JP2003284559A - Method for identifying species of bacillus - Google Patents
Method for identifying species of bacillusInfo
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- JP2003284559A JP2003284559A JP2002090214A JP2002090214A JP2003284559A JP 2003284559 A JP2003284559 A JP 2003284559A JP 2002090214 A JP2002090214 A JP 2002090214A JP 2002090214 A JP2002090214 A JP 2002090214A JP 2003284559 A JP2003284559 A JP 2003284559A
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- artificial sequence
- dna
- sequence
- 16srrna
- probe
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、ある細菌種の同定
をいくつかの特異的なプローブの組合せにより、擬陽性
及び偽陰性を回避しながら、高精度に細菌種を同定する
ための細菌検出用チップ、菌種同定方法 およびこれに
利用する塩基配列に関する。TECHNICAL FIELD The present invention relates to the identification of a bacterial species with a combination of several specific probes, which is used for bacterial detection to identify a bacterial species with high accuracy while avoiding false positives and false negatives. The present invention relates to a chip, a bacterial species identification method, and a nucleotide sequence used for the method.
【０００２】[0002]
【従来の技術】菌種同定方法としては、１６ＳｒＲＮＡ
の特定領域について、２段階のＰＣＲ（Nested PCR）
を行い、生成する増幅産物を検出・遺伝子配列を決定す
ることにより、マイコプラズマ属、ウレアプラズマ属の
菌種同定を行う方法がある。しかし、これはあくまで、
特異的な領域を増幅して検出し、塩基配列を決定の上系
統解析にあてはめて菌種を同定するにすぎないものであ
った。2. Description of the Related Art 16S rRNA is used as a bacterial species identification method.
Two-step PCR (Nested PCR) for specific region of
Then, the amplification product produced is detected and the gene sequence is determined to identify the bacterial species of the genus Mycoplasma and Ureaplasma. However, this is just
It was only to amplify and detect a specific region, determine the nucleotide sequence and apply it to phylogenetic analysis to identify the bacterial species.
【０００３】[0003]
【発明が解決しようとする課題】本発明の課題は、細菌
の１６ＳｒＲＮＡを対象とし、細菌種の同定のためいく
つかの特異的なプローブを組合せ、擬陽性及び偽陰性を
回避しながら、高精度に細菌種を同定する方法を提供す
ることである。The object of the present invention is to target bacterial 16S rRNA, combine several specific probes for the identification of bacterial species, and avoid false positives and false negatives with high accuracy. It is to provide a method for identifying bacterial species.
【０００４】[0004]
【課題を解決するための手段】本発明は、食中毒検出用
チップとして有用な試薬の提供のため、各菌種に特異的
なプローブと、系統解析での各細菌種もしくは細菌群へ
の分岐点に位置するプローブを組合わせることで、擬陽
性及び偽陰性を回避しながら、高精度に細菌種を同定す
る方法を提供することに成功し本発明を完成した。The present invention provides a probe useful for a food poisoning detection chip, a probe specific to each bacterial species, and a branch point to each bacterial species or bacterial group in phylogenetic analysis. The present invention has been completed by successfully providing a method for identifying a bacterial species with high accuracy while avoiding false positives and false negatives by combining probes located at.
【０００５】すなわち、本発明は、
１．細菌の16SrRNAを対象とする細菌種同定方法であっ
て、系統解析での各細菌種もしくは細菌群への分岐点に
位置するプローブ、及び各細菌種に特異的なプローブを
少なくとも組合わせて用いることを特徴とする細菌検出
用チップ。
２．配列番号59〜68の塩基配列、これら塩基配列にスト
リンジェントな条件下でハイブリダイズする塩基配列、
またはこれら塩基配列の相補鎖から選択されるプローブ
の少なくとも１の配列を組み合わせてなる前項１の細菌
検出用チップ。
３．配列番号1〜58の塩基配列、これら塩基配列にスト
リンジェントな条件下でハイブリダイズする塩基配列、
またはこれら塩基配列の相補鎖から選択されるプローブ
の少なくとも１の配列との併用で利用される前項２の細
菌検出用チップ。
４．前項１〜３の何れか一に記載の細菌検出用チップに
よる菌種同定方法。
５．食中毒細菌検出用である前項４の菌種同定方法。
６．以下から選択される塩基配列；
１）配列表の配列番号１〜１６、１９〜２３、２７〜６
８において特定される塩基配列、
２）１）の各配列にストリンジェントな条件下でハイブ
リダイズする塩基配列、
３）１）又は２）の各配列への相補鎖。からなる。That is, the present invention is as follows: A method of identifying a bacterial species for 16S rRNA of a bacterium, comprising using at least a combination of a probe located at a branch point to each bacterial species or bacterial group in phylogenetic analysis, and a probe specific to each bacterial species. Bacteria detection chip characterized by: 2. SEQ ID NOs: 59 to 68, nucleotide sequences that hybridize to these nucleotide sequences under stringent conditions,
Alternatively, the bacterium detection chip according to item 1 above, which comprises a combination of at least one sequence of a probe selected from complementary strands of these base sequences. 3. SEQ ID NOs: 1 to 58, nucleotide sequences that hybridize to these nucleotide sequences under stringent conditions,
Alternatively, the bacterium detection chip according to item 2 above, which is used in combination with at least one sequence of a probe selected from complementary strands of these base sequences. 4. A method for identifying a bacterial species using the chip for detecting a bacterium according to any one of items 1 to 3 above. 5. The method for identifying a bacterial species according to the above item 4 for detecting food poisoning bacteria. 6. A base sequence selected from the following: 1) SEQ ID NOs: 1 to 16, 19 to 23, 27 to 6 in the sequence listing
8) a base sequence specified in 8), 2) a base sequence that hybridizes to each sequence of 1) under stringent conditions, and 3) a complementary strand to each sequence of 1) or 2). Consists of.
