JPH04297489A - Detection of vero toxin gene by type and primer to be used therefor - Google Patents
Detection of vero toxin gene by type and primer to be used thereforInfo
- Publication number
- JPH04297489A JPH04297489A JP3087611A JP8761191A JPH04297489A JP H04297489 A JPH04297489 A JP H04297489A JP 3087611 A JP3087611 A JP 3087611A JP 8761191 A JP8761191 A JP 8761191A JP H04297489 A JPH04297489 A JP H04297489A
- Authority
- JP
- Japan
- Prior art keywords
- primer
- gene
- type
- sequence
- vt2vp
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108010017898 Shiga Toxins Proteins 0.000 title abstract description 17
- 238000001514 detection method Methods 0.000 title abstract description 6
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 42
- 238000000034 method Methods 0.000 claims description 20
- 230000000295 complement effect Effects 0.000 claims description 6
- 108700012359 toxins Proteins 0.000 claims description 2
- 238000004587 chromatography analysis Methods 0.000 claims 1
- 150000007523 nucleic acids Chemical group 0.000 description 17
- 108020004414 DNA Proteins 0.000 description 15
- 230000000692 anti-sense effect Effects 0.000 description 13
- 108091028043 Nucleic acid sequence Proteins 0.000 description 10
- 239000012634 fragment Substances 0.000 description 8
- 108020004707 nucleic acids Proteins 0.000 description 8
- 102000039446 nucleic acids Human genes 0.000 description 8
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 3
- 208000032759 Hemolytic-Uremic Syndrome Diseases 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 230000008506 pathogenesis Effects 0.000 description 3
- 238000011895 specific detection Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 108020003215 DNA Probes Proteins 0.000 description 2
- 230000004544 DNA amplification Effects 0.000 description 2
- 239000003298 DNA probe Substances 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 108010006785 Taq Polymerase Proteins 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 230000002934 lysing effect Effects 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 244000000010 microbial pathogen Species 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- 241000510930 Brachyspira pilosicoli Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 206010014896 Enterocolitis haemorrhagic Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 101001128634 Homo sapiens NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 2, mitochondrial Proteins 0.000 description 1
- 102100032194 NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 2, mitochondrial Human genes 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 108010079723 Shiga Toxin Proteins 0.000 description 1
- 241000607768 Shigella Species 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
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- 235000019322 gelatine Nutrition 0.000 description 1
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- MCJGNVYPOGVAJF-UHFFFAOYSA-N quinolin-8-ol Chemical compound C1=CN=C2C(O)=CC=CC2=C1 MCJGNVYPOGVAJF-UHFFFAOYSA-N 0.000 description 1
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- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
Abstract
Description
【０００１】0001
【産業上の利用分野】本発明は、ＰＣＲ（ＤＮＡ増幅）
法によるＶｅｒｏ毒素遺伝子の型別検出方法およびそれ
に用いるプライマ−に関する。[Industrial Application Field] The present invention is directed to PCR (DNA amplification)
The present invention relates to a method for detecting Vero toxin genes by type and primers used therein.
【０００２】0002
【従来の技術】Ｖｅｒｏ毒素産生性大腸菌（Ｖｅｒｏｔ
ｏｘｉｎ−ｐｒｏｄｕｃｉｎｇ Ｅｓｃｈｅｒｉｃｈｉ
ａ ｃｏｌｉ， ＶＴＥＣ）による出血性大腸炎や溶血
性***症侯群の報告例は、我が国においても年々増加
の傾向にある。下痢と腹痛を伴う症状から短期間で溶血
性***症侯群を続発し、死亡する例も報告されており
、ＶＴＥＣの迅速な検出・同定法の確立が急務とされて
いる。[Prior Art] Vero toxin-producing Escherichia coli
oxin-producing Escherichi
The number of reported cases of hemorrhagic colitis and hemolytic uremic syndrome caused by A. coli, VTEC) is increasing year by year in Japan. There have been reports of cases in which hemolytic uremic syndrome develops in a short period of time due to symptoms accompanied by diarrhea and abdominal pain, resulting in death, and there is an urgent need to establish a method for rapid detection and identification of VTEC.
【０００３】本菌感染において重要な病原的役割を果た
すと考えられているＶｅｒｏ毒素（ＶＴ）は、現在まで
に少なくとも５種類が報告されている。すなわち、志賀
赤痢菌の産生する志賀毒素と分子構造が全く同一のＶＴ
１、生物学的性状はＶＴ１と類似しているが免疫学的物
理化学的性状が全く異なるＶＴ２、ＶＴ２と一部共通抗
原性を有する豚浮腫病由来の大腸菌が産生するＶＴ２ｖ
ｐ、ＶＴ２と一部共通抗原性を有し溶血性***症侯群
患者由来の大腸菌の産生するＶＴ２ｖｈａ、ＶＴ２ｖｈ
ｂの５種類である。At least five types of Vero toxin (VT), which are thought to play an important pathogenic role in infection with this fungus, have been reported to date. In other words, VT has exactly the same molecular structure as Shiga toxin produced by Shiga Shigella.
1. VT2, which has similar biological properties to VT1 but completely different immunological and physicochemical properties, and VT2v produced by Escherichia coli derived from swine edema disease, which has some common antigenicity with VT2.
p, VT2vha, VT2vh produced by Escherichia coli derived from patients with hemolytic uremic syndrome and having some common antigenicity with VT2.
There are five types of b.
