers 104 to 205
In the region No. 2, a nucleotide sequence of the above-mentioned nucleotide No. 104 to which a promoter sequence of T7 RNA polymerase was added at the 5 ′ end.
PCR was carried out using a forward primer having a sequence homologous to SEQ ID NOs. To 122 and a reverse primer having a sequence complementary to the aforementioned base numbers 2029 to 2052. (2) Using the above PCR product as a template, a standard RN is obtained by a transcription reaction using T7 RNA polymerase (manufactured by Takara Shuzo Co., Ltd.).
A was adjusted. Thereafter, the PCR product of the template was decomposed by DNA polymerase (manufactured by Takara Shuzo Co., Ltd.),
Standard RNA was purified using MA SPIN 100 (trade name, manufactured by Toyobo Co., Ltd.). (3) After quantifying the standard RNA by ultraviolet absorption at 260 nm, an RNA diluent (10 mM Tris-HCl (pH
8.0), 0.1 mM EDTA, 1 mM DTT,
It diluted so that it might become 0.45 pmol / microliter using 0.5U / microliter RNase Inhibitor. (4) 9.0 μl of a reaction solution having the following composition was placed in a PCR tube (volume: 0.5 ml: Gene Amp Thin-W).
alled Reaction Tubes (product name,
PerkinElmer).

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 0.9 μM Standard RNA 2.0 μM Oligonucleotide (Oligo-1): SEQ ID NO: 1 (oligo-2): SEQ ID NO: 2 (oligo-3): SEQ ID NO: 3 (oligo-4): SEQ ID NO: 4 (oligo- 5): SEQ ID NO: 5 (oligo-6): SEQ ID NO: 6 (oligo-7): SEQ ID NO: 7 (oligo-8): SEQ ID NO: 8 (oligo-9): SEQ ID NO: 9 (oligo-10): SEQ ID NO: 10 (Oligo-11): SEQ ID NO: 11 (Oligo-12): SEQ ID NO: 12 Distilled water for volume adjustment (5) After keeping the above reaction solution at 41 ° C. for 5 minutes, 0.1 U
/ Μl of RNase H (manufactured by Takara Shuzo Co., Ltd.) was added thereto (RNase H is a DNA / RNA double-stranded R
It is an enzyme that cleaves NA). (6) Subsequently, the PCR tube was kept at 41 ° C. for 15 minutes. (7) Urea-denatured polyacrylamide gel (acrylamide concentration: 6%, urea: 7M) electrophoresis was performed to confirm the cleavage fragments after the reaction. Staining after electrophoresis is SYB
R Green II (trade name, manufactured by Takara Shuzo Co., Ltd.). When the oligonucleotide binds to a specific sequence of the standard RNA (target RNA), DNase is generated by RNaseH.
The A / RNA double-stranded RNA is cleaved and a specific band is observed.

The results of the electrophoresis are shown in FIG. If the oligonucleotide specifically binds to the standard RNA, the standard RNA is degraded in that region, resulting in a degradation product of a particular length. Table 1 shows the position of the oligonucleotide when it specifically bound to the standard RNA and the expected chain length of the band. For oligo-1 to oligo-12, cleavage at the expected position was confirmed. From the above, these oligonucleotides can be used as standard RNAs at 41 ° C. and in a constant state.
That is, it was shown that it strongly binds to the Salmonella toxin gene invA mRNA.

[0025]

[Table 1] Example 2 An RNA amplification reaction was performed using an oligonucleotide probe that specifically binds to the Salmonella toxin gene invA. (1) The above Salmonella toxin gene invA mRNA was diluted with an RNA diluent (10 mM Tris-HCl (pH 8.
0) 1 mM EDTA, 0.5 U / μl RNase
Inhibitor (manufactured by Takara Shuzo Co., Ltd.), 5 mM D
Using TT), the mixture was diluted to 10 ⁴ copies / 5 μl. For the control (negative) test group 0, only the diluent was used. (2) 20.8 μl of a reaction solution having the following composition was placed in a 0.5 ml PCR tube (Gene Amp Thin-Wal).
Dispensed into red Reaction Tubes (trade name, manufactured by PerkinElmer),
1 was added.

