KR101413121B1 - Method for detecting Acidovorax citrulli - Google Patents

Abstract

The present invention relates to a method for detecting ex-deborosceliuli, a bacterial pathogen of watermelon, in leaf, stem, seed, etc. of watermelon, comprising the steps of: preparing primer sets AC158F and AC158R from a specific DNA fragment of YD repeat protein gene; Amplifying the DNA fragments of the exsidovirus sheetuli of the sample using the AC158F and AC158R primer sets; And identifying the amplification product.

Description

Method for detecting Acidovorax citrulli < RTI ID = 0.0 >

The present invention relates to a method for detecting Acidovorax citrulli , a bacterial pathogen of watermelon, in leaves, stems, seeds and the like of watermelons.

Bacterial fruit blotch rot (BFB) is a disease that is typical of fruit dark brown speckles. Fruit grows gradually and grows with a round concave spots about 3-5mm in diameter. It covers the entire fruit in the bay. The initial lesion becomes more concave and eventually punctures the skin. Then the inside of the fruit is corrupted, and the contents leak out. If the lesion is treated without perforating the pericardium, the lesion becomes dry. The leaf is formed with the small spots of the first acupuncture point and grows irregularly gradually, and the central part of the lesion is dissolved in the tissue to form the transparent epidermal spot pattern. In the seedlings, the inside of the cotyledon forms a needle-shaped lesion.

Watermelon fruit rot disease is transmitted to seeds and begins to develop in seedlings. It is well tolerated in high temperature and high humidity environment and there are no external symptoms but it is transmitted by infected seedlings. An infected plant does not die immediately after planting but shows typical symptoms immediately before the fruit grows and matures. In Korea, the details of the occurrence and extent of damage have not yet been investigated.

Pathogens causing watermelon fruit rot disease include Acidovorax citrulli ,, Pseudomonas pickettii, Pseudomonas pseudoalcaligenes are known.

In order to detect the fruit rot disease of the watermelon, the method of detecting the exsidovorax strain was carried out using the IMS-PCR method and the Acidovorax avenae subsp . A method for detecting a citrulli strain has been known. Sensitivity can be detected up to 10 CFU / ml and IMS (immunomagnetic separation) method is used. (RR Walcott and RD Gitaitis. 2000. Detection of Acidovorax avenae subsp. citrull i in Watermelon Seed Using Immunomagnetic Separation and the Polymerase Chain Reaction. Plant Disease / Vol. 84, No. 4, 470-474).

In order to overcome the non-specific detection problems of the currently-developed assay and molecular diagnostic methods, a super-sensitive real-time bio-PCR assay using nano-analysis technology was developed.

(R. R. Walcott and R. D. Gitaitis. 2000. Detection of Acidovorax avenae subsp. Citrulli in Watermelon Seed Using Immunomagnetic Separation and the Polymerase Chain Reaction. Plant Disease / Vol.84 No.4, 470-474.)

The present invention seeks to provide a precise diagnostic molecular labeling and assay method for the ex-observe lactase infected seeds and crops by improving existing non-specific assay methods.

The present invention relates to a method for preparing a primer set, comprising the steps of: preparing primer sets AC158F and AC158R from a specific DNA fragment of YD repeat protein gene; Amplifying the DNA fragments of the exsidovirus sheetuli of the sample using the AC158F and AC158R primer sets; And confirming the amplification product. ≪ Desc / Clms Page number 2 >

The present invention provides a composition for detecting ex vivo lactase comprising a nucleic acid sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2.

The present invention also provides an exdodobrose citrusy detection kit comprising the composition.

The present invention provides a method for detecting ex-deborosceliuli which is a germ pathogen of watermelon in leaves, stem and seeds of watermelon, and can be used for the disease test in the field of seed industry and quarantine, It can contribute to breed cultivation.

FIG. 1 is a graph showing the relationship between YD repeat protein This is the result of analysis of gene through blastn program.
FIG. 2 is a graph showing the relationship between YD repeat protein The result of electrophoresis of the PCR product amplified the gene is shown.
FIG. 3 shows real-time PCR results of watermelon leaves, stems, and seeds infected with ex-deboroc saccharide using a primer set according to the present invention.

The present invention relates to a method for preparing a primer set, comprising the steps of: preparing primer sets AC158F and AC158R from a specific DNA fragment of YD repeat protein gene; Amplifying the DNA fragment of the sample Acidovorax citrulli using the AC158F and AC158R primer sets; And confirming the amplification product. ≪ IMAGE >

The AC158F primer is a nucleic acid sequence represented by SEQ ID NO: 1 (AC158F primer: 5'-CTTG GTGC TCCA TGCT CGA-3 '19mer), AC158R primer is SEQ ID NO: 2 (AC158R primer: 5'- GGCT TGGT TGCG AATT CACT- 3 '20mer).