【０００４】この５種類のＶＴのＤＮＡ配列は、以下の
通り、既に報告されている（図１および図２参照）。
ＶＴ１： Ｍｉｃｒｏｂｉａｌ Ｐａｔｈｏｇｅｎｅｓ
ｉｓ １９８７， ２： １４７−１５３Ｊ． Ｂａｃ
ｔｅｒｉｏｌ．， Ｓｅｐｔ． １９８７， ｐ． ４
３１３−４３１９， Ｖｏｌ． １６９， Ｎｏ． ９
Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳ
Ａ， Ｖｏｌ． ８４，ｐｐ． ４３６４−４３６８，
Ｊｕｌｙ １９８７
Ｍｉｃｒｏｂｉａｌ Ｐａｔｈｏｇｅｎｅｓｉｓ １９
８８， ５： ３５７−３６９ＶＴ２： ＦＥＭＳ Ｍ
ｉｃｒｏｂｉｏｌｏｇｙ Ｌｅｔｔｅｒｓ ４４ （１
９８７） １０９−１１４（配列番号：１）
ＶＴ２ｖｐ： Ｍｉｃｒｏｂｉａｌ Ｐａｔｈｏｇｅｎ
ｅｓｉｓ １９８８，５： ４１９−４２６Ｊ． Ｂａ
ｃｔｅｒｉｏｌ．， Ｓｅｐｔ． １９８８， ｐ．
４２２３−４２３０， Ｖｏｌ． １７０， Ｎｏ．
９
ＶＴ２ｖｈａ、ＶＴ２ｖｈｂ： Ｍｉｃｒｏｂｉａｌ
Ｐａｔｈｏｇｅｎｅｓｉｓ １９９０， ８： ４７−
６０[0004] The DNA sequences of these five types of VT have already been reported as follows (see FIGS. 1 and 2). VT1: Microbial Pathogenes
is 1987, 2: 147-153J. Bac
teriol. , Sept. 1987, p. 4
313-4319, Vol. 169, No. 9 Proc. Natl. Acad. Sci. US
A, Vol. 84, pp. 4364-4368,
July 1987 Microbial Pathogenesis 19
88, 5: 357-369VT2: FEMS M
icrobiology Letters 44 (1
987) 109-114 (SEQ ID NO: 1) VT2vp: Microbial Pathogen
esis 1988, 5: 419-426J. Ba
cteriol. , Sept. 1988, p.
4223-4230, Vol. 170, No.
9 VT2vha, VT2vhb: Microbial
Pathogenesis 1990, 8: 47-
60
【０００５】また、ＶＴ１遺伝子およびＶＴ２遺伝子の
それぞれをＰＣＲ法により増幅したという報告が既にな
されている。
ＶＴ１： Ｔｈｅ Ｊｏｕｒｎａｌ ｏｆ Ｉｎｆｅｃ
ｔｉｏｕｓ Ｄｉｓｅａｓｅｓ １９９０；１６２：１
１９５−１１９８
ＶＴ２： Ｊｏｕｒｎａｌ ｏｆ Ｃｌｉｎｉｃａｌ
Ｍｉｃｒｏｂｉｏｌｏｇｙ， Ｏｃｔ． １９９０，
ｐ． ２３５１−２３５３，
Ｖｏｌ． ２８， Ｎｏ． １０[0005] Furthermore, there have already been reports that the VT1 gene and VT2 gene were each amplified by the PCR method. VT1: The Journal of Infec
tious Diseases 1990;162:1
195-1198 VT2: Journal of Clinical
Microbiology, Oct. 1990,
p. 2351-2353, Vol. 28, No. 10
【０００６】このように、ＶＴ１遺伝子およびＶＴ２遺
伝子のＰＣＲ法による増幅の報告はあるものの、これが
特異的なものかどうかは不明である。また、それ以外の
ＶＴ２ｖｐ、ＶＴ２ｖｈａ、ＶＴ２ｖｈｂ遺伝子の増幅
に関する報告は無い。[0006] Although there have been reports of amplification of the VT1 and VT2 genes by PCR, it is unclear whether this is specific. Furthermore, there are no reports regarding amplification of other VT2vp, VT2vha, and VT2vhb genes.
【０００７】[0007]
【発明が解決しようとする課題】上記の通り、ＶＴ１、
ＶＴ２、ＶＴ２ｖｐ、ＶＴ２ｖｈａ、およびＶＴ２ｖｈ
ｂ遺伝子を型別に検出する方法は未だ報告されておらず
、これらの型別検出方法、およびこれらを型別にＰＣＲ
法で特異的に増幅しうるプライマ−が待望されていた。[Problem to be solved by the invention] As mentioned above, VT1,
VT2, VT2vp, VT2vha, and VT2vh
A method for detecting the b gene by type has not yet been reported, and these detection methods and PCR methods for each type are necessary.
There has been a long-awaited need for primers that can be specifically amplified by this method.
【０００８】[0008]
【課題を解決するための手段】本発明は、ＰＣＲ法によ
って、ＶＴ１、ＶＴ２、ＶＴ２ｖｐ、およびＶＴ２ｖｈ
遺伝子のいずれか１つのみを特異的に検出し得るプライ
マ−を提供する。本明細書中、「ＶＴ２ｖｈ遺伝子」と
は、ＶＴｖｈａ遺伝子およびＶＴｖｈｂ遺伝子の両方を
意味する。従って、「ＶＴ２ｖｈ遺伝子を特異的に検出
しうるプライマ−」は、ＶＴｖｈａ遺伝子およびＶＴｖ
ｈｂ遺伝子の両方を増幅しうるプライマ−を意味する。[Means for Solving the Problems] The present invention provides VT1, VT2, VT2vp, and VT2vh by PCR method.
Provided are primers that can specifically detect only one of the genes. As used herein, "VT2vh gene" means both the VTvha gene and the VTvhb gene. Therefore, "a primer capable of specifically detecting the VT2vh gene" refers to the VTvha gene and the VTvh gene.
It means a primer that can amplify both hb genes.
【０００９】本発明のプライマ−は、ＶＴ１、ＶＴ２、
ＶＴ２ｖｐ、およびＶＴ２ｖｈ遺伝子のいずれか１つと
のみ同一であり、他の３つとは異なる塩基配列を有する
ものである。
ＶＴ２ｖｈ遺伝子と同一の塩基配列を有するということ
は、ＶＴｖｈａ遺伝子およびＶＴｖｈｂ遺伝子の塩基配
列が異なる部分については、いずれか一方と同一である
ことを意味する。本発明のプライマ−の鎖長は１５〜４
０塩基であり、これはＰＣＲ法に用いられるプライマ−
の通常の長さである。The primers of the present invention are VT1, VT2,
It is identical to only one of the VT2vp and VT2vh genes and has a different base sequence from the other three genes. Having the same base sequence as the VT2vh gene means that the portions where the base sequences of the VTvha gene and VTvhb gene differ are the same as either one. The chain length of the primer of the present invention is 15 to 4
0 base, which is the primer used in the PCR method.
This is the normal length.
【００１０】具体的には、５つの遺伝子の配列を比較し
て（図１および図２参照）、それぞれに特異的な領域を
検索し、図３に示す領域から本発明のプライマ−がデザ
インされた。Specifically, the sequences of five genes were compared (see FIGS. 1 and 2), specific regions were searched for each, and the primers of the present invention were designed from the regions shown in FIG. Ta.
