JPH11243996A - Detection of foreign dna fragment insertion generated in verotoxin gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new oligouncleotide comprising plural DNA fragments having continuous nucleic acid sequences having specific continuous base numbers and selected from among genes encoding verotoxin produced by verotoxin- producing Escherichia coil and useful for amplifying verotoxin genes, etc. SOLUTION: This oligonucleotide is selected from among genes having nucleic acid sequence of formula I or formula II encoding verotoxin produced by verotoxin-producing Escherichia coil, contains a nucleic acid sequence comprising at least 15 continuous base, and is useful for amplifying a verotoxin gene comprising at least two kinds of DNA fragments. The oligonucleotide enables the simple, rapid and specific detection of the verotoxin gene of verotoxin-producing Escherichia coil from the excrement of many organisms, foods or environments. The oligonucleotide is obtained by synthesizing at least two kinds of DNA fragments by use of a DNA synthesizer, etc. The two kinds of DNA fragments are selected from among the VT1 gene of formula I and the VT2 gene of formula II which encode the verotoxin produced by the verotoxin-producing Escherichia coil.

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 a mutant gene thereof, and a verotoxin gene (abbreviated as VT gene) using the oligonucleotide. And a method for detecting a foreign DNA fragment inserted into a VT gene, and a reagent therefor.
The present invention makes it possible to detect VT gene inactivation due to transfer of a foreign DNA fragment (IS).

[0002]

2. Description of the Related Art VTEC is used for enterohemorrhagic Escherichia coli (Entero).
hemorrhagic Escherichia coli, abbreviated as EHEC)
It is a pathogenic E. coli characterized by producing VT as a virulence factor. At present, the same toxin is referred to by several names such as VT or Shiga-like toxin (Shiga-like toxin, abbreviated as SLT) due to the history of discovery, and the name of the toxin has been changed for the purpose of avoiding confusion occurring there. Shiga toxin (abbreviated as STX)
In accordance with this, the category name of the toxin-producing bacteria was changed to VTE
C or EHEC to produce Shiga toxin-producing Escherichia coli (Shiga to
xin-producing Escherichia coli (abbreviated as STEC). But,
Since unification of these names has not yet reached global agreement, the present invention uses VT as the toxin name and VTEC as the category name of the toxin-producing bacteria. Accordingly, the terms 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, and in some cases, hemolytic uremic syndrome (Hemolytic uremic syndrome,
HUS), and in the worst case, death.

[0004] VT is a virulence factor causing a series of these symptoms. Vero toxin type 1 (abbreviated as VT1) having the same structure as Shiga toxin produced by Shigella dysenteriae, and biological properties similar to VT1. However, there are Verotoxin type 2 (abbreviated as VT2) whose immunological properties are greatly different, and variants with partially different antigenicity and amino acid sequence for each are known. For VT1, T. Takao et al., Microb. Pathog., 5, 357-369,
1988, and for VT2, see MPJackson et al.
al., FEMS Microbio., Lett., 44, 109-114, 1987.

At present, the VT gene of VTEC present in the faeces or food or environment of any organism is
(Polymerase chain reaction) Amplification using a method has attracted attention. Although the PCR method is very effective in that VT, which is a virulence factor, can be detected quickly and easily,
As a problem peculiar to the PCR method for detecting a gene,
It has been pointed out that there may be VTECs that carry the VT gene but do not produce VT.

The present inventors have conducted intensive studies on the VTEC VT gene and its mutant gene, and
A VTEC strain that harbors one gene and the VT2 gene and produces VT1 but not VT2 was discovered. As a result of a detailed study on the strain, foreign D
By the insertion of the NA fragment, the VT2 producing ability of the strain has been lost, that is, the VT2 gene has been inactivated, and the foreign DNA fragment is a DNA fragment which does not originally exist on the VT gene. A foreign DNA fragment is an insertion sequence (insertion sequence) which is a kind of transposable genetic element.
ce, IS).

[0007] The insertion sequence (IS) is usually one of the transposable genetic elements that move between various replicons such as chromosomes, plasmids, and phage DNAs, and several types are known to exist in Escherichia coli. Therefore, it is considered that the inactivation of the VT gene by the transfer of IS can sufficiently occur in VTEC other than the strain, regardless of the type of VT gene possessed. In addition, when the inserted IS is completely removed by further transposition, it is known that a functional protein is synthesized again and returns to a wild type. That is, Escherichia coli having the VT gene inactivated by insertion of IS has a possibility that VT production ability will be restored by removal of the molecule, and is determined to be VT negative by various toxin production tests. It is possible that even strains that have potential have the potential to produce VT.

[0008]

SUMMARY OF THE INVENTION An object of the present invention is to provide a VT
It is an object of the present invention to provide a method for detecting whether or not an exogenous DNA fragment, ie, one of the transposable genetic elements, IS, has been inserted into the VT gene of EC, and a reagent therefor.

