KR20140064206A - Method of identifying xanthomonas cucurbitae - Google Patents
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Abstract
Description
The present invention relates to a method for identification of Xanthomonas cucurbitae, and more particularly, to a method for identifying a base of 23S rRNA isolated from a sample to identify Xanthomonas cucurbitae.
The genus Santomonas (Xanthomonas) is a bacteria involved in the denitrification of soil nitrogen. These include pathogenic bacteria such as peach tree bacterial pericarditis, non-black rot, citrus peptic ulcer disease, rice blight white leaf blight,
Dot-blot, PCR (PCR), etc. are used for gene identification of plant pathogenic bacteria. In particular, PCR is a widely used method because it is economical in terms of time, effort, and manpower than the dot-blot method. PCR (polymerase chain reaction) is a method for identifying a DNA fragment (primer) consisting of 10-20 bp in the genomic DNA of a bacterium and then using heat-resistant DNA polymerase The PCR amplification product is electrophoresed on an agarose gel or an acrylamide gel and ethidium bromide is added to the reaction mixture, bromide, and silver stain. The DNA polymorphism of the bacterium, such as the presence or absence of DNA bands, is identified and identified. Currently, the identification method of bacteria by PCR is mainly used for identification of bacterial diseases in humans and animals. However, in recent years, PCR identification method has been developed to identify plant pathogens and is used for identification of diseases and quarantine of agricultural products . In addition, the PCR identification method is shorter in time than other methods, is low in cost for identification, and is also economical because many samples can be assayed at the same time. Recently, PCR identification method that amplifies nucleic acid which is a genetic material among the identification methods of Santomonas (Xanthomonas) is most preferred.
However, existing PCR identification methods are difficult to classify due to genetic similarity, and a solution to overcome this problem is needed.
Among the existing microorganisms of the genus Santomonas, Xanthomonas oryzae pv. Oryzae, Xanthomonas campestris pv. Vesicatoria, Xanthomonas campestris pv. Campestris, Xanthomonas axonopodis pv. citric acid, and Xanthomonas axonopodis pv. glycines. However, it has been reported that the individual PCR primers used for identification of Xanthomonas cucurbitae from the genus Xanthomonas (Xanthomonas cucurbitae) The method has not yet been studied. It is required to develop a method for identification of Xanthomonas cucurbitae through the combination of species or pathogenic type specific bases and bases between plant pathogenic bacteria.
Accordingly, the inventors of the present invention have found out the difference from the 23s rRNA base sequence of another species of Santomonas bacteria while studying to identify Santomonas cucurbitae more accurately and quickly from Xanthomonas genus And developed a method of identification using it.
Accordingly, an object of the present invention is to provide a method for detecting (i) isolating 23S rRNA from a sample; (b) confirming the nucleotide sequence of the 23S rRNA isolated in the step (a); (C) the nucleotide sequence identified in step (b) is G, the 78th base is G, the 86th base is C, the 740th base is A, the 1177th base is C, the 1735th base is T, 2649 (Xanthomonas cucurbitae) which comprises the step of judging the first base as T, the 2715th base as C, and the 2737th base as G as Xanthomonas cucurbitae.
It is still another object of the present invention to provide a polynucleotide consisting of 20-100 consecutive DNA sequences comprising the 78th base G of SEQ ID NO: 1, a polynucleotide consisting of the 86th base C , A polynucleotide consisting of 20-100 consecutive DNA sequences comprising the 740th base A of SEQ ID NO: 1, a polynucleotide consisting of 20-100 consecutive DNA sequences comprising the 740th base A of SEQ ID NO: 1, a polynucleotide consisting of the 1177th nucleotide C A polynucleotide consisting of 20-100 consecutive DNA sequences comprising SEQ ID NO: 1, a polynucleotide consisting of 20-100 consecutive DNA sequences comprising 1735th base T of SEQ ID NO: 1, a polynucleotide consisting of 20-100 consecutive DNA sequences comprising SEQ ID NO: , A polynucleotide consisting of 20-100 consecutive DNA sequences containing the 2715th base C of SEQ ID NO: 1, and 20-100 consecutive DNA sequences Polynucleotides, polynucleotides consisting of 20-100 consecutive DNA sequences comprising nucleotide 2737 of SEQ ID NO: 1, and complementary polynucleotides thereof. ≪ RTI ID = 0.0 > (Xanthomonas cucurbitae) identification probe.
