KR20170054925A - Quantitative analysis method of Cylindrocarpon destructans in the soil using RT-PCR and radicicol selective medium - Google Patents
Quantitative analysis method of Cylindrocarpon destructans in the soil using RT-PCR and radicicol selective medium Download PDFInfo
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Abstract
Description
The present invention relates to a method for quantitative analysis of ginseng root rot disease bacteria in soil using a radishcorn selective medium, and more particularly, to a method for quantitatively analyzing ginseng root rot disease bacteria in a soil using a radicicol selective medium, The present invention relates to a method for effectively quantifying ginseng root rot disease bacteria through real-time polymerase chain reaction after extracting the DNA of the root rot bacteria.
Ginseng ( Panax ginseng ) is a perennial semi-silky ginseng root, which belongs to the acanthaceae family. Its optimum growth temperature is around 20 ℃. It grows or grows mainly in the Far East region of the northern hemisphere. Korea's main region is Gyeonggi region, Poongsan and Geumsan provinces. The main pharmacological component of ginseng is saponin, its consumption has been on the rise due to its excellent efficacy as nutritional supplement, immunity enhancement, fatigue recovery, central nervous system regulation, antistress, antidiabetic and antitumor activity. Consumption is expected to steadily increase as excellence is revealed.
However, in spite of the excellent efficacy and consumption desire of ginseng, ginseng is a perennial ophthalmic plant, which is a semi-oily plant. Therefore, ginseng is the most important growth limiting factor and is a low temperature plant which inhibits growth at high temperature. It is a weak plant with high light intensity. In addition, ginseng was evaluated as a plant which is difficult to cultivate because of its weak tolerance and effective control method in addition to the above-mentioned growth inhibition factors.
In addition, the plant of ginseng consists of root and head under the ground, stem, leaf, flower (fruit) on the ground, and it is a perennial, so every spring the bud emerges from the root in the ground. do. Ginseng should be cultivated for 4 ~ 6 years in the same packaging for a long time, and after the ginseng is harvested, it is cultivated again for 1 ~ 2 years without cultivation of other crops, It is a plant with a high probability of occurrence of large and continuous obstacles.
The main pathogens of root rot caused by the damage in ginseng include Cylindrocarpon destructans , Fusariumsolani , Rhizoctonia solani, Botryis cinerea ), Phytophthora ( Phytophthora cactorum , Pythium sp ., and Alternaria panax have been reported. These pathogens are known to occur either alone or in combination.
According to recent research results from the Agricultural Research Institute of Korea, the causes of ginseng rooting for 4 years or longer are 35% for root rot, 32% for gray mold, and 15% for spotted fungus, In particular, C. destructans causes a direct disease on the roots while wintering in the soil, so it is difficult to control after the planting of ginseng, and the damage caused by the disease is very short in 4 years (21.8%) and 6 years (50% It is estimated to be the biggest enemy in the production of ginseng such as 30 ~ 80% in small 4 year old muscle.
The initial symptom of ginseng root rot disease is reddish leaf edge, leaf is inward, and symptoms show symptoms. Root showing such symptom forms reddish brown or black brown lesion at the end or middle part and discoloration of hairy root is reddish brown Become corrupt. Also, ginseng root rot disease is the most serious problem in Korean ginseng cultivation farms, such as from seedling ginseng to 6 years old, occurring in all the years and becoming more and more generations to high birth rate.
Root rot fungi, a major causal organism of Korean ginseng root rot disease, is a Cylindrocone desatta tans, which has been isolated from typical malignant lesion tissue of pathogenic fungi, and its pathogenicity has been confirmed. Since then, studies on physiological characteristics and culture of fungi have been actively conducted have. Korean Patent Laid-open Publication No. 10-2013-0098792 (published on Sep. 05, 2013) discloses a method for identifying a species and a genus specific primer capable of diagnosing four soil pathogenic fungi causing disease in ginseng . However, other pathogens were detected in addition to the ginseng root rot fungi, so that the density of the ginseng root rot fungus in soil could not be detected effectively because it was not a primer specific for ginseng root rot.
