KR20170059090A - Specific primer set for diagnosing Curtobacterium flaccumfaciens pv. flaccumfaciens, quarantine plant pathogen and uses thereof - Google Patents
Specific primer set for diagnosing Curtobacterium flaccumfaciens pv. flaccumfaciens, quarantine plant pathogen and uses thereof Download PDFInfo
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Abstract
The present invention relates to a method for the treatment and / The present invention relates to a primer set for specifically diagnosing plaque pachysense and a use thereof. In the present invention, a total of five primer sets are used to isolate quarantine bacteria pQ. Curtobacterium flaccumfaciens pv. flaccumfaciens ) can be diagnosed specifically. Accordingly, the present invention provides a method for producing a microorganism belonging to the genus Plasmodium pv. Since a specific diagnosis for Plurkpathians can be performed easily and quickly, it is possible to use a plasmid selected from the group consisting of Kurtobacterium plucum pasifens pv. It is very useful because it can thoroughly prevent the introduction of the Pseudomonas sp. Strains into Korea.
Description
The present invention relates to a method for the treatment and / The present invention relates to a primer set and a use thereof for specifically diagnosing Plurkpathians. More particularly, the present invention relates to a primer set for the diagnosis of Plurkpathians pv. A primer set for specifically diagnosing Curtobacterium flaccumfaciens pv. Flaccumfaciens , a plasmid containing the above-mentioned primer set, A kit for specifically diagnosing Plurkpathians, and a kit for the diagnosis of Plasmopathia pv. Pv. The present invention relates to a method of specifically diagnosing Plurkfaciens.
In order to prevent the spread of pests infesting and settling into other areas, plant quarantine is prohibited from inspecting the target plants, prohibiting the movement, disinfection of plants with germs and pests (chemical spraying, fumigation, water immersion, etc.) ) · We take measures such as disposal (incineration, crushing, landfill). Just as animals and plants are unique in every country on the planet, the distribution of pests varies from region to region. The reason that pests live only in certain areas is that the climate and environment of the new settlement is not suitable for survival, but most pests are difficult to move by themselves even if there are many other places where they live.
The migration of pests generally consists of the following: (1) lurking or buried in host plants and their products, (2) moving on a transportation means such as packaging materials, vessels, aircraft, ships or automobiles, (3) Or scattering), and in the body such as insects or birds, or moving on the body, or (4) moving by wind, water, or air currents.
However, recently, with the rapid increase of international trade volume due to the development of transportation and industry and the frequent passage of travelers, many kinds of pests have been rapidly moved by artificial factors. Once a new pest invaded from foreign countries is suitable for climate and environmental conditions and there are abundant host plants, there are many examples that cause more damage than the origin. Therefore, in order to stabilize the production of agriculture and forestry industry by preventing national disasters that may occur when new pest infestation is introduced, to protect the ecosystem, to make the nature beautify and to make human life comfortable, .
The fundamental reason for the policy strengthening of such phytosanitary measures by governments around the world is that the expense and inconvenience of preventing the entry of new pests is less than the cost or damage to be spent in controlling the infested pests It is very economical.
Korean Pat. No. 0905224 discloses a method of quarantine and clean a plant parasitic nematode. However, as in the present invention, the quarantine bacterium Kurtobacterium plucum pasifens pv. A primer set and a use thereof for specifically diagnosing Plurkpathians have not been disclosed.
DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a method for producing a Pseudomonas sp. Curtobacterium flaccumfaciens pv. flaccumfaciens ) was developed in order to develop a primer capable of specifically diagnosing. After genomic sequencing of Plurkpathians was carried out from ATCC, the genomic region of the genome was found to be homologous to other bacteria. Curtobacterium flaccumfaciens pv. flaccumfaciens ) were designed. Among the 14 primer sets, finally, five types of primer sets were selected from the group consisting of Clostobacillus plumjumplasmus pv. The present inventors have completed the present invention by confirming that there is a PCR reaction only in Plasmapassien and that there is no PCR reaction in the soil microbial DNA complex.
In order to solve the above problems, the present invention provides oligonucleotide primer sets of SEQ ID NOS: 3 and 4; An oligonucleotide primer set of SEQ ID NOS: 5 and 6; An oligonucleotide primer set of SEQ ID NOs: 9 and 10; An oligonucleotide primer set of SEQ ID NOs: 23 and 24; And an oligonucleotide primer set of SEQ ID NOs: 25 and 26; and a set of at least one primer selected from the group consisting of Clostrobacillus plicum pasifens pv. Curtobacterium flaccumfaciens pv. The present invention provides a primer set for specifically diagnosing flaccumfaciens .
