KR20150005878A - Method of identifying Xanthomonas euvesicatoria - Google Patents

Abstract

The present invention relates to a method for identifying Xanthomonas euvesicatoria and, more specifically, to a method for identifying Xanthomonas euvesicatoria by checking bases of 23S rRNA separated from a specimen. Xanthomonas euvesicatoria can be effectively identified from Xanthomonas genus, when using the method for identifying Xanthomonas euvesicatoria and an identification probe of the present invention.

Description

Method of identifying Xanthomonas euvesicatoria}

The present invention relates to a method for identifying Xanthomonas euvesicatoria, and more particularly, to a method for identifying Xanthomonas euvesicatoria by confirming the base of 23S rRNA isolated from a sample.

Xanthomonas are bacteria involved in denitrification of soil nitrogen. There are pathogenic bacteria that cause peach tree bacterial hole disease, radish black rot, citrus ulcer disease, rice white leaf blight, and gangnam bean burn blight.

Xanthomonas euvesicatoria is a highly infectious pathogenic bacterium that causes bacterial spotting mainly on strawberries. Small water needle-like spots appear on the leaves, and when progressing, they are enlarged to angled lesions by leaf veins. Susceptible varieties have a yellow edge, and resistant varieties have small spots.

Methods such as dot-blot and polymerase chain reaction (PCR) are used to identify the genes of plant pathogens. In particular, PCR is a method that is widely used recently because it is more economical in time, effort, and manpower than the dot-blot method. The PCR (polymerase chain reaction) identification method involves annealing a specific DNA fragment (primer) consisting of 10-20 bp to the bacterial genomic DNA, and then using Taq polymerase. If the synthesis reaction is repeated after addition, the region to which the primer is bound is rapidly amplified, and the PCR amplified product is electrophoresed on an agarose gel or acrylamide gel, and then ethidium bromide is used. It is a method to identify and identify DNA polymorphisms of bacterial species such as the presence or absence of DNA bands or differences in morphology that appear after DNA is stained with bromide) and silver stain. Currently, this method of identification of bacteria by PCR is mainly used to identify bacterial diseases of humans or animals, but recently, PCR identification methods have been developed to identify plant pathogens and are used for quarantine of imported and exported agricultural products along with identification of diseases. Has become. In addition, the PCR identification method is economical because the time required for identification is shorter than that of other methods, the cost for identification is inexpensive, and it is possible to simultaneously assay many samples. In recent years, among the methods for identifying Xanthomonas, a PCR identification method that amplifies a nucleic acid, which is a genetic material, is the most preferred.

However, the existing PCR identification method has difficulty in distinguishing between species due to genetic similarity, so a solution to overcome this problem is needed.

Among the existing microorganisms in the genus Santomonas, rice white leaf blight pathogen (Xanthomonas oryzae pv. oryzae), bacterial spot pattern pathogen (Xanthomonas campestris pv. citri) and Xanthomonas axonopodis pv. glycines, etc., individual PCR primers used for identification of various microorganisms have been studied, but Xanthomonas euvesicatoria has been identified from Xanthomonas. The method of paying has not been studied yet. There is a need to develop a method to identify Xanthomonas euvesicatoria through species or pathogen-specific bases and base combinations between plant pathogens.

Accordingly, the inventors of the present invention were studying to more accurately and quickly identify Xanthomonas euvesicatoria from the genus Santomonas (Xanthomonas). And developed an identification method using it.

Accordingly, an object of the present invention is to: (a) separating 23S rRNA from a sample; (b) confirming the base sequence of the 23S rRNA isolated in step (a); And (c) the nucleotide sequence identified in step (b) is the 78th base of SEQ ID NO: 1 is G, the 86th base is C, the 698th base is C, the 740th base is A, the 1177th base is C, 1735. The first base is T, the 1966th base is A, the 1967th base is T, the 1969th base is C, the 1973th base is C, the 1977 to 1979th base is CTC, the 2048 to 2050th base is GAG, and the 2053th base is G , When the 2056th base is A, the 2058th base is A, the 2060th base is A, the 2061th base is T, the 2626th base is C, the 2649th base is T, the 2715th base is C, and the 2737th base is G It is to provide a method for identification of Santo Monas Yuvesicatoria (Xanthomonas euvesicatoria) comprising the step of judging the Santo Monas Yuvesicatoria (Xanthomonas euvesicatoria).