Composition of reaction solution (each concentration is 30
Concentration in μl) 60 mM Tris-HCl buffer (pH 8.6) 13 mM Magnesium chloride 90 mM Potassium chloride 39 U RNase Inhibitor 1 mM DTT 0.25 mM dATP, dCTP, dGTP, dT
TP 3.6 mM ITP 3.0 mM ATP, CTP, GTP, UTP 0.16 μM first oligonucleotide 1.0 μM second oligonucleotide 1.0 μM third oligonucleotide 13% DMSO Distilled water for volume adjustment ( 3) As described below, an RNA amplification reaction was performed using oligonucleotides having the sequences shown in Table 2 as first, second, and third oligonucleotides. (4) A solution was prepared in (2) so that the combination of the first, second, and third oligonucleotides became the combination shown in Table 2. (5) After keeping the above reaction solution at 41 ° C. for 5 minutes, 4.2 μl of an enzyme solution having the following composition was added.

Composition of enzyme solution (each concentration is 30
(concentration in μl) 1.7% sorbitol 3 μg bovine serum albumin 142 U T7 RNA polymerase (manufactured by Gibco) 8 U AMV reverse transcriptase (manufactured by Takara Shuzo Co., Ltd.) Distilled water for volume adjustment (6) Subsequently, keep the PCR tube at 41 ° C. for 30 minutes. did. (7) An agarose gel (agarose concentration: 4%) electrophoresis was performed to confirm the RNA amplification portion after the reaction.
Staining after electrophoresis was performed using SYBR Green II (trade name, manufactured by Takara Shuzo Co., Ltd.). When the oligonucleotide binds to the specific site of the target RNA, the RNA between the second and third oligonucleotides is amplified,
Specific bands are observed.

FIG. 2 shows the results of the electrophoresis. The chain length of the specific band amplified in this reaction is as shown in Table 2. From this, in the RNA amplification reaction using the combinations of oligonucleotides shown in Table 2, specific bands could be confirmed in any of the combinations, indicating that these are effective in detecting the target RNA.

[0029]

[Table 2] Table 2 shows the combinations of the first, second, and third oligonucleotides used in this example, and the chain lengths of specific bands amplified when an RNA amplification reaction was performed using the combinations. In the base sequence of the first oligonucleotide, the 3′-terminal hydroxyl group is aminated. "A" at the first 5 'end of the base sequence of the second oligonucleotide
The portion from to the 22nd "A" is the T7 promoter sequence, and the portion from the 23rd "G" to the 28th "A" is the enhancer sequence.

First oligonucleotide 2S (SEQ ID NO: 2) 3S (SEQ ID NO: 3) 5S (SEQ ID NO: 5) Second oligonucleotide 2F5 (SEQ ID NO: 19) 3F5 (SEQ ID NO: 20) 5F5 (SEQ ID NO: 21) 2F10 (Sequence) No. 22) 3F10 (SEQ ID NO: 23) Third oligonucleotide 4R (SEQ ID NO: 4) 5R (SEQ ID NO: 5) 8R (SEQ ID NO: 8) Example 3 Salmonella toxin gene invA mRNA using a combination of oligonucleotides according to the present invention At various initial copy numbers. (1) The same standard RNA as in Example 1 was prepared using an RNA diluent (1
0 mM Tris-HCl (pH 8.0), 1 mM ED
TA, 0.5 U / μl RNase Inhibito
r (Takara Shuzo Co., Ltd.), 5 mM DTT,
⁵ was diluted to a copy / 5 [mu] l to 10 ² copies / 5 [mu] l. In the control test group (negative), only the diluent was used. (2) 20.8 μl of a reaction solution having the following composition was added to a 0.5 ml PCR tube (Gene Amp Thin-W).
alled Reaction Tubes (product name,
(Perkin Elmer), and 5 μl of the above RNA sample was added thereto.

