KR101937390B1 - Primer set for detecting Peronospora destructor Berk., Diagnostic kit, and Detecting method using the same - Google Patents

Abstract

The present invention relates to a primer set for onion bacteriophage detection, a detection kit comprising the same, and a detection method using the primer set. In particular, the present invention includes a primer consisting of the nucleotide sequences of SEQ ID NOS: 5 to 8, A primer set for onion nosocomial bacterium detection capable of effectively detecting onion fungus infection because it is capable of detecting a small amount of onion fungus bacteria within 2 hours as well as being able to detect in natural light by naked eye without any auxiliary equipment , A detection kit including the same, and a detection method using the same.

Description

TECHNICAL FIELD [0001] The present invention relates to a primer set for detecting onion nodule bacteria, a detection kit including the same, and a detection method using the same.

The present invention relates to a primer set for detecting Peronospora destructor Berk., A detection kit comprising the same, and a detection method using the same. More particularly, the present invention relates to a primer set, a detection kit and a method for effectively controlling onion fungi .

Allium crops such as onion, green onion, garlic, and so on are known to cause various diseases, but the disease is caused by airborne infectious disease, It is becoming a cause. In particular, the damage caused by onion nosocomial infestation is persistent in the onion juvenile complex. However, due to the nature of the onion nociceptor ( Peronospora destructor Berk.), Which is an Obligate parasite, The method has not been established yet. Peronospora destructor Berk.) Was found in the soil after harvesting, and it was found that the damage caused by the first infectious disease which occurred in the early spring of the following winter after wintering, and by the conidiospore derived from the first infectious source, (Choi, et al., 2011: Jung, 1964). However, it is difficult to distinguish between the two types of infection. Therefore, if the density of conidia sprouting from the primary strain is increased, secondary infection may become worse. Therefore, the pathogen causing the primary infection should be diagnosed early and appropriately controlled.

In general, the diagnosis method of onion fungus depends on the questionnaire survey which confirms whether or not spore formation on the surface of onion. Even if it is diagnosed by this method, it is difficult for effective control to be done since it is already after spreading on other onion around. Recently, onion nosocomial bacterium detection technology has been developed, but there is a drawback in that it requires expensive specialized equipment and takes a long time.

Therefore, it is urgently required to develop a technique capable of detecting the onion nosocomiasis rapidly and accurately.

Korean Patent Registration No. 10-1608576

It is an object of the present invention to provide a primer set, a detection kit and a method which can more easily, quickly and visually detect onion fungus causing onion fungal disease.

In addition, an object of the present invention is to provide a primer set, a detection kit and a method capable of specifically detecting only Onionpan pollinosis.

It is another object of the present invention to provide a primer set, a detection kit and a method which can visually confirm the presence or absence of onion nosocomycetes without any additional equipment.

It is also an object of the present invention to provide a primer set, a detection kit and a method capable of early diagnosis of onion nosocomial fungi in an agricultural field and effectively controlling nasopharyngeal diseases.

1. A primer set for detecting onion bacillus comprising the primers of SEQ ID NOs: 5 to 8 or the primers of SEQ ID NOs: 9 to 12.

2. A primer set for detecting onion bacteriology comprising the primers of SEQ ID NOS: 5 to 8,

3. A primer set for detecting onion fungi, comprising the primers of SEQ ID NOS: 5 to 8 and the primers of SEQ ID NOS: 9 to 12,

4. An onion bacterium detection kit comprising the primer set of any one of 1 to 3 above.

5. The detection kit according to 4 above, wherein said kit further comprises hydroxyl naphthol blue.

6. A method for detecting onion fungi comprising the step of amplifying genomic DNA of Onion fungus strain with a primer set of any one of 1 to 3 above.

7. The method according to 6 above, wherein the amplification is by Loop-mediated isothermal amplification.

8. The method of 6 above, further comprising the step of mixing the product obtained from the amplification with hydroxyl naphthol blue.