Another object of the present invention is, in the polynucleotide consisting of SEQ ID NO: 1, a polynucleotide consisting of 20-100 consecutive DNA sequences including the 78th base G of SEQ ID NO: 1, the 86th base C of SEQ ID NO: 1 Polynucleotide consisting of 20-100 consecutive DNA sequences including, polynucleotide consisting of 20-100 consecutive DNA sequences including 698 base C of SEQ ID NO: 1, 740 base A of SEQ ID NO: 1 A polynucleotide consisting of 20-100 consecutive DNA sequences including, a polynucleotide consisting of 20-100 consecutive DNA sequences including the 1177th base C of SEQ ID NO: 1, the 1735th base T of SEQ ID NO: 1 Polynucleotide consisting of 20-100 consecutive DNA sequences including, polynucleotide consisting of 20-100 consecutive DNA sequences including 1966th base A of SEQ ID NO: 1, 1967th base T of SEQ ID NO: 1 A polynucleotide consisting of 20-100 consecutive DNA sequences including, a polynucleotide consisting of 20-100 consecutive DNA sequences including the 1969 base C of SEQ ID NO: 1, the 1973 base C of SEQ ID NO: 1 Polynucleotide consisting of 20-100 consecutive DNA sequences including, 1977 to 1979 of SEQ ID NO: 1 Polynucleotide consisting of 20-100 consecutive DNA sequences including CTC, 2048 to SEQ ID NO: 1 A polynucleotide consisting of 20-100 consecutive DNA sequences including the 2050th base GAG, a polynucleotide consisting of 20-100 consecutive DNA sequences including the 2053th base G of SEQ ID NO: 1 A polynucleotide consisting of 20-100 consecutive DNA sequences including the 2056th base A, a polynucleotide consisting of 20-100 consecutive DNA sequences including the 2058th base A of SEQ ID NO: 1, A polynucleotide consisting of 20-100 consecutive DNA sequences including the 2060th base A of SEQ ID NO: 1, a polynucleotide consisting of 20-100 consecutive DNA sequences including the 2061th base T of SEQ ID NO: 1, A polynucleotide consisting of 20-100 consecutive DNA sequences including the 2626th base C of SEQ ID NO: 1, a polynucleotide consisting of 20-100 consecutive DNA sequences including the 2649th base T of SEQ ID NO: 1, A polynucleotide consisting of 20-100 consecutive DNA sequences including the 2715th base C of SEQ ID NO: 1, a polynucleotide consisting of 20-100 consecutive DNA sequences including the 2737th base G of SEQ ID NO: 1, and To provide a probe for identification of Xanthomonas euvesicatoria comprising one or more polynucleotides selected from the group consisting of complementary polynucleotides thereof.

In order to achieve the above object, the present invention provides a method for identifying Xanthomonas euvesicatoria.

In order to achieve another object of the present invention, the present invention provides a probe for identification of Xanthomonas euvesicatoria.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for identifying Xanthomonas euvesicatoria.

That is, the present invention secures the base combination of the 23S rRNA gene of the genus Santomonas (Xanthomonas) to identify Santomonas euvesicatoria (Xanthomonas euvesicatoria).

More specifically, the method for identifying Xanthomonas euvesicatoria of the present invention includes the steps of: (a) separating 23S rRNA from a sample; (b) confirming the base sequence of the 23S rRNA isolated in step (a); And (c) the nucleotide sequence identified in step (b) is the 78th base of SEQ ID NO: 1 is G, the 86th base is C, the 698th base is C, the 740th base is A, the 1177th base is C, 1735. The first base is T, the 1966th base is A, the 1967th base is T, the 1969th base is C, the 1973th base is C, the 1977 to 1979th base is CTC, the 2048 to 2050th base is GAG, and the 2053th base is G , When the 2056th base is A, the 2058th base is A, the 2060th base is A, the 2061th base is T, the 2626th base is C, the 2649th base is T, the 2715th base is C, and the 2737th base is G There is a feature that includes the step of judging as Xanthomonas euvesicatoria.