The technique of testing the density of pathogens in the soil is a useful method for the development and prediction of soil diseases caused by plant pathogens. In some countries, studies on soil diseases of major crops are progressing considerably. Is necessary.
As one of the methods for quantifying bacteria in a specimen. Although the phytosecination method can be used, it has a disadvantage in that it is difficult to accurately discriminate the inside of the strain. In addition, the above method is generally performed on bacteria existing in plants and the like, and is not suitable for quantifying bacteria present in the soil. In particular, there is little research on methods for quantifying pathogenic microorganisms in soil with high accuracy.
Accordingly, the present inventors have completed the present invention by developing a radishcor selection medium which selectively proliferates ginseng roots rot fungi only and a method of quantifying a trace amount of ginseng root rot fungi in soil.
Accordingly, an object of the present invention is to provide a method for quantifying ginseng root rot disease bacteria in soil.
Another object of the present invention is to provide a kit for quantitative analysis of ginseng root rot disease bacteria in soil.
In order to achieve the above object, the present invention provides a method for producing a soil sample, comprising the steps of: (a) separating DNA from a soil sample; (b) performing a real-time PCR using a mixture solution containing the DNA obtained in the step (a) and a primer for identification of a ginseng root rot disease virus; And (c) analyzing the results of the real-time PCR reaction performed in the step (b) to quantify the ginseng root rot disease bacteria in the soil sample.
According to another preferred embodiment of the present invention, the soil sample may be a soil for growing ginseng.
According to another preferred embodiment of the present invention, the step (a) of separating the DNA may further comprise the step of culturing the soil sample in the radishcor selection medium.
According to another preferred embodiment of the present invention, the soil sample may be cultured in a radish call selective medium for 36 to 60 hours, more preferably 42 To < RTI ID = 0.0 > 54 < / RTI >
According to another preferred embodiment of the present invention, the selective medium may be a commercially available radishcorn or a rice culture extract of ginseng roots rot fungi.
According to another preferred embodiment of the present invention, the selective medium may contain radicicol at a concentration of 30 to 70 mg / L.
According to another preferred embodiment of the present invention, the primer for identification of the ginseng root rot disease vaccine may be any two or more selected from the group consisting of SEQ ID NOS: 1 to 18, A primer set consisting of a primer set, SEQ ID NOS: 3 and 4, a primer set consisting of SEQ ID NOS: 5 and 6, and a primer set consisting of SEQ ID NOS: 7 and 8 .
According to another preferred embodiment of the present invention, the step (c) of analyzing the results of the polymerase chain reaction comprises the steps of: preparing a calibration curve with a standard sample of known concentration; And calculating the number of gene copies detected from the generated calibration curve.
The present invention also relates to a primer set for identifying ginseng root rot disease bacteria in soil; Radishcorn selection medium; And a kit for quantitative analysis of ginseng root rot disease bacteria in soil containing a reagent for polymerase chain reaction.
According to another preferred embodiment of the present invention, the reagent for the polymerase chain reaction comprises at least one selected from the group consisting of a dNTP mixture (dATP, dCTP, dGTP, dTTP), a DNA polymerase and a buffer solution .
According to another preferred embodiment of the present invention, the primer for identifying ginseng root rot disease vaccine may be any two or more selected from the group consisting of SEQ ID NOS: 1 to 18, preferably a primer consisting of SEQ ID NOS: 1 and 2 A primer set consisting of SEQ ID NOS: 3 and 4, a primer set consisting of SEQ ID NOS: 5 and 6, and a primer set consisting of SEQ ID NOS: 7 and 8.
Hereinafter, terms of the present invention will be described.
"Primer" of the present invention refers to a single-stranded oligonucleotide sequence complementary to a nucleic acid strand to be duplicated and can serve as a starting point for the synthesis of a primer extension product. The length and sequence of the primer should allow the synthesis of the extension product to begin. The preferred length of the primer is 5 to 50 nucleotides. The specific length and sequence 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.
The term " real-time PCR " of the present invention is a method for simultaneously amplifying a target DNA molecule and measuring the amount thereof. For one or more specific sequences in a DNA sample, a real- It is a method that can detect and measure quantities. Weighing can count absolute number of copies or relative amount.