9. The method according to 6 above, further comprising the step of separating the product obtained from the amplification by electrophoresis on an agarose gel.

According to one embodiment of the present invention, the primer set, the detection kit and the method of the present invention can more easily and quickly detect the onion fungus causing onion fungus disease easily and visually.

Further, according to one embodiment of the present invention, the primer set, the detection kit and the method of the present invention can specifically detect onionparis bacteria only.

In addition, according to one embodiment of the present invention, the primer set, the detection kit and the method of the present invention can visually confirm the presence or absence of onion nosopharyngioma bacteria without any additional equipment.

In addition, according to one embodiment of the present invention, the primer set, the detection kit and the method of the present invention enable the early diagnosis of onion nosocomial fungi in an agricultural field, thereby effectively preventing nociceptive diseases.

Fig. 1 shows a healthy onion and an onion susceptible to nosocomial infections. The left side shows a case of onion and onion nosocomial infections, and the right side shows onion nosocomial infections.
Fig. 2 is a photomicrograph of a spore and sporangia of onion nodule bacteria ( Peronospora destructor Berk.).
3 shows a nucleotide sequence for preparing a primer set for LAMP for onion bacterium detection.
Figure 4 shows the results of LAMP PCR experiments with five designed primer sets.
5 shows a primer set for LAMP comprising the primers of SEQ ID NOS: 5 to 8 for onion bacteriology detection.
FIG. 6 shows the results of establishing optimal reaction conditions with a primer set for LAMP comprising the primers of SEQ ID NOS: 5 to 8.
Fig. 7 shows the detection limit of the primer set for LMAP.
Figure 8 shows the specificity of the LMAP primer set for onion fungi: M-Marker, 1: distilled water, 2: onion nociceptor spores, 3: onion black rot fungi, 4: black fungi, 5: distilled water , 6: onion nociceptor spore, 7: normal onion, 8: onion infected with nociceptive disease.

The present invention relates to a primer set for onion bacteriophage detection, a detection kit comprising the same, and a detection method using the primer set. In particular, the present invention includes a primer consisting of the nucleotide sequences of SEQ ID NOS: 5 to 8, A primer set for onion nosocomial bacterium detection capable of effectively detecting onion fungus infection because it is capable of detecting a small amount of onion fungus bacteria within 2 hours as well as being able to detect in natural light by naked eye without any auxiliary equipment , A detection kit including the same, and a detection method using the same.

Hereinafter, the present invention will be described in detail.

A "primer" refers to a single strand oligonucleotide sequence that is complementary to a nucleic acid strand, ie, a template strand, to be copied, and can act as a synthetic start point for cloning the template strand. The length and sequence of the primer should allow the template strand to begin replication. 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 specification, "primer of SEQ ID NO: x to y" is used in the same sense as "primer consisting of polynucleotide of SEQ ID NO: x to y". Here, x and y are each independently an integer of 1 to 20.

The term " complementary " when used in reference to a nucleic acid means a nucleic acid having a polarity in one direction containing a polynucleotide sequence in which the bases bind to bases of a polynucleotide of another nucleic acid of opposite polarity . For example, a nucleic acid having a sequence GCAT in the 5 'to 3' direction is complementary to a nucleic acid having a sequence CGTA in the 3 'to 5' direction. As used herein, the term complementary is used to include substantially complementary nucleic acids. Substantially complementary means that not all nucleotides are paired with the nucleotides of another nucleic acid, but nevertheless both nucleic acids have a sequence capable of forming a stable hybrid under certain conditions.

&Quot; Polynucleotide " means a polymer in which several or more deoxyribonucleotides or ribonucleotides are polymerized in the form of a single strand or a double strand, and unless otherwise specified, an analog of a natural polynucleotide Phosphorothioates, alkylphosphorothioates or peptide nucleic acids), intercalators, and the like. &Quot; Polynucleotide " has the same meaning as " base sequence ".