In addition, the present invention provides a probe for identification of Xanthomonas euvesicatoria.

That is, the present invention secures the base combination of the 23S rRNA gene of the genus Santomonas (Xanthomonas) to identify Santomonas euvesicatoria (Xanthomonas euvesicatoria).

More specifically, the probe for identification of Xanthomonas euvesicatoria of the present invention is a polynucleotide consisting of SEQ ID NO: 1, consisting of 20-100 consecutive DNA sequences including the 78th base G of SEQ ID NO: 1. Polynucleotide, consisting of 20-100 consecutive DNA sequences including the 86th base C of SEQ ID NO: 1, polynucleotide consisting of 20-100 consecutive DNA sequences, including the 698th base C of SEQ ID NO: 1 Polynucleotide, consisting of 20-100 consecutive DNA sequences including 740th base A of SEQ ID NO: 1, polynucleotide consisting of 20-100 consecutive DNA sequences including 1177th base C of SEQ ID NO: 1 Polynucleotide, consisting of 20-100 consecutive DNA sequences containing the 1735th base T of SEQ ID NO: 1, polynucleotide consisting of 20-100 consecutive DNA sequences containing the 1966th base A of SEQ ID NO: 1 Polynucleotide, a polynucleotide consisting of 20-100 consecutive DNA sequences containing the 1967th base T of SEQ ID NO: 1, a polynucleotide consisting of 20-100 consecutive DNAs containing the 1969th base C of SEQ ID NO: 1 Nucleotide, a polynucleotide consisting of 20-100 consecutive DNA sequences including the 1973th base C of SEQ ID NO: 1, consisting of 20-100 consecutive DNA sequences including the 1977 to 1979th base CTC of SEQ ID NO: 1 Polynucleotide consisting of 20-100 consecutive DNA sequences including the 2048 to 2050 base GAG of SEQ ID NO: 1, 20-100 consecutive DNA sequences including the 2053 base G of SEQ ID NO: 1 A polynucleotide consisting of, a polynucleotide consisting of 20-100 consecutive DNA sequences including the 2056th base A of SEQ ID NO: 1, the 2058th of SEQ ID NO: 1 Polynucleotide consisting of 20-100 consecutive DNA sequences containing base A, 2060th of SEQ ID NO: 1 polynucleotide consisting of 20-100 consecutive DNA sequences containing base A, 2061th of SEQ ID NO: 1 Polynucleotide consisting of 20-100 consecutive DNA sequences including base T, 2626th polynucleotide consisting of 20-100 consecutive DNA sequences including base C of SEQ ID NO: 1, 2649th in SEQ ID NO: 1 Polynucleotide consisting of 20-100 consecutive DNA sequences containing base T, 2715th polynucleotide consisting of 20-100 consecutive DNA sequences containing base C of SEQ ID NO: 1, 2737th of SEQ ID NO: 1 It is characterized by including one or more polynucleotides selected from the group consisting of a polynucleotide consisting of 20-100 consecutive DNA sequences including base G and complementary polynucleotides thereof.

In the present invention, the separation of the nucleic acid in step (a) may be performed according to a method commonly used in the art, and may be performed using a commercially available extraction kit.

In addition, in step (b), the nucleotide sequence may be performed according to an automatic or manual nucleotide sequencing method known in the art, and preferably, PCR corresponding to 23S rRNA is performed by performing PCR on a test subject or sample. After preparing the product, the sequence thereof can be analyzed and performed according to a known nucleotide sequence analysis method.

In step (c), based on SEQ ID NO: 1, the base sequence corresponding to the determination point in the microorganism of the present invention may be checked to determine whether it corresponds to the microorganism of the present invention.

On the other hand, the present invention provides a microarray for detection of microorganisms of the present invention, characterized in that the probe of the present invention is integrated on a substrate. The microarray is made of a conventional microarray, except for including the polynucleotide of the present invention, and a method of preparing a microarray by immobilizing a polynucleotide used as a marker on an organ is well known in the art.

