KR102000469B1 - Tomato spotted wilt virus disease resistance gene Tsw specific primer set for pepper cultivar and uses thereof - Google Patents

Abstract

The present invention relates to a primer set specific to Tsw, a gene resistant to a tomato spotted wilt virus (TSWV), and to a usage thereof, and more specifically to a primer set for identifying red pepper varieties resistant or sensitive to TSWV, a kit comprising the primer set, and a method for identifying red pepper varieties resistant or sensitive to TSWV using the primer set. According to the present invention, a PCR execution using the primer set specific to Tsw genes enable specific detection of the Tsw genes, genes resistant against TSVW in a red pepper plant, thereby being used in identifying red pepper varieties resistant or sensitive to TSWV. Accordingly, since there is no need for a progeny test which should be executed for each generation for growing red peppers resistant to TSWV, time, costs and efforts for growing can be reduced.

Description

The pepper TSWV disease resistance gene Tsw-specific primer set and its use {Tomato spotted wilt virus disease resistance gene Tsw specific primer set for pepper cultivar and uses thereof}

The present invention relates to a primer set specific to Tsw , a Tomato spotted wilt virus (TSWV) disease resistance gene, and its use. More particularly, the present invention relates to a primer set for TSWV disease-susceptible or susceptible pepper variety identification, a kit including the primer set, and a method for discriminating TSWV disease-susceptible or susceptible pepper cultivars using the primer set.

Tomato spotted wilt virus (TSWV) belongs to the Bunyaviridae family, Tospovirus genus, and was first discovered in the 1920s. The genome of the TSWV is a segmented genome divided into three parts and consists of RNAs of about 80-110 nm in size. TSWV has a very broad host range and can infect about 900 plants. Specifically, TSWV violates crops such as tobacco, tomato, celery, peanut, pepper, soybean, potato, pea, eggplant, spinach, lettuce, cucumber, cabbage and leafy vegetables. I will. Thripidae (Thripidae), Thysanoptera) of various species, such as tobacco thrush ( Frankliniella fusca), flower thrips (F. occidentalis), blossom thrips (F. schultzei), cucumber thrips (Thrips palmi), thistle thrips (T. setosus), par thrips (T. tabaci), the convex side dish Scirtothrips and many other infections. Therefore, the removal of thrips is one of the most important control measures.

On the other hand, in the red pepper, the flower yellow thrips ( F. occidentalis ) is the main parameter, and the red pepper transmitted to the TSWV decreases the yield and the quality of the fruit decreases. Viruses such as TSWV are highly resistant to chemical or biological control, and can be transmitted by domestic worms, so it is urgent to cultivate TSWV resistant varieties.

I studied the resistance to TSWV in several research groups, three Chinen capsicum (Capsicum Chinense) and capsicum Bakar Tomb (C. baccatum) species' PI152225 ',' PI159236 ', ' CNPH-275 ',' PI-15 ',' C00943 ',' 7204 ', and' ECU-973 '. As a result of the allelism test, it was confirmed that TSWV resistance of 'PI159236', 'PI152225', 'CNPH-275', and '7204' belonging to Capsicum annuinense was inherited by a single dominant gene. It is confirmed that the locus is located in one locus ' Tsw '. The major resistance gene for TSWV, Tsw, was mapped in Capsicum Chinense and its cDNA sequence was identified in 2014.

In addition, recent molecular genetic studies have shown that the Tsw gene is located on chromosome 10, and two research groups have used random amplified polymorphic DNA (RAPD) and restriction fragment (RFLP) for efficient breeding using the Tsw- length polymorphism (Moury et al. , Genome , 2000, 43 (1): 137-142, 2000). One group of researchers developed the SCAC568, a PCR-based molecular marker that is relatively easy to analyze with near genetic distance. However, SCAC568 is more than 1 cM away from the Tsw locus, so there is a limit to analyze the TSWV resistance genotype of breeding material.

Korean Patent No. 10-0984169 discloses a primer set, method and kit for selecting TMV resistant pepper cultivars. Korean Patent No. 10-1242426 discloses a primer set, method and kit for selecting TMM resistant pepper cultivars. Primer set, method and kit '. However, no primer sets, methods, and kits for discriminating tomato-spotted- fowl virus resistant or susceptible varieties capable of specifically detecting TSWV disease resistance gene Tsw as described in the present invention have been disclosed.

