KR101785094B1 - Composition for detecting puccinia horiana on chrysanthemum and detecting method using thereof - Google Patents

Abstract

The present invention relates to a composition for the detection of Chrysanthemum spp., And a method for detecting Chrysanthemum spp. When the primer set and / or probe of the present invention is used, it is possible to detect up to 20pg of genomic DNA of Chrysanthemum morifolium viridis, and up to 6pg of genomic DNA can be detected when real-time PCR is performed.

Description

TECHNICAL FIELD The present invention relates to a composition for detecting a white rust bacterium, and to a method for detecting a white rust bacterium using the same,

The present invention relates to a method of specifically diagnosing Chrysanthemonica white rust bacterium using primers and probes capable of specifically detecting chrysanthemum white rust bacteria.

Chrysanthemum is a familiar flower for the world, spread to China, Korea, Japan, Europe and America around the 1800s. The cultivation area of cut flowers and chrysanthemums in Korea has been steadily increasing since 1995. The cultivation area reached 805ha in 2006 and has been steadily decreasing since then. However, among the cut flowers, the sales volume is the second highest at 70.5 billion won , Which accounts for a large portion of domestic flower crops. In addition, Chrysanthemum exports are mainly exported to Japan. In 2011, exports were 1.9 times higher than the 2001 level, which was a major export item of USD 13,802 thousand. However, in recent years, chrysanthemum cultivation area, production volume and exports have been on the decline due to the inflow of low-priced chrysanthemums in China and the decrease in export volume due to the increase in domestic production costs. In addition, competitors such as Malaysia and China are increasing the volume of imports to Japan, so export farmers in Korea are having difficulties. In order to overcome these difficulties, it is necessary to develop high-quality cutflower varieties reflecting the preference of domestic consumers and exporting countries. Chrysanthemum quality standards vary slightly depending on the type of chrysanthemums, but one of the things that is commonly applied is whether they are insect pests. Chrysanthemum pests and diseases are not only deteriorating in domestic shipments but also impeding expansion of overseas exports. In Korea, chrysanthemum white rust is one of the most serious damages. Chrysanthemum White rust was first discovered worldwide in Japan in 1895 and then spread to China and South Africa. Since 1963, it has been found in many European countries, including the United Kingdom. Currently, white rust is occurring in all regions where chrysanthemums are cultivated. It is still occurring despite the strict regulation of movement between countries. In Korea, it is occurring in chrysanthemum plantation area nationwide every year until now. Especially, it adopts a greenhouse-based cultivation method which is difficult to control the environment, and the daily varieties are mainly occurred in spring and fall, and it occurs most especially in May. Chrysanthemum white rust, once it has developed, is infected with the entire vinyl house within a short time due to the nature of the pathogenic bacteria. Therefore, it is difficult to control the chemical pesticide, and the cost of the treatment pesticide is high. Current methods to minimize the cost are to control the humidity of the vinyl house to 80% or less and to monitor the occurrence of the disease continuously to remove the infectious agent from the plantation prematurely. However, this method requires much labor and time, and there are limitations in eradicating the disease. The fundamental solution is development of resistant varieties, but it takes a lot of time, so if you develop a technique that can test for the presence of chrysanthemum white rust before you identify the lesion with naked eyes in the short term, you can minimize the disease occurrence more efficiently and effectively There will be.

Real-time quantitative polymerase chain reaction (qPCR), one of the most effective PCR techniques, has been developed as a fast, simple and convenient method by real-time fluorescence analysis for accurate quantification of each gene , Because this method has specificity and sensitivity that can be quantified while amplifying the target DNA molecule. Particularly, in the TaqMan probe method, a fluorescence substance is synthesized at the 5 'end and a quencher dye is synthesized at the 3' end. If the activity reducing agent is nearby, energy is not emitted during the excited state of the fluorescent material , And when it is more than a certain distance, activity decrease due to the activity reducing agent is not generated, and energy is generated, and then the PCR amplification product can be measured by detecting.