In addition, the present invention is a marker for detection of the microorganism of the present invention, characterized in that in the nucleotide having the nucleotide sequence represented by SEQ ID NO: 1, each base corresponding to the determination point of the microorganism of the present invention is substituted. Provides. The judgment points are as described in Table 3.

The nucleotide, probe or marker of the present invention may be DNA or RNA, and in the case of RNA, it is obvious to those skilled in the art that thymine described in the sequence is replaced by uracil.

In the case of using the method for identification of Santomonas euvesicatoria and the probe for identification of the present invention, it is possible to effectively identify Xanthomonas euvesicatoria from Xanthomonas genus.

FIG. 1 shows the results of a comparative analysis of nucleotide sequences of Santomonas campestris campestris (Xanthomonas campestris pv. campestris) and Santomonas euvesicatoria.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

<Example 1>

23s rRNA analysis of X. euvesicatoria

Through the following experimental method, a probe for identification of bacteria of the genus Xanthomonas was designed. Total RNA isolation, nucleotide sequence amplification, and analysis methods used in the present invention are as follows.

<1-1> 23s rRNA amplification of bacteria

After separating the DNA of X. euvesicatoria, PCR was performed using primer pairs as shown in [Table 1] below that can isolate 23s rRNA of bacteria of the genus Xanthomonas.

Primer name Primer sequence Xan23SF5 (Omnidirectional) 5'-TGGTCAAGCCGCACGGATCATTAGTAT-3' Xan23SR6 (reverse) 5'-ACGTGGATAGCCTGCGAAAAGTGTC-3' Xan23SF7 (Omnidirectional) 5'-GAGACCGCCCCAGTCAAACTAC-3' Xan23SR7 (reverse) 5'-ACCTTTTGTATAATGGGTCAACG-3' Xan23SF9 (Omnidirectional) 5'-GTCAAGCCGCACGGATCATTAGTAT-3' Xan23SR9 (reverse) 5'-GTCGGGGAGCTGGCAACAAG-3'

PCR reaction was performed with 20 ng of the total DNA isolated, 10 pmoles of downstream primers, 10 pmoles of upstream primers, 0.2 U of Taq DNA polymerase, 10 times polymerase chain reaction buffer (100 mM Tris-HCl, 500 5 μl of mM KCl, 15 mM MgCl ₂ , pH 8.3) was added, and distilled water was added to make the reaction solution 50 μl.

The reaction solution was heated at 95℃ for 5 minutes, then amplified 30 times at 95℃ for 30 seconds, 65℃ for 60 seconds, and 72℃ for 180 seconds, and finally processed at 72℃ for 10 minutes in a PCR machine (DNA Engine PTC-200). I did. After amplification, PCR products were subjected to electrophoresis using 1× TBE buffer and 1.5% agarose gel, stained with ethidium bromide, and checked in an ultraviolet lamp.

<1-2> Analysis of the amplified 23s rRNA sequence

The nucleotide sequence of the PCR products obtained in Example <1-1> was analyzed using BioEdit Sequence Alignment Editor.

After analysis, X. campestris pv. campestris 23s rRNA sequence was compared. X. campestris pv. campestris 23s rRNA sequence was used information published in Genbank (NC_003902.1). The 23s rRNA sequences of the bacteria used in this experiment were organized by sequence number as shown in Table 2 below.

Sequence number Bacteria One X. campestris pv. campestris 2 X. euvesicatoria

<Example 2>

Comparative analysis of 23s rRNA sequence between bacteria

The nucleotide sequence differences were compared between SEQ ID NOs: 1 to 2 in [Table 2]. Base sequences were compared using BioEdit Sequence Alignment Editor.

The analysis results are described in [Fig. 1], and X. campestris pv. Campestris's base sequence (SEQ ID NO: 1) is summarized in the following [Table 3] for the location of the base sequence difference with X. euvesicatoria.

Bacteria Position / Base Changed X. euvesicatoria 78/g 86/c 698/c 740/a 1177/c 1735/t 1966/a 1967/t 1969/c 1973/c 1977-
1979/ctc 2048-
2050/gag 2053/g 2056/a 2058/a 2060/a 2061/t 2626/c 2649/t 2715/c 2737/g

The number indicating the position in the table refers to the nth digit of SEQ ID NO: 1. Therefore, bacteria can be identified using the difference in base shown in [Table 3].