Composition of reaction solution (each concentration is 30
60 mM Tris-HCl buffer (pH 8.6) 17 mM Magnesium chloride 90 mM Potassium chloride 39 U RNase Inhibitor 1 mM DTT 0.25 mM each dATP, dCTP, dGTP, dT
TP 3.6 mM ITP 3.0 mM ATP, CTP, GTP, UTP 0.16 μM first oligonucleotide (3S in Table 2)
(SEQ ID NO: 3) The hydroxyl group at the 3 ′ end is aminated. ) 1.0 μM of the second oligonucleotide (3F10 in Table 2)
(SEQ ID NO: 23)) 1.0 μM third oligonucleotide (4R in Table 2 (SEQ ID NO: 4)) Oligonucleotide labeled with 25 nM intercalating fluorescent dye (SEQ ID NO: 28, where the intercalating fluorescent dye is , "A" at the 13th position and 14th "A" at the 5 'end of the oligonucleotide of SEQ ID NO: 28
Is labeled between. Further, the hydroxyl group at the 3 ′ end is modified with a glycol group. 13% distilled water for DMSO volume adjustment (3) The above reaction solution was kept at 41 ° C. for 5 minutes, and then 4.2 μl of an enzyme solution having the following composition and previously kept at 41 ° C. for 2 minutes was added.

Composition of enzyme solution (each concentration is 30
(concentration in μl) 1.7% sorbitol 3 μg bovine serum albumin 142U T7 RNA polymerase (manufactured by Gibco) 8U AMV reverse transcriptase (manufactured by Takara Shuzo) distilled water for volume adjustment (4) Temperature at which PCR tube can be directly measured Using a fluorescence spectrophotometer with an adjustable function, keep the temperature at 41 ° C,
The reaction solution was measured over time at an excitation wavelength of 470 nm and a fluorescence wavelength of 510 nm. Assuming that the time of enzyme addition is 0 minutes,
FIG. 3 (upper) shows the change over time in the fluorescence intensity ratio of the sample (the fluorescence intensity value at a predetermined time / the fluorescence intensity value of the background). In addition, the logarithmic value of the initial RNA amount and the detection time (the value obtained by adding three times the standard deviation to the average value of the negative fluorescence intensity ratio: 1.2
FIG. 3 (lower) shows the result of the relationship with the time (before reaching). The initial RNA amount is 10 ¹ copy / 30 μl to 10 ⁵ copies / 30 μl.

[0033] From FIG. 3, 10 ² copies were detected in about 13 minutes. A fluorescence profile and a calibration curve depending on the initial concentration of the labeled RNA are obtained, and the target RN present in the unknown sample is obtained.
It was shown that quantification of A was possible. As described above, it was shown that the present method enables rapid and highly sensitive detection of invA mRNA.

Example 4 For the Salmonella toxin gene stn mRNA, 41
Oligonucleotides that specifically bind at C were selected. (1) Nucleotide sequence of Salmonella toxin gene stn (Cho
prA. K. Et al., Microb. Patho
g. , 16, 85-98 (1994), U.S. GenBa.
NK accession number L16014) base numbers 346 to 109
In the region of No. 346, the base sequence No. 346 was added with a promoter sequence of T7 RNA polymerase at the 5 ′ end.
PCR was carried out using a forward primer having a sequence homologous to 369 to 369 and a reverse primer having a sequence complementary to bases 1076 to 1092 described above. (2) Using the above PCR product as a template, a standard RN is obtained by a transcription reaction using T7 RNA polymerase (manufactured by Takara Shuzo Co., Ltd.).
A was adjusted. Thereafter, the PCR product of the template was decomposed by DNA polymerase (manufactured by Takara Shuzo Co., Ltd.),
Standard RNA was purified using MA SPIN 100 (trade name, manufactured by Toyobo Co., Ltd.). (3) After quantifying the standard RNA by ultraviolet absorption at 260 nm, an RNA diluent (10 mM Tris-HCl (p
H8.0), 0.1 mM EDTA, 1 mM DTT,
It diluted so that it might become 0.45 pmol / microliter using 0.5U / microliter RNase Inhibitor. (4) 9.0 μl of a reaction solution having the following composition was placed in a PCR tube (volume: 0.5 ml: Gene Amp Thin-W).
alled Reaction Tubes (product name,
PerkinElmer).