【０００６】[0006]
【発明の実施の形態】本発明は、細菌の16SrRNAを対象
とする細菌種同定方法である。菌体RNAの抽出は、自体
公知の方法でおこない、自体公知の方法で増幅して同定
に用いうる。検体の調製は、臨床検査法提要（金原出版
株式会社）に記載の方法で行いうる。調製された検体に
本発明のプローブに標識化した試薬を反応させ、目的遺
伝子の検出を行う。プローブの標識は、通常化学合成す
る場合、ビオチン、ジゴキシゲニン、DNP等である。ま
た、RI標識法によってもよい。BEST MODE FOR CARRYING OUT THE INVENTION The present invention is a method for identifying a bacterial species which targets 16S rRNA of a bacterium. The microbial cell RNA can be extracted by a method known per se, amplified by a method known per se, and used for identification. The preparation of the sample can be carried out by the method described in the Clinical Laboratory Method Recommendation (Kanehara Publishing Co., Ltd.). The prepared sample is reacted with the reagent labeled with the probe of the present invention to detect the target gene. The label of the probe is usually biotin, digoxigenin, DNP or the like when chemically synthesized. Alternatively, RI labeling may be used.
【０００７】図１は、害微生物の16S rRNAに基づく系統
解析と検出用プローブの位置付けを示す。図は、微生物
名を記載し、その次に当該微生物検出用のプローブ番号
を記す。例えば、最上部のS.shomron（微生物名）はAE0
01のプローブ（図４：配列番号１）と27のプローブ（図
５：配列番号６８）が有用であることを示す。図中カッ
コ内の数字は塩基のミスマッチ数を示す。FIG. 1 shows phylogenetic analysis based on 16S rRNA of harmful microorganisms and positioning of detection probes. The figure describes the name of the microorganism and then the probe number for detecting the microorganism. For example, S.shomron (microorganism name) at the top is AE0.
It shows that probe 01 (FIG. 4: SEQ ID NO: 1) and probe 27 (FIG. 5: SEQ ID NO: 68) are useful. The numbers in parentheses in the figure indicate the number of base mismatches.
【０００８】本発明の各細菌種に特異的なプローブと
は、細菌の特異的配列部分を同定しそれをプローブにす
ればよい。本発明では、配列表の配列番号１〜５８がこ
れに該当する。なお、配列番号１（プローブ名：AE00
1）は、全てeubacteriaに共通である。本発明の特徴で
ある系統解析での各細菌種もしくは細菌群への分岐点に
位置するプローブとは、図１の分枝部が該当する。この
具体例は配列表の配列番号５９〜６８（図５）が該当す
る。この２種類を少なくとも組合わせたプローブを用い
ることで従来にない有用な細菌検出用チップが提供され
る。The probe specific to each bacterial species of the present invention may be that a specific sequence portion of bacteria is identified and used as a probe. In the present invention, this corresponds to SEQ ID NOs: 1 to 58 in the sequence listing. Sequence number 1 (probe name: AE00
1) is common to all eubacteria. The branch portion in FIG. 1 corresponds to the probe located at the branch point to each bacterial species or bacterial group in the phylogenetic analysis, which is a feature of the present invention. This specific example corresponds to SEQ ID NOs: 59 to 68 (FIG. 5) in the sequence listing. By using a probe in which at least these two types are combined, a useful chip for detecting bacteria, which has never been available, is provided.
【０００９】細菌の系統発生学上（系統解析）のグルー
プに特異的なプローブとしては、例えばプロテオバクテ
リアのα-、β-、γ-、ε-の各サブクラスに特異的なプ
ローブが好適に利用できる（図１）[0009] As a probe specific to the group of the phylogeny of bacteria (phylogenetic analysis), for example, a probe specific to each subclass of α-, β-, γ-, and ε- of Proteobacteria is preferably used. Yes (Figure 1)
【００１０】本発明の各細菌種もしくは細菌群への分岐
点に位置するプローブに相当する具体的な配列は、配列
番号５９〜６８の塩基配列が例示される（図５）。また
これら塩基配列にストリンジェントな条件下でハイブリ
ダイズする塩基配列も好適に用いられる。ハイブリダイ
ゼーションの条件は、例えば、Molecular Cloning;ALab
oratory Manual、第２版、Sambrookら編、コールド・ス
プリング・ハーバー・ラボラトリー・プレス、コールド
・スプリング・ハーバー、ニューヨーク、1989年等に従
うことができる。ストリンジェントなハイブリダイゼー
ション条件は一般に知られたものであるが、その一例と
しては、５０％ホルムアミド、５×ＳＳＣ（１５０ｍＭ
ＮａＣｌ、１５ｍＭ クエン酸三ナトリウム）、５０ｍ
Ｍ リン酸ナトリウム，ｐＨ７．６、５×デンハーツ溶
液、１０％デキストラン硫酸、および２０μｇ／ｍｌの
変性剪断サケ・***ＤＮＡを含む溶液中、４２℃で一
晩、次いで、約６５℃において０．１×ＳＳＣ中での洗
浄といった条件であってもよい。これらのポリヌクレオ
チドは目的のポリヌクレオチド、特に配列表の配列番号
５９〜６８の塩基配列からなるポリヌクレオチドまたは
その相補鎖にハイブリダイズするものであれば必ずしも
相補的配列でなくとも良い。例えば、配列表の配列番号
５９〜６８の塩基配列またはその相補的配列に対する相
同性において、少なくとも約４０％、例えば、約７０％
以上、好ましくは約８０％以上、より好ましくは約９０
％以上、さらに好ましくは約９５％以上である。または
これら塩基配列の相補鎖も当然に利用できる。その相補
鎖は、本発明に係る配列表の配列番号５９〜６８に記載
の配列に対する相補鎖を意味する。Specific sequences corresponding to the probes located at branch points to each bacterial species or bacterial group of the present invention are exemplified by the nucleotide sequences of SEQ ID NOs: 59 to 68 (FIG. 5). A base sequence that hybridizes to these base sequences under stringent conditions is also preferably used. Hybridization conditions include, for example, Molecular Cloning; ALab
Oratory Manual, Second Edition, edited by Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Stringent hybridization conditions are generally known, and one example thereof is 50% formamide, 5 × SSC (150 mM).