As described above, when using the identification method and probe for identification of Santomonas euvesicatoria of the present invention, since it is possible to identify Santomonas euvesicatoria from the genus Santomonas (Xanthomonas), It has high industrial applicability.

<110> REPUBLIC OF KOREA(MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Method of identifying Xanthomonas euvesicatoria <130> NP14-0108 <160> 8 <170> KopatentIn 2.0 <210> 1 <211> 2883 <212> DNA <213> Artificial Sequence <220> <223> X. campestris pv. campestris NC_003902 <400> 1 atggtcaagc cgcacggatc attagtatca gttagctcaa tacattgctg tacttacaca 60 cctgacctat caaccacata gtctatatgg ttcctttagg gggcttgtgc cccgggaagt 120 ctcatcttga ggcgcgcttc ccgcttagat gctttcagcg gttatcgctt ccgaacatag 180 ctacccggca atgccactgg cgtgacaacc ggaacaccag aggttcgtcc actccggtcc 240 tctcgtacta ggagcagccc ctctcaaact tccaacgccc atggcagata gggaccgaac 300 tgtctcacga cgttctgaac ccagctcgcg taccacttta aatggcgaac agccataccc 360 ttgggaccga ctacagcccc aggatgtgat gagccgacat cgaggtgcca aacaccgccg 420 tcgatatgaa ctcttgggcg gtatcagcct gttatccccg gagtaccttt tatccgttga 480 gcgatggccc ttccatacag aaccaccgga tcactaagac ctactttcgt acctgcttga 540 tccgtcgatc ttgcagtcaa gcacgcttat gcctttgcac acagtgcgcg atgtccgacc 600 gcgctgagcg taccttcgtg ctcctccgtt actctttagg aggagaccgc cccagtcaaa 660 ctacccacca tacacggtcc ctgatccgga taacggatct aggttagaac gtcaagcacg 720 acagggtggt atttcaaggt tggctccact gcagctagcg ccacagtttc atagcctccc 780 acctatccta cacagacgaa ctcaacgttc agtgtaaagc tatagtaaag gttcacgggg 840 tctttccgtc ttgccacggg aacgctgcat cttcacagcg atttcaattt cactgagtct 900 cgggtggaga cagcgccgct gtcgttacgc cattcgtgca ggtcggaact tacccgacaa 960 ggaatttcgc taccttagga ccgttatagt tacggccgcc gtttactggg gcttcgatca 1020 agagcttcgc cttgcggctg accccatcaa ttaaccttcc agcaccgggc aggcgtcaca 1080 ccctatacgt ccactttcgt gtttgcagag tgctgtgttt ttgataaaca gtcgcagcgg 1140 cctggtttct gcgaccctct tcagctatag ctcgcatgag ccaccaaaaa gggtgcacct 1200 tctcccgaag ttacggtgcc atgttgccta gttccttcac ccgagttctc tcaagcgcct 1260 gagaattctc atcctaccca cctgtgtcgg tttacggtac ggtcttcgtg agctgaagct 1320 taggagcttt tcctggaagc gtggtatcag tgacttcgcc ataaaggctc gtctcggtgc 1380 tcggtcttaa aggatcccgg atttgccaaa gatccaaacc taccgccttt ccccgggaca 1440 accaacgccc ggtacaccta accttctccg tccctccatc gcactcacgc gaggtgcagg 1500 aatattaacc tgcttcccat cgactacggc tttcgccctc gccttaggga ccgactaacc 1560 ctgcgtcgat taacgttgcg caaggaaacc ttgggctttc ggcgtgcggg cttttcaccc 1620 gcattatcgt tactcatgtc agcattcgca cttccgatac ctccagcaga cttctcaatc 1680 caccttcgca ggcttacgga acgctcctct accgcgcata aaaccgaagt tttacgcacc 1740 ccaagcttcg