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 0.9 μM Standard RNA 2.0 μM Oligonucleotide (Oligo-13): SEQ ID NO: 13 (oligo-14): SEQ ID NO: 14 (oligo-15): SEQ ID NO: 15 (oligo-16): SEQ ID NO: 16 (oligo- 17): SEQ ID NO: 17 (oligo-18): SEQ ID NO: 18 Distilled water for volume adjustment (5) After keeping the above reaction solution at 41 ° C. for 5 minutes, 0.1
1 μl of U / μl RNase H (manufactured by Takara Shuzo) was added (RNase H is a DNA / RNA double-stranded R
It is an enzyme that cleaves NA). (6) Subsequently, the PCR tube was kept at 41 ° C. for 15 minutes. (7) Urea-denatured polyacrylamide gel (acrylamide concentration: 6%, urea: 7M) electrophoresis was performed to confirm the cleavage fragments after the reaction. Staining after electrophoresis is SYB
R Green II (trade name, manufactured by Takara Shuzo Co., Ltd.). When the oligonucleotide binds to a specific site of the target RNA (target RNA), DNase is generated by RNaseH.
A / RNA Double-stranded RNA is cleaved, and a specific band is observed.

FIG. 4 shows the results of the electrophoresis. If the oligonucleotide specifically binds to the standard RNA, the standard RNA is degraded in that region, resulting in a degradation product of a particular length. Table 3 shows the position of the oligonucleotide when it specifically bound to the standard RNA and the expected chain length of the band. For oligo-13 to oligo-18, cleavage at the expected position was confirmed. From the above, it was shown that these oligonucleotides strongly bind to the Salmonella toxin gene stn mRNA at a constant temperature of 41 ° C.

[0037]

[Table 3] Example 5 Detection of various initial copy numbers of the Salmonella toxin gene stn mRNA was performed using the oligonucleotide combination according to the present invention. (1) The same salmonella toxin gene stn as in Example 4
mRNA standard RNA was diluted with RNA diluent (10 mM Tri).
s-HCl (pH 8.0), 1 mM EDTA, 0.5 U
/ Μl RNase Inhibitor (manufactured by Takara Shuzo Co., Ltd., 5 mM DTT) and 10 ⁴ copies / 5
Diluted to make μl. Control test plot (negative)
Only the diluent was used. (2) 20.8 μl of a reaction solution having the following composition was added to a 0.5 ml PCR tube (Gene Amp Thin-W).
alled Reaction Tubes (product name,
(Perkin Elmer), and 5 μl of the above RNA sample was added thereto.

Composition of reaction solution (each concentration is 30
60 mM Tris-HCl buffer (pH 8.6) 17 mM Magnesium chloride 90 mM Potassium chloride 39 U RNase Inhibitor 1 mM DTT 0.25 mM each dATP, dCTP, dGTP, dT
TP 3.6 mM ITP 3.0 mM ATP, CTP, GTP, UTP 0.16 μM first oligonucleotide (combination in Table 4; hydroxyl group at 3 ′ end is aminated) 1.0 μM second oligo Nucleotides (combination in Table 4) 1.0 μM third oligonucleotide (combination in Table 4) Oligonucleotide labeled with 25 nM intercalating fluorescent dye (SEQ ID NO: 29, where the intercalating fluorescent dye is SEQ ID NO: 29) 12th "A" and 13th "A" at the 5 'end of the oligonucleotide
Is labeled between. Further, the hydroxyl group at the 3 ′ end is modified with a glycol group. 13% distilled water for DMSO volume adjustment (3) The above reaction solution was kept at 41 ° C. for 5 minutes, and then 4.2 μl of an enzyme solution having the following composition and previously kept at 41 ° C. for 2 minutes was added.

Composition of enzyme solution (each concentration is 30
(concentration in μl) 1.7% sorbitol 3 μg bovine serum albumin 142U T7 RNA polymerase (manufactured by Gibco) 8U AMV reverse transcriptase (manufactured by Takara Shuzo) distilled water for volume adjustment (4) Temperature at which PCR tube can be directly measured Using a fluorescence spectrophotometer with an adjustable function, keep the temperature at 41 ° C,
The reaction solution was measured over time at an excitation wavelength of 470 nm and a fluorescence wavelength of 510 nm.

FIG. 5 shows the time-dependent change of the fluorescence increase ratio of the sample (fluorescence intensity value at a predetermined time / background fluorescence intensity value), with the time at the time of addition of the enzyme being 0 minute.

[0041] From FIG. 5, the fluorescence increase ratio is the detection time period exceeding 1.2, ¹⁰⁴ copies were detected in about 11 to 15 minutes. From the above, stn mRN can be obtained by this method.
It was shown that rapid and highly sensitive detection of A was possible.

