JPH11137298A - Oligonucleotide for amplifying bacteriotoxin gene and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new oligonucleotide containing at least a nucleic acid sequence consisting of a specific number of consecutive bases among specific nucleic acid sequences or nucleic acid sequences complementary thereto, and usable e.g. for the detection or type judgment of the vero toxin gene of vero toxin-productive Escherichia coli. SOLUTION: This new oligonucleotide which contains at least a nucleic acid sequence consisting of consecutive 15 bases among nucleic acid sequences of formula I or formula II or nucleic acid sequences complementary thereto, is useful as e.g. a primer for amplifying bacteriotoxin gene capable of type judgment of VT1 or VT2 gene as well as detection of the above gene produced by vero toxin (VT1 or 2)-productive Escherichia coli (VTEC) in a specimen. This new oligonucleotide is obtained by designing a nucleic acid sequence so as to enable amplified gene fragments to be separated based on strand length in consideration of the presence of strains bearing VT1 and VT2 genes and variant genes thereof and synthesizing the aimed nucleic acid by phosphoamidite method using a DNA synthesizer.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a verotoxin-producing Escherichia coli, VTE
C), an oligonucleotide capable of specifically amplifying a verotoxin (abbreviated as VT) and its mutant gene, a reagent for amplifying the VT gene using the oligonucleotide, and the amplification method And a method for detecting the gene.

[0002]

2. Description of the Related Art VTEC is used for enterohemorrhagic Escherichia coli (Enterohe).
morrhagic Escherichia coli (EHEC), which is a pathogenic Escherichia coli characterized by producing VT as a pathogenic factor. At present, VT or Shiga-like toxin (Sh
iga-like toxin, abbreviated as SLT). To avoid confusion, the name of the toxin is changed to Shiga toxin (Shiga toxin, abbreviated as STX). Category name from VTEC or EHEC to Shiga toxin-prod
ucing Escherichia coli, abbreviated as STEC). However, since unification of these names has not yet reached a global agreement, the present invention uses VT as the toxin name and VTEC as the category name of the toxin-producing bacteria. Therefore, the names VT and VTEC in the present invention include all names that can be used in the same meaning.

[0003] The main symptom of an infection caused by VTEC is a food poisoning symptom typified by hemorrhagic colitis.
Hemolytic Uremic Syndrome, H
(Abbreviated as US).
In recent years, many cases of large-scale VTEC outbreaks occurring in a short period of time in Japan have been reported from HUS onset to death, and a rapid VTEC detection method is desired.

[0004] Until now, VTs isolated and identified in Japan
The majority of ECs are serotype O157: H7, but O2
6: Outbreaks of other serotypes of VTEC, such as H11, have also been reported. Therefore, VT by serotype
When identifying EC, it is necessary to perform a test using antisera against various serotypes previously identified as VTEC, not limited to O157: H7.

[0005] Furthermore, since identification of H antigen requires several days, these methods have a high speed and a high simplicity.
It is hard to say that it is an EC detection method. In addition, since the serotype and toxin production may not coincide with each other, it is considered that a method for detecting the virulence factor VT is more effective in identifying VTEC.

[0006] VT includes Vero toxin type 1 (VT1) having the same structure as Shiga toxin produced by Shigella dysenteriae, and Vero toxin 2 whose biological properties are similar to VT1 but have greatly different immunological properties. There is a type (VT2), and variants with partially different antigenicity and amino acid sequence for each are known. Analysis of the gene sequences encoding these has also been advanced, and at least 7 types of mutant (including variants) genes have been reported for VT1 and at least 21 types for VT2. Therefore, VT present in the faeces or food or environment of any organism
The VT gene of EC is converted into a PCR (polymerase chain react).
Attention has been focused on a method of amplifying using the ion) method.

[0007] Oligonucleotides for VT gene amplification reported to date (JP-A-4-297488, JP-A-4-297489, JP-A-7-828)
No. 0), VT1 and VT2 are obtained by one amplification.
Detection of the VT gene (without typing) by amplification of the common sequence of both genes, detection of the VT1 gene by amplification of the specific sequence of the VT1 gene, or detection of the VT2 gene by amplification of the specific sequence of the VT2 gene Or do. Therefore, when the type of the VT1 gene or the VT2 gene is detected at the same time as the detection of the VT gene in the sample using these oligonucleotides, it is necessary to amplify the gene with at least two samples of the reaction solution per sample. .