gttcactgct tagccccgtt aaatcttccg cgcagaccga ctcgaccagt 1800 gagctattac gctttcttta aagggtggct gcttctaagc caacctcctg gctgtctatg 1860 cctttccaca tcgttttcca cttagcagtg aatttgggac cttagctgtg ggtctgggtt 1920 gtttcccttt tcacgacgga cgttagcacc cgccgtgtgt ctcccggata gtacgtactg 1980 gtattcggag tttgcaatgg tttggtaagt cgcgatgacc ccctagccat aacagtgctc 2040 tacccccagt agtattcgtc cgaggcgcta cctaaatagc tttcgaggag aaccagctat 2100 ctccgggttc gattagcttt tcactcctaa tcacagctca tccccgtctt ttgcaacaga 2160 cgtgggttcg ggcctccagt acctgttacg gcaccttcac cctggccatg actagatcac 2220 ccggtttcgg gtctactgcc cgcgactatg cgcccttatc agactcggtt tcccttcgcc 2280 tcccctatac ggttaagctt gccacgaaca gtaagtcgct gacccattat acaaaaggta 2340 cgcagtcact cttgcgagct cctactgctt gtacgcacac ggtttcagga tctatttcac 2400 tcccctctcc ggggttcttt tcgcctttcc ctcacggtac tggttcacta tcggtcggtc 2460 aggagtattt agccttggag gatggtcccc ccatattcag acagggtttc acgtgccccg 2520 ccctactcgt cttcactgga gtggcccttt taaatacagg gctatcacct tctatggcca 2580 atctttccag attgtttttc taaagccatt ccagcttaag ggctgttccc cgttcgctcg 2640 tcactactca gggaatctcg gttgatttct tttcctccgg ttacttagat atttcagttc 2700 accgggttcg cttcaagcag ctatgaattc actgcaagat actgccgaag cagtgggttt 2760 ccccattcgg atattgccgg atcaaagctt gttgccagct ccccgacact tttcgcaggc 2820 taccacgtcc ttcatcgcct ctgaccgcct aggcatccac cgtgtgcgct tattcgcttg 2880 acc 2883 <210> 2 <211> 2714 <212> DNA <213> Artificial Sequence <220> <223> X cassavae BC620 <400> 2 acacacctga cctatcaacc acgtagtcta catggttcct ttagggggct tgtgccccgg 60 gaagtctcat cttgaggcgc gcttcccgct tagatgcttt cagcggttat cgcttccgaa 120 catagctacc cggcaatgcc actggcgtga caaccggaac accagaggtt cgtccactcc 180 ggtcctctcg tactaggagc agcccctctc aaacttccaa cgcccatggc agatagggac 240 cgaactgtct cacgacgttc tgaacccagc tcgcgtacca ctttaaatgg cgaacagcca 300 tacccttggg accgactaca gccccaggat gtgatgagcc gacatcgagg tgccaaacac 360 cgccgtcgat atgaactctt gggcggtatc agcctgttat ccccggagta ccttttatcc 420 gttgagcgat ggcccttcca tacagaacca ccggatcact aagacctact ttcgtacctg 480 cttgatccgt cgatcttgca gtcaagcacg cttatgcctt tgcacacagt gcgcgatgtc 540 cgaccgcgct gagcgtacct tcgtgctcct ccgttactct ttaggaggag accgccccag 600 tcaaactacc caccatacac ggtccctgat ccggataacg gatctaggtt agaacgtcaa 660 gcacgacagg gtggtatttc aaggatggct cccctgcagc tagcgccaca gtttcatagc 720 ctcccaccta tcctacacag acgaactcaa cgttcagtgt aaagctatag taaaggttca 780 cggggtcttt ccgtcttgcc acgggaacgc tgcatcttca cagcgatttc aatttcactg 840 agtctcgggt ggagacagcg ccgctgtcgt tacgccattc gtgcaggtcg gaacttaccc 900 gacaaggaat ttcgctacct taggaccgtt atagttacgg ccgccgttta ctggggcttc 960 gatcaagagc ttcgccttgc ggctgacccc atcaattaac cttccagcac cgggcaggcg 