In addition, the present invention relates to a kit for the treatment of Kurtobacterium plucum pasiens pv. Provided is a kit for specifically diagnosing plaque feathers.
In addition, the present invention provides a method for screening for the presence or absence of the above-mentioned primer set. The present invention provides a method for specifically diagnosing Plurkfaciens.
In the present invention, a total of five primer sets selected lastly were used to isolate the quarantine bacteria Kurtobacterium plucum pasifens pv. Curtobacterium flaccumfaciens pv. flaccumfaciens ) can be diagnosed specifically. Accordingly, the present invention provides a method for producing a microorganism belonging to the genus Plasmodium pv. Since a specific diagnosis for Plurkpathians can be easily and quickly carried out, It is possible to effectively prevent the introduction of the Pseudomonas sp. Strain into the domestic market, which is a very useful invention.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. Curtobacterium flaccumfaciens pv. flaccumfaciens ) using 14 pairs of primers specially designed for the first time to select the primers that specifically react with flaccumfaciens . The result is the result of performing PCR using the Plasmapassian genomic DNA as a template. The size (bp) in the table on the right indicates the expected size of the PCR product, while the red indicates the set of primers excluded from the first selection with 7 primer sets in which the expected PCR product is not generated.
FIG. 2 is a graph showing the results obtained by the method of the present invention. In order to secondarily select primers that specifically react with Plasmapassense, a total of 7 primer sets were used as a negative control and PCR was performed using a DNA complex extracted from humus in an acid having high microbial diversity as a template. The size (bp) in the table on the right represents the expected size of the PCR product, while the red color represents the second set of primers selected from the five primer sets in which the expected PCR product is not generated.
FIG. 3 is a result of PCR using a DNA complex extracted from humus in an acid having a high microbial diversity as a template. The final five primer sets selected do not produce PCR products for the humectant DNA complex, Plurkum Passens pv. It is specific to Plurkpathians.
In order to achieve the object of the present invention, the present invention provides oligonucleotide primer sets of SEQ ID NOS: 3 and 4; An oligonucleotide primer set of SEQ ID NOS: 5 and 6; An oligonucleotide primer set of SEQ ID NOs: 9 and 10; An oligonucleotide primer set of SEQ ID NOs: 23 and 24; And an oligonucleotide primer set of SEQ ID NOs: 25 and 26; and a set of at least one primer selected from the group consisting of Clostrobacillus plicum pasifens pv. Curtobacterium flaccumfaciens pv. The present invention provides a primer set for specifically diagnosing flaccumfaciens .
The primers include at least 16, at least 17, at least 18, at least 19, at least 18, at least 18, at least 18, , And oligonucleotides consisting of fragments of 20 or more consecutive nucleotides. For example, the primer of SEQ ID NO: 1 may comprise an oligonucleotide consisting of fragments of 16 or more, 17 or more, 18 or more, 19 or more, 20 or more consecutive nucleotides in the sequence of SEQ ID NO: 1, The primer of SEQ ID NO: 2 may comprise oligonucleotides consisting of fragments of 16 or more, 17 or more, 18 or more, 19 or more, or 20 or more consecutive nucleotides in the sequence of SEQ ID NO: 2. The primers may also include additional, deleted or substituted sequences of the nucleotide sequences of SEQ ID NOS: 3, 4, 5, 6, 9, 10, 23, 24, 25 and 26.
An oligonucleotide primer set according to one embodiment of the present invention comprises oligonucleotide primer sets of SEQ ID NOS: 3 and 4; An oligonucleotide primer set of SEQ ID NOS: 5 and 6; An oligonucleotide primer set of SEQ ID NOs: 9 and 10; An oligonucleotide primer set of SEQ ID NOs: 23 and 24; And an oligonucleotide primer set of SEQ ID NOS: 25 and 26; Curtobacterium flaccumfaciens pv. flaccumfaciens ). < / RTI >
The simultaneous use of the oligonucleotide primer sets of SEQ ID NOS: 3, 4, 5, 6, 9, 10, 23, 24, 25 and 26 resulted in the mutation of Clostrobacellum plucum pasciens pv. Plockum Passens (Curtobacterium flaccumfaciens pv. flaccumfaciens) Can be more efficiently diagnosed, predicted, or detected.
