KR101913475B1 - Diagnosis method for Rhizoctonia solani - Google Patents

Abstract

The present invention relates to a probes and a primer for diagnosing Rhizoctonia solani, which is a pathogenic bacterium, and the method for diagnosing a bacterium of the plant using the probe and the primer set according to the present invention can detect DNA up to 1 fg, And it is possible to confirm the infection, so that the spread of the disease can be suppressed through early diagnosis.

Description

{Diagnosis method for Rhizoctonia solani}

The present invention relates to a method for diagnosing malignant germs.

Ginseng ( Panax ginseng ) belonging to the Araliaceae grows in the same place for 3 ~ 5 years, causing various pest damage.

Rhizoctonia solani ( Rhizoctonia solani AG2-1) is a typical soil infectious disease known as the main cause of ginseng extract. Melanosis occurs from mid-April to early May in Bongpo (more than two years old), in which the seedling is transplanted and cultivated. The stem of the soil surface is infected by pathogens and turns dark brown and falls down as it becomes constricted. It also shows that the disease spreads roundly around the affected plant.

The average incidence of drowning was 4 ~ 7% in 4 ~ 6 year olds, and the incidence of drowning in first 2 years was 18%. The damage area increases every year (Non-Patent Document 1).

Mongolian disease caused by the same pathogenic bacterium, Laijononia Solani, is also common in seedlings where seeds are sown and seedlings are cultivated nationwide.

The seedling exhibits symptoms of two aspects, which, after infestation by Raiwononia Solani in the middle of April, the seed does not originate from the beginning, or after the seed emerges, it becomes constricted and collapsed as the rice cake grows.

It is recommended that the seeds be treated with chemical agents before the seeds are planted for the control of the mitochondria. However, in the case of drought, there is no currently recommended control agent. Therefore, it is important to diagnose Laryngophysiophilus solani quickly and accurately in order to prevent the spread of the disease.

Accordingly, the present inventors have studied a method for efficiently detecting a trace amount of Raionia solani and confirmed that it is possible to diagnose Laionia solani by designing probes and primers specific to Laionia solani Thus completing the present invention.

Journal of Ginseng Research, Vol. 29, No. 4, pp. 185-191 (2005)

DISCLOSURE OF THE INVENTION The present inventors have focused on the problem that the conventional method of controlling glazes is not possible only after the infection spreads and thus a method of quickly detecting even a small amount of Laionia solani from the plant Respectively.

In order to solve the above-mentioned problems, the present invention provides a primer set for the diagnosis of Ulcerous germ of a plant comprising the primer of SEQ ID NO: 4 and the primer of SEQ ID NO:

In addition, the present invention provides a composition for detecting Ulcerous germ of a plant comprising the primer set.

In addition, the present invention provides a kit for detecting Ulcerous germ of a plant comprising the composition for detection.

In addition, the present invention provides a method for producing a mixture comprising: 1) preparing a mixture by mixing a template DNA derived from a plant or a cultivated soil sample with the primer set of claim 1; And 2) PCR-amplifying the resultant mixture.

The method for diagnosing a germplasm of a plant using the probe and the primer set according to the present invention can detect the DNA concentration up to 1 fg and can collect the plant during the germination period to confirm the infection so that the spread of the disease can be suppressed through early diagnosis There is an effect.

FIG. 1 shows the results of confirming the sensitivity of a laryngopharyngeal-specific probe and a primer set, which are the causative agents of the bruch.
Fig. 2 shows the standard curves of a Lyophyllon Solani-specific probe and a primer set, which are Ulcerous germs according to the amount of template DNA.
Fig. 3 shows the results of real time PCR using DNA isolated from Raiotonia solani and ginseng main pathogens, which are anchovy, as template DNA.
FIG. 4 shows the results of DNA isolation from a plant tissue infected with Staphylococcus aureus and amplification with a RH-R-TM probe and a RH-RF / RH-RR primer set.
5 shows the result of real-time PCR using a probe / primer set (set 1) other than the probe / primer set developed in the present invention.

