KR101767371B1 - Primer set for detecting of Mycosphaerella nawae and uses thereof - Google Patents
Primer set for detecting of Mycosphaerella nawae and uses thereof Download PDFInfo
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Abstract
The present invention relates to a primer set and a use thereof for detecting or diagnosing a persimmon foliar infection, and more particularly, to a primer set capable of specifically detecting or diagnosing persimmon round foliar foliar disease bacteria. A composition for diagnosing a persimmon round discolor; Diagnostic kit; And to a method for detecting a deciduous flower leaf of the persimmon using the same. The primer set for PCR reaction according to the present invention can specifically amplify a gene region of Mycosphaerella nawae , and when used, it can effectively and efficiently isolate persimmon round-headed deciduous germs in a short time from the specimen by species- Can be detected. Therefore, it is expected that the Mycosphaerella nawae ) can be detected at an early stage, so that a more rapid and efficient diagnosis system can be constructed. In addition, a Mycosphaerella It is expected to prevent the economic loss due to infection.
Description
The present invention relates to a primer set and a use thereof for detecting or diagnosing a persimmon foliar infection, and more particularly, to a primer set capable of specifically detecting or diagnosing persimmon round foliar foliar disease bacteria. A composition for diagnosing a persimmon round discolor; Diagnostic kit; And to a method for detecting a deciduous flower leaf of the persimmon using the same.
Circular leaf spot (CLS) is the most severe disease in the persimmon tree. It infects the leaves in the form of ascospores from the middle of May to early August, and leaves the leaves early after the end of August. It occurs mainly on the leaves and rarely occurs on the throat. The first black round spot is formed on the leaf, and as the lesion enlarges, the inside of the lesion is light brown or reddish-brown, and the border of the lesion is black green. Thereafter, the circular lesion fuses and grows into an irregular lesion. Leaves with severe disease occur early and fall off. This greatly affects the growth and yield of trees and is susceptible to the East Sea.
Mycosphaerella nawae is a pathogenic bacterium that causes rot fungi of the persimmon tree. It belongs to the fungal genus of the fungus, and forms a conidia and conidia. Cryptocyte is a dark brown to egg-shaped, and contains several sacs. The optimum temperature for growth of pathogenic bacteria is 20 ~ 25 ℃. Artificial cultivation is possible but it is very difficult to separate. The inventive conditions and transmission route of persimmon leaves are known to swell in the form of hyphae or mycelium in deciduous diseased lesions and then spread to the pseudothecium in April and scattered in May to July , And the symptom is manifested in September-October. The pathogens that enter the persimmon leaves develop after a certain latency period, and they are believed to have a long latency period of 2 to 4 months, and not to transmit the second infection. June to July, many occur in rainy weather and rainfall management is poor.
Persimmon leaves of persimmon are most common diseases in persimmon growing fields in Korea. Persimmon leaves are the most harmful diseases in persimmon cultivation areas in the southern region. This disease has a very long time until the time when the infection becomes symptomatic and once the disease has occurred, there is no way to control it, so there are many farmers who are suffering from damage each year without establishing clear control measures until now. When the disease is caught, the severely infected leaves are deciduous leaves, which is a terrible disease that leads to the degradation of fruit trees, which leads to a decrease in productivity, such as immature fruit and decolorization. Most of these diseases are infected by primary infectious agents, and the latency period from infection to symptoms is very long (latency period of about 90-120 days after infection), which makes it difficult to control the disease. Therefore, it is necessary to treat pre-existing fungicides in accordance with the timing of aspergillus spores in order to control the effective deciduous disease of persimmon leaves. In order to control the disease, persimmon growers are spraying more than 6 disinfectants a year, which increases the economic burden on growers.
Therefore, in order to minimize the damage of the persimmon leaves, it is indispensable to prevent the disease before the disease develops. Therefore, it is urgently required to develop a technology capable of quickly and quickly diagnosing the infection of the persimmon leaves .
