KR101940286B1 - Primer Set for Identifying Maternal origin of Ilex x wandoensis and Uses Thereof - Google Patents

Abstract

The present invention relates to a primer set for identifying mother line of ilex x wandoensis and use thereof. According to the present invention, only by differences in length of a PCR product, the mother tree of ilex x wandoensis can be easily identified from ilex, which is genetically identical to the ilex x wandoensis. In addition, the primer set can more accurately identify the mother tree than the existing methods so that the primer set can be used for breed protection and resolution for seed conflict.

Description

[0001] The present invention relates to a primer set for identification of a mother line of wandering ferns and to a use thereof.

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a primer set for identification of mother line of domestic wandering thorns and its use.

Ilex x wandoensis is a natural hybrid of Ilex cornuta and Ilex integra, which is native to the southwest coast of Korea. Its leaf edge has an intermediate form of two trees, And the fruit that ripens red in autumn is also in winter, so it has excellent value. Red fruit of Ilex genus trees is attracting attention as an environmental species because it can attract birds.

Plant cells have a genome in the nucleus, chloroplast, and mitochondria. Chloroplasts are the main organ responsible for photosynthesis, and generally produce maternal heredity. The size of the chloroplast genome is 120 ~ 217kb, which is conserved and maintained as a mutation with about 130 genes. However, relatively large numbers of single nucleotide polymorphisms (SNPs), insertions and deletions in intergenic spacers (IGS) (InDel), inversion, and translocation. The chloroplasts are circular in all plant cells and there are hundreds of copies in one cell.

However, in the case of plants, various interspecific and interspecific hybrids are frequently reported, and it is difficult to understand the mother lineage and evolution for accurate identification of plant taxa due to complicated aspects of drainage. Therefore, it is desired to develop DNA capable of grasping the lineage of the mother line.

Currently, no genetic markers have been developed to accurately identify only the mother line species of Wanthosaurus thunbergii, which is native to Korea, from potato plants having many genetic identities.

Among these nuclear DNA markers, there are rDNA and intergenic transcribed spacer (ITS) sections, but there are few differences between species and interspaces in rRNA genes, while there are thousands of copies per single genome in ITS section, It can provide characteristics with useful information for analysis.

In the case of wandering thorns, Ilex cornuta and Ilex integra have all the ITS nucleotide sequences inherited from their parents.

Since chloroplasts contain hundreds of copies in a single cell, they can be used as evolutionary studies or genetic markers for identification of the same mother line through hybridization between two trees by chloroplast IGS (intergenic spacers) sequencing.

Under these circumstances, the present inventors have sought to develop a method for discriminating the mother tree of Wanthora thaliana. As a result, the present inventors have developed four kinds of chloroplast marker primers targeting the genes of ycf3-rps4 IGS, rpl16-rps3 IGS, ndhA and ndhC-atpB IGS genes, which are known to be highly mutated in the chloroplast DNA of the Chinese cabbage plants, The present inventors have completed the present invention by confirming the identification of the mother tree of Wanthosaurus thornb.

Therefore, one object of the present invention is to provide a primer set for maternal origin identification of Ilex x wandoensis.

Another object of the present invention is to provide a composition for identifying a maternal origin of Ilex x wandoensis comprising the primer set.

It is still another object of the present invention to provide a kit for identifying a maternal origin of Ilex x wandoensis comprising the primer set.

In addition, another object of the present invention is to provide a method for identifying a maternal origin of Ilex x wandoensis.

Hereinafter, the present invention will be described in detail.

According to one aspect of the present invention, there is provided a primer set comprising a primer represented by the nucleotide sequence of SEQ ID NO: 1 and a primer represented by the nucleotide sequence of SEQ ID NO: 2; A primer set consisting of a primer represented by the nucleotide sequence of SEQ ID NO: 3 and a primer represented by the nucleotide sequence of SEQ ID NO: 4; A primer set consisting of a primer represented by the nucleotide sequence of SEQ ID NO: 5 and a nucleotide sequence represented by SEQ ID NO: 6; And a primer set consisting of a primer represented by the nucleotide sequence of SEQ ID NO: 7 and a primer represented by the nucleotide sequence of SEQ ID NO: 8, and a primer set for identifying the maternal origin of Ilex x wandoensis Lt; / RTI >

The present inventors used a gene in the chloroplast of a plant transferred in the mother line as a target to prepare a primer corresponding to the nucleotide sequence of the gene.

The selected target genes are at least one selected from the group consisting of ycf3-rps4 IGS, rpl16-rps3 IGS, ndhA and ndhC-atpB IGS, which are known to be mutated in the pot plants.

As used herein, the term " primer " refers to a nucleic acid sequence having a short free 3 'hydroxyl group and capable of forming a base pair with a complementary template, Refers to a short nucleic acid sequence that functions as a starting point. The primers can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures.

As used herein, the term " primer set " means a set consisting of a forward primer and a reverse primer used for amplifying a desired gene through a polymerase chain reaction, and is compatible with the term " primer pair ".

For the purpose of the present invention, the primer set for detection of ycf3-rps4 IGS in the primer sets of the present invention can be used for detection of chlorophyll genes of Ilex latifolia NCBI accession number: KX426465), and can be composed of a forward primer from 45964 to 45983 from the 5 'end and a reverse primer from 47016 to 47035 in the same gene, A primer represented by the nucleotide sequence of SEQ ID NO: 1 and a primer represented by the nucleotide sequence of SEQ ID NO: 2.

