KR101834763B1 - Composition for diagnosing soybean wild fire and use thereof - Google Patents

Abstract

The present invention relates to: a primer set capable of diagnosing the infection of Pseudomonas syringae pv. tabaci, which is an infectious disease strain of soybean wild fire disease; a composition for diagnosing the soybean wild fire disease comprising the primer set; a kit for diagnosing the soybean wild fire disease comprising the composition; and a method for diagnosing the soybean wild fire disease using the composition or the kit. By using the composition for diagnosing the soybean wild fire disease of the present invention, it is possible to easily confirm the infection of the soybean wild fire disease, thereby being widely used for the effective control of the soybean wild fire disease.

Description

[0001] Composition for diagnosis of bean curd disease and use thereof [0002]

The present invention relates to a composition for diagnosing bean curd disease and a use thereof. More particularly, the present invention relates to a composition for diagnosing bean curd disease and a method for diagnosing the infection of Pseudomonas syringae pv. Tabaci, A primer set, a primer set, a kit for diagnosing a bean curd disease comprising the composition, and a method for diagnosing a bean curd disease using the composition or the kit.

Soybeans are important food crops in the world, and are important crops in terms of cropping systems for maintaining and promoting intellect. These soybeans are grown in large quantities. The biggest problem in the cultivation of such soybeans is the infection caused by bacteria or viruses.

These soybean and plant diseases can be classified into diseases caused by virus infection and diseases caused by bacterial infection. The diseases caused by virus infection are generally bean mosaic necrosis virus (BCMNV), alfalfa mosaic virus The viruses such as Alfalfa mosaic virus (AMV), soybean yellow mottle mosaic virus (SYMMV), soybean yellow common mosaic virus (SYCMV), peanut stunt virus (PSV) It is known to be caused by viruses such as soybean dwarf virus (SbDV) and soybean mosaic virus (SMV). Diseases caused by bacterial infections include Xanthomonas axonopodis pv. soybean flour blight caused by infection of glycines strain, Pseudomonas syringae pv. and tuberculosis caused by infections of tabaci strains.

In order to effectively control such soybean plant diseases, various methods have been developed. For example, Korean Patent No. 1250388 discloses a multi-diagnostic primer set comprising a primer capable of diagnosing AMV, SYMMV, SYCMV, PSV, SbDV and SMV and a combination thereof, and its use, 1357852 discloses a marker composition for diagnosing resistance to soybean flour disease.

On the other hand, among the above diseases, bean curd disease is a disease occurring in the leaves of legumes and plants, and lesions are widely and irregularly generated in the leaves. It is known that some of the symptoms of yellowish or brownish blotches are gradually changed to dark brown or black with the passage of time. Especially around the lesion, exhibits the characteristic of being accompanied by a band of no color. Such a bean sickle disease is known to be caused by the infection of Pseudomonas syringae pv. Tabaci strain. After the strain is infected through the damaged area of the soybean plant, a specific toxin (tabtoxin) , And the toxin exhibits an effect of inhibiting photosynthesis and photorespiration of the plant. As a result, it is known that it induces symptoms such as chlorophyll breakdown, sulphurization symptoms and plant necrosis of the plant. However, the method of diagnosing such a bean sickness disease has not yet been developed.

Under these circumstances, the present inventors have made extensive efforts to develop a method for effectively diagnosing bean curd disease, and as a result, they have found that a primer set capable of diagnosing the infection of a causative organism using a LAMP (Loop-mediated isothermal amplification) And completed the present invention.

One object of the present invention is to provide a method for producing Pseudomonas sp. Pseudomonas sp. syringae pv. The present invention provides a primer set capable of diagnosing infection of tabaci.

Another object of the present invention is to provide a composition for diagnosing bean curd disease comprising the primer set.

It is still another object of the present invention to provide a kit for diagnosing bean curd disease comprising the composition.

It is another object of the present invention to provide a method for diagnosing bean curd disease using the composition or kit.