The present inventors have established a technique for producing Bacillus pertussis in the laboratory according to the necessity of early diagnosis of the above-mentioned C. fungi, and have completed the present invention by selecting a gene that selects only fungi of the genus Chrysanthemum among the fungal pathogens developed in the fungi.

1. Pedley KF (2009) PCR-based assays for the detection of Puccinia horiana on chrysanthemums. Plant Dis 93: 1252-1258 2. Alaei H (2009) Molecular detection of Puccinia horiana in Chrysanthemum x morifolium through conventional and real-time PCR. J Microbiol Methods. 2009 Feb; 76 (2): 136-45.

It is an object of the present invention to provide a composition for detecting chrysanthemonous white rust bacteria comprising the primers of SEQ ID NO: 1 and SEQ ID NO: 2 and the probe of SEQ ID NO:

It is another object of the present invention to provide a method for detecting chrysanthemum white rust bacteria.

Yet another object of the present invention is to provide a kit for detecting chrysanthemum white rust bacteria.

The present invention provides a primer set for detecting Chrysanthemum White Rust Bacteria comprising the primer set of SEQ ID NO: 1 and SEQ ID NO: 2.

The present invention also provides a probe for detecting Chrysanthemum spp., A base sequence consisting of SEQ ID NO: 3.

The present invention also provides a composition for detecting mungbean white rust bacteria, comprising a primer set for detecting a mungbean white rust bacterium comprising a primer set of SEQ ID NO: 1 and SEQ ID NO: 2 and / or a probe comprising a nucleotide sequence consisting of SEQ ID NO: 3 do.

The primer set of the present invention, or the composition comprising the primer set and the probe, can specifically detect the gene of Chrysanthemum White Rust bacterium represented by SEQ ID NO: 4.

The term "primer" of the present invention refers to a single-stranded oligonucleotide sequence complementary to a nucleic acid strand to be copied, and may serve as a starting point for the 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. Oligonucleotides used as primers can also include nucleotide analogs such as phosphorothioates, alkylphosphorothioates or peptide nucleic acids or they can contain an intercalating agent ).

The term " probe "of the present invention is a single-stranded nucleic acid molecule comprising a sequence that is substantially complementary to the target nucleic acid sequence.

The primers used in the present invention include a hybridization nucleotide sequence complementary to the target nucleic acid. The term complementary means that under a given annealing or hybridization condition the primer or probe is sufficiently complementary to selectively hybridize to the target nucleic acid sequence and includes all substantially complementary and perfectly complementary , And preferably means completely complementary.

The present invention also relates to

1) extracting DNA from the sample;

2) performing PCR (polymerase chain reaction) using the extracted DNA of step 1) as a template and using the primer set of claim 1; And

3) detecting the amplified product as a result of amplification by PCR in the step 2).

The term amplification reaction according to the present invention means a reaction for amplifying a nucleic acid molecule. A variety of amplification reactions have been reported in the art, including polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159), reverse-transcription polymerase chain reaction (RT-PCR) (Sambrook et al., Molecular Cloning. (LCR) (see, for example, A Laboratory Manual , 3rd Ed. Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al (EP 329,822) 17,18), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA) 19 (WO 88/10315) (US Ser. No. 6,410, 276), self-sustained sequence replication (20) (WO 90/06995), selective amplification of target polynucleotide sequences (U.S. Patent No. 6,410,276), consensus sequence priming polymerase chain The consensus sequence primed polymerase chain reaction (CPPCR) (U.S. Patent No. 4,437,975), random (US Pat. Nos. 5,413,909 and 5,861, 245), nucleic acid sequence based amplification (NASBA) (US Pat. No. 5,130,238, 5,409,818, 5,554,517, and 6,063,603), strand displacement amplification (21,22), and loop mediated isothermal amplification. (LAMP) 23, but is not limited thereto. Other amplification methods that may be used are described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617 and U.S. Patent No. 09 / 854,317.

