KR101681645B1 - Primer set for detecting Fusarium oxysporum f. sp. raphani and method for detecting F. oxysporum f. sp. raphani using the same - Google Patents
Primer set for detecting Fusarium oxysporum f. sp. raphani and method for detecting F. oxysporum f. sp. raphani using the same Download PDFInfo
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Abstract
Description
The present invention relates to a primer set for detecting Fusarium oxysporum f. Raphani causing fusarium wilt and a method for detecting fusarium oxysporum f. Sp. Using the same.
Fusarium oxysporum is a pathogenic bacterium that causes serious diseases to many kinds of plants in the world. It has high specificity of host, and it has more than 120 f. (formae specialis) (Agrios, 2005). In other words, since the pathogens of melons, cucumbers and watermelcs are different even in the same foams and crops, melon vinegar and fungus can not be used as fusarium oxysporum melonice oxysporum f. sp. melonis ), and Watermelon sp . Kim, Byung-kyun, were identified as Fusarium spp. oxysporum f. sp. niveum ) and they are generally not infected with each other. These pathogens can form dormant spores in the soil and can remain dormant for years without host plants. Then, when the environment improves, the thick spore germinates and invades the roots of the host plant causing a fusarium wilt or a fusarium wilt (Van Peeret al., 1988).
Wiltless disease is an infectious disease caused by soil infectious disease, formerly known as a fungus infestation . Fusarium oxysporum f. Sp. Raphani is the pathogen causing wilt. Wiltless disease was first reported in 1934 in the White Chinese Winter Radish seedling in San Benito, California, USA, and it has now occurred in various parts of the United States after the occurrence of wilt in Waukesha, Wisconsin, Wisconsin (Pound, 1959; Pound and Fowler, 1953). In 1981, it was first discovered in the rice farming complex in Cheongwon-gun, Korea, and the widespread occurrence of wilt disease has been increasing gradually (Moon et al., 2001; Nam, 1994).
In order to effectively control the wiltless disease, it is important to develop a technology for rapidly and precisely diagnosing pathogens causing wilt disease, to quickly identify the disease occurrence and establish a rapid control measure. In addition, many researchers have reported that non-pathogenic fusarium oxysporum (Fusarium oxysporum) is present in a variety of plants. Also known is the non-pathogenic fuarium oxysporum, where other plant pathogens first enter the plant and then invade secondary and survive byproducts. Therefore, there are many cases of the non-pathogenic fungal oxysporum even if the fusarium oxysporum species is isolated from diseased plants exhibiting wilting symptoms.
Molecular markers capable of diagnosing fusarium oxysporum have been developed, but Fusarium sp. oxysporum f. sp. No molecular markers have been reported to diagnose raphani . Therefore, in order to diagnose a disease caused by radishes as a wilted disease, pathogens were isolated from radishes and confirmed to be pseudomonas spores by a molecular marker for assaying fosarium oxysporum. If it is pathogenic, it has been identified as fuarium oxysporum lappani. However, this method takes longer than 8 weeks to be performed by a skilled expert, and in the case of non-experts, the pathogenicity test fails and it takes longer time to identify the pathogen.
In order to identify the pathogens infected with the plant as described above, the pathogens have been isolated from the plants and the fungus infected with the plant-free fungus has been identified through a fungal indomethacin molecular marker and a pathogenicity test. Therefore, the procedure was cumbersome and time consuming. However, by using the molecular marker of the present invention, it is possible to isolate the DNA from the radish and perform the PCR reaction, so that it is possible to identify pathogens infected with radish in a short and simple time, as well as to identify a plant that has been infected with fusarium oxysporum lappani It is also possible to confirm the site of. Therefore, it is considered that the technique of the present invention capable of specifically detecting the pathogenic fuzarium oxysporum lepani, which is not a non-pathogenic fusarium oxysporum strain, is very useful.
On the other hand, PCR (polymerase chain reaction) analysis technique for amplifying a specific nucleotide sequence of a pathogen is known as an excellent method for detecting pathogens with high sensitivity and accuracy because cultivation and propagation of pathogens are not required. The pathogen-specific detection technique should be able to clearly distinguish between other pathogens and contaminants and the targeted pathogens. For this purpose, it is important to obtain a primer having a specific nucleotide sequence of the target pathogen in the PCR detection method.
