KR101491776B1 - Specific primer pair for rapid detection of Fusarium verticillioides, a causal agent of corn ear rot, and method for detecting Fusarium verticillioides using the same - Google Patents

Abstract

The present invention relates to a primer pair for detecting maize corn rot fungi, and a method for detecting maize corn rot fungi. More particularly, the present invention relates to a primer pair capable of specifically detecting a maize corn bacterium in a sample and a corn ear rot A kit for detecting bacteria and a kit for detecting corn sprouting bacteria. The primer pair, detection method, and detection kit of the present invention are effective in quickly, easily and accurately confirming the presence of corn borer disease bacteria in an unknown sample.

Description

A PCR primer pair for detection of maize rot disease and a method for detecting corn spider rot disease using the same primer pair for rapid detection of Fusarium verticillioides, a causal agent of corn ear rot, and detecting Fusarium verticillioides using the same method.

The present invention relates to a method for producing corn sprouts, The present invention relates to a primer pair for detecting corn borer disease, a pair of primers for detecting corn borer disease having two nucleotide sequences selected from the group consisting of SEQ ID NOS: 1 to 4, And a method for detecting corn spider rot disease by PCR method using the same.

Fusarium verticillioides may cause corn blight disease (rot), root rot (root disease), stem rot (neck disease), ear rot (Root-, stalk-, and ear-infecting Fusarium species on corn in the USA. (Fusarium moniliforme, Canadian Journal of Botany 74: 31-36), is a plant pathogenic fungus that infects nutritional and reproductive organs without distinct disease symptoms (Fusarium fisorum, Fusarium diisese, Biology and Taxanomy, eds Nelson PE, 1195-1202). Infected young plants typically die early, exhibiting counterfeit symptoms. The leaves become blurred green when growing, the lower stem becomes dark green - straw straw color, the inner part of the stem becomes red color, and because it shows softness symptoms, loss of water is caused by obstruction of movement of water and nutrients. In corn fruit, the fruit husks and lignin part rotten with pink or reddish brown color, and mycelia of mold may appear as disease progresses. Worldwide, host plants include corn, sorghum, sugar cane, wheat, cotton, bananas, pineapples and tomatoes. F. verticillioides is also known as a fungus that can cause various infections such as keratitis, necrotic fungus, skin infection, arthritis and peritonitis in immunosuppressed or trauma patients as well as plants (a case of nail fungus caused by Fusarium verticillioides) 13 (1): 26-30). Many strains of pathogens produce a variety of toxins that are toxic to humans and livestock, and in particular Fumonisin has been shown to be a useful agent in the treatment of horseshoe leukoencephalitis (Fusarium moniliforme and fumonisins in Australian maize in relation to animal disease. In Toxic Plants and Other Natural Toxicants, eds T. Garland and AC Barr, pp. 474-478, Fumonisin B1 production and vegetative compatibility of strains from Gibberella fujikuroi mating population A (Fusarium moniliforme), Mycopathologia 117, 37-45) (Fumonisin occurrence in corn from high- and low-risk areas for human esophageal cancer in China. Appl. Environ. Microbiol. 60: 1626-1629).

Pathways of pathogens in corn vary. The most common methods of infection are mycelium in the remains of the infected crops on the surface of the soil or soil, forming spores and then spreading by wind or rain to infect corn. Another infection pathway is systemic infection of the corn by Fusarium moniliforme. Phytopathology 52: 870-872. Infections originate from the seed surface or inside the seeds or mycelium. The fungus develops from the inside of the young plants, moves from the roots, and eventually reaches the corn and the ears (Fumonisins in maize: can we reduce their occurrence Plant Dis. 81: 556-565). Systemic infections are also likely to be caused by inocula that are alive in the soil sick leave. Insects such as corn and moths act as vectors that carry pathogens, or scratches and bodily stresses can promote the onset of disease (Factors that affect the occurrence of fumonisin. Environmental Health Perspectives, 109: 321-324).

