JPH0870896A - New nucleic acid fragment and detection of corynebacterium sp. j1 strain using the fragment - Google Patents

Abstract

PURPOSE: To provide a new nucleic acid fragment composed of the 16SrRNA gene of Corynebacterium sp.J1 strain having a specific base sequence, capable of metabolizing organic compounds and enabling the quick and easy detection of bacteria e.g. for the cleaning of environmental pollution in high detection accuracy and sensitivity. CONSTITUTION: This new nucleic acid fragment is composed of the 16SrRNA gene of Corynebacterium sp.J1 strain having the base sequence expressed by formula. Bacteria capable of metabolizing a wide variety of organic compounds, especially having excellent decomposition activity of various aromatic compounds and useful e.g. for the cleaning of the pollution with organic compounds in soil caused by environmental pollution can quickly and easily be detected in high specificity and sensitivity by using the nucleic acid fragment or a part of the gene as a primer or a probe. The nucleic acid fragment can be produced by extracting an rRNA from Corynebacterium sp.J1 strain and amplifying the base sequence by PCR method using a synthesized primer.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel nucleic acid fragment and its use. For more information, visit Corynebacterium
A nucleic acid fragment that can be used as a primer or a probe for detecting the um sp. J1 strain, a partial sequence of the base sequence that constitutes the nucleic acid fragment, and these were used.
The present invention relates to a method for detecting Corynebacterium sp. J1 strain.

[0002]

Corynebacterium is a Gram-positive bacillus and is known to have the ability to metabolize a wide variety of organic compounds, and in particular to be excellent in decomposing various aromatic compounds.

In recent years, environmental pollution due to aromatic compounds, aliphatic hydrocarbons such as paraffin and naphthene, or organic chlorine compounds such as trichloroethylene has become a problem, and the already polluted environment can be purified. There is a strong demand for the establishment of technology to restore such conditions. Various physicochemical methods have been used as this environmental restoration technology, but there are difficulties related to cost, operability, amount of energy released, treatment range, etc., simple extraction of pollutants,
It is not necessarily a practically satisfactory technique from the viewpoint that it is not recovered and converted into a harmless chemical substance.

Therefore, with respect to the above physicochemical method,
It is expected that the treatment using microorganisms can provide a practical method for repairing a contaminated environment. Here, many of the pollutants are synthetic substances used in the chemical industry,
Depending on the microorganisms that live in the natural world, many of them are not easily decomposed and therefore not easily treated. However, C
As mentioned above, the bacteria of the genus orynebacterium have the ability to metabolize a wide variety of organic compounds, and are said to be persistent compounds such as aromatic hydrocarbons and organochlorine compounds that cause soil pollution problems. However, many strains that can be decomposed have been isolated. In particular, the development of trichlorethylene-degrading bacteria has been actively pursued, and the application of genetically modified bacteria to the soil, which has improved its degrading activity or made the inducer of trichlorethylene-degrading enzyme unnecessary, has begun to be considered. .

[0005] Here, the inventors of the present invention, Corynebact
erium sp. J1 strain (deposit number: P-1433
No. 2) decomposes trichlorethylene, and such Corynebacterium
In order to disseminate the environmental purification technology using genus bacteria and to establish this technology as a practical and socially effective technology, in addition to the development of bacteria with excellent decomposing ability, in the soil where it was introduced I am studying to fully understand the activity, growth, transmission, survival of bacteria, that is, the dominance of bacteria in soil and the growth status. In order to solve these problems, it is essential to establish a method that can selectively detect the target bacteria.

Various separation culture methods are known as a method for detecting a bacterium belonging to the genus Corynebacterium , and in general, selective culture in a special medium is often performed.
This method is widely used because it is simple, but it is not always the case that only the target bacteria are selectively cultured.
For example, Coryne , a fungus that decomposes phenol
In the case of the Bacterium sp. J1 strain, the yellow coloring of 2-hydroxymuconic semialdehyde, which is a degradation product of catechol by catechol 2,3 oxygenase, can be used as a simple index. However, there may be other bacteria that can metabolize catechol by meta-cleavage, and strictly speaking, it is not possible to use only yellow coloring as an index, and more detailed morphological or biochemical properties are not possible. You also need to inspect.

However, it requires a lot of skill and experience to detect the target bacterium from the morphological or biochemical properties required here, and the method is complicated and time-consuming. However, there were various problems in practical use.

[0008] There is also known a method in which an antibody specific to a target bacterium is prepared, labeled with a radioisotope or a fluorescent dye and used for detection, but this method requires a great deal of labor to obtain the antibody. In addition to this point, the fact that the specificity of the antibody varies greatly from lot to lot is a major drawback.

As an alternative to these methods, a method of detecting a biological species by paying attention to the base sequence of a nucleic acid specific to the biological species and utilizing an oligonucleotide primer or probe having a sequence complementary thereto Has been announced. In particular, ribosomal RNA (rRNA) is an essential cell constituent in organisms, and its structure is relatively well conserved during the evolution of organisms. r
Among RNAs, 16S rRNA has been relatively well studied, and its nucleotide sequence has been identified for many biological species.

It is known that 16S rRNA has a region commonly conserved in various organisms and a variable region having a different sequence depending on the organism species. A DNA primer or probe corresponding to this variable region was used. Bacteria detection and identification methods have been developed in recent years. For example, rRNA
Is more than 10 ⁴ in a cell and can be very advantageously used as a target of a hybridization method from the viewpoint of sensitivity, but here, a probe or primer for detecting a target bacterium is used. How to select is the most important and difficult issue, and in order to use this method,
It is necessary to correctly know the base sequence of S rRNA or its gene.

[0011]

As described above, when the purification treatment of pollutants in the soil is carried out by utilizing a specific microorganism, in order to optimize the treatment condition and the growth condition of the microorganism, Therefore, it is necessary to know the predominance of the microorganism in the soil and the growth condition. Its specific detection of microorganisms in order, but there is a need for a method that can measure, Coryne ever
Detection of bacterium bacterium and culture with a selective medium whose only carbon source phenol, those due antibody method, the specificity, sensitivity, simplicity, many problems in terms of the time required for detection, in soil It was not possible to monitor the behavior of the bacteria in real time.

The present invention has been made in view of the above problems of the prior art, and the purpose thereof is to provide a quick, simple and specific method.
Moreover, Corynebacterium s with good sensitivity
It is to provide a method for detecting and counting the p.J1 strain.

[0013]

The present invention provides Corynebacterium s represented by SEQ ID NO: 1 described below.
p. J1 strain 16S rRNA gene. The present invention also relates to a nucleic acid represented by the above-mentioned gene sequence, a nucleic acid having a complementary base sequence thereof, and a nucleic acid fragment selected from the nucleic acids represented by these mutant sequences.

The present invention also relates to a nucleic acid fragment comprising a nucleic acid which is a partial sequence in any of the above nucleic acids and can be used as a primer or a probe. In particular,
The nucleic acid that can be used as a primer has a length of 10 to 50 bases, and the nucleic acid that can be used as a probe has a length from 10 bases to an upper limit of a total number of bases of each nucleic acid or less.

The present invention also relates to a primer or probe using the above nucleic acid fragment. That is, the primer or probe according to the present invention is a nucleic acid fragment as described above, which is a nucleic acid fragment which may have a site capable of binding to a label or / and a solid phase carrier introduced. .

The present invention also provides Corynebacterium utilizing the above-mentioned primer or probe.
The present invention relates to a method for detecting m sp. That is, Corynebact according to the present invention using a primer
The erium sp. J1 strain can be detected by using the primer according to the present invention as described above, preferably a primer composed of a combination of two kinds, and the following (1) to (4):
It is characterized by carrying out the step of. (1) Corynebacterium sp. J1
Prepare a sample for which the presence or absence of a strain is to be detected, (2) crush cells in the sample as necessary, (3) add the above primer to the sample, and perform an extension reaction of the primer, (4)
A detection operation is performed on the extension reaction product obtained in step (3).

C according to the present invention using a probe
The method for detecting the orynebacterium sp. J1 strain is characterized by using at least one kind of probe as described above.

The present invention will be described in detail below. The Corynebacterium sp. J1 strain referred to here also includes its mutant strains which exhibit the same action according to the common knowledge of genetic engineering as long as they meet the purpose of the present invention. Is.

Corynebacterium sp.
Cloning of J1 strain 16S rRNA gene, determination of total nucleotide sequence Comparison of rRNA nucleotide sequences of various known aerobic bacteria,
The consensus sequence region was estimated. Next, a primer DNA is synthesized from this region, and Coryneb is synthesized by PCR.
16S in the DNA of the S. bacterium sp.
A fragment of about 1100 bases within the rRNA gene region was amplified and cloned into the HincII cleavage site of plasmid pUC119. By determining the nucleotide sequence of the obtained gene and comparing it with the known 16S rRNA nucleotide sequence,
It was confirmed that this fragment was the 16S rRNA gene.

Using this gene fragment, a full-length PC
The 16S rRNA gene that did not pass through R was cloned. First, Corynebacterium s
The p.J1 strain DNA was cleaved with Sau3AI and inserted into the BamHI site of the λ phage vector DASHII to prepare a library. This was screened by the plaque hybridization method using the above-mentioned 16S rRNA gene partial fragment as a probe to obtain full-length 16S rRNA.
A clone containing the RNA gene was selected.

The nucleotide sequence was determined by the deletion method and subcloning using an appropriate restriction enzyme cleavage site. The deletion was performed by recloning the full-length 16S rRNA gene at the HincII cleavage site of plasmid pUC119, followed by ExonuclearaseIII and MungBe.
It was introduced stepwise by an Nuclease. The nucleotide sequence was determined by the dideoxy method using these step deletion clones and subclones utilizing restriction enzymes.

Corynebacterium sp.
Determination of specific region of J1 strain 16S rRNA gene The specific region was determined by comparing the nucleotide sequence determined as described above with a known 16S rRNA gene sequence of other species of bacteria. In this case, EMBL nucleic acid sequence data library or the like was used to perform analysis using a personal computer.

The specific region thus determined is a novel nucleotide sequence and has applicability for the purpose of the present invention, and therefore is claimed in the scope of claim 3.

Nucleic Acid Fragment The nucleic acid fragment according to the present invention is a nucleic acid fragment represented by SEQ ID NO: 1, a nucleic acid having a complementary base sequence thereof, and a nucleic acid fragment represented by these mutant sequences,
In addition, it is as described above that it is a partial sequence in any of the above-mentioned nucleic acids and is composed of a nucleic acid that can be used as a primer or a probe.