NaCl, 15 mM trisodium citrate), 50 m
M sodium phosphate, pH 7.6, 5x Denhardt's solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA in a solution at 42 ° C overnight, then 0.1% at about 65 ° C. It may be a condition of washing in × SSC. These polynucleotides may not necessarily be complementary sequences as long as they hybridize to the desired polynucleotide, particularly the polynucleotide consisting of the nucleotide sequences of SEQ ID NOs: 59 to 68 in the sequence listing or its complementary strand. For example, the homology to the nucleotide sequences of SEQ ID NOs: 59 to 68 in the sequence listing or its complementary sequence is at least about 40%, for example, about 70%.
Or more, preferably about 80% or more, more preferably about 90%
% Or more, more preferably about 95% or more. Alternatively, the complementary strands of these base sequences can naturally be used. The complementary strand means a complementary strand to the sequences described in SEQ ID NOs: 59 to 68 of the sequence listing according to the present invention.
【００１１】本発明の細菌検出用チップは、上記記載の
配列から選択されるプローブの少なくとも１の配列を組
み合わせることによって構成される。The bacterial detection chip of the present invention is constituted by combining at least one sequence of probes selected from the sequences described above.
【００１２】本発明の各細菌種に特異的なプローブと
は、具体的には配列番号１〜５８の塩基配列が該当する
（図４）。これら塩基配列にストリンジェントな条件下
でハイブリダイズする塩基配列も該当し、さらにこれら
塩基配列の相補鎖を該当する。これらから選択されるプ
ローブの少なくとも１の配列と前記分枝部に該当する配
列の併用によって、本発明の目的とする細菌検出用チッ
プが提供される。The probe specific to each bacterial species of the present invention specifically corresponds to the nucleotide sequences of SEQ ID NOs: 1 to 58 (FIG. 4). The base sequences that hybridize to these base sequences under stringent conditions also correspond, and further the complementary strands of these base sequences correspond. The combination of at least one sequence of the probe selected from these and the sequence corresponding to the branching portion provides the chip for detecting bacteria, which is the object of the present invention.
【００１３】かくして、上記に記載したような配列（プ
ローブ）を使い、擬陽性、偽陰性を回避しながら細菌検
出用チップによる菌種同定方法が可能となる。そして、
この方法は、より正確な食中毒細菌検出のための菌種同
定方法を提供する。Thus, it becomes possible to use the above-described sequence (probe) to carry out a method for identifying a bacterial species by a bacterial detection chip while avoiding false positives and false negatives. And
This method provides a method for identifying a bacterial species for more accurate detection of food poisoning bacteria.
【００１４】本発明で新規な配列は、以下から選択され
る塩基配列である。
１）配列表の配列番号１〜１６、１９〜２３、２７〜６
８において特定される塩基配列、
２）１）の各配列にストリンジェントな条件下でハイブ
リダイズする塩基配列、
３）１）又は２）の各配列への相補鎖。The sequence novel in the present invention is a base sequence selected from the following. 1) SEQ ID NOs: 1 to 16, 19 to 23, 27 to 6 in the sequence listing
8) a base sequence specified in 8), 2) a base sequence that hybridizes to each sequence of 1) under stringent conditions, and 3) a complementary strand to each sequence of 1) or 2).
【００１５】図３、図３−２、図３−３は、危害微生物
の系統解析に用いた16S rRNA配列リスト（一部環境菌も
含む）であり、左から微生物名、Strain、Accesion N
o.を表示する。DNA Data Bank of Japan(DDBJ）のSRS
（Sequence Retrieval System）からDDBJおよびDDBJ N
EWを検索データベースとして用い、各種の細菌のシーク
エンスを抽出後、太字の該当物のシークエンスを系統解
析に用いた。AccesionNo.はDDBJおよびDDBJ NEWの検索
番号を示す。FIG. 3, FIG. 3-2, and FIG. 3-3 are 16S rRNA sequence lists (including some environmental fungi) used for phylogenetic analysis of harmful microorganisms. From the left, the microorganism name, Strain, Accesion N
Display o. DNA Data Bank of Japan (DDBJ) SRS
(Sequence Retrieval System) to DDBJ and DDBJ N
Using EW as a search database, the sequences of various bacteria were extracted, and then the sequences of the corresponding substances in bold were used for phylogenetic analysis. Accesion No. indicates the search number of DDBJ and DDBJ NEW.
【００１６】図４、図４−２は、16SrRNAに基づく危害
微生物（標準株）検出用DNAプローブリストであり、左
から配列番号、微生物名、プローブ名、配列、長さ及び
Tm（℃）を示す。Tmは、Breslauer et al., Proc. Nat.