Fusarium moniliforme has been reported as a causative agent of corn borer rot in Korea, but F. moniliforme has not been used as a species complex containing several morphologically similar species. As a result of molecular biology, the corn borer was identified as F. verticillioides , and recently Choi et al. (Identification of Fusarium spp. Involved in corn borer disease. Korean Journal of Infectious Diseases 37 (2): 121-129) F. verticillioides as well as Fusarium graminearum and Fusarium subglutinans were identified as cornborne rot fungi, among which F. verticillioides was the highest isolate. F. verticillioides has been reported to be effective against Fusarium fujikuroi, Fusarium fujikuroi, Fusarium proliferatum, and F. subglutinas , which are corn stalks, as well as Fusarium andiyazi causing stem rot in millet. . Health Perspect 109:. 239-243) and the Fusarium thapsinum Klittich et al (Aggressiveness of Gibberella fujikuroi (Fusarium moniliforme) Isolates to Grain Sorghum Under Greenhouse Conditions Plant Disease 76 (9):.. 897-900) as transient morphological very It is known to be similar.

With the development of molecular biology, a variety of bacteria detection methods have been developed. In this PCR method using a speci fi c primer, a DNA fragment (genomic DNA) to be analyzed is analyzed with a DNA polymerase and DNA When dNTP (deoxyribonucleotide triphosphate), a substance necessary for synthesis, is added and the synthesis reaction is repeatedly performed, the region to which the primer is bound is rapidly amplified. The PCR amplification product is electrophoresed on an agarose gel, and ethidium bromide Ethidium bromide staining with DNA bands. F. verticillioides- specific primers are designed to react with similar Fusarium species that are morphologically similar and difficult to distinguish by microscopy, so that F. verticillioides can be specifically detected. In addition, the present invention solves the problems encountered in the conventional microscopic observation for detecting F. verticillioides , which makes it easier and more accurate to diagnose, and the time required for diagnosing pathogens infection in plant tissues as well as in seeds . In order to solve the conventional problems, there is a need for rapid and accurate diagnosis of cornborne rot disease. Therefore, a specific primer capable of detecting the pathogen in a species-specific manner has been developed, and a method for detecting a bacterium in corn- Is required.

Accordingly, the present inventors have conducted a series of studies to develop a novel method for specifically detecting F. verticillioides , and as a result, have developed a new pair of primers capable of specifically detecting corn spider rot disease , And it was confirmed that corn sprouting bacteria can be effectively detected in an unknown sample using the same, thus completing the present invention.

Accordingly, an object of the present invention is to provide a pair of primers for detecting corn borer disease and a method for detecting corn borer disease.

In order to achieve the above object, the present invention provides a primer pair for detecting F. verticillioides having two nucleotide sequences selected from the group consisting of SEQ ID NOS: 1 to 4.

In order to achieve another object of the present invention,

(a) separating a DNA sample from the corn seed to be discriminated;

(b) carrying out PCR using the DNA isolated in (a) as a template and using the primer pair of claim 1; and

(c) detecting the amplified product in step (b);

( F. verticillioides ), which comprises the steps of:

In order to accomplish still another object of the present invention, the present invention provides a kit for detecting F. verticillioides comprising the pair of primers for detection of the present invention.

In order to accomplish still another object of the present invention, the present invention provides a method for producing a polynucleotide comprising a marker specifically detecting F. verticillioides comprising a polynucleotide represented by SEQ ID NO: 5, And a microarray for detecting F. verticillioides .

Hereinafter, the present invention will be described in detail.

The invention corn ears having two sequences selected from the group consisting of SEQ ID NO: SEQ ID NO: 1 to 4 rot fungus (Fusarium verticillioides ) detection primer pairs.

The primer pair for detecting F. verticillioides of the present invention specifically detects maize corn rot fungi and not only does not detect other fungi belonging to the genus Fusarium due to corn sprouting, Is not detected. Accordingly, the primer pair for detecting corn borer disease of the present invention can specifically detect the corn borer disease.

The primer pair for detecting corn sprout disease bacteria of the present invention may preferably be a primer pair for detecting the corn borer disease selected in SEQ ID NO: More preferably, the pair of primers for detecting corn borer disease of the present invention may be a pair of primers represented by SEQ ID NOS: 1 and 2 or a pair of primers represented by SEQ ID NOS: 3 and 4, respectively. The primer pair does not show specific amplification with other fungi of the genus Fusarium and can detect all the isolates present in the species and thus can be used for more accurate detection.

The primer pair of the present invention can be usefully used in the detection of corn spider rot disease bacteria in a sample. Accordingly, the present invention provides a method for detecting corn borer disease from a sample comprising performing PCR using the primer pair.