The present invention provides a primer set consisting of the nucleotide sequences of SEQ ID NOS: 5 to 8 or a primer set of the onion pollinator comprising the primers of SEQ ID NOS: 9 to 12.

Peronospora destructor Berk. Is an absolute active parasite producing onion fungus. It spreads onion through air and causes continuous serious loss of yield in onion fungus.

The primer set of the present invention can quickly detect and early diagnose onion nosocomiasis, thereby reducing the number of primary injuries, thereby preventing a serious decrease in yield.

The primers consisting of the nucleotide sequences of SEQ ID NOS: 5 to 8 and the nucleotide sequences of SEQ ID NOS: 9 to 12 are designed to amplify polynucleotides or complementary polynucleotides constituting genomic DNA of onion bacillus.

The primer set of the present invention is for loop-mediated isothermal amplification (LAMP), and when the isothermal amplification method is performed with the primer, the onion nodule bacterium can be detected in a short time without specialized equipments.

In the isothermal amplification method of the present invention, a DNA polymerase and a primer set of the present invention are used. First, an internal primer binds to DNA and is extended, followed by extension of an external primer outside thereof, . The loop structure is formed from the 5'-end of the single strand that is separated from the loop, which repeats the same process at the 3'-end and elongates the loop structure.

Unlike conventional polymerase chain reaction (PCR), the isothermal amplification method does not require temperature control to amplify the gene, so it can amplify the gene without specialized equipments and it is possible to amplify the gene at a high concentration in a short time.

According to one embodiment of the present invention, when the isothermal amplification method and the primer set of the present invention are used, detection of onion nosocomial bacterium can be performed within 2 hours. Rapid detection is possible, which can prevent damage to onion's serious quantity loss.

In order to utilize loop-mediated isothermal amplification (LAMP), four primers (F3, B3, FIP, and BIP) must function as one set. F3 (outer-forward primer) and FIP (inner-forward primer). B3 (outer-reverse primer) and BIP (inner-reverse primer) are primers that bind in the reverse direction in the 3 'direction. In addition, FIP and BIP are primers that contain the nucleotide sequence of F2 (or B2) and F1c (or B1c).

The primer set of the present invention is a primer set having all of the above-mentioned four primers, and the present example describes the role of the primer included in the primer set of the present invention (Tables 2 to 3).

The primer set of the present invention can be modified (for example, addition, deletion, substitution, etc.) within a range that does not affect the specific detection of onion bacterium.

The primers of SEQ ID NOS: 5 to 8 or the primers of SEQ ID NOS: 9 to 12 can stably amplify the onion fungus genomic DNA in various temperature ranges and stably show color upon addition of hydroxylnaphthol blue, The presence or absence of detection can be confirmed. In addition, it has high specificity for Onion fungus (specifically genomic DNA of onion fungus) and it has high detection accuracy and can amplify Onion fungus genome DNA by isothermal amplification method, Bacteria can be detected.

According to one embodiment of the present invention, a primer set for detecting onion bacillus can include the primers of SEQ ID NOS: 5 to 8.

The primer set containing the primers of SEQ ID NOS: 5 to 8 can amplify the genomic DNA of the onion fungus at various temperatures in the same manner as the primer set containing the primers of SEQ ID NOS: 9 to 12, Then, the detection result can be visually confirmed. In addition, it is possible to amplify the genomic DNA of onion nodule bacteria by the registered amplification method, and it is possible to detect onion nodule bacteria within a short time.

According to one embodiment of the present invention, a primer set for detecting onion bacillus can include the primers of SEQ ID Nos: 5 to 8 and the primers of SEQ ID Nos. 9 to 12.

The primers of SEQ ID NOS: 5 to 8 and the primers of SEQ ID NOS: 9 to 12 can detect onion bacillus, and when both of them are used, it is possible to perform the same effect as the duplication test of onion bacillus.

That is, when the amplification of the onion nosocomial bacterium by a primer set does not occur due to the damage of the primer or the like and the presence or absence of detection of the onion bacterium can not be confirmed, the presence or absence of the onion bacterium can be detected by performing amplification with another primer set have.