[0008] Performing typing of VT produced by VTEC at the same time as its detection is very effective as a means for identifying infectious bacteria and the source of infection. Furthermore, in view of the need for quick and simple detection of VTEC, it is often necessary to amplify the gene using an oligonucleotide optimized for performing the typing while simultaneously detecting the VT gene. , Considered very effective.

[0009]

SUMMARY OF THE INVENTION An object of the present invention is to provide a VT
Novel oligonucleotide for specifically amplifying EC VT and its mutant gene, and a method for simultaneously detecting the VT gene in a sample and simultaneously typing the VT1 gene or VT2 gene using the oligonucleotide Is to provide.

[0010]

Means for Solving the Problems The present inventors have developed VTEC.
As a result of intensive studies on VT and its mutant genes, a nucleic acid sequence almost specific to the VT1 gene and its mutant gene and a nucleic acid sequence almost specific to the VT2 gene and its mutant gene were found. The gene amplification method used was established, and the present invention was completed.

That is, the present invention relates to an oligonucleotide characterized by containing a nucleic acid sequence consisting of at least 15 consecutive nucleotides among the nucleic acid sequence shown in SEQ ID NO: 1 or 2 or a nucleic acid sequence complementary to the sequence. is there.

[0012] The present invention also relates to a nucleic acid sequence represented by SEQ ID NO: 3 or 4 or a nucleic acid sequence complementary thereto.
The oligonucleotide is characterized by containing a nucleic acid sequence consisting of at least 15 consecutive bases.

According to the present invention, at least two kinds of primers
A Vero toxin type 1 gene produced by Vero toxin-producing Escherichia coli containing a nucleic acid sequence consisting of at least 15 consecutive nucleotides among the nucleic acid sequences shown in SEQ ID NOs: 1 and 2 or a nucleic acid sequence complementary to the sequence ( VT1 gene), when the extension product of one primer is separated from its complement,
Primer serving as template for the other primer, heat stability D
A VT1 gene amplification reagent comprising NA polymerase, dNTPs and a buffer.

The present invention also relates to the present invention, wherein at least two kinds of primers have at least one of the nucleic acid sequences shown in SEQ ID NOs: 3 and 4 or a nucleic acid sequence complementary thereto.
Verotoxin type 2 gene (VT2 gene) produced by verotoxin-producing Escherichia coli containing a nucleic acid sequence consisting of 5 bases
Which comprises a primer, a thermostable DNA polymerase, a dNTP, and a buffer that serves as a template for the other primer when the extension product of one primer is separated from its complement. A reagent for amplifying a VT2 gene.

Further, the present invention relates to (i) a method in which at least two kinds of primers comprise a nucleic acid sequence consisting of at least 15 consecutive nucleotides among the nucleic acid sequences shown in SEQ ID NOs: 1 and 2 or a nucleic acid sequence complementary to said sequence. An oligonucleotide for amplifying the VT1 gene, the primer serving as a template for the other primer when the extension product of one primer is separated from its complement,
And (ii) amplifying the VT2 gene, wherein at least two kinds of primers contain a nucleic acid sequence consisting of at least 15 consecutive nucleotides among the nucleic acid sequences shown in SEQ ID NOs: 3 and 4 or a nucleic acid sequence complementary to the sequences. A primer that becomes a template for the other primer when the extension product of one primer is separated from its complement, (iii) a thermostable DNA polymerase, (iv) dNTP and (v) A VT1 gene, a VT2 gene or a reagent for amplifying these genes, which comprises a buffer.

According to the present invention, at least two consecutive primers selected from the nucleic acid sequences shown in SEQ ID NOs: 1 and 2 or the complementary nucleic acid sequences
An oligonucleotide for amplifying the VT1 gene containing a nucleic acid sequence consisting of bases is used, and when the extension product of one primer is separated from its complement, it becomes a template for the other primer. Characteristic V
This is a method for amplifying T1 gene.

Further, the present invention comprises, as at least two kinds of primers, a nucleic acid sequence consisting of at least 15 consecutive bases among the nucleic acid sequences shown in SEQ ID NOs: 3 and 4 or a nucleic acid sequence complementary to the sequences. A method for amplifying a VT2 gene, comprising using an oligonucleotide for amplifying a VT2 gene, wherein when an extension product of one primer is separated from its complement, it becomes a template for the other primer. is there.

