KR101947677B1 - LAMP primer set for detecting Erwinia amylovora and uses thereof - Google Patents

Abstract

The present invention relates to a LAMP primer set for detecting Erwinia amylobora and a method for detecting Erwinia amylobora using the primer set, wherein the primer set for LAMP of the present invention is a primer set for Erwinia amylobora And it is possible to detect Erwinia amylobora directly from the field without expensive equipments, so that it can be effectively used for prevention of fruit burn disease through a simple diagnosis process.

Description

[0001] The present invention relates to a LAMP primer set for detection of Erwinia amylolbora,

The present invention relates to a loop-mediated isothermal amplification (LAMP) primer set for detection of Erwinia amilobora and uses thereof.

Fire blight is a fatal disease caused by bacteria in plants such as pears and apples. It is known that Erwinia amylovora pathogen is the cause of this burning disease, and it is called a burn sickness in Korea because it is said that the fire, the flower of the apple tree, the branch and the fruit are blackened as if it was burned. Fruit-borne illnesses, which are already known to occur in 54 countries, including the United States and Canada, are the top-ranked insolvency recipients of the Korean Phytosanitary Act.

Fruit burns occur mainly in plants, but also in leaves, branches, stems, flowers and fruits. It begins to occur mainly in the stigma of a flower and spreads to a petal. It becomes a needle-like shape as if it is poached in water and then it is shrunk and turns into a dark brown color or it hangs on a tree with a blackish brown little fruit. In fruit, spots appear like hot water and gradually turn black. In the leaves, black lesions begin to appear at the point where they meet with petiole, and develop like falling down along the veins, eventually the leaves turn black and die. Symptoms of the disease develop into flower-like branches or develop into adjacent branches, resulting in black-brown lesions along the veins. When the symptoms are severe, bacterial effusions come out along the stem and become brown when they come into contact with air. As the disease progresses, the diseased leaf dries and shrinks, usually hanging on the branches, and the tip of the diseased twigs showing signs of bending. The bark of the diseased branch is turned dark brown, then contracted after being shrunk, and the bark of the branch showing the ulcer symptoms on the stem represents a water-borne lesion on the stalk, and darkened with dark color.

Fruit bottle bottles, which have been pointed out to be infected with pathogens in foreign agricultural products that have increased imports since the conclusion of a free trade agreement (FTA), are likely to be transmitted through lumps or pores in high temperature and high humidity conditions, It can be transmitted by insects such as birds, mites, bees, and butterflies in the flowering season (flowering period), and it can be spread by wind and rain. It can be transmitted by insect pests such as aphids, lepidoptera, lepidoptera, It is known to spread through wounds.

In the event of a fruit burning sickness, the occurrence area within a radius of 100m, the control area within a radius of 2km, the control area within a radius of 5km shall be controlled. In order to prevent the spread of the disease in the controlled area, periodic disease outbreaks should be monitored and radiation of pollen-borne insects including bees should be stopped.

Once you have a strong infectious disease, you do not have any remedy, so if you are diagnosed as having an infection, your fruit should be picked up and buried in the soil within a radius of 100m. In addition, once a sickle sickle can not grow fruit trees for the next five years. Until now, there is no clear preventive method, so there is no treatment in developed countries including the United States.

Fruit burns are important to prevent disease spreading through rapid diagnosis. However, in general, the diagnosis of fruit sickness is first confirmed by visual observation of the symptoms on the tree and then confirmed by close inspection in the laboratory. It is difficult to diagnose fruit sickness in a short time by carrying out instant inspection of diseased samples at the field of disease.

More recently, loop-mediated isothermal amplification (LAMP) has been developed over PCR-based diagnostic methods. LAMP reaction can be annealed and stretched at a constant temperature without changing the temperature, unlike the case where the amplification reaction is performed in three steps of denaturation, annealing and extension in the conventional PCR, so that the reaction time is 1 hour , And there is no DNA damage or damage due to temperature change. Since it is necessary to maintain only a certain temperature, it is possible to perform experiments using only a simple thermostat without requiring expensive equipment, so that the application is high in the field. In addition, since four specific primers are used to recognize six regions of the gene sequence to be amplified, the specificity of the target gene is very high.