[0009]

Means for Solving the Problems The present inventors have found a VT gene inactivated by insertion of IS, and as a result of intensive studies on the gene, as a result, the VT gene upstream and downstream of the insertion site of IS has been found. The inventors have found nucleic acid sequences, established a gene amplification method using them, and have completed the present invention.

The present inventors have also conducted intensive studies in consideration of the possibility that several types of IS present in Escherichia coli may be transferred and inserted into any region of the VT gene.
A nucleic acid sequence almost specific to one gene and its mutant gene,
In addition, the inventors have found a nucleic acid sequence almost specific to the VT2 gene and its mutant gene, established a method for amplifying almost the entire VT gene using them, and have completed the present invention.

[0011] That is, the present invention relates to at least 15 contiguous genes selected from the gene encoding verotoxin produced by verotoxin-producing Escherichia coli, specifically the gene encoding verotoxin type 1 or verotoxin type 2 (SEQ ID NO: 1 or 2). It is an oligonucleotide for amplifying at least two kinds of DNA fragments, a verotoxin gene, containing a nucleic acid sequence consisting of bases.

The present invention also relates to an oligonucleotide for amplifying the above-mentioned Verotoxin type 1 or Verotoxin type 2 gene as at least two kinds of primers, wherein an extension product of one of the primers is obtained from its complement. A method for amplifying a verotoxin gene, which comprises using an oligonucleotide that becomes a template for the other primer when separated.

Further, the present invention provides an oligonucleotide for amplifying a verotoxin type 1 or verotoxin type 2 gene produced by the above-mentioned verotoxin-producing Escherichia coli, wherein one of the primers is extended. A reagent for amplifying a verotoxin gene, which comprises a primer serving as a template for the other primer when the product is separated from its complement, a thermostable DNA polymerase, dNTPs and a buffer.

[0014] The present invention also provides a method for exogenous DN to verotoxin gene based on the chain length of the amplified gene fragment obtained by the above amplification method.
A method for detecting the insertion of a foreign DNA fragment generated in a verotoxin gene, comprising detecting the insertion of an A fragment.

Further, the present invention provides an oligonucleotide for amplifying a verotoxin type 1 or verotoxin type 2 gene produced by a verotoxin-producing Escherichia coli, wherein at least two kinds of primers are formed by extension of one of the primers. Thing is,
A primer that becomes a template for the other primer when separated from its complement, a thermostable DNA polymerase,
A reagent for detecting insertion of a foreign DNA fragment generated in a verotoxin gene, comprising dNTP and a buffer.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The oligonucleotide of the present invention comprises
A gene encoding a verotoxin produced by a verotoxin-producing E. coli, selected from the VT1 gene (SEQ ID NO: 1);
At least two types of DNA fragments or at least two types of DNA fragments selected from the VT2 gene (SEQ ID NO: 2), each containing a nucleic acid sequence consisting of at least 15 consecutive nucleotides, preferably 20 to 35 nucleotides. The oligonucleotide is a Vero toxin gene (VT gene) produced by Vero toxin-producing Escherichia coli (VTEC),
Amplify T1 gene or VT2 gene. At least one of the oligonucleotides is located upstream of the insertion site of the foreign DNA fragment, and the other is located downstream thereof.

The oligonucleotides of the present invention are, specifically, nucleic acid sequences shown in SEQ ID NOs: 3 to 6 and 7 to 10 (where A is adenine, C is cytosine, G is guanine, and T is thymine. In addition, T at any position may be substituted with uracil (U).) The nucleic acid sequence comprises at least 15 consecutive nucleotides, preferably 20 to 35 nucleotides.

Although the oligonucleotide of the present invention is used as a primer, it can be particularly used 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.

The oligonucleotide of the present invention can also be used as a primer for 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 region of the nucleotide sequence shown in SEQ ID NO: 3 to 10 or the complementary sequence thereof. An appropriate length sequence derived from the base sequence in the sequence listing and its complementary sequence can be determined by calculating the dissociation temperature (Tm value) or the like according to the PCR reaction conditions and the like. However, in order for primers to have sufficient specificity, at least 15 consecutive
A base, more preferably 20 to 35 bases, is required.

In order for the primer to have sufficient specificity, the base at the 3 'end must be 3' as shown in the nucleic acid sequence in the sequence listing.
It is desirable to use a terminal base. Depending on the conditions used, purpose, etc., some substitutions may be made in the oligonucleotide. In addition, the 5′-terminal of the primer may be further extended according to the nucleotide sequence of the VT1 gene and its mutant gene or the VT2 gene and its mutant gene to such an extent that the specificity of the primer is not affected. .