The present inventors have carried out various studies in order to develop a method for more effectively diagnosing the onset of bean curd disease caused by the infection of Pseudomonas sylin G. vivata bacterium strain. Among them, LoP-mediated isothermal amplification (LAMP) I came to notice the method. The LAMP method refers to a gene amplification method in which a specific gene is amplified in a loop structure at isothermal temperature. The advantage of amplifying DNA in a single tube is that it can amplify a target gene simply and effectively in a field other than a laboratory .

Accordingly, the inventors of the present invention have developed a primer capable of amplifying the gene of the Pseudomonas silifen V. vulnificus strain by the LAMP method, and using the primer, By easily amplifying the gene of the strain, it was possible to confirm whether or not the strain was infected.

As described above, a primer set capable of amplifying the gene of the Pseudomonas syllin G. pvitravaki strain by the LAMP method has not been known at all and has been developed for the first time by the present inventor.

As one embodiment for achieving the above object, the present invention provides a method for producing Pseudomonas sp. Pseudomonas sp. syringae pv. The present invention provides a primer set comprising the nucleotide sequences of SEQ ID NOS: 1 to 4 capable of diagnosing infection of tabaci.

Terms of the present invention, "Pseudomonas ache dog blood V other Bakı (Pseudomonas syringae pv. tabaci) "means a strain that causes an outbreak of bean curd disease.

The term "diagnosis" of the present invention means that a gene of an individual suspected of infection is amplified with a primer set of the present invention to judge whether or not the infection is caused. In the present invention, the suspect of the infection may be soybean and plant itself. For example, if the soybean and plant itself is judged to be infected, the plant may be removed.

The primer set includes a specific sequence of the Pseudomonas silifen vivatavaki gene, and the specific sequence of these primers is as follows:

Pt-FIP: 5'-GATTCGCCGAGAGCAAGACCAGTTTTCTGCAGGCCGAAACTTCG-3 '(SEQ ID NO: 1)

Pt-BIP: 5'-GACCATACGTTCTTGCAGCGCTTTTTAGTCACTTCCGGGACAGG-3 '(SEQ ID NO: 2)

Pt-F3: 5'-GCCAATGCCCAAGTGAGG-3 '(SEQ ID NO: 3)

Pt-B3: 5'-GCGGATGCGATACCGATT-3 '(SEQ ID NO: 4)

The term "specific sequence" of the present invention means a series of nucleotide sequences capable of synthesizing a PCR product using as a template only when a gene to be diagnosed exists without synthesizing a PCR product when the gene is reacted with another gene.

In the present invention, the primer set can be used for the purpose of confirming the infection of Pseudomonas sylin P. vivatavaki infected with soybean plants through the LAMP method.

The term "LAMP (Loop-mediated isothermal amplification)" of the present invention is one of the PCR methods. Unlike the conventional PCR method in which temperature is varied at a high temperature, a gene amplification .

The LAMP method can be performed through a commercially available kit. As an example of the kit, Loopamp DNA Amplification kit (Lot No.: 5Y002 / EIKEN CHEMICAL CO., LTD.) Can be used.

The term "a pair of primers" of the present invention refers to a base sequence for amplifying a gene to be diagnosed by RT-PCR, wherein a forward primer and a reverse primer are set and reacted with one gene to be diagnosed PCR products are synthesized. At this time, if the forward and reverse primer sets are determined, the size of the PCR product synthesized therefrom can be predicted.

In another embodiment, the present invention provides a composition for diagnosing bean curd disease comprising the primer set.

The term " wild fire " of the present invention means a plant disease caused by an infection of Pseudomonas syl. The above-mentioned strain produces a specific toxin (tabtoxin) after being infected through the damaged area of the plant. The toxin has an effect of inhibiting photosynthesis and photorespiration of the plant. As a result, chlorophyll breakdown, And necrosis of the nervous system. It has been reported that the wild boar disease is caused in tobacco crops, but also in soya beans and crops.