&Quot; Radicicol "of the present invention means a compound represented by the following formula (1).
[Chemical Formula 1]
The " calibration curve " of the present invention refers to a curve for the relationship between the known concentration and the reaction of the device of the substance to be analyzed. The calibration curve is drawn up to measure the response of the analyzer at several concentration levels of the standard solution, assuming that the analyte, standard solution of the compound to be analyzed, and workplace sample are reacted to the analyzer in the same manner.
Accordingly, the present invention provides a quantitative analysis method of the ginseng root rot disease bacteria in the soil using the radishcor selection medium, and a quantitative analysis kit comprising the radishcorn selective medium, the primer set, and the reagent for the polymerase chain reaction. The present invention is effective for detecting ginseng root rot caused by a trace amount of ginseng in soil, which can be quantified with high sensitivity.
1 is a schematic diagram of the entire process of the present invention. The soil pre - treatment process for stably extracting the germplank roots rot fungus DNA in the soil using radishcorse selective medium and the real - time PCR method after extracting the germplank roots rot fungus DNA from the soil, Is the method of testing the density.
FIG. 2 is a quantitative curve for quantitative analysis or quantitative analysis of ginseng root rot disease bacteria in soil.
Hereinafter, the present invention will be described in more detail.
As described above, there has not been established a method for quantifying bacteria present in the soil to date, and further, there is little research on a method for quantitatively determining ginseng root rot disease bacteria present in trace amounts in soil with high accuracy.
Accordingly, the present invention provides a quantitative analysis method of ginseng root rot disease bacteria in a soil using a radish call selective medium and a quantitative analysis kit including a radish call selective medium, a primer set, and a reagent for a polymerase chain reaction, We have sought a solution to this problem.
As a result, it is possible to quantify the ginseng root rot caused by ginseng root in soil with high sensitivity.
Accordingly, the present invention provides a method for detecting DNA in a soil sample comprising the steps of: (a) separating DNA from a soil sample; (b) performing a real-time PCR using a mixture solution containing the DNA obtained in the step (a) and a primer for identification of a ginseng root rot disease virus; And (c) analyzing the results of the real-time PCR reaction performed in the step (b) to quantify the ginseng root rot disease bacteria in the soil sample.
The soil sample of the present invention may be one separated from ginseng, preferably ginseng root rot caused by ginseng root or the surrounding soil. The sample was obtained from Rhizoctonia < RTI ID = 0.0 > solani , Botrytis cinerea , Alternaria but are not limited to, one or more fungi selected from the group consisting of panax , Sclerotinia sclerotiorum , and Pythium ultimum .
The step (a) of isolating the DNA of the present invention may further include the step of culturing the soil sample in a radish call selective medium.
The selection medium may be prepared by adding an effective amount of radishcor to a base medium which can be used for the cultivation of a carcass decontamination tank. The "base medium" is not limited to any kind that can be used for culturing the Cylindrocone Desoldering Tans. In one embodiment of the present invention, the base medium may be a liquid or a solid medium. In one embodiment of the present invention, the basal medium may be a medium such as MM (minimal medium), PDA (potato dextrose agar), CM (complete medium), CDA (czapek dox agar) broth) or CMC (carboxylmethyl cellulose) medium, most preferably MM (minimal medium) medium, but is not limited thereto.
The "effective amount" as used herein refers to an addition amount of radishcor contained in the selective medium, which exhibits antibacterial and / or antifungal activity against microorganisms other than the Cylindrocodystartans tans contained in the sample, and the culture conditions, The most suitable amount to selectively cultivate the carcass decontamination truck can be selected. Radishcorn of the selective medium may be prepared by adding commercially available radicicol, but it may also be prepared by adding a rice culture extract of ginseng root rot disease virus.
The selective medium of the present invention may contain radicicol at a concentration of 30 to 70 mg / l, more preferably 40 to 60 mg / l, and may contain radicicol at a concentration of 40 to 60 mg / More preferably 45 to 55 mg / l.