[0042]

[Table 4] Table 4 shows combinations of the first, second, and third oligonucleotides used in the present example, and the chain lengths of specific bands amplified when an RNA amplification reaction was performed using the combinations. In the base sequence of the first oligonucleotide, the 3′-terminal hydroxyl group is aminated. "A" at the first 5 'end of the base sequence of the second oligonucleotide
The portion from to the 22nd "A" is the T7 promoter sequence, and the portion from the 23rd "G" to the 28th "A" is the enhancer sequence.

First oligonucleotide B1S (SEQ ID NO: 13) B3S (SEQ ID NO: 14) Second oligonucleotide B1F5 (SEQ ID NO: 24) B3F5 (SEQ ID NO: 25) B1F10 (SEQ ID NO: 26) B3F10 (SEQ ID NO: 27) Third oligo Nucleotide B4R (SEQ ID NO: 15)

[0044]

As described above, the present invention provides an oligonucleotide that binds complementarily to the intramolecular structure-free region of the Salmonella toxin gene invA and stnmRNA, and a detection method using the same. The present invention also relates to the Salmonella toxin genes invA and stn.
An object of the present invention is to provide an oligonucleotide for detecting mRNA, that is, an oligonucleotide primer or an oligonucleotide probe used in a nucleic acid amplification method. Since the oligonucleotide provided by the present invention can specifically bind to a target RNA even at a relatively low temperature and a constant temperature, it is particularly suitable as a primer used in a target RNA amplification step.

The amplification step and the detection method provided by the present invention use an oligonucleotide which is preferable for the target RNA amplification step as described above. As a result, in performing the amplification process at a relatively low and constant temperature,
This achieves the effect that there is no need to heat denature the target RNA in advance.

[0046]

[Sequence List] SEQUENCE LISTING <110> Tosoh Corporation <120> Oligonucleotides for Salmonella detection and detection method <130> PA211-0678 <160> 29 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide capable of specifically binding to Salmonella toxin gene invA mRNA <400> 1 agacgactgg tactgatcga 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223 > Oligonucleotide capable of specifically binding to Salmonella toxin gene invA mRNA <400> 2 aggaaccgta aagctggctt 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Salmonella toxin gene invA mRNA <400> 3 taatgatgcc ggcaatagcg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Specific binding to Salmonella toxin gene invA mRNA Oligonucleotide <400> 4 atcaacaatg c ggggatctg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> that can specifically bind to salmonella toxin gene invA mRNA <400> 5 atttacgcgg gtcacgataa 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide capable of specifically binding to salmonella toxin gene invA mRNA <400> 6 ctgcgtcatg atattccgcc 20 <210> 7 <211> 20 <212 > DNA <213> Artificial Sequence <220> <223> Oligonucleotide capable of specifically binding to salmonella toxin gene invA mRNA <400> 7 ccgataaaat aacaaaaacc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide capable of specifically binding to Salmonella toxin gene invA mRNA <400> 8 tgcttcacgg aatttaaaat 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223 > Specific binding to Salmonella toxin gene invA mRNA Possible oligonucleotide <400> 9 tttgctggtt ttaggtttgg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400 capable of specifically binding to Salmonella toxin gene invA mRNA > 10 tttttcctca atactgagcg 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide capable of specifically binding to salmonella toxin gene invA mRNA <400> 11 ccgtaaattg ttcaacacgg 20 < 210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide capable of specifically binding to salmonella toxin gene invA mRNA <400> 12 gacttcatcg gaataattta 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide that can specifically bind to salmonella toxin gene stn mRNA <400> 13 aaggtgaaaa gtattgaggg 20 <210> 14 <211> 20 <212> DNA < 213> Artificial Sequence <220> <223> Monkey Oligonucleotide <400> 14 gatagcggga aagggatcgc 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Salmonella toxin gene stn mRNA Oligonucleotide capable of specific binding <400> 15 aggctgactc aggtgctgtt 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Specific binding to salmonella toxin gene stn mRNA Oligonucleotide <400> 16 atattattac tcactccctg 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 17 that can specifically bind to salmonella toxin gene stn mRNA ggggcatctg gcggcgggcg 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> that can specifically bind to the salmonella toxin gene stn mRNA <400> 18 atgaagcgta aagaaaagct 20 <210> 19 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Second oligonucleotide <400> 19 aattctaata cgactcacta tagggagatt cctttgacgg tgcgatga 48 <210> 20 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Second oligonucleotide <400> 20 aattctaata cgactcacta tagggagagg catcattatt atctttgt 48 <210> 21 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Second oligonucleotide <400> 21 aattctaata cgactcacta tagggagata aatggcgata cggataat 48 <210> 22 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Second oligonucleotide <400> 22 aattctaata cgactcacta tagggagata cggttccttt gacggtgc 48 <210> 23 <211 > 48 <212> DNA <213> Artificial Sequence <220> <223> Second oligonucleotide <400> 23 aattctaata cgactcacta tagggagaca ttattatctt tgtgaact 48 <210> 24 <211> 48 <212> DNA <213> Artificial Sequence < 220> <223> Second oligonucleotide <400> 24 aattctaata cgactcacta tagggagaac cttaatcgcg ccgccatg 48 <210> 25 < 211> 48 <212> DNA <213> Artificial Sequence <220> <223> Second oligonucleotide <400> 25 aattctaata cgactcacta tagggagact atcggtaaca gtgatgat 48 <210> 26 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Second oligonucleotide <400> 26 aattctaata cgactcacta tagggagatt ttcaccttaa tcgcgccg 48 <210> 27 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Second oligonucleotide < 400> 27 aattctaata cgactcacta tagggagatc ccgctatcgg taacagtg 48 <210> 28 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Detection probe for detecting salmonella toxin gene invA <400> 28 tcagcatggt ataagtagac agggcg 26 < 210> 29 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Detection probe for detecting salmonella toxin gene stn mRNA <400> 29 agcgtagagg caaaagaaag tgggac 26