Under these circumstances , the inventors of the present invention have found that the TSWV disease resistance gene Tsw ( TSWV) can be easily transformed by the polymerase chain reaction A primer set specific to the Tsw gene capable of determining the presence or absence of a gene and a detection method including the primer set have been completed.

Korean Patent No. 10-0984169 Korean Patent No. 10-1242426

Moury et al., Genome, 2000, 43 (1): 137-142, 2000

The object of the present invention is to provide a primer set for TSWV disease resistance or susceptible pepper variety discrimination which is hybridized specifically to Tsw , a Tomato spotted wilt virus (TSWV) disease resistance gene, a kit comprising the primer set, Set of the present invention is to provide a TSWV disease resistance or susceptible pepper variety discrimination method.

In order to achieve the above object, the present invention provides a primer set for Tomato spotted wilt virus (TSWV) disease resistant or susceptible pepper variety identification.

In another aspect, the present invention provides a kit for discriminating tomato-spotted-fungus virus-resistant or susceptible pepper cultivars, comprising the primer set and a reagent for carrying out an amplification reaction.

In another embodiment, the present invention provides a method for producing a pepper sample comprising the steps of: a) separating genomic DNA from a pepper sample; b) amplifying the target sequence by performing PCR (polymerase chain reaction) using the separated genomic DNA as a template and using the primer set of SEQ ID NO: 3 and SEQ ID NO: 4; And c) detecting the PCR amplification product of step b). The present invention also provides a method for identifying tomato susceptible viral disease resistant or susceptible pepper cultivars.

The Tsw Since the TSWV disease resistance gene, Tsw gene, can be specifically detected in pepper plants through PCR using a primer set specific for the gene, TSWV disease resistance or susceptible pepper varieties can be effectively used for discrimination. As a result, it is not necessary to carry out a follow-up test for each generation of resistant pepper breeding to TSWV, which can save time, cost and effort required for breeding.

FIG. 1 shows a gradient PCR performed to clone a region containing the first intron region of Tsw / tsw in ' Kola- chan' and ' Green Kwangyu ' pepper cultivars according to an embodiment of the present invention It is an electrophoresis photograph.
FIG. 2 is a diagram showing the nucleotide sequences analyzed for cloned amplified products of 'Kola-chan' and 'Green Kwang' red pepper varieties according to an embodiment of the present invention. Here, the red arrow indicates Tsw derived from the above-described nucleotide sequence comparison analysis result Specific primer sequences.
FIG. 3 is a graph showing the results of using gDNA of ' Kola- chan' and 'Kwangwang' red pepper varieties as a template according to an embodiment of the present invention, and Tsw 1 is an electrophoresis image showing gradient PCR performed using a resistance gene specific primer set (Tsw_2595F (SEQ ID NO: 3) and Tsw_Intron_114R (SEQ ID NO: 4)).
FIG. 4 is a graph showing the results of using gDNA of 58 pepper plants of the Tsw / tsw mating group as a template according to an embodiment of the present invention, and Tsw 1 is an electrophoresis image showing the result of PCR performed using a resistance gene specific primer set (Tsw_2595F (SEQ ID NO: 3) and Tsw_Intron_114R (SEQ ID NO: 4)).

The present invention relates to a primer set specific to Tsw , a Tomato spotted wilt virus (TSWV) disease resistance gene and its use, and more particularly to a primer set for TSWV disease resistance or susceptible pepper variety identification, Kit and a method for discriminating TSWV disease-susceptible or susceptible pepper cultivars using the primer set.

In one embodiment, the present invention provides a primer set for Tomato spotted wilt virus (TSWV) disease-resistant or susceptible pepper varieties identified by SEQ ID NO: 3 and SEQ ID NO: 4.