In Korea, there are many studies on black shoot blight, which is very similar to that of fruit sickness, but there is a lack of studies on fruit sickness. Accordingly, the present inventors have developed a method for quickly and accurately diagnosing fruit sickness on the spot by using a LAMP primer set having excellent specificity to Erwinia amylobora, a pathogenic bacterium of fruit sickness.

Korean Patent No. 1572143 discloses a DNA extraction method of pine tree reed from a piece of wood, a LAMP primer set of pine tree creeper and a method for detecting pine tree reed from a piece of wood, and Korean Patent Publication No. 2017-0030190 Discloses a primer for detection of Clostridium perfringens using LAMP and a use thereof. However, the LAMP primer set for detection of Erwinia amilobora of the present invention and its use are not disclosed.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and the present inventors have developed a primer set for detecting Erwinia amylovora that causes a fruit-burning disease using LAMP reaction, See, E. pyrifoliae and Pseudomonas silvin pv. As a result of performing LAMP reaction on Pseudomonas syringae pv. Syringae , it was confirmed that the LAMP primer set of the present invention specifically and precisely detects only Erwinia amyloborans, thereby completing the present invention.

In order to solve the above-mentioned problems, the present invention provides oligonucleotide primers of SEQ ID NO: 1; An oligonucleotide primer of SEQ ID NO: 2; An oligonucleotide primer of SEQ ID NO: 3; And a set of LAMP (Loop-mediated isothermal amplification) primers for detection of Erwinia amylovora comprising an oligonucleotide primer of SEQ ID NO: 4.

In addition, the present invention provides a kit for detecting Erwinia amylobora comprising the LAMP primer set and a reagent for carrying out an amplification reaction.

The present invention also provides a method for detecting Erwinia amylobora using the LAMP primer set.

The LAMP primer set for detecting Erwinia amylobora according to the present invention and the Erwinia amylobora detection method using the same can amplify a gene under isothermal conditions, so that the reaction time is relatively short, and DNA damage and loss The amplification efficiency is excellent and reaction and detection can be performed in a thermostat in which a constant temperature is maintained, so that it is possible to diagnose the fruit sickness bottle quickly and accurately at the site where the disease occurs. In addition, the primer set of the present invention can be applied to a pseudomonas silvin pv. It is a primer set with excellent specificity and sensitivity that does not react to Pseudomonas syringae pv. Syringae and Erwinia pyrifoliae . It is a useful primer set which is useful for the diagnosis of fruit sickness disease through detection of Erwinia amylobora It will be possible.

Brief Description of the Drawings Fig. 1 is a diagram showing primer positions and base sequences of SEQ ID NOS: 1 to 5 for LAMP reaction.
Fig. 2 is a graph showing the results of measurement of the fluorescence intensity of Erwinia amylovora (abbreviation Ea), E. pyrifoliae (abbreviation Ep) and Pseudomonas syllin pv. The results of LAMP reaction on several strains of Pseudomonas syringae pv. Syringae (abbreviation Pss).
FIG. 3 is a graph showing the changes in the expression level of Erwinia amylobora (Ea), Erwinia pyrioperias (Ep) and Pseudomonas sylivia pv. The results of the LAMP reaction on various strains of Pseudomonas spp.
Fig. 4 is a graph showing the changes in the expression of Erwinia amilobora (Ea), Erwinia piri polyA (Ep) and Pseudomonas syllin pv. As a result of LAMP reaction on several strains of Pseudomonas spp . , (A), Pseudomonas sp . (C) and (D) are the results for Erwinia polypolyes.

In order to accomplish the objects of the present invention, the present invention provides oligonucleotide primers of SEQ ID NO: 1; An oligonucleotide primer of SEQ ID NO: 2; An oligonucleotide primer of SEQ ID NO: 3; And a set of LAMP (Loop-mediated isothermal amplification) primers for detection of Erwinia amylovora comprising an oligonucleotide primer of SEQ ID NO: 4.