First, terms used in the present invention will be described.

The term "primer" as used herein 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 primers below are designed to allow the synthesis of elongation products to begin. The specific length and sequence of the primer can be determined according to the primer usage conditions such as temperature and ionic strength, as well as the complexity of the desired DNA or RNA target.

The term "complementary ", as used herein, means that under certain annealing or hybridization conditions, the primer or probe is sufficiently complementary to selectively hybridize to the target nucleic acid sequence and is substantially complementary and / Quot; is meant to encompass all that is perfectly complementary, and is preferably entirely complementary.

The term "PCR" as used herein means a process of amplifying a target nucleic acid by repeating the steps of denaturation, annealing, and extension reaction using a target nucleic acid as a template and using a primer specific to the target nucleic acid.

As used herein, the term "specific" means binding specifically to germs without binding to other pathogens.

Hereinafter, the present invention will be described in detail.

The present invention provides a primer set for the diagnosis of Staphylococcus aureus comprising a primer of SEQ ID NO: 4 and a primer of SEQ ID NO: 5.

The phytophthora of the plant may be any one or more selected from the group consisting of Rhizoctonia cerealis , Rhizoctonia oryzae and Rhizoctonia solani , The phytopathogenic fungus of the plant is Rhizoctonia solani .

The primer used in the present invention includes a hybridization nucleotide sequence complementary to the target nucleic acid. Since the primer can be applied to the present invention as long as it has sufficient complementarity to hybridize with the template and can perform a function inherent to the primer, the primer does not need to have a sequence perfectly complementary to the template nucleotide sequence. In addition, the primer of the present invention can be modified (e.g., added, deleted, substituted) within a range that does not affect the specific detection of Laioniana solani. For example, the primers include oligonucleotides consisting of fragments of at least 14, at least 15, at least 16, at least 17 contiguous nucleotides in the sequence of SEQ ID NO: 4 or SEQ ID NO: 5, depending on the sequence length of each primer can do.

Oligonucleotides used as primers in the present invention may also include nucleotide analogs such as phosphorothioates, alkylphosphorothioates or peptide nucleic acids, intercalating agent.

The primer set may further comprise a probe of SEQ ID NO: 3.

Probes can be modified to the extent that the hybridization specificity is not impaired. For example, a reporter or a quantizer may be attached to the distal end of the probe.

Fam may be attached as a reporter to the 5 'end of the probe, and other fluorescent material showing fluorescence may be attached, but is not limited thereto. For example, the reporter may be selected from the group consisting of fluorescein, fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethylrhodamine, FITC , Oregon green, alexa Fluor, ROX, HEX, texas red, TET, TRITC, TAMRA, cyanine dyes and thiadicarbocyanine dyes. And the like.

At the 3 'end of the probe, Black Hole Quencher-1 (BHQ-1) may be attached as a quencher, and other materials that can be used as a quencher may be attached thereto, No. For example, the quencher may be a BHQ-2, BH3-3, carboxy-X-rhodamine, Dabcyl, TAMRA, Eclipse, DDQ, QSY, Blackberry Quencher, Qxl, Iowa Black Iowa black) FQ, Iowa black RQ, and IRDye QC-1.

In one embodiment of the present invention, the probe is labeled with Fam at the 5 'terminus and hybridized to the target sequence by the action of BHQ-1 present at the 3' end as a quencher, It does not emit fluorescence. However, in the extension step of the PCR, the probe hybridized to the template is degraded by the 5 '→ 3' exonuclease activity of the thermostable DNA polymerase such as Taq polymerase, and the fluorescent substance at the 5 ' Separated from the probe to emit fluorescence away from fluorescence inhibition by the quanta.

The primer set and probe may be for use in real time polymerase chain reaction (PCR), and the primer set may be for use in PCR.

The plant may be any one selected from the group consisting of ginseng, radish, green onion, watermelon, cabbage, cucumber, amber, eggplant, melon, pepper, onion and tomato, I never do that.