The oligonucleotides of the present invention shown in SEQ ID NOs: 3 and 4 have high homology to the portion corresponding to the oligonucleotide (plus strand) in the VT1 gene and its mutant gene. There is no homology to the extent that it can be amplified with respect to the VT1 gene and its full-length mutant gene except for the VT1 gene. Furthermore, the oligonucleotide does not show any amplifiable homology to the VT2 gene and its full-length mutant gene.

The oligonucleotides shown in SEQ ID NOs: 5 and 6 have high homology to a portion (plus strand) complementary to the oligonucleotide in the VT1 gene and its mutant gene. The removed VT1 gene and its full-length mutant gene do not show any degree of homology that can be amplified. Furthermore, the oligonucleotide does not show any amplifiable homology to the VT2 gene and its full-length mutant gene.

These facts indicate that the amplification using the oligonucleotides shown in SEQ ID NOs: 3 and 5 as primers resulted in the VT1 gene, its mutant gene and VT1 gene.
This means that even when two genes and their mutant genes are present at the same time, the only amplified DNA fragment (1,226 bp) derived from the VT1 gene and its mutant genes can be generated. When the oligonucleotides shown in SEQ ID NOs: 4 and 6 are used as primers, amplified DNA
A fragment (1,276 bp) can be generated.

The oligonucleotides shown in SEQ ID NOs: 7 and 8 correspond to a portion (positive strand) corresponding to the oligonucleotide in the VT2 gene and its mutant gene.
VT which has high homology to
There is no homology that can be amplified between the two genes and their full length mutant genes. Furthermore, the oligonucleotide does not show any amplifiable homology to the VT1 gene and its full-length mutant gene.

The oligonucleotides shown in SEQ ID NOs: 8 and 10 have a high homology to the VT2 gene and a portion complementary to the oligonucleotide (positive strand) in the mutant gene and the VT2 gene. The VT2 gene and its mutant gene have no homology to the extent that they can be amplified. Furthermore, the oligonucleotide does not show any amplifiable homology to the VT1 gene and its full-length mutant gene.

These facts indicate that the amplification using the oligonucleotides shown in SEQ ID NOs: 7 and 9
This means that even when the gene and its mutant gene and the VT2 gene and its mutant gene are present at the same time, only an amplified DNA fragment (1,231 bp) derived from the VT2 gene and its mutant gene can be generated. I have.
When the oligonucleotides shown in SEQ ID NOs: 8 and 10 were used as primers, the amplified DNA fragment (1, 29
1 bp).

Specifically, the oligonucleotide of the present invention comprises an oligonucleotide containing a nucleic acid sequence consisting of at least 15 consecutive bases among the nucleic acid sequences shown in SEQ ID NOs: 3 to 6 or a nucleic acid sequence complementary thereto. It is an oligonucleotide containing a nucleotide or a nucleic acid sequence consisting of at least 15 consecutive bases of the nucleic acid sequence shown in SEQ ID NOs: 7 to 10 or a nucleic acid sequence complementary to the sequence.

In the present invention, the above-mentioned at least two kinds of oligonucleotides are used as amplification primers, and the extension product of one primer, when separated from its complement, is sequenced so as to become a template for the other primer. You need to choose.

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 the VT1 gene and its mutant gene from various samples, combinations of the oligonucleotides shown in SEQ ID NOs: 3 to 6 can be suitably used. However, by re-amplifying the DNA amplified by the primers outside of the oligonucleotides shown in SEQ ID NOs: 3 to 6 using the oligonucleotides shown in SEQ ID NOs: 3 to 6, higher sensitivity can be obtained. It is expected that amplification will be possible. The outer primer used at this time was VT1
Desirably, the primers are specific for the gene and its mutant gene, but primers with low specificity may be used.

For the amplification of the VT2 gene and its mutant gene from various samples, combinations of the oligonucleotides shown in SEQ ID NOs: 7 to 10 can be suitably used. However, by further re-amplifying the DNA amplified by the primers outside the oligonucleotides shown in SEQ ID NOs: 7 to 10 using the oligonucleotides shown in SEQ ID NOs: 7 to 10, higher sensitivity can be obtained. Amplification becomes possible. The outer primer used at this time is desirably specific to the VT2 gene and its mutant gene, but a primer with low specificity may be used.

In the amplification of the VT1 gene and its mutant gene, it is necessary to use a combination of the oligonucleotides shown in SEQ ID NOs: 3 to 6, respectively. In addition, in amplification of the VT2 gene and its mutant gene,
It is necessary to use a combination of the oligonucleotides shown in SEQ ID NOs: 7 to 10, 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.

[0034] Specifically, the reagent for amplifying the VT1 gene of the present invention comprises at least two kinds of primers having SEQ ID NOs: 3 to
Vero toxin type 1 gene (VT1 gene) produced by Vero toxin-producing Escherichia coli containing a nucleic acid sequence consisting of at least 15 consecutive bases among the nucleic acid sequence shown in SEQ ID NO: 6 or a nucleic acid sequence complementary to said sequence. Oligonucleotides for amplification, comprising a primer, a thermostable DNA polymerase, dNTPs and a buffer that, when the extension product of one primer is separated from its complement, becomes a template for the other primer .

