JP2001340088A - Method for detecting toxin gene of vibrio parahaemolyticus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a combination of oligonucleotides useful for specifically amplifying and/or sensitively detecting and/or identifying an RNA derived from a tdh-related hemolysin gene (trh2) of Vibrio parahaemolyticus. SOLUTION: This method is a method for the detection utilizing an RNA- amplifying process of an RNA derived from a tdh-related hemolysin gene (trh2) of Vibrio parahamolyticus, wherein, the oligonucleotide shown by sequence 1 of the specification is used as a first primer and the oligonucleotide shown by sequence 2 of the specification is used as a second primer.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for detecting Vibrio parahaemolyticus in clinical tests, public health, food tests or food poisoning tests.

[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. This suggests that Vibrio parahaemolyticus and the Kanagawa phenomenon are deeply involved.

[0003] Subsequently, this Kanagawa phenomenon was caused by the thermostable toxic toxin of Vibrio parahaemolyticus.
ct hemolysin (tdh) has been found to be a phenomenon that occurs because it is released outside the cells, and tdh has attracted attention as a virulence factor of Vibrio parahaemolyticus.

[0004] More recently, hemolytic toxins having a nucleotide sequence similar to tdh and having a partial antigenicity (a thermotolerant hemotoxin-like hemolytic toxin) have been recently selected from strains which are pathogenic despite being a Kanagawa phenomenon-negative strain. TDH-related hemo
lysin: trh)) was also confirmed.

[0005]

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 these tdh and trh genes has been attempted, but it is difficult to obtain sufficient detection sensitivity in food inspection and the like. there were.

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, the emergence of a quick and highly sensitive detection method required for food inspection and the like is desired. Furthermore, in order to make inspection easier, it is also desired to develop an automated inspection device.

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 a specific sequence in the following (hereinafter, these are referred to as target nucleic acids) and then detect the amplified sequence.

[0008] 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 thermal 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 a 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. In addition, in order to amplify the specific sequence by the PCR method, an oligonucleotide having high specificity to the specific sequence is required. Further, in order to perform the detection and identification with high sensitivity, specificity to the target DNA is required. Are required. Furthermore, it is necessary to consider the optimal combination of those oligonucleotides.

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 3S
The R method and the like are known. In this method, a double-stranded DNA containing a promoter sequence is synthesized with respect to a specific sequence of a target RNA using a primer containing a promoter sequence, a reverse transcriptase, and ribonuclease H, and using the double-stranded DNA as a template, An RNA containing the specific sequence is synthesized by polymerase, and the RNA is subsequently subjected to a chain reaction as a template for the synthesis of a 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 RNA
May form an intramolecular structure, inhibit the binding of the primer, and reduce the reaction efficiency. Therefore,
Performing thermal denaturation of the target RNA before the amplification reaction destroys the internal structure of the target RNA and requires an operation for improving the binding efficiency of the primer.

Therefore, the present invention relates to an RN derived from a thermotolerant hemotoxin-like hemotoxin gene (trh2) of Vibrio parahaemolyticus.
It is an object of the present invention to provide a suitable combination of oligonucleotides useful for specifically amplifying A at a relatively low temperature (for example, 41 ° C.) and performing detection and identification with high sensitivity.

[0011]

Means for Solving the Problems According to the present invention, which has been made to achieve the above object, the present invention relates to a method for measuring the presence of t in a sample.
Using a specific sequence of RNA derived from rh2 as a template, a first primer having a sequence complementary to the specific sequence and a second primer having a sequence homologous to the specific sequence (here, the first or second primer One of the primers has a sequence in which the promoter sequence of RNA polymerase is added to the 5 ′ side), and forms an RNA-DNA duplex by generating cDNA by RNA-dependent DNA polymerase. RNA-DNA Double-strand RNA is degraded by Nuclease H to produce single-stranded DNA.
Producing a double-stranded DNA having a promoter sequence capable of transcribing the RNA sequence or RNA consisting of a sequence complementary to the RNA sequence by dependent DNA polymerase,
Then, the double-stranded DNA is converted to R in the presence of RNA polymerase.
Producing an NA transcript, wherein said RNA transcript is subsequently a single-stranded DNA by said RNA-dependent DNA polymerase
In a detection method using an RNA amplification step as a production template, an oligonucleotide of SEQ ID NO: 1 is used as a first primer, and an oligonucleotide of SEQ ID NO: 2 is used as a second primer.