Acad. Sci. 83, 3746-50, 1986 に基づき、プローブ濃
度50nM,、塩濃度50nMで算出した。図５は、16SrRNAに基
づく危害微生物（標準株）検出用DNAプローブ（分岐
部）リストであり、配列番号、空欄、プローブ名、配
列、長さ及びTm（℃）を示す。Tmは、Breslauer et a
l., Proc. Nat. Acad. Sci. 83, 3746-50, 1986 に基づ
き、プローブ濃度50nM,、塩濃度50nMで算出した。FIGS. 4 and 4-2 are DNA probe lists for detecting harmful microorganisms (standard strains) based on 16S rRNA. From the left, the sequence number, microorganism name, probe name, sequence, length and
Indicates Tm (° C). Tm is from Breslauer et al., Proc. Nat.
It was calculated based on Acad. Sci. 83, 3746-50, 1986 at a probe concentration of 50 nM and a salt concentration of 50 nM. FIG. 5 is a DNA probe (branching part) list for detecting harmful microorganisms (standard strain) based on 16S rRNA, and shows the sequence number, blank, probe name, sequence, length and Tm (° C.). Tm is Breslauer et a
It was calculated at a probe concentration of 50 nM and a salt concentration of 50 nM based on I., Proc. Nat. Acad. Sci. 83, 3746-50, 1986.
【００１７】[0017]
【実施例】以下に実施例で本発明を説明するが、本発明
はこれに限定されるものではない。EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited thereto.
【実施例１】（プローブDNAのFASTA解析による特異性の
確認）図２は、各微生物とプローブとの関係を示す概念
図である。黒塗り部分が、FASTA解析の結果、各微生物
の核酸と各プローブが反応（ハイブリダイズ）すると考
えられる関係を示す。図２−１〜図２−１５は、その詳
細を示し、各数字は、プローブDNAがターゲットとハイ
ブリダイズする１６S rRNA上の位置を示す。プローブAE
001（配列番号１）は、全てのeubacteriaの共通プロー
ブであり、殆どの微生物との反応がある。プローブShSP
002（配列番号２）は、微生物Shigellasppとハイブリダ
イズし、その位置は56-76である。以下、図は図２−１
から図２−３が各微生物名を標記し、図２−１は図２−
４、図２−７、図２−１０、図２−１３と続き、図２−
２は図２−５、図２−８、図２−１１、図２−１４と続
き、図２−３は図２−６、図２−９、図２−１２、図２
−１５へと続く。また、図２−１と図２−４と図２−７
と図２−１０と図２−１３に横欄に各プローブ名を表示
し、ShSP002（図２−１）〜Hin001（図２−１３）が各
微生物特異的プローブであり、プローブ1（図２−１
３）〜プローブ27（図２−１３）が分枝部に相当するプ
ローブである。Example 1 (Confirmation of Specificity by FASTA Analysis of Probe DNA) FIG. 2 is a conceptual diagram showing the relationship between each microorganism and the probe. As a result of FASTA analysis, the black-painted portion shows the relationship in which the nucleic acid of each microorganism and each probe are considered to react (hybridize). 2-1 to 2-15 show the details thereof, and each numeral indicates the position on the 16S rRNA where the probe DNA hybridizes with the target. Probe AE
001 (SEQ ID NO: 1) is a common probe for all eubacteria and is reactive with most microorganisms. Probe ShSP
002 (SEQ ID NO: 2) hybridizes with the microorganism Shigellas pp and its position is 56-76. Below, the figure is shown in Figure 2-1.
Fig. 2-3 shows the names of each microorganism, and Fig. 2-1 shows Fig. 2-.
4, Fig. 2-7, Fig. 2-10, Fig. 2-13, and Fig. 2-
2 is continued from FIG. 2-5, FIG. 2-8, FIG. 2-11, and FIG. 2-14, and FIG. 2-3 is FIG. 2-6, FIG. 2-9, FIG. 2-12, and FIG.
Continue to -15. Also, FIGS. 2-1, 2-4, and 2-7
Each probe name is displayed in the horizontal column in Fig. 2-10 and Fig. 2-13, and ShSP002 (Fig. 2-1) to Hin001 (Fig. 2-13) are each microorganism-specific probes, and probe 1 (Fig. 2 -1
3) to probe 27 (FIGS. 2 to 13) are probes corresponding to the branch portion.
【００１８】[0018]
【発明の効果】本発明は、細菌種の同定のために細菌の
系統発生学上のグループに特異的なプローブ又は細菌群
への分岐点に位置するプローブを使う手段を提供し、擬
陽性、偽陰性を回避しながら高精度に細菌種を同定する
ことに成功した。INDUSTRIAL APPLICABILITY The present invention provides means for using a probe specific to a phylogenetic group of bacteria or a probe located at a branch point to a bacterial group for the identification of bacterial species, false positive, false We succeeded in identifying the bacterial species with high accuracy while avoiding the negative.
【図１】危害微生物の16S rRNAに基づく系統解析であ
る。FIG. 1 is a phylogenetic analysis of harmful microorganisms based on 16S rRNA.
【図２】プローブDNAのFASTA解析による特異性の確認の
概略図である。FIG. 2 is a schematic diagram of confirmation of specificity by FASTA analysis of probe DNA.
【図２−１】プローブDNAのFASTA解析による特異性の確
認の１図である。FIG. 2-1 is a diagram showing confirmation of specificity by FASTA analysis of probe DNA.
【図２−２】プローブDNAのFASTA解析による特異性の確
認の２図である。FIG. 2-2 is a diagram showing confirmation of specificity by FASTA analysis of probe DNA.