Accordingly, the present inventors have sought to find a method for quickly and accurately diagnosing the infection of the persimmon leaves of the Japanese persimmon tree. As a result, Mycosphaerella nawae gene specific sequence, and that when the PCR is carried out by using the primer, it is possible to accurately diagnose the persimmon round-leaf lance disease, and the present invention has been completed.
Accordingly, an object of the present invention is to provide a primer set capable of effectively diagnosing persimmon round-headed leaves.
Another object of the present invention is to provide a composition for diagnosing persimmon round leaf litter comprising the primer set.
It is still another object of the present invention to provide a kit for diagnosing a persimmon round leaf litter comprising the composition.
It is still another object of the present invention to provide a method for effectively detecting persimmon round foliar germs using the primer set.
In order to achieve the above object, the present invention provides a primer pair comprising a first primer pair represented by SEQ ID NO: 1 and SEQ ID NO: 2; Or a second pair of primers represented by SEQ ID NO: 3 and SEQ ID NO: 4.
In one embodiment of the present invention, the first pair of primers can amplify 599 bp of the specific PCR product of persimmon fungus, which is a polynucleotide of SEQ ID NO: 6.
In one embodiment of the present invention, the second pair of primers can amplify a 204 bp round-tailed deciduous germ-specific PCR product comprising the polynucleotide of SEQ ID NO: 7.
In addition, the present invention provides a composition for diagnosing persimmon round leaf litter comprising the primer set.
In one embodiment of the present invention, the composition comprises a first primer pair represented by SEQ ID NO: 1 and SEQ ID NO: 2; Or a DNA polymerase for PCR reaction, dNTPs, a reaction buffer, and distilled water in addition to the second primer pair shown in SEQ ID NO: 3 and SEQ ID NO: 4.
In addition, the present invention provides a diagnostic kit for a persimmon round leaf litter containing the composition.
(A) extracting genomic DNA from a plant specimen; (b) a first primer pair shown in SEQ ID NO: 1 and SEQ ID NO: 2, using the extracted genomic DNA as a template; Or amplifying the target sequence by performing a PCR reaction using a second primer pair represented by SEQ ID NO: 3 and SEQ ID NO: 4; And (c) detecting the amplification product. The present invention also provides a method for detecting persimmon round foliar germs.
In one embodiment of the present invention, the step of detecting the amplification product may be performed by electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement.
The primer set for PCR reaction according to the present invention can specifically amplify a gene region of Mycosphaerella nawae , and when used, it can effectively and efficiently isolate persimmon round-headed deciduous germs in a short time from the specimen by species- Can be detected. Therefore, it is expected that the Mycosphaerella nawae ) can be detected at an early stage, so that a more rapid and efficient diagnosis system can be constructed. In addition, a Mycosphaerella It is expected to prevent the economic loss due to infection.
FIG. 1 is a schematic diagram showing the reaction conditions for the PCR method for diagnosing a round foliar disease.
2 is a photograph showing a sample used in the present experiment.
FIG. 3 shows the results of PCR performed using the primer sets (MN-F1 / MN-R1 and MN-F2 / MN-R2) for detection of deciduous germs of the present invention: M ( Mycosphaerella nawae ), DL (diseased leaf), HF (healthy leaf).
FIG. 4 is a graph showing the results of PCR using a primer set (MN-F1 / MN-R1) for detection of the deciduous germs of persimmon leaves of the present invention in nine different M. nawae and 7 pathogens The results are as follows: AA ( Amycosphaerella africana ), PM ( Paramycospherella marksii ), PI ( Paramycospherella intermedia ), ZP ( Zasmidium parkii ), MM ( Mycosphaerella medeirae ), LA ( Lecanosticta acicola ), PF ( Passalora fulva ) Mycospherella graminicola), MM (Mycospherella musae ), BC (Botrytis cinerea), PD (Pestalotiopsis diospyri), PE (Penicillium expansum), PhD (Phomopsis diospyri), CC (Cladosporium cladosporioides), CG (Colletotrichum gloeosporioides), PK (Pseudocercospora kaki ), Po (Positive control: Mycosphaerella nawae ).