Further, the present invention contains, as at least two kinds of primers, a nucleic acid sequence consisting of at least 15 consecutive nucleotides among the nucleic acid sequences shown in SEQ ID NOs: 1 and 2 or a nucleic acid sequence complementary to the sequences. An oligonucleotide for amplifying the VT1 gene is used, and as at least two primers, at least 15 consecutive nucleotides of the nucleic acid sequence shown in SEQ ID NOs: 3 and 4 or a nucleic acid sequence complementary thereto. Oligonucleotides for amplifying the VT2 gene, each containing a nucleic acid sequence of the following formula: When the extension product of one primer is separated from its complement, each becomes a template for the other primer. A method for amplifying a VT1 gene, a VT2 gene, or a gene characterized by the characteristics.

According to the present invention, based on the chain length of the amplified gene fragment obtained by the above amplification method, the amplified gene fragment
A method for detecting a bacterial toxin gene, comprising detecting which of the VT2 gene and both genes corresponds to the gene.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION The oligonucleotide of the present invention comprises
The nucleic acid sequences shown in SEQ ID NOs: 1 to 4 (where A represents adenine, C represents cytosine, G represents guanine, and T represents thymine. In addition, T at any position may be substituted with uracil (U). ) Contains a nucleic acid sequence consisting of at least 15 consecutive bases.

Although the oligonucleotide of the present invention is used as a primer, it can be used particularly as a primer for gene amplification. In that case, at least two oligonucleotides are used, the two primers define both ends of the nucleic acid sequence to be amplified, and when the extension product of one primer is separated from its complement, the other The base sequence is selected so as to serve as a template for the primer. In addition, the oligonucleotide of the present invention can be used as a primer when performing sequence analysis. Furthermore, it can also be used as a detection probe.

It is not necessary for the oligonucleotide of the present invention to use the entire sequence shown in SEQ ID NOS: 1 to 4 or the complementary sequence thereof. An appropriate length sequence derived from the sequence in the sequence listing and its complementary sequence can be determined by calculating the dissociation temperature (Tm value) and the like in accordance with the PCR reaction conditions and the like. However, in order for the primer to have sufficient specificity, it must have a length of at least 15 consecutive bases, more preferably at least 20 consecutive bases.

In order for the primer to have sufficient specificity, it is desirable to use the 3′-terminal base shown in the sequence in the sequence listing as the 3′-terminal base. Depending on the conditions used, purpose, etc., some substitutions may be made in the oligonucleotide. In addition, VT1 and its mutant gene or VT2 were selected so as not to affect the specificity of the primer.
5 ′ of the primer according to the sequence of the
The terminal side may be further extended and used.

In the present invention, when designing an oligonucleotide, considering that there is also a mutant in which a base in the target gene complementary to the 3 ′ end of the oligonucleotide is mutated, a base having a low probability of mutation is considered. Is set as the base at the 3 ′ end of the oligonucleotide.
The base at the 3 'end of the oligonucleotide serving as a primer for the polymerase serves as a starting point for the polymerase reaction. Therefore, when the sequence of the target gene is mutated, the efficiency of the polymerase reaction is extremely reduced.

The base G (plus strand) of the VT1 gene corresponding to the base (G) at the 3 ′ end of the oligonucleotide shown in SEQ ID NO: 1 is a sequence of VT1 which has already been determined.
When the base (G) is mutated to A or C or T, the glycine encoded by GGC becomes serine (AGC) or arginine (CGC) or cysteine (TGC). Will be translated as Furthermore, the glycine encoded by this GGC is also conserved at the amino acid level in VT1 and its mutant toxin, which have already been sequenced.

The base G (plus strand) of the VT1 gene complementary to the base (C) at the 3 'end of the oligonucleotide shown in SEQ ID NO: 2 is the same as that of VT1 whose sequence has already been determined and that of its mutant gene. When the base (G) is mutated to A or C or T, GCA
Is translated as threonine (ACA) or proline (CCA) or serine (TCA). Furthermore, alanine encoded by this GCA is also conserved at the amino acid level in VT1 and its mutant toxin, which have already been sequenced.

Furthermore, the base A (plus strand) of the VT2 gene corresponding to the base (A) at the 3 'end of the oligonucleotide shown in SEQ ID NO: 3 is between the VT2 whose sequence has already been determined and its mutant gene. When the base (A) is mutated to C or G or T, ACG
Is translated as proline (CCG) or alanine (GCG) or serine (TCG). At this time, the VT2 coding sequence (ACG) of the amino acid (threonine) is ACA or ACC.
Although there are mutant genes, there is no difference in the types of amino acids that can be translated by the mutation of the base (A). In addition, the threonine encoded by this ACG is also conserved at the amino acid level in VT2 and its mutant toxins, which have already been sequenced.