【図２−３】プローブDNAのFASTA解析による特異性の確
認の３図である。FIG. 2-3 is a diagram showing confirmation of specificity by FASTA analysis of probe DNA.
【図２−４】プローブDNAのFASTA解析による特異性の確
認の１図の続き４図である。[Fig. 2-4] Fig. 2-4 is a continuation of Fig. 4 of Fig. 2 for confirming the specificity of the probe DNA by FASTA analysis.
【図２−５】プローブDNAのFASTA解析による特異性の確
認の２図の続き５図である。[Fig. 2-5] Fig. 2-5 is a sequel to Fig. 2 confirming the specificity of the probe DNA by FASTA analysis.
【図２−６】プローブDNAのFASTA解析による特異性の確
認の３図の続き６図である。[Fig. 2-6] Fig. 2-6 is a continuation of Fig. 3 of Fig. 3 for confirming the specificity of the probe DNA by FASTA analysis.
【図２−７】プローブDNAのFASTA解析による特異性の確
認の４図の続き７図である。[Fig. 2-7] Fig. 2-7 is a continuation of Fig. 7 of Fig. 4 for confirming the specificity of the probe DNA by FASTA analysis.
【図２−８】プローブDNAのFASTA解析による特異性の確
認の５図の続き８図である。[Fig. 2-8] Fig. 2-8 is a continuation of Fig. 8 of Fig. 5 for confirming the specificity of the probe DNA by FASTA analysis.
【図２−９】プローブDNAのFASTA解析による特異性の確
認の６図の続き９図である。[Fig. 2-9] Fig. 9 is a continuation of Fig. 9 of Fig. 6 of confirmation of specificity by FASTA analysis of probe DNA.
【図２−１０】プローブDNAのFASTA解析による特異性の
確認の７図の続き１０図である。FIG. 2-10 is a sequel to FIG. 7 showing confirmation of specificity by FASTA analysis of probe DNA.
【図２−１１】プローブDNAのFASTA解析による特異性の
確認の８図の続き１１図である。FIG. 2-11 is a sequel to FIG. 8 showing confirmation of specificity by FASTA analysis of probe DNA.
【図２−１２】プローブDNAのFASTA解析による特異性の
確認の９図の続き１２図である。FIG. 2-12 is a sequel to FIG. 9 showing confirmation of specificity by FASTA analysis of probe DNA.
【図２−１３】プローブDNAのFASTA解析による特異性の
確認の１０図の続き１３図である。[Fig. 2-13] Fig. 2-13 is a continuation of 13 views of Fig. 10 for confirmation of specificity by FASTA analysis of probe DNA.
【図２−１４】プローブDNAのFASTA解析による特異性の
確認の１１図の続き１４図である。[Fig. 2-14] Fig. 2-14 is a continuation of Fig. 11 of Fig. 11 of confirmation of specificity by FASTA analysis of probe DNA.
【図２−１５】プローブDNAのFASTA解析による特異性の
確認の１２図の続き１５図である。FIG. 2-15 is a sequel to FIG. 12 showing confirmation of specificity by FASTA analysis of probe DNA.
【図３】危害微生物の系統解析に用いた１６SrRNA配列
リストである。DNA DataBank of Japan(DDBJ）のSRS（S
equence Retrieval System）からDDBJおよびDDBJNEWを
検索データベースとして用い、各種の細菌のシークエン
スを抽出後、太字のシークエンスを系統解析に用いた。
Accesion No.はDDBJおよびDDBJ NEWの検索番号を示す。FIG. 3 is a 16S rRNA sequence list used for phylogenetic analysis of harmful microorganisms. DNA DataBank of Japan (DDBJ) SRS (S
Sequence Retrieval System) was used as a search database, sequences of various bacteria were extracted, and sequences in bold type were used for phylogenetic analysis.
Accesion No. indicates the search number of DDBJ and DDBJ NEW.
【図３−２】図３の続きである。FIG. 3-2 is a continuation of FIG. 3.
【図３−３】図３−２の続きである。FIG. 3-3 is a continuation of FIG. 3-2.
【図４】16S rRNAに基づく危害微生物（標準株）検出
用DNAプローブリストを示す。図中、Tmは、Breslauer e
t al., Proc. Nat. Acad. Sci. 83, 3746-50, 1986 に
基づき、プローブ濃度50nM,、塩濃度50nMで算出した。FIG. 4 shows a list of DNA probes for detecting harmful microorganisms (standard strain) based on 16S rRNA. In the figure, Tm is Breslauer e
It was calculated at a probe concentration of 50 nM and a salt concentration of 50 nM based on T. al., Proc. Nat. Acad. Sci. 83, 3746-50, 1986.
【図４−２】図４の続きである。4-2 is a continuation of FIG. 4.
【図５】16S rRNAに基づく危害微生物（標準株）検出
用DNAプローブ（分枝部）リストを示す。図中、Tmは、B
reslauer et al., Proc. Nat. Acad. Sci. 83, 3746-5
0, 1986 に基づき、プローブ濃度50nM,、塩濃度50nMで
算出した。FIG. 5 shows a list of DNA probes (branching part) for detecting harmful microorganisms (standard strain) based on 16S rRNA. In the figure, Tm is B
reslauer et al., Proc. Nat. Acad. Sci. 83, 3746-5
Based on 0, 1986, the probe concentration was 50 nM, and the salt concentration was 50 nM.