In the above method, the step 2) may be performed by further including the probe of the second aspect, but the present invention is not limited thereto.

In addition, the PCR in the step 2) may be performed according to a real-time PCR, and may preferably be performed using a Taqman probe method, but is not limited thereto.

The term " real-time PCR " of the present invention is a technique for monitoring and analyzing the increase of PCR amplification products in real time (Levak KJ, et al., PCR Methods Appl., 4 (6): 357-62 (1995)). The PCR reaction can be monitored by recording fluorescence emission in each cycle during the exponential phase, during which the increase in PCR product is proportional to the initial amount of target template. The higher the starting copy number of the nucleic acid target, the faster the fluorescence increase is observed and the lower the threshold value cycle. A pronounced increase in fluorescence above the baseline value measured between 3-15 cycles implies detection of accumulated PCR products. Compared to conventional PCR methods, real-time PCR has the following advantages: (a) conventional PCR is measured in a plateau, while real-time PCR provides data during the exponential growth phase have; (b) the increase in the reporter fluorescence signal is directly proportional to the number of amplicons generated; (c) The degraded probe provides permanent record amplification of the amplicon; (d) increase in detection range; (e) requires at least 1,000 times less nucleic acid than conventional PCR methods; (f) detection of amplified DNA without separation by electrophoresis is possible; (g) using a small amplicon size can achieve increased amplification efficiency; And (h) the risk of contamination is low.

When the amount of PCR amplified acid reaches a detectable amount by fluorescence, the amplification curve begins to occur, and the signal rises exponentially to reach the stagnation state. The larger the initial amount of DNA, the faster the amplification curve appears because the number of cycles with which the amount of amplified product reaches the detectable amount is smaller. Therefore, when real-time PCR is performed using a stepwise diluted standard sample, an amplification curve is obtained in which the initial DNA amounts are arranged in the order of the same intervals. Here, if a threshold is set at an appropriate point, the CT value at which the threshold and the amplification curve intersect is calculated.

In real-time PCR, PCR amplification products are detected through fluorescence. The detection methods are largely an interchelating method (SYBR Green I method) and a method using a fluorescent label probe (TaqMan probe method). Since the intercalating method detects double stranded DNA, it is not necessary to prepare a probe for each gene, so that a reaction system can be constructed at a low cost. The method using a fluorescent label probe is costly, while the detection specificity is high, so even the similar sequence can be detected.

TaqMan probes, on the other hand, typically use a probe with a fluorophore at the 5-terminus and a quencher at the 3-terminus (e.g., TAMRA or non-fluorescent quencher (NFQ)). The TaqMan probes are designed to anneal to internal parts of the PCR product. Preferably, the TaqMan probe can be designed as an internal sequence of the 16S rRNA gene fragment amplified by the first and second sequences of the Sequence Listing.

The TaqMan probe specifically hybridizes to the template DNA in the annealing step, but the fluorescence is inhibited by the quencher on the probe. During the extension reaction, the TaqMan probe hybridized to the template is degraded by the 5to 3 nuclease activity of the Taq DNA polymerase, so that the fluorescent dye is released from the probe and the inhibition by the quencher is released, indicating fluorescence. At this time, the 5-terminal of the TaqMan probe should be located downstream of the 3-terminal of the primer. That is, when the 3-terminus of the primer is extended by a template-dependent nucleic acid polymerase, the 5-terminal 3-nucleotide of the TaqMan probe is cleaved by the 5to 3 nuclease activity of the polymerase to generate a fluorescent signal of the reporter molecule .

Both the reporter molecule and the quencher molecule attached to the TaqMan probe are fluorescent materials. Fluorescent reporter molecules and quencher molecules that may be used in the present invention may be any of those known in the art and are not limited thereto.

The present invention also provides a kit for detecting chrysanthemum white rust bacteria comprising the primers of SEQ ID NO: 1 and SEQ ID NO: 2 and the probe of SEQ ID NO: 3.