In the present invention, a "primer" refers to a single strand oligonucleotide sequence complementary to a nucleic acid strand to be copied, and may serve as a starting point for synthesis of a primer extension product. The length and sequence of the primer should allow the synthesis of the extension product to begin. The specific length and sequence of the primer will depend on the primer usage conditions such as temperature and ionic strength, as well as the complexity of the desired DNA or RNA target.
As used herein, an oligonucleotide used as a primer may also include a nucleotide analogue, such as phosphorothioate, alkylphosphorothioate, or peptide nucleic acid, or alternatively, And may include an intercalating agent.
In order to achieve still another object of the present invention, the present invention provides a kit comprising at least one set of oligonucleotide primers according to the present invention; And a reagent for carrying out an amplification reaction. ≪ RTI ID = 0.0 > pC. ≪ / RTI > Curtobacterium flaccumfaciens pv. The present invention provides a kit for specifically diagnosing flaccumfaciens .
In the kit of the present invention, the reagent for carrying out the amplification reaction may include DNA polymerase, dNTPs, buffer and the like. In addition, the kit of the present invention may further include a user guide describing optimal reaction performing conditions. The manual is a printed document that explains how to use the kit, for example, how to prepare PCR buffer, the reaction conditions presented, and so on. The brochure includes instructions on the surface of the package including the brochure or leaflet in the form of a brochure, a label attached to the kit, and a kit. In addition, the brochure includes information that is disclosed or provided through an electronic medium such as the Internet.
In order to achieve still another object of the present invention,
Separating the genomic DNA from the sample to be analyzed;
Amplifying the target sequence by using the separated genomic DNA as a template and carrying out an amplification reaction using the oligonucleotide primer set; And
And detecting the amplification product. ≪ RTI ID = 0.0 > pC. ≪ / RTI > Curtobacterium flaccumfaciens pv. flaccumfaciens ). < / RTI >
The method of the present invention comprises separating genomic DNA from a sample to be analyzed. As a method for separating the genomic DNA from the sample, a method known in the art can be used. For example, the CTAB method may be used, or a wizard prep kit (Promega) may be used. The target sequence may be amplified by performing amplification reaction using the separated genomic DNA as a template and using one or more oligonucleotide primer sets according to one embodiment of the present invention as primers. Methods for amplifying a target nucleic acid include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, transcription-based amplification system, Strand displacement amplification or amplification with Q [beta] replicase, or any other suitable method for amplifying nucleic acid molecules known in the art. Among them, PCR is a method of amplifying a target nucleic acid from a pair of primers that specifically bind to a target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used.
In the method of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. In one embodiment, the labeling material can be, but is not limited to, a fluorescent, phosphorescent or radioactive substance. Preferably, the labeling substance is Cy-5 or Cy-3. When the target sequence is amplified, PCR is carried out by labeling the 5'-end of the primer with Cy-5 or Cy-3, and the target sequence may be labeled with a detectable fluorescent labeling substance. When the radioactive isotope such as 32P or 35S is added to the PCR reaction solution, the amplification product is synthesized and the radioactivity is incorporated into the amplification product, so that the amplification product can be labeled as radioactive. The set of one or more oligonucleotide primers used to amplify the target sequence is as described above.
The method of the present invention comprises detecting said amplification product. The detection of the amplification product can be performed by DNA chip, gel electrophoresis, radioactive measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto. As one method of detecting the amplification product, gel electrophoresis can be performed. Gel electrophoresis can be performed using agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product. Also, in the fluorescence measurement method, Cy-5 or Cy-3 is labeled at the 5'-end of the primer. When PCR is carried out, the target is labeled with a fluorescent label capable of detecting the target sequence. The labeled fluorescence is measured using a fluorescence meter can do. In addition, in the case of performing the PCR, the radioactive isotope such as 32P or 35S is added to the PCR reaction solution to mark the amplification product, and then the radioactivity is measured using a radioactive measuring device such as a Geiger counter or a liquid scintillation counter A liquid scintillation counter can be used to measure radioactivity.
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.