Recently, it has been known that restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), sequence characterized amplified region (SCAR) marker, intergenic spacer of the nuclear ribosomal DNA (IGS), internal spacer of the nuclear ribosomal DNA And a PCR analysis technique using the molecular markers are widely used in the identification and pathogenesis of Fusarium spp. (See, for example, Baeen et al., 1997; Kawabe et al., 2005; Li et al., 1994; Migheli et al., 1998; Waalwijk et al., 1996). However, the fusarium worms, Fusarium spp. oxysporum f. sp. Primers that specifically diagnose raphani ) and PCR detection methods using the primers have not yet been developed.
In attempting to develop a specific molecular marker for fusarium oxysporum laponii to detect wilt disease pathogens infected with radish and to identify the pathogenic fungi that have been isolated, By comparing and analyzing the nucleotide sequences of genomes with other species of genus Fusarium which are very genetically similar to each other, specific primers of Fusarium oxysporum laponia species were prepared and their specificity was confirmed using PCR technique Thus completing the present invention.
It is an object of the present invention to provide a primer set for detection of Fusarium oxysporum f. Sp. Raphani causing wilt in radish and a method for detecting fusarium oxysporum f.
In order to achieve the above object, the present invention provides a primer comprising a forward primer consisting of the nucleotide sequence of SEQ ID NO: 1 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2 and a Fusarium oxysporum f. sp. A primer set for detection of raphani is provided.
In addition, the present invention provides a composition for detecting puarium oxysporum lanfani comprising the primer set.
In addition, the present invention provides a kit for detecting puarium oxysporum lanfani comprising the primer.
In addition,
1) extracting genomic DNA from fuzarium oxysporum species isolated from radish or radish directly infected with wilt fungus;
2) detecting Fusarium oxysporum f. Sp. Raphani by polymerase chain reaction (PCR) using the primer set according to
3) the DNA PCR amplification and quantification of the amplification product.
The Fusarium < RTI ID = 0.0 > oxysporum f. sp. (PCR) (polymerase chain reaction) using a primer set capable of detecting the specific DNA fragment sequence of the fusarium oxysporum leponia strain , It is possible to distinguish fusarium oxysporum lappani from other similar fungi, as well as effectively detect fusarium oxysporum lappani in a non-plant sample infected with wilt disease, It can be useful for diagnosis.
Figure 1A shows the Raphani F / Raphani R primer set of the present invention (primer set containing the nucleotide sequence shown in SEQ ID NO: 1 and 2) and the Fo F / FoR primer set (the nucleotide sequence shown in SEQ ID NO: 3 and 4 PCR amplification was carried out using genomic DNA extracted from 25 strains of fusarium oxysporum species isolated from melon, cucumber, watermelon, tomato, cabbage and radish, which are various vesicle host plants, , Followed by electrophoresis and development and staining with ethidium bromide (EtBr).
Fig. 2 is a graph showing the relationship between the genomic DNA extracted from the susceptible fungus infected individuals of the susceptible non-cultivars 'Cheongsu court' and the 'non-fungus infestant' against the puarium oxysporum lafana as a template and the Raphani F / Raphani R primer set (SEQ ID NOS: 1 and 2) and the FoF / FoR primer set (SEQ ID NOS: 3 and 4), amplified by electrophoresis, and amplified with ethidium bromide (EtBr) Fig.
Hereinafter, the present invention will be described in detail.
The present invention provides a primer set for detecting Fusarium oxysporum f. Sp. Raphani comprising a forward primer consisting of the nucleotide sequence of SEQ ID NO: 1 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2.
Preferably, the primer set amplifies a 450 bp fusarium oxysporum lapanus-specific DNA PCR product, and the primer set is selected from the group consisting of a hu- manarium incompatibility protein (HET) functional gene Is amplified.
In addition, the present invention provides a composition for detecting puarium oxysporum lanfani comprising the primer set.