In one embodiment of the present invention, the primer set and the probe of the present invention, the respective DNAs isolated from the roots and the healthy roots of the ginseng cultivated in cultivation, and other materials necessary for real-time PCR are mixed and the mixture is subjected to real- As a result, it was confirmed that the reaction appeared only in the dead-rooted root (see FIG. 4).

In addition, the present invention provides a composition for detecting Ulcerous germ of a plant comprising the primer set. The composition for detection may further include, but is not limited to, the probe of SEQ ID NO: 3.

In addition, the present invention provides a kit for detecting Ulcerous germ of a plant comprising the composition for detection. The kit may further include, but is not limited to, the probe of SEQ ID NO: 3.

The kit of the present invention may further include other components in addition to the above components. For example, if the kit of the present invention is applied to a PCR amplification procedure, the kit of the present invention may optionally comprise a reagent, such as a buffer, a DNA polymerase (e.g., Thermus aquaticus (Taq), Thermus thermophilus Thermostable DNA polymerases obtained from Thermus filiformis , Thermis flavus , Thermococcus literalis or Pyrococcus furiosus (Pfu)), DNA polymerase joins and dNTPs. The kit of the present invention can be produced in a number of separate packages containing the reagent components described above.

The present invention also relates to a method for preparing a mixture comprising: 1) mixing a template DNA derived from a plant or plant cultivated soil sample with the primer set of claim 1 to prepare a mixture; And 2) PCR-amplifying the resultant mixture.

The probe of SEQ ID NO: 3 may be further added to the mixture of step 1), and real-time PCR may be used to detect the causal organism of the plant, but is not limited thereto.

PCR is the most well-known nucleic acid amplification method, and many variations and applications thereof have been developed. For example, conventional PCR procedures have been modified to enhance the specificity or sensitivity of PCR, such as touchdown PCR, hot start ) PCR, nested PCR and booster PCR. In addition, real-time PCR, differential display PCR, DD-PCR, rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction chain reaction, IPCR), vectorette PCR and thermal asymmetric interlaced PCR (TAIL-PCR) can be used for specific applications.

When carrying out the PCR, the reaction vessel may be provided with an excessive amount of components necessary for the reaction. The excess amount of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially restricted to the concentration of the component. Attachments such as Mg ²⁺ , dATP, dCTP, dGTP and dTTP can be provided to the reaction mixture to such an extent that the desired degree of amplification can be achieved. All enzymes used in the amplification reaction may be active under the same reaction conditions. Buffers can be added and prepared so that all enzymes are close to optimal reaction conditions.

The method may further include detecting the amplification product after the step 2). The detection of the amplification product can be performed by DNA chip, gel electrophoresis, radiation measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto.

In one embodiment of the present invention, the primers of SEQ ID NO: 4 and SEQ ID NO: 5 and the probe of SEQ ID NO: 3 are used, and the probes of Raiotonia solani, Sclerotinian varis, Cylindrocapone desatuntus, When the specificities of the primers and probes were confirmed by real-time PCR using DNA isolated from Cineera, Fusarium lysolony, Fusarium oxysporum, Altanaria panax, and Collectotrichum panacicola as the template, respectively, It does not show any specificity for pathogenic bacteria, but shows specificity only for Laryngotonia solani (see FIG. 3).

In another embodiment of the present invention, the RNA polymerase II region was amplified by each strain using the genomic DNA of Laionia solani as a template and SEQ ID NO: 1 and SEQ ID NO: 2, followed by sequencing. A probe and a primer set (hereinafter referred to as set 1) other than the probe and primer set developed in the present invention were prepared using Designer Program (Premier Biosoft). As a result of confirming the sensitivity of the prepared set 1, signals were also shown to other strains other than Laietonia solani, and compared with the primers of SEQ ID NO: 4 and SEQ ID NO: 5 and the probe set of SEQ ID NO: 3 of the present invention, (See Fig. 5).