In this specification, the term 'Loop-mediated isothermal amplification (LAMP)' is a method capable of performing an amplification reaction under an isothermal condition, unlike the conventional PCR (polymerase chain reaction) method. Four kinds of primers (F3, B3, FIP, BIP) are basically required for the LAMP reaction, and two types of primers are added to improve the reaction speed. The final six kinds of oligonucleotide primers having different base sequences It is necessary for the reaction.

The four basic primers are composed of two types of outer primers and two inner primers. The outer primers include a forward outer (F3) primer and a backward outer (B3) primer And serves to release DNA duplexes during the non-cyclic step of the reaction. The internal primers consist of forward internal primer (FIP) and backward inner primer (BIP), and are designed to bind to both forward and reverse base sequences so as to form a loop necessary for the ring-mediated isothermal amplification reaction. And consists of the corresponding nucleotides. The additional two primers are composed of forward loop (LF) primer and backward loop (LB) primer and attached to the base sequence that does not bind the inner primer to accelerate ring-mediated isothermal amplification reaction .

The set of LAMP primers according to one embodiment of the present invention may be a set of 5 kinds of oligonucleotide primers including oligonucleotide primers of SEQ ID NO: 5, but is not limited thereto.

The primer set of the present invention is preferably used for the LAMP reaction, and is a set composed of an internal primer, an external primer, and a ring primer. Specifically, the primer of SEQ ID NO: 1 is F3 which is a forward external primer, the primer of SEQ ID NO: 2 is B3 which is a reverse external primer, the primer of SEQ ID NO: 3 is FIP which is a forward internal primer and the primer of SEQ ID NO: Primer BIP, and the primer of SEQ ID NO: 5 is a forward link primer LF.

The oligonucleotide primer of the present invention may comprise 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more And more preferably at least 35, at least 37, at least 38, at least 39, at least 40, at least 41 contiguous nucleotides in SEQ ID NOS: 3 and 4, RTI ID = 0.0 > oligonucleotides < / RTI > For example, a primer (22 oligonucleotides) of SEQ ID NO: 1 may comprise at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 nucleotides in the sequence of SEQ ID NO: RTI ID = 0.0 > oligonucleotides < / RTI >

In the present invention, the term " primer " refers to a single strand of oligonucleotide sequence complementary to the nucleic acid strand to be duplicated and may serve as a starting point for the synthesis of the 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.

In the present invention, the oligonucleotide used as a primer may also include a nucleotide analogue such as phosphorothioate, alkylphosphorothioate, or peptide nucleic acid, or And may include an intercalating agent.

The present invention also provides a kit for detection of Erwinia amylobora comprising a set of LAMP primers for detecting Erwinia amylobora and a reagent for carrying out an amplification reaction.

In the kit of the present invention, the LAMP primer set is as described above, and the reagent for performing the amplification reaction may include a DNA polymerase, dNTPs, a 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 make the 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.

The present invention also relates to

Isolating the genomic DNA from the suspect sample of the fruit bottle;

Amplifying the target sequence by performing amplification reaction using the separated genomic DNA as a template and using the LAMP primer set; And

And detecting the product of the amplification step. The method of detecting Erwinia amilobora is also provided.

The method of the present invention includes isolating genomic DNA from a suspect sample of the fruit bottle. The suspect sample of the fungal infection is not limited thereto, but may be an apple or a pear flower, branch, fruit, and the like. Methods for isolating genomic DNA from the sample can be performed by a method known in the art. For example, a CTAB method may be used, or a Wizard prep kit (Promega) may be used. Using the separated genomic DNA as a template, an amplification reaction can be performed using a LAMP primer set according to an embodiment of the present invention to amplify the target sequence.

In the detection method according to an embodiment of the present invention, the step of amplifying the target sequence may be carried out at a temperature of 63 to 67 ° C, preferably at a temperature of 65 ° C, but is not limited thereto.

In the method of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. When the target sequence is amplified, Cy-5 or Cy-3 is labeled at the 5'-end of the primer and the amplification reaction is performed. When the target sequence is amplified, Can be labeled with a detectable fluorescent labeling substance. When the radioactive isotope is added to the amplification reaction solution, a radioactive isotope such as ³² P or ³⁵ S may be incorporated into the amplification product while the amplification product is synthesized, so that the amplification product can be labeled as radioactive .