In order to achieve the above object, the present invention provides a method for identification of Santomonas cucurbitae.
In order to accomplish still another object of the present invention, the present invention provides a probe for identification of Xanthomonas cucurbitae.
Hereinafter, the present invention will be described in detail.
The present invention relates to a method for identifying Xanthomonas cucurbitae.
That is, the present invention secures a base combination of the 23S rRNA gene of Xanthomonas to identify Xanthomonas cucurbitae.
More specifically, the method for identifying Xanthomonas cucurbitae of the present invention comprises the steps of: (a) separating 23S rRNA from a sample; (b) confirming the nucleotide sequence of the 23S rRNA isolated in the step (a); (C) the nucleotide sequence identified in step (b) is G, the 78th base is G, the 86th base is C, the 740th base is A, the 1177th base is C, the 1735th base is T, 2649 The second base is T, the 2715th base is C, and the 2737th base is G, it is judged to be Santomonas cucurbitae.
The present invention also provides a probe for identification of Xanthomonas cucurbitae.
That is, the present invention secures a base combination of the 23S rRNA gene of Xanthomonas to identify Xanthomonas cucurbitae.
More specifically, the probe for identifying Xanthomonas cucurbitae of the present invention comprises a polynucleotide consisting of 20 to 100 consecutive DNA sequences comprising the 78th base G of SEQ ID NO: 1, A polynucleotide consisting of 20-100 consecutive DNA sequences comprising the 86th base C of SEQ ID NO: 1, a polynucleotide consisting of 20-100 consecutive DNA sequences comprising the 740th base A of SEQ ID NO: 1, A polynucleotide consisting of 20-100 consecutive DNA sequences comprising nucleotide 1177 of SEQ ID NO: 1, a polynucleotide consisting of 20-100 consecutive DNA sequences comprising nucleotide 1735 of SEQ ID NO: Nucleotide, a polynucleotide consisting of 20-100 consecutive DNA sequences comprising the 2649th base T of SEQ ID NO: 1, a polynucleotide consisting of the 2715th nucleotide of SEQ ID NO: 1 A polynucleotide consisting of 20-100 consecutive DNA sequences comprising base C, a polynucleotide consisting of 20-100 consecutive DNA sequences comprising 2737th base G of SEQ ID NO: 1 and their complementary polynucleotides ≪ / RTI > and at least one < RTI ID = 0.0 > polynucleotide < / RTI >
In the present invention, the nucleic acid may be separated in step (a) according to a method commonly used in the art, and may be carried out using a commercially available extraction kit.
Also, the base sequence in step (b) may be performed according to automatic or manual base sequence analysis methods known in the art. Preferably, PCR is performed on a test specimen or a sample to perform PCR corresponding to 23S rRNA And the sequence of the product can be analyzed and analyzed according to a known nucleotide sequence analysis method.
In the step (c), the nucleotide sequence corresponding to the judgment point in the microorganism of the present invention can be identified based on SEQ ID NO: 1 to determine whether the microorganism corresponds to the present invention.
Meanwhile, the present invention provides a microarray for detecting microorganisms of the present invention, wherein the probe of the present invention is integrated on a substrate. The microarray consists of a conventional microarray except that it contains a polynucleotide of the present invention, and a method for producing a microarray by immobilizing a polynucleotide used as a marker on an organ is well known in the art.