The selective medium of the present invention may further include a substance exhibiting an antibacterial or antifungal activity against a microorganism other than Cylindrocone desatta tans.
The soil sample of the present invention may be cultured in the radishcorn selective medium for 36 to 60 hours, more preferably for 42 to 54 hours. In one embodiment of the present invention, the soil samples were cultured for 0 hours, 24 hours, 48 hours, 60 hours, and 82 hours, and then the Cylindrocone desatanthan germs were quantified. However, there was a problem that a low concentration of bacteria was not detected at 0 hour, and a low concentration of bacteria was detected at 24 hours. However, there was a problem that the difference in the density of the bacteria was not apparent and the quantitation was not correct. In addition, when cultured for 60 hours or more, there was a problem that the difference in the density of bacteria was not apparent. Thus, the soil sample of the present invention may be cultured for 36 to 60 hours, more preferably for 42 to 54 hours. Still more preferably for 46 to 50 hours.
The selective medium exhibits antimicrobial or antifungal activity against fungi including microorganisms other than Cylindrone dextrin tans, preferably fungi, more preferably entomopathogenic fungi, incomplete fungi and egg fungi. Examples of the pathogenic fungus in which the selective medium exhibits antifungal activity include Rhizoctonia solani, Botrytis cinerea, Alternaria panax, Sclerotinia sclerotioca, Room (Sclerotinia sclerotiorum), and Pythium ultimum.
The primer for identification of the ginseng root rot disease virus of the present invention may be any oligonucleotide having a sequence complementary to the gDNA root rot germ plasmid gDNA sequence. Preferably, the primer for amplifying the microsatellite DNA contained in the sample DNA Base sequence. More preferably two or more selected from the group consisting of SEQ ID NOS: 1 to 18, preferably a primer set consisting of SEQ ID NOS: 1 and 2, a primer set consisting of SEQ ID NOS: 3 and 4, And 6, and a primer set consisting of SEQ ID NOS: 7 and 8, and a primer set selected from the group consisting of SEQ ID NOS: 7 and 8.
The real-time PCR reaction of the present invention may preferably be a quantitative real-time PCR. "Quantitative real-time polymerase chain reaction" generally means a series of reactions using PCR to quantify the amount of template DNA at the start of the amplification reaction. Quantitative PCR includes internal standard methods using standard reference standards and competitive methods using molecules competing in the amplification reaction. In order to accurately and easily determine the number of molecules of each sequence, a PCR method using a template DNA sequence as a standard called a standard sample is used, and the progress of the reaction, that is, the degree of amplification is checked at any time during the reaction . Therefore, in the present invention, "quantitative real-time PCR" means a PCR for quantifying the amount of template DNA at the start of the amplification reaction, and means that the amplification can be confirmed with respect to the molecule to be amplified at any time during the reaction. For quantification, such a PCR is generally combined with a calibration curve that correlates the number of template DNA molecules at the start of the reaction with a signal that is an index of the amplification. This calibration curve can be prepared using standard samples with a known number of molecules.
The step (c) of analyzing the results of the polymerase chain reaction of the present invention comprises the steps of preparing a calibration curve as a standard sample with known concentration; And calculating the number of gene copies detected from the generated calibration curve.
The present invention also relates to a primer set for identifying ginseng root rot disease bacteria in soil; Radishcorn selection medium; And a kit for quantitative analysis of ginseng root rot disease bacteria in soil containing a reagent for polymerase chain reaction.
The term "kit " in the present invention means a tool that enables absolute quantification of a target microorganism by confirming the number of copies of a gene representing a microorganism to be detected or quantified in the sample by real time quantitative PCR. The kit for absolute quantification of a microorganism according to the present invention is characterized by comprising an artificial base containing at least one primer and / or a probe site specific to a gene representing a microorganism to be detected or quantified as a standard sample for the check- In addition to the sequence, one or more other component compositions, solutions, or devices suitable for the assay method may be included.
The kit for absolute quantification of microorganisms according to the present invention comprises one or more other component compositions, solutions or devices suitable for the assay method. Specifically, the real-time PCR kit can be used to test the presence of a test tube or other appropriate container, a dNTP mixture (dATP, dCTP, dGTP, dTTP), a primer specific for ginseng roots rot fungi, a DNA polymerase, Various), sterilized water, and the like.