Materials and methods
1. Genome sequencing, identification of strains and specific primer design
(1) Genome sequencing and identification of strains
In the present invention, Curtobacterium flaccumfaciens pv. Genomic sequencing was performed on flaccumfaciens ATCC51875. Genomic sequencing was performed by NGS sequencing using an ion torrent PGM sequencer. For each genome segment obtained as a result of the sequencing, the quality of each sequence was confirmed for subsequent genome assembly.
In addition, the genome assembly was used to construct scaffolds close to the dielectric length based on the sequencing result sequence, using the CLC genomics workbench v6.5 and gsAssembler v2.9 assemble system. The randomly copied genomes are randomly cut, sorted according to size, read out by a certain length, assembled the first pieces of the read sequences to create a contig, and generate a scaffolding process to create a longer genome sequence I have.
The genome annotation process consisted of gene prediction and annotation stages. Glimmer 3.0 was used for genome prediction, and RNAmmer and tRNAscan-SE were used for tRNA and rRNA analysis. The predicted genes were compared with NCBI-NR, Uniref90, COG, and KEGG databases using BLAST, and gene annotation was performed.
(2) Design of specific primer
Since the specific primer design for each species should be carried out using sequences that are not similar to other species, the region of similarity is analyzed through BLAST among all the bacteria in which the gene information is known and the sequence of the region is extracted. Primers were designed using the extracted sequences.
- Primer size (bp): 18-27, optimum 20
- Tm (占 폚): 57.0 to 63.0, optimum 60.0
- GC%: 20.0-80.0
- PCR product size (bp): 100-1000, optimum 200
The primer designed through the above procedure was designed in the area where there is no similarity with other bacteria. Kurtobactherium plukum passense pv. Curtobacterium flaccumfaciens pv. flaccumfaciens ), and BLAST was performed between the designed primer and the NCBI-NT database. After filtering the designed primer using the BLAST results between the designed primer and NCBI-NT database, the list of specific primers was finally obtained. The filtering procedure and the filtering criteria are as follows.
The criteria for filtering were divided into three stages. First, if the primer sequence and the sequence of other species are exactly the same as the result of BLAST, and if the sequence of the primer sequence and the sequence of the other species (primer sequence length) is 1 , And the degree to which the sequence of the other species matches the sequence of the primer sequence (the length of the primer sequence) as a result of BLAST is 2.
In the filtering process, the BLAST results were checked for each of the three filtering standards, and the primers found in other species were removed. After removal, the forward and reverse primers must be present in pairs. Therefore, only one of the forward primer or the reverse primer Was also removed.
2. Genomic DNA extraction of soil bacteria
Soil samples were collected from humus birds with high microbial diversity, ie, many kinds of microbes, and gDNA was extracted from 400 mg humus soil using Macherey-Nagel NucleoSpin soil kit (Germany). The gDNA concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo).
3. In the present invention, the conditions for performing the PCR using the design primer
Bioneer AccuPower® HotStart PCR Premix tubes (BIONEER, MyGenie96 Thermal black) were mixed with PCR mixtures as shown in Table 1 and PCR was performed under the conditions shown in Table 2.
Example 1. In the present invention, Kurtobacterium Plakumfathians pv . Plockum Passens ( Curtobacterium flaccumfaciens pv . flaccumfaciens ) Specific Primer design
In the present invention, Curtobacterium flaccumfaciens pv. flaccumfaciens ) ATCC51875. Since the specific primer design should be performed using sequences that are not similar to other species, it is necessary to analyze the regions having no similarity through BLAST among all the bacteria with known gene information, The sequence was extracted. Particularly, in the present invention, Curtobacterium flaccumfaciens pv. 14 pairs of primers were designed as shown in Table 3 for the genomic sequence extracted from flaccumfaciens ATCC51875.