The kit can specifically detect the gene of Chrysanthemum White Rust bacterium represented by SEQ ID NO: 4. The kit may be, but is not limited to, a PCR kit or a kit for real-time PCR.

In the specific examples of the present invention, PCR was carried out using the primer set of SEQ ID NO: 1 and SEQ ID NO: 2, and as a result, DNA of 184 bp in size was found for the Chrysanthemum morifolium collected in four regions of the Korean Chrysanthemum plantation area, , Pathogenic fungus such as sclerotinia, black rot, and wilt disease were not amplified. Therefore, it was confirmed that the composition of the present invention can specifically detect only the Chrysanthemum White Rust. Also, it was confirmed that 20pg of genomic DNA of Chrysanthemum spp. Was detected by the PCR method of the present invention, and the genomic DNA of Chrysanthemum spp. Was detected up to 6pg by Taqman real-time PCR method.

The chrysanthemum white rust bacteria specific primers and probes of the present invention can specifically detect only the Chrysanthemum white rust. Therefore, it is possible to diagnose early white rust caused by chrysanthemum.

FIG. 1 shows the result of amplifying the ITS region of rDNA against genomic DNA of five species of moxibustion pathogens generated from four species of Chrysanthemum White Rusty Bacterium collected from four regions of Korea;
lane 1. Chrysanthemum morifolium , lane 2. Puccinia horiana collected from Gumi, lane 3. Puccinia horiana collected from Changwon, lane 4. Puccinia horiana collected from Muan , lane 5. Puccinia horiana collected from Boryeong, lane 6. Phytophthora sp . (KACC 40914), lane 7. Sclerotinia sclerotiorum (KACC 42223), lane 8. Thanatephorus sp . (KACC 2225), lane 9. Septoria obesa (KACC 43858), lane 10. Verticillium dahliae (KACC 45724).
Figure 2 is a graph horriana and Puccinia graminis f. sp . tritici CRL. < / RTI >
FIG. 3 is a PCR result of the detection of Chrysanthemum White Rust Bacteria using a Chrysanthemum White Rust Bacteria-specific primer according to the present invention;
lane 1. Puccinia horiana collected from Gumi, lane 2. Puccinia horiana collected from Changwon, lane 3. Puccinia horiana collected from Muan , lane 4. Puccinia horiana collected from Boryeong, lane 5. Phytophthora sp . (KACC 40914), lane 6. Sclerotinia sclerotiorum (KACC 42223), lane 7. Thanatephorus sp . (KACC 2225), lane 8. Septoria obesa (KACC 43858), lane 9. Verticillium dahliae (KACC 45724)
Figure 4 compares the results of PCR electrophoresis using three primers specific for Chrysanthemum onset pathogen (lane 1. Puccinia horiana collected from Gumi, lane 2. Puccinia horiana collected from Changwon, lane 3. Puccinia horiana collected from Boryeong, lane 4. Sclerotinia sclerotiorum (KACC 42223), lane 5. Phythium ultimum (KACC 42224), lane 6. Thanatephorus sp . (KACC 2225), lane 7. Phythium helicoides (KACC 42227), lane 8. Septoria chrysanthemella (KACC 43113), lane 9. Septoria obesa (KACC 43858), lane 10. Verticillium dahliae (KACC 45724)
Fig. 5 compares PCR electrophoresis results using three kinds of matsutake white rust-bacillus specific primers (lane 1 to 8 genomic DNA amounts: 20 ng, 10 ng, 5 ng, 1 ng, 100 pg, 50 pg, 20 pg and 10 pg)
Figure 6 shows the results of PCR and real-time PCR analysis using Chrysanthemum morifolium-specific primers (Part B: CP value (crossing point values) for the number of copies of T-Ph182; Part C: Puccinia CP value for the amount of DNA of horiana genome).