Therefore, the presence or absence of the onion bacterium can be more accurately detected when the primer set is included.

In another aspect, the present invention provides an onion bacterium infection detection kit comprising the primer set of the present invention.

The detection kit of the present invention comprises the primers of SEQ ID Nos: 5 to 8 or the primers of SEQ ID Nos. 9 to 12.

The primers of SEQ ID NOS: 5 to 8 or the primers of SEQ ID NOS: 9 to 12 can stably amplify the Onion fungus genomic DNA in various temperature ranges, and when the hydroxyl naphthol blue is added, the color development is stably visible and the onion nosocomial fungus Can be detected. In addition, it has high specificity for onion bacillus, which is very high in detection accuracy, and can amplify onion bacillus genome DNA by isothermal amplification method, so that onion bacillus can be detected within a short time compared to conventional PCR.

According to one embodiment of the present invention, the primers of SEQ ID NOS: 5 to 8 may be included.

According to one embodiment of the present invention, the primers of SEQ ID Nos: 5 to 8 and the primers of SEQ ID Nos. 9 to 12 may be included.

According to an embodiment of the present invention, hydroxyl naphthol blue may be further included. In this case, the presence or absence of onion nosocomial bacillus can be judged visually without accompanying equipment (fluorescence reader, electric synchronization, etc.).

The detection kit of the present invention is not limited to a specific form, but may be implemented in various forms. According to one embodiment, the detection kit may comprise a lyophilized primer contained in a container (e.g., a disposable PCR tube, etc.).

For example, in the case of containing two or more primer sets, a plurality of primer sets may be mixed and contained in a PCR tube, and each primer set may be individually contained in each PCR tube. For example, in the case of a detection kit comprising two primer sets, a disposable PCR tube in which the primer sets of SEQ ID NOS: 5 to 8 are contained in the form of lyophilized powder, and the primer sets of SEQ ID NOS: 9 to 12 are contained in the form of lyophilized powder A disposable PCR tube may be included in the detection kit. In the case of a detection kit including three or more primer sets, it may be provided in a similar manner. A primer set that can be used for detection of onion bacillus other than the primer set of the present invention may also be included.

The detection kit of the present invention may include a polymerase, a reaction buffer, a polymerase, a dNTP (dATP, dCTP, dGTP and dTTP), a joiner such as Mg ²⁺ and a buffer in addition to the above primer set. The polymerase and the buffer may be contained in separate containers, or the mixture of the polymerases may be contained in one container, the buffer may be contained in a separate container, or both the polymerase and the buffer may be mixed in a container have.

In the detection kit of the present invention, a reaction mixture containing the above primers, a polymerase and a buffer may be contained in a container (for example, a disposable PCR tube) in the form of a liquid. It can be stored frozen and thawed if necessary. In the case of such a detection kit, the convenience of the evaluation and the speed of the evaluation can be increased since a separate mixing process is not required except for adding the sample at the time of evaluation.

In another aspect, the present invention provides a method for detecting onion fungi comprising the step of amplifying the genomic DNA of Onion fungus strain with the primer set of the present invention.

In the detection method of the present invention, the DNA of the sample obtained from the site of the onion is amplified with the primer set of the present invention to confirm the presence or absence of the onion bacterium genomic DNA. The genomic DNA of the onion bacterium can be detected by the root, stem, Leaves, cells, etc. can be obtained from all the parts constituting the onion.

Methods for extracting DNA from onion nosopharyngeal genomic DNA or onion include phenol / chloroform extraction, SDS extraction (Tai et al., Plant Mol. Biol. Reporter, 8: 297-303, 1990) (Cetyl Trimethyl Ammonium Bromide; Murray et al., Nuc. Res., 4321-4325, 1980) or commercially available DNA extraction detection kits.