FIG. 5 is a graph showing the results of PCR diagnosis of 13 different persimmon leaves of a susceptible variety using the primer sets (MN-F1 / MN-R1, MN-F2 / MN-R2) 6 (Hansan Sushi), 7 (Suhong), 8 (Qingdao Banshi), 9 (Shaanxi Province), 4 (Shaolin Province), 5 (DDW: Negative control), 15 (Dongguk city), 10 (single phase), 11 (moon time), 12
The present invention is characterized by providing a primer set capable of diagnosing persimmon round-headed deciduous diseases. The primer set according to the present invention can specifically detect or diagnose only Mycosphaerella nawae , which causes persimmon round- headed deciduous disease.
The term " specific " in the present invention refers to a pathogenic fungus such as Mycosphaerella nawae ) of the species.
In the present invention, the term " primer " refers to a single strand oligonucleotide sequence complementary to a nucleic acid strand to be copied, and may serve as a starting point for synthesis of a primer extension product. The appropriate length of the primer may vary depending on the purpose of use, but is generally comprised of 15 to 30 bases. The primer sequence need not be completely complementary to the template, but should be sufficiently complementary to hybridize with the template.
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.
Meanwhile, the primer set provided in the present invention is a forward primer and a reverse primer designed to amplify a partial region of the
The primers of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution of one or more natural nucleotides with an analogue, and modification between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, Amidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).
When the first primer pair shown in SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention is used, the specific PCR product of the persimmon foliar fungus of about 600 bp can be amplified and the PCR product of SEQ ID NO: 3 and SEQ ID NO: When the second primer pair is used, about 200 bp of the persimmon fungus specific PCR-PCR product can be amplified.
In one embodiment of the present invention, the PCR product of 599 bp of the persimmon fungus-infested with fungus, which is amplified using the first primer pair, may be a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 6, The 204 bp squid circular deciduous bacillus-specific PCR product amplified using the polynucleotide sequence may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7.
In order to develop a primer set for detection of persimmon leaves, the present inventors first performed a random sequencing by collecting Mycosphaerella nawae , a pathogenic bacterium, Each of the genes registered in Mycosphaerella fungi registered in the database of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov) was collected and multiple alignments , And a species-specific primer was prepared based on the species-specific sequence (SEQ ID NO: 5). As described above, the primer thus constructed comprises a first primer pair shown in SEQ ID NO: 1 and SEQ ID NO: 2; Or a second primer pair represented by SEQ ID NO: 3 and SEQ ID NO: 4.
In the following examples of the present invention, PCR was carried out using the prepared persimmon-fungus-type deciduous germ-specific primers and 9 pathogenic fungi of the persimmon leaves and 7 pathogens derived from 7 persimmons. As a result, the bands were amplified only in the persimmon leaves of the persimmon leaves, while the bands were not detected in the nine species and seven other pathogens. From these results, it was confirmed that the primer set of the present invention was species-specific for persimmon leaves by specifically amplifying persimmon round-headed deciduous germs.
The present invention also provides a first primer pair represented by SEQ ID NO: 1 and SEQ ID NO: 2; Or a second primer pair represented by SEQ ID NO: 3 and SEQ ID NO: 4.
The primer can be used to amplify a DNA fragment by hybridizing to a DNA sequence containing a species-specific base region present in the gene region of the persimmon leaves. In one embodiment of the present invention, when the first primer pair shown in SEQ ID NO: 1 and SEQ ID NO: 2 is used, 599 bp of the polynucleotide of SEQ ID NO: 6 can be amplified in a specific PCR product of persimmon fungus, In case of using the second primer pair shown in SEQ ID NO: 3 and SEQ ID NO: 4 of the invention, a 204 bp round-tailed deciduous bacterium-specific PCR product consisting of the polynucleotide of SEQ ID NO: 7 can be amplified.
In one embodiment of the present invention, the composition comprises a first primer pair represented by SEQ ID NO: 1 and SEQ ID NO: 2; Or a DNA polymerase for PCR reaction, dNTPs, a reaction buffer and distilled water in addition to the second primer pair shown in SEQ ID NO: 3 and SEQ ID NO: 4.