In this way, the bean curd disease that occurs in soybean is caused by irregularly growing lesions on leaves, It is known that some of the symptoms of yellowish or brownish blotches are gradually changed to dark brown or black with the passage of time. Especially around the lesion, exhibits the characteristic of being accompanied by a band of no color.

In another embodiment, the present invention provides a kit for diagnosing bean curd disease comprising the primer set or the composition for diagnosing soybean curd disease.

The diagnostic kit of the present invention is characterized in that it specifically diagnoses whether Pseudomonas sylin P. vivatavaki is infected by using a primer set comprising the nucleotide sequences of SEQ ID NOS: 1 to 4.

The diagnostic kit of the present invention may further include a reverse transcription enzyme, a DNA polymerase and a buffer solution thereof and a PCR reaction buffer solution having the composition described above so as to facilitate the reverse transcription reaction and the PCR reaction, The components or diagnostic criteria necessary for performing the electrophoresis to confirm whether amplification of the PCR product can be further included in the kit of the present invention.

In another embodiment, the present invention provides a method of diagnosing bean curd disease using the composition or kit.

Specifically, the method for diagnosing bean curd disease comprises the steps of: (a) obtaining a sample derived from a soybean plant; (b) adding a primer set provided in the present invention to the sample and performing LAMP (Loop-mediated isothermal amplification) amplification; And (c) confirming the result of performing LAMP amplification.

In the above method, the sample obtained in step (a) is not particularly limited in the case of a sample obtained from soybean plants, but may be, for example, a leaf, a root, a stem, a flower, a juice. The method may further include a step of grinding each of the samples to more effectively identify the gene of the Pseudomonas sylin P. vivatavarki strain contained in the sample. However, in the case of the juice, it is not necessary to crush the sample. Therefore, when the juice is used as the sample, the above method can be more effectively carried out.

In the above method, step (b) may be carried out under appropriate temperature and time conditions. At this time, the temperature condition to be used is not particularly limited as long as the LAMP amplification can be performed, but it may be, for example, 50 to 70 캜, as another example, 56 to 67 캜, For example 63 < 0 > C. In addition, the time conditions to be used are not particularly limited as long as the LAMP amplification can be performed, but may be, for example, 40 to 80 minutes, as another example, 50 to 70 minutes, , And 60 minutes.

In the above method, step (c) may be performed using a conventional electrophoresis method or a coloring agent. A conventional electrophoresis method is a method of confirming whether an amplification product is generated by LAMP amplification through electrophoresis. When a coloring agent is used, the coloring agent to be used may be, for example, SYBR Green I as long as it can confirm the result of LAMP amplification. In order to confirm using the color developer, a method of adding the above-mentioned coloring agent to the LAMP amplification product under dark condition, and then adding a UV lamp can be confirmed. When the color development of green color was confirmed after applying the UV lamp, it can be interpreted that the sample contains the gene of Pseudomonas silifen V. vivata bacterium. As a result, .

The use of the composition for diagnosing bean curd disease of the present invention can be used to effectively control the infection of bean curd disease because the infection of soybean curd disease can be easily confirmed.

FIG. 1 is an electrophoresis image showing that the diagnostic method of bean curd disease using the LAMP amplification provided in the present invention is a specific method for the infectious agent causing diseases of other legume plants. In FIG. 1, The lane 2 represents the gene for soybean flour infection, the lane 3 represents the gene for the soybean bacterium, and the lane 4 represents the gene for the soybean bacterium.
FIG. 2 is an electrophoresis image showing the optimum reaction time for performing the diagnostic method of bean curd disease using the LAMP amplification provided in the present invention. In FIG. 2, lane 1 is 10 minutes, lane 2 is 20 minutes, 30 minutes, 40 minutes for lane 4, 50 minutes for lane 5, 60 minutes for lane 6, 70 minutes for lane 7 and 80 minutes for lane 8.
FIG. 3 is an electrophoresis image showing the optimum reaction temperature for performing the method for diagnosing bean curd disease using the LAMP amplification provided in the present invention, wherein the lane 1 is 53 ° C., the lane 2 is 54.2 ° C., 56.3 캜, 59.4 캜 for lane 4, 63.2 캜 for lane 5, 66.6 캜 for lane 6, 68.7 캜 for lane 7, and 70 캜 for lane 8.
FIG. 4 is a schematic diagram showing a method of performing a diagnosis method of bean curd disease using LAMP amplification provided in the present invention. FIG.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: beans Wildfire bottle  For diagnostic purposes Primer  Production and specificity test