Further, the reagent for amplifying the VT2 gene of the present invention comprises at least two kinds of primers having SEQ ID NOs: 7 to 10.
Verotoxin 2 produced by Verotoxin-producing Escherichia coli, comprising a nucleic acid sequence consisting of at least 15 consecutive bases among the nucleic acid sequence shown in (1) or a nucleic acid sequence complementary thereto.
For amplifying a gene of the type (VT2 gene), a primer that becomes a template for the other primer when the extension product of one primer is separated from its complement, a thermostable DNA Contains polymerase, dNTPs and buffer.

The primer, thermostable DNA polymerase, and dNTP are included in amounts sufficient to perform the amplification step. For example, DNA polymerase is used in an amount of 1 to 200 U / m2.
l, dNTP: 50-500 μM, primer: 1-1
000 nM is included.

The reagent of the present invention further comprises a VT1 gene,
A buffer suitable for the step for isolating the VT2 gene and a buffer suitable for amplifying the gene may also be included. Further, various combinations of reagents for detecting the amplified gene can be used in accordance with the detection method.

Further, according to the present invention, a foreign D
By detecting the insertion of the NA fragment, it becomes possible to detect the insertion mutation generated in VT.

Specifically, the method of the present invention for detecting an insertion mutation in VT1 comprises detecting the insertion of a foreign DNA fragment into the VT1 gene from the chain length of the amplified gene fragment obtained by the above amplification method. It is characterized by. DN amplified using primers having the nucleotide sequences shown in SEQ ID NOs: 3 to 6
The A fragment was 1,226 bp (SEQ ID NOS: 3 and 5),
If it is larger than 76 bp (SEQ ID NOS: 4 and 6), 1,252 bp (SEQ ID NOs: 3 and 6) or 1,250 bp (SEQ ID NOs: 4 and 5), it means that a foreign DNA fragment has been inserted.

The method of the present invention for detecting an insertion mutation occurring in VT2 is characterized in that the insertion of a foreign DNA fragment into the VT2 gene is detected from the chain length of the amplified gene fragment obtained by the above amplification method. I do. DNA fragments amplified using primers having the nucleotide sequences shown in SEQ ID NOs: 7 to 10 were 1,232 bp (SEQ ID NOs: 7 and 9), 1,291
If it is larger than bp (SEQ ID NOS: 8 and 10), 1,266 bp (SEQ ID NOs: 7 and 10), and 1,256 bp (SEQ ID NOs: 8 and 9), it means that a foreign DNA fragment has been inserted.

Further, the amplification D obtained by this amplification method
According to the chain length of the NA fragment, the DNA fragment was determined to be the VT1 gene or the VT2 gene or the V
It is possible to determine which of the T1 gene and the VT2 gene into which the foreign DNA fragment has been inserted.

In the present invention, the gene to be amplified is the VTE present in the faeces of any organism or in food or the environment.
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 various materials described above, or nucleic acids roughly extracted or purified therefrom can also be used as samples.

[0044]

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 nucleic acid sequences shown in SEQ ID NOs: 3, 5, 7 and 9 using the A synthesizer type 392 by the phosphoramidite method (Primer 1: 1-33 of SEQ ID NO: 3, Primer 2 :
It was obtained by synthesizing (SEQ ID NO: 5, 1-227, primer 3: 1-31, SEQ ID NO: 7, primer 4: SEQ ID: 9, 1-32).
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 was performed by HPLC (manufactured by Beckman) using a reversed-phase chromatography column (manufactured by Nacalai Tesque).

(2) Preparation of Sample A VTEC strain having the VT1 gene, a VTEC strain having the VT2 gene, a VTEC strain having the VT1 and VT2 genes, and a VTEC having the VT1 gene and the VT2 gene having the IS inserted therein. Each of the four bacterial colonies of the strain was used directly as a PCR sample (see Table 1). A VTEC colony (one platinum loop) on an agar medium was suspended in 100 μl of water, and 1 μl thereof was suspended.
1 was used for PCR.

(3) PCR The oligonucleotides shown in SEQ ID NOs: 3 and 5 (primers 1 and 2) or the oligonucleotides shown in SEQ ID NOs: 7 and 9 (primers 3 and 4)
Was used as a primer for PCR (see Table 1).