The present invention also relates to a first primer pair represented by SEQ ID NO: 1 and SEQ ID NO: 2; Or a second primer pair represented by SEQ ID NO: 3 and SEQ ID NO: 4, for diagnosing persimmon round-headed decidua.
The kit of the present invention comprises a first primer pair represented by SEQ ID NO: 1 and SEQ ID NO: 2; Or one or more other component compositions, solutions or devices suitable for the assay method, as well as a set of primers for diagnosing persimmon round-bottomed leaflets, comprising a second primer pair represented by SEQ ID NO: 3 and SEQ ID NO: 4 .
For example, the kit of the present invention may be a kit containing essential elements necessary for performing PCR. The PCR kit may contain, in addition to the primer set of the present invention (the first primer pair shown in SEQ ID NO: 1 and SEQ ID NO: 2, or the second primer pair shown in SEQ ID NO: 3 and SEQ ID NO: 4) Can include enzymes such as buffers (varying in pH and magnesium concentration), deoxynucleotides (dNTPs), Taq polymerase and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water and sterile water have.
In addition, the kit may further include a user's manual describing optimal reaction performing conditions. The instructions include instructions on the surface of the package including the brochure or flyer-shaped brochure, the label attached to the kit, and the kit. The manual also includes information that is disclosed or provided through an electronic medium such as the Internet.
The present invention also provides a method for producing a plant, comprising: (a) extracting genomic DNA from a plant sample; (b) the primer set of the present invention (the first primer pair shown in SEQ ID NO: 1 and SEQ ID NO: 2; or the second primer pair shown in SEQ ID NO: 3 and SEQ ID NO: 4) Amplifying the target sequence by performing PCR using the primers; And (c) detecting the amplification product. The present invention also provides a method for detecting persimmon round foliar germs.
The present invention relates to a method for rapidly and accurately detecting a persimmon leaf-leaf litter by amplifying a Mycosphaerella nawae species-specific gene region using a PCR reaction as a molecular genetic technique.
The method of the present invention comprises isolating genomic DNA from a plant sample. A method known in the art can be used to isolate the genomic DNA, and the plant sample may be a persimmon leaf susceptible to infection.
Using the separated genomic DNA as a template and using the primer set of the present invention (the first primer pair shown in SEQ ID NO: 1 and SEQ ID NO: 2, or the second primer pair shown in SEQ ID NO: 3 and SEQ ID NO: 4) PCR can be performed to amplify Mycosphaerella nawae species-specific gene target sequence of persimmon leaves.
The target sequence amplified by the PCR may be labeled with a detectable labeling substance. In one specific example, the labeling material can be, but is not limited to, a material that emits fluorescence, phosphorescence, or radiation. Preferably, the labeling substance is Ethidium Bromide (EtBr), Cy-5 or Cy-3. When the target sequence is amplified, PCR is carried out by labeling the 5'-end of the primer with Cy-5 or Cy-3, and the target sequence may be labeled with a detectable fluorescent labeling substance. In addition, if the radioactive isotope such as 32P or 35S is added to the PCR reaction solution, the amplification product may be synthesized and the radiation may be incorporated into the amplification product and the amplification product may be labeled with the radiation.
Wherein the amplified product is detected by a DNA chip. Such as, but not limited to, gel electrophoresis, radiation measurement, fluorescence measurement, or phosphorescence measurement. 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. Also, in the fluorescence measurement method, Cy-5 or Cy-3 is labeled at the 5'-end of the primer. When PCR is carried out, the target is labeled with a fluorescent label capable of detecting the target sequence. The labeled fluorescence is measured using a fluorescence meter can do. In addition, when the PCR is performed, the radioactive isotope such as 32P or 35S is added to the PCR reaction solution to mark the amplification product, and then the radioactivity is measured using a radiation measuring device such as a Geiger counter or a liquid scintillation counter A liquid scintillation counter can be used to measure radiation.
Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.