The term " Tomato spotted wilt virus (TSWV) disease "in the present invention is a disease caused by tomato spotting virus (TSWV), also referred to as a color disease. TSWV has a wide range of host strains and can infect about 900 plants. In detail, TSWV can infect about 900 species of plants, including pepper, tobacco, tomato, celery, peanut, soybean, potato, pea, eggplant, spinach, lettuce, cucumber, cabbage, In addition to infringing crops, we also use various kinds of flowers and weeds as a host. It is known that the TSWV disease is mainly transmitted by 10 kinds of worms possessing TSWV in the body. TSWV is infected with young seedlings, resulting in necrosis and death of plants. Typical round spots and rugged anomalies occur in the leaves and fruits, and the stems also have brown spots or browning symptoms.

The term "resistance" in the present invention refers to the nature of the pepper varieties resistant to tomato spotting virus (TSWV), and the term "susceptibility" refers to the properties of pepper varieties that are susceptible to tomato spotting virus .

The term "discrimination" in the present invention means judging or distinguishing or selecting the resistance or susceptibility to tomato spotting virus (TSWV) from the pepper cultivar sample by the primer set of the present invention, Can be used in combination.

The term "primer " as used herein refers to a single stranded oligonucleotide sequence complementary to a nucleic acid strand to be copied, and may serve as a starting point for the synthesis of a primer extension product. The length and sequence of the primer should allow for the start of synthesis of the extended product and the specific length and sequence of the primer will depend on the complexity of the desired DNA or RNA target as well as the primer usage conditions such as temperature and ionic strength something to do.

In the present invention, the primer may further include, but is not limited to, a substance that emits intercalating fluorescence, phosphorescence, or radioactive. Preferably, the labeling substance is FAM or HEX. When the target sequence is amplified, FAM or HEX is labeled at the 5'-end of the allele-specific primer, and PCR is carried out, whereby the target sequence can be labeled with a detectable fluorescent labeling substance.

The term "primer set" in the present invention means a plurality of primers. In addition, the primer set may further include a container for receiving the primer. Primer refers to a short nucleotide sequence that can form a base pair with a complementary template with a short free 3 'hydroxyl group and serves as a starting point for template strand copying . The primer can initiate DNA synthesis in the presence of a reagent for polymerization and four nucleoside triphosphates at the appropriate buffer solution and temperature. The primer may be an oligonucleotide and the oligonucleotide may comprise a nucleotide analogue such as phosphorothioate, alkylphosphorothioate or peptide nucleic acid, intercalating agent. The primer can be produced through a chemical synthesis method using a phosphoramidite method, a phosphodiester method, a diethylphosphoramidite method, or the like. In addition, the primer sequence may be modified by means known in the art.

The primer set of the present invention is composed of a forward primer (Tsw_2595F) having a nucleotide sequence of SEQ ID NO: 3 and a reverse primer (Tsw_Intron_114R) having a nucleotide sequence of SEQ ID NO: 4 and is resistant to or susceptible to tomato spotting virus It is used as a marker to select the pepper cultivars.

The primer set may specifically amplify the Tsw gene which is a resistance gene for tomato spotted viral disease and may be a primer set for amplifying a 216 bp nucleic acid fragment consisting of the nucleotide sequence of SEQ ID NO:

In the present invention, TSWV resistant pepper cultivars can be selected when the 216 bp product is amplified by the primer set, and TSWV susceptible pepper cultivars can be selected when the product having a size different from the size of 216 bp is amplified.

The " Tsw gene" is a dominant gene found in Capsicum Chinense , which was found to be located in pepper-associated group map 10, and resistance to TSWV in pepper is determined by the gene.

In the present invention, the " Tsw gene" may be the one consisting of the nucleotide sequence of SEQ ID NO: 5, which is commercially available as a Tsw / tsw genotype, that is, a variant of ' Kalachan ' pepper, which is a TSWV resistant strain .

In one embodiment of the present invention, Tsw / tsw genotype as commercially available is "color chan pepper varieties and TSWV (Tomato spotted wilt virus) which is known as susceptible to disease, tsw / tsw genotype is" nokgwang "Pepper Varieties (FIG. 2), and based on the result of the above-described analysis, Tsw Resistant gene-specific primer sets were prepared (Table 2). PCR amplification of each of the genomic DNAs of 'Kalachan' and 'Green' was carried out using the primer set. As a result, in the case of TSWV resistant 'Kalachan', the 216 bp product was amplified and TSWV It was confirmed that the amplification product was clearly distinguished from the susceptible varieties 'green light' (FIG. 3).