Amplification may be performed using a variety of techniques, such as loop-mediated isothermal amplification, PCR, real-time PCR, ligase chain reaction, nucleic acid sequence- A transcription-based amplification system, a strand displacement amplification or amplification by a replication enzyme, or any other suitable method for amplifying a nucleic acid molecule known in the art.

According to one embodiment of the present invention, amplification may be by isothermal amplification, but is not limited thereto. When amplification is performed by isothermal amplification method, amplification can be performed faster than amplification by conventional PCR, and since the amplification can be performed quickly, the diagnosis time can be greatly reduced.

The detection of the product obtained from the amplification can be performed by, but not limited to, detection with an indicator (e.g., hydroxylnaphthol blue), DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement, and phosphorescence measurement.

According to one embodiment of the present invention, the method of the present invention may further comprise mixing hydroxyl naphthol blue with the amplification product. When the amplification product and the hydroxyl naphthol blue are mixed, the blue color develops when the primer and the target sequence are both present, that is, when the onion bacterium is present. Thus, the presence of the onion bacterium can be judged visually.

The hydroxylnaphthol blue may be mixed before the amplification product is generated (e.g., before the start of amplification).

Further, according to one embodiment of the present invention, the method of the present invention may further comprise separating the amplification product by agarose gel electrophoresis. The presence or absence of onion bacterium can be judged by judging whether a ladder-shaped band and the presence of DNA having a size corresponding to the amplified DNA are detected by electrophoresis on an agarose gel.

According to one embodiment of the present invention, amplification products can be separated by acrylamide gel electrophoresis, and amplification products can be detected by confirming the presence of DNA having a size corresponding to the DNA amplified by the primer pair used have.

In the method of the present invention, the product obtained from the amplification (the same meaning as the amplification product) can be used as an enzyme, an enzyme substrate, a radioactive substance, a fluorescent dye, a chromophore, a chemiluminescent label, an electrochemiluminescent label, donor and a fluorescence acceptor, or a ligand having a binding partner, such as a fluorescence resonance energy transfer (FRET) pair.

Hereinafter, the present invention will be described in more detail with reference to the following examples. The following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

Example

1. Materials and Methods

Isolation and storage of onion nodule bacteria

The onion stalk of black onion nodule spore was collected from the onion seedlings of Muan County onion cultivation farm, which is an onion farming farm in Chonnam National University, located in Gwangju Metropolitan City. And stored at -80 ° C.

Spore morphology of onion nodule bacteria

In order to observe the morphology of the onion nodule spores, the spores were attached to the cellophane tape and observed on a microscope. Infected plants were cut and observed on a microscope to observe the onion nodule bacteria in onion tissue (Fig. 1 2).

Genomic DNA isolation of onion nodosa from diseased tissue

DNA extraction detection kit was used for genomic DNA extraction of isolated onion nodule bacteria. The extraction method was performed according to the manufacturer's instructions, and the extracted genomic DNA samples were quantified using a spectrophotometer.

Production of LAMP primer

For the primer set to be used in this experiment, a LAMP primer for detecting onion nodule bacteria was prepared using the LAMP Primer Explorer V4 website (http://primerexplorer.jp/e/index.html). All five were purchased from Marcrogen (Korea) and purified using MOPC. The Tm values relating to F1c, B1c, F2, B2, F3 and B3 constituting each primer and the dG value involved in the stability of the primer were considered. The Tm value proposed by the production program development company is F1c / B1c: 64 ° C to 66 ° C; F2 / B2 and F3 / B3: 59 to 61 DEG C; The 3'dG value of F2 / B2, the 5'dG value of F1c / B1c, and the 3'dG value of F3 / B3 were -4.0 Kcal / mol or less. The prepared primer sequences are shown in Tables 1 to 5 below. Table 1 shows the primers (SEQ ID NOs: 1 to 4), Table 2 shows the primers (SEQ ID NOs: 5 to 8), Table 3 shows the primers (SEQ ID NOs: 9 to 12) , Table 4 shows the primers (SEQ ID NOs: 13 to 6) and Table 5 shows the primers (SEQ ID NOs: 17 to 20).