These nucleic acid fragments can be used especially as probes, and the partial base sequences can be used as primers or probes. Nucleic acids that can be used as primers or probes are Corynebacterium
Any primer may be used as long as it functions as a primer or a probe for detecting the um sp. J1 strain. Here, when a partial base sequence is used, Coryneba
other bacteria specific for the C. sp. J1 strain,
For example, P. cepacia , P. putida , ae
Pseudomonas bacteria such as ruginosa , better isolated from soil, Alcaligenes
Genus bacterium, Xanthomonas genus bacterium, Agroba
bacterium of the genus Cterium , Enterobacteria
It is preferable to select a portion having a low homology to a bacterium of the genus ceae . Specifically, the partial base sequence has a length of 10 to 50 bases in the case of a primer, and a length of 10 bases to the total number of bases of each nucleic acid or less in the case of a probe.

These nucleic acid fragments or their partial base sequences can be prepared by any purposeful method. For example, all or part of the sequences may be chemically synthesized according to the method described in Examples below. . Or Cory
Nebacterium sp. excising from the genes for strain J1 directly using, for example, restriction enzymes are also possible, also, E those genes. It is also possible to clone the plasmid into a plasmid such as E. coli , collect the plasmid after growth of the bacterium, cut out and use it.

Examples of the mutation in the nucleic acid fragment of the present invention include deletion, substitution and addition of some bases or base sequences. When using a nucleic acid fragment as a primer here, it is preferable that there is no mutation near the 3'end, which is considered to have a great influence on the extension reaction of the primer, or even if there is, it should be minimized. It is suitable for the purpose of the present invention to make mutations preferably near the 5'end.

The base sequence of the nucleic acid fragment which can be preferably used herein is, of course, a base sequence which has all or most of its origin in the specific region and has a small contiguous portion, and has a base length of a degree convenient for the purpose. As the inventor, the inventor submits what is shown in claim 6 of the claims. For the sake of understanding, these base sequences are listed below together with the base numbers shown in Sequence Listing 1.