In addition, as a result of performing PCR by practically applying the primer set to the pepper plants of the Tsw / tsw mating group in which the actual TSWV disease response was known, the pepper plants having resistance to the actual TSWV And amplified product of 216 bp was observed in all of them. On the other hand, in the case of the susceptible pepper plants, it was confirmed that the band was formed at a position slightly deviated from the size of 216 bp. That is, it was confirmed that the primer set according to the present invention can clearly distinguish the presence or absence of the Tsw resistance gene (FIG. 4).

Thus, the Tsw Resistance gene specific primer sets (Tsw_2595F (SEQ ID NO: 3) and Tsw_Intron_114R (SEQ ID NO: 4)) can be usefully used to identify TSWV disease resistant and susceptible varieties.

In another aspect, the present invention provides a kit for discriminating tomato-spotted-fungus virus-resistant or susceptible pepper cultivars, comprising the primer set and a reagent for carrying out an amplification reaction.

In the kit of the present invention, the reagent for carrying out the amplification reaction may include a DNA polymerase, dNTPs and a buffer. The dNTP mixture may include dATP, dCTP, dGTP, dTTP, and the heat-resistant DNA polymerase may be a commercially available heat-resistant DNA polymerase such as Taq DNA polymerase or Tth DNA polymerase. In addition, the kit of the present invention may further include a user guide describing optimal reaction performing conditions. The manual is a printed document that explains how to use the kit, for example, how to prepare PCR buffer, the reaction conditions presented, and so on. The manual includes instructions on the surface of the package including a brochure or leaflet in the form of a brochure, a label attached to the kit, and a kit. In addition, the brochure includes information that is disclosed or provided through an electronic medium such as the Internet.

In another embodiment, the present invention provides a method for producing a pepper sample comprising the steps of: a) separating genomic DNA from a pepper sample; b) amplifying the target sequence by performing PCR (polymerase chain reaction) using the separated genomic DNA as a template and using the primer set of SEQ ID NO: 3 and SEQ ID NO: 4; And c) detecting the PCR amplification product of step b). The present invention also provides a method for identifying tomato susceptible viral disease resistant or susceptible pepper cultivars.

The method of the present invention comprises isolating genomic DNA from pepper samples. The method for isolating the genomic DNA from the sample can be carried out by a conventional method known in the art and can be performed by a phenol / chloroform extraction method, a SDS extraction method (Tai et al., Plant Mol. Biol. Reporter, 8: 303, 1990), Cetyl Trimethyl Ammonium Bromide (Murray et al., Nuc. Res., 4321-4325, 1980), or commercially available DNA extraction kits.

The target sequence can be amplified by performing amplification reaction using the genomic DNA of the separated pepper samples as a template and using a primer set according to an embodiment of the present invention. Methods for amplifying a target nucleic acid include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, transcription-based amplification system, Strand displacement amplification or amplification with Q [beta] replicase, or any other suitable method for amplifying nucleic acid molecules known in the art. Among them, PCR is a method of amplifying a target nucleic acid from a pair of primers that specifically bind to a target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used. The PCR can be carried out using a PCR reaction mixture containing various components known in the art necessary for the PCR reaction. The PCR reaction mixture includes a suitable amount of DNA polymerase, dNTP, PCR buffer solution, and water in addition to the genomic DNA extracted from the pepper samples to be analyzed and the primer set provided in the present invention. The PCR buffer comprises Tris -HCl (Tris-HCl), MgCl 2, KCl and the like.

In the method of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. The labeling substance may be a fluorescent, phosphorescent or radioactive substance, but is not limited thereto. The labeling substance may be FAM or HEX. When the target sequence is amplified, PCR is carried out by labeling FAM or HEX at the 5'-end of the primer, and the target sequence may be labeled with a detectable fluorescent labeling substance. When the radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution, the amplification product may be synthesized and the radioactive substance may be incorporated into the amplification product and the amplification product may be labeled as radioactive. The primer set used to amplify the target sequence is as described above.