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.

Extraction of DNA from published strains

Rhizoctonia solani and other strains used in the present invention were isolated strains isolated from KGC Ginseng Research Institute Korea Ginseng Corporation. The isolates were inoculated into potato dextrose agar (Difco) and cultured at 20 ℃. Genomic DNA isolation from the cultured strains was performed using MACHEREY-NAGEL's NucleoSpin  Plant II protocol, and the separated DNA was stored at -20 ° C. The Laietonia solani strains and other strains used in the present invention are shown in Table 1 below.

Pathogen Strain number Separation area Rhizoctonia solani AG2-1

KGC-RH9801 Suwon-si, Gyeonggi-do KGC-RH0203 Chungnam Budget City KGC-RH0702 Hwaseong, Gyeonggi-do Sclerotinia nivalis

KGC-S0608 Hongcheon-gun, Gangwon-do KGC-S0707 Gangwon-do Cheolwon-gun KGC-S1001 Yanggu-gun, Gangwon-do Cylindrocarpon destructans

KGC-CY9801 Suwon-si, Gyeonggi-do KGC-CY1405 Dangjin city, Chungnam KGC-CY1412 Jeonnam Jangsu-gun Botrytis cinerea

KGC-BC0407 Suwon-si, Gyeonggi-do KGC-BC0725 Yeoju-gun KGC-BC0736 Chungnam Budget City Fusarium solani KGC-F0503 Suwon-si, Gyeonggi-do Fusarium oxysporum KGC-F1410 Hongcheon-gun, Gangwon-do Alternaria panax KGC-AP0507 Suwon-si, Gyeonggi-do Colletotrichum panacicola KGC-C1040 Wonju city, Gangwon province

Rai Thonias Solani Specific Probe and primer design

The RNA polymerase II region was amplified by strain using the genomic DNA of each strain isolated in Example 1 as a template and primer pairs fRPB2-7F (SEQ ID NO: 1) and fRPB2-11aR (SEQ ID NO: 2).

(ATG GG (C / T) AA (A / G) CAA GC (C / T) ATG GG) fRPB2-7F

GC (A / G) TGG ATC TT (A / G) TC (A / G) TC (C / G) ACC) fRPB2-11aR

Sequence analysis of each strain was performed and TaqMan probe RH-R-TM and primer RH-RF / RH-RR, specific for Laryngotracanthonia solani, were used to design the strain, using Beacon Designer Program (Premier Biosoft) . Designed probes and primers are shown in Table 2 below.

Probe and primer name The base sequence (5 '- > 3') RH-R-TM (SEQ ID NO: 3) 5'-Fam-CTTGTTCCGTAGCATGTACTATCGCAG-BHQ-1-3 ' RH-R-F (SEQ ID NO: 4) 5'-CCGTGATTATGAACCAGAG-3 ' RH-R-R (SEQ ID NO: 5) 5'-TCCTCCAGATTCAAGACG-3 '

Sensitivity of the probe and primer prepared in Example 2

In order to examine the sensitivity of the probes and primer set of Example 2 to the Rhizoctonia solani, the amplified RNA polymerase II region sequence was inserted into the pBHA vector in the above Example 2 to obtain the plasmid Respectively. The copy number of the synthesized plasmid DNA was calculated as follows (using the program posted at http://cels.uri.edu/gsc/cndna.html, Andrew Staroscik, 2004).

Number of copies = (amount x 6.022 x 10 ²³ ) / (length x 1 x 10 ⁹ x 650)

Plasmid DNA was continuously diluted in 8 steps from 10 ng to 1 fg (4.39 × 10 ⁹ to 4.39 × 10 ² copies / μl) per 1 μl and used for PCR reaction. A real-time PCR reaction composition of the designed probe and primer set was prepared by adding sterilized water to 0.5 μl each of a real time master mix (Toyobo, Japan), 1 μl of a probe (2 pmol) and 5 μl of a primer . After the initial denaturation at 95 ° C for 1 min, the denaturation at 95 ° C for 15 sec and the binding at 60 ° C for 1 min were repeated 40 times. For real-time PCR analysis, the ABI 7500 Fast Real Time PCR System was used. The results of the analysis are shown in Table 3.