【００１１】すなわち、ＶＴ１遺伝子のみを特異的に検
出しうるプライマ−としては、
５’ＧＣＡＧＴＴＣＧＴＧＧＣＡＡＧＡＧＣＧ ３’お
よび５’ＧＣＧＴＣＧＣＣＡＧＣＧＣＡＣＴＴＧ ３’
が例示される。[0011] Specifically, examples of primers that can specifically detect only the VT1 gene include 5'GCAGTTCGTGGCAAGAGCG 3' and 5'GCGTCGCCAGCGCACTTG 3'.
【００１２】ＶＴ２遺伝子のみを特異的に検出しうるプ
ライマ−としては、
５’ＡＡＴＴＴＡＴＡＴＧＴＧＧＣＡＧＧＧＴＴＣ ３
’および５’ＣＴＴＣＡＣＴＧＴＡＡＡＴＧＴＧＴＣＡ
ＴＣ ３’
が例示される。[0012] As a primer that can specifically detect only the VT2 gene, 5'AATTTATATGTGGCAGGGTTC 3
' and 5'CTTCACTGTAAATGTGTCA
TC 3' is exemplified.
【００１３】ＶＴ２ｖｐ遺伝子のみを特異的に検出しう
るプライマ−としては、
５’ＡＡＧＣＣＴＴＡＣＧＧＴＴＣＡＧＧＣＡＡ ３’
および５’ＣＡＧＴＴＡＡＡＣＴＴＣＡＣＣＴＧＧＧＣ
３’
が例示される。[0013] As a primer that can specifically detect only the VT2vp gene, 5'AAGCCTTACGGTTCAGGCAA 3'
and 5'CAGTTAAACTTCACCTGGGC
3' is exemplified.
【００１４】ＶＴ２ｖｈ遺伝子のみを特異的に検出しう
るプライマ−としては、
５’ＧＡＡＡＴＡＴＣＧＡＣＴＣＣＴＣＴＴＧＡＧ ３
’および５’ＧＴＡＴＴＣＴＴＴＣＣＣＧＧＣＣＡＣＴ
３’
が例示される。[0014] As a primer that can specifically detect only the VT2vh gene, 5'GAAATATCGACTCCTCTTGAG 3
' and 5'GTATTCTTTCCCGGCCACT
3' is exemplified.
【００１５】これらのプライマ−に対する相補鎖も、も
ちろん本発明のプライマ−として用いることができる。
また、これらのプライマ−を数塩基短縮またはかなり延
長しても、１５〜４０塩基にわたり、各遺伝子に対して
特異的なものとなることは図１および図２から明らかで
あり、そのようなものも本発明の範囲に含まれる。Of course, complementary strands to these primers can also be used as primers in the present invention. Furthermore, it is clear from Figures 1 and 2 that even if these primers are shortened by a few bases or considerably lengthened, they will span 15 to 40 bases and will be specific to each gene. Also included within the scope of the present invention.
【００１６】通常、ＰＣＲ法に用いるプライマ−は、１
５塩基以上であると、所望の特異性が得られる。従って
、本発明のプライマ−も１５塩基以上であることが望ま
しく、上記の配列のうち、少なくとも連続した１５塩基
またはその相補鎖を有するプライマ−も本発明に包含さ
れる。[0016] Usually, the primers used in the PCR method are 1
When the number of bases is 5 or more, the desired specificity can be obtained. Therefore, the primer of the present invention is also desirably 15 bases or more, and the present invention also includes a primer having at least 15 consecutive bases or its complementary strand among the above sequences.
【００１７】本発明のプライマ−は、ＰＣＲ法によるＶ
Ｔ遺伝子の型別検出用プライマ−として用いられるだけ
でなく、ＶＴ遺伝子の型別検出用プロ−ブとして用いる
こともできる。従って、本発明のプライマ−と同一の塩
基配列を有するオリゴヌクレオチドは、その用途に限定
されず、すべて本発明に包含される。[0017] The primer of the present invention can be used for V
It can be used not only as a primer for type-specific detection of T genes, but also as a probe for type-specific detection of VT genes. Therefore, any oligonucleotide having the same base sequence as the primer of the present invention is included in the present invention without being limited in its use.
【００１８】本発明のプライマ−を用いるＰＣＲ法によ
るＶＴ遺伝子の型別検出は、通常の方法で行なえばよい
。例えば、被検菌を溶菌させたのち、Ｔａｑ ＤＮＡポ
リメラ−ゼを加え、変性（９４℃、１分）→アニ−リン
グ（６２℃、１．５分）→合成（７２℃、１．５分）の
反応を約３０回繰り返し、増幅したＤＮＡ断片をポリア
クリルアミドゲル電気泳動法により検出すればよい。用
いるプライマ−は、センス／アンチセンスの２種のうち
、両方が特異性の高いものである必要はなく、一方のみ
でもよい。VT gene type detection by PCR using the primers of the present invention may be carried out by a conventional method. For example, after lysing the test bacteria, add Taq DNA polymerase, denaturation (94℃, 1 minute) → annealing (62℃, 1.5 minutes) → synthesis (72℃, 1.5 minutes). ) may be repeated approximately 30 times, and the amplified DNA fragments may be detected by polyacrylamide gel electrophoresis. It is not necessary that both sense and antisense primers have high specificity, and only one of them may be used.
【００１９】[0019]
【実施例】プライマ−：それぞれのＶＴをコ−ドする遺
伝子の塩基配列を比較して（図１および図２参照）、そ
れぞれに特異的な領域を検索し、図３に示す領域から、
以下の配列を有するオリゴヌクレオチドを合成した。[Example] Primer: Compare the base sequences of the genes encoding each VT (see Figures 1 and 2), search for each specific region, and from the regions shown in Figure 3,
An oligonucleotide having the following sequence was synthesized.