In addition, the present invention provides a kit for detecting puarium oxysporum lanfani comprising the primer.
The kit preferably further comprises a DNA polymerase or a buffer solution.
In addition,
1) extracting genomic DNA from fuzarium oxysporum species isolated from radish or radish directly infected with wilt fungus;
2) detecting Fusarium oxysporum f. Sp. Raphani by polymerase chain reaction (PCR) using the primer set according to
3) the DNA PCR amplification and quantification of the amplification product.
The DNA PCR amplification product is preferably analyzed using gel electrophoresis.
In a specific embodiment of the present invention, PCR (polymerase chain reaction) was performed using the primers of SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention. As a result, among the various fusarium oxysporum strains, (See Table 4, Table 5, and Fig. 1), it is possible to distinguish fusarium oxysporum lappani from other similar fungi, as well as to isolate plants free from wilt disease The sample can also be used effectively to detect pseudomonas aeruginosa spp. Effectively and to rapidly diagnose wilt disease.
The Raphani F / Raphani R primer set according to the present invention is prepared so as to amplify a gene region of a functional region of a fusarium oxysporum lapanii nucleus incompatibility protein (HET), and an IGS (intergenic spacer of the nuclear ribosomal DNA), and ITS (internal spacer of the nuclear ribosomal DNA).
In the present specification, the term " primer " refers to a complementary single-stranded oligonucleotide sequence of a template nucleic acid strand amplified by PCR and can be used as a starting point for the synthesis of an amplification product of DNA PCR. The length and sequence design of the primer are dependent on the primer usage conditions, depending on the ratio of G, A, T, C of the primer, the formation of dimer between the primers, and the complexity of the template nucleic acid strand. The primers can initiate DNA synthesis in the presence of DNA polymerase, four different nucleotide triphosphates (dNTPs), and template nucleic acid strands at appropriate buffer solutions containing Mg2 +, at reaction temperatures and reaction times.
The kit of the present invention may contain the components necessary for the PCR reaction of detection of puarium oxysporum lanfani using the above primer set. Specifically, in addition to the primer set, a suitable amount of DNA polymerase (Thermus aquaticus, Taq), four different nucleotide triphosphates (dNTPs), a suitable buffer solution containing Mg2 + and water (ddH2O) are contained.
In addition, the kit may further include a component and a guide for performing electrophoresis to confirm the presence or absence of the PCR amplification product. Specifically, it includes a booklet in the form of a flyer containing information required for electrophoresis (Tris-acetate-EDTA, TAE 50X) and information published or provided through electronic media such as agarose and the Internet.
In addition, in the step of extracting the genomic DNA of the analytical sample, the genomic DNA may be separated according to a method commonly used in the art, and a commercially available DNA extraction kit may be used.
In addition, the nucleic acid sample may be used as a template and polymerase chain reaction (PCR) may be performed using the primer set
In the PCR reaction, DNA PCR amplification products may be separated by electrophoresis on an agarose gel stained with ethidium bromide (EtBr) to confirm DNA PCR amplification products.
The sample may include other species belonging to fusarium oxysporum and strains suspected of containing fusarium oxysporum lavonia, for example, other species belonging to fusarium oxysporum and fusarium oxysporum It may be a crop expected to contain lepani itself or other species belonging to oxysporum by infection and fuarium oxysporum lepani.
DNA PCR amplification products of the PCR reaction can be confirmed by the presence or absence of the band of the PCR product and the size of the PCR product according to the result of agarose gel electrophoresis.
Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.
However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.
< Example 1> No Wilt ( Fusarium oxysporum f. sp . raphani ) For detection primer design
<1-1> Wilt Selection of genes for detection
Fujairah Solarium Room oxy Spokane La Trapani (Fusarium oxysporum f. sp. raphani ) is very similar morphologically and genetically to other fusarium oxysporum species. GenBank (http://www.ncbi.nlm.nih.gov) published F. oxysporum f. Sp. Raphani Genes of 54 005), and Fusarium species and different soknae genomic nucleotide sequence of Fusarium Comparative Database (http://www.broadinstitute.org) compared to the specific DNA sequence variation is high whole genome shotgun sequence 316 using a program area of the ( FOQG17869).