The base T (plus strand) of the VT2 gene which is complementary to the base (A) at the 3 ′ end of the oligonucleotide shown in SEQ ID NO: 4
Conserved between T2 and its mutant gene, when the base (T) is mutated to A or C or G, serine encoded by TCT becomes threonine (ACT) or proline (CCT) or alanine (GCT). ). Furthermore, serine encoded by this TCT is also conserved at the amino acid level in VT2 and its mutant toxin, which have already been sequenced. These facts indicate that the amino acids play an important role in maintaining the function of the protein.If these amino acids are mutated to other amino acids by mutation, the clone may be eliminated by natural selection. It means high.

Therefore, almost all VT1 that can exist and its mutant gene can be specifically amplified by the amplification method using the oligonucleotides shown in SEQ ID NOS: 1 and 2. In addition, almost all possible VT2 and its mutant genes can be specifically amplified by the amplification method using the oligonucleotides shown in SEQ ID NOs: 3 and 4.

In the present invention, in designing an oligonucleotide, the type of VT gene possessed by VTEC differs depending on the strain, that is, VTEC possesses a strain possessing VT1 and its mutant gene or a strain possessing VT2 and its mutant gene. In consideration of the existence of strains or strains having both genes, even when VT1 and its mutant gene amplification oligonucleotide and VT2 and its mutant gene amplification oligonucleotide are used in combination,
T1 and its mutant gene and VT2 and its mutant gene are independently amplified, and an oligonucleotide sequence is set which can easily separate and classify each amplified gene fragment by chain length.

The oligonucleotide of the present invention shown in SEQ ID NO: 1 has a high homology to the portion corresponding to the oligonucleotide (positive strand) in VT1 and its mutant gene, but this portion was excluded. VT1 and its full-length mutant gene have no homology that can be amplified. Furthermore, the oligonucleotide has no homology to the extent that it can be amplified with VT2 and the full length of its mutant gene.

The oligonucleotide shown in SEQ ID NO: 2 has high homology to VT1 and a portion (plus strand) complementary to the oligonucleotide in VT1 and its mutant gene. There is no homology that can be amplified with the full length of the mutant gene. Furthermore, the oligonucleotide has no homology to the extent that it can be amplified with VT2 and the full length of its mutant gene. These facts indicate that VT1 was amplified by amplification using the oligonucleotides shown in SEQ ID NOs: 1 and 2.
And that the mutant gene and VT2 and the mutant gene are present at the same time, it means that VT1 and the only amplified DNA fragment (658 bp) derived from the mutant gene can be generated.

The oligonucleotide shown in SEQ ID NO: 3 has high homology to the portion corresponding to the oligonucleotide (positive strand) in VT2 and its mutant gene, but VT2 and its There is no homology that can be amplified to the full length of the mutant gene. Furthermore, the oligonucleotide does not show any degree of homology that can be amplified with VT1 and the full length of its mutant gene.

The oligonucleotide shown in SEQ ID NO: 4 has a high homology to VT2 and a portion (plus strand) complementary to the oligonucleotide in the mutant gene, but VT2 and VT2 excluding this portion have a high homology. There is no homology that can be amplified with the full length of the mutant gene. Furthermore, the oligonucleotide does not show any degree of homology that can be amplified with VT1 and the full length of its mutant gene. These facts indicate that VT1 is amplified by amplification using the oligonucleotides shown in SEQ ID NOs: 3 and 4.
This means that even when VT2 and its mutant gene and VT2 and its mutant gene are present at the same time, only amplified DNA fragment (429 bp) derived from VT2 and its mutant gene can be generated.

Therefore, the type of VT gene possessed by VTEC, ie, VTEC possesses VT1 and its mutant gene is obtained from the length of the amplified DNA fragment obtained by the amplification method using the oligonucleotides shown in SEQ ID NOs: 1 to 4 in combination. (658 bp), VT2 and its mutant gene (429 bp), or both (658 bp and 429 bp)
Can be determined.