【００２０】ＶＴ１用：
センスプライマ−：５’ＧＣＡＧＴＴＣＧＴＧＧＣＡＡ
ＧＡＧＣＧ ３’ （配列番号：２）
アンチセンスプライマ−：５’ＧＣＧＴＣＧＣＣＡＧＣ
ＧＣＡＣＴＴＧ３’ （配列番号：３）[0020] For VT1: Sense primer: 5'GCAGTTCGTGGCAA
GAGCG 3' (SEQ ID NO: 2) Antisense primer: 5'GCGTCGCCAGC
GCACTTG3' (SEQ ID NO: 3)
【００２１】ＶＴ２用：
センスプライマ−：５’ＡＡＴＴＴＡＴＡＴＧＴＧＧＣ
ＡＧＧＧＴＴＣ ３’ （配列番号：４）
アンチセンスプライマ−：５’ＣＴＴＣＡＣＴＧＴＡＡ
ＡＴＧＴＧＴＣＡＴＣ ３’ （配列番号：５）[0021] For VT2: Sense primer: 5'AATTTATATGTGGC
AGGGTTC 3' (SEQ ID NO: 4) Antisense primer: 5'CTTCACTGTAA
ATGTGTCATC 3' (SEQ ID NO: 5)
【００２２】ＶＴ２ｖｐ用：
センスプライマ−：５’ＡＡＧＣＣＴＴＡＣＧＧＴＴＣ
ＡＧＧＣＡＡ３’ （配列番号：６）
アンチセンスプライマ−：５’ＣＡＧＴＴＡＡＡＣＴＴ
ＣＡＣＣＴＧＧＧＣ ３’ （配列番号：７）[0022] For VT2vp: Sense primer: 5'AAGCCTTACGGTTC
AGGCAA3' (SEQ ID NO: 6) Antisense primer: 5'CAGTTAAACTT
CACCTGGGC 3' (SEQ ID NO: 7)
【００２３】ＶＴ２ｖｈ用：
センスプライマ−：５’ＧＡＡＡＴＡＴＣＧＡＣＴＣＣ
ＴＣＴＴＧＡＧ ３’ （配列番号：８）
アンチセンスプライマ−：５’ＧＴＡＴＴＣＴＴＴＣＣ
ＣＧＧＣＣＡＣＴ ３’ （配列番号：９）[0023] For VT2vh: Sense primer: 5'GAAATATCGACTCC
TCTTGAG 3' (SEQ ID NO: 8) Antisense primer: 5'GTATTCTTTCC
CGGCCACT 3' (SEQ ID NO: 9)
【００２４】使用菌株：各ＶＴの陽性コントロ−ルとし
て、ＶＴ１、ＶＴ２、ＶＴ２ｖｈａ、ＶＴ２ｖｈｂにつ
いては、クロ−ニング株を、ＶＴ２ｖｐについては、オ
リゴプロ−ブでＶＴ２ｖｐ以外のＶＴは保持していない
ことを確認した菌株を用いた。試験菌株は、日本、アメ
リカ、イギリス、カナダ、ミャンマなどの患者および家
畜から分離された５６株のＶＴＥＣである。Bacterial strains used: As positive controls for each VT, cloning strains were used for VT1, VT2, VT2vha, and VT2vhb, and oligoprobes were used for VT2vp to confirm that no VTs other than VT2vp were retained. The confirmed bacterial strain was used. The test strains were 56 strains of VTEC isolated from patients and livestock in Japan, the United States, the United Kingdom, Canada, Myanmar, etc.
【００２５】ＰＣＲ：ＤＮＡの増幅反応は、１０ ｍＭ
Ｔｒｉｓ−ＨＣｌ （ｐＨ８．５）、５０ ｍＭ Ｋ
Ｃｌ、１．５ ｍＭ ＭｇＣｌ２、０．０１％（Ｗ／Ｖ
） ゼラチン、２００ μＭ各ｄＮＴＰ、および１ μ
Ｍ各 プライマ−を含む溶液に、被検菌の培養液１μｌ
を加えた反応溶液１００μｌを９４℃、５分間で溶菌さ
せた後、Ｔａｑ ＤＮＡポリメラ−ゼ２．５単位を加え
、変性（９４℃、１分）→アニ−リング（６２℃、１．
５分）→合成（７２℃、１．５分）の反応を３０回繰り
返した。増幅したＤＮＡ断片は、６％ポリアクリルアミ
ドゲル電気泳動法により検出した。[0025] PCR: DNA amplification reaction is carried out at 10 mM
Tris-HCl (pH 8.5), 50 mM K
Cl, 1.5 mM MgCl2, 0.01% (W/V
) Gelatin, 200 μM each dNTP, and 1 μM
Add 1 μl of the culture solution of the test bacteria to the solution containing each M primer.
After lysing 100 μl of the reaction solution at 94°C for 5 minutes, 2.5 units of Taq DNA polymerase was added, followed by denaturation (94°C, 1 minute) → annealing (62°C, 1 minute).
5 minutes) → synthesis (72°C, 1.5 minutes) was repeated 30 times. The amplified DNA fragments were detected by 6% polyacrylamide gel electrophoresis.
【００２６】結果：各ＶＴ遺伝子に特異的なプライマ−
と陽性コントロ−ル株を用いてＰＣＲを行なった結果、
それぞれに特異的なＤＮＡ断片のみが増幅された（ＶＴ
１：５０３ｂｐ、ＶＴ２：８０６ｂｐ、ＶＴ２ｖｐ：６
７０ｂｐ、ＶＴ２ｖｈ：９８０ｂｐ、図４および図５参
照）。ＶＴ２ｖｈを産生する菌株は、９９％の相同性を
持つ２種のＶＴ２ｖｈオペロン（ｖｔｘ２ｖｈａ、ｖｔ
ｘ２ｖｈｂ）を持つが、両者を特異的に検出する事は不
可能であったので、両オペロンからそれぞれ１ケ所ずつ
プライマ−用の配列を選出し、両オペロンを同時に検出
した。増幅したＤＮＡ断片が目的の断片であることは、
制限酵素での消化により、計算値と一致するＤＮＡ断片
に分離することで確認した。その結果、それぞれのプラ
イマ−を用いるＰＣＲが、各遺伝子に対して特異的なも
のであることが確認された。Results: Primers specific to each VT gene
As a result of PCR using the positive control strain and
Only specific DNA fragments for each were amplified (VT
1:503bp, VT2:806bp, VT2vp:6
70bp, VT2vh: 980bp, see Figures 4 and 5). The strain producing VT2vh has two VT2vh operons (vtx2vha, vt
x2vhb), but since it was impossible to specifically detect both, one primer sequence was selected from each of both operons, and both operons were detected simultaneously. The fact that the amplified DNA fragment is the desired fragment means that
This was confirmed by separating DNA fragments that matched the calculated values by digestion with restriction enzymes. As a result, it was confirmed that PCR using each primer was specific for each gene.