<1-2> No Wilt Specific for detection primer design
The specific primers for the detection of fosarium oxysporum leponi were designed based on the nucleotide sequence analysis of the gene. The primer sequences are shown as forward primer and reverse primer as shown in Table 1. < tb > < TABLE >
Primers were designed so that no dimer or internal loop structure was formed between the hairpin loops structure and the primers inside the primer using the Oligonucleotide Properties Calculator (http://www.basic.northwestern.edu). The nucleotide sequence of the displayed primers of SEQ ID NOS: 3 and 4 in Table 1 below is a primer specific to Fusarium oxysporum reported in the prior art (Lin et al., 2010).
< Experimental Example 1> Various Fuarium Oxyspolom Specific Primer Used Fuarium Oxyspolom Lafani's detection
<1-1> Fuarium Oxyspolom ( Fusarium oxysporum ) Securing 25 strains
Fusarium, a pathogenic fusarium isolated from melons, cucumbers, watermelons, tomatoes, cabbages, radishes, oxysporum ) were collected and used. As shown in Table 2 below, F. oxysporum f. Melonis , which is a melon root, F. oxysporum f. Sp. Melonis , has 15 strains,
<1-2> Fuarium Oxyspolom From 25 strains Genomic DNA ( Genomic DNA) separation
The fusarium oxysporum strains thus obtained were transplanted into potato dextrose agar and cultured at 25 ° C for 7 days. Mycelia were scraped with sterile water (20 ml) into a 2 ml eppendorf tube and centrifuged to freeze the pellet at 70 ° C. A grinding the frozen mycelium using a glass bead and then in 400㎕ of extraction buffer [200mM Tris-HCl ( pH 8.0), 200mM NaCl, 30mM EDTA, 0.5% SDS] and proteinase K 37 o C by the addition of (50㎍) 1 hour. Next, add 400 μl of 2 × solution [2% CTAB (w / v), 100 mM Tris HCl (pH 8.0), 20 mM EDTA (pH 8.0), 1.4 M NaCl and 1% PVP (polyvinylpyrrolidone) After extraction with chloroform: isoamylalcohol (24: 1) and centrifugation at 12,000 rpm for 10 minutes, the supernatant was added to a 1.5 ml tube. 0.7 part of isopropanol was added to the supernatant, and the mixture was allowed to stand at room temperature for 10 minutes and then centrifuged at 12,000 rpm for 10 minutes. The precipitated DNA was washed with 70% ethanol in the same manner. After removing the ethanol in the tube, the DNA was dissolved in 50 μl of TE buffer, and 2 μl (10 mg / ml) of RNase was added to remove the RNA. The DNA was stored at 20 ° C.
<1-3> Fuarium Oxyspolom Rafani Specific Primer Used PCR detection
In Table 1, Raphani F / Raphani R, a primer set specific for pseudomonas spp., Which contains the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2, and the specific primer set for pseudomonas spp. PCR amplification and electrophoresis were performed on 25 fusarium oxysporum strains isolated from melon, cucumber, watermelon, tomato, cabbage and radish using the primer set Fo F / Fo R. The PCR reaction conditions were initial denaturation at 94 ° C for 5 min, denaturation at 94 ° C for 20 sec, annealing at 57 ° C for 19 sec and extension at 72 ° C / 35 cycles per second and final extension at 72 ° C / 5 min. The composition conditions of the PCR reaction are shown in Table 3 below.
As a result, as shown in Table 4 and FIG. 1A, in the case of the PCR amplification reaction using the base sequence shown in SEQ ID NOs: 1 and 2 in Table 1, that is, a specific primer set of Fusarium oxysporum lavonia, A specific 450 bp single amplification product was found only in the pathogenic fuzarium oxysporum laponi strain. PCR amplification product of the size of 327 bp was confirmed in all the Fusarium oxysporum strains tested by the specific primer set of fusarium oxysporum (Fo primer set consisting of the nucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 4) (Table 4 and Figure 1A).