The primer for identification of the ginseng root rot disease virus of the present invention may be any oligonucleotide having a sequence complementary to the gDNA root rot germ plasmid gDNA sequence. Preferably, the primer for amplifying the microsatellite DNA contained in the sample DNA Base sequence. More preferably two or more selected from the group consisting of SEQ ID NOS: 1 to 18, preferably a primer set consisting of SEQ ID NOS: 1 and 2, a primer set consisting of SEQ ID NOS: 3 and 4, And 6, and a primer set consisting of SEQ ID NOS: 7 and 8, and a primer set selected from the group consisting of SEQ ID NOS: 7 and 8.
According to Example 6 of the present invention, ginseng roots rot germs in soil samples were quantitatively determined under various culture conditions. As a result, when a medium other than the MM medium containing radishcor is used, the SQ value becomes high as a result of real time PCR, which is not easy to quantify. Therefore, when inspecting soil using actual ginseng farming pavement, many microorganisms are present in the soil. Therefore, it is necessary to use an effect to inhibit the growth of other microbes by using radishcore selective medium. A radish call selection medium capable of accurately detecting root rot bacteria is provided.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.
Soil pretreatment process using radishcore selective medium
The present invention is a method for stably extracting the DNA of C. destructans in soil, and the soil samples collected in the soil were removed with 1 mm of impurities. 5 g is taken from a soil sample filtered with impurities, and a selective medium containing 50 mg / l of radishcor is added, followed by shaking culture at 20 ° C for 48 hours.
Soil preparation and soil DNA extraction
The samples cultured for 48 hours in radishcor selection medium were centrifuged to remove supernatant and lyophilized. DNA was extracted using 0.3 g of dried soil samples and a soil DNA extraction kit commonly used. The extracted gDNA was used for PCR amplification and real-time PCR.
Real-time gene analyzer
1 μl of the forward primer (10 pmol), 1 μl of the reverse primer (10 pmol) shown in SEQ ID NO: 2, 2 μl of the gDNA extracted in Example 2, 6 μl of sterilized water, Sso advanced 10 μl of TM universalsybr ® green supermix were mixed to prepare a mixed solution. Then, 20 μl of the mixed solution was used for real-time PCR.
Specifically, 20 μl of the mixed solution was subjected to initial denaturation at 98 ° C. for 3 minutes, denaturation at 95 ° C. for 10 seconds, annealing at 61 ° C. for 15 seconds, extension at 72 ° C. for 30 seconds The cycle for repeating 40 cycles with one cycle as an extension cycle was followed by a process for preparing a melt curve.
Then, the result obtained in the real-time PCR was confirmed by using the provided program to determine the melt curve of the primer and the number of amplified genetic sites (SQ).
Determination of optimal culture period for selective medium selection for pathogen density in soil
As shown in FIG. 1 or Table 2, in order to test the density of the ginseng root rot disease bacteria in the soil, a radishcore selective medium was added to the artificial soil inoculated with the spores of pathogenic bacteria and cultured, Respectively.
First, real - time gene analysis showed no spores of 10 3 or less in the sample (0 day) that was not cultured in the radish calle selective medium. In order to detect ginseng root rot disease bacteria in soil by real-time polymerase chain reaction (PCR), a culture period of at least one day is required. The amplification number of PCR fragments by the spore concentration of pathogens in soil samples cultured for 48 hours in radishcore selective medium SQ, starting quantity was the best.
Therefore, quantitative analysis of ginseng root rot disease by conventional ginseng showed that soil DNA was not obtained when the density of pathogens in the soil was low (below 10 3 ), and therefore, it was not suitable to perform density analysis using a real - time gene analyzer. The problem can be solved by adding the soil pretreatment process using the radish call selective medium proposed in the present invention.