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 Isolation of Chrysanthemum White Rust Bacteria and Genomic DNA for PCR Assays

Chrysanthemum chrysanthemum was isolated from Byeongju, Gumi facility. In addition, we used Chrysanthemum spp. Isolated from Bongwooju, which originated from Changwon, Boryeong, and Muan Chrysanthemum plantations. The five pathogenic microbial pathogens from other chrysanthemums were purchased from the Agricultural Microbiological Bank of Korea (RAC) (Table 1). Lee Byeong - ju of Chrysanthemum White Rust Bacteria used Chrysanthemum varieties susceptible to Chrysanthemum White Rust which was distributed from Gumi Facilities Corporation and Changwon, Boryeong, and Muan Chrysanthemum plantations. The host plants that were sold were propagated through cuttings and used for inoculation. The seeds were cut and scraped for 2 weeks at 23 ° C. The roots were transferred to a flowerpot after controlling the water content so that the cuttings did not dry out. The leaves were used for inoculation after 2 weeks of growth in the greenhouse. Two weeks after the disease was confirmed by naked eyes, it was used as an inoculum. The plants inoculated with the pathogens were grown on the inoculation phase to the proliferative growth phase and cultivated for 4 weeks at 23 ° C, 16 h light grown plant growth to propagate the pathogens.

For mycelial DNA isolation of Chrysanthemum White Rust Bacteria, the mycelium from the leaves of the leaves was used after 4 weeks of inoculation with a sharp razor blade. Genomic DNA isolation was performed according to the fungal pathogen genomic DNA isolation method of NucleoSpin plant kit (Macherey-Nagel products).

Example 2. Chrysanthemum white rust bacterium ITS PCR amplification

PCR amplification of Chrysanthemum White Rust ITS (Internal Transcribed Spacer) and Chrysanthemum ITS was performed using primers 5'-TGCATGAATTTTTGAAAGGT-3 '(PhrF; SEQ ID NO: 5), 5'-CAAAAATTATTTTGTGAGAGGG 3 '(PhrR; SEQ ID NO: 6) and Chrysanthemum ITS primer used 5'-GTCGATGCGCATTTAC TCGA-3' (CmF; SEQ ID NO: 7), 5'-TTCGGCCAACCACGCCATGA-3 '(CmR; SEQ ID NO: 8) Respectively.

10 ng of template DNA (Chrysanthemum White Rusty Bacterium) used for PCR amplification for ITS assay was used. The PCR reaction was carried out at 94 ° C for 2 minutes, 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1 minute Was repeated 30 times, and the final elongation reaction was carried out at 72 ° C for 10 minutes. The DNA amplified by the combination of the primers of SEQ ID NO: 5 and SEQ ID NO: 6 was cloned into the pTOP BluntV2 (Enzynomics products) vector and then the DNA cloned using primers M13 (SEQ ID NO: 9) and M13 (SEQ ID NO: 10) The nucleotide sequence was analyzed.

name 5'-3 ' SEQ ID NO: 9  M13 reverse priming site 5'-cag gaa aca gct atg a-3 ' SEQ ID NO: 10   M13 forward (-20) priming site 5'-gta aaa cga cgg cca gt-3 '

The PCR amplified fragments of ITS of Chrysanthemum morifolium ITS isolated from Lee, Byeongju, which was distributed from Gumi Facilities Corporation, were 340bp, and the nucleotide sequence was 100% identical to the published rDNA sequence (Pedley 2009) (Non-Patent Document 1) It was confirmed that the strain used was Chrysanthemum White Rust.