In the method of the present invention, the detection of the amplification product can be performed through fluorescence measurement, DNA chip, gel electrophoresis, radioactive 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. In the fluorescence measurement method, when Cy-5 or Cy-3 is labeled at the 5'-end of the primer and the amplification reaction is performed, the target sequence is labeled with a fluorescent label capable of detecting the target. Can be measured. In addition, in the radioactive measurement method, a radioactive isotope such as ³² P or ³⁵ S is added to the amplification reaction solution to label the amplified product, and then a radioactive measurement instrument, for example, a Geiger counter or liquid The radioactivity can be measured using a liquid scintillation counter. In addition, the detection method may be a method including measuring the detection time to quantify Erwinia amiloborrhoea. The quantification may be performed to quantify the detection time of the fluorescent substance or the like according to the DNA concentration by comparing with the standard curve when the amplification reaction using the primer set according to the present invention is performed.

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. Preparation of strain

(YKB12324, YKB12325, YKB12326, YKB12327, YKB12328, KACC13945, KACC13946, KACC13947, KACC13948, YAC12318, YKB12318, YKB12318, YKB12318, YKB12221, YKB12321, YKB12322, YKB12323), Erwinia amilobolus strain KACC13949, KACC13951, KACC13952), Pseudomonas sp. Pv . (SHPW, WSPS), and the Center for Microbial Biotechnology (KACC) from the National Institute of Crop Science (RDA), the Department of Crop Protection (YKB), RDA and SHP0056, WSPS039, WSPS056, WSPS039, Were used.

2. LAMP Primer  making

In order to detect ERWINIA AMILORBORA, a fruit-borne pathogen pathogen, through the LAMP reaction, primer-Explorer V4 software (http://primerexplorer.jp/e/index.html), which is a primer design program for LAMP, (FIP: F1c + F2, BIP: B1c + B2) and a loop primer (LF) for accelerating the amplification reaction were prepared.

The specific base sequence of the Erwinia amyloborrhoea-specific LAMP primer (Ea-primer) set is shown in Table 1 below.

room Sime  One. For LAMP primer  Set of Erwinia Look at Amilo  Specific detection confirmation

1 μl each of the F3 and B3 primers at a concentration of 5 μM, 1 μl each of the FIB and BIP primers at a concentration of 40 μM, 1 μl of the 10 μM concentration of the LF primer, 15 μl of the isothermal master mix (OptiGene, UK), 4 μl of distilled water, Were mixed to prepare a final volume of 25 μl, followed by amplification reaction at 65 ° C for 30 minutes. In addition, the amplification reaction was confirmed in real time using Instrument Genie Ⅲ (OptiGene), a portable real-time fluorescent photometer capable of detecting the fluorescent dye contained in the isothermal master mix in real time.

As a result, E. amylovora , E. pyrifoliae , and Pseudomonas syllin pv . Pseudomonas syringae pv . syringae ), it was confirmed that the amplification reaction was performed only on Erwinia amylobora (Fig. 2). The strain used in the LAMP reaction of the present invention and the corresponding LAMP result (amplification peak time) are summarized in Table 2 below. It was confirmed from the above results that the Ea primer set of the present invention can specifically detect Erwinia amyloborrha, a pathogenic bacterium of the fruit flask.

+: DNA amplification reaction occurs

-: DNA amplification reaction does not occur

*: Amplification peak time

Example 2. Buhlmann - Primer  Specificity analysis

A primer set (Buhlmann-primer) set forth in a previous report (Andreas B. J Microbiol Methods 2013, 92 (3): 332-9) describing a LAMP primer set for Erwinia amylobora was synthesized and used in Example 1 The LAMP reaction was performed on the strains used and the specificity or sensitivity was analyzed.