The composition of the reaction solution was such that primers 1 and 2 or primers 3 and 4 were each 0.2 μM and dA
0.2 mM each of TP and dGTP and dCTP and dTTP, thermostable DNA polymerase (KODD)
ash: Toyobo) 25 units / ml, and a 1-fold concentration buffer for amplification (Toyobo). For actual use, a sample solution (VTEC colony suspension) 1
μl was added to make the reaction solution volume 100 μl and subjected to the following reaction. The reaction conditions are as follows: Thermal denaturation: 94 ° C., 20 seconds Annealing: 65 ° C., 2 seconds Extension reaction: 74 ° C., 90 seconds The above thermal 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 1% agarose gel, stained with ethidium bromide, and the fluorescence under ultraviolet irradiation was detected. 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 The length of the DNA fragment amplified by PCR using the primers 1 and 2 is usually 1,226 bp. The length of a DNA fragment amplified by PCR using the primers 3 and 4 is usually 1,231 bp. FIG. 1 shows agarose gel electrophoresis used for detection of the amplified DNA fragment.

[0050]

[Table 1]

As is apparent from FIG. 1, primers 1 and 2 (lanes 1, 3, 5, 7, and 9) were VT1
Only the gene of the strain carrying the gene was amplified, and did not react at all with the gene of the strain not carrying the VT1 gene. That is, it indicates that the primers 1 and 2 can specifically amplify the VT1 gene and accurately detect VTEC carrying the VT1 gene.

As is apparent from FIG. 1, primers 3 and 4 (lanes 2, 4, 6, 8, and 10) were VT
Amplifying only the genes of the strains carrying the two genes, VT2
It did not react at all with the gene of a strain that does not carry the gene. That is, it shows that the oligonucleotide specifically amplifies the VT2 gene and can accurately detect VTEC carrying the VT2 gene.

Further, as is clear from FIG. 1, the strain having the VT1 gene without IS inserted thereinto is:
With the primers 1 and 2, a DNA fragment having a length of 1,226 bp was amplified (lanes 1, 5, 7). From the strains having the VT2 gene without IS inserted, 231 bp long D
The NA fragment was amplified (lanes 4, 6). On the other hand, from the strain having the VT2 gene into which IS was inserted (lane 8), about 2,500 bp
Was amplified. In other words, it shows that the method of the present invention can accurately detect a VTEC carrying a VT gene into which a foreign DNA fragment has been inserted.

[0054]

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
Simultaneously with the detection of the VT gene possessed by the TEC, the insertion mutation caused by the insertion of a foreign DNA fragment into the gene can be detected more simply and quickly than the length of the amplified DNA fragment.

In the present invention, insertion of a foreign DNA fragment into the VT1 gene or VT2 gene causes the VT1 or VT2 producing ability of the strain to disappear, ie, VT1 or VT2.
It can be detected that one gene or the VT2 gene has been inactivated, and can be effectively applied to the diagnosis of food poisoning symptoms represented by hemorrhagic colitis.

[0056]

[Sequence list] SEQ ID NO: 1 Sequence length: 1369 Sequence type: Nucleic acid Topology: Linear Sequence type: Genomic DNA Sequence characteristics Origin: Verotoxin-producing Escheri that produces VT1
chia coli sequence TATCGTATGG TGCTCAAGGA GTATTGTGTA ATATGAAAAT AATTATTTTT AGAGTGCTAA 60 CTTTTTTCTT TGTTATCTTT TCAGTTAATG TGGTGGCGAA GGAATTTACC TTAGACTTCT 120 CGACTGCAAA GACGTATGTA GATTCGCTGA ATGTCATTCG CTCTGCAATA GGTACTCCAT 180 TACAGACTAT TTCATCAGGA GGTACGTCTT TACTGATGAT TGATAGTGGC ACAGGGGATA 240 ATTTGTTTGC AGTTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCGGTTT AATAATCTAC 300 GGCTTATTGT TGAACGAAAT AATTTATATG TGACAGGATT TGTTAACAGG ACAAATAATG 360 TTTTTTATCG CTTTGCTGAT TTTTCACATG TTACCTTTCC AGGTACAACA GCGGTTACAT 420 TGTCTGGTGA CAGTAGCTAT ACCACGTTAC AGCGTGTTGC AGGGATCAGT CGTACGGGGA 480 TGCAGATAAA TCGCCATTCG TTGACTACTT CTTATCTGGA TTTAATGTCG CATAGTGGAA 540 CCTCACTGAC GCAGTCTGTG GCAAGAGCGA TGTTACGGTT TGTTACTGTG ACAGCTGAAG 600 CTTTACGTTT TCGGCAAATA CAGAGGGGAT TTCGTACAAC ACTGGATGAT CTCAGTGGGC 660 GTTCTTATGT AATGACTGCT GAAGATGTTG ATCTTACATT GAACTGGGGA AGGTTGAGTA 720 GCGTCCTGCC TGACTATCAT GGACAAGACT CTGTTCGTGT AGGAAGAATT TCTTTTGGAA 780 GCATTAATGC AATTCTGGGA AGCGTGGCAT TAATACTGAA TTGTCATCAT CATGCATCGC 840 GAGT TGCCAG AATGGCATCT GATGAGTTTC CTTCTATGTG TCCGGCAGAT GGAAGAGTCC 900 GTGGGATTAC GCACAATAAA ATATTGTGGG ATTCATCCAC TCTGGGGGCA ATTCTGATGC 960 GCAGAACTAT TAGCAGTTGA GGGGGTAAAA TGAAAAAAAC ATTATTAATA GCTGCATCGC 1020 TTTCATTTTT TTCAGCAAGT GCGCTGGCGA CGCCTGATTG TGTAACTGGA AAGGTGGAGT 1080 ATACAAAATA TAATGATGAC GATACCTTTA CAGTTAAAGT GGGTGATAAA GAATTATTTA 1140 CCAACAGATG GAATCTTCAG TCTCTTCTTC TCAGTGCGCA AATTACGGGG ATGACTGTAA 1200 CCATTAAAAC TAATGCCTGT CATAATGGAG GGGGATTCAG CGAAGTTATT TTTCGTTGAC 1260 TCAGAATAGC TCAGTGAAAA TAGCAGGCGG AGATTCATAA ATGTTAAATA CATCTCAATT 1320 CAGTCAGTTG TTGCCGGTTC TGATAATAGA TGTGTTAGAA AATTCTTGC 1369