More specifically, the present invention relates to

(a) separating a DNA sample from the corn seed to be discriminated;

(b) carrying out PCR using the DNA isolated in (a) as a template and using the primer pair of claim 1; and

(c) detecting the amplified product in step (b);

( F. verticillioides ), which comprises the steps of:

The DNA may be separated in step (a) according to a method commonly used in the art, and may be carried out using a commercially available DNA extraction kit.

In the step (b), PCR is performed using the separated DNA as a template, and a PCR reaction is carried out using SEQ ID NO: 1 and SEQ ID NO: 3 of the primer pair of the present invention as a forward primer and SEQ ID NO: 2 and SEQ ID NO: 4 as a reverse primer Can be performed.

In the step (c), the DNA of the PCR product can be isolated by size according to a method well known in the art. Preferably by agarose gel or polyacrylamide gel electrophoresis or an ABI prism 3100 genetic analyzer-electropherogram. In this case, in order to use the fluorescence analyzer, PCR is performed in step (b) using a pair of primers having a fluorescent dye well known in the art. The PCR amplification result can preferably be confirmed by agarose gel electrophoresis. After electrophoresis, electrophoresis results can be analyzed by ethidium bromide staining. Methods for performing general PCR and analyzing the results are well known in the art.

The present invention also provides a kit for detecting F. verticillioides comprising a pair of primers according to the present invention. In order to facilitate the PCR reaction in addition to the primer pair, the kit may further include a PCR reaction buffer solution of the DNA polymerase and the composition described above, and may be subjected to electrophoresis The necessary components may be further included in the kit of the present invention.

The kit of the present invention includes various reagents necessary for the detection (for example, PCR) and confirmation of the results of PCR for detecting corn borer disease by using the primer pair, for example, PCR reagents, restriction enzymes, agarose, A buffer solution necessary for migration, and the like. In more detail, the PCR composition may be prepared by mixing a suitable DNA polymerase (eg, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, and the like) in addition to the pair of complementary DNAs synthesized by the reverse transcription reaction and the PCR primers provided in the present invention. Thermostable DNA polymerases from Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu), dNTPs, PCR buffer solutions and water (dH2O). The PCR buffer solution includes Tris-HCl, MgCl2, KCl, and the like. At this time, the MgCl2 concentration greatly affects the specificity and yield of amplification. Generally, if an excess of the Mg ^{2 +} increase the non-specific PCR amplification products, and reduce the yield of a PCR product if the Mg ^{2 +} insufficient. The PCR buffer solution includes, but is not limited to, Triton X-100, dimethylsufoxide (DMSO), Tween 20, nonidet P40, PEG 6000, formamide and bovine serum albumin bovine serum albumin (BSA)) may be further included.

In addition, the kit may further include a DNA polymerase and a PCR reaction buffer solution having the above composition in order to facilitate the PCR reaction, and a kit for performing electrophoresis, May be further included in the kit of the present invention.

Meanwhile, the present invention provides a marker that specifically detects F. verticillioides with the polynucleotide of SEQ ID NO: 5.

The present invention also provides a microarray for detecting F. verticillioides comprising polynucleotides of SEQ ID NO: 5 or complementary polynucleotides thereof.

The marker and the microarray of the present invention are characterized by specifically detecting the corn borer disease.

The microarray may be one comprising a DMA or an RNA polynucleotide. The microarray is made of a conventional microarray except that it contains the polynucleotide of the present invention, and a method for producing a microarray by immobilizing a polynucleotide used as a marker on an organ is well known in the art.

As described above, the present invention provides a pair of primers capable of specifically detecting F. verticillioides in a sample, a method for detecting corn borer disease bacteria and a kit for detecting corn borer disease. The primer pair, detection method, and detection kit of the present invention can be used in industrial and quarantine sites because it is possible to quickly, easily, and accurately identify the presence of corn borer disease bacteria in an unknown sample.