EXCHIBOLUX SHEET Specifically (GenBank Accession No. NC_008752, gi | 120608714: 2237201-2242513) for production of a primer set for detection of YD repeat protein Genes were identified by blastn and blastx blast programs and primers were constructed.

The present invention provides a method for detecting ex-shock lactase using any one of DNA extracts selected from leaf, stem and seed of watermelon as a sample.

Also, the DNA amplification method of the present invention provides a method for detecting ex vivo lactase using Bio-PCR.

PCR amplification can be carried out by conventional PCR, but Bio-PCR analysis can be performed using water immersed in water for 30 minutes to 40 minutes after isolating a portion of the sample without DNA extraction for the target sample.

In one embodiment of the present invention, the sensitivity of the assay for pathogen was about 2 CFU / ul (colony forming unit) for DNA analysis and about 7 CFU / ul (colony forming unit) for cell analysis, You can check up to the number of marie.

The present invention provides a composition for detecting ex vivo lactase comprising a nucleic acid sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2.

The present invention also provides an exdodobrose citrusy detection kit comprising the composition.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> Axidobolax  Strain DNA  extraction

Other microorganisms, including Acidovorax citrulli , which are used in the present invention, can be obtained from the Belgian Co-ordinated Collections of Micro-organisms (BCCM ^TM ), Korean Agricultural Culture Collection (KACC) and National Collection of Plant Pathogenic Bacteria (NCPPB), the sources of which are summarized in Table 1. Acidovorax strains were cultured in NA medium (Peptone: 0.5%, NaCl: 0.5%, Yeast extract: 0.2%, Lab-Lemco beef extract: 0.1%, Agar: 1.5%: 1L).

No. Scientific Name Source ^a Geographic origin One Acidovorax citrulli NCPPB 3679 ^T USA 2 Acidovorax citrulli NCPPB 3244 USA 3 Acidovorax citrulli NCPPB 4203 Brazil 4 Acidovorax citrulli NCPPB 4204 Brazil 5 Acidovorax citrulli LMG 2254 USA 6 Acidovorax citrulli LMG 5483 7 Acidovorax avenae NCPPB 1011 ^T USA 8 Acidovorax cattleyae NCPPB 961 ^T 9 Acidovorax conjugate NCPPB 3698 ^T Japan 10 Acidovorax valerianellae NCPPB 4283 ^T 11 Acidovorax delafieldii LMG 5943 ^T USA 12 Acidovorax facilis LMG 6598 13 Acidovorax temperance LMG 7169 ^T Sweden 14 Burkholderia glumae LMG 2196 ^T Japan 15 Burkholderia gladoli LMG 2216 ^T USA 16 Burkholderia platarii LMG 9035 ^T Japan 17 Burkholderia cepacia LMG 1222 ^T USA 18 Burkholderia cartophyophvlli LMG 2155 ^T USA 19 Burkholderia andropogonis LMG 2129 ^T USA 20 Burkholderia cenocepacia KACC 12021 ^T UK 21 Pseudomonas libanensis KACC 10809 22 Pseudomonas fuscovaginae KACC 10676 ^T Japan 23 Pseudomonas graminis LMG 21661 ^T Germany 24 Pseudomonas ludensis LMG 13517 ^T 25 Pseudomonas aeruginosa KACC 2386 ^T 26 Pectobacterium atrosepticum KACC 10477 ^T UK 27 Erwinia tracheiphila KACC 2707 ^T USA 28 Pantoea ananatis LMG 2676 USA 29 Pantoea agglomerans LMG 2565 Canada 30 Rhizobium RadioBacter KACC 10736 31 Xanthomonas campestris pv. vesicatoria LMG 921 USA 32 Xanthomonas campestris pv. oryzae KACC 10331 Republic of Korea 33 Xanthomonas campestris pv. campestris KACC 10913 ^T UK 34 Ralstonia solanasearum KACC 10814 ^T USA 35 Ralstonia pickettii LMG 5942 USA

The total DNA of the cultured strains was extracted by CTAB method, and the total DNA of other microorganisms was extracted using genomic-tips kit of Qiagen (Hilden, Germany).

< Example  2> primer  making

Acidovorax citrulli is a Specifically Acidovorax , registered at Genbank ( www.ncbi.nlm.nih.gov/ ) of the National Center for Biotechnology Information (NCBI) for the production of primer sets for detection, Citrulli genomic information (registration number: GenBank Accession No. NC_008752, gi | 120608714: 2237201-2242513) YD repeat protein The genes were identified by blastn and blastx programs and primers were prepared (Fig. 1).