SEQ ID NO: 2 Sequence length: 1664 Sequence type: nucleic acid Topology: linear Sequence type: genomic DNA Sequence characteristics Origin: Verotoxin-producing Escheri producing VT2
chia coli sequence ATCGCATAGC TCATCGGAAC AAGCTCAAGC GGTCTCCGGT CGAGTCCTCA TGCGTCCATT 60 ATCTGCATTA TGCGTTGTTA GCTCAGCCGG ACAGAGCAAT TGCCTTCTGA GCAATCGGTC 120 ACTGGTTCGA ATCCAGTACA ACGCGCCATA TTTATTTACC AGGCTCGCTT TTGCGGGCCT 180 TTTTTATATC TGCGCCGGGT CTGGTGCTGA TTACTTCAGC CAAAAGGAAC ACCTGTATAT 240 GAAGTGTATA TTATTTAAAT GGGTACTGTG CCTGTTACTG GGTTTTTCTT CGGTATCCTA 300 TTCCCGGGAG TTTACGATAG ACTTTTCGAC CCAACAAAGT TATGTCTCTT CGTTAAATAG 360 TATACGGACA GAGATATCGA CCCCTCTTGA ACATATATCT CAGGGGACCA CATCGGTGTC 420 TGTTATTAAC CACACCCCAC CGGGCAGTTA TTTTGCTGTG GATATACGAG GGCTTGATGT 480 CTATCAGGCG CGTTTTGACC ATCTTCGTCT GATTATTGAG CAAAATAATT TATATGTGGC 540 CGGGTTCGTT AATACGGCAA CAAATACTTT CTACCGTTTT TCAGATTTTA CACATATATC 600 AGTGCCCGGT GTGACAACGG TTTCCATGAC AACGGACAGC AGTTATACCA CTCTGCAACG 660 TGTCGCAGCG CTGGAACGTT CCGGAATGCA AATCAGTCGT CACTCACTGG TTTCATCATA 720 TCTGGCGTTA ATGGAGTTCA GTGGTAATAC AATGACCAGA GATGCATCCA GAGCAGTTCT 780 GCGTTTTGTC ACTGTCACAG CAGAAGCCTT ACGCTTCAGG CAGATACAGA GAGAATTTCG 840 TCAG GCACTG TCTGAAACTG CTCCTGTGTA TACGATGACG CCGGGAGACG TGGACCTCAC 900 TCTGAACTGG GGGCGAATCA GCAATGTGCT TCCGGAGTAT CGGGGAGAGG ATGGTGTCAG 960 AGTGGGGAGA ATATCCTTTA ATAATATATC AGCGATACTG GGGACTGTGG CCGTTATACT 1020 GAATTGCCAT CATCAGGGGG CGCGTTCTGT TCGCGCCGTG AATGAAGAGA GTCAACCAGA 1080 ATGTCAGATA ACTGGCGACA GGCCTGTTAT AAAAATAAAC AATACATTAT GGGAAAGTAA 1140 TACAGCTGCA GCGTTTCTGA ACAGAAAGTC ACAGTTTTTA TATACAACGG GTAAATAAAG 1200 GAGTTAAGCA TGAAGAAGAT GTTTATGGCG GTTTTATTTG CATTAGCTTC TGTTAATGCA 1260 ATGGCGGCGG ATTGTGCTAA AGGTAAAATT GAGTTTTCCA AGTATAATGA GGATGACACA 1320 TTTACAGTGA AGGTTGACGG GAAAGAATAC TGGACCAGTC GCTGGAATCT GCAACCGTTA 1380 CTGCAAAGTG CTCAGTTGAC AGGAATGACT GTCACAATCA AATCCAGTAC CTGTGAATCA 1440 GGCTCCGGAT TTGCTGAAGT GCAGTTTAAT AATGACTGAG GCATAACCTG ATTCGTGGTA 1500 TGTGGGTAAC AAGTGTAATC TGTGTCACAA TTCAGTCAGT TTGACAGTTG CCTGTCAGAC 1560 TGAGCATTTG TTAAAAAAAT TTCGCATGGT GAATCCCCCT GTGTGGGGCG ACTGGTGAAA 1620 AATCCTTGCT TGTGATTCAT TATCGACACG GGTTCGGTGG TACC 1664