< Example 1>
Persimmon Round-headed For species-specific gene amplification primer set
In order to develop a primer set for detection of the persimmon round leaf discolor of the present invention, Mycosphaerella nawae , which is a pathogenic bacterium , was collected and subjected to random sequencing. For comparison of their nucleotide sequences, each gene registered in the Mycosphaerella genus fungus registered in the database of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov) After the collection, the specific sequence sequence of the M. nawae species was analyzed through multiple alignment with these genes. As a result, the species-specific sequence was found to be a DNA fragment of about 2,036 bp (SEQ ID NO: ), Which was converted to protein and confirmed by partial sequence of
Using the M. nawae species-specific sequence of SEQ ID NO: 5 identified by the above process, the inventors of the present invention performed PCR amplification that can detect and diagnose the persimmon fungus of the present invention A primer set for reaction was prepared as shown in Table 1 below.
≪ Example 2 >
Collection of specimens
The whole genomic DNA was extracted from round leaves, leaves, and persimmon leaves and used as a template for PCR reaction.
In detail, round leaf discolorage infected leaves and healthy leaves were randomly collected, a certain portion was collected from the collected leaves, and a Biofact TM Total DNA was extracted according to the manufacturer's method using Genomic DNA Prep Kit (Cat. No. GD141-050 Biofact, Korea) and the extracted product was used as template DNA.
< Example 3>
The persimmon of the present invention Round-headed deciduous germ For detection primer Check the availability of the set
A PCR primer composition was prepared in order to confirm that the PCR primer set of the present invention prepared in Example 1 can be used to specifically detect only persimmon round-headed deciduous germs. Its composition and reaction conditions are shown in Table 2 below.
The amplification reaction composition prepared in the composition of Table 2 was subjected to pre-denaturation at 95 ° C for 1 minute, followed by 35 cycles at 95 ° C for 30 seconds, 60 ° C for 60 seconds, and 72 ° C for 90 seconds , Followed by extension at 72 ° C for 5 minutes (see FIG. 1). The amplification product of the PCR reaction was analyzed by agarose gel electrophoresis.
<3-1> Round patterned deciduous germ , round patterned deciduous bottle PCR in infected leaves and healthy persimmon leaves result
PCR was carried out using the compositions of Table 2, which were prepared from the round-headed deciduous germs, round-headed leaf-infected leaves and whole genomic DNA of healthy persimmon leaves.
As a result, the target DNA fragments of about 600 bp and 200 bp of the MN-F1 / MN-R1 and MN-F2 / MN-R2 primer sets were confirmed in the round leaves of the deciduous and round foliage infected leaves . As a result of PCR with MN-F2 / MN-R2 using the product amplified with MN-F1 / MN-R1, the desired fragments were confirmed in the remaining samples except healthy persimmon leaf.
<3-2> persimmon
leaves round pattern germs
(Mycosphaerella
PCR
results
at 7 different species of pathogenic bacteria occurring in
closely related species,
and
In order to confirm the species specificity of the primer set of the present invention, 9 kinds of M. nawae and 7 kinds of other pathogenic microorganisms were obtained and PCR reaction was performed using the MN-F1 / MN-R1 primer set Respectively.
Seven species of fungi from persimmon; Botrytis cinerea , Pestalotiopsis diospyri, Penicillium expansum, Phomopsis diospyri , Cladosporium cladosporioides , Colletotrichum gloeosporioides and Pseudocercospora kaki were purchased from the Korean Center for Microorganism Genetic Resources (KACC), and nine species of fungi were reported to be phylogenetically similar to M. nawae ; Amycosphaerella africana , Paramycospherella marksii , Paramycospherella intermedia , Zasmidium parkii , Mycospherella medeirae , Lecanosticta acicola , Passalora fulva , Mycospherella graminicola , Mycospherella musae were used to bataheun pre-sale pre-sale center in Holland strains (Fungal biodiversity centre, CBS).
As a result, as shown in Fig. 4, bands were amplified in the persimmon leaves of the persimmon leaves, while none of the nine species and seven other pathogens were amplified in the target region.