SEQ ID NO: Sequence position Nucleotide sequence 1 1547-1570: GGTAAGAATCCACAACAAGTCGTT 2 1529-1552: AACGAAAGATTGACGATTGGTAAG 3 1440-1458: AGGAGGACGCTTACCACGG 4 1428-1449: AGTCTAACCGCAAGGAGGACGC 5 1428 -1GA: 132132 1AG: AGTCGAACCAGGC TCGGAATCGCTAGTAATCGC 8 1321 - 1341: GTCGGAATCGCTAGTAATCGC 9 1321 - 1340: GTCGGAATCGCTAGTAATCG 10 1320 - 1341: AGTCGGAATCGCTAGTAATCGC 11 1320 - 1340: AGTCGGAATCGCTAGTAATCG 12 1319 - 1341: AAGTCGGAATCGCTAGTAATCGC 13 1319 - 1340: AAGTCGGAATCGCTAGTAATCG 14 1319 - 1339: AAGTCGGAATCGCTAGTAATC 15 1319 - 1338: AAGTCGGAATCGCTAGTAAT 16 1319-1336: AAGTCGGAATCGCTAGTA 17 1318-1341: GAAGTCGGAATCGCTAGTAATCGC 18 1318-1340: GAAGTCGGAATCGCTAGTAATCG 19 1318 -1339: GAAGTCGGAATCGCTAGTAATC 20 1318 -AGC 13 ATGATCATC 13TAG -13AG -16AG -ATGA 13GC -GAAGTCTCAATAG 13AG- 1335: CATGAAGTCGGAATCGCTAGT 25 1314 -1335: CCATGAAGTCGGAATCGCTAGT 26 1314-1334: CCATGAAGTCGGAATCGCTAG 27 1314-1332: CCATGAAGTCGGAATCGCT 28 1303-1321: GCAACTCGACTCCATGAAG 29 1303-1320: GCAACTCGACTCCATGAGA30 302-1321: TGCAACT1CAT13GA1 : CTGCAACTCGACTCCATGAA 34 1301-1319: CTGCAACTCGACTCCATGA 35 1300-1321: TCTGCAACTCGACTCCATGAAG 36 1300-1320: TCTGCAACTCGACTCACTCACTGACAT 40 381300-1318: TCTGCAACTCGACTCACTGACTCGA38 1300-1318: TCTGCAACTCGACTCACTGACATC 38G 300 24 42 1201-1218: CGACCAGGGCTACACACG 43 1200-1218: ACGACCAGGGCTACACACG 44 1199-1219: TACGACCAGGGCTACACACGT 45 1198-1219: TTACGACCAGGGCTACACACGT 46 1198-12GC: 11TAC-12CA: 11GC-11CA-11CA-11GC-12GC: 11TAC-12CA: 11AG -1127: TTTGCCAGCGGGTTAAGC 51 1109-1128: ATTTGCCAGCGG GTTAAGCC 52 1109-1127: ATTTGCCAGCGGGTTAAGC 53 1109-1126: ATTTGCCAGCGGGTTAAG 54 1108-1128: TATTTGCCAGCGGGTTAAGCC 55 1108-1 1107-1126: TTATTTGCCAGCGGGTTAAG 61 1107-1125: TTATTTGCCAGCGGGTTAA 62 1107-1124: TTATTTGCCAGCGGGTTA 63 1106-1128: CTTATTTGCCAGCGGGTTAAGCC 64 1106TT-1GT: CGTATTAGC 65 1106TGCCAG 1CTG-1AG: CTTATTTGCCAGCGGGTTAAGC 65 1106-1CC-1CC: 1126-1126 1123: CTTATTTGCCAGCGGGTT 69 1105-1128: CCTTATTTGCCAGCGGGTTAAGCC 70 1105-1127: CCTTATTTGCCAGCGGGTTAAGC 71 1105-1126: CCTTATTTGCCAGCGGGTTAAG 72 1105 -1GT: CC112CCCCTTATTTGCCAGCGGGTTAA 73 1105 -1CC: CCTTATTTGCCAGCGGGTTAA 73 1105 -1CC: CCGTATT105CC74CG1 TCCTTATTTGCCAGCGGGTTAAGC 77 1104-1126: TCCTTATTTGCCAGC GGGTTAAG 78 1104 - 1125: TCCTTATTTGCCAGCGGGTTAA 79 1104 - 1124: TCCTTATTTGCCAGCGGGTTA 80 1104 - 1123: TCCTTATTTGCCAGCGGGTT 81 1104 - 1122: TCCTTATTTGCCAGCGGGT 82 1104 - 1121: TCCTTATTTGCCAGCGGG 83 1103 - 1126: TTCCTTATTTGCCAGCGGGTTAAG 84 1103 - 1125: TTCCTTATTTGCCAGCGGGTTAA 85 1103 - 1124: TTCCTTATTTGCCAGCGGGTTA 86 1103-1123: TTCCTTATTTGCCAGCGGGTT 87 1103-1121: TTCCTTATTTGCCAGCGGG 89 1102-1125: TTTCCTTATTTGCCAGCGGGTTAA 90 1010-1029: GTGCCTTCGGGAACTTACAT 91 100GA -1000GGAC 102GAC 102GAC 102GAC 102GAG-1002-1022: CCTGGAGA 100 100-1022: CCTGGAGA 100 100-1022: CCTGCGAGA 1021: GAGATGGATTGGTGCCTTCGGG 96 1000-1020: GAGATGGATTGGTGCCTTCGG 97 999-1022: AGAGATGGATTGGTGCCTTCGGGA 98 999 -1021: AGAGATGGATTGGTGCCTTCGGG 99 999 -TG CG: TGCATGTG: 10AG: TGCATG: 10AG: 999 -TG: 10G: TAG-10G: 999-10G CAGAGATGGATTGGTGCCTTCG 10 4 998-1018: CAGAGATGGATTGGTGCCTTC 105 984-1004: TAGTAAGAACTTTCCAGAGAT 106 828-846: CTTTGAGGCTTTAGTGGCG 107 732-750: CTAATACTGACGCTGAGGT 108 TG 732 -GAC ACT -GAC ACT ACT -GAC ACT -GAC ACT ACT -GAC ACT -GAC ACT -GAC ACT ACT -GAG- -748: GCCTAATACTGACGCTGAG 113 729-750: GGCCTAATACTGACGCTGAGGT 114 729-749: GGCCTAATACTGACGCTGAGG 115 728-750: TGGCCTAATACTGACGCTGAGGT 116 GA -CT 728 -CT ACT TAG-TG ACT-CG ACT-ACT-ACT-ACT-ACT-ACT-- : CTGGCCTAATACTGACGCTGAGG 121 727 - 748: CTGGCCTAATACTGACGCTGAG 122 726 - 749: TCTGGCCTAATACTGACGCTGAGG 123 726 - 748: TCTGGCCTAATACTGACGCTGAG 124 726 - 747: TCTGGCCTAATACTGACGCTGA 125 726 - 746: TCTGGCCTAATACTGACGCTG 126 726 - 744: TCTGGCCTAATACTGACGC 127 725 - 748: ATCTGGCCTAATACTGACGCTGAG 128 725 - 747: ATCTGGCCTAATACTGACGCTGA 129 725-746: ATCTGGCCTAATACTGACGCTG 130 72 5-745: ATCTGGCCTAATACTGACGCT 131 724-747: CATCTGGCCTAATACTGACGCTGA 132 724-746: CATCTGGCCTAATACTGACGCTG 133 GC -GC: CATCTGGCCTAATACTGACGCT AG AG CG AG AG CG AG CG TAG AG CG TAG AG CG TAG AG CG TAG CG TAG CG TAG CG TAG CG TAG AG CG TAG CG TAG CG TAG CG TAG CG TAG CG TAG CG TAG ACT AG ACTUAL ACTIVITIES 721: AATACCGATGGCGAAGGCA 139 702-723: GAATACCGATGGCGAAGGCAGC 140 702-722: GAATACCGATGGCGAAGGCAG 141 702 -GC: GAATACCGATGGCGAAGGCA 142 702-720: GACCAGGAAGC 147 GA: GAATACCGATGGCGAAGGC 143 701 -723: GAAGCCGACGCAG143 AGGAATACCGATGGCGAAGGCAGC 147 700 - 722: AGGAATACCGATGGCGAAGGCAG 148 700 - 721: AGGAATACCGATGGCGAAGGCA 149 700 - 720: AGGAATACCGATGGCGAAGGC 150 699 - 722: GAGGAATACCGATGGCGAAGGCAG 151 699 - 721: GAGGAATACCGATGGCGAAGGCA 152 699 - 720: GAGGAATACCGATGGCGAAGGC 153 699 - 719: GAGGAATACCGATGGCGAAGG 154 699 - 717: GAGGAATACCGATGGCGAA 155 699-716: GAGGAATACCGATGGCGA 156 698- 721: GGAGGAATACCGATGGCGAAGGCA 157 698 - 720: GGAGGAATACCGATGGCGAAGGC 158 698 - 719: GGAGGAATACCGATGGCGAAGG 159 698 - 718: GGAGGAATACCGATGGCGAAG 160 698 - 717: GGAGGAATACCGATGGCGAA 161 698 - 716: GGAGGAATACCGATGGCGA 162 698 - 715: GGAGGAATACCGATGGCG 163 697 - 720: TGGAGGAATACCGATGGCGAAGGC 164 697 - 719: TGGAGGAATACCGATGGCGAAGG 165 697 - 718: TGGAGGAATACCGATGGCGAAG 166 697 - 717: TGGAGGAATACCGATGGCGAA 167 697 - 716: TGGAGGAATACCGATGGCGA 168 697 - 715: TGGAGGAATACCGATGGCG 169 697 - 714: TGGAGGAATACCGATGGC 170 696 - 719: CTGGAGGAATACCGATGGCGAAGG 171 696 - 718: CTGGAGGAATACCGATGGCGAAG 172 696 - 717: CTGGAGGAATACCGATGGCGAA 173 696 - 716: CTGGAGGAATACCGATGGCGA 174 696 - 715: CTGGAGGAATACCGATGGCG 175 696 - 714: CTGGAGGAATACCGATGGC 176 695 - 718: TCTGGAGGAATACCGATGGCGAAG 177 695 - 717: TCTGGAGGAATACCGATGGCGAA 178 695 - 716: TCTGGAGGAATACCGATGGCGA 179 695 - 715: TCTGGAGGAATACCGATGGCG 180 695 - 714: TCTGGAGGAATACCGATGGC 181 695 - 713: TCTGGAGGAATACCGATGG 182 694-717: ATCTGGAGGAATACCGATGGCGAA 183 694 - 716: ATCTGGAGGAATACCGATGGCGA 184 694 - 715: ATCTGGAGGAATACCGATGGCG 185 694 - 714: ATCTGGAGGAATACCGATGGC 186 694 - 713: ATCTGGAGGAATACCGATGG 187 693 - 716: GATCTGGAGGAATACCGATGGCGA 189 693 - 715: GATCTGGAGGAATACCGATGGCG 190 693 - 714: GATCTGGAGGAATACCGATGGC 191 693 - 713: GATCTGGAGGAATACCGATGG 192 693 - 712: GATCTGGAGGAATACCGATG 193 693 - 710: GATCTGGAGGAATACCGA 194 675 - 696: AGCGGTGAAATGCGTAGAGATC 195 674 - 696: TAGCGGTGAAATGCGTAGAGATC 196 674 - 695: TAGCGGTGAAATGCGTAGAGAT 197 673 - 696: GTAGCGGTGAAATGCGTAGAGATC 198 673 - 694: GTAGCGGTGAAATGCGTAGAGA 199 672 - 695: TGTAGCGGTGAAATGCGTAGAGAT 200 668 - 689: AGGTTGTAGCGGTGAAATGCGT 201 667 - 690: CAGGTTGTAGCGGTGAAATGCGTA 202 667 - 689: CAGGTTGTAGCGGTGAAATGCGT 203 667 - 688: CAGGTTGTAGCGGTGAAATGCG 204 666 - 689: CCAGGTTGTAGCGGTGAAATGCGT 205 666 - 688: CCAGGTTGTAGCGGTGAAATGCG 206 666 - 687: CCAGGTTGTAGCGGTGAAATGC 207 665 - 688: TCCAGGTTGTAGCGGTGAAATGCG 208 644 - 662: TATGGGAGAGGATGGT AGA 209 643-663: GTATGGGAGAGGATGGTAGAA 210 643-662: GTATGGGAGAGGATGGTAGA 211 643-661: GTATGGGAGAGGATGGTAG 212 643 -GA 216 GA: GAGAGAGA TAG 213 GA GG TAG: GAGAGAGAGAGAATGA 642 TAG: GAGAGAGAGAGAGAGAGAGA 216 GA 641 - 664: GAGTATGGGAGAGGATGGTAGAAT 218 641 - 662: GAGTATGGGAGAGGATGGTAGA 219 641 - 661: GAGTATGGGAGAGGATGGTAG 220 641 - 660: GAGTATGGGAGAGGATGGTA 221 641 - 659: GAGTATGGGAGAGGATGGT 222 641 - 658: GAGTATGGGAGAGGATGG 223 640 - 663: AGAGTATGGGAGAGGATGGTAGAA 224 640 - 662: AGAGTATGGGAGAGGATGGTAGA 225 640 - 661: AGAGTATGGGAGAGGATGGTAG 226 640 - 660: AGAGTATGGGAGAGGATGGTA 227 640 - 659: AGAGTATGGGAGAGGATGGT 228 640 - 658: AGAGTATGGGAGAGGATGG 229 640 - 657: AGAGTATGGGAGAGGATG 230 639 - 662: TAGAGTATGGGAGAGGATGGTAGA 231 639 - 661: TAGAGTATGGGAGAGGATGGTAG 232 639 - 660: TAGAGTATGGGAGAGGATGGTA 233 639 - 659: TAGAGTATGGGAGAGGATGGT 234 639-658: TAGAGTATGGGAGAGGATGG 235 639-657 : TAGAGTATGGGAGAGGATG 236 638 - 661: CTAGAGTATGGGAGAGGATGGTAG 237 638 - 660: CTAGAGTATGGGAGAGGATGGTA 238 638 - 659: CTAGAGTATGGGAGAGGATGGT 239 638 - 658: CTAGAGTATGGGAGAGGATGG 240 638 - 657: CTAGAGTATGGGAGAGGATG 241 638 - 656: CTAGAGTATGGGAGAGGAT 242 638 - 655: CTAGAGTATGGGAGAGGA 243 637 - 660: GCTAGAGTATGGGAGAGGATGGTA 244 637 - 659: GCTAGAGTATGGGAGAGGATGGT 245 637 - 658: GCTAGAGTATGGGAGAGGATGG 246 637 - 657: GCTAGAGTATGGGAGAGGATG 247 637 - 655: GCTAGAGTATGGGAGAGGA 248 636 - 659: AGCTAGAGTATGGGAGAGGATGGT 249 636 - 658: AGCTAGAGTATGGGAGAGGATGG 250 636 - 657: AGCTAGAGTATGGGAGAGGATG 251 636 - 656: AGCTAGAGTATGGGAGAGGAT 252 636 - 655: AGCTAGAGTATGGGAGAGGA 253 636 - 654: AGCTAGAGTATGGGAGAGG 254 635 - 658: AAGCTAGAGTATGGGAGAGGATGG 255 635 - 657: AAGCTAGAGTATGGGAGAGGATG 256 635 - 656: AAGCTAGAGTATGGGAGAGGAT 257 635 - 655: AAGCTAGAGTATGGGAGAGGA 258 635 - 654: AAGCTAGAGTATGGGAGAGG 259 635 - 653: AAGCTAGAGTATGGGAGAG 260 625 - 643 : CGATACTGGGAAGCTAGAG 261 625-642: CGA TACTGGGAAGCTAGA 262 624 - 642: TCGATACTGGGAAGCTAGA 263 624 - 641: TCGATACTGGGAAGCTAG 264 590 - 610: TGTGAAATCCCTGAGCTTAAC 265 590 - 609: TGTGAAATCCCTGAGCTTAA 266 590 - 608: TGTGAAATCCCTGAGCTTA 267 589 - 610: ATGTGAAATCCCTGAGCTTAAC 268 589 - 609: ATGTGAAATCCCTGAGCTTAA 269 589 - 608: ATGTGAAATCCCTGAGCTTA 270 589-607: ATGTGAAATCCCTGAGCTT 271 589-606: ATGTGAAATCCCTGAGCT 272 465-485: GTGGACGTTACTCGCAGAATA 273 465-483: GTGGACGTTACTCGCAGAA 274 465 GG -AG 447 TAG-TAG 426 TAG-TAG 426 TAG-426 TAG-426 TAG- 228: GCTAATAGATGAGCCTAAGTCA 279 194-213: CTTCGGACCTTGCGCTAATA 280 193 -212: TCTTCGGACCTTGCGCTAAT 281 193-211: TCCTTCGGACCTTGCGCTAA 282 193 -210: TCCTTCGGTCTTACCGGACCTTGCGCTATA 283 192 -212: ATCTTCGGACCTTGCGCTATA 283 192 -212: ATCTTCGGACCTTGCTATA GGATCTTCGGACCTTGCGCTAATA 287 185-206: GCAGGGGATCTTCGGACCTTGC 289 184-205: AG CAGGGGATCTTCGGACCTTG 290 184-204: AGCAGGGGATCTTCGGACCTT 291 184-202: AGCAGGGGATCTTCGGACC 292 161-178: ACCGCATACGCCCTACTACGCGCGCGCTACCG CAT CG: GC CG CAT CG: GC CAT CG: GC CAT CG: GC CAT: GC CAT: GC CAT: GC CAT: GC CAT: GC CAT: GC TAC: GC CAT: GC TAC: GC TAC: GC 158-178: AATACCGCATACGCCCTACGG 299 158-177: AATACCGCATACGCCCTACG 300 158-176: AATACCGCATACGCCCTAC 301 158-175: AATACCGCATACGCCCTA 302 157-178: TAATACCGCATACGCCCTACGG 303 157-177: CCATACGGCATACGCCCTACGG 303 157-177: CCATAGGC 304CG 174: TAATACCGCATACGCCCT 307 156-178: CTAATACCGCATACGCCCTACGG 308 156-177: CTAATACCGCATACGCCCTACG 309 156-176: CTAATACCGCATACGCCCTAC 310 156-174: CTAATACCGCATAG CG: CGAATACCCC 177 GCTAATACCGCATACGCCCTAC 315 155-175: GCTAATACCGCATACGCCCTA 316 155-174 : GCTAATACCGCATACGCCCT 317 155 - 173: GCTAATACCGCATACGCCC 318 147 - 165: AAAGGAATGCTAATACCGC 319 146 - 164: GAAAGGAATGCTAATACCG 320 146 - 163: GAAAGGAATGCTAATACC 321 145 - 165: CGAAAGGAATGCTAATACCGC 322 144 - 165: CCGAAAGGAATGCTAATACCGC 323 137 - 155: CAACATTCCGAAAGGAATGC 324 124 - 144: TATTAGTGGGGGACAACATTC 325 124-142: TATTAGTGGGGGACAACAT 326 89-110: GCGGCGGACGGGTGAGTAATGC 327 89-109: GCGGCGGACGGGTGAGTAATG 328 89-107: GCGGCGGACGGGTGAGTAA 329 88 -111: GGCGGCGGACGGGTGAGT88AG- -106: AGCGGCGGACGGGTGAGTA 334 87-110: TAGCGGCGGACGGGTGAGTAATGC 335 87-109: TAGCGGCGGACGGGTGAGTAATG 336 87-108: TAGCGGCGGACGGGTGTGAGTAAT 337 87GG -TAG: CG87CG -AG: CG87CG -AG: CG87AG -AG: CG87GT -AG: CG87CG -AG: CG87AG -AG: CG87CG -AG: CG87AG-CG: CG: CG: 87AG-CG: CG: 87AG : CTAGCGGCGGACGGGTGAGTAA 342 86-106: CTAGCGGCGGACGGGTGAGTA 343 85-108: CCTAGCGGCGGACGGGTGAGTAAT 344 85 -107: CCTAGCGGCGGACGGGTGAGTAA 345 85 -106: CCTAGCGGCGGACGGGTGAGTA 346 85 -105: CCTAGCGGCGGACGGGTGAGT 347 78 -95: TACATTTCCTAGCGGCGG 348 56-77: CGAGCGGGGAGATGTAGCT If necessary, the nucleic acid fragment or partial base sequence that can be used as a primer or probe may have a site capable of binding to a label or a solid phase carrier.
Here, in the case of performing detection using a primer, the position at which a site capable of binding to a label or a solid phase carrier may be introduced may be any position as long as it does not interfere with the extension reaction of the primer, but if possible 5 ′ The ends are most preferred. In the case of performing detection using a probe, the position at which a site capable of binding to a label or a solid phase carrier may be introduced is at the 3'-terminal or 5'-terminal hydroxyl groups, as well as the base moiety or phosphodiester moiety. Although conceivable, it is desirable to consider the nucleotide sequence and length of the probe so as not to interfere with hybridization.