The method of the present invention for amplifying VT1 and its mutant gene is characterized by using the oligonucleotides shown in SEQ ID NOS: 1 and 2. The method for amplifying VT2 and its mutant gene of the present invention is characterized by using the oligonucleotides shown in SEQ ID NOS: 3 and 4. VT1 of the present invention and its mutant gene or VT2
And a method for amplifying the mutant gene or both genes are characterized in that oligonucleotides shown in SEQ ID NOs: 1 to 4 are mixed and used. However, in these amplification methods, at least two types of oligonucleotides are used as amplification primers, and the extension product of one primer, when separated from its complement, is sequenced so that it becomes a template for the other primer. You need to choose.

Further, based on the chain length of the amplified gene fragment obtained by this method, the gene fragment
It is possible to determine which of the T2 gene or both genes corresponds.

In the present invention, the gene to be amplified is the VTE present in the faeces or food or environment of any organism.
C-derived gene, but also includes amplifying the gene of isolated and cultured bacterial cells. The sample of the present invention includes, in addition to the above-mentioned various materials and cultured cells, nucleic acids isolated and purified therefrom.

That is, as the sample in the present invention,
In addition to using the above-mentioned various materials directly, a culture solution of the above-mentioned various materials, or a nucleic acid extracted or purified therefrom may be used. Furthermore, cells on an agar medium or cultures thereof isolated from the above-mentioned various materials, or nucleic acids extracted or purified therefrom can also be used as samples.

As primers used for gene amplification,
All oligonucleotides described above are included. In general, PCR (polymerase chain r) is used for this gene amplification.
eaction) method is used. As described above, for amplification of VT1 and its mutant gene from various samples, SEQ ID NO: 1
And the combination of the oligonucleotides shown in 2 can be suitably used. However, by re-amplifying the DNA amplified by the primers outside of the oligonucleotides shown in SEQ ID NOs: 1 and 2 using the oligonucleotides shown in SEQ ID NOs: 1 and 2, respectively, a higher sensitivity can be obtained. It is expected that amplification will be possible. The outer primer used at this time is preferably specific to VT1 and its mutant gene, but a primer with low specificity may be used.

For amplification of VT2 and its mutant gene from various samples, combinations of the oligonucleotides shown in SEQ ID NOs: 3 and 4 can be suitably used. However, by re-amplifying the DNA amplified by the primers outside of the oligonucleotides shown in SEQ ID NOs: 3 and 4, respectively, using the oligonucleotides shown in SEQ ID NOs: 3 and 4, respectively, higher sensitivity can be obtained. Amplification becomes possible. The outer primer used at this time is desirably specific to VT2 and its mutant gene, but a primer with low specificity may be used.

In the amplification of VT1 and its mutant gene, it is necessary to use a combination of the oligonucleotides shown in SEQ ID NOS: 1 and 2, respectively. In addition, in the amplification of VT2 and its mutant gene, it is necessary to use a combination of the oligonucleotides shown in SEQ ID NOS: 3 and 4, respectively. This is because the synergistic effect of the two oligonucleotides used is important for specificity in the amplification reaction. In order to further increase the specificity, it is important to keep the concentration of the primer used for amplification as low as possible.

When VT1 and its mutant gene and VT2 and its mutant gene are simultaneously amplified, that is, when the VTEC to be detected has two types of VT genes, SEQ ID NOs: 1 to 4 respectively show It is necessary to use a combination of oligonucleotides to be used. This is because the independence of the different genes in the simultaneous amplification reaction requires the use of oligonucleotides that do not interfere with each other's amplification reaction. Further, the combination of the oligonucleotides is used when only one of VT1 and its mutant gene or VT2 and its mutant gene is amplified, that is, the VTEC to be detected has two types of VTEC.
Even when only one of the T genes is possessed, it can be suitably used.

Conventional methods for amplifying the VT gene (JP-A-4-297488, JP-A-4-297489, and JP-A-7-8280) disclose VT1 and VT.
(2) Detection of VT gene (without typing) by PCR amplification using an oligonucleotide having a sequence common to both genes as a primer, or PC using an oligonucleotide having a sequence specific to the VT1 gene as a primer
Either detection of the VT1 gene by R amplification or detection of the VT2 gene by PCR amplification using an oligonucleotide having a sequence specific to the VT2 gene as a primer is performed by one PCR. Therefore,
When the VT1 gene or the VT2 gene is typed simultaneously with the detection of the VT gene in the sample, it was necessary to perform PCR using at least two samples of the reaction solution per sample.