Genomic DNAs of four species of Chrysanthemum spp. And four chrysanthemum species collected from four regions of Korea (Gumi, Changwon, Boryung, Muan) using primers (SEQ ID NOS: 5 and 6) As a result of amplifying the ITS region, DNA of the same size was confirmed for five kinds of fungus other than fungus, which are different from each other in different regions (Fig. 1). Pedley (2009) (Non-Patent Document 1) is a case of confirmation the same primers used (SEQ ID NO: 5 and 6), chrysanthemum huinnok pathogens and chrysanthemum result chrysanthemum white PCR amplified DNA only rust amplifies the black rust (Puccinia chrysanthemi) by PCR Two species of pathogens were identified. But the chrysanthemum station germs isolated in Korea (Phytophthora sp.), Gyunhaekbyeong fungus (Sclerotinia sclerotiorum ), Septoria obesa , chrysanthemum leaf rot ( thanatephorus sp.), and chrysanthemum halves wilt (verticillium dahliae) 5 jong chrysanthemum fungus (Table 1) having been verified by PCR amplifying DNA of the same size using SEQ ID NO: 5 and SEQ ID NO: 6 primers at the chrysanthemum is endemic in chrysanthemum white rust and chrysanthemum It was not possible to distinguish fungal pathogens except white rust bacillus. Therefore, in the present invention, an attempt was made to develop a primer of a specific gene for Chrysanthemum morifus.

Example 3. Primer design and PCR amplification for specific detection of Chrysanthemum spp.

<3-1> Chrysanthemum white rust bacillus-specific primer design

In order to select the specific primers of Chrysanthemum morifolium, 10 kinds of genes were selected by using genomic information of Chrysanthemum White Rust Bacteria, and one kind of genes which amplify only the Chrysanthemum White Rustic bacterium was selected by carrying out PCR using more than 30 primer combinations ). The selected gene (SEQ ID NO: 4) is Puccinia graminis f. sp . The gene of Tritici CRL was 67.7% homologous to the DNA sequence and was unknown.

The primer of Chrysanthemum White Rust Bacteria was selected from 5'-GGGATTCAATGGCGAA-3 '(PhRTF; SEQ ID NO: 1) by selecting a region different from the wheat rust bacterium gene such as William Pearson's lalign program ), 5'-GAGGGTGGGTTTTGAGA-3 '(PhRTR; SEQ ID NO: 2) was designed (FIG. 5'-CGCGACTTGTCTCATCAAAC-3 '(PhTRT; SEQ ID NO: 3) was designed and used as a chrysanthemum white rust bacterium-specific probe.

<3-2> Chrysanthemum White Rust Bacteria-Specific PCR Amplification

10 ng of template DNA (chrysanthemum bacteria, fungi, rot fungi, chrysanthemum leaf rot, and wilt disease bacteria) used in PCR amplification for chrysanthemum white rust bacillus specific test was used and the detection limit concentration of chrysanthemum white rust bacterium DNA amount was The amount of template DNA was diluted 1/2 fold starting from 10 ng and genomic DNA of up to 0.25 ng was used. The PCR reaction was repeated 30 times at 94 ° C for 2 minutes, at 94 ° C for 30 seconds, at 65 ° C for 30 seconds, and at 72 ° C for 1 minute, and the final extension reaction was carried out at 72 ° C for 10 minutes.

PCR was carried out at an annealing temperature of 65 ° C with primers (SEQ ID NO: 1 and SEQ ID NO: 2) of the chrysanthemum white rope bacteria of the present invention, DNA of 184 bp was confirmed, but 184 bp of DNA was amplified in the pathogen of chrysanthemum chrysanthemum (Chrysanthemum spp., Klebsiella spp., Black rot, leaf rot, and wilt disease) (Fig. 3). Therefore, it was confirmed that PCR was carried out using the combination of primers of SEQ ID NO: 1 and SEQ ID NO: 2 to specifically detect the infection of Chrysanthemum white rust.

<Comparative Example>

3 '(Ph155F: SEQ ID NO: 11) and 5'-TTGCTACTTTCCTTGATGAA-3' (Ph157R: SEQ ID NO: 12) (Non-Patent Document 2) and 5'-CCCTTTTAAATATATCACCCAAACTAT-3 '(Ph260F : SEQ ID NO: 13) and 5'-GATTAATTTTGGGTTTTTAGAAGTCTT-3 '(Ph256R: SEQ ID NO: 14) (Non-Patent Document 2) A primer set was used for comparison with the primer pairs of SEQ ID NO: 1 and SEQ ID NO: 2 (Fig. 4).