SEQ ID NO: 3 Sequence length: 33 Sequence type: Number of nucleic acid chains: Both forms Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Location: 1. . 33 Method of determining the characteristics: S Other characteristics: having a sequence complementary to the sequence of the VT1 gene produced by VTEC. Sequence ATGAAAATAA TTATTTTTA GAGTGCTAAC TTT 33

SEQ ID NO: 4 Sequence length: 27 Sequence type: Number of nucleic acid chains: Both forms Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Location: 1. . 27 Method of determining the characteristics: S Other characteristics: having a sequence complementary to the sequence of the VT1 gene produced by VTEC. Sequence GGTGCTCAAG GAGTATTGTG TAATATG 27

SEQ ID NO: 5 Sequence length: 27 Sequence type: Number of nucleic acid chains: Both forms Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Location: 1. . 27 Method of determining the characteristics: S Other characteristics: having a sequence complementary to the sequence of the VT1 gene produced by VTEC. Sequence CAACGAAAAA TAACTTCGCT GAATCCC 27

SEQ ID NO: 6 Sequence length: 28 Sequence type: Number of nucleic acid chains: Both forms Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Location: 1. . 28 Method of determining the characteristics: S Other characteristics: having a sequence complementary to the sequence of the VT1 gene produced by VTEC. Sequence GCTATTTTCA CTGAGCTATT CTGAGTCA 28

SEQ ID NO: 7 Sequence length: 31 Sequence type: Number of nucleic acid chains: Both forms Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Location: 1. . 31 Method of determining the characteristics: S Other characteristics: having a sequence complementary to the sequence of the VT2 gene produced by VTEC. Sequence ATGAAGTGTA TATTATTTAA ATGGGTACTG T 31

SEQ ID NO: 8 Sequence length: 28 Sequence type: Number of nucleic acid chains: Both forms Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Location: 1. . 28 Method for determining the characteristics: S Other characteristics: A sequence complementary to the sequence of the VT2 gene produced by VTEC. Array CTTCAGCCAA AAGGAACACC TGTATATG 28

SEQ ID NO: 9 Sequence length: 32 Sequence type: Number of nucleic acid chains: Both forms Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Location: 1. . 32 Method for determining characteristics: S Other characteristics: having a sequence complementary to the sequence of the VT2 gene produced by VTEC. Sequence TTAAACTGCA CTTCAGCAAA TCCGGAGCCT GA 32

SEQ ID NO: 10 Sequence length: 28 Sequence type: Number of nucleic acid chains: Both forms Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Location: 1. . 28 Method for determining the characteristics: S Other characteristics: A sequence complementary to the sequence of the VT2 gene produced by VTEC. Sequence CACATACCAC GAATCAGGTT ATGCCTCA 28

[Brief description of the drawings]

FIG. 1 is an electrophoretic photograph instead of a drawing, showing the results of amplifying genes in cell colonies of various bacterial VTEC strains.

Claims (20)