<3-3> PCR results using healthy persimmon leaves (total 13 species)
In this experiment, experiments were conducted with whole genomic DNA of 13 different varieties of persimmon leaves. First, the PCR reaction was performed using the MN-F1 / MN-R1 primer set, and then the PCR product was used as the MN-F2 / MN-R2 primer set.
As a result, as shown in Fig. 5, the fragments of the target region were amplified in the leaves infected with the persimmon leaves of the persimmon leaves, whereas the fragments of the target region were not amplified in the healthy persimmon leaves of 13 different varieties .
As a result, the primer set of the present invention proved to be species-specific that can specifically detect only the persimmon leaves of the Japanese persimmon tree (MN-F1 / MN-R1 and MN-F2 / MN-R2) Respectively.
The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.
<110> Kyongsangbuk-do Agricultural Technology Administration <120> Primer set for detection of Mycosphaerella nawae and uses thereof <130> PN1512-344 <160> 7 <170> KoPatentin 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MN-F1 forward primer <400> 1 gcggtgaact cactaaagct 20 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> MN-R1_reverse primer <400> 2 cttatcgtat gcagtgatct cc 22 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> MN-F2 forward primer <400> 3 cggatgaaga agaatacaag c 21 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> MN-F2_reverse primer <400> 4 gattgaatac cagaaccgtc ggg 23 <210> 5 <211> 2036 <212> DNA <213> Artificial Sequence <220> <223> Mycosphaerella nawae_partial sequence of glycoside hydrolase family 3 gene <400> 5 ggtgtgccag aggatggaga gtcagatgca tggtatcctt cgcctttggg aggaacgcta 60 gatgcgtggg tggagagcta tcgaaaagct ggggagctgg tcgatcagat gacgcttgtg 120 gagaaggtga acatcacctc aggcacaggc tggcagatgg gaccttgtgt gggcaatact 180 ggaccagtcg atcgtctagg cttcccgagc ttgtgtttgc aggacggacc actgggcatg 240 cgattcgtgg acaacgggac tgcttggcca gctggagtta ctgtgggcgc aacctggaac 300 aaggacttga tgtaccagcg aggcaaggca catgggtttg aggctaagtt gaagggcatc 360 aatgtcattc ttgggccctc aatgggaggc atcggacgat tgcctgctgg tggtcggaac 420 tgggaagcat tcgggccgga cccggtcctc caaggaatcg cttcagcgca gacgatcaaa 480 ggtatccagg agaacggcgt gatcgctaca gcgaaacatt tcgtggggaa tgagcaagag 540 cacttccggc aagcctggga gtgggccaac cctaacgcca tctcttcgaa tatcggagac 600 agagcgcttc atgaaatcta cgcgtggccc ttcgcggact caataaaggc tggagtggca 660 agcgtcatgt gttcctacaa ccaagtgaac aactcgtacg cctgccagaa cagcaagctc 720 atgaacggcg tgctcaagga cgagatgggc ttccaaggtt tcatccagag cgattggctg 780 gcgcagcgat ctggtgtcgc aagcgcgctg gcaggtcttg acatgacaat gcctggagac 840 ggtttgagat ggcaggatgg caagacactt tggggcggtg aactcactaa agctgcgctg 900 aacggctccg tgcccatgaa gcgcttgaat gacgctgtac tgcgcattgt ggcggcttgg 960 tatcagcttc gtcaagataa cgagacagcc tggccgagaa agacgctcag cagctcggca 1020 actttcagca gctggacgga tgaagaagaa tacaagcttc accctggctc gcctggcagc 1080 gaggagaatg gcaaaattaa cgagtatgtt ccagtccggg aaaccgagga gggtggcaat 1140 cacgacgatt tggcccgaaa gattgctgct gaaggcattg ttatggtcaa gaatgacgac 1200 aatgtactgc ccatcaagcc cgacggttct ggtattcaat cttcgagagc cggccagagc 1260 tttggaaaga aattcaagat cggcattttt ggagaagacg ccttcgccaa cccgaaaggt 1320 