However, in the present invention, the VT1 gene and its mutated or VT2 gene are detected by a single PCR using a plurality of oligonucleotides optimized for combination, based on the chain length of the amplified gene fragment at the same time as detection of the VT gene. And its mutant gene can be classified.
That is, in the present invention, SEQ ID NOs: 1 to 4
By using PCR using the oligonucleotides shown in Tables 1 and 2, it is possible to simultaneously detect the VT gene of VTEC and simultaneously classify VT1 and its mutant gene or VT2 and its mutant gene.

The reagent for amplifying the VT1 gene of the present invention, VT2
The reagent for gene amplification and the VT1 gene, VT2 gene or a reagent for amplifying the same may comprise at least two oligonucleotides for amplifying the VT1 gene or two or more oligonucleotides for amplifying the VT2 gene.
Included as seed primers, plus thermostable DNA polymerase, dNTPs and buffers.

Primers, thermostable DNA polymerases and dNTPs are included in amounts sufficient to carry out the amplification step.
l, dNTP: 50-500 μM, primer: 1-1
000 nM is included. Further, the reagent of the present invention may further contain a buffer suitable for the step of isolating the VT1 gene and the VT2 gene and a buffer suitable for amplifying the gene. Further, various combinations of reagents for detecting the amplified gene can be used in accordance with the detection method.

[0048]

The present invention will be specifically described below with reference to examples. Example 1 (1) Synthesis of Oligonucleotide Oligonucleotide for VT gene amplification was manufactured by ABI DN
Oligonucleotides having the sequences shown in SEQ ID NOs: 1 to 4 (primer 1: 1 to 33 of SEQ ID NO: 1, primer 2: SEQ ID NO: 2) by the phosphoramidite method using A synthesizer type 392 home,
1st to 21st, primer 3: 1 to 1 of SEQ ID NO: 3
31st, primer 4: 1 to 22 of SEQ ID NO: 4
Th) was synthesized. The procedure was performed according to the manual of ABI, and the deprotection of various oligonucleotides was carried out at 55 ° C. overnight with aqueous ammonia. Purification is HPLC
(Manufactured by Beckman) using a reversed-phase chromatography column (manufactured by Nacalai Tesque).

(2) Preparation of Samples A total of 37 cell colonies of VTEC strains isolated from patients, livestock, foods, etc. were directly subjected to PCR.
Was used as a sample. In all VTEC strains, the type of VT (VT1 or VT2 or both) produced by various conventional toxin production tests has already been identified. A VTEC colony (one platinum loop) on an agar medium was suspended in 100 μl of water, and 1 μl of the colony was used for PCR.

(3) PCR The oligonucleotides shown in SEQ ID NOs: 1 and 2 (primers 1 and 2) or the oligonucleotides shown in SEQ ID NOs: 3 and 4 (primers 3 and 4)
Alternatively, the oligonucleotides shown in SEQ ID NOs: 1 to 4 (primer 1, primer 2, primer 3, primer 4) were used as primers for PCR. The composition of the reaction solution was such that primers 1 and 2, or 3 and 4, or 1-4 were each 0.2 μM, dATP and dG
TP, dCTP and dTTP are each 0.2 mM, thermostable DNA polymerase (KOD Dash: manufactured by Toyobo) 25 units / ml, and 1-fold concentration buffer solution (manufactured by Toyobo). In actual use, 1 μl of a sample solution (VTEC colony suspension) was added to the reaction solution, and the reaction volume was adjusted to 100 μl, followed by the following reaction.

The reaction conditions are as follows: Thermal denaturation: 94 ° C., 20 seconds Annealing: 65 ° C., 2 seconds Extension reaction: 74 ° C., 30 seconds The above heat denaturation, annealing and extension reaction were repeated 30 times. These operations were performed using a DNA thermal cycler (GeneAmp2400) manufactured by PerkinElmer.

(4) Detection 10 μl of the reaction solution after the amplification reaction was electrophoresed on a 2% agarose gel, stained with ethidium bromide, and the fluorescence was detected under ultraviolet irradiation. The electrophoresis was performed at a constant voltage of 100 V for 30 minutes. The procedure and other conditions are described in Maniatis et al.
The technique described in Molecular Cloning (1982) was followed. In addition to the reaction solution, a molecular weight marker was simultaneously electrophoresed, and the length of the detected DNA fragment was calculated by comparing the relative mobility.