Copies / μl Ct + SD (n = 3) 4.39 × 10 ⁹ 11.84 + 0.08 4.39 × 10 ⁸ 15.17 ± 0.06 4.39 × 10 ⁷ 18.44 + 0.07 4.39 × 10 ⁶ 21.59 ± 0.06 4.39 × 10 ⁵ 24.97 + 0.04 4.39 × 10 ⁴ 28.23 + 0.02 4.39 × 10 ³ 31.14 + - 0.10 4.39 × 10 ² 34.28 + - 0.33

As a result, as shown in FIG. 1, when the probe and the primer set were used, reactions were observed up to a plasmid DNA concentration of ¹ fg (4.39 × 10 ² copies / μl), and each Ct (Threshold cycle) Respectively. The standard curve is shown in Fig. 2 (slope: -3.211, Y-inter: 15.181, R ² : 1, Efficiency: 104.865%).

From these results, it can be seen that the probe and primer set prepared in Example 2 can detect a specific amount of Laionia solani and a trace amount of Laionia solani.

Identification of specificity of probe RH-R-TM and primer RH-R-F / RH-R-R for ginseng main pathogen DNA

The specificity of the probe and the primer set of Example 2 for ginseng main pathogens was determined by the following method. Panax and Colletotrichum panacicola strains were also confirmed. Real-time PCR reaction conditions were confirmed in the same manner as in Example 3 except that the genomic DNA of each strain isolated in Example 1 was used.

As a result, the probes and primer sets developed in the present invention specifically reacted only with the strain Rhizoctonia solani (KGC-RH9801, KGC-RH0203, KGC-RH0702 in Table 1) It was confirmed that the ginseng main pathogens other than Solanii did not show any specificity (Fig. 3).

Diagnosis of Laietonia solani using direct plant tissue infected with ginseng melancholy

In order to diagnose the infectious disease of the plant by using the developed probes and primer sets, MACHEREY-NAGEL's NucleoSpin  DNA was isolated according to the plant II protocol. The real-time PCR reaction composition was prepared by adding sterilized water to a total of 20 쨉 l, to each 1 ㎕ of DNA, 0.5 각 each of primer (4 pmol) and 1 프로 of real time master mix (Toyobo), 1 프로 of probe (2 pmol) 95 ° C for 1 min. After the initial denaturation, 15 sec denaturation at 95 ° C and 1 min at 60 ° C were repeated 40 times.

As a result, as shown in Fig. 4, the reaction could be confirmed only in the ginseng melanocytic tissue. Therefore, real time PCR was performed using the probes and primer sets of the present invention using ginseng tissues suspected of being infected, and it was found that infection with Raiomania solani can be easily confirmed.

Comparison of probe / primer sets other than the probe / primer set developed in the present invention

A set other than the probe and primer set of Example 2 (hereinafter referred to as "set 1") was designed through the Beacon Designer Program to compare the degree of difference in the detection specificity with respect to the strain Laioniae Solani. Specifically, the RNA polymerase II region was amplified using primers of SEQ ID NO: 1 and SEQ ID NO: 2, using the genomic DNA of Laionia solani as a template and sequenced. A probe and a primer set other than the probe and primer set developed in the present invention were prepared using the Beacon Designer Program (Premier Biosoft). Table 4 shows the set 1 probe and primer set. Eight representative strains were used in the experiment and ginseng plant DNA was also used. The real-time PCR reaction composition and reaction conditions of the designed probe and primer sets (RH-1-TM, RH-1-F / R) were performed in the same manner as in Example 3 above.