When the nucleic acid is used as a probe or a primer, either a radioactive substance or a non-radioactive substance may be used as the label. As the non-radioactive label, a fluorescein derivative, which can be directly labeled,
Examples include fluorescent substances such as rhodamine and its derivatives, chemiluminescent substances, and delayed fluorescent substances.

It is also possible to indirectly detect the labeled substance by using a substance that specifically binds to the labeled substance. Examples of such labeled substances include biotin and hapten. In the case of biotin, avidin or streptavidin is used, and in the case of hapten, an antibody that specifically binds to this is used. As the hapten, a compound having a 2,4-dinitrophenyl group, digoxigenin, or the like can be used. Any of these labeled substances can be introduced into a probe or a primer alone or in combination of two or more kinds as necessary.

When it is necessary to specifically bind a specific fragment of a nucleic acid to a solid phase carrier such as sandwich hybridization, the site capable of binding to the solid phase carrier is such that it can selectively bind to the carrier. It can be anything. Examples thereof include biotin or fluorescein, compounds having a 2,4-dinitrophenyl group, and haptens such as digoxigenin, and these can be introduced into a probe or a primer alone or in combination of two or more kinds as necessary.

Detection method using a probe Corynebacte according to the present invention using a probe
The method for detecting the rum sp. J1 strain comprises a nucleic acid fragment having the nucleotide sequence of the nucleic acid fragment, which may have a site capable of binding to a label or / and a solid phase carrier. It is characterized by using at least one kind of probe.

In general, detection methods using a probe include dot hybridization, Southern hybridization, and in situ hybridization. In any of these methods, the labeled nucleic acid fragment is used and hybridization is carried out in the usual manner. You can

Recently, in order to improve the sensitivity of hybridization, rRN in which a large number of copies exist per cell
Although a method using A as a detection target has been developed, the nucleic acid fragment in the present invention is a nucleic acid fragment containing a region encoding 16S rRNA, and can be used for rRNA detection by the same method.

In addition, in the in situ hybridization as well, detection with a good S / N ratio is generally possible by using rRNA as the detection target, but the nucleic acid fragment of the present invention can be used for detection in the same manner as above. is there.

In addition, methods based on sandwich hybridization have been developed in order to simplify the operation, and these methods also utilize the nucleic acid fragment of the present invention to produce Corynebacterium sp.
The J1 strain can be detected.

Detection Method Using a Primer Corynebact According to the Present Invention Using a Primer
The method for detecting erium sp. J1 strain comprises a nucleic acid fragment having a base sequence of the nucleic acid fragment, which may have a site capable of binding to a label or / and a solid phase carrier. It is characterized by using at least one kind of primer, and as a more preferable example, PCR (Polymerase Chain React) for amplifying a minute amount of nucleic acid fragment in a sample by a gene amplification reaction by two kinds of different primers as described below.
Ion) method.

Here, the basic form of the two kinds of primers is, for example, that the two kinds of primers are not modified at all, or at least one of the two kinds of primers has a detectable label or a solid phase carrier. Introducing a moiety capable of binding to a solid phase carrier, one of the two types of primers having a label introduced, and the other having a site capable of binding to a solid phase carrier Examples include those into which a bondable moiety has been introduced.

Corynebacterium according to the present invention
The method for detecting the um sp. J1 strain can be carried out as follows using the primer according to the present invention as described above, preferably a primer composed of a combination of two kinds. (1) Corynebacterium sp. J1
Prepare a sample for which the presence or absence of a strain is to be detected, (2) crush cells in the sample as necessary, (3) add the above primer to the sample, and perform an extension reaction of the primer, (4)
A detection operation is performed on the extension reaction product obtained in step (3).

Here, for the detection of the nucleic acid fragment amplified by the primer extension reaction, a commonly used method such as electrophoresis or hybridization may be used, or different primers may be used for each primer in the gene amplification reaction. It is also possible to use a method in which a label is introduced, the product after the amplification reaction is adsorbed on a solid phase carrier, and the product is selectively detected.

Here, as the solid phase carrier, polystyrene balls, agarose beads, polyacrylic beads, latex, microtiter wells, or other solid phase materials capable of capturing the binding site introduced in the primer, for example, streptavidin. , Those into which an antibody or the like has been introduced. For example, to capture a PCR product from a biotin-introduced primer, a carrier to which streptavidin is bound to a solid phase is used, and to capture an extension reaction product from a fluorescein-introduced primer, an antibody against each is used. A carrier in which is bound to a solid phase is used.

Further, by using fine particles as the solid phase carrier, the target nucleic acid can be easily determined by the presence or absence of aggregation or precipitation. In addition, one primer has a site capable of binding to the solid-phase carrier, and the other primer has a label introduced therein. There is also a method in which the target nucleic acid is washed and removed with an appropriate solvent, and the target nucleic acid is immobilized on the solid phase carrier in a form having a label and specifically detected.

For the actual detection of these labeling substances, a general method may be used depending on the labeling substances used. For example, if the labeling substance is a radioisotope, the activity may be measured as it is. For example, in the case of biotin, an avidin-enzyme conjugate, and in the case of a hapten, an antibody-
The reaction may be performed with a substrate such as AMPPD using an enzyme conjugate or the like, and detection may be performed by optical or fluorescent means.

Although various methods have been devised for the enzyme used in the gene amplification reaction, reaction conditions, etc., the nucleic acid fragment of the present invention has a length and base sufficient for use in various PCR methods. It has a sequence, and by using it, Corynebacterium sp. J
One strain can be detected reliably and easily.

[0046]

【Example】

[Example 1] Corynebacterium s
Cloning of 16S rRNA gene of p. J1 strain and determination of nucleotide sequence Known aerobic bacteria ( Pseudomonas testo)
steroni , Acinetobacter cal
coaceticus , Alcaligenes xy
losoxidans and Alcaligenes
faecalis ) and 16S rRNA was obtained, and the following two common nucleotide sequences were selected. Sequence 1: 5'AGAGTTTTGATCCTGGCTCA
G3 'Sequence 2: 5'GTGTCGTGAGATGTGTGGT
T3 ′ Regarding the above sequence, the main chain was synthesized for Sequence 1 and its complementary strand was synthesized for Sequence 2. These synthesized DN
Corynebacterium with A as a primer
PCR was performed using the m sp. J1 strain of DNA.
The generated DNA fragment of about 1100 base pairs was inserted into the HincII cleavage site of plasmid pUC119, and Escherichia coli JM109 was transformed.

Transformants were treated with ampicillin, IPTG,
Selection was performed on an agar medium containing X-gal, a plasmid was prepared from the resulting white colonies, and the presence or absence of the inserted fragment was examined. A recombinant plasmid in which a DNA fragment of about 1100 base pairs was inserted was selected, and the base sequence at the end of the PCR fragment was determined by the dideoxy method. When this was compared with the known 16S rRNA nucleotide sequences of various bacteria, the homology was very high, and this insert fragment was Coryne.
16S rRNA of Bacterium sp. J1 strain
Presumed to be a partial sequence.

Full length 16S r without PCR
Cloning of the RNA gene was performed as follows. C
DN of orynebacterium sp. J1 strain
A was partially digested with the restriction enzyme Sau3AI, and a 9-23 kilobase pair fragment was separated and purified by agarose gel electrophoresis, and these DNA fragments were combined with λDASHII (STRAT).
AGENE) BamHI digestion product. Giga
packII Gold extract (STRAT
AGENE) was used to prepare phage particles by the in vitro packaging method to prepare a DNA library. Here, the above 16S rRNA gene partial fragment was used as a probe to screen positive phages from the DNA library by the plaque hybridization method. About 1 in 500 positive phages could be obtained. After culturing the positive phage by a conventional method, the phage DNA was purified by the cesium chloride density gradient method.

The nucleotide sequence was determined by using the step deletion clone and 1
It was carried out using a subclone obtained by utilizing a restriction enzyme cleavage site in the 6S rRNA gene. The deletion is the full-length 16S at the HincII cleavage site of plasmid pUC119.
Exonucl after recloning rRNA gene
easeIII and Mung Bean Nuclea
It was introduced stepwise by se. The nucleotide sequence was determined by the dideoxy method using these step deletion clones and subclones utilizing restriction enzymes. Corynebacterium sp. Thus determined
The base sequence of 16S rRNA of J1 strain is the base sequence of SEQ ID NO: 1.

[Example 2] Preparation of primer In the present invention, a primer having a site capable of binding to a labeled substance or a solid phase carrier or a primer having no site introduced therein was prepared by the chemical synthesis method shown below. .

First, a DNA automatic synthesizer (Applied Biosystems In
c. Model 381A) was used to synthesize by a phosphoramidite method on a 0.2 μmol scale, and an OPC cartridge (Applied BiosystemsIn) was used.
c. ).