(5) Results As described above, the nucleotide sequences of the VT1 gene and VT2 gene of VTEC have already been determined, and they have been amplified by PCR using the oligonucleotide of the present invention as a primer (primers 1 to 4). The length of the DNA fragment can be easily estimated. According to that, VT1
If the gene is present, a 658 bp long D
If the NA fragment is amplified and the VT2 gene is present, the oligonucleotides indicated by primers 3 and 4 should amplify a 429 bp long DNA fragment. If the length of the amplified DNA fragment matches these estimates, these oligonucleotides, ie, primers, have correctly amplified the target region in the gene, and the strain in the sample is the amplified DNA fragment. It was determined to have a VT gene corresponding to the length of the fragment.

Table 1 shows the results of PCR amplification experiments performed on colonies of 37 VTEC strains of different origins.

[0055]

[Table 1]

FIG. 1 shows a part of agarose gel electrophoresis used for detection of the amplified gene fragment.

[0057]

[Table 2]

As is clear from Table 1 and FIG. 1 (lanes 1 to 4), primers 1 and 2 amplify only the genes of the strains determined to be VT1-positive in the conventional various toxin production tests and have a length of 658 bp. DNA fragment
It did not react at all with the gene of the strain determined to be T1 negative. That is, this shows that the oligonucleotide specifically amplifies the VT1 gene and can accurately detect VTEC carrying the VT1 gene.

As is clear from Table 1 and FIG. 1 (lanes 5 to 8), the oligonucleotides shown in primers 3 and 4 have V
Only the gene of the strain determined to be T2 positive was amplified to 42
A 9-bp DNA fragment was generated, and did not react at all with the gene of a strain determined to be VT2-negative. That is,
This shows that the oligonucleotide specifically amplifies the VT2 gene and can accurately detect VTEC carrying the VT2 gene.

Further, Table 1 and FIG. 1 (lanes 9 to 1)
As is clear from 2), even when the oligonucleotides represented by the primers 1 to 4 are mixed and used, no adverse effect is observed on the specific amplification by the oligonucleotides, and the VT1 gene or the VT2 gene or those genes are not affected. Was able to be accurately detected. That is, by using the combination of the oligonucleotides, the detection of VTEC and the amplified DNA
It shows that the type of VT produced by the bacterium can be classified into either VT1 or VT2 or both based on the chain length of the fragment.

[0061]

Industrial Applicability According to the present invention, VTE can be easily, rapidly and specifically extracted from feces of many organisms or foods or the environment.
C may amplify the VT gene. According to the present invention, V
At the same time as detecting the VT gene possessed by TEC,
That is, the classification of the case of possessing VT1 and its mutant gene (658 bp), the case of possessing VT2 and its mutant gene (429 bp), or the case of possessing both genes (658 bp and 429 bp) is based on the classification of the amplified DNA fragment. It can be performed more easily and quickly than the chain length.

[0062]

[Sequence list]

SEQ ID NO: 1 Sequence length: 33 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Location: 1 ... 33 Characteristic determined Method: S Other features: Has a sequence complementary to the sequence of the VT1 gene produced by VTEC. Sequence CAGGAGGTAC GTCTTTACTG ATGATTGATA GTG 33

SEQ ID NO: 2 Sequence length: 21 Sequence type: Number of nucleic acid strands: Both forms Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence features Location: 1..21 Determined method: S Other characteristics: It has a sequence complementary to the sequence of the VT1 gene produced by VTEC. Sequence CATTCTGGCA ACTCGCGATG C 21

SEQ ID NO: 3 Sequence length: 31 Sequence type: Number of nucleic acid strands: Both forms Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Location: 1..31 Method determined: S Other characteristics: Sequence complementary to the sequence of VT2 gene produced by VTEC. Sequence CAGGCACTGT CTGAAACTGC TCCTGTGTAT A 31

SEQ ID NO: 4 Sequence length: 22 Sequence type: Number of nucleic acid strands: Both forms Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Location: 1..22 Method determined: S Other characteristics: Sequence complementary to the sequence of VT2 gene produced by VTEC. Array CCGCCGCCAT TGCATTAACA GA 22

[Brief description of the drawings]

FIG. 1 is an electrophoretic photograph instead of a drawing showing the result of electrophoresis of an amplified gene on an agarose gel after amplifying a gene in a cell colony of 37 VTEC strains using the primers of the present invention.