【配列表】 SEQUENCE LISTING <110> IRC <120> BACTERIA PROBE <130> NP01-1147 <140> <141> <160> 68 <170> PatentIn Ver. 2.1 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 1 tgctgcctcc cgtaggagtc 20 <210> 2 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 2 atgcagctat taactacact accttcctca c 31 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 3 cactccagcg tctccgctag a 21 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 4 cgtctccgct ggcttctctg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 5 tgctttgctc ttgcgaggtt 20 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 6 aaaagatacc gctattaacg atacctcctt 30 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 7 caacattatg cggtattaat cttcctttc 29 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 8 accttgtccc cgaaggattt cc 22 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 9 aatccaacaa cgtattaagt tattggcctt 30 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 10 aatcacaaag gttattaacc tttatgcctt 30 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 11 tggcatgcgc cacacttt 18 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 12 gcgaaacagc aagctgcttc c 21 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 13 tctttcaaaa gcgtggcatg c 21 <210> 14 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 14 gggtaacgtc aatgccacta ggtattaact agt 33 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 15 taacttatcc aaccgcctgc gtg 23 <210> 16 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 16 gccagcagat attagctact gacctt 26 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 17 agagaagcaa gcttctcgtc cg 22 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 18 ggctatcccc cactttgagg c 21 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 19 ccccgaaggg cttcacctat ct 22 <210> 20 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 20 aacttcataa gagcaagctc ttaatccatt 30 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 21 tctccactaa ccgcgaccgg 20 <210> 22 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 22 gacgcaggct aatcttaaag cgc 23 <210> 23 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 23 gagcaaaggt attaacttta ctcccttcct 30 <210> 24 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 24 aattgatgaa cgtattaagc tcaccacct 29 <210> 25 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 25 aatcgccaag gttattaacc ttaacg 26 <210> 26 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 26 caattgacaa gggtattaac cttatcacc 29 <210> 27 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 27 cagcaaggta ttaacttact gcccttc 27 <210> 28 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 28 ctcatcttag gtattaacta agagagcctc ctc 33 <210> 29 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 29 tcatgactca gggttattaa ccctaagct 29 <210> 30 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 30 gaacaaccag gtattaaccg gct 23 <210> 31 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 31 cagaacaacc gagtattaat cgattgc 27 <210> 32 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 32 acctctcagc aggattccga c 21 <210> 33 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 33 caaatctctt cggctttcca gac 23 <210> 34 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 34 tctaagttct agcaagctag caccctct 28 <210> 35 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 35 tcctctatct ctaaaggatt ccgtacatg 29 <210> 36 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 36 tccatctctg gaaacttcct gcc 23 <210> 37 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 37 acaatgtctc cactgcccaa ac 22 <210> 38 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 38 tcagatgcaa tttaagcccg g 21 <210> 39 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 39 ccagcttatt cgtagtacat ttaagtcttg g 31 <210> 40 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 40 aaatcaaaac catgcggttt cgat 24 <210> 41 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 41 actcttatgc catgcggcat aaac 24 <210> 42 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 42 attctgtccc cgaagaacag ct 22 <210> 43 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 43 gtctccgatg taccgaagta aaactcta 28 <210> 44 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 44 cagaggagca agctcctcgt ct 22 <210> 45 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 45 ccctactcaa cttgtgttaa gcaggag 27 <210> 46 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 46 ggatgcatag ttacttacat cct 23 <210> 47 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 47 tgcaggtcca ttgctggaag agat 24 <210> 48 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 48 cccgaactca agatcttcag tatca 25 <210> 49 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 49 tctgccatac tcaagacttc cagtatca 28 <210> 50 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 50 tccggtctca agccaaccag ta 22 <210> 51 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 51 ccgatgttca agcaatccag tc 22 <210> 52 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 52 tgcagcccga caactagcaa t 21 <210> 53 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 53 aaccgaagtt tcatcccccc tg 22 <210> 54 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 54 gtgccagagt taaaccccaa ccc 23 <210> 55 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 55 tttgtcaccg gcagtcacct taga 24 <210> 56 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 56 tgctgcggtt attaaccaca aca 23 <210> 57 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 57 accgtagcat gctgatctac gatt 24 <210> 58 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 58 cacgtgttac tcacccgtcc gc 22 <210> 59 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 59 acgtagttag ccgtcccttt ctggt 25 <210> 60 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 60 gaatatctac gaatttcacc tctacactcg 30 <210> 61 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 61 ggtgagccct tacctcacca acta 24 <210> 62 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 62 cgaaggcact aaagcatctc tgctaaa 27 <210> 63 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 63 ccctttaaac ccaataaatc cggata 26 <210> 64 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 64 ccccaggcgg tcaacttcac 20 <210> 65 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 65 gcacctgagc gtcagtcttt gtc 23 <210> 66 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 66 tctcatctct gaaaacttcc cgtg 24 <210> 67 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 67 ccctctacag tactctagtc tgccagtttc 30 <210> 68 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:designed DNA based on 16SrRNA <400> 68 gcaccaatcc atctctggaa ag 22[Sequence list] SEQUENCE LISTING <110> IRC <120> BACTERIA PROBE <130> NP01-1147 <140> <141> <160> 68 <170> PatentIn Ver. 2.1 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 1 tgctgcctcc cgtaggagtc 20 <210> 2 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 2 atgcagctat taactacact accttcctca c 31 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 3 cactccagcg tctccgctag a 21 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 4 cgtctccgct ggcttctctg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 5 tgctttgctc ttgcgaggtt 20 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 6 aaaagatacc gctattaacg atacctcctt 30 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 7 caacattatg