ccgaacgcct gcgaggaccg aggctgcaac gagtacacac ttggtagcgg atggggatct 1380 ggtgcagcag actttccgca tctggtcgca cctttcgatg ctttgaattc gaccttcaac 1440 cattcggctg tggagatcac tgcatacgat aagcaggcgg ctaagcatgc cggagggatt 1500 gctgagaacc aggacctgtg cattgtcttt gccaactcgg atgctggaga aggcttcctc 1560 agttggggtc ctgtgaaggg agaccgacca gatcttaaga cacagaagaa tggcgatgca 1620 ctgatcaccg aggttgcgaa gaagtgtggg aacgcactgc aagctcatgg caacgcagca 1680 aacaccattg tcgtgttgca cacggtgggt gcggtcatca tggaacactg gatcgacctc 1740 ccgggcgtga agacagttct ggtcgcgcat cttcctggac aagaatccgg caatgcgctc 1800 gcggatgtca tctttggcaa aatcaacccg tctggcagac tgccatacac cgttgccaaa 1860 gacgaggagg attacggccc gacgagcaag atcctcacca ttcctaacca cgttgtacca 1920 caacagaact tcacagaagg catctacttc gactatcgat acttcgacaa gcacaacatt 1980 actcctcgat acgaattcgg ctacggcctc tcatacacgg acttccatct gtcttc 2036 <210> 6 <211> 599 <212> DNA <213> Artificial Sequence <220> <223> Mycosphaerella nawae specific PCR product (599 bp) <400> 6 gcggtgaact cactaaagct gcgctgaacg gctccgtgcc catgaagcgc ttgaatgacg 60 ctgtactgcg cattgtggcg gcttggtatc agcttcgtca agataacgag acagcctggc 120 cgagaaagac gctcagcagc tcggcaactt tcagcagctg gacggatgaa gaagaataca 180 agcttcaccc tggctcgcct ggcagcgagg agaatggcaa aattaacgag tatgttccag 240 tccgggaaac cgaggagggt ggcaatcacg acgatttggc ccgaaagatt gctgctgaag 300 gcattgttat ggtcaagaat gacgacaatg tactgcccat caagcccgac ggttctggta 360 ttcaatcttc gagagccggc cagagctttg gaaagaaatt caagatcggc atttttggag 420 aagacgcctt cgccaacccg aaaggtccga acgcctgcga ggaccgaggc tgcaacgagt 480 acacacttgg tagcggatgg ggatctggtg cagcagactt tccgcatctg gtcgcacctt 540 tcgatgcttt gaattcgacc ttcaaccatt cggctgtgga gatcactgca tacgataag 599 <210> 7 <211> 204 <212> DNA <213> Artificial Sequence <220> The Mycosphaerella nawae specific PCR product (204 bp) <400> 7 cggatgaaga agaatacaag cttcaccctg gctcgcctgg cagcgaggag aatggcaaaa 60 ttaacgagta tgttccagtc cgggaaaccg aggagggtgg caatcacgac gatttggccc 120 gaaagattgc tgctgaaggc attgttatgg tcaagaatga cgacaatgta ctgcccatca 180 agcccgacgg ttctggtatt caat 204
Claims (8)
The first primer pair amplifies the specific PCR product of the persimmon round-headed deciduous germ including the polynucleotide of SEQ ID NO: 6,
Wherein the second primer pair comprises a polynucleotide of SEQ ID NO: 7, wherein the primer set amplifies the specific PCR product of the persimmon leaves.
Wherein the composition further comprises DNA polymerase for PCR reaction, dNTPs, reaction buffer, and distilled water.
(b) performing PCR using the extracted genomic DNA as a template and using the primer set according to claim 1 to amplify the target sequence; And
(c) detecting the amplified product.
Wherein the step of detecting the amplification product is performed by electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement.
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Non-Patent Citations (4)
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| Eur. J. Plant Pathol., Vol. 137, pp. 237-281 (2013.06.23.)* |
| GenBank Accession No. XM_007921724.1 (2014.12.22.)* |
| Korean J. Plant Pathol., Vol. 13, pp. 215-218 (1997.) |
| Res. Plant Dis., Vol. 10, pp. 209-216 (2004.) |
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