Figure 1 using the primers of SEQ ID NOS: 6 and 7 illustrating the PCR amplification products obtained from F. verticillioides and Fusarium similar bacteria (L: DNA ladder, 1: F. annulatum, 2: F. fusikuroi, 3: F. nygamai, 4:. F. proliferatum 5 : F. sacchari, 6: F. subglutinans, 7: F. thapsinum, 8: F. verticilloides, 9: F. condolor, 10: F. globosum, 11: F. nisikadoi, 12: F. asiaticum, 13: F. solani, 14: F. oxysporum, 15: F. graminearum, 16: F. lactis, 17: F. succisae, 18: F. circinatum, 19: F. concentrichum, 20: F. napiforme, 21: F. oxysporum f sp lycopersici, 22:.... F. oxysporum f sp radicis - lycopersici).
FIG. 2 shows PCR results of F. verticillioides using the primers of SEQ ID NO: 1 [FVF (155-174)] and SEQ ID NO: 2 [FVR (834-853)]. Here, genomic DNA of Table 2 was used as a template.
FIG. 3 shows PCR diagnosis results of F. verticillioides using the primers of SEQ ID NO: 3 [FVF (511-530)] and SEQ ID NO: 4 [FVR (833-852)]. Here, genomic DNA of Table 2 was used as a template.
FIG. 4 shows the result of PCR amplification according to the annealing temperature of two pairs of maize corn borer specific primers (L: DNA ladder, 1-5: FVF (155-174) / FVR (834-853) 6-10: FVF (511-530) / FVR (833-852) primer pair, 1 and 6: 50 ℃, 2 and 7: 55 ℃, 3 and 8: 58 ℃, 4 and 9: 10: 64 ° C).
FIG. 5 shows the results of PCR amplification of DNA pairs (l: DNA ladder, 1-8: FVF (155-174) / FVR (834-853)) of the template DNA concentration (ng / 1 and 9: 2 ng, 2 and 10: 1 ng, 3 and 11: 0.5 ng, 4 and 12: 0.25 ng, and 5 and 9-16: FVF (511-530) / FVR (833-852) 13: 0.125 ng, 6 and 14: 0.0625 ng, 7 and 15: 0.03125 ng, 8 and 16: 0 ng).
FIG. 6 is a PCR amplification result of detecting maize corn rot fungi from corn seeds infected with cornstalks (L: DNA ladder, 1-6: FVF (155-174) / FVR (834-853) -12: FVF (511-530) / FVR (833-852) primer pairs, 1-4, 7-10: diseased corn seeds, 5,6,11,12: healthy corn seeds).

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 >

Corn sprout rot disease Fusarium verticillioides ) Design of detection primer

<1-1> Selection of genes for detection of maize corn rot fungi

Corn sprout rot fungi are morphologically similar and have various genetically related species. Genes used for phylogenetic classification are not suitable for species specific primer design due to very low sequence variation. Based on the nucleotide sequence in Chromosome 6 supercontig 2 of the F. verticillioides 7600 strain of the Fusarium comparative database (http://www.broadinstitute.org/annotation/genome/fusarium_group/MultiHome.htm), a pair of oligonucleotide primer pairs The primers shown in SEQ ID NOs: 6 and 7 were prepared, and F. verticillioides PCR was performed on NRRL22001 and similar species (Fig. 1). The PCR reaction conditions were predenaturation at 94 ° C for 5 min, denaturation at 94 ° C for 1 min, annealing at 58 ° C for 1 min, extension at 72 ° C for 2 min, and finally post-extension at 72 ° C for 7 min. The nucleotide sequence of PCR amplification bands of F. verticillioides NRRL22001 was analyzed and used for the development of species specific primers. As a result of analyzing the similarity of base sequences using the Fusarium comparative database, it was found that the analyzed sequence (SEQ ID NO: 5) was 99% homologous with Chromosome 6 supercontig 2 1793627-1794653 of F. verticillioides 7600 strain and F. verticillioides Gene.

<1-2> Corn corn rot fungi Fusarium verticillioides ) Design and amplification product of primer for detection

The primer for the detection of maize corn rot fungi was designed based on the nucleotide sequence analysis of the above gene and designed to detect the corn borer disease. Therefore, two forward primers and two reverse primers were designed as primer sequences for detecting corn sprout disease bacteria as shown in Table 1 below.

