JP2003284559A - Method for identifying species of bacillus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for highly accurately identifying the species of a bacillus by targeting 16SrRNA of the bacillus, while evading false positive reaction and false negative reaction by combining some specific probes for identifying the species of the bacillus. <P>SOLUTION: The method for highly accurately identifying the species of the bacillus while evading the false positive reaction and the false negative reaction comprises combining a probe specific to each species, with a probe positioned at the branch point to each species of the bacilli or a bacterial group in phylogenetic analysis. The method is used for producing a reagent useful as a chip for detecting food poisoning. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

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]

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]

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]

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.

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]

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.

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.

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] 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)

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.

The bacterial detection chip of the present invention is constituted by combining at least one sequence of probes selected from the sequences described above.

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.

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.

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).

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.

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]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited thereto.

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]

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.

[Brief description of drawings]

FIG. 1 is a phylogenetic analysis of harmful microorganisms based on 16S rRNA.

FIG. 2 is a schematic diagram of confirmation of specificity by FASTA analysis of probe DNA.

FIG. 2-1 is a diagram showing confirmation of specificity by FASTA analysis of probe DNA.

FIG. 2-2 is a diagram showing confirmation of specificity by FASTA analysis of probe DNA.

FIG. 2-3 is a diagram showing confirmation of specificity by FASTA analysis of probe DNA.

[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.

[Fig. 2-5] Fig. 2-5 is a sequel to Fig. 2 confirming the specificity of the probe DNA by FASTA analysis.

[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.

[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.

[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.

[Fig. 2-9] Fig. 9 is a continuation of Fig. 9 of Fig. 6 of confirmation of specificity by FASTA analysis of probe DNA.

FIG. 2-10 is a sequel to FIG. 7 showing confirmation of specificity by FASTA analysis of probe DNA.

FIG. 2-11 is a sequel to FIG. 8 showing confirmation of specificity by FASTA analysis of probe DNA.

FIG. 2-12 is a sequel to FIG. 9 showing confirmation of specificity by FASTA analysis of probe DNA.

[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.

[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.

FIG. 2-15 is a sequel to FIG. 12 showing confirmation of specificity by FASTA analysis of probe DNA.

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.

FIG. 3-2 is a continuation of FIG. 3.

FIG. 3-3 is a continuation of FIG. 3-2.

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.

4-2 is a continuation of FIG. 4.

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.

[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

   ─────────────────────────────────────────────────── ─── 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)

[Claims] 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. 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. 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. 4. A method for identifying a bacterial species using the chip for detecting bacteria according to any one of claims 1 to 3. 5. The method for identifying a bacterial species according to claim 4, which is 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).