SEQ ID NO: Primer set 1 Sequence 5 '-> 3' direction One F3 ACG TTC TTC CTG CTA TGG C (19mer) F (Forward) 2 B3 GCG GGT AAT CTT GCC TGA T (19mer) R (Reverse) 3 FIP CGC AAC AGC AAA GCC GAT TCA ATT TTG GTA CGA ACT GGT GAA CCG (45mer) F 4 BIP GAC AGT TTC GGC TGT CGA GTG TTT TTA GGT CCA ATT GAG ATG CCA C (46mer) R

SEQ ID NO: Primer set 2 Sequence 5 '-> 3' direction 5 F3 TCG ATT CGC GGT ATG ATT GG (20mer) F 6 B3 CGA AGT CGC ACG CAC AAT (18mer) R 7 FIP ACG GTT CAC CAG TTC GTA CCG
TTT TCG GCT AAA CAG GCG CTTA (43mer) F 8 BIP TTG AAT CGG CTT TGC TGT TGC GTT
TTC CAA ATG GGT CGA CAC TCG (45mer) R

SEQ ID NO: Primer set 3 Sequence 5 '-> 3' direction 9 F3 ACG TTC TTC CTG CTA TGG C (19mer) F 10 B3 GCG GGT AAT CTT GCC TGA T (19mer) R 11 FIP TTC GCA ACA GCA AAG CCG ATT CTT
TTA CGA ACT GGT GAA CCG TAG T (46mer) F 12 BIP GAC AGT TTC GGC TGT CGA GTG
TTT TTA GGT CCA ATT GAG ATG CCA C (46mer) R

SEQ ID NO: Primer set 4 Sequence 5 '-> 3' direction 13 F3 ACA GGC GCT TAT TGA ACG T (19mer) F 14 B3 GCG GGT AAT CTT GCC TGA T (19mer) R 15 FIP CGC AAC AGC AAA GCC GAT TCA ATT
TTG GCG GTA CGA ACT GGT GA (44mer) F 16 BIP GCG ACA GTT TCG GCT GTC GAG
TTT TGG TCC AAT TGA GAT GCC ACC (45mer) R

SEQ ID NO: Primer set 5 Sequence 5 '-> 3' direction 17 F3 AGT GTT CGA TTC GCG GTA TG (20mer) F 18 B3 CGA AGT CGC ACG CAC AAT (18mer) R 19 FIP GTT CAC CAG TTC GTA CCG CCA TTT TTG GCT TCG GCT AAA CAG GC (44mer) F 20 BIP TTG AAT CGG CTT TGC TGT TGC GTT TTC CAA
ATG GGT CGA CAC TCG (45mer) R

Establishment of optimum condition of LAMP method

In order to confirm the detection limit of LAMP for onion nosocomial infection, genomic DNA extracted from onion nad bacteria was diluted 10-fold in a stepwise manner to determine the optimal conditions of template, and the LAMP composition for onion nodule bacteria detection was constructed as follows. 10pM F3 primer, 40pM FIP (forward inner primer), BIP (Backward inner primer), 8U Bst DNA polymer large fragment (Enzynomics, Korea), 10x reaction buffer [200mM Tris, 100 mM KCl, 100 mM (NH ₄ ) ₂ SO ₄ , 40 mM MgSO ₄ , 0.02% Tween 20, HNB 1200 mM], 10 mM dNTP and distilled water. The optimal temperature for DNA synthesis was between 55 ° C and 65 ° C. After each LAMP reaction, the amplified product was electrophoresed on 3.0% agarose gel at 100 V for 30 min and then agarose gel was added to ethidium bromide Stained and observed on UV.

Identification of specificity of LAMP primer set for detection of onion bacterium

LAMP was performed using genomic DNA of other fungal pathogens such as Venturia nashicola and Screlotium cepivorum as a template in order to confirm that the LAMP primer set for onion nodule detection was specific to onion nodule bacteria.