Thus, it was determined that the Raphani primer set containing the nucleotide sequences of SEQ ID NOS: 1 and 2 of the present invention was able to specifically detect Fusarium oxysporum laferni, a non-wilt disease pathogen.
primer
primer
Detection of 25 strains of fusarium oxysporum using two kinds of primer sets
+: Primer-specific PCR products, -: No primer-specific PCR products
< Experimental Example 2> Fuarium Oxyspolom Pathogenic investigations of 15 strains and detection of puarium oxysporum lappani using specific primers
<2-1> separated from radish Fuarium Oxyspolom Pathogenicity assays for radish of 15 strains
Among the 15 fusarium oxysporum isolates isolated from radishes representing the wilting symptom collected from various regions, these strains were identified as 'Fusarium oxysporum laminitis', which is a pathogenic pathogen, And the pathogenicity was investigated. Fifteen strains of fusarium oxysporum were inoculated on potato agar medium (PDA) and cultured at 25 ° C for 7 days. Six mycelium pieces were detached from the pathogen microflora formed on the PDA medium, 100 ml of malt extract broth was inoculated and cultured at 25 ° C for 7 days with shaking at 150 rpm. Then, the cultured medium was filtered with 4 layers of gauze to remove mycelia, and the spores were harvested by centrifugation. The precipitated spores were suspended in sterilized water, and the spore concentration was measured using a hemocytometer, diluted with sterilized water, and adjusted to a concentration of 1 × 10 7 conidia / ml to prepare a spore suspension to be used as an inoculum.
The seedlings were inoculated with 40 ml of spore suspension prepared by inoculating the seedlings and removing the soil of the host plants ('Cheongwoo court' wilt disease susceptible non-cultivar) roots grown in the greenhouse for 12 days and inoculated for 30 minutes. The immersed seedlings were transferred to a new pot with horticultural soil and treated for 24 hours at 25 ° C. After 25 o C growth, the plants were cultivated under light irradiation for 12 hours a day, and the occurrence of wilt was investigated 25 days after inoculation. The severity of the disease was determined by the following disease index of 0-5: 0 = healthy, 1 = browning in the lower part, no symptoms in the upper part, 2 = browning in the lower part, 3) The underground part was browned, the growth part of the ground part was suppressed, and the part was slightly sulphated. 4) The undergrowth was browned, the growth was suppressed in both the underground part and the ground part, and 6) The mean disease incidence was 1.0 or less, and non-pathogenic.
Of the 15 strains tested, 7 fusarium oxysporum strains (57, 58, 59, 60, 147, 151, DN-2) were browned at the root of the plants after 25 days of inoculation, A typical symptomless vesicular disease was observed which was sulphated and showed a high degree of wilt disease of 3.3 or more. Therefore, these seven strains were identified as fusarium oxysporum lappani. The remaining 8 strains were found to be less than 0.8 in the inoculated seedlings, indicating that they were non-pathogenic fosarium oxysporum strains other than fusarium oxysporum lappani (Table 5).
+: Pathogenic, -: Pathogenic
<2-2> Fuarium Oxyspolom From 15 strains Genomic ( Genomic ) DNA Isolation
To isolate genomic DNA, each strain was inoculated on a potato agar medium and cultured at 25 ° C for 7 days. Mycelia of the cultivated strains were scraped together with sterilized water (20 ml), placed in a 2 ml eppendorf tube, and centrifuged to freeze the pellet at 70 ° C. A grinding the frozen mycelium using a glass bead and then in 400㎕ of extraction buffer [200mM Tris-HCl ( pH 8.0), 200mM NaCl, 30mM EDTA, 0.5% SDS] and proteinase K 37 o C by the addition of (50㎍) 1 hour. Next, add 400 μl of 2 × solution [2% CTAB (w / v), 100 mM Tris HCl (pH 8.0), 20 mM EDTA (pH 8.0), 1.4 M NaCl and 1% PVP (polyvinylpyrrolidone) After extraction with chloroform: isoamylalcohol (24: 1) and centrifugation at 12,000 rpm for 10 minutes, the supernatant was added to a 1.5 ml tube. 0.7 part of isopropanol was added to the supernatant, and the mixture was allowed to stand at room temperature for 10 minutes and then centrifuged at 12,000 rpm for 10 minutes. The precipitated DNA was washed with 70% ethanol in the same manner. After removing the ethanol in the tube, the DNA was dissolved in 50 μl of TE buffer, and 2 μl (10 mg / ml) of RNase was added to remove the RNA. The DNA was stored at 20 ° C.