[Brief description of the drawings]

FIG. 1 shows oligo-1 to oligo-1 obtained in Example 1.
2 is an electrophoresis photograph (black and white reversal) of urea-denatured 6% PAGE of a sample after performing a cutting experiment of a Salmonella toxin gene invA mRNA standard product at 41 ° C. using RNaseH and RNaseH.

FIG. 2 shows the Salmonella toxin gene in Example 2
Initial RNA amount of vA mRNA standard 10 ⁴ copies / 3
4% agarose gel electrophoresis photograph when an RNA amplification reaction was performed at 0 μl using the oligonucleotide probes of combinations (a) to (h) in Table 2. Lanes 1 and 2
Are combination (a), lanes 3 and 4 are combination (b), lanes 5.6 are combination (c), lane 7
8 is combination (d), lanes 9 and 10 are combination (e), lanes 11 and 12 are combination (f), lanes 13 and 14 are combination (g), and lanes 15 and 16 are combination (h). And lane 2,
4, 6, 8, 10, 12, 14, and 16 are controls (using only a diluent instead of the RNA sample). A specific band could be confirmed using any of the combinations.

FIG. 3 is a graph showing the relationship between the reaction time and the fluorescence intensity ratio that increases with the production of RNA when the initial RNA amount of the salmonella toxin gene invA mRNA standard prepared in Example 3 is 10 ¹ copy / 30 μl to 10 ⁵ copies / 30 μl. It is a graph (upper) and a calibration curve (lower) obtained between the logarithmic value of the initial RNA amount and the detection time (time when the fluorescence intensity ratio becomes 1.2). An initial copy number of 10 ² copies / 30 μl of RNA could be detected in about 13 minutes of the reaction, indicating a correlation between the initial RNA amount and the detection time.

FIG. 4 shows the results obtained from oligo-13 to oligo-
18 is an electrophoretic photograph of urea-denatured 6% PAGE of a sample after performing a cleavage experiment of a standard sample of Salmonella toxin gene stn mRNA at 41 ° C. using RNase H and No. 18 (white inverted).