Name of the primer Base sequence SEQ ID NO: Amplification Size (bp) Forward FVF (155-174) 5'-GGTTGGCTCAGATGTGTATG-3 ' One 699 Reverse FVR (834-853) 5'-GTCAAGGATACCTAGCTAGC-3 ' 2 Forward FVF (511-530) 5'-GCAATGTCAGTCAGAACTAG-3 ' 3 342 Reverse FVR (833-852) 5'-TCAAGGATACCTAGCTAGCT-3 ' 4

< Example  2>

Maize corn rot fungus  For detection Primer  Species specificity test

In order to test the species specificity of corn sprouting bacteria for each of the above primer pairs FVF (155-174) / FVR (834-853) and FVF (511-530) / FVR (833-852) The strains purchased from the US ARS collection center were used. Table 2 below shows the number of 24 Fusarium species used for identification of primers for the detection of maize corn rot fungi Bell And strain number. Genomic DNA of Fusarium was inoculated into Potato dextrose broth (PDB) medium and cultured at 25 ℃ for 3 days. The mycelium was collected with miracloth, lyophilized, and then extracted with DNeasy kit (QIAGEN, Germany). Then, PCR was carried out using the above two primer pairs. The results are shown in FIG. 2 and FIG. The PCR reaction conditions were: predenaturation at 94 ° C for 5 min, denaturation at 94 ° C for 1 min, annealing at 61 ° C for 1 min, extension at 72 ° C for 2 min, and finally post-extension at 72 ° C for 7 min. Bands appeared only in lanes 8, 9, and 10, which were PCR-primed with corn borer rot fungus in both primer pairs. These results confirmed that the two primer pairs were not amplified in Fusarium spp. Other than F. verticillioides .

lane Class name Strain number lane Class name Strain number One F. annulatum NRRL13614 13 F. nisikadoi NRRL25308 2 F. fusikuroi NRRL22010 14 F. asiaticum NRRL13818 3 F. nygamai NRRL25449 15 F. solani NRRL26156 4 F. proliferatum NRRL20983 16 F. oxysporum NAAS1205 5 F. sacchari NRRL22006 17 F. graminearum CF535 6 F. subglutinans NRRL13588 18 F. lactis SPF01 7 F. thapsinum NRRL22007 19 F. succicae F1 8 F. verticilloides NRRL22001 20 F. circinatum NRRL25331 9 F. verticilloides NRRL20956 21 F. concentrichum NRRL25181 10 F. verticilloides CF449 22 F. napiforme NRRL13604 11 F. condolor NRRL13994 23 F. o f. sp . lycopersici CBS306.91 12 F. globosum NRRL25190 24 F. o f. sp . radicis - lycopersici KACC40031

< Example  3>

Maize corn rot fungus  For detection Primer pair PCR  Establish condition

In order to optimize the PCR reaction conditions of the two primer pairs selected in the present invention, the PCR reaction according to the annealing temperature was examined. At this time, the mold was placed in the US ARS collection center prevalent strain ( Fusarium verticillioides NRRL22001), respectively, and the PCR analysis results are shown in FIG. As shown in FIG. 4, the FVF (155-174) / FVR (834-853) primer pair was diagnosed in the temperature range of 55 ° C and 64 ° C, but at 50 ° C and 58 ° C, nonspecific bands appeared The optimal annealing temperature was in the range of 61 ℃ to 64 ℃ rather than the temperature range. PCR was performed at 50 ℃ ~ 64 ℃ according to annealing temperature using FVF (511-530) / FVR (833-852) primer pair.

< Example  4>

Maize corn rot fungus  For detection Primer pair  template DNA  By concentration PCR  reaction

In order to examine the sensitivities of the two primer pairs selected in the present invention according to the template DNA concentration, usarium DNA was isolated from the verticillioides NRRL22001 strain, and PCR was performed at 20 ng / μl to 0.625 ng / μl concentration. The results are shown in FIG. As shown in FIG. 5, the concentration of the band of FVF (155-174) / FVR (834-853) primer and FVF (511-530) / FVR (833-852) Except for the case that DNA was not included, a band appeared at the lowest concentration of 0.625 ng / μl, and diagnosis was possible.

&Lt; Example 5 >

Selected two The primer pair  Establishment of detection method of bacteria in used corn seeds

The selected primers were used to establish the method to investigate the infection of maize seeds with maize seed rot. The healthy and diseased seeds were finely ground and DNA was extracted with extraction buffer (200 mM Tris-HCl (pH 8.0), 300 mM NaCl, 25 mM EDTA (pH 8.0), 1% sodium dodecylsulfate) As a template. The results of PCR amplification using the two selected primer pairs are shown in FIG. No band appeared in healthy seeds but band appeared from diseased seeds. Through the above procedure, a specific primer for detecting corn sprouting bacteria of the present invention and a method for detecting bacteria using the same were completed using two pairs of primers.