cggtattaat cttcctttc 29 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 8 accttgtccc cgaaggattt cc 22 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 9 aatccaacaa cgtattaagt tattggcctt 30 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 10 aatcacaaag gttattaacc tttatgcctt 30 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 11 tggcatgcgc cacacttt 18 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 12 gcgaaacagc aagctgcttc c 21 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 13 tctttcaaaa gcgtggcatg c 21 <210> 14 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 14 gggtaacgtc aatgccacta ggtattaact agt 33 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 15 taacttatcc aaccgcctgc gtg 23 <210> 16 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 16 gccagcagat attagctact gacctt 26 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 17 agagaagcaa gcttctcgtc cg 22 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 18 ggctatcccc cactttgagg c 21 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 19 ccccgaaggg cttcacctat ct 22 <210> 20 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 20 aacttcataa gagcaagctc ttaatccatt 30 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 21 tctccactaa ccgcgaccgg 20 <210> 22 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 22 gacgcaggct aatcttaaag cgc 23 <210> 23 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 23 gagcaaaggt attaacttta ctcccttcct 30 <210> 24 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 24 aattgatgaa cgtattaagc tcaccacct 29 <210> 25 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 25 aatcgccaag gttattaacc ttaacg 26 <210> 26 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 26 caattgacaa gggtattaac cttatcacc 29 <210> 27 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 27 cagcaaggta ttaacttact gcccttc 27 <210> 28 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 28 ctcatcttag gtattaacta agagagcctc ctc 33 <210> 29 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 29 tcatgactca gggttattaa ccctaagct 29 <210> 30 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 30 gaacaaccag gtattaaccg gct 23 <210> 31 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 31 cagaacaacc gagtattaat cgattgc 27 <210> 32 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 32 acctctcagc aggattccga c 21 <210> 33 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 33 caaatctctt cggctttcca gac 23 <210> 34 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 34 tctaagttct agcaagctag caccctct 28 <210> 35 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 35 tcctctatct ctaaaggatt ccgtacatg 29 <210> 36 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 36 tccatctctg gaaacttcct gcc 23 <210> 37 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 37 acaatgtctc cactgcccaa ac 22 <210> 38 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 38 tcagatgcaa tttaagcccg g 21 <210> 39 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 39 ccagcttatt cgtagtacat ttaagtcttg g 31 <210> 40 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 40 aaatcaaaac catgcggttt cgat 24 <210> 41 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 41 actcttatgc catgcggcat aaac 24 <210> 42 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 42 attctgtccc cgaagaacag ct 22 <210> 43 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 43 gtctccgatg taccgaagta aaactcta 28 <210> 44 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 44 cagaggagca agctcctcgt ct 22 <210> 45 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 45 ccctactcaa cttgtgttaa gcaggag 27 <210> 46 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 46 ggatgcatag ttacttacat cct 23 <210> 47 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 47 tgcaggtcca ttgctggaag agat 24 <210> 48 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 48 cccgaactca agatcttcag tatca 25 <210> 49 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 49 tctgccatac tcaagacttc cagtatca 28 <210> 50 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 50 tccggtctca agccaaccag ta 22 <210> 51 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 51 ccgatgttca agcaatccag tc 22 <210> 52 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 52 tgcagcccga caactagcaa t 21 <210> 53 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 53 aaccgaagtt tcatcccccc tg 22 <210> 54 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 54 gtgccagagt taaaccccaa ccc 23 <210> 55 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 55 tttgtcaccg gcagtcacct taga 24 <210> 56 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 56 tgctgcggtt attaaccaca aca 23 <210> 57 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 57 accgtagcat gctgatctac gatt 24 <210> 58 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 58 cacgtgttac tcacccgtcc gc 22 <210> 59 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 59 acgtagttag ccgtcccttt ctggt 25 <210> 60 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 60 gaatatctac gaatttcacc tctacactcg 30 <210> 61 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 61 ggtgagccct tacctcacca acta 24 <210> 62 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 62 cgaaggcact aaagcatctc tgctaaa 27 <210> 63 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 63 ccctttaaac ccaataaatc cggata 26 <210> 64 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 64 ccccaggcgg tcaacttcac 20 <210> 65 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 65 gcacctgagc gtcagtcttt gtc 23 <210> 66 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 66 tctcatctct gaaaacttcc cgtg 24 <210> 67 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 67 ccctctacag tactctagtc tgccagtttc 30 <210> 68 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: designed DNA based on 16SrRNA <400> 68 gcaccaatcc atctctggaa ag 22
───────────────────────────────────────────────────── フロントページの続き (72)発明者 池田 昌郁 神戸市西区室谷１丁目１−２ 国際試薬株 式会社研究開発センター内 (72)発明者 佐守 友博 京都府久世郡久御山町大橋辺16−10 株式 会社日本医学臨床検査研究所内 (72)発明者 藤原 由美 兵庫県高砂市米田町米田1149−１ 株式会 社日本食品エコロジー研究所分析センター 検査部検査課内 Ｆターム(参考） 4B024 AA11 BA80 CA04 CA09 CA11 HA12 4B063 QA01 QA18 QQ06 QQ54 QR32 QR55 QR66 QR75 QS34 QX02 ─────────────────────────────────────────────────── ─── Continued front page (72) Inventor Masakazu Ikeda 1-2, Muroya, Nishi-ku, Kobe International reagent stock In ceremony company R & D center (72) Inventor Tomohiro Samoru 16-10 Ohashibe, Kumiyama-cho, Kuse-gun, Kyoto Prefecture Stock Company Japan Medical Laboratory Laboratory (72) Inventor Yumi Fujiwara 1149-1 Yoneda Yoneda Town, Takasago City, Hyogo Prefecture Stock Association Japan Food Ecology Research Center Inspection Department Inspection Section F-term (reference) 4B024 AA11 BA80 CA04 CA09 CA11 HA12 4B063 QA01 QA18 QQ06 QQ54 QR32 QR55 QR66 QR75 QS34 QX02
Claims (6)
法であって、細菌の系統解析での各細菌種もしくは細菌
群への分岐点に位置するプローブ、及び各細菌種に特異
的なプローブを少なくとも組合わせて用いることを特徴
とする細菌検出用チップ。1. A method for identifying a bacterial species for 16S rRNA of a bacterium, comprising a probe located at a branch point to each bacterial species or group in a bacterial phylogenetic analysis, and a probe specific to each bacterial species. A chip for detecting bacteria, which is characterized by using at least the following in combination.