The pathogen detection method was the same as in Example 3-2 except that the primer set used was a primer set of SEQ ID NOs: 11 and 12 and a primer set of SEQ ID NOs: 13 and 14 instead of SEQ ID NOs: 1 and 2 Respectively.

As a result, it was confirmed that the primer pair of SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention specifically amplified the 184-bp-sized nucleic acid fragment only in the case of the Chrysanthemum white rust bacterium. However, the PCR results of the primer pairs of SEQ ID NO: 11 and SEQ ID NO: 12 and the primer pairs of SEQ ID NO: 13 and SEQ ID NO: 14 in the non-patent document 2 showed amplified fragments of various sizes even in pathogens other than the chrysanthemum white rust bacterium, , And it was confirmed that the specificity was lower than that of the primer of the present invention (Fig. 4).

Example 4. Taqman real-time PCR and DNA detection limit analysis

A 5'-CGCGACTTGTCTCATCAAAC-3 '(PhTRT; SEQ ID NO: 3) was designed and used as a specific Taqman real-time PCR probe for Chrysanthemum White Rust Bacteria. Two fluorescence staining reagents, Texas Red, and matching black hole quencher (BHQ2), were used for the probe 5 'and the 3' probe. The recombinant DNA (named T-Ph184) inserted with 184 bp amplified by the PCR primer combination of SEQ ID NO: 1 and SEQ ID NO: 2 in the pTOP BluntV2 vector was diluted 10-fold to 10 to 102 (the number of copies of recombinant DNA) The relationship between the value and the number of gene copies was investigated and used as template DNA. To investigate the correlation between genomic DNA and CP value of chrysanthemum white rust bacterium, up to 6 pg of genomic DNA was used as template DNA. The real-time PCR reaction was carried out at 95 ° C for 4.5 minutes, followed by 15 cycles of reaction at 95 ° C for 15 seconds and reaction at 65 ° C for 15 seconds. The template DNA corresponding to each dilution was replicated three times in Light Cycler 480 II (Roche, Germany ) Instrument to amplify the DNA.

The ultimate goal of the present invention is to test for the infection of the plant before the pathogen appears in the plant tissue and before the pathogen appears. For this purpose, the detection limit of the genomic DNA of Chrysanthemum spp. At this time, the template DNA was separated from the leaves of Bacillus thuringiensis, and DNA isolated from pure bacillus spores confirmed by microscopy was used. PCR was carried out at an annealing temperature of 65 캜 by diluting 2-fold to 10 pg from 20 ng of genomic DNA. As a result, it was possible to detect up to 20 pg of genomic DNA of chrysanthemum white rust bacteria (Fig. 5).

In addition, Taqman real-time PCR was performed to improve the accuracy and precision of the Chrysanthemum White Rust Bacterial Assay (FIG. 6). The number of copies of T-Ph184 including the 184 bp PCR product amplified by the primer combination of SEQ ID NO: 1 and SEQ ID NO: 2 was measured to prepare a standard curve. The coefficient of determination (R2) of the standard curve showing the correlation between the number of copies of T-Ph184 and the CP value is 0.99 or more, indicating a very high level of reliability (Fig. 6B). Also, the graph showing the correlation between the CP value and the amount of genomic DNA of Chrysanthemum White Rust Bacteria also showed a very high level of reliability at R2> 0.99 (FIG. 6C). Therefore, it was confirmed that the Taqman real-time PCR method using the primer combinations of SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention can detect the genomic DNA of Chrysanthemum White Rox.