As a result, the Buhlmann-primer set was found to contain not only Erwinia amylobolas but also Erwinia piri polytensis YKB 12325 and KACC 13951, Pseudomonas chinensis pv . It was confirmed that the amplification reaction also occurs in the serine-derived WSPS 039 (FIG. 3). The strain used in the LAMP reaction using the Buhlmann-primer set and the corresponding LAMP result (amplification peak time) are summarized in Table 3 below. Since the Buhlmann primer set showed a nonspecific response to strains other than Erwinia amylobora, it was confirmed that the EA primer set for LAMP of the present invention was superior to the Buhlman primer set for Erwinia amylobora .

Example 3. CN - Primer  Specificity analysis

A primer set (CN-primer) presented in a previous report (Chinese Patent Laid-Open No. 104388577) describing a LAMP primer set for Erwinia amylobora was synthesized and the E-primer set of the present invention and the strain Were subjected to LAMP reactions and compared for specificity or sensitivity.

As a result, in the E-primer set of the present invention, it was confirmed that the amplification reaction was performed only in the strain of Erwinia amiloborrhoea. In addition, in the CN-primer set, in addition to Erwinia amilobora, amplification reaction was performed in the sample of Erwinia polyphea strain (Fig. 4 and Table 4).

Thus, since the CN-primer set exhibited a nonspecific response to the Erwinia polyphea strain in addition to the target pathogen to be detected, the E-primer set for LAMP of the present invention is more effective than the CN- primer set for Erwinia amilobor And that the specificity of the drug is high

Example 4 . Through field diagnosis Erwinia Look at Amilo  Detection analysis

In order to confirm whether diagnosis of a fruit-burning disease can be made on the spot using the EA-primer set for LAMP of the present invention, whether or not the detection of Erwinia amylobora in the fruit orchard, Respectively.

As a result, Erwinia amylobora was detected in the twig canker showing symptoms of blooming of the orchid bloom in the orchard, and it was not detected in the healthy twig. In addition, it was confirmed that Erwinia amylobora was not detected in the healthy branches of the fruit birds without fruit bruising, and that Erwinia amylobora was not detected in the branches exhibiting symptoms of blooming of fruit trees susceptible to fruit blooms (Table 5 ).

Thus, it was confirmed that the Ea primer set of the present invention can accurately diagnose fruit sickness in the field by accurately detecting Erwinia amylobora.

*: Date of fruit tree diagnosis

**: fruit orchards occur in fruit orchards

***: No fruit bloom in the fruit orchard

****: Suspected fruit orchards occur in fruit orchards

1: Branches showing symptoms of warts

2: healthy branches

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> LAMP primer set for detecting Erwinia amylovora and uses thereof <130> PN17188 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> F3 primer <400> 1 ataataagag aatggcgcta tg 22 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> B3 primer <400> 2 tctacatctc cacctttgg 19 <210> 3 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> FIP primer <400> 3 taatgaagtt gaatctcagg catgagaaaa aatccattgt aaaaccttcg 50 <210> 4 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> BIP primer <400> 4 gatggattgc ttagtgagct cagccaatct ctccacaacc g 41 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> LF primer <400> 5 aaagttgttt tcatcccacg ga 22

Claims (6)

An oligonucleotide primer of SEQ ID NO: 1; An oligonucleotide primer of SEQ ID NO: 2; An oligonucleotide primer of SEQ ID NO: 3; And a LAMP (Loop-mediated isothermal amplification) primer set for detection of Erwinia amylovora comprising the oligonucleotide primer of SEQ ID NO: 4. The LAMP primer set according to claim 1, wherein the LAMP primer set for detecting Erwinia amylobora further comprises an oligonucleotide primer of SEQ ID NO: 5. A kit for detection of Erwinia amylobora comprising the LAMP primer set of claim 1 or 2 and a reagent for carrying out an amplification reaction. 4. The kit according to claim 3, wherein the reagent for carrying out the amplification reaction comprises a DNA polymerase, dNTPs and a buffer. Isolating the genomic DNA from the suspect sample of the fruit bottle;
Amplifying the target sequence by performing amplification reaction using the separated genomic DNA as a template and using the oligonucleotide primer set of claim 1 or 2; And
And detecting the product of said amplification step. 6. The method of claim 5, wherein the LAMP reaction is performed at 65 &lt; 0 &gt; C.

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