The invention of claim 2 of the present application is directed to the invention of claim 1, wherein the first primer is an oligonucleotide consisting of at least 10 consecutive nucleotides or more in the sequence of SEQ ID NO: 1. The invention of claim 3 of the present application is directed to the invention of claim 1, wherein the second primer is an oligonucleotide consisting of at least 10 consecutive nucleotides or more in the sequence of SEQ ID NO: 2.
The invention of claim 4 of the present application is directed to the invention of claim 1,
Performing the RNA amplification step in the presence of an oligonucleotide probe labeled with an intercalating fluorescent dye, wherein the sequence of the probe is complementary to at least a portion of the RNA transcript, and Due to the complementary binding with the RNA transcript, the fluorescence characteristics are changed as compared with the case where no complex is formed.
This is a method for detecting a toxin gene of Vibrio parahaemolyticus, comprising measuring the fluorescence intensity of a reaction solution. Hereinafter, the present invention will be described in detail.

According to the present invention, a relatively low temperature and a constant temperature (3
(5 ° C. to 50 ° C., preferably 41 ° C.) to provide an oligonucleotide combination for detecting RNA from trh2 of Vibrio parahaemolyticus, ie trh2
By providing a combination of an oligonucleotide primer for amplification of RNA derived from and an oligonucleotide probe for detection, a simple, rapid and highly sensitive thermotolerant hemolytic toxin gene of Vibrio parahaemolyticus utilizing the same. The present invention provides a detection method and a detection kit for (trh2) for food inspection, food poisoning inspection, and the like.

In one embodiment of the present invention, the first primer (3 of the specific sequence of the target RNA) is used as a template with the specific sequence of the RNA derived from the thermotolerant hemotoxin-like hemotoxin gene (trh2) present in the sample. 'A sequence complementary to the terminal region)
Are complementary to each other and produce cDNA from the extension reaction by RNA-dependent DNA polymerase, whereby RNA-D
An RNA-DNA double-stranded RNA is degraded by ribonuclease H to form a single-stranded DNA. Thereafter, a second primer (having a sequence homologous to the 5′-end region of the target RNA and having a promoter sequence of RNA polymerase added to the 5′-end) complementarily binds to the single-stranded DNA, and is dependent on DNA. Double-stranded DNA having a promoter capable of transcribing RNA consisting of a sequence homologous to the target RNA sequence is produced by sex DNA polymerase. Then, in the presence of the RNA polymerase, the double-stranded DNA amplifies an RNA transcript having a sequence homologous to the target RNA. And this invention is characterized by using the oligonucleotide of SEQ ID NO: 1 as the first primer and the oligonucleotide of SEQ ID NO: 2 as the second primer. The first and second primers may be the full-length nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively, but use a combination of oligonucleotides consisting of at least 10 consecutive nucleotides or more in each sequence. Can also.

In one embodiment of the present invention, the target RN
A needs to be truncated at the 5 'end of the specific sequence. As a method for cleaving the target RNA in this manner, a target RNA is added by adding an oligonucleotide having a sequence complementary to a region overlapping and adjacent to the 5 ′ end of the specific sequence (cleavage oligonucleotide). Is preferably cleaved with ribonuclease H or the like. It is desirable to use a 3′-end of the cleavage oligonucleotide that has been treated so as not to function as an oligonucleotide primer, for example, an aminated one.

In one embodiment of the present invention, the amplification step is carried out in the presence of an oligonucleotide probe labeled with an intercalating fluorescent dye having a sequence complementary to at least a part of the sequence of the RNA transcript to be amplified. Is preferably performed. At this time, the probe is RN
The fluorescence characteristics change as compared with the case where no complex is formed due to the complementary binding with the A transcript, and the fluorescence intensity of the reaction solution may be measured. Furthermore, when a labeled oligonucleotide probe is allowed to coexist during the amplification step, modification such as addition of glycolic acid to the 3 ′ end of the probe is performed so that the probe does not function as a primer for the extension reaction. Especially desirable. As the oligonucleotide probe, use of an oligonucleotide consisting of at least 10 consecutive nucleotides or more of the sequence of SEQ ID NO: 6 can be exemplified.

In another aspect of the present invention, a specific sequence of trh2-derived RNA present in a sample is used as a template to form a first primer (a sequence complementary to a target RNA;
Complementary to the 5 ′ end of the RNA polymerase, a cDNA is formed from an extension reaction by the RNA-dependent DNA polymerase to form a double-stranded RNA-DNA, and then a ribonucleic acid is formed. Ace H degrades RNA-DNA double-stranded RNA to produce single-stranded DNA. Then, the single-stranded D
A double-stranded strand having a promoter capable of transcribing RNA comprising a sequence complementary to the target RNA sequence by DNA-dependent DNA polymerase, to which a second primer (a sequence homologous to the target RNA) complementarily binds to NA DNA
Generate Then, the double-stranded DNA generates an RNA transcript having a sequence complementary to the target RNA in the presence of RNA polymerase. Then, the second primer complementarily binds to the RNA transcript (a sequence complementary to the target RNA), generates cDNA by RNA-dependent DNA polymerase, and forms a double-stranded RNA-DNA. Then, RNA-D was prepared using ribonuclease H.
Decomposes NA double-stranded RNA to produce single-stranded DNA,
A first primer complementarily binds to the single-stranded DNA;
The target RNA is obtained by DNA-dependent DNA polymerase.
A double-stranded DNA having a promoter capable of transcribing RNA consisting of a sequence complementary to the sequence is generated. Then, in the presence of the RNA polymerase, the double-stranded DNA amplifies an RNA transcript having a sequence complementary to the target RNA. And this invention is characterized by using the oligonucleotide of SEQ ID NO: 1 as the first primer and the oligonucleotide of SEQ ID NO: 2 as the second primer. The first and second primers may be the full-length nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively, but use a combination of oligonucleotides consisting of at least 10 consecutive nucleotides or more in each sequence. Can also.