FIG. 5 is a graph showing the reaction time and the fluorescence intensity ratio that increases with the generation of RNA when the initial RNA amount of the salmonella toxin gene stn mRNA standard product in Example 5 was 10 ⁴ copies / 30 μl. With each primer combination, 10 ⁴ copies / 30 μl of RNA could be detected in about 11 to 15 minutes of the reaction.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (Reference) G01N 33/58 C12R 1:42 // (C12Q 1/68 C12N 15/00 A C12R 1:42) F-term (Reference) 2G045 AA28 CB21 DA12 DA14 FB02 FB07 FB12 GC15 4B024 AA13 BA80 CA01 CA09 CA12 HA12 4B063 QA01 QA18 QA19 QQ01 QQ06 QQ16 QQ18 QQ53 QR08 QR42 QR55 QR62 QS25 QS34 QS36 QX02

Claims (8)

[Claims] 1. A Salmonella toxin gene invA mRNA
An oligonucleotide comprising at least 10 consecutive nucleotides or more in any one of the sequences shown in SEQ ID NOS: 1 to 12, which can specifically bind to Salmonella gene invA mRNA. 2. An oligonucleotide for detecting the Salmonella toxin gene stn mRNA, which comprises at least one of the sequences shown in SEQ ID NOs: 13 to 18 which can specifically bind to the Salmonella toxin gene stn mRNA. Oligonucleotide comprising at least 10 bases. 3. A salmonella gene inv present in a sample.
A Using a specific sequence of mRNA as a template, RNA-dependent D
CDNA is synthesized by NA polymerase, and RNase in RNA / DNA hybrid is synthesized by ribonuclease H.
A is decomposed to produce a single-stranded DNA, and the single-stranded DNA is used as a template to perform R-consisting of the specific sequence or a sequence complementary to the specific sequence by DNA-dependent DNA polymerase.
Double-stranded DN having promoter sequence capable of transcribing NA
A in the RNA amplification step, wherein the double-stranded DNA produces an RNA transcript in the presence of RNA polymerase, and the RNA transcript subsequently serves as a template for cDNA synthesis by the RNA-dependent DNA polymerase. A first oligonucleotide consisting of at least 10 consecutive nucleotides or more in any of the sequences shown in SEQ ID NOS: 1 to 12 that can specifically bind to the Salmonella gene invA mRNA; Salmonella gene invA mRN to be amplified, comprising at least 10 consecutive bases in the sequence
A second oligonucleotide having a sequence homologous to a part of the A sequence (where either one of the first and second oligonucleotides contains a promoter sequence of RNA polymerase on its 5 'side). A step of amplifying Salmonella gene invA mRNA. 4. A salmonella gene stn present in a sample.
Using a specific sequence of mRNA as a template, RNA-dependent DN
CDNA synthesized by polymerase A and RNA in RNA / DNA hybrids by ribonuclease H
To produce a single-stranded DNA, and using the single-stranded DNA as a template, an RN comprising the specific sequence or a sequence complementary to the specific sequence by DNA-dependent DNA polymerase.
Double-stranded DNA having a promoter sequence capable of transcribing A
And the double-stranded DNA produces an RNA transcript in the presence of the RNA polymerase, and the RNA transcript is subsequently c-linked by the RNA-dependent DNA polymerase.
In the RNA amplification step as a template for DNA synthesis, a first sequence consisting of at least 10 consecutive nucleotides or more in any of the sequences shown in SEQ ID NOS: 13 to 18 that can specifically bind to the Salmonella gene stn mRNA Salmonella gene stn mRNA to be amplified, comprising an oligonucleotide and at least 10 consecutive nucleotides in any of the sequences shown in SEQ ID NOS: 24 to 27
Using a second oligonucleotide having a sequence homologous to a part of the sequence (where either one of the first and second oligonucleotides includes an RNA polymerase promoter sequence on its 5 'side). The step of amplifying the Salmonella gene stn mRNA. 5. An oligonucleotide probe capable of specifically binding to an RNA transcript generated by amplification in the amplification step according to claim 3 or 4, and labeled with an intercalating fluorescent dye. A detection method characterized in that the method is carried out in the presence and measures a change in the fluorescence property of the reaction solution (wherein the labeled oligonucleotide has a different sequence from the first oligonucleotide and the second oligonucleotide). is there). 6. The probe is designed so as to complementarily bind to at least a part of the sequence of an RNA transcript, and has a change in fluorescence characteristics as compared to a case where no complex is formed. The detection method according to claim 5, wherein 7. The probe for detecting invA mRNA,
The detection method according to claim 6, comprising at least 10 consecutive nucleotides or more in the sequence shown in SEQ ID NO: 28 or its complementary sequence. 8. The detection method according to claim 6, wherein the stn mRNA detection probe comprises at least 10 or more consecutive nucleotides in the sequence shown in SEQ ID NO: 29 or its complementary sequence.