Using the LAMP amplification provided in the present invention, a primer for amplifying the gene of Pseudomonas sylin G. vivata bacterium strain infected with soybean plants was prepared as follows.

Pt-FIP: 5'-GATTCGCCGAGAGCAAGACCAGTTTTCTGCAGGCCGAAACTTCG-3 '(SEQ ID NO: 1)

Pt-BIP: 5'-GACCATACGTTCTTGCAGCGCTTTTTAGTCACTTCCGGGACAGG-3 '(SEQ ID NO: 2)

Pt-F3: 5'-GCCAATGCCCAAGTGAGG-3 '(SEQ ID NO: 3)

Pt-B3: 5'-GCGGATGCGATACCGATT-3 '(SEQ ID NO: 4)

In order to confirm whether the prepared primer is specific to the gene of Pseudomonas sylin P. vivatavaki strain, which is an infectious disease of bean curd disease, the strain that is commonly infected with soybean and plant bean infectious disease strain, , Soybean bacterium spotted flower infectious strain, and soybean bacterium brown spot fungus infectious disease were subjected to LAMP amplification using the primers (FIG. 1). To perform LAMP amplification, Loopamp® DNA Amplification kit (Lot No.: 5Y002 / EIKEN CHEMICAL CO., LTD.) Was used, and 12.5 μl of Reaction Mix, 1 μl of Enzyme Mix, 1 μl of each of four primers, Using DW 2.5 [mu] l, LAMP amplification was performed.

FIG. 1 is an electrophoresis image showing that the diagnostic method of bean curd disease using the LAMP amplification provided in the present invention is a specific method for an infectious agent causing diseases of other legume plants.

As shown in Fig. 1, when the primer set provided in the present invention was used to amplify LAMP of each gene of bean-to-bean flour infection strain, soybean flour infection strain, soybean bacterial flour infection infection strain and soybean- It was confirmed that only the genes of wild - type infectious disease were amplified specifically.

Example  2: Soybean Wildfire bottle  Establish optimal conditions for LAMP amplification used in diagnostic methods

In order to amplify the gene of Pseudomonas sylin P. vivatavaki strain infected with soybean plants using LAMP amplification provided in the present invention, the optimum temperature and time conditions were established.

Example  2-1: Establishment of time condition

20, 30, 40, 50, 60, 70, or 80 minutes in which LAMP amplification using the primers of SEQ ID NOS: 1 to 4 was carried out using the gene of Pseudomonas sylin G. pvitravaki as a template, Was confirmed by electrophoresis (Fig. 2).

FIG. 2 is an electrophoresis image showing the optimum reaction time for performing the diagnostic method of bean curd disease using LAMP amplification provided in the present invention. FIG.

As shown in FIG. 2, LAMP amplification was performed after at least 40 minutes elapsed, and it was confirmed that no difference was observed even when LAMP amplification was performed for 60 minutes or more.

Thus, the optimum reaction time was found to be 60 minutes.

Example  2-2: Establishment of temperature condition

The LAMP amplification using the primers of SEQ ID NOS: 1 to 4 was carried out at 53, 54.2, 56.3, 59.4, 63.2, 66.6, 68.7, or 70 ° C with the gene of Pseudomonas silifen V. tabaci strain as a template, Was confirmed by electrophoresis (Fig. 3).

FIG. 3 is an electrophoresis image showing an optimal reaction temperature for performing a diagnostic method of bean curd disease using LAMP amplification provided in the present invention. FIG.

As shown in FIG. 3, LAMP amplification was performed at a temperature of at least 56.3 ° C., and LAMP amplification was not performed at a temperature of 68.7 ° C. or higher.