A primer having a site capable of binding to a label or a solid phase carrier is first synthesized as an oligonucleotide having an amino group introduced at its 5'end, and then labeled with an appropriate reagent. A site capable of binding to a substance or a solid phase carrier was introduced. An example is shown below.

An oligonucleotide (5'-GCTAATATAGATGAGCCTA having an amino group introduced at the 5'end)
AGTCA-3 ') is synthesized by adding a final base (G in this case) to the 5'end by a synthetic reaction as described above, followed by Aminolink II ^™ (Applied Bios).
systems Inc. ) Was further added, and after the synthesis was completed, the product was purified with an OPC cartridge.

Biotinylation was performed as follows.
1O. D. Aminated oligonucleotide aqueous solution of 10μ
l, 10 μl of 1M NaHCO ₃ aqueous solution, 30 μl of water
1 and 20 μg / μl of biotinyl-N-hydroxysuccinimide ester (BRL) in DMF at 50.
μl was added, mixed and left at room temperature. After 4 hours, it was subjected to gel filtration using Sephadex G-50 as a carrier to obtain 50 mM.
It was eluted with TEAB (triethylammonium bicarbonate) buffer (pH 7.5), the first peak was collected, dried and dissolved in TE buffer (pH 8.0).

Dinitrophenyl group (DNP) at the 5'end
Introduced an oligonucleotide (5'-CTTACCA
ATCGTCAATCTTTCGGT-3 ') was first synthesized and purified as an oligonucleotide having an amino group introduced at its 5'end, as in the case of biotin labeling. The 2O. D. 180 μl of aminated oligonucleotide aqueous solution of 1M NaHCO ₃
20 μl of an aqueous solution was added, and 100 μl of an ethanol solution of 5% (v / v) dinitrofluorobenzene was added thereto, followed by heating at 37 ° C. for 2 hours to carry out a reaction. Purification is performed by gel filtration as with biotinylated oligonucleotides,
After drying to dryness, it was dissolved in TE buffer (pH 8.0).

[Example 3] Preparation of probe Oligonucleotide (5'-AAGCGAGGAGGAGGCTACTTA) having a biotin label introduced at the 3'end.
GATTAATACTCTAGGATAGG-3 ')
Was prepared. 0.5'mol scale 3'Biotin-O with 3'end labeled in advance with biotin
Oligonucleotides were synthesized by the phosphoramidite method using a N CPG column (CLONTECH), purified by an OPC cartridge by a conventional method, and dried to dryness.
It was dissolved in TE buffer (pH 8.0).

[Example 4] Evaluation of specificity of primer Corynebacterium sp. J1 strain,
P. cepacia KK01 strain (FERM BP-4
235), P. P. putida BH strain, P. aerug
inosa (IFO 3080), P. fluores
cens (IFO14160), Alcaligene
s faecalis (IFO 14479), Xan
thomonas maltophilia (IFO
14161), Enterobacter cloac
ae (IFO 13535), E. coli JM10
Using 9 strains of 9 species, D
Using the prepared NA as a sample, the specificity of the primer was evaluated by the PCR method. The primers were the two types of primers prepared in Example 2 (5'-Biotin-GCTA).
ATAGATGAGCCTAAGTCA-3 ', 5'-
DNP-CTTACCAATCGTCAATCTTTC
GTT-3 ') was used. PCR was performed under the following reaction solution composition and conditions.

1 μl of each of the above primers at a concentration of 30 pmol / μl and 5 μl of the reaction buffer attached to the enzyme
l, 5 μl of the dNTP mixed solution attached to the enzyme, sample DN
Add 10 ng of A, and further add water to bring the total amount of the reaction solution to 5
The volume was 0 μl. To this, AmpliTaq DNA p
Add 1 unit of olmerase (Takara Shuzo), 90
After heating to ℃ and holding for 5 minutes, 90 ℃ · 60 seconds, 55
The reaction was carried out for 30 cycles with one cycle consisting of 45 ° C. for 45 seconds and 72 ° C. for 90 seconds, and after the reaction was further incubated at 72 ° C. for 5 minutes. After the reaction, 10 μl is taken from 50 μl, agarose gel electrophoresis and ethidium bromide staining are performed,
The amplified nucleic acid chain was detected. As a result, Coryneb
Lactobacillus sp. Only in the J1 strain, a clear single band was confirmed at the expected length of about 1400 base pairs. Here, in the other 8 strains, no amplified nucleic acid chain could be detected at all. Example 5 Corynebacterium s
p. Detection Using Primer of J1 Strain (1) In the same manner as in Example 4, DNA was prepared from 9 kinds of various bacteria. PC with the following reaction solution composition and reaction conditions
It was subjected to R.

20 pmol / μl prepared in Example 2
Concentration of primer (5'-Biotin-GCTAAT
AGATGAGCCTAAGTCA-3 ', 5'-DN
P-CTTACCAATCGTCAATCTTTTCGT
T-3 ′) 1 μl each, reaction buffer 5 μl attached to the enzyme, dNTP mixed solution 2 μl attached to the enzyme
l, sample DNA is Corynebacterium s
p. 10 pg for J1 strain, 10 ng for other bacteria
Was added, and further water was added to make the total amount of the reaction solution 50 μl. In addition to this, AmpliTaq DNA polymer
Add 1 unit of Rase (Takara Shuzo), heat to 90 ° C and hold for 5 minutes, then 90 ° C for 60 seconds, 55 ° C for 45
The reaction was carried out for 35 cycles, with one cycle consisting of 72 ° C. and 90 seconds for 72 seconds, and finally incubated at 72 ° C. for 5 minutes. After the reaction,
Centrifuge tube with filter for removing primer SUPR
The unreacted primer was removed three times with EC-02 (Takara Shuzo).

On a streptavidin-immobilized microplate, 0.15 M NaCl, 0.05% Tween2 was added.
Tris-Cl buffer (pH 7.5) containing 0 to 100
Add 10 μl of the above mixture after reaction to this.
In addition, after leaving it at room temperature for 30 minutes, it was washed three times with 500 μl of the above buffer solution. To this, 100 μl of an alkaline phosphatase-labeled anti-DNP antibody diluted 2000-fold with the above buffer was added, and the mixture was allowed to stand at room temperature for 30 minutes, and then washed with 500 μl of the above buffer 3 times. To this, p- was dissolved in 1M diethanolamine buffer at a concentration of 4 mg / ml.
Add 100 μl of nitrophenyl phosphoric acid solution, room temperature 30
After standing for 40 minutes, use a microplate reader to
Absorbance at 5 nm was measured. The results are shown in Table 1.

[0061]

[Table 1] [Example 6] Corynebacterium s
p. Detection using J1 strain primer (2) Corynebacterium was treated in the same manner as in Example 4.
m sp. DNA was prepared from the J1 strain. This was subjected to PCR with the following reaction solution composition and reaction conditions.

20 pmol / μl prepared in Example 2
Concentration of primer (5'-Biotin-GCTAAT
AGATGAGCCTAAGTCA-3 ', 5'-DN
P-CTTACCAATCGTCAATCTTTTCGT
T-3 ′) 1 μl each, reaction buffer 5 μl attached to the enzyme, dNTP mixed solution 2 μl attached to the enzyme
l, Corynebacterium s as a sample
p. DNA prepared from strain J1 was added to 10 pg, 1 pg,
100 fg and 10 fg were added, and further water was added to make the total amount of the reaction solution 50 μl. To this, AmpliTaq D
Add 1 unit of NA polymerase (Takara Shuzo), heat to 90 ° C and hold for 5 minutes, then 90 ° C ・ 60
Seconds, 55 ° C./45 seconds, 72 ° C./90 seconds as one cycle, 40 cycles of reaction were carried out, and finally the temperature was kept at 72 ° C. for 5 minutes. After the reaction, the unreacted primer was removed in the same manner as in Example 5.

On a streptavidin-immobilized microplate, 0.15M NaCl, 0.05% Tween2 was added.
Tris-Cl buffer (pH 7.5) containing 0 to 100
Add 10 μl of the above mixture after reaction to this.
In addition, after leaving it at room temperature for 30 minutes, it was washed three times with 500 μl of the above buffer solution. To this, 100 μl of an alkaline phosphatase-labeled anti-DNP antibody diluted 2000-fold with the above buffer was added, and the mixture was allowed to stand at room temperature for 30 minutes, and then washed with 500 μl of the above buffer 3 times. To this, p- was dissolved in 1M diethanolamine buffer at a concentration of 4 mg / ml.
Add 100 μl of nitrophenyl phosphoric acid solution, room temperature 30
After standing for 40 minutes, use a microplate reader to
Absorbance at 5 nm was measured. Table 2 shows the results.

[0064]

[Table 2] [Example 7] Corynebacterium s
p. J1 strain primer and P. cepacia KK0
Simultaneous detection of both strains using one strain of primer Corynebacterium prepared in Example 4
sp. J1 strain DNA and P. cepacia KK01
Eight samples prepared by mixing any one of the other eight kinds of DNA such as strains were prepared, and the possibility of simultaneous detection of these J1 strain and KK01 strain by the PCR method was evaluated using these samples as samples.

The primers were the two types of primers prepared in Example 2 (5'-Biotin-GCTAATAGAT).
GAGCCTAAGTCA-3 ', 5'-DNP-CT
TACCAATCGTCAATCTTTCGTT-
3 ') and already P described in Patent Application already Tokutaira 6-51739. Two kinds of primers (5′-Biotin-AA) which specifically react with the cepacia KK01 strain
ATCCTTGGCTCTAATACAGTCG-
3 ', 5'-DNP-AGCACTCCCCACCTCT
CAGCAG-3 'was used. PCR was performed under the following reaction solution composition and reaction conditions.

1 μl of each of the above primers at a concentration of 15 pmol / μl and 5 μl of the reaction buffer attached to the enzyme
l, 5 μl of dNTP mixed solution attached to the enzyme, J1 strain DN
5 ng of A and 5 ng of one kind of other eight kinds such as the KK01 strain used in the experiment in Example 4 were added to 5 ng, and water was further added to make the whole reaction solution 50 μl. Am this
1 μl of pliTaq DNA polymerase
In addition, the amplification reaction was performed under the same temperature and time conditions as in Example 4 including nit. After the reaction, electrophoresis and staining were performed as in Example 4. As a result, a clear single band was confirmed in all samples at a length of about 1400 base pairs, which is expected only in the J1 strain. In addition, only the sample in which the DNA of the KK01 strain was mixed, the expected amount of the KK01 strain was about 58.
Another clear band was confirmed at a length of 0 base pairs. Here, in the other seven kinds of samples, any amplified nucleic acid chain other than the band of about 1400 base pairs could not be detected.