【００２７】患者および家畜由来の５６株のＶＴＥＣに
ついてＰＣＲ法で調べた結果、ＶＴ１、ＶＴ２、ＶＴ２
ｖｐ、ＶＴ２ｖｈそれぞれ単独で陽性を示したものが９
、４、１２、及び３株であった。ＶＴ１とＶＴ２、ＶＴ
１とＶＴ２ｖｈの両方で陽性を示したものが、それぞれ
１０および２株であった。いずれのプライマ−でもＤＮ
Ａの増幅が認められどれとも反応しなかったものが１６
株存在した。ｂｅａｄ−ＥＬＩＳＡ法およびＤＮＡプロ
−ブ法の結果と比較したところ、これらの方法では陽性
であったにもかかわらず、ＰＣＲ法で陰性を示した菌株
が存在した。またＰＣＲ法もしくは、ＤＮＡプロ−ブで
陰性であったにもかかわらず、ＶＴ２ｖｈに特異的なｂ
ｅａｄ−ＥＬＩＳＡ法で陽性を示した菌株が存在した。
したがって、この菌株は４種のＶＴと異なる新しいＶｅ
ｒｏ毒素を産生していると考えられた。[0027] As a result of investigating 56 strains of VTEC derived from patients and livestock by PCR method, VT1, VT2, VT2
9 cases showed positive for vp and VT2vh alone.
, 4, 12, and 3 strains. VT1 and VT2, VT
10 and 2 strains were positive for both VT2vh and VT2vh, respectively. DN with either primer
16 showed amplification of A but did not react with any of them.
There were stocks. When the results were compared with those of the bead-ELISA method and the DNA probe method, there were strains that were positive in these methods but negative in the PCR method. In addition, even though PCR or DNA probe results were negative, VT2vh-specific b
There were strains that showed positive results by the ead-ELISA method. Therefore, this strain has four types of VT and a new Ve
It was thought that they were producing ro toxin.
【００２８】[0028]
【発明の効果】本発明のプライマ−を用いるＶＴ遺伝子
の型別検出方法によると、ＶＴ１、ＶＴ２、ＶＴ２ｖｐ
、ＶＴ２ｖｈ遺伝子のそれぞれを特異的に検出すること
が可能であり、ＶＴＥＣの迅速で詳細な型別検出が可能
になる。Effects of the Invention According to the method for detecting VT genes by type using the primers of the present invention, VT1, VT2, VT2vp
, it is possible to specifically detect each of the VT2vh genes, and rapid and detailed type-specific detection of VTEC is possible.
【００２９】配列番号：１
配列の長さ：１２４１
配列の型：核酸
配列
ＡＴＧＡＡＧＴＧＴＡ ＴＡＴＴＡＴＴＴＡＡ ＡＴＧ
ＧＧＴＡＣＴＧ ＴＧＣＣＴＧＴＴＡＣ ＴＧＧＧＴＴ
ＴＴＴＣ ＴＴＣＧＧＴＡＴＣＣ ６０ＴＡＴ
ＴＣＣＣＧＧＧ ＡＧＴＴＴＡＣＧＡＴ ＡＧＡＣＴＴ
ＴＴＣＧ ＡＣＣＣＡＡＣＡＡＡ ＧＴＴＡＴＧＴＣＴ
Ｃ ＴＴＣＧＴＴＡＡＡＴ １２０ＡＧＴＡＴＡ
ＣＧＧＡ ＣＡＧＡＧＡＴＡＴＣ ＧＡＣＣＣＣＴＣＴ
Ｔ ＧＡＡＣＡＴＡＴＡＴ ＣＴＣＡＧＧＧＧＡＣ Ｃ
ＡＣＡＴＣＧＧＴＧ １８０ＴＣＴＧＴＴＡＴＴ
Ａ ＡＣＣＡＣＡＣＣＣＣ ＡＣＣＧＧＧＣＡＧＴ Ｔ
ＡＴＴＴＴＧＣＴＧ ＴＧＧＡＴＡＴＡＣＧ ＡＧＧＧ
ＣＴＴＧＡＴ ２４０ＧＴＣＴＡＴＣＡＧＧ Ｃ
ＧＣＧＴＴＴＴＧＡ ＣＣＡＴＣＴＴＣＧＴ ＣＴＧＡ
ＴＴＡＴＴＧ ＡＧＣＡＡＡＡＴＡＡ ＴＴＴＡＴＡＴ
ＧＴＧ ３００ＧＣＣＧＧＧＴＴＣＧ ＴＴＡＡ
ＴＡＣＧＧＣ ＡＡＣＡＡＡＴＡＣＴ ＴＴＣＴＡＣＣ
ＧＴＴ ＴＴＴＣＡＧＡＴＴＴ ＴＡＣＡＣＡＴＡＴＡ
３６０ＴＣＡＧＴＧＣＣＣＧ ＧＴＧＴＧＡＣ
ＡＡＣ ＧＧＴＴＴＣＣＡＴＧ ＡＣＡＡＣＧＧＡＣＡ
ＧＣＡＧＴＴＡＴＡＣ ＣＡＣＴＣＴＧＣＡＡ
４２０ＣＧＴＧＴＣＧＣＡＧ ＣＧＣＴＧＧＡＡＣＧ
ＴＴＣＣＧＧＡＡＴＧ ＣＡＡＡＴＣＡＧＴＣ ＧＴ
ＣＡＣＴＣＡＣＴ ＧＧＴＴＴＣＡＴＣＡ ４８
０ＴＡＴＣＴＧＧＣＧＴ ＴＡＡＴＧＧＡＧＴＴ ＣＡ
ＧＴＧＧＴＡＡＴ ＡＣＡＡＴＧＡＣＣＡ ＧＡＧＡＴ
ＧＣＡＴＣ ＣＡＧＡＧＣＡＧＴＴ ５４０ＣＴ
ＧＣＧＴＴＴＴＧ ＴＣＡＣＴＧＴＣＡＣ ＡＧＣＡＧ
ＡＡＧＣＣ ＴＴＡＣＧＣＴＴＣＡ ＧＧＣＡＧＡＴＡ
ＣＡ ＧＡＧＡＧＡＡＴＴＴ ６００ＣＧＴＣＡ
ＧＧＣＡＣ ＴＧＴＣＴＧＡＡＡＣ ＴＧＣＴＣＣＴＧ
ＴＧ ＴＡＴＡＣＧＡＴＧＡ ＣＧＣＣＧＧＧＡＧＡ
ＣＧＴＧＧＡＣＣＴＣ ６６０ＡＣＴＣＴＧＡＡ
ＣＴ ＧＧＧＧＧＣＧＡＡＴ ＣＡＧＣＡＡＴＧＴＧ
ＣＴＴＣＣＧＧＡＧＴ ＡＴＣＧＧＧＧＡＧＡ ＧＧＡ
ＴＧＧＴＧＴＣ ７２０ＡＧＡＧＴＧＧＧＧＡ
ＧＡＡＴＡＴＣＣＴＴ ＴＡＡＴＡＡＴＡＴＡ ＴＣＡ