Detection of onion nodule by HNB (Hydroxyl Naphthol Blue)

Detection of onion nodule bacillus In order to confirm the amplification products of LAMP by electrophoresis without color change, the tube containing HNB was checked for color change by visual inspection. After LAMP reaction including HNB, the solution was amplified when positive control, that is, both the primer and the template, confirmed the color to be blue.

Results

Develop primer set for LAMP and establish conditions

A primer set for LAMP for onion nodosis detection was designed for a total of 274 bp (SEQ ID NO: 21) including the ITS2 region and some regions of 28S rRNA (Fig. 3). Therefore, the Forward Inner Primer (FIP) consists of the complementary base sequence (on-sense) of two oncodia antisense sequences (F1c, F2), the TTTT spacer and the loop- The backward inner primer (BIP) is also designed to combine the complementary nucleotide sequence of the two oncocin sense sequences (B1c, B2) with the TTTT spacer, a loop-antisense ). The F3 and B3 primers were designed to be located outside the inner primer, respectively.

As a result of LAMP experiment using 5 kinds of primer set, 2 and 3 primer sets showed a ladder-shaped band characteristic of LAMP PCR and blue color with a ladder-shaped band. However, the remaining primers 1, 4 and 5 had no ladder-like bands as well as no color development, indicating that the LAMP did not proceed (FIG. 4).

In particular, the primer set 2 was stably amplified in a relatively wide range of temperatures than that of the primer set 3, so that the color development was stable. The primer sets 2 and 3 were selected as a primer set for onion bacterium detection (FIG. 5).

Therefore, in order to confirm the optimal reaction temperature and the LAMP reaction time including HNB in the LAMP primer set of the LAMP method for onion nodule bacillus detection using the No. 2 primer set, the range from 54.5 ° C to 65.5 ° C, Minute, and the color development was visually confirmed.

As a result, a ladder-shaped band characteristic of PCR was stably amplified in the range of 58.9 ° C to 65 ° C, and the reaction time including HNB was visualized by visual observation after 45 minutes with a ladder band as well as a ladder band ).

Identify the sensitivity and specificity of the LAMP primer set for detection of onion nodule bacteria

In order to confirm the detection limit of LAMP for onion nosocomial infection, genomic DNA extracted from onion nad bacteria was diluted stepwise from 7.25ng / ㎕ to 7.25fg / ㎕. As a result, it was confirmed that the reactivity was lowered gradually as the dilution ratio was increased, and it was confirmed that the amplification was performed from 7.25 ng / μl to 7.25 fg / μl. Therefore, it was judged that LAMP for onion nociceptor detection could detect up to 7.25 fg / ㎕ per reaction. In addition, as a result of the experiment in the same condition experiment as the conventional conventional PCR primer, it is found that the onion nodule detection method using the LAMP method has higher detection ability than the conventional PCR method (FIG. 7).

In order to confirm that the LAMP primer set for onion nosopharyngeal infection was specific to onion nodule bacteria, genomic DNA of other fungal pathogens such as Venturia nashicola and Sclerotium cepivorum was used as a template Respectively. As a result, the LAMP primer set for onion onion nosocomial bacterium detection did not react with the black rot fungi and onion black rot fungi, but only the onion bacillus was specifically reacted and developed. It was also confirmed by electrophoresis (Fig.