[Claims] 1. At least two kinds of DNA fragments containing a nucleic acid sequence consisting of at least 15 consecutive bases selected from a gene (SEQ ID NO: 1 or 2) encoding verotoxin produced by verotoxin-producing Escherichia coli. Oligonucleotide for amplifying the Vero toxin gene. 2. The oligonucleotide according to claim 1, wherein at least two types of DNA fragments are selected from upstream and downstream of the insertion site of the foreign DNA fragment in the verotoxin gene. 3. At least two kinds of DNA fragments containing a nucleic acid sequence consisting of at least 15 consecutive bases selected from a gene encoding Vero toxin type 1 (SEQ ID NO: 1) produced by Vero toxin-producing Escherichia coli. Vero toxin 1
Oligonucleotide for amplifying type gene. 4. The oligonucleotide according to claim 3, wherein at least one of the at least two DNA fragments is selected from upstream and downstream of the insertion site of the foreign DNA fragment in the verotoxin gene. 5. At least two kinds of DNA fragments containing a nucleic acid sequence consisting of at least 15 consecutive bases selected from a gene encoding Vero toxin type 2 produced by Vero toxin-producing Escherichia coli (SEQ ID NO: 2). Verotoxin 2
Oligonucleotide for amplifying type gene. 6. The oligonucleotide according to claim 5, wherein at least one of the at least two DNA fragments is selected from upstream and downstream of the insertion site of the foreign DNA fragment in the verotoxin gene. 7. An oligonucleotide for amplifying a verotoxin gene according to any one of claims 1 to 6 as at least two kinds of primers, wherein an extension product of one of the primers is A method for amplifying a verotoxin gene, which comprises using an oligonucleotide that becomes a template for the other primer when separated from a complement. 8. An oligonucleotide for amplifying a verotoxin gene produced by the verotoxin-producing Escherichia coli according to claim 1, wherein the at least two primers are:
Vero toxin gene amplification, characterized in that when the extension product of one primer is separated from its complement, it contains a primer serving as a template for the other primer, a thermostable DNA polymerase, dNTPs and a buffer. reagent. 9. An exogenous DNA to a vero toxin gene from a chain length of an amplified gene fragment obtained by the amplification method according to claim 7.
A method for detecting insertion of a foreign DNA fragment generated in a verotoxin gene, comprising detecting insertion of the fragment. 10. An oligonucleotide for amplifying a verotoxin gene produced by the verotoxin-producing Escherichia coli according to claim 1, wherein at least two kinds of primers are an extension product of one of the primers, Detecting the insertion of a foreign DNA fragment generated in the verotoxin gene, which comprises a primer serving as a template for the other primer when separated from its complement, a thermostable DNA polymerase, dNTPs and a buffer. reagent. 11. An oligonucleotide comprising a nucleic acid sequence consisting of at least 15 consecutive bases among the nucleic acid sequences shown in SEQ ID NOs: 3 to 6 or a nucleic acid sequence complementary to said sequence. 12. An oligonucleotide comprising a nucleic acid sequence consisting of at least 15 consecutive bases of the nucleic acid sequence shown in SEQ ID NOs: 7 to 10 or a nucleic acid sequence complementary to said sequence. 13. The at least two kinds of primers,
An oligonucleotide for amplifying a Vero toxin type 1 gene, comprising a nucleic acid sequence consisting of at least 15 consecutive nucleotides among the nucleic acid sequences shown in SEQ ID NOs: 3 to 6 or a nucleic acid sequence complementary to said sequence. When the extension product of one primer is separated from its complement,
A method for amplifying a verotoxin type 1 gene, comprising using an oligonucleotide serving as a template for the other primer. 14. As at least two kinds of primers,
An oligonucleotide for amplifying a Vero toxin type 2 gene, comprising a nucleic acid sequence consisting of at least 15 consecutive nucleotides among the nucleic acid sequences shown in SEQ ID NOs: 7 to 10 or a nucleic acid sequence complementary to the nucleic acid sequence, A method for amplifying a verotoxin type 2 gene, comprising using an oligonucleotide serving as a template for the other primer when the extension product of one primer is separated from its complement. 15. Verotoxin 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: 3 to 6 or a nucleic acid sequence complementary to the sequence. An oligonucleotide for amplifying a Vero toxin type 1 gene produced by Escherichia coli, which, when an extension product of one primer is separated from its complement, a primer serving as a template for the other primer; Stable DNA polymerase, dNT
A reagent for amplifying a Vero toxin type 1 gene, comprising P and a buffer. 16. Verotoxin 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: 7 to 10 or a nucleic acid sequence complementary to the sequences. An oligonucleotide for amplifying a Vero toxin type 2 gene produced by a bacterium Escherichia coli, wherein when an extension product of one primer is separated from its complement, a primer serving as a template for the other primer; Stable DNA polymerase, dN
A reagent for amplifying a verotoxin type 2 gene, comprising TP and a buffer. 17. A verotoxin type 1 gene characterized by detecting the insertion of a foreign DNA fragment into a verotoxin type 1 gene from the chain length of the amplified gene fragment obtained by the amplification method according to claim 13. A method for detecting the resulting insertion of a foreign gene. 18. A verotoxin type 2 gene, characterized in that insertion of a foreign DNA fragment into a verotoxin type 2 gene is detected from the chain length of the amplified gene fragment obtained by the amplification method according to claim 14. A method for detecting the resulting insertion of a foreign gene. 19. Verotoxin 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 to 6 or a nucleic acid sequence complementary thereto. An oligonucleotide for amplifying a Vero toxin type 1 gene produced by Escherichia coli, which, when an extension product of one primer is separated from its complement, a primer serving as a template for the other primer; Stable DNA polymerase, dNT
A reagent for detecting insertion of a foreign gene generated in a verotoxin type 1 gene, comprising P and a buffer. 20. Verotoxin 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: 7 to 10 or a nucleic acid sequence complementary thereto. An oligonucleotide for amplifying a Vero toxin type 2 gene produced by a bacterium Escherichia coli, wherein when an extension product of one primer is separated from its complement, a primer serving as a template for the other primer; Stable DNA polymerase, dN
A reagent for detecting insertion of a foreign gene generated in a verotoxin type 2 gene, comprising TP and a buffer.