列にストリンジェントな条件下でハイブリダイズする塩
基配列、またはこれら塩基配列の相補鎖から選択される
プローブの少なくとも１の配列を組み合わせてなる請求
項１の細菌検出用チップ。2. A combination of the nucleotide sequences of SEQ ID NOs: 59 to 68, a nucleotide sequence hybridizing to these nucleotide sequences under stringent conditions, or at least one sequence of a probe selected from complementary strands of these nucleotide sequences in combination. The chip for detecting bacteria according to claim 1.
列にストリンジェントな条件下でハイブリダイズする塩
基配列、またはこれら塩基配列の相補鎖から選択される
プローブの少なくとも１の配列との併用で利用される請
求項２の細菌検出用チップ。3. A combination of the nucleotide sequences of SEQ ID NOs: 1 to 58, a nucleotide sequence that hybridizes to these nucleotide sequences under stringent conditions, or at least one sequence of a probe selected from a complementary strand of these nucleotide sequences. The chip for detecting bacteria according to claim 2, which is used in.
用チップによる菌種同定方法。4. A method for identifying a bacterial species using the chip for detecting bacteria according to any one of claims 1 to 3.
定方法。5. The method for identifying a bacterial species according to claim 4, which is for detecting food poisoning bacteria.
８において特定される塩基配列、 ２）１）の各配列にストリンジェントな条件下でハイブ
リダイズする塩基配列、 ３）１）又は２）の各配列への相補鎖。6. A base sequence selected from the following: 1) SEQ ID NOs: 1 to 16, 19 to 23, 27 to 6 in the sequence listing
8) a base sequence specified in 8), 2) a base sequence that hybridizes to each sequence of 1) under stringent conditions, and 3) a complementary strand to each sequence of 1) or 2).
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| JP2002090214A JP2003284559A (en) | 2002-03-28 | 2002-03-28 | Method for identifying species of bacillus |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2002090214A JP2003284559A (en) | 2002-03-28 | 2002-03-28 | Method for identifying species of bacillus |
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|---|---|
| JP2003284559A true JP2003284559A (en) | 2003-10-07 |
Family
ID=29235564
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1788093A1 (en) * | 2004-08-04 | 2007-05-23 | Suntory Limited | Instrument for detecting bacterium, method of detecting bacterium and kit for detecting bacterium |
| US8637249B2 (en) | 2008-11-14 | 2014-01-28 | Gen-Probe Incorporated | Compositions, kits and methods for detection of Campylobacter nucleic acid |
| JP2015231362A (en) * | 2014-05-12 | 2015-12-24 | 三菱レイヨン株式会社 | Bacterial flora analysis method and bacterial flora analysis device |
| WO2018209398A1 (en) * | 2017-05-17 | 2018-11-22 | Microbio Pty Ltd | Biomarkers and uses thereof |
-
2002
- 2002-03-28 JP JP2002090214A patent/JP2003284559A/en not_active Withdrawn
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1788093A1 (en) * | 2004-08-04 | 2007-05-23 | Suntory Limited | Instrument for detecting bacterium, method of detecting bacterium and kit for detecting bacterium |
| EP1788093A4 (en) * | 2004-08-04 | 2009-02-11 | Suntory Ltd | Instrument for detecting bacterium, method of detecting bacterium and kit for detecting bacterium |
| US8637249B2 (en) | 2008-11-14 | 2014-01-28 | Gen-Probe Incorporated | Compositions, kits and methods for detection of Campylobacter nucleic acid |
| US9175353B2 (en) | 2008-11-14 | 2015-11-03 | Gen-Probe Incorporated | Compositions, kits and methods for detection of campylobacter nucleic acid |
| US10829824B2 (en) | 2008-11-14 | 2020-11-10 | Gen-Probe Incorporated | Compositions, kits and methods for detection of campylobacter nucleic acid |
| JP2015231362A (en) * | 2014-05-12 | 2015-12-24 | 三菱レイヨン株式会社 | Bacterial flora analysis method and bacterial flora analysis device |
| JP2018086016A (en) * | 2014-05-12 | 2018-06-07 | 三菱ケミカル株式会社 | Bacterial flora analysis method and bacterial flora analysis device |
| JP2020188799A (en) * | 2014-05-12 | 2020-11-26 | 三菱ケミカル株式会社 | Bacterial flora analysis method and bacterial flora analysis device |
| JP7040562B2 (en) | 2014-05-12 | 2022-03-23 | 三菱ケミカル株式会社 | Bacterial flora analysis method and device for bacterial flora analysis |
| WO2018209398A1 (en) * | 2017-05-17 | 2018-11-22 | Microbio Pty Ltd | Biomarkers and uses thereof |
| US11739389B2 (en) | 2017-05-17 | 2023-08-29 | Microbio Pty Ltd | Biomarkers and uses thereof |
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