1020 tcacacccta tacgtccact ttcgtgtttg cagagtgctg tgtttttgat aaacagtcgc 1080 agcggcctgg tttctgcgac cctcttcagc tatagctcgc atgagccacc aaaaagggtg 1140 caccttctcc cgaagttacg gtgccatgtt gcctagttcc ttcacccgag ttctctcaag 1200 cgcctgagaa ttctcatcct acccacctgt gtcggtttac ggtacggtct tcgtgagctg 1260 aagcttagga gcttttcctg gaagcgtggt atcagtgact tcgccataaa ggctcgtctc 1320 ggtgctcggt cttaaaggat cccggatttg ccaaagatcc aaacctaccg cctttccccg 1380 ggacaaccaa cgcccggtac acctaacctt ctccgtccct ccatcgcact cacgcgaggt 1440 gcaggaatat taacctgctt cccatcgact acggctttcg ccctcgcctt agggaccgac 1500 taaccctgcg tcgattaacg ttgcgcaagg aaaccttggg ctttcggcgt gcgggctttt 1560 cacccgcatt atcgttactc atgtcagcat tcgcacttcc gatacctcca gcagacttct 1620 caatccacct tcgcaggctt acggaacgct cctctaccgc gcataaaaca agttttatgc 1680 accccaagct tcggttcact gcttagcccc gttaaatctt ccgcgcagac cgactcgacc 1740 agtgagctat tacgctttct ttaaagggtg gctgcttcta agccaacctc ctggctgtct 1800 atgcctttcc acatcgtttt ccacttagca gtgaatttgg gaccttagct gtgggtctgg 1860 gttgtttccc ttttcacgac ggacgttagc acccgccgtg tgtctcccgg atagtacgta 1920 ctggtattcg gagtttgcaa tggtttggta agtcgcgatg accccctagc cataacagtg 1980 ctctaccccc agtagtattc gtccgaggcg ctacctaaat agctttcgag gagaaccagc 2040 tatctccggg ttcgattagc ttttcactcc taatcacagc tcatccccgt cttttgcaac 2100 agacgtgggt tcgggcctcc agtacctgtt acggcacctt caccctggcc atgactagat 2160 cacccggttt cgggtctact gcccgcgact atgcgccctt atcagactcg gtttcccttc 2220 gcctccccta tacggttaag cttgccacga acagtaagtc gctgacccat tatacaaaag 2280 gtacgcagtc actcttgcga gctcctactg cttgtacgca cacggtttca ggatctattt 2340 cactcccctc tccggggttc ttttcgcctt tccctcacgg tactggttca ctatcggtcg 2400 gtcaggagta tttagccttg gaggatggtc cccccatatt cagacagggt ttcacgtgcc 2460 ccgccctact cgtcttcact ggagtggccc ttttaaatac agggctatca ccttctatgg 2520 ccaatctttc cagattgttt ttctaaagcc attccagctt aagggctgtt ccccgttcgc 2580 tcgtcactac ttagggaatc tcggttgatt tcttttcctc cggttactta gatatttcag 2640 ttcaccgggt tcgcttccag cagctatgaa ttcactgcag gatactgccg aagcagtggg 2700 tttccccatt cgga 2714 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Xan23SF5 <400> 3 tggtcaagcc gcacggatca ttagtat 27 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Xan23SR6 <400> 4 acgtggatag cctgcgaaaa gtgtc 25 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Xan23SF7 <400> 5 gagaccgccc cagtcaaact ac 22 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Xan23SR7 <400> 6 accttttgta taatgggtca acg 23 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Xan23SF9 <400> 7 gtcaagccgc acggatcatt agtat 25 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Xan23SR9 <400> 8 gtcggggagc tggcaacaag 20