Probe and primer name The base sequence (5 '- > 3') RH-R-1-TM (SEQ ID NO: 6) 5'-Fam-ACACCACACTCCGTATGAAACACGG-BHQ-1-3 ' RH-R-1-F (SEQ ID NO: 7) 5'-CAGCTACATGGATATCGAGAA-3 ' RH-R-1-R (SEQ ID NO: 8) 5'-CTTGCCGATGATAATGTCTTC-3 '

Pathogen Strain number Separation area Rhizoctonia solani AG2-1 KGC-RH9801 Suwon-si, Gyeonggi-do Sclerotinia nivalis KGC-S0608 Hongcheon-gun, Gangwon-do Cylindrocarpon destructans KGC-CY9801 Suwon-si, Gyeonggi-do Botrytis cinerea KGC-BC0407 Suwon-si, Gyeonggi-do Fusarium solani KGC-F0503 Suwon-si, Gyeonggi-do Fusarium oxysporum KGC-F1410 Hongcheon-gun, Gangwon-do Alternaria panax KGC-AP0507 Suwon-si, Gyeonggi-do Colletotrichum panacicola KGC-C1040 Wonju city, Gangwon province

As a result, in the case of the set 1 (RH-1-TM, RH-1-F / R), the signal was also observed in the Cylindrocone desaturation in addition to the Laryngotonia solanii, and the specificity was low (FIG.

From the above results, it can be seen that the probes and primer sets developed in the present invention have a higher specificity to the Laijononia solani strains as compared with the probes and primer sets containing other sequences, Able to know.

<110> KOREA GINSENG CORP. <120> Diagnosis method for Rhizoctonia solani <130> 2016-DPA-1429 <160> 8 <170> KoPatentin 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> fRPB2-7F <400> 1 atgggyaarc aagcyatggg 20 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> fRPB2-11aR <400> 2 gcrtggatct trtcrtcsac c 21 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> RH-R-TM <400> 3 cttgttccgt agcatgtact atcgcag 27 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> RH-R-F <400> 4 ccgtgattat gaaccagag 19 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> RH-R-R <400> 5 tcctccagat tcaagacg 18 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> RH-R-1-TM <400> 6 acaccacact ccgtatgaaa cacgg 25 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> RH-R-1-F <400> 7 cagctacatg gatatcgaga a 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> RH-R-1-R <400> 8 cttgccgatg ataatgtctt c 21

Claims (15)

A primer set of SEQ ID NO: 4 and a primer set forth in SEQ ID NO: 5,
Wherein said Staphylococcus aureus is Rhizoctonia solani AG2-1. delete delete The method according to claim 1,
Wherein the primer set further comprises the probe of SEQ ID NO: 3. The method of claim 4,
Wherein the primer set and the probe are for use in a real time polymerase chain reaction (PCR). The method according to claim 1,
Wherein the primer set is for use in PCR. The method according to claim 1,
Wherein the plant is any one selected from the group consisting of ginseng, radish, persimmon, fresh, watermelon, cabbage, cucumber, zucchini, eggplant, melon, pepper, onion and tomato. The method of claim 7,
Wherein said plant is ginseng. A composition for detecting a glazed germ of a plant comprising the primer set of claim 1,
Wherein the Staphylococcus aureus is Rhizoctonia solani AG2-1. The method of claim 9,
Wherein the detecting composition further comprises the probe of SEQ ID NO: 3. A kit for detecting Ulcerative germ of a plant comprising the composition for detection according to claim 9,
The kit according to claim 1, wherein the Staphylococcus aureus is Rhizoctonia solani AG2-1. The method of claim 11,
Wherein the kit further comprises the probe of SEQ ID NO: 3. 1) preparing a mixture by mixing template DNA derived from a plant or plant cultivated soil sample with the primer set of claim 1; And
2) PCR amplifying the resultant mixture, wherein the method comprises the steps of:
Wherein said Staphylococcus aureus is Rhizoctonia solani AG2-1. 14. The method of claim 13,
Wherein the probe of SEQ ID NO: 3 is further added to the mixture of step 1). 14. The method of claim 13,
Wherein said PCR is real-time PCR.

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