ＧＣＧＡＴＡＣ ＴＧＧＧＧＡＣＴＧＴ ＧＧＣＣＧＴ
ＴＡＴＡ ７８０ＣＴＧＡＡＴＴＧＣＣ ＡＴＣ
ＡＴＣＡＧＧＧ ＧＧＣＧＣＧＴＴＣＴ ＧＴＴＣＧＣ
ＧＣＣＧ ＴＧＡＡＴＧＡＡＧＡ ＧＡＧＴＣＡＡＣＣ
Ａ ８４０ＧＡＡＴＧＴＣＡＧＡ ＴＡＡＣＴＧ
ＧＣＧＡ ＣＡＧＧＣＣＴＧＴＴ ＡＴＡＡＡＡＡＴＡ
Ａ ＡＣＡＡＴＡＣＡＴＴ ＡＴＧＧＧＡＡＡＧＴ
９００ＡＡＴＡＣＡＧＣＴＧ ＣＡＧＣＧＴＴＴＣ
Ｔ ＧＡＡＣＡＧＡＡＡＧ ＴＣＡＣＡＧＴＴＴＴ Ｔ
ＡＴＡＴＡＣＡＡＣ ＧＧＧＴＡＡＡＴＡＡ ９
６０ＡＧＧＡＧＴＴＡＡＧ ＣＡＴＧＡＡＧＡＡＧ Ａ
ＴＧＴＴＴＡＴＧＧ ＣＧＧＴＴＴＴＡＴＴ ＴＧＣＡ
ＴＴＡＧＣＴ ＴＣＴＧＴＴＡＡＴＧ １０２０Ｃ
ＡＡＴＧＧＣＧＧＣ ＧＧＡＴＴＧＴＧＣＴ ＡＡＡＧ
ＧＴＡＡＡＡ ＴＴＧＡＧＴＴＴＴＣ ＣＡＡＧＴＡＴ
ＡＡＴ ＧＡＧＧＡＴＧＡＣＡ １０８０ＣＡＴＴ
ＴＡＣＡＧＴ ＧＡＡＧＧＴＴＧＡＣ ＧＧＧＡＡＡＧ
ＡＡＴ ＡＣＴＧＧＡＣＣＡＧ ＴＣＧＣＴＧＧＡＡＴ
ＣＴＧＣＡＡＣＣＧＴ １１４０ＴＡＣＴＧＣＡ
ＡＡＧ ＴＧＣＴＣＡＧＴＴＧ ＡＣＡＧＧＡＡＴＧＡ
ＣＴＧＴＣＡＣＡＡＴ ＣＡＡＡＴＣＣＡＧＴ ＡＣ
ＣＴＧＴＧＡＡＴ １２００ＣＡＧＧＣＴＣＣＧＧ
ＡＴＴＴＧＣＴＧＡＡ ＧＴＧＣＡＧＴＴＴＡ ＡＴ
ＡＡＴＧＡＣＴＧ Ａ
１２４１SEQ ID NO: 1 Sequence length: 1241 Sequence type: Nucleic acid sequence ATGAAGTGTA TATTATTTAA ATG
GGTACTG TGCCTGTTAC TGGGTT
TTTC TTCGGTATCC 60TAT
TCCCGGG AGTTTACGAT AGACTT
TTCG ACCCAACAAA GTTATGTCT
CTTCGTTAAAT 120AGTATA
CGGA CAGAGATATC GACCCCTCT
T GAACATATAT CTCAGGGGAC C
ACATCGGTG 180TCTGTTATT
A ACCACACCCC ACCGGGCAG T
ATTTTGCTG TGGATATACG AGGG
CTTGAT 240GTCTATCAGG C
GCGTTTTGA CCATCTTCGT CTGA
TTATTG AGCAAAATAA TTTATAT
GTG 300GCCGGGTTCGTTAA
TACGGC AACAAAATACT TTCTACC
GTT TTTCAGATTT TACACATATA
360TCAGTGCCCGGTGTGAC
AAC GGTTTCCATG ACAACGGACA
GCAGTTATAC CACTCTGCAA
420CGTGTCGCAG CGCTGGAACG
TTCCGGAATG CAAATCAGTC GT
CACTCACT GGTTTCATCA 48
0TATCTGGCGTTAATGGAGTT CA
GTGGTAAT ACAAT GACCA GAGAT
GCATC CAGAGCAGTT 540CT
GCGTTTTG TCACTGTCAC AGCAG
AAGCC TTACGCTTCA GGCAGATA
CA GAGAGAATTT 600CGTCA
GGCAC TGTCTGAAAC TGCTCCTG
TG TATACGATGA CGCCGGGAGA
CGTGGACCTC 660ACTCTGAA
CT GGGGGCGAAT CAGCAATGTG
CTTCCGGAGT ATCGGGGAGA GGA
TGGTGTC 720AGAGTGGGGA
GAATATCCTTT TAATAATATA TCA
GCGATAC TGGGGACTGT GGCCGT
TATA 780CTGAATTGCC ATC
ATCAGGG GGCGCGTTCT GTTCGC
GCCG TGAATGAAGA GAGTCAACC
A 840GAATGTCAGATAACTG
GCGA CAGGCCTGTT ATAAAAAAATA
A ACAATACATT ATGGGAAAGT
900AATACAGCTG CAGCGTTTTC
T GAACAGAAAG TCACAGTTTT T
ATATACAAAC GGGTAAATAA 9
60AGGAGTTAAG CATGAAGAAAG A
TGTTTATGG CGGTTTATT TGCA
TTAGCT TCTGTTAATG 1020C
AATGGCGGC GGATTGTGCT AAAG
GTAAAA TTGAGTTTTC CAAGTAT
AAT GAGGAT GACA 1080CATT
TACAGT GAAGGTTGAC GGGAAAG
AAT ACTGGACCAG TCGCTGGAAT
CTGCAACCGT 1140TACTGCA
AAG TGCTCAGTTG ACAGGAATGA
CTGTCACAAT CAAATCCAGT AC
CTGTGAAT 1200CAGGCTCCGG
ATTTGCTGAA GTGCAGTTTA AT
AATGACTG A
1241
【００３０】配列番号：２
配列の長さ：１９
配列の型：核酸
配列の種類：他の核酸 合成ＤＮＡ
アンチセンス：Ｎｏ
配列
ＧＣＡＧＴＴＣＧＴＧ ＧＣＡＡＧＡＧＣＧ
１９SEQ ID NO: 2 Sequence length: 19 Sequence type: Nucleic acid Sequence type: Other nucleic acid Synthetic DNA Antisense: No Sequence GCAGTTCGTG GCAAGAGCG
19
【００３１】配列番号：３
配列の長さ：１８
配列の型：核酸
配列の種類：他の核酸 合成ＤＮＡ
アンチセンス：Ｙｅｓ
配列
ＧＣＧＴＣＧＣＣＡＧ ＣＧＣＡＣＴＴＧ