In the method of the present invention, a method for TSWV disease resistance or susceptible pepper cultivar identification comprises detecting the amplification product, wherein the detection of the amplification product is performed by sequencing, DNA chip, gel electrophoresis, Or phosphorescence measurement, but is not limited thereto. 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. In addition, in the fluorescence measurement method, FAM or HEX is labeled at the 5'-end of the primer and when the PCR is performed, the target sequence is labeled with a fluorescent label capable of detecting the fluorescence, and the fluorescence thus labeled can be measured using a fluorescence analyzer. In addition, in the case of performing the PCR, the radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution to mark the amplification product, and then a radioactive measurement device such as a Geiger counter or liquid scintillation counter The radioactivity can be measured using a liquid scintillation counter.

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  One: Tomato spotted virus  Disease resistance gene Tsw on  Specific for hemp Kerr Development

Tomato spotted wilt virus (TSWV) To develop a specific marker for Tsw , a disease resistance gene, the ' Tsh / tsw' genotype, 'Kalla Chan' varieties and TSWV disease known as sensitivity, tsw / tsw genotype of 'nokgwang' pepper varieties used to analyze the nucleotide sequence of the gene regions Tsw and Tsw based on this resistance A gene-specific primer set was prepared.

Example  1-1: Tomato spotted virus  Sequence analysis of disease-resistant and susceptible varieties

Genomic DNA (gDNA) was isolated from the ' tsw / tsw ' genotype of 'green' pepper, which is known to be susceptible to ' Tsw / tsw' genotypes and 'TSWV (Tomato spotted wilt virus) .

Then, in order to clone a region containing the first intron region of Tsw / tsw in ' Kalachan ' and ' Green Kwangyoo ' varieties, the first intron of the known Tsw cDNA region The primer set of the following Table 1 was prepared from the region expected to contain the intron portion and the gDNA of the two cultivars was annealed at an annealing temperature of 57 DEG C to 68 DEG C ) PCR was performed (Fig. 1). The PCR product was subjected to PCR under the temperature condition of 68 ° C. After amplification product of 1,997 bp was cloned, the base sequence was completely analyzed.

SEQ ID NO: primer  designation primer  direction The sequence (5 '- > 3') One Tsw_2544F Forward 5`-CACACACTTTTACAGTGACACTGTTGAG-3` (28mer) 2 Tsw_3088R Reverse 5`-GCATCTTCATTTCAGGGCAGTTACG-3` (25 mer)

Example  1-2: Tomato spotted virus  To identify disease-resistant and susceptible varieties Tsw Resistant gene specific primer  design

The nucleotide sequences analyzed for the cloned amplified products of the 'Kola-chan' and 'Green Kwangyu' varieties in Example 1-1 were compared with each other (FIG. 2) Tsw Resistant gene specific primer set.

SEQ ID NO: primer  designation primer  direction The sequence (5 '- > 3') 3 Tsw_2595F Forward 5`-CCATTATATAGAGTCCCCTAATGGTC-3` (26mer) 4 Tsw_Intron_114R Reverse 5`-CAATATTATAAAAGTTGGAACAAAGAGAGTAAC-3` (33 mer)

On the other hand, gDNA isolated from 'Kola-chan' and ' Green Kwangju ' varieties was used as a template, and Tsw Gradient PCR was performed under annealing temperature conditions ranging from 57 ° C to 68 ° C using a set of resistant gene primers.

As a result, as shown in Fig. 3, Tsw It was confirmed that amplification was specific to the resistance gene. Specifically, in the case of the ' Tsh / tsw ' genotype of ' Kola- chan' pepper cultivars under the annealing temperature of 57 ℃, 216 bp product was amplified and the ' tsw / tsw ' .

Through this, it is possible to identify a variety having resistance or susceptibility to Tomato spotted wilt virus (TSWV) disease, that is, Tsw / tsw In distinguishing genotypes, the Tsw It was found that a resistance gene specific primer set (Tsw_2595F (SEQ ID NO: 3) and Tsw_Intron_114R (SEQ ID NO: 4)) could be used advantageously.