<Comparative Example>

3 '(Ph155F: SEQ ID NO: 11) and 5'-TTGCTACTTTCCTTGATGAA-3' (Ph157R: SEQ ID NO: 12) (Non-Patent Document 2) and 5'-CCCTTTTAAATATATCACCCAAACTAT-3 '(Ph260F (SEQ ID NO: 13) and 5'-GATTAATTTTGGGTTTTTAGAAGTCTT-3 '(Ph256R: SEQ ID NO: 14) (Non-patent document 2) were compared with the primer pairs of SEQ ID NO: 1 and SEQ ID NO: 2 prepared in the present invention (Fig. 5).

The pathogen detection method was carried out in the same manner as in <Example 4> except that the primer set used was a primer set of SEQ ID NOs: 11 and 12 and a primer set of SEQ ID NOs: 13 and 14 instead of SEQ ID NOs: 1 and 2 .

Pedley (2009) (Non-Patent Document 1) has a sensitivity of about 50 times higher than the detection limit concentration of 1 ng obtained by PCR amplification of rDNA with SEQ ID NO: 9 and SEQ ID NO: 10 using the primer set and / or probe of the present invention Respectively. Further, Alaei H (2009) (non-patent reference 2) has a detection limit concentration of 100 pg obtained by PCR amplification of rDNA with SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, The sensitivity was about 5 times higher (Figure 5).

<110> Republic of Korea <120> COMPOSITION FOR DETECTING PUCCINIA HORIANA ON CHRYSANTHEMUM AND          DETECTING METHOD USING THEREOF <130> P15R12D0377 <160> 14 <170> Kopatentin 2.0 <210> 1 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> PhRTF primer <400> 1 gggattcaat ggcgaa 16 <210> 2 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> PhRTR primer <400> 2 gagggtgggt tttgaga 17 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PhTRT probe <400> 3 cgcgacttgt ctcatcaaac 20 <210> 4 <211> 184 <212> DNA <213> Puccinia horiana <400> 4 gggattcaat ggcgaagagc gcgggatgaa cgtccacacc ctactaccca tcgacattct 60 ccaacacccc gcgacttgtc tcatcaaact tctcccagta aataacacac tcatgaagaa 120 aatgctcaca aggatcggtg aacaacttta tacatcgatc tccaccctct caaaacccac 180 cctc 184 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PhrF primer <400> 5 tgcatgaatt tttgaaaggt 20 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PhrR primer <400> 6 caaaaattat tttgtgagag gg 22 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CmF primer <400> 7 gtcgatgcgc atttactcga 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CmR primer <400> 8 ttcggccaac cacgccatga 20 <210> 9 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> M13R primer <400> 9 caggaaacag ctatga 16 <210> 10 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> M13F primer <400> 10 gtaaaacgac ggccagt 17 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ph155F primer <400> 11 cttggttgca tgaatttttg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ph157R primer <400> 12 ttgctacttt ccttgatgaa 20 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Ph260F primer <400> 13 cccttttaaa tatatcaccc aaactat 27 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Ph256R primer <400> 14 gattaatttt gggtttttag aagtctt 27

Claims (9)

Chrysanthemum comprising a primer set consisting of SEQ ID NO: 1 and SEQ ID NO: 2, white rust (Puccinia horiana ) detection primer set. delete A composition for detecting mungbean white rust bacteria, comprising the primer set of claim 1 and the probe of SEQ ID NO: 3. [Claim 4] The composition according to claim 3, wherein the composition is capable of specifically detecting the gene of the mungbean white rust bacterium of SEQ ID NO: 4. 1) extracting DNA from the sample;
2) performing PCR (polymerase chain reaction) using the extracted DNA of step 1) as a template and using the primer set of claim 1; And
3) detecting the amplified product as a result of PCR amplification in step 2). [6] The method according to claim 5, wherein the step 2) further comprises the probe of SEQ ID NO: 3. [6] The method according to claim 5, wherein the PCR in step 2) is performed according to real-time PCR. 8. The method according to claim 7, wherein the real-time PCR is performed by a Taqman probe method. A primer set consisting of the primers of SEQ ID NO: 1 and SEQ ID NO: 2, and a probe of SEQ ID NO: 3.

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