１８SEQ ID NO: 3 Sequence length: 18 Sequence type: Nucleic acid Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence GCGTCGCCAG CGCACTTG
18
【００３２】配列番号：４
配列の長さ：２１
配列の型：核酸
配列の種類：他の核酸 合成ＤＮＡ
アンチセンス：Ｎｏ
配列
ＡＡＴＴＴＡＴＡＴＧ ＴＧＧＣＡＧＧＧＴＴ Ｃ
２１Sequence number: 4 Sequence length: 21 Sequence type: Nucleic acid Sequence type: Other nucleic acid Synthetic DNA Antisense: No Sequence AATTTATATG TGGCAGGGTT C
21
【００３３】配列番号：５
配列の長さ：２１
配列の型：核酸
配列の種類：他の核酸 合成ＤＮＡ
アンチセンス：Ｙｅｓ
配列
ＣＴＴＣＡＣＴＧＴＡ ＡＡＴＧＴＧＴＣＡＴ Ｃ
２１SEQ ID NO: 5 Sequence length: 21 Sequence type: Nucleic acid Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence CTTCACTGTA AATGTGTCAT C
21
【００３４】配列番号：６
配列の長さ：２０
配列の型：核酸
配列の種類：他の核酸 合成ＤＮＡ
アンチセンス：Ｎｏ
配列
ＡＡＧＣＣＴＴＡＣＧ ＧＴＴＣＡＧＧＣＡＡ
２０SEQ ID NO: 6 Sequence length: 20 Sequence type: Nucleic acid Sequence type: Other nucleic acid Synthetic DNA Antisense: No Sequence AAGCCTTACG GTTCAGGCAA
20
【００３５】配列番号：７
配列の長さ：２０
配列の型：核酸
配列の種類：他の核酸 合成ＤＮＡ
アンチセンス：Ｙｅｓ
配列
ＣＡＧＴＴＡＡＡＣＴ ＴＣＡＣＣＴＧＧＧＣ
２０SEQ ID NO: 7 Sequence length: 20 Sequence type: Nucleic acid Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence CAGTTAAACT TCACCTGGGC
20
【００３６】配列番号：８
配列の長さ：２１
配列の型：核酸
配列の種類：他の核酸 合成ＤＮＡ
アンチセンス：Ｎｏ
配列
ＧＡＡＡＴＡＴＣＧＡ ＣＴＣＣＴＣＴＴＧＡ Ｇ
２１Sequence number: 8 Sequence length: 21 Sequence type: Nucleic acid Sequence type: Other nucleic acid Synthetic DNA Antisense: No Sequence GAAATATCGA CTCCTCTTGAG
21
【００３７】配列番号：９
配列の長さ：１９
配列の型：核酸
配列の種類：他の核酸 合成ＤＮＡ
アンチセンス：Ｙｅｓ
配列
ＧＴＡＴＴＣＴＴＴＣ ＣＣＧＧＣＣＡＣＴ
１９SEQ ID NO: 9 Sequence length: 19 Sequence type: Nucleic acid Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes Sequence GTATTCTTTTC CCGGCCACT
19
【図１】ＶＴ１、ＶＴ２、ＶＴ２ｖｐ、ＶＴ２ｖｈａ、
およびＶＴ２ｖｈｂ遺伝子の配列を比較した図である。
「−」はＶＴ２遺伝子の配列と同一であることを示し、
「＋」は欠失を示す。四角で囲った部分は、プライマ−
の設計に選択された部分である。[Figure 1] VT1, VT2, VT2vp, VT2vha,
and VT2vhb gene sequences. "-" indicates the same sequence as the VT2 gene,
"+" indicates deletion. The squared area is the primer
This is the part selected for the design.
【図２】ＶＴ１、ＶＴ２、ＶＴ２ｖｐ、ＶＴ２ｖｈａ、
およびＶＴ２ｖｈｂ遺伝子の配列を比較した図であり、
図１の続きである。「−」はＶＴ２遺伝子の配列と同一
であることを示し、「＋」は欠失を示す。四角で囲った
部分は、プライマ−の設計に選択された部分である。[Figure 2] VT1, VT2, VT2vp, VT2vha,
It is a diagram comparing the sequences of the and VT2vhb genes,
This is a continuation of FIG. "-" indicates the same sequence as the VT2 gene, and "+" indicates deletion. The boxed area is the area selected for the primer design.
【図３】本発明のプライマ−がデザインされた領域示す
。FIG. 3 shows the area where the primers of the present invention were designed.
【図４】本発明の方法により増幅したＤＮＡ断片を、６
％ポリアクリルアミドゲル電気泳動法により検出した結
果を示す図である。FIG. 4: DNA fragments amplified by the method of the present invention are
% polyacrylamide gel electrophoresis.