Example  2: Tomato spotted virus  Disease-resistant and susceptible varieties

The Tsw < RTI ID = 0.0 > Resistance gene-specific primer set (Tsw_2595F (SEQ ID NO: 3) and Tsw_Intron_114R (SEQ ID NO: 4)) Tsw / tsw genotype of using, Tsw / tsw mating groups were divided.

Specifically, the actual TSWV (Tomato spotted wilt virus) After removing the genomic DNA (genomic DNA, gDNA) from 58 pepper plants of Tsw / tsw mating group know the reaction bottle, the Tsw PCR was carried out under an annealing temperature condition of 57 캜 using a resistance gene specific primer set (Tsw_2595F and Tsw_Intron_114R). After PCR, electrophoresis was performed to confirm whether a 216 bp Tsw- specific gene-specific amplification product appeared.

As a result, as shown in Fig. 4, the pepper plants having resistance to the actual TSWV And amplified product of 216 bp was observed in all of them. On the other hand, in the case of the susceptible pepper plants, it was confirmed that the band was formed at a position slightly deviated from the size of 216 bp.

In the Tsw / tsw mating group, which knows the actual TSWV disease response, Tsw PCR was carried out by practically applying the resistance gene specific primer sets (Tsw_2595F (SEQ ID NO: 3) and Tsw_Intron_114R (SEQ ID NO: 4)) to determine the presence or absence of the Tsw resistance gene in the actual pepper plants using the primer set In the case of

Thus, the Tsw Resistance gene specific primer sets (Tsw_2595F (SEQ ID NO: 3) and Tsw_Intron_114R (SEQ ID NO: 4)) were found to be useful for discriminating TSWV disease resistant and susceptible varieties.

<110> Republic of Korea <120> Tomato spotted wilt virus disease resistance gene Tsw specific          primer sets and uses thereof <130> P18G10C0337 <160> 5 <170> KoPatentin 3.0 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Tsw_2544F <400> 1 cacacacttt tacagtgaca ctgttgag 28 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Tsw_3088R <400> 2 gcatcttcat ttcagggcag ttacg 25 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Tsw_2595F <400> 3 ccattatata gagtccccta atggtc 26 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Tsw_Intron_114R <400> 4 caatattata aaagttggaa caaagagagt aac 33 <210> 5 <211> 216 <212> DNA <213> Artificial Sequence <220> <223> Tsw gene <400> 5 ggtaatatat ctcaggggat taccagagtt ccaaaatttc tggccaacag ccaacaatgc 60 tatcaccgac tcaaatcctc tttttaatga aaaggtttgc ttctacacga ataagttttt 120 tttttgaagg ggttttatga attaatattt tagctttaac ttgagcaatc atatttataa 180 gatgttactc tctttgttcc aacttttata atattg 216

Claims (7)

Tomato spotted wilt virus (TSWV) as set forth in SEQ ID NO: 3 and SEQ ID NO: 4, primer set for the identification of susceptible or susceptible pepper cultivars.
The primer set for discriminating tomato-spotted- fowl virus-resistant or susceptible pepper cultivars according to claim 1, wherein the primer set specifically amplifies the Tsw gene of SEQ ID NO: 5 which is a tomato spotted viral disease resistance gene.
A kit for discriminating tomato-spotted-pox virus-resistant or susceptible pepper cultivars, comprising the primer set of claim 1 or 2 and a reagent for carrying out an amplification reaction.
[Claim 4] The kit for discriminating tomato-spotted-fowl virus-resistant or susceptible pepper cultivars according to claim 3, wherein the reagent for performing the amplification reaction comprises DNA polymerase, dNTPs and a buffer.
a) separating the genomic DNA from the pepper sample;
b) amplifying the target sequence by performing PCR (polymerase chain reaction) using the separated genomic DNA as a template and using the primer set of claim 1 or 2; And
and c) detecting the PCR amplification product of step b). &lt; Desc / Clms Page number 19 &gt;
6. The method according to claim 5, wherein the amplified target sequence is labeled with a fluorescent, phosphorescent or radioactive substance.
6. The method according to claim 5, wherein the detection of the amplification product is performed by sequencing, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement.

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