<2-3> Fuarium Oxyspolom Rafani Specific Primer Used PCR detection
Using the primer set containing the nucleotide sequences shown in SEQ ID NOs: 1 and 2 and SEQ ID NOs: 3 and 4 in Table 2 above, PCR amplification and electrophoresis were performed on 15 Fusarium oxysporum isolates isolated from radish Respectively. The PCR reaction conditions are the same as those in Experimental Example 1-3, and the composition conditions of the PCR reaction are shown in Table 6 below.
As a result, as shown in Table 7 and FIG. 1B, in the case of the PCR amplification reaction using the primer set of the nucleotide sequence shown in SEQ ID NOs: 1 and 2 in Table 1, the fucosyltransferase A single 450 bp amplification product was detected only in Phanomyces spp., But no primer - specific amplification products were detected in 8 non - pathogenic fuarium oxysporum strains. In addition, PCR amplification products of 327 bp in size were confirmed in all of the tested puarium oxysporum strains by a specific primer set of fuarium oxysporum (Fo primer set consisting of the nucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 4) (Table 7 and Figure 1B).
primer
+: Primer-specific PCR products, -: No primer-specific PCR products
Therefore, it has been determined that the Raphani primer set containing the nucleotide sequences of SEQ ID NOS: 1 and 2 of the present invention can be species-specifically detected for the causative pathogenic wiltless fungus strain, i.e., fuarium oxysporum lappani.
< Experimental Example 3> Wilt fungus Detection of pathogens in infected plants
≪ 3-1 > Genomic DNA ( Genomic DNA) separation
Genomic DNA was isolated to detect pathogens from non - plants infected with wilt disease. The stems (the portion corresponding to 2 cm above the border of the upper part of the soil and the soil at the border of the upper part of the soil and the soil) of the seeds of 'Cheongsu court' and 'Ungangunjang' (wicker susceptible non-susceptible varieties) cultivated for 17 days after the inoculation of the wilt- After washing with water and removing the water, it was put in a 2 ml eppendorf tube and freezed at 70 ° C and then ground using a glass bead. Then, 400 μl of extraction buffer [200 mM Tris-HCl (pH 8.0), 200 mM NaCl, 30 mM EDTA, 0.5% SDS ] And proteinase K (50)) were added and treated at 37 ° C for 1 hour. Then, 400 μl of 2 × solution [2% CTAB (w / v), 100 mM Tris HCl (pH 8.0), 20 mM EDTA (pH 8.0), 1.4 M NaCl and 1% PVP (polyvinylpyrrolidone) The supernatant was extracted with chloroform: isoamylalcohol (24: 1) and centrifuged at 12,000 rpm for 10 minutes. 0.7 part of isopropanol was added to the supernatant, and the mixture was allowed to stand at room temperature for 10 minutes and then centrifuged at 12,000 rpm for 10 minutes. The precipitated DNA was washed in the same manner with 70% ethanol. After removing the ethanol in the tube, the DNA was dissolved in 50 μl of TE buffer, and 2 μl (10 mg / ml) of RNase was added to remove the RNA. The DNA was stored at 20 ° C. Genomic DNA was isolated in the same manner also in healthy non-plants ('Cheongsu court' and 'non-infested' varieties) that did not infect wilt disease with control.
<3-2> Wilt fungus Two infected non-breed From seedlings Species-specific primer Using a set Fuarium Oxyspolom Lafani's detection
Using the genomic DNA isolated in Experimental Example <3-1> as a template, primers set forth in SEQ ID NOs: 1 and 2 and SEQ ID NOs: 3 and 4 in Table 1 were used for PCR amplification and Electrophoresis was performed. The PCR reaction conditions are the same as those in Experimental Example 1-3, and the composition conditions of the PCR reaction are shown in Table 8 below.