[Embodiment 8] Corynebacterium
ium sp. Detection using probe of J1 strain (1) In the same manner as in Example 4, DNA was prepared from 9 kinds of various bacteria. After denaturing each DNA with an alkali, 1 μg of each DNA was dot-coated on a nylon membrane (Tropilon-45, Tropix) using a dot blot apparatus (BRL). After drying at 80 ° C for 2 hours, the nylon membrane was placed in a vinyl bag and the prehybridization solution (6
X SSC, 5 x Denhardt's solution, 0.5% SDS, 10
Add 3 ml of 0 μg / ml denatured salmon sperm DNA) to 60
Prehybridization was performed for 1 hour at ° C. To this, a hybridization solution (prehybridization solution was added to the biotin-labeled oligonucleotide probe (5'-AAGCGAGGGAGG prepared in Example 3)).
AGGCTACTTAGATTAATACTCTAGG
ATAGTG-Biotin-3 ') is heat-denatured and then 1
3 ml (of which 100 ng was added) was added, and hybridization was carried out at 60 ° C. for 2 hours. The nylon membrane was taken out from the vinyl bag and washed with 6 × SSC, 0.5% SDS solution at 60 ° C. for 5 minutes 3 times. Detection was performed using Southern light (Tropix) using the chemiluminescence method with alkaline phosphatase-labeled streptavidin and AMPPD ^™ according to the attached protocol.

As a result, Corynebacterium
m sp. An extremely strong positive reaction could be confirmed only for the J1 strain DNA dots. Here, no positive reaction could be detected in the other 8 strains.

[Embodiment 9] Corynebacterium
ium sp. Detection using probe of J1 strain (2) Nine kinds of various bacteria described in Example 4 were cultured by a conventional method. After washing with 0.1 M sodium phosphate buffer (pH 8.0), the number of each bacterium was 2 × 1 using the above buffer.
It was adjusted to 0 ⁷ cells / ml. To 50 μl of the thus-prepared bacterial suspension, 50 μl of 6% formaldehyde solution was added to fix the bacterial cells, and 30 μl was added dropwise to a slide glass coated with 0.1% gelatin and 0.01% chromium alum, and air-dried. did. The slide glass on which the sample was fixed was immersed in 90% methanol, 3% formaldehyde solution for 10 minutes to re-fix the cells, and then washed with pure water.

The slide glass which has been subjected to the above treatment is treated with 50
It was immersed in a 10 mM Tris-Cl buffer solution (pH 8.0) containing mM NaBH ₄ at room temperature for 30 minutes in a light-shielded state, washed with pure water and air-dried. The probe was a biotin-labeled oligonucleotide probe (5-A prepared in Example 3).
ACGGAGGAGGAGGCTACTTAGATTA
ATACTCTAGGATAGTG-Biotin-
3 '), FITC (Fluorescein iso)
Thiocyanato) Labeled streptavidin was used in advance. This labeled probe was added to a hybridization solution (0.1M Tr
is-Cl buffer (pH 8.0), 0.75M NaC
1, 5 mM EDTA, 10% dextran sulfate, 0.
2% BSA (Bovine Serum Albumi
n), 0.01% polyadenylic acid) to a concentration of 5 ng / μl, and 30 μl was added dropwise. The slide glass was placed in an airtight container, and the reaction was carried out at 45 ° C. for 1 hour in the light-shielded state.

After the reaction, SET buffer solution (Tris-Cl
(PH 8.0), 0.2 mM EDTA, 30 mM N
The slide glass was washed with aCl), air-dried in a light-shielded state, and then examined with an Olympus epi-illumination fluorescence microscope to examine the presence or absence of fluorescence. A mercury lamp was used as an excitation light source, and observation was performed by B excitation. As a result of the speculum, Corynebac
terium sp. Fluorescence was observed in the cells of the J1 strain, but fluorescence could not be observed in the other 8 strains.

[0072]

The nucleic acid fragment of the present invention is Coryneba.
cterium sp. It is a gene encoding 16S rRNA of the J1 strain, and if the constituent bases or the base sequences of a part thereof are used as a primer or a probe, Corynebacterium sp. J1
The strain can be specifically detected.

Further, Corynebact according to the present invention
erium sp. The method for detecting the J1 strain uses the above-mentioned nucleic acid fragment or a part thereof as a primer or a probe, and is excellent in terms of sensitivity, specificity, simplicity, and speed, and is used in fields such as environmental purification. It makes a great contribution.

[0074]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 1566 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: rRNA gene DNA Origin organism name: Corynebacterium sp. Strain name: J1 sequence 1: AGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCAGGCTTAACACATG 51: CAAGTCGAGCGGGGAGATGTAGCTTGCTACATTTCCTAGCGGCGGACGGG 101: TGAGTAATGCTTAGGAATCTGCCTATTAGTGGGGGACAACATTCCGAAAG 151: GAATGCTAATACCGCATACGCCCTACGGGGGAAAGCAGGGGATCTTCGGA 201: CCTTGCGCTAATAGATGAGCCTAAGTCAGATTAGCTAGTTGGTGGGGTAA 251: AGCCTACCAAGGCGACGATCTGTAGCGGGTCTGAGAGGATGATCCGCCAC 301: ACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAA 351: TATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGA 401: AGGCCTTTTGGTTGTAAAGCACTTTAAGCGAGGAGGAGGCTACTTAGATT 451: AATACTCTAGGATAGTGGACGTTACTCGCAGAATAAGCACCGGCTAACTC 501: TGTGCCAGCAGCCGCGGTAATACAGAGGGTGCGAGCGTTAATCGGATTTA 551: CTGGGCGTAAAGCGTGCGTAGGCGGCTTTTTAAGTCGGATGTGAAATCCC 601: TGAGCTTAACTTAGGAATTGCATTCGATACTGGGAAGCTAGAGTATGGGA 651: GAGGATGGT AGAATTCCAGGTTGTAGCGGTGAAATGCGTAGAGATCTGGA 701: GGAATACCGATGGCGAAGGCAGCCATCTGGCCTAATACTGACGCTGAGGT 751: ACGAAAGCATGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCG 801: TAAACGATGTCTACTAGCCGTTGGGGCCTTTGAGGCTTTAGTGGCGCACT 851: AACGCGATAAGTAGACCGCCTGGGGAGTACGGTCGCAAGACTAAAACTCA 901: AATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCG 951: ATGCAACGCGAAGAACCTTACCTGGTCTTGACATAGTAAGAACTTTCCAG 1001: AGATGGATTGGTGCCTTCGGGAACTTACATACAGGTGCTGCATGGCTGTC 1051: GTCAGCTGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCC 1101: TTTTCCTTATTTGCCAGCGGGTTAAGCCGGGAACTTTAAGGATACTGCCA 1151: GTGACAAACTGGAGGAAGGCGGGGACGACGTCAAGTCATCATGGCCCTTA 1201: CGACCAGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCTACC 1251: TAGCGATAGGATGCTAATCTCAAAAAGCCGATCGTAGTCCGGATTGGAGT 1301: CTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCGGATCAGAA 1351: TGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCA 1401: TGGGAATTTGTTGCACCAGAAGTAGGTAGTCTAACCGCAAGGAGGACGCT 1451: TACCACGGTGTGGCCGATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTA 1501 : GGGGAACCTGCGGCTGGATCACCTCCTTA ACGAAAGATTGACGATTGGTA 1551: AGAATCCACAACAAGT

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C12Q 1/06 ZAB 6807-4B // (C12N 15/09 ZNA C12R 1:15) C12R 1:15 )

Claims (18)