Finally, LAMP method was carried out from onion nodule spores and onion nodule, respectively. Then, the color of the tube containing HNB was visually examined for change in color development. As a result, the reaction was not observed in the healthy onion, but it was only visible on the onion which was exposed to onion nociceptor spores and onion necrosis, and was confirmed by the naked eye and confirmed by electrophoresis (FIG.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Primer set for detecting Peronospora destructor Berk., Diagnostic          kit, and Detecting method using the same <130> 17P03018 <160> 21 <170> KoPatentin 3.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer set 1 F3 <400> 1 acgttcttcc tgctatggc 19 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer set 1 B3 <400> 2 gcgggtaatc ttgcctgat 19 <210> 3 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Primer set 1 FIP <400> 3 cgcaacagca aagccgattc aattttggta cgaactggtg aaccg 45 <210> 4 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Primer set 1 BIP <400> 4 gacagtttcg gctgtcgagt gtttttaggt ccaattgaga tgccac 46 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer set 2 F3 <400> 5 tcgattcgcg gtatgattgg 20 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer set 2 B3 <400> 6 cgaagtcgca cgcacaat 18 <210> 7 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Primer set 2 FIP <400> 7 acggttcacc agttcgtacc gttttcggct aaacaggcgc tta 43 <210> 8 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Primer set 2 BIP <400> 8 ttgaatcggc tttgctgttg cgttttccaa atgggtcgac actcg 45 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer set 3 F3 <400> 9 acgttcttcc tgctatggc 19 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer set 3 B3 <400> 10 gcgggtaatc ttgcctgat 19 <210> 11 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Primer set 3 FIP <400> 11 ttcgcaacag caaagccgat tcttttacga actggtgaac cgtagt 46 <210> 12 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Primer set 3 BIP <400> 12 gacagtttcg gctgtcgagt gtttttaggt ccaattgaga tgccac 46 <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer set 4 F3 <400> 13 acaggcgctt attgaacgt 19 <210> 14 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer set 4 B3 <400> 14 gcgggtaatc ttgcctgat 19 <210> 15 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Primer set 4 FIP <400> 15 cgcaacagca aagccgattc aattttggcg gtacgaactg gtga 44 <210> 16 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Primer set 4 BIP <400> 16 gcgacagttt cggctgtcga gttttggtcc aattgagatg ccacc 45 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer set 5 F3 <400> 17 agtgttcgat tcgcggtatg 20 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer set 5 B3 <400> 18 cgaagtcgca cgcacaat 18 <210> 19 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Primer set 5 FIP <400> 19 gttcaccagt tcgtaccgcc atttttggct tcggctaaac aggc 44 <210> 20 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Primer set 5 BIP <400> 20 ttgaatcggc tttgctgttg cgttttccaa atgggtcgac actcg 45 <210> 21 <211> 274 <212> DNA <213> Artificial Sequence <220> <223> Primer set for LAMP comprising ITS2 region and a part of 26S rRNA          region <400> 21 tctgctatgg tatcaatgga ggagtgttcg attcgcggta tgattggctt cggctaaaca 60 ggcgcttatt gaacgttctt cctgctatgg cggtacgaac tggtgaaccg tagttcatgc 120 atgacttggc ttttgaatcg gctttgctgt tgcgaagtag agcgacagtt tcggctgtcg 180 agtgtcgacc catttgggaa attgtgcgtg cgacttcggt cgggtggcat ctcaattgga 240 cctgatatca ggcaagatta cccgctgaac ttaa 274

Claims (9)

A primer set of SEQ ID NOS: 5 to 8 or a primer set forth in SEQ ID NOS: 9 to 12;
The primer set according to claim 1, comprising the primers of SEQ ID NOS: 5 to 8.
[Claim 6] The primer set of claim 1, wherein the primers of SEQ ID NOS: 5 to 8 and the primers of SEQ ID NOS: 9 to 12 are used.
An onion bacterium infection detection kit comprising the primer set of any one of claims 1 to 3.
5. The detection kit according to claim 4, wherein the kit further comprises hydroxyl naphthol blue.
And amplifying the genomic DNA of onion nad bacterium with the primer set of any one of claims 1 to 3.
7. The method of claim 6, wherein the amplification is by Loop-mediated isothermal amplification.
7. The method of claim 6, further comprising mixing the product obtained from the amplification with hydroxyl naphthol blue.
7. The method of claim 6, further comprising the step of separating the product obtained from the amplification by electrophoresis on an agarose gel.

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