/0.3g soil
* nd: not detected
Quantitative curve using real-time gene analyzer
As shown in FIG. 2, a quantitative curve for testing the density of the ginseng root rot disease bacteria in the soil was prepared. Real-time gene analysis was performed using artificial soil inoculated with soil pathogen spores through a soil sample pretreatment (48 hours incubation) using the radishcor selection medium determined in Example 4 and Table 3. It has a pathogen spore concentrations SQ value obtained through four times repeatedly subjected to statistical analysis to the trend line was obtained (Y = 160.74X 1 .74, Y : number of spores: SQ, X).
As can be seen from the examples, the soil pretreatment process utilizing the radishcor selection medium of the present invention can provide a stable soil sample DNA for real-time PCR, and can be used for detecting the density of the germ root rot disease bacteria in soil It was confirmed that it provides a proper identification method.
/0.3g soil
* Avr: average
# SD: standard deviation
Quantitative determination of ginseng root rot disease in soil using various media
Ginseng roots rot fungi present in 1 ~ 3 soil of farmhouse were quantified by using different media.
The media conditions were MM medium, medium supplemented with radishcor on MM medium, PDB medium and CM medium, respectively. Real time PCR was performed under the same conditions using the primers of SEQ ID NOS: 1 and 2 after different conditions of the medium were different. The results are shown in Table 4 below.
In the experimental group using MM medium, it was not detected by PCR because of insufficient growth of ginseng root rot disease bacteria. In the experimental group treated with radicicol in MM medium, the ginseng root rot bacteria were selectively grown, so that a relatively accurate quantification was possible . On the other hand, in the experimental group using the PDB medium or CM medium, not only the ginseng rot rot but also all the microorganisms in the soil were grown. Therefore, the SQ (scarcification quantity) value was high and the accurate quantification was difficult.
/0.3g soil
* nd: not detected
That is, if other medium is used, the real-time PCR result value SQ becomes high and it is not easy to quantify. Since there is a lot of microorganisms in the soil when using the soil of actual ginseng farming pavement, it is necessary to use the radishcore selective medium to inhibit the growth of other microorganisms. Accordingly, the present invention provides a method for selectively growing ginseng root rot disease bacteria to accurately quantify ginseng root rot disease bacteria.
<110> REPUBLIC OF KOREA <120> Quantitative analysis method of Cylindrocarpon destructans in the soil using RT-PCR and radicicol-selective medium <130> 1042462 <160> 18 <170> Kopatentin 2.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Ccms105-AC51 forward <400> 1 tgggtgtggg tcggatctat ttct 24 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Ccms105-AC51 reverse <400> 2 gtcgcgggaa aggaaaaaga aag 23 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Ccms66-GCA51 forward <400> 3 atcgagacgg ggatgcgaca a 21 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Ccms66-GCA51 reverse <400> 4 tttgcgctgt gcggactagg tt 22 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Ccms57-1 forward <400> 5 aagcccacag gattgatg 18 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ccms57-1 reverse <400> 6 gactacttga ctccgtgatt 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Ccms75-2 forward <400> 7 cagcgacaac agtttatttg a 21 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Ccms 75-2 reverse <400> 8 cttgaggact atgcgacc 18 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ITS1 <400> 9 tccgtaggtg aacctgcgg 19 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ITS4 <400> 10 tcctccgctt attgatatgc 20 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Dest1 <400> 11 ttgttgcctc ggcggtgcct g 21 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Dest4 <400> 12 ggtttaacgg cgtggccgcg ctgtt 25 <210> 13 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Destruc2F <400> 13 gtgcctgytt cggcagc 17 <210> 14 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Destruc2R <400> 14 ctgtttycca gtgcgaggtg tgc 23 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CylF01 <400> 15 catgcgtgag attgtaagtt 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CylR625 <400> 16 tgacccttgg cccagttgtt 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CylF146 <400> 17 acgacgtgat tttgggacaa 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CylR407 <400> 18 tcgttgaagt agacgctcat 20
Claims (13)
(b) performing a real-time PCR on a mixture of the DNA obtained in the step (a) and a primer set for identification of C. destructans ; And
(c) analyzing the results of the real-time PCR reaction performed in the step (b) to quantify the ginseng root rot disease bacteria in the soil sample, and determining the ginseng root rot disease bacteria in the soil.
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