[Claims] 1. Cor represented by the nucleotide sequence of SEQ ID NO: 1
16S of ynebacterium sp. J1 strain
rRNA gene. 2. A novel nucleic acid fragment selected from the nucleic acid represented by the sequence of claim 1, a nucleic acid having a complementary base sequence thereof, and a nucleic acid represented by these mutant sequences. 3. The novel nucleic acid fragment according to claim 2, which comprises, as a part, any one of the specific sequence numbers 1 to 8 listed below or any one of these sequences. Specific sequence 1 (SEQ 174 bases from nucleotide numbers 56 in number 1 to nucleotide number 226) CGAGCGGGGAGATGTAGCTTGCTACATTTCCTAGCGGCGGACGGGTGAGTAATGCTT AGGAATCTGCCTATTAGTGGGGGACAACATTCCGAAAGGAATGCTAATACCGCATAC GCCCTACGGGGGAAAGCAGGGGATCTTCGGACCTTGCGCTAATAGATGAGCCTAAGT CAG specific sequence 2 (72 bases from nucleotide numbers 419 in SEQ ID NO: 1 to base No. 490) GCACTTTAAGCGAGGAGGAGGCTACTTAGATTAATACTCTAGGATAGTGGACGTTAC TCGCAGAATAAGCAC specific sequence 3 ( base in 187 bases) TGCGTAGGCGGCTTTTTAAGTCGGATGTGAAATCCCTGAGCTTAACTTAGGAATTGC ATTCGATACTGGGAAGCTAGAGTATGGGAGAGGATGGTAGAATTCCAGGTTGTAGCG GTGAAATGCGTAGAGATCTGGAGGAATACCGATGGCGAAGGCAGCCATCTGGCCTAA TACTGACGCTGAGGTA specific sequence 4 (from nucleotide numbers 810 in SEQ ID NO: 1 to base No. 865 56 bases) TishitieishitieijishishijititijijijijishishitititijieijijishitititieiGTGGCGCACTAACGCGATAAGTAGA specific sequence 5 (SEQ ID NO: 1 from nucleotide numbers 565 to nucleotide number 751 in SEQ ID NO: 1 56 bases from number 973 to base number 1031) TGGTCTTG EishieitieijitieieijieieishitititishishieijieijieitiGGATTGGTGCCTTCGGGAACTTACA TA-specific sequence 6 (40 bases from nucleotide numbers 1101 in SEQ ID NO: 1 to base No. 1140) TTTTCCTTATTTGCCAGCGGGTTAAGCCGGGAACTTTAAG (170 bases from nucleotide numbers 1186 in SEQ ID NO: 1 to base No. 1355) specific sequences 7 TCATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGTCGGTACAAAGGG TTGCTACCTAGCGATAGGATGCTAATCTCAAAAAGCCGATCGTAGTCCGGATTGGAG TCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCGGATCAGAATGCCG specific sequence 8 ( 54 bases from base number 1408 to base number 1461 in SEQ ID NO: 1) TTGTTGCACCAGAAGTAGGTAGTCTAACCGCAAGGAGGACGCTTACCACGGTGT 4. A novel nucleic acid fragment comprising a nucleic acid which is a partial sequence in any of the nucleic acids according to claim 3 and can be used as a primer or a probe. 5. A nucleic acid usable as a primer is 10
The novel nucleic acid fragment according to claim 4, which has a length of -50 bases and a nucleic acid that can be used as a probe is from 10 bases to the total number of bases of the nucleic acid according to claim 2. 6. A partial sequence No. 1 in which the partial sequence is arranged next.
To 348, the novel nucleic acid fragment according to claim 4. SEQ ID NO: nucleotide sequence 1 GGTAAGAATCCACAACAAGTCGTT 2 AACGAAAGATTGACGATTGGTAAG 3 AGGAGGACGCTTACCACGG 4 AGTCTAACCGCAAGGAGGACGC 5 AGTCTAACCGCAAGGAGGA 6 AGTCTAACCGCAAGGAGG 7 TCGGAATCGCTAGTAATCGC 8 GTCGGAATCGCTAGTAATCGC 9 GTCGGAATCGCTAGTAATCG 10 AGTCGGAATCGCTAGTAATCGC 11 AGTCGGAATCGCTAGTAATCG 12 AAGTCGGAATCGCTAGTAATCGC 13 AAGTCGGAATCGCTAGTAATCG 14 AAGTCGGAATCGCTAGTAATC 15 AAGTCGGAATCGCTAGTAAT 16 AAGTCGGAATCGCTAGTA 17 GAAGTCGGAATCGCTAGTAATCGC 18 GAAGTCGGAATCGCTAGTAATCG 19 GAAGTCGGAATCGCTAGTAATC 20 GAAGTCGGAATCGCTAGTAA 21 TGAAGTCGGAATCGCTAGTAATCG 22 ATGAAGTCGGAATCGCTAGTAATC 23 ATGAAGTCGGAATCGCTAG 24 CATGAAGTCGGAATCGCTAGT 25 CCATGAAGTCGGAATCGCTAGT 26 CCATGAAGTCGGAATCGCTAG 27 CCATGAAGTCGGAATCGCT 28 GCAACTCGACTCCATGAAG 29 GCAACTCGACTCCATGAA 30 TGCAACTCGACTCCATGAAG 31 TGCAACTCGACTCCATGA 32 CTGCAACTCGACTCCATGAAG 33 CTGCAACTCGACTCCATGAA 34 CTGCAACTCGACTCCATGA 35 TCTGCAACTCGACTCCATGAAG 36 TCTGCAACTCGACTCCATGAA 37 TCTGCAACTCGACTCCATGA 38 TCTGCAACTCGACTCCATG 39 CAATGGTCGGTACAAAGGG 40 ACGTGCTACAATGGT CGGT 41 CGACCAGGGCTACACACGT 42 CGACCAGGGCTACACACG 43 ACGACCAGGGCTACACACG 44 TACGACCAGGGCTACACACGT 45 TTACGACCAGGGCTACACACGT 46 TTACGACCAGGGCTACACACG 47 TTACGACCAGGGCTACACA 48 TTGCCAGCGGGTTAAGCC 49 TTTGCCAGCGGGTTAAGCC 50 TTTGCCAGCGGGTTAAGC 51 ATTTGCCAGCGGGTTAAGCC 52 ATTTGCCAGCGGGTTAAGC 53 ATTTGCCAGCGGGTTAAG 54 TATTTGCCAGCGGGTTAAGCC 55 TATTTGCCAGCGGGTTAAGC 56 TATTTGCCAGCGGGTTAAG 57 TATTTGCCAGCGGGTTAA 58 TTATTTGCCAGCGGGTTAAGCC 59 TTATTTGCCAGCGGGTTAAGC 60 TTATTTGCCAGCGGGTTAAG 61 TTATTTGCCAGCGGGTTAA 62 TTATTTGCCAGCGGGTTA 63 CTTATTTGCCAGCGGGTTAAGCC 64 CTTATTTGCCAGCGGGTTAAGC 65 CTTATTTGCCAGCGGGTTAAG 66 CTTATTTGCCAGCGGGTTAA 67 CTTATTTGCCAGCGGGTTA 68 CTTATTTGCCAGCGGGTT 69 CCTTATTTGCCAGCGGGTTAAGCC 70 CCTTATTTGCCAGCGGGTTAAGC 71 CCTTATTTGCCAGCGGGTTAAG 72 CCTTATTTGCCAGCGGGTTAA 73 CCTTATTTGCCAGCGGGTTA 74 CCTTATTTGCCAGCGGGTT 75 CCTTATTTGCCAGCGGGT 76 TCCTTATTTGCCAGCGGGTTAAGC 77 TCCTTATTTGCCAGCGGGTTAAG 78 TCCTTATTTGCCAGCGGGTTAA 79 TCCTTATTTGCCAGCGGGTTA 80 TCCTTATTTGCCAGCGGGTT 81 TCCTTATTTGCCAGCGGGT 82 TCCTTATTTGCCAGCGGG 83 TTCCTTATTTGCCAGCGGGTTAAG 84 TTCCTTATTTGCCAGCGGGTTAA 85 TTCCTTATTTGCCAGCGGGTTA 86 TTCCTTATTTGCCAGCGGGTT 87 TTCCTTATTTGCCAGCGGG 89 TTTCCTTATTTGCCAGCGGGTTAA 90 GTGCCTTCGGGAACTTACAT 91 GATGGATTGGTGCCTTCGGGA 92 AGATGGATTGGTGCCTTCGGGA 93 AGATGGATTGGTGCCTTCGGG 94 GAGATGGATTGGTGCCTTCGGGA 95 GAGATGGATTGGTGCCTTCGGG 96 GAGATGGATTGGTGCCTTCGG 97 AGAGATGGATTGGTGCCTTCGGGA 98 AGAGATGGATTGGTGCCTTCGGG 99 AGAGATGGATTGGTGCCTTCGG 100 AGAGATGGATTGGTGCCTTCG 101 CAGAGATGGATTGGTGCCTTCGGG 102 CAGAGATGGATTGGTGCCTTCGG 103 CAGAGATGGATTGGTGCCTTCG 104 CAGAGATGGATTGGTGCCTTC 105 TAGTAAGAACTTTCCAGAGAT 106 CTTTGAGGCTTTAGTGGCG 107 CTAATACTGACGCTGAGGT 108 CTAATACTGACGCTGAGG 109 CCTAATACTGACGCTGAGG 110 GCCTAATACTGACGCTGAGGT 111 GCCTAATACTGACGCTGAGG 112 GCCTAATACTGACGCTGAG 113 GGCCTAATACTGACGCTGAGGT 114 GGCCTAATACTGACGCTGAGG 115 TGGCCTAATACTGACGCTGAGGT 116 TGGCCTAATACTGACGCTGAGG 117 TGGCCTAATACTGACGCTGAG 118 TGGCCTAATACTGACGCTG 119 CTGGCCTAATACTGACGCTGAGGT 120 CTGGCCTAATACTGACGCTGAGG 121 CTGGCCTAATACTG ACGCTGAG 122 TCTGGCCTAATACTGACGCTGAGG 123 TCTGGCCTAATACTGACGCTGAG 124 TCTGGCCTAATACTGACGCTGA 125 TCTGGCCTAATACTGACGCTG 126 TCTGGCCTAATACTGACGC 127 ATCTGGCCTAATACTGACGCTGAG 128 ATCTGGCCTAATACTGACGCTGA 129 ATCTGGCCTAATACTGACGCTG 130 ATCTGGCCTAATACTGACGCT 131 CATCTGGCCTAATACTGACGCTGA 132 CATCTGGCCTAATACTGACGCTG 133 CATCTGGCCTAATACTGACGCT 134 CATCTGGCCTAATACTGACGC 135 TACCGATGGCGAAGGCAGC 136 ATACCGATGGCGAAGGCAG 137 AATACCGATGGCGAAGGCAGC 138 AATACCGATGGCGAAGGCA 139 GAATACCGATGGCGAAGGCAGC 140 GAATACCGATGGCGAAGGCAG 141 GAATACCGATGGCGAAGGCA 142 GAATACCGATGGCGAAGGC 143 GGAATACCGATGGCGAAGGCAGC 144 GGAATACCGATGGCGAAGGCAG 145 GGAATACCGATGGCGAAGGCA 146 AGGAATACCGATGGCGAAGGCAGC 147 AGGAATACCGATGGCGAAGGCAG 148 AGGAATACCGATGGCGAAGGCA 149 AGGAATACCGATGGCGAAGGC 150 GAGGAATACCGATGGCGAAGGCAG 151 GAGGAATACCGATGGCGAAGGCA 152 GAGGAATACCGATGGCGAAGGC 153 GAGGAATACCGATGGCGAAGG 154 GAGGAATACCGATGGCGAA 155 GAGGAATACCGATGGCGA 156 GGAGGAATACCGATGGCGAAGGCA 157 GGAGGAATACCGATGGCGAAGGC 158 GGAGGAATACCGATGGCGAAGG 159 G GAGGAATACCGATGGCGAAG 160 GGAGGAATACCGATGGCGAA 161 GGAGGAATACCGATGGCGA 162 GGAGGAATACCGATGGCG 163 TGGAGGAATACCGATGGCGAAGGC 164 TGGAGGAATACCGATGGCGAAGG 165 TGGAGGAATACCGATGGCGAAG 166 TGGAGGAATACCGATGGCGAA 167 TGGAGGAATACCGATGGCGA 168 TGGAGGAATACCGATGGCG 169 TGGAGGAATACCGATGGC 170 CTGGAGGAATACCGATGGCGAAGG 171 CTGGAGGAATACCGATGGCGAAG 172 CTGGAGGAATACCGATGGCGAA 173 CTGGAGGAATACCGATGGCGA 174 CTGGAGGAATACCGATGGCG 175 CTGGAGGAATACCGATGGC 176 TCTGGAGGAATACCGATGGCGAAG 177 