Therefore, PCR can be carried out using a specific primer pair for detection of corn borer disease bacteria, so that it is possible to detect species-specific, rapidly and accurately whether or not the maize seeds have been infected with corn borers.

Accordingly, the present invention provides a primer pair capable of specifically detecting Fusarium verticillioides in a sample, a method for detecting corncorn rot disease bacteria and a kit for detecting corncorn rot disease using the same. The primer pair, detection method, and detection kit of the present invention are effective in fast, simple, and accurate confirmation of the presence of corn borer disease bacteria in an unknown sample, and thus are highly industrially applicable.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Specific primer pair for rapid detection of Fusarium          verticillioides, a causal agent of corn ear rot, and method for          detecting Fusarium verticillioides using the same specific primer          pair for rapid detection of Fusarium verticillioides, a causal          agent of corn ear rot, and method for detecting Fusarium          verticillioides using the same <130> NP12-0072 <160> 7 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FVF (155-174) <400> 1 ggttggctca gatgtgtatg 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FVR (834-853) <400> 2 gtcaaggata cctagctagc 20 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > FVF (511-530) <400> 3 gcaatgtcag tcagaacta 19 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FVR (833-852) <400> 4 tcaaggatac ctagctagct 20 <210> 5 <211> 1026 <212> DNA <213> Artificial Sequence <220> <223> Fusarium verticillioides specific marker <400> 5 gaggaagagt tgcgactttc ctcgcccggt cgagctggas ccacccgatg ggtgcaggtg 60 tgatatatgt ccccgggctt ggctggaagc aggtcgatgg cagtgaaaag gtaggtaact 120 acttctttgc tgtgcgtaat ccgaggcctc atagggttgg ctcagatgtg tatgattcat 180 gttgctatac ggttacacaa aagtttgggg tcgcaaatga tcgaatgaca gtcaattgta 240 atatagtgta aaatgattag gtagttcgcc agctgaaagg ctcttactat gtaaagtata 300 atccgatagg ggatttgtta actattaaat tggcgtgatt ccacataaag ctcctgtaac 360 agggcatgcg tcatgcgagg atcaaacttg gtcctgctag agactaccaa tgggcatttt 420 ggttggtgag aactattctc ttcgctcagc ttacctattg tagaaagag cgtacctgta 480 tactgtaagg ctggggagtt gtcgtcgttt gcaatgtcag tcagaactag cagttcctaa 540 tgtcggaatg ggcaccaccg aaccagtaac atctcacagc atgttgcggc tctgcgagct 600 gatacagctt catgctcctg gatccctacc actaataagg atttacagct taccgggata 660 tgatccttgg cgtaacaggt tttcgcgttg cacgtcaact cgtacagaac aaacagtgcc 720 tcacgttgag catgaggtat ctatttggtg tatttgttct gagcaacagc gaatcgaatc 780 tagaagacct caattatcta agagggtctt cacatgtttt gataggtacc ttagctagct 840 aggtatcctt gaccagacaa ttacgaggca ctcactgtac gtcgggcttc atacagctac 900 ccgagacaac cccgcccact aagatccaat gccttagccg acamcgccag ggcagattcc 960 gctaattaag agggctgaca gactccaaaa cgccctatga tgagcctccg atacagtgca 1020 agacca 1026 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FVUP 12F <400> 6 ggtcttgcac tgtatcggag 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FVUP1021R <400> 7 acgaggaaga gttgcgaact 20

Claims (6)

A pair of primers for detecting Fusarium verticillioides which is a primer pair represented by SEQ ID NO: 1 or 2 or a primer pair represented by SEQ ID NO: 3 or 4.
delete (a) separating DNA from a sample;
(b) carrying out PCR using the DNA isolated in (a) as a template and using the primer pair of claim 1; and
(c) detecting the amplified product in step (b);
( Fusarium &lt; RTI ID = 0.0 &gt; verticillioides ) detection method.
A kit for detecting Fusarium verticillioides comprising the primer pair of claim 1.
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