In one embodiment of the present invention, the step is carried out in the presence of an oligonucleotide probe labeled with an intercalating fluorescent dye having a sequence complementary to at least a part of the sequence of the RNA transcript to be amplified. Is preferred. At this time, if the fluorescence characteristics of the probe are changed by the complementary binding with the RNA transcript as compared with the case where no complex is formed, the fluorescence intensity of the reaction solution may be measured. Here, when the labeled oligonucleotide probe is co-present during the amplification step, so that the probe does not function as a primer for the extension reaction,
For example, it is particularly desirable to perform modification such as adding glycolic acid to the 3 ′ end. As an oligonucleotide probe, use of an oligonucleotide having a sequence complementary to the sequence described in SEQ ID NO: 6 can be exemplified.

[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 Specific amplification of a target RNA was performed using a combination of oligonucleotide primers according to the present invention. Note that t
rh2-RNA is RNA synthesized and purified by in vitro transcription using a double-stranded DNA containing the nucleotide sequence of trh2 as a template. Similarly, trh1-RNA, tdh2
-RNA was also prepared by in vitro transcription.

(1) trh2-RNA of Vibrio parahaemolyticus
Base numbers 1 to 610 (base numbers of RNA are Nishibuchi et al., A
ppl. Environ. Microbiol. , 5
8, 2449 to 2457 (1992)), and after quantification by ultraviolet absorption at 260 nm, an RNA diluent (10 mM T
ris-HCl (pH 8.0), 0.1 mM EDT
A, 0.5 U / μl RNase Inhibito
r, 5.0 mM DTT) and diluted to 1.0 × 10 ⁴ copies / 5 μl and 1.0 × 10 ³ copies / 5 μl. In the control test group (Nega), only the diluent was used.

(2) 20.8 μl of the reaction solution having the following composition was placed in a commercially available 0.5 ml PCR tube (trade name: Gen)
e Amp Thin-Walled Reactio
n Tubes, manufactured by PerkinElmer Co., Ltd.), and 5.0 μl of the above RNA sample was added thereto.

Composition of reaction solution (concentration is final concentration of reaction system after addition of enzyme solution) 60.0 mM Tris-HCl buffer (pH 8.6) 13.0 mM magnesium chloride 90.0 mM potassium chloride 1.0 mM DTT 0 each .25 mM dATP, dCTP, dGTP, dT
TP 3.0 mM ATP, CTP, UTP 2.25 mM GTP 3.6 mM ITP 1.0 μM each of the first primer and the second primer (Primer combinations are as shown in Table 1, where SEQ ID NO: 4 and SEQ ID NO: In the sequence of No. 5, the portion from the 1st “A” at the 5 ′ end to the 28th “A” is a sequence added as a promoter sequence of T7 polymerase. 0.16 μM oligonucleotide primer for cleavage (SEQ ID NO: 3) Oligonucleotide for cleaving target RNA at a position where the second primer can bind, 3 'end is aminated) 39U Ribonuclease inhibitor (Takara Shuzo Co., Ltd.) 15.0% for DMSO volume adjustment Distilled water

[0024]

[Table 1]

(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 (re-final concentration during reaction) 1.7% sorbitol 8 units AMV reverse transcriptase (Takara Shuzo Co., Ltd.) 142 units T7 RNA polymerase (GIB)
(Manufactured by CO) 3 μg bovine serum albumin distilled water for adjusting the volume (4) Subsequently, the PCR tube was kept at 41 ° C. for 30 minutes, and then a specific amplification product was analyzed by electrophoresis using a 4% agarose gel.

(5) For staining after electrophoresis, a commercially available staining solution (trade name: SYBR Green II, Takara Shuzo Co., Ltd.)
Was used.