Further, the present invention is a marker for detecting a microorganism of the present invention, wherein a nucleotide having a nucleotide sequence shown in SEQ ID NO: 1 is substituted for each of the bases corresponding to the judgment point of the microorganism of the present invention. Lt; / RTI > The above judgment points are as shown in Table 3.
The nucleotide, probe or marker of the present invention may be DNA or RNA, and it is obvious to those skilled in the art that the thymine described in the sequence is replaced by uracil in the case of RNA.
When the identification method of the Xanthomonas cucurbitae of the present invention and the identification probe are used, it is possible to effectively identify Xanthomonas cucurbitae from Xanthomonas.
Figure 1 shows the results of a comparison of base sequence analysis of Santomonas campestris pv. Campestris and Xanthomonas cucurbitae.
Hereinafter, the present invention will be described in detail with reference to examples.
However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
≪ Example 1 >
23s rRNA analysis of X. cucurbitae
Through the following experimental procedure, a probe for identification of bacteria belonging to the genus Xanthomonas was designed. The total RNA isolation, base sequence amplification and analysis methods used in the present invention are as follows.
<1-1> 23s rRNA amplification of bacteria
After the DNA of X. cucurbitae was isolated, PCR was carried out using primer pairs as shown in Table 1 below, which can isolate 23s rRNA of bacteria of the genus Xanthomonas.
The PCR reaction consisted of 20 ng of isolated total DNA, 10 pmoles of downstream primer, 10 pmole of upstream primer, 0.2 U of Taq DNA polymerase, 10 times Polymerase chain reaction buffer (100 mM Tris-HCl, 500 mM KCl, 15 mM MgCl 2 , pH 8.3) was added and distilled water was added to make 50 μl of the reaction solution.
The reaction solution was heated at 95 ° C. for 5 minutes and then amplified 30 times at 95 ° C. for 30 seconds, 65 ° C. for 60 seconds, and 72 ° C. for 180 seconds. Finally, the reaction solution was treated at 72 ° C. for 10 minutes with a PCR machine (DNA Engine PTC-200) Respectively. After amplification, the PCR product was electrophoresed using 1 × TBE buffer and 1.5% agarose gel, stained with ethidium bromide, and then confirmed by ultraviolet lamp.
<1-2> Amplified 23s rRNA sequence analysis
The nucleotide sequences of the PCR products obtained in Example <1-1> were analyzed using BioEdit Sequence Alignment Editor.
After analysis, X. campestris pv. We compared the 23s rRNA sequences of campestris. X. campestris pv. For the campestris 23s rRNA sequence, the information (NC_003902.1) published in Genbank was used. The 23s rRNA sequences of the bacteria used in this experiment are summarized in the sequence numbers as shown in Table 2 below.
< Example 2>
Comparative analysis of 23s rRNA sequence between bacteria
The nucleotide sequence differences between SEQ ID NOS: 1 and 2 in Table 2 were compared. The nucleotide sequences were compared using the BioEdit Sequence Alignment Editor.
The results of the analysis are shown in Fig. 1, and X. campestris pv. Table 3 summarizes the positions where base sequence differences with X. cucurbitae are based on the nucleotide sequence of Campestris (SEQ ID NO: 1).
The number indicating the position in the above table means the n-th digit of SEQ ID NO: 1. Therefore, the bacterium can be identified using the difference of the bases shown in [Table 3] above.
As described above, when the identification method and identification probe of Xanthomonas cucurbitae of the present invention are used, it is possible to identify Xanthomonas cucurbitae from Xanthomonas, It is highly available.
<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Method of identifying Xanthomonas cucurbitae <130> NP12-0066 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 2883 <212> DNA <213> Xanthomonas campestris pv. campestris <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> X cucurbitae <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 ccactgcagc 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 acgagccacc 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 (4)
(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 78th base is G, the 86th base is C, the 740th base is A, the 1177th base is C, the 1735th base is T, (Xanthomonas cucurbitae), wherein the base is judged to be T, when the 2715th base is C, and when the 2737th base is G, it is judged to be Xanthomonas cucurbitae.
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