Claims (3)

(a) separating 23S rRNA from the sample;
(b) confirming the nucleotide sequence of the 23S rRNA isolated in the step (a); And
(c) the nucleotide sequence identified in step (b) is G, the nucleotide at position 78 is G, the nucleotide at position 86 is C, the nucleotide at position 698 is C, the nucleotide at position 740 is A, the nucleotide at position 1177 is C, The base is T, the 1966th base is A, the 1967th base is T, the 1969th base is C, the 1973th base is C, the 1977th to 1979th base is CTC, the 2048th to 2050th base is GAG, the 2053th base is G, The 2056th base is A, the 2058th base is A, the 2060th base is A, the 2061th base is T, the 2626th base is C, the 2649th base is T, the 2715th base is C, the 2737th base is G, (Xanthomonas euvesicatoria), comprising the step of determining the presence or absence of a disease of the genus Xanthomonas euvesicatoria.
A polynucleotide consisting of 20-100 consecutive DNA sequences comprising G substituted at the 78th base of SEQ ID NO: 1, 20-100 comprising a C substituted at the 86th base of SEQ ID NO: 1 Polynucleotide consisting of 20-100 consecutive DNA sequences consisting of consecutive DNA sequences of SEQ ID NO: 1, C substituted with 698th base of SEQ ID NO: 1, polynucleotide consisting of 20-100 consecutive DNA sequences consisting of substituted A A polynucleotide consisting of 20-100 consecutive DNA sequences comprising SEQ ID NO: 1, a polynucleotide consisting of 20-100 consecutive DNA sequences comprising C substituted at position 1177 of SEQ ID NO: 1, 1735 Polynucleotide consisting of 20-100 consecutive DNA sequences comprising the T base substituted with the base T, 20-100 consecutive DNAs comprising the A nucleotide substituted at position 1966 of SEQ ID NO: 1 A polynucleotide consisting of 20-100 consecutive DNA sequences comprising the T at position 1967 substituted with the 1967 base of SEQ ID NO: 1, a polynucleotide consisting of 20 &lt; RTI ID = 0.0 &gt; A polynucleotide consisting of 20-100 consecutive DNA sequences comprising a polynucleotide consisting of -100 consecutive DNAs, C substituted with the 1973 th base of SEQ ID NO: 1, a polynucleotide consisting of the 1977 to 1979 base of SEQ ID NO: 1 A polynucleotide consisting of 20-100 consecutive DNA sequences comprising a substituted CTC, a polynucleotide consisting of 20-100 consecutive DNA sequences comprising a GAG substituted at positions 2048 to 2050 of SEQ ID NO: 1, A polynucleotide consisting of 20-100 consecutive DNA sequences comprising G substituted at position 2053 of SEQ ID NO: 1, a polynucleotide consisting of a nucleotide at position 2056 of SEQ ID NO: 1 substituted A, a polynucleotide consisting of 20-100 consecutive DNA sequences containing the A at position 2058 of SEQ ID NO: 1, a polynucleotide consisting of 20-100 consecutive DNA sequences comprising A substituted at position 2058 of SEQ ID NO: 1, A polynucleotide consisting of 20-100 consecutive DNA sequences comprising 2060th base substituted A, 20-100 consecutive DNA sequences comprising T substituted at position 2061 of SEQ ID NO: 1, A polynucleotide consisting of 20-100 consecutive DNA sequences comprising C substituted at position 2626 of SEQ ID NO: 1, 20-100 consecutive nucleotides comprising T substituted at position 2649 of SEQ ID NO: 1 A polynucleotide consisting of 20-100 consecutive DNA sequences comprising a polynucleotide consisting of a DNA sequence of SEQ ID NO: 1, C substituted at position 2715 of SEQ ID NO: 1, a polynucleotide consisting of SEQ ID NO: 1 at position 2737 (Xanthomonas euvesicatoria) comprising at least one polynucleotide selected from the group consisting of polynucleotides consisting of 20-100 consecutive DNA sequences containing a base substituted G and complementary polynucleotides thereof. Probe.
In the nucleotide having the nucleotide sequence shown in SEQ ID NO: 1, the 78th nucleotide of SEQ ID NO: 1 is G, the 86th nucleotide is C, the 698th nucleotide is C, the 740th nucleotide is A, the 1177 nucleotide is C, T is 1966th base A, 1967th base is T, 1969th base is C, 1973th base is C, 1977th to 1979th is CTC, 2048th to 2050th base is GAG, 2053th base is G, 2056 The second base is A, the 2058th base is A, the 2060th base is A, the 2061th base is T, the 2626th base is C, the 2649th base is T, the 2715th base is C, and the 2737th base is G Marker for detection of Santomonas eubicictoria (Xanthomonas euvesicatoria).

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