 ──────────────────────────────────────────────────の Continuing on the front page (72) Inventor Fumiyoshi Kawakami 10-24 Toyo-cho, Tsuruga-shi, Fukui Toyobo Co., Ltd. Inside Tsuruga Bio-Research Laboratory (72) Inventor Yoshihisa Kawamura 10-24 Toyo-cho, Tsuruga-shi, Fukui Toyobo Co., Ltd., Tsuruga Bio Research Laboratory

Claims (9)

[Claims] 1. An oligonucleotide comprising a nucleic acid sequence consisting of at least 15 consecutive bases of the nucleic acid sequence shown in SEQ ID NO: 1 or 2 or a nucleic acid sequence complementary to the sequence. 2. An oligonucleotide comprising a nucleic acid sequence consisting of at least 15 consecutive bases of the nucleic acid sequence shown in SEQ ID NO: 3 or 4 or a nucleic acid sequence complementary to said sequence. 3. Vero toxin production, wherein at least two kinds of primers contain a nucleic acid sequence consisting of at least 15 consecutive nucleotides among the nucleic acid sequences shown in SEQ ID NOS: 1 and 2 or a nucleic acid sequence complementary to the sequences. An oligonucleotide for amplifying a Vero toxin type 1 gene (VT1 gene) produced by bacterium Escherichia coli, and when an extension product of one primer is separated from its complement, it is used as a template for the other primer. A reagent for amplifying a VT1 gene, comprising a primer, a thermostable DNA polymerase, dNTPs and a buffer. 4. Vero toxin production, wherein at least two kinds of primers contain a nucleic acid sequence consisting of at least 15 consecutive bases among the nucleic acid sequences shown in SEQ ID NOs: 3 and 4 or a nucleic acid sequence complementary to said sequences. Is an oligonucleotide for amplifying the Vero toxin type 2 gene (VT2 gene) produced by bacterium Escherichia coli, and when the extension product of one primer is separated from its complement, it is used as a template for the other primer. VT2 gene amplification reagent comprising a primer, a thermostable DNA polymerase, dNTPs and a buffer. 5. The method according to claim 1, wherein (i) at least two kinds of primers are
An oligonucleotide for amplifying the VT1 gene, which comprises a nucleic acid sequence consisting of at least 15 consecutive nucleotides among the nucleic acid sequences shown in SEQ ID NOs: 1 and 2 or a nucleic acid sequence complementary to the nucleic acid sequence; When the extension product of is separated from its complement, the primer serving as a template for the other primer, and (ii) at least two primers are selected from the nucleic acid sequence shown in SEQ ID NO: 3 and 4 or the nucleic acid sequence shown in SEQ ID NO: 3 or 4. An oligonucleotide for amplifying the VT2 gene, which comprises a nucleic acid sequence consisting of at least 15 consecutive nucleotides among complementary nucleic acid sequences, wherein an extension product of one primer is separated from its complement A primer serving as a template for the other primer, (iii) a thermostable DNA polymerase,
(iv) A VT1 gene, a VT2 gene, or a reagent for amplifying the gene, which comprises dNTP and (v) a buffer. 6. A VT1 gene containing, as at least two kinds of primers, a nucleic acid sequence comprising at least 15 consecutive nucleotides of the nucleic acid sequences shown in SEQ ID NOS: 1 and 2 or a nucleic acid sequence complementary to said sequences. A method for amplifying a VT1 gene, wherein an oligonucleotide for amplification is used, and when an extension product of one primer is separated from its complement, it serves as a template for the other primer. 7. A VT2 gene containing, as at least two kinds of primers, a nucleic acid sequence comprising at least 15 consecutive nucleotides among the nucleic acid sequences shown in SEQ ID NOs: 3 and 4 or a nucleic acid sequence complementary to said sequence. A method for amplifying a VT2 gene, wherein an oligonucleotide for amplification is used, and when an extension product of one primer is separated from its complement, it serves as a template for the other primer. 8. A VT1 gene containing, as at least two kinds of primers, a nucleic acid sequence consisting of at least 15 consecutive nucleotides of the nucleic acid sequences shown in SEQ ID NOs: 1 and 2 or a nucleic acid sequence complementary to said sequences. Use oligonucleotides for amplification and at least 2
As a species primer, a nucleic acid sequence comprising at least 15 consecutive bases among the nucleic acid sequences shown in SEQ ID NOs: 3 and 4 or a nucleic acid sequence complementary to the sequence,
An oligonucleotide for amplifying the VT2 gene is used, and when the extension product of one primer is separated from its complement, it serves as a template for the other primer. Genes or methods for amplifying those genes. 9. The amplified gene fragment according to claim 8, wherein the amplified gene fragment is a VT1 gene,
A method for detecting a bacterial toxin gene, comprising detecting whether the gene corresponds to the VT2 gene or those genes.