TCTGGAGGAATACCGATGGCGAA 178 TCTGGAGGAATACCGATGGCGA 179 TCTGGAGGAATACCGATGGCG 180 TCTGGAGGAATACCGATGGC 181 TCTGGAGGAATACCGATGG 182 ATCTGGAGGAATACCGATGGCGAA 183 ATCTGGAGGAATACCGATGGCGA 184 ATCTGGAGGAATACCGATGGCG 185 ATCTGGAGGAATACCGATGGC 186 ATCTGGAGGAATACCGATGG 187 GATCTGGAGGAATACCGATGGCGA 189 GATCTGGAGGAATACCGATGGCG 190 GATCTGGAGGAATACCGATGGC 191 GATCTGGAGGAATACCGATGG 192 GATCTGGAGGAATACCGATG 193 GATCTGGAGGAATACCGA 194 AGCGGTGAAATGCGTAGAGATC 195 TAGCGGTGAAATGCGTAGAGATC 196 TAGCGGTGAAATGCGTAGAGAT 197 GTAGCGGTGAAATGCGTAGAGATC 198 GTAGCGGTGAAATGCGTAGAGA 199 TGTAGCGGTGAAATGCGTAGAGAT 200 AGGTTGTAGCGGTGAAATGCGT 201 CAGGTTGTAGCGGTGAAATGCGTA 202 CAGGTTGTAGCGGTGAAATGCGT 203 CAGGTTGTAGCGGTGAAATGCG 204 CCAGGTTGTAGCGGTGAAATGCGT 205 CCAGGTTGTAGCGGTGAAATGCG 206 CCAGGTTGTAGCGGTGAAATGC 207 TCCAGGTTGTAGCGGTGAAATGCG 208 TATGGGAGAGGATGGTAGA 209 GTATGGGAGAGGATGGTAGAA 210 GTATGGGAGAGGATGGTAGA 211 GTATGGGAGAGGATGGTAG 212 GTATGGGAGAGGATGGTA 213 AGTATGGGAGAGGATGGTAGAA 214 AGTATGGGAGAGGATGGTAGA 215 AGTATGGGAGAGGATGGTAG 216 AGTATGGGAGAGGATGGTA 217 GAGTATGGGAGAGGATGGTAGAAT 218 GAGTATGGGAGAGGATGGTAGA 219 GAGTATGGGAGAGGATGGTAG 220 GAGTATGGGAGAGGATGGTA 221 GAGTATGGGAGAGGATGGT 222 GAGTATGGGAGAGGATGG 223 AGAGTATGGGAGAGGATGGTAGAA 224 AGAGTATGGGAGAGGATGGTAGA 225 AGAGTATGGGAGAGGATGGTAG 226 AGAGTATGGGAGAGGATGGTA 227 AGAGTATGGGAGAGGATGGT 228 AGAGTATGGGAGAGGATGG 229 AGAGTATGGGAGAGGATG 230 TAGAGTATGGGAGAGGATGGTAGA 231 TAGAGTATGGGAGAGGATGGTAG 232 TAGAGTATGGGAGAGGATGGTA 233 TAGAGTATGGGAGAGGATGGT 234 TAGAGTATGGGAGAGGATGG 235 TAGAGTATGGGAGAGGATG 236 CTAGAGTATGGGAGAGGATGGTAG 237 CTAGAGTATGGGAGAGGATGGTA 238 CTAGAGTATGGGAGAGGATGGT 239 CTAGAGTATGGGAGAGGATGG 240 CTAGAGTATGGGAGAGGATG 241 CTAGAGTATGGGAGAGGAT 242 CTAGAGTATGGGAGAGGA 243 GCTAGAGTATGGGAGAGGATGGTA 244 GCTAGAGTATGGGAGAGGATGGT 245 GCTAGAGTATGGGAGAGGATGG 246 GCTAGAGTATGGGAGAGGATG 247 GCTAGAGTATGGGAGAGGA 248 AGCTAGAGTATGGGAGAGGATGGT 249 AGCTAGAGTATGGGAGAGGATGG 250 AGCTAGAGTATGGGAGAGGATG 251 AGCTAGAGTATGGGAGAGGAT 252 AGCTAGAGTATGGGAGAGGA 253 AGCTAGAGTATGGGAGAGG 254 AAGCTAGAGTATGGGAGAGGATGG 255 AAGCTAGAGTATGGGAGAGGATG 256 AAGCTAGAGTATGGGAGAGGAT 257 AAGCTAGAGTATGGGAGAGGA 258 AAGCTAGAGTATGGGAGAGG 259 AAGCTAGAGTATGGGAGAG 260 CGATACTGGGAAGCTAGAG 261 CGATACTGGGAAGCTAGA 262 TCGATACTGGGAAGCTAGA 263 TCGATACTGGGAAGCTAG 264 TGTGAAATCCCTGAGCTTAAC 265 TGTGAAATCCCTGAGCTTAA 266 TGTGAAATCCCTGAGCTTA 267 ATGTGAAATCCCTGAGCTTAAC 268 ATGTGAAATCCCTGAGCTTAA 269 ATGTGAAATCCCTGAGCTTA 270 ATGTGAAATCCCTGAGCTT 271 ATGTGAAATCCCTGAGCT 272 GTGGACGTTACTCGCAGAATA 273 GTGGACGTTACTCGCAGAA 274 GTGGACGTTACTCGCAGA 275 ATTAATACTCTAGGATAGTG 276 AAGCGAGGAGGAGGCTACTTAGAT 277 TTTAAGCGAGGAGGAGGC 278 GCTAATAGATGAGCCTAAGTCA 279 CTTCGGACCTTGCGCTAATA 280 TCTTCGGACCTTGCGCTAAT 281 TCTTCGGACCTTGCGCTAA 282 TCTTCGGACCTTGCGCTA 283 ATCTTCGGACCTTGCGCTAAT 284 ATCTTCGGACCTTGCGCTA 285 GATCTTCGGACCTTGCGCTAAT 286 GGATCTTCGGACCTTGCGCTAATA 287 GCAGGGGATCTTCGGACCTTGC 289 AGCAGGGGATCTTCGGACCTTG 290 AGCAGGGGATCTTCGGACCTT 291 AGCAGGGGATCTTCGGACC 292 ACCGCATACGCCCTACGG 293 TACCGCATACGCCCTACGG 294 TACCGCATACGCCCTACG 295 ATACCGCATACGCCCTACGG 296 ATACCGCATACGCCCTACG 297 ATACCGCATACGCCCTAC 298 AATACCGCATACGCCCTACGG 299 AATACCGCATACGCCCTACG 300 AATACCGCATACGCCCTAC 301 AATACCGCATACGCCCTA 302 TAATACCGCATACGCCCTACGG 303 TAATACCGCATACGCCCTACG 304 TAATACCGCATACGCCCTAC 305 TAATACCGCATACGCCCTA 306 TAATACCGCATACGCCCT 307 CTAATACCGCATACGCCCTACGG 308 CTAATACCGCATACGCCCTACG 309 CTAATACCGCATACGCCCTAC 310 CTAATACCGCATACGCCCT 311 CTAATACCGCATACGCCC 312 GCTAATACCGCATACGCCCTACGG 313 GCTAATACCGCATACGCCCTACG 314 GCTAATACCGCATACGCCCTAC 315 GCTAATACCGC ATACGCCCTA 316 GCTAATACCGCATACGCCCT 317 GCTAATACCGCATACGCCC 318 AAAGGAATGCTAATACCGC 319 GAAAGGAATGCTAATACCG 320 GAAAGGAATGCTAATACC 321 CGAAAGGAATGCTAATACCGC 322 CCGAAAGGAATGCTAATACCGC 323 CAACATTCCGAAAGGAATGC 324 TATTAGTGGGGGACAACATTC 325 TATTAGTGGGGGACAACAT 326 GCGGCGGACGGGTGAGTAATGC 327 GCGGCGGACGGGTGAGTAATG 328 GCGGCGGACGGGTGAGTAA 329 AGCGGCGGACGGGTGAGTAATGCT 330 AGCGGCGGACGGGTGAGTAATGC 331 AGCGGCGGACGGGTGAGTAATG 332 AGCGGCGGACGGGTGAGTAAT 333 AGCGGCGGACGGGTGAGTA 334 TAGCGGCGGACGGGTGAGTAATGC 335 TAGCGGCGGACGGGTGAGTAATG 336 TAGCGGCGGACGGGTGAGTAAT 337 TAGCGGCGGACGGGTGAGTAA 338 TAGCGGCGGACGGGTGAGT 339 CTAGCGGCGGACGGGTGAGTAATG 340 CTAGCGGCGGACGGGTGAGTAAT 341 CTAGCGGCGGACGGGTGAGTAA 342 CTAGCGGCGGACGGGTGAGTA 343 CCTAGCGGCGGACGGGTGAGTAAT 344 CCTAGCGGCGGACGGGTGAGTAA 345 CCTAGCGGCGGACGGGTGAGGC346CGTAGGGGAGA 347 TAGGTTGACCTAG347 7. The mutant sequence according to claim 2, wherein a part of the base sequence of the nucleic acid is deleted, or the base sequence is replaced or added with another base or base sequence. Novel nucleic acid fragment of. 8. The nucleic acid fragment according to any one of claims 2 to 7, which comprises a nucleic acid fragment into which a site capable of binding to a label or / and a solid phase carrier may be introduced. Primer. 9. The nucleic acid fragment according to any one of claims 2 to 7, which comprises a nucleic acid fragment into which a site capable of binding to a label or / and a solid phase carrier may be introduced. probe. 10. A primer comprising a combination of two kinds of nucleic acid fragments, wherein at least one nucleic acid fragment is selected from the nucleic acid fragments according to claim 8, and each nucleic acid fragment has a label or ( And) A primer which may have a site capable of binding to the solid phase carrier. 11. The variant sequence according to claim 8, wherein a part of the nucleotide sequence of the nucleic acid fragment is deleted, or the nucleotide sequence is replaced or added with another nucleotide or nucleotide sequence. Primer. 12. A site capable of binding to a labeled substance or a solid phase carrier is introduced at the 5′-end side of a nucleic acid fragment.
11. The primer or probe according to any one of 1 to 11. 13. The site capable of binding to the labeled substance or the solid phase carrier is any one of a biotin residue, a 2,4-dinitrophenyl group and a digoxigenin residue. Primers or probes. 14. At least one kind of nucleic acid fragment having the nucleotide sequence of the nucleic acid according to any one of claims 2 to 7, 9, 12 or 13 is used as a probe.
Method for detecting Corynebacterium sp. J1 strain. 15. A Cor using at least one nucleic acid fragment having a nucleotide sequence of the nucleic acid according to any one of claims 2 to 8 and 10 to 13 as a primer.
A method for detecting a ynebacterium sp. J1 strain. 16. The primer according to claim 8, 10 to 13 or 15 is used, and the following (1) to (4) are used.
Corynebac characterized by carrying out the steps of
Method for detecting terium sp. J1 strain. (1) Corynebacterium sp. J1
Prepare a sample to detect the presence or absence of the strain, (2) if necessary, crush the bacterial cells in the sample, (3) add the above-mentioned primer to the sample, and perform an extension reaction of the primer, (4)
A detection operation is performed on the extension reaction product obtained in step (3). 17. The Corynebacterium according to claim 16, wherein the primer according to claim 10 is used.
Method for detecting sp. J1 strain. 18. The extension reaction of the primer is PCR (P
18. The method according to claim 16 or 17, which is an polymerase chain reaction.
Method for detecting Corynebacterium sp. J1 strain.