The results of the electrophoresis are shown in FIG. In both combinations, trh2-RNA
In the system to which was added, specific RNA amplification products (Fig.
1 (arrow part) was obtained. On the other hand, tdh2-RNA,
No amplification product was obtained in the system to which trh1-RNA was added. This indicates that the combination of these oligonucleotide primers is useful for the amplification and detection of RNA derived from a thermotolerant hemotoxin (trh2) similar to Vibrio parahaemolyticus.

[0029]

As described above, according to the present invention,
Even under relatively low temperature and constant temperature (35 ° C. to 50 ° C., preferably 41 ° C.), the RNA in the sample forms an intramolecular structure and may inhibit the binding of primers and probes.
Thermotolerant hemotoxin-like hemolytic toxin gene of Vibrio parahaemolyticus (tr
h2) specifically binds to RNA derived from
Is useful as a combination of an oligonucleotide primer and an oligonucleotide probe for rapidly amplifying and detecting etc.

In addition to the above, the combination of the oligonucleotides of the present invention is not limited to trh2-RNA. To detect cDNA obtained by reverse transcription of RNA, the complementary sequence of the above-mentioned oligonucleotide is also useful. is there.

The base length of the oligonucleotide in the combination of the present invention is not limited to the specifically described length, and includes oligonucleotides consisting of at least 10 consecutive bases or more in these sequences. This is apparent from the fact that a base sequence of about 10 bases is sufficient to ensure sufficient specificity of the primer or probe for the target nucleic acid.

[0032]

[Sequence List] SEQUENCE LISTING <110> Tosoh Corporation <120> Method for detecting toxin gene of Vibrio parahaemolyticus <130> PA211-0192 <160> 6 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 1 gtgaccattg atgttgactg 20 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 tatctaaatc attcgcgatt gatctgcca 29 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 gatttagata ttgaaaatat 20 <210> 4 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 aattctaata cgactcacta tagggagata tctaaatcat tcgcgattg 49 <210> 5 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 aattctaata cgactcacta tagggagaaa atcattcgcg attgatctgc ca 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 cgattgaccg tatacatctt 20

[Brief description of the drawings]

FIG. 1 shows an initial RNA amount of 10 ⁴ copies / performed in Example 1.
This is the result of performing an RNA amplification reaction using two sets of primers at 30 μl and 10 ³ copies / 30 μl. Nega is a sample using only a diluent instead of an RNA sample.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/58 C12R 1:63) // (C12N 15/09 ZNA (C12Q 1/68 A C12R 1:63) ) C12R 1:63) (C12Q 1/68 C12N 15/00 ZNAA C12R 1:63) C12R 1:63) F term (reference) 2G045 AA25 AA28 AA35 CB21 DA12 DA13 DA14 DA77 FB01 FB02 FB07 FB12 GC15 4B024 AA13 CA11 HA14 4B063 QA01 QA18 QQ16 QQ52 QR08 QR35 QR56 QR62 QS25 QS34 QX02

Claims (4)

[Claims] 1. An RNA derived from a thermotolerant hemotoxin-like hemotoxin gene (trh2) of Vibrio parahaemolyticus present in a sample.
Using a specific sequence as a template, a first primer having a sequence complementary to the specific sequence and a second primer having a sequence homologous to the specific sequence (where either one of the first or second primer is used) Primer has an R
RNA-DNA double strand is formed by generating cDNA by RNA-dependent DNA polymerase, and RNA-DNA2 is formed by ribonuclease H.
A promoter capable of degrading single-stranded RNA to generate single-stranded DNA, and using the single-stranded DNA as a template to transcribe the RNA sequence or RNA comprising a sequence complementary to the RNA sequence by DNA-dependent DNA polymerase. To generate a double-stranded DNA having the sequence
An RNA transcript is generated in the presence of NA polymerase, and the RNA transcript is subsequently used as a template for the production of single-stranded DNA by the RNA-dependent DNA polymerase.
A detection method using an NA amplification step, wherein an oligonucleotide of SEQ ID NO: 1 is used as a first primer and an oligonucleotide of SEQ ID NO: 2 is used as a second primer. 2. The method according to claim 1, wherein said first primer is an oligonucleotide consisting of at least 10 consecutive nucleotides in the sequence of SEQ ID NO: 1. 3. The detection method according to claim 1, wherein the second primer is an oligonucleotide consisting of at least 10 consecutive nucleotides or more in the sequence of SEQ ID NO: 2. 4. The method according to claim 1, wherein the RNA amplification step is performed in the presence of an oligonucleotide probe labeled with an intercalating fluorescent dye.
Here, the sequence of the probe is complementary to at least a part of the RNA transcript, and the probe has a fluorescent property that is changed by complementary binding to the RNA transcript as compared with a case where no complex is formed. A method for detecting a toxin gene of Vibrio parahaemolyticus, comprising measuring the fluorescence intensity of a reaction solution.