【図５】本発明の方法により増幅したＤＮＡ断片を、６
％ポリアクリルアミドゲル電気泳動法により検出した結
果を示す図であり、図４の続きである。FIG. 5: DNA fragments amplified by the method of the present invention are
% polyacrylamide gel electrophoresis, and is a continuation of FIG. 4.
Claims (7)
２ｖｈ遺伝子のいずれか一つのみを特異的に検出しうる
プライマ−。Claim 1: VT1, VT2, VT2vp, and VT
A primer that can specifically detect only one of the 2vh genes.
プライマ−。2. The primer according to claim 1, having a length of 15 to 40 bases.
ＡＡＧＡＧＣＧ ３’または５’ＧＣＧＴＣＧＣＣＡＧ
ＣＧＣＡＣＴＴＧ ３’のうち、少なくとも連続した１
５塩基またはその相補鎖を有し、ＶＴ１遺伝子のみを特
異的に検出しうる請求項１または２記載のプライマ−。[Claim 3] Base sequence: 5'GCAGTTCGTGGC
AAGAGCG 3' or 5'GCGTCGCCAG
At least consecutive 1 of CGCACTTG 3'
The primer according to claim 1 or 2, which has 5 bases or a complementary strand thereof and is capable of specifically detecting only the VT1 gene.
ＧＣＡＧＧＧＴＴＣ ３’または５’ＣＴＴＣＡＣＴＧ
ＴＡＡＡＴＧＴＧＴＣＡＴＣ ３’のうち、少なくとも
連続した１５塩基またはその相補鎖を有し、ＶＴ２遺伝
子のみを特異的に検出しうる請求項１または２記載のプ
ライマ−。Claim 4: Base sequence: 5'AATTTATATGTG
GCAGGGTTC 3' or 5'CTTCACTG
The primer according to claim 1 or 2, which has at least 15 consecutive bases of TAAATGTGTCATC 3' or a complementary strand thereof, and is capable of specifically detecting only the VT2 gene.
ＴＣＡＧＧＣＡＡ ３’または５’ＣＡＧＴＴＡＡＡＣ
ＴＴＣＡＣＣＴＧＧＧＣ ３’のうち、少なくとも連続
した１５塩基またはその相補鎖を有し、ＶＴ２ｖｐ遺伝
子のみを特異的に検出しうる請求項１または２記載のプ
ライマ−。Claim 5: Base sequence: 5'AAGCCTTACGGT
TCAGGCAA 3' or 5'CAGTTTAAAC
The primer according to claim 1 or 2, which has at least 15 consecutive bases of TTCACCTGGGC 3' or its complementary strand, and is capable of specifically detecting only the VT2vp gene.
ＣＣＴＣＴＴＧＡＧ ３’または５’ＧＴＡＴＴＣＴＴ
ＴＣＣＣＧＧＣＣＡＣＴ ３’のうち、少なくとも連続
した１５塩基またはその相補鎖を有し、ＶＴ２ｖｈ遺伝
子のみを特異的に検出しうる請求項１または２記載のプ
ライマ−。Claim 6: Base sequence: 5'GAAATATCGACT
CCTCTTGAG 3' or 5'GTATTCTT
The primer according to claim 1 or 2, which has at least 15 consecutive bases of TCCCGGCCACT 3' or its complementary strand, and is capable of specifically detecting only the VT2vh gene.
−を用いることを特徴とする、ＰＣＲ法によるＶｅｒｏ
毒素遺伝子の型別検出方法。7. Vero chromatography by PCR method, characterized in that the primer according to any one of claims 1 to 6 is used.
Method for detecting toxin genes by type.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3087611A JP2905945B2 (en) | 1991-03-26 | 1991-03-26 | Vero toxin gene type detection method and primer used therefor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3087611A JP2905945B2 (en) | 1991-03-26 | 1991-03-26 | Vero toxin gene type detection method and primer used therefor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH04297489A true JPH04297489A (en) | 1992-10-21 |
| JP2905945B2 JP2905945B2 (en) | 1999-06-14 |
Family
ID=13919771
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3087611A Expired - Fee Related JP2905945B2 (en) | 1991-03-26 | 1991-03-26 | Vero toxin gene type detection method and primer used therefor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2905945B2 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH078279A (en) * | 1993-06-15 | 1995-01-13 | Shimadzu Corp | Oligonucleotide for detection of vibriocholerae and detection thereby |
| EP0669399A2 (en) * | 1994-02-28 | 1995-08-30 | Shimadzu Corporation | Oligonucleotides and method for detecting bacteria |
| JP2005218317A (en) * | 2004-02-03 | 2005-08-18 | Tosoh Corp | Detection reagent for shiga toxin group gene of enterohemorrhagic escherichia coli |
| EP3156501A4 (en) * | 2014-06-11 | 2017-12-13 | Toyo Seikan Group Holdings, Ltd. | Method for detecting escherichia coli and carrier for detecting escherichia coli |
-
1991
- 1991-03-26 JP JP3087611A patent/JP2905945B2/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH078279A (en) * | 1993-06-15 | 1995-01-13 | Shimadzu Corp | Oligonucleotide for detection of vibriocholerae and detection thereby |
| EP0669399A2 (en) * | 1994-02-28 | 1995-08-30 | Shimadzu Corporation | Oligonucleotides and method for detecting bacteria |
| EP0669399A3 (en) * | 1994-02-28 | 1996-05-22 | Shimadzu Corp | Oligonucleotides and method for detecting bacteria. |
| US5795717A (en) * | 1994-02-28 | 1998-08-18 | Shimadzu Corporation | Oligonucleotides for detecting bacteria and detection process |
| US6218110B1 (en) | 1994-02-28 | 2001-04-17 | Shimadzu Corporation | Oligonucleotides for detecting verotoxin-producing E. coli and detection process |
| JP2005218317A (en) * | 2004-02-03 | 2005-08-18 | Tosoh Corp | Detection reagent for shiga toxin group gene of enterohemorrhagic escherichia coli |
| JP4501443B2 (en) * | 2004-02-03 | 2010-07-14 | 東ソー株式会社 | Detection reagent for Shiga toxin group gene of enterohemorrhagic Escherichia coli |
| EP3156501A4 (en) * | 2014-06-11 | 2017-12-13 | Toyo Seikan Group Holdings, Ltd. | Method for detecting escherichia coli and carrier for detecting escherichia coli |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2905945B2 (en) | 1999-06-14 |
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