Example 2. Designed in the present invention As a primer Done PCR Lt; RTI ID = 0.0 > Plakumfathians pv . Plockum Passens ( Curtobacterium flaccumfaciens pv . flaccumfaciens ) Specific diagnosis
In the present invention, Clostrobacillum plucum pasiens pv. Curtobacterium flaccumfaciens pv. flaccumfaciens ) using 14 pairs of primers specially designed for the first time to select the primers that specifically react with flaccumfaciens . PCR was performed using Plurkpathian genomic DNA as a template (Fig. 1). Kurtobactherium plukum passens pv. The PCR product size was compared with the PCR product size predicted by performing PCR using the Plurkpathian genomic DNA template as a template, and the PCR product size as a result of the actual experiment was found to be identical to that of the C. pneumoniae genome pv. As a result of the first selection, primer sets 1 to 5, 12 and 13 were properly amplified and found to match the predicted PCR product size, and the primer set 6 , 7, 8, 9 and 10 were not amplified, and primer sets 11 and 14 were found to be greatly out of the predicted size. This was excluded from the first selection process because primer sets 6, 7, 8, 9, 10, 11, and 14 were found to be unresponsive to the bacterium.
In addition, in the present invention, a total of seven primer sets selected from the first line are selected from the group consisting of Kurtobacterium plucum pasifens pv. In order to analyze whether or not it specifically reacted with Plurkpathians, genomic DNA complexes were extracted from humic bulb containing various microorganisms and the concentration was found to be 319.6 ng / μl. PCR was performed using the extracted soil genomic DNA complex as a template as a negative control. As a result, when PCR was carried out using the soil genomic DNA as a template, five amplified fragments were obtained from the strains of C. tuberculosis pv. (Fig. 2), primer sets 2, 3, 5, 12, and 13 were not amplified in the soil DNA complexes, and as shown in Fig. 1, Tobac Terium Plumbach Passions pv. It was confirmed that the specific reaction was only observed in Plasmapassien. Thus, through this second round of selection, A total of five primer sets that specifically reacted with Plasmapassien were selected. A total of two primer sets excluded from the secondary selection can be found to contain the sequence that the fragment amplified by the corresponding primer has in common in other microorganisms. Such a primer can be obtained from the corresponding Kurtobacterium plum Fassians pv. Since it can not be said to be a primer that specifically reacts only with Plasmapassien, it is excluded from the selection process.
Finally, Clostrobacterium plucum pasciens pv. Primer sets 2, 3, 5, 12 and 13 (FIG. 2) were selected (FIG. 3) while generating PCR products in Plurkum passens (FIG. 1) and at the same time not producing PCR products in soil DNA complexes.
<110> REPUBLIC OF KOREA (Ministry of Agriculture, Food and Rural Affairs, Animal and Plant Quarantine Agency) <120> Specific primer set for diagnosing Curtobacterium flaccumfaciens pv. flaccumfaciens, quarantine plant pathogen and uses thereof <130> PN15255 <160> 28 <170> KoPatentin <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 ggtctggaag agcgactcac 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 aagttctcga tgcaggtgct 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gtctggaaga gcgactcacc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 aagttctcga tgcaggtgct 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gagactcatg tgggtccgtt 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gtccgttcct gaacaacgat 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 agacgtagct gccgatcagt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 cgtcgagtcg aacttcatca 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 aacaggacct cgaacacgac 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 cgtcgagtcg aacttcatca 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ccttcgctga ctttccactc 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 ggaccaacta cactcggcat 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 gaagtgcgtc cgagagtttc 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 aaccggtcta tgcactacgg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 agatcatcgc aaggctcact 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 cgctcttact tgcctatgcc 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 gccgtccgat agtcgtacat 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 ctgtatgccg agcacttcaa 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 cacaaagtgt tcggggagtt 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 gtaccgtcga gcgagatagc 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 attcactacg ggtactggcg 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 catgctggct cattcgagta 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 gtcgacctct acacggtggt 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 gtcctggtac ttcccgttga 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 gtgacacgag acatggttgg 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 tcactcgtca agataccccc 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 attcactacg ggtactggcg 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 catgctggct cattcgagta 20
Claims (6)
Amplifying the target sequence by performing amplification reaction using the separated genomic DNA as a template and using an oligonucleotide primer set according to claim 1 or 2; And
And detecting the amplification product. ≪ RTI ID = 0.0 > pC. ≪ / RTI > Curtobacterium flaccumfaciens pv. RTI ID = 0.0 > flaccumfaciens ). < / RTI >
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20180055248A (en) * | 2016-11-16 | 2018-05-25 | 대한민국(농촌진흥청장) | Method of identifying Curtobacterium flaccumfaciens or pathotypes thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20180055248A (en) * | 2016-11-16 | 2018-05-25 | 대한민국(농촌진흥청장) | Method of identifying Curtobacterium flaccumfaciens or pathotypes thereof |
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