AC158F primer and AC158R primer set amplifying a fragment of 158 bp in size were prepared. The sequence of the primer is as follows.

1) AC158F primer: 5'-CTTG GTGC TCCA TGCT CGA-3 '19mer

2) AC158R primer: 5'-GGCT TGGT TGCG AATT CACT-3 '20mer

The primers were prepared by confirming the specificity of the sequence information of each YD repeat protein gene registered in GenBank through blastn program.

PCR amplification reactions were performed to identify primers.

PCR amplification reaction was carried out using a ^{PTC-200 TM thermocycler (MJ research} , Watertown, mass), each reaction is Tris-HCl 20 mM, KCl 100 mM, 50% Glycerol, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20, 0.5% Nonidet P-40, 1 mM PMSF and 0.2 mM dNTP, 20 pM each of primers and Taq polymerase 2unit (SolGent, Deajeon, Repulic of Korea) . The amount of genomic DNA of some microorganisms added to the PCR mixture was about 50 ng. The reaction was denatured at 95 ° C for 5 minutes, repeated 35 times at 95 ° C for 60 seconds, 56 ° C for 30 seconds, and 72 ° C for 60 seconds, followed by reaction at 72 ° C for 10 minutes. After amplification reaction, 10 μl of the PCR product was electrophoresed on 1.5% agarose gel, stained with ethidium bromide, and confirmed on a UV transilluminator (FIG. 2).

It was confirmed that a 158 bp nucleic acid fragment was specifically amplified only in Acidovorax citrulli . Also as in Figure 3, Acidovorax citrulli genomic DNA, cell suspension, exsteboraxe citruli, and part of the stem and seeds.

Therefore, it can be seen that the AC158F / R primer can amplify only a specific DNA fragment of exsidovirus sheetuli, so that it can be used as a PCR primer for diagnosing the infection of exsidovorus sheetuli, and the specificity of the YD repeat protein gene It was confirmed that a primer set prepared from a DNA fragment can specifically detect ex-deborosquatriuli.

< Example  3> Infected Seeds and Plants Real - time PCR  Amplification reaction

In order to measure the real-time PCR diagnostic sensitivity from the seeds, the watermelon leaves (about 1 cm x 1 cm), part of stem and seeds were soaked in 1.0 ml of sterilized water for 30 minutes, Respectively. Real-time PCR amplification was performed using a CFX96 real-time PCR system (Bio-Rad Laboratories, Inc., USA). SYBR green PCR mixtures containing 5 pM of each primer Lt; / RTI &gt; The amount of genomic DNA of some microorganisms added to the PCR mixture was about 5 ng. The reaction was repeated for 45 seconds at 95 ° C for 5 seconds and at 56 ° C for 30 seconds. The reaction was continued for 10 seconds at 65 ° C from 0.5 ° C intervals, and then the reaction mixture was allowed to react at 95 ° C until a melting curve and a melting peak . As a result, as shown in Fig. 3, specific curves and peaks of each of the leaves, stems, and seeds were confirmed.

FIG. 3 is a photograph showing the results of real-time PCR using primers AC158F and AC158R of the present invention against exsidovirus sheetuli, and 1 lane was Acidovorax citrulli genomic DNA , lane 2 is Acidovorax &lt; RTI ID = 0.0 &gt; citrulli cell suspension, lanes 3 to 17 belong to Acidovorax citrulli and lanes 18 to 20 belong to Acidovorax citrulli ) and the 21 lane is a negative control containing only distilled water. From this, it can be seen that lanes 1-17 have amplification corresponding to each exsidovirus sheet gene, and amplification does not occur in lanes 18-21.

<110> The Foundation of Agri. Tech. Commercialization & Transfer <120> Method for detecting Acidovorax citrulli <130> P120276 <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 19 <212> DNA <213> AC158F <400> 1 cttggtgctc catgctcga 19 <210> 2 <211> 20 <212> DNA <213> AC158R <400> 2 ggcttggttg cgaattcact 20

Claims (7)

1) dipping one sample selected from the group consisting of leaves, stems and seeds of watermelon into sterilized water;
2) performing a real-time PCR by adding a primer set consisting of the nucleic acid sequence of SEQ ID NO: 1 and the nucleic acid sequence of SEQ ID NO: 2 to the sterile water containing the sample of the step 1); And
3) A method for diagnosing a bacterial fruit blotch rot (BFB), comprising the step of detecting the presence of an amplified product amplified by the primer set and detecting ex-boost PCR.
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