From the results of Fig. 3, it was deduced that the temperature at which most amplification is effectively performed is about 63 캜.

It was confirmed that a commercially available thermos bottle could be used as means for maintaining the temperature.

That is, the reaction solution at 70 캜 was placed in a commercially available thermos bottle, and the temperature change of the reaction solution was measured at intervals of 1 hour (Table 1).

Effect of maintaining temperature of reaction liquid using thermos bottle Elapsed time (hr) 0 One 2 3 3.5 4.5 Temperature (℃) 70 64 59 56 53 48

As shown in Table 1, it was confirmed that when the thermos bottle was used, the temperature required for LAMP amplification could be maintained for at least 3 hours.

Example  3: Field verification

First, use Loopamp® DNA Amplification kit (Lot No.: 5Y002 / EIKEN CHEMICAL CO., LTD.). 12.5 μl of Reaction Mix, 1 μl of Enzyme Mix, 1 μl of each of four primers, and 2.5 μl of DW were added to prepare a diagnostic tube.

Then, the plant leaf area suspected of bean curd infestation was repeatedly stuck to a sufficient amount of juice using a toothpick to obtain a black circle. The toothpick thoroughly immersed in the juice was immersed in the prepared diagnostic tube for about 5 minutes, the toothpick was removed, and the lid was closed. Then, the toothpick was allowed to react for 1 hour in a thermos bottle. After the reaction was completed, the tubes were taken out from the thermos bottle, and SYBR Green I nucleic acid gel stain was added to the tubes using 1 ml of the injection.

FIG. 4 is a schematic diagram showing a method of performing a diagnosis method of bean curd disease using LAMP amplification provided in the present invention. FIG.

As shown in FIG. 4, when the tube was put on a black cloth or a cloth and then the flashlight was illuminated, it was found to be fluorescent in color when infected with an outbreak disease, and not reacted with the uninfected virus.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Composition for diagnosing soybean wild fire and use thereof <130> KPA170155-KR <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gattcgccga gagcaagacc agttttctgc aggccgaaac ttcg 44 <210> 2 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gaccatacgt tcttgcagcg ctttttagtc acttccggga cagg 44 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gccaatgccc aagtgagg 18 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gcggatgcga taccgatt 18

Claims (12)

Pseudomonas ache more blood V Bakı other (Pseudomonas syringae pv. The present invention relates to a primer set comprising a nucleotide sequence of SEQ ID NOS: 1 to 4,
The method according to claim 1,
Wherein the primer set is used in a LAMP (Loop-mediated isothermal amplification) amplification method.
A composition for diagnosing bean curd disease comprising the primer set of claim 1.
A diagnostic kit for bean curd disease comprising the primer set of claim 1 or the composition for diagnosing soybean curd disease of claim 3.
(a) obtaining a sample derived from a soybean plant;
(b) adding the primer set of claim 1 or the composition for diagnosing bean bull's disease of claim 3 to the sample and performing LAMP (Loop-mediated isothermal amplification) amplification; And
(c) identifying the result of performing LAMP amplification.
6. The method of claim 5,
Wherein the sample of step (a) is leaf, roots, stem, flower or juice of soybean and plant.
6. The method of claim 5,
Wherein the LAMP amplification in step (b) is carried out at 50 to 70 &lt; 0 &gt; C.
6. The method of claim 5,
Wherein the LAMP amplification of step (b) is performed for 40 to 80 minutes.
6. The method of claim 5,
Wherein the step (c) is performed by electrophoresis or treatment of a coloring agent.
10. The method of claim 9,
Wherein the coloring agent is SYBR Green I.
10. The method of claim 9,
Wherein identification of said coloring agent is carried out by irradiating UV lamps under dark conditions.
12. The method of claim 11,
A method for diagnosing a bean curd disease wherein the bean curd disease is diagnosed in the soybean plants obtained from the sample when the green color is developed by irradiating the UV lamp under the dark condition.

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