As a result, as shown in Table 9 and FIG. 2, in the PCR amplification reaction using the primer set including the nucleotide sequences shown in SEQ ID NOS: 1 and 2 and SEQ ID NOS: 3 and 4 in Table 1, A single amplification product of 450 bp, a specific product of fuarium oxysporum laponia, appeared in the non - planted plants of 'Cheongsu court' and 'Non - coniferous seeds' (worm - sensitive susceptible non - cultivars), and healthy whole seedlings (Fig. 9 and Fig. 2), no specific amplification product was observed in the case of the " Cheongsu court " and " Thus, using the primer set of the present invention, it is possible to obtain Fusarium < RTI ID = 0.0 > oxysporum f. sp. raphani ) can be diagnosed.
primer
<110> Korea Research Institute of Chemical Technology <120> Primer set for detecting Fusarium oxysporum f. sp. raphani and method for detecting F. oxysporum f. sp. raphani using the same <130> 15P-09-002 <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 tggatcagcc aggagataag cctct 25 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gctatcaacc catagatggc tgatctgt 28 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 taccacttgt tgcctcggcg gatcag 26 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ttgaggaacg cgaattaacg cgagtc 26
Claims (8)
2) detecting Fusarium oxysporum f. Sp. Raphani by polymerase chain reaction (PCR) using the primer set according to claim 1 as a template of the nucleic acid sample; And
3) the DNA PCR amplification and quantification of the amplification product.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| KR101913742B1 (en) | 2017-08-21 | 2018-10-31 | 경상대학교산학협력단 | Specific primer set for detecting Fusarium oxysporum f.sp fragariae and uses thereof |
| WO2022102726A1 (en) * | 2020-11-11 | 2022-05-19 | 国立研究開発法人理化学研究所 | Method of diagnosing fusarium oxysporum soil by pathogen genomics |
| CN116083630A (en) * | 2022-12-31 | 2023-05-09 | 昆明理工大学 | Primer group for detecting pathogenic bacteria Fusarium oxysporum of root rot of pseudo-ginseng by real-time fluorescent LAMP |
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| JPH10234380A (en) * | 1997-02-28 | 1998-09-08 | Shinkinrui Kinou Kaihatsu Kenkyusho:Kk | Nucleic acid sequence for detecting fungus of genus fusarium |
| KR101361296B1 (en) * | 2013-02-05 | 2014-02-12 | 충남대학교산학협력단 | Primer set for binding marker involving in fusarium wilt resistance and selection method using them |
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| JPH10234380A (en) * | 1997-02-28 | 1998-09-08 | Shinkinrui Kinou Kaihatsu Kenkyusho:Kk | Nucleic acid sequence for detecting fungus of genus fusarium |
| KR101361296B1 (en) * | 2013-02-05 | 2014-02-12 | 충남대학교산학협력단 | Primer set for binding marker involving in fusarium wilt resistance and selection method using them |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101913742B1 (en) | 2017-08-21 | 2018-10-31 | 경상대학교산학협력단 | Specific primer set for detecting Fusarium oxysporum f.sp fragariae and uses thereof |
| WO2019039653A1 (en) * | 2017-08-21 | 2019-02-28 | 경상대학교산학협력단 | Primer set for specifically detecting fusarium oxysporum f. sp fragariae and use thereof |
| WO2022102726A1 (en) * | 2020-11-11 | 2022-05-19 | 国立研究開発法人理化学研究所 | Method of diagnosing fusarium oxysporum soil by pathogen genomics |
| CN116083630A (en) * | 2022-12-31 | 2023-05-09 | 昆明理工大学 | Primer group for detecting pathogenic bacteria Fusarium oxysporum of root rot of pseudo-ginseng by real-time fluorescent LAMP |
| CN116083630B (en) * | 2022-12-31 | 2024-04-12 | 昆明理工大学 | Primer group for detecting pathogenic bacteria Fusarium oxysporum of root rot of pseudo-ginseng by real-time fluorescent LAMP |
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