In addition, the primer set for detection of rpl16-rps3 IGS can be composed of forward and reverse primers based on the chloroplast gene (NCBI accession number: KX426465) of broad-leaved rhizome trees, and the primers set at positions 84725 to 84745 And a primer represented by the nucleotide sequence of SEQ. ID. NO. 3 and a primer represented by the nucleotide sequence of SEQ. ID. NO. 4, which may be composed of a forward primer of SEQ ID NO: 3 and a reverse primer of 85588 to 85607 of the same gene.

Also, the primer set for detection of ndhA can be constituted of forward and reverse primers based on the chloroplast gene (NCBI accession number: KX426465) of the broad-leaved rhizome, and forward primer from 123367 to 123387 from the 5 ' And reverse primers 124481 to 124498 in the same gene, and may be composed of a primer represented by the nucleotide sequence of SEQ ID NO: 5 and a primer represented by the nucleotide sequence of SEQ ID NO: 6.

In addition, the primer set for detection of ndhC-atpB IGS can be constituted of forward and reverse primers based on the chloroplast gene (NCBI accession number: KX426465) of broad-leaved thornbird, and from 52809 to 52828 from the 5 ' And a reverse primer from the 536th to 53658th positions in the same gene. Most preferably, the primer is composed of a primer represented by the nucleotide sequence of SEQ ID NO: 7 and a primer represented by the nucleotide sequence of SEQ ID NO: .

In the specific primers of the present invention, some of the constitutive base sequences may be changed. Such modifications may be accomplished by partial deletion, addition, substitution, or a combination thereof, of the constitutive nucleotide sequence, but some modifications of such constitutive nucleotide sequences are specific for the template (cDNA or PCR amplification products) of the specific primers It requires considerable care in application because it can damage the bond. At this time, it must be experimentally investigated and changed to the extent that the specificity is not impaired. Since these are general matters to those skilled in the art, a detailed description thereof will be omitted. It is most preferred to maintain the sequences of the primers presented in the present invention.

According to a preferred embodiment of the present invention, the primer set of the present invention is a set of primers for carrying out polymerase chain reaction (PCR).

The primer set provided in the present invention was developed to be suitable for PCR amplification and sequencing, and the four primer pairs can be used to identify the mother tree of Wanthora thaliana.

According to a preferred embodiment, the primer set is Wando holly (Ilex x wandoensis), holly (Ilex cornuta), gamtang tree (Ilex integra), yanghorang thorns (Ilex aquifolium), Chinese holly (Ilex chinensis), Holly (Ilex crenata Thunb. var. crenata ), one Rex Deki Dua (Ilex decidua), daepaetjip trees (Ilex macropoda Miq. for. macropoda ), Min daepaetjip trees (Ilex macropoda for. pseudomacropoda (Loes . ) H.Hara), American holly (Ilex opaca) and mate (Ilex paraguariensis), Office buildings trees (Ilex pedunculosa), Ilex rotunda (Ilex rotunda Thunb.), Ilex serrata (Ilex serrata Thunb.), one Rex Berti Aquila other (Ilex verticillata) and one Rex Bomi thoria (Ilex vomitoria) by which to identify the mother tree of gamtang wood in plants is selected from the group consisting of, and more preferably Wando holly (Ilex x wandoensis), holly Ile x cornuta , or Ilex integra , and most preferably Wando and Thornbird.

The primer annealing temperature of the primer set may be preferably 45 ° C to 55 ° C, and most preferably 50 ° C.

According to another aspect of the present invention, there is provided a kit for identifying a maternal origin and a maternal origin identification kit for Ilex x wandoensis comprising the above-described primer set.

In addition, the compositions and kits of the present invention may further comprise one or more other component compositions, solutions or devices suitable for the assay method.

Preferably, the compositions and kits may comprise the necessary elements to perform the PCR.

The compositions and kits of the present invention may further comprise DNA polymerase, tubes or other suitable containers for amplifying complementary DNA, reaction buffers (varying in pH and magnesium concentration), deoxynucleotides (dNTPs), enzymes such as Hot start Taq polymerase and reverse transcriptase, sterile water, and the like.

The components included in each tube in the present invention include, but are not limited to, DNA polymerase, Tris-HCl, MgCl2, KCl, dATP, dTTP, dGTP, dCTP, Triton X-100 and BSA. These should be included in the appropriate concentration range. 2.5 units of DNA polymerase, 100 mM Tris-HCl, 15 mM MgCl2, 500 mM KCl, 2 mM dATP, 2 mM dTTP, 2 mM dGTP, 2 mM Of dCTP.

On the other hand, the components added to each tube may be prepared in a liquid form or in a dried form in order to improve the stability of the product by lowering the degree of freedom of contained components. In order to produce such a dried form, it is necessary to apply a drying step, and it can be used in the form of warm drying, natural drying, vacuum drying, freeze drying, or a combination thereof.

Since the compositions and kits of the present invention utilize the present primer sets described above, their description is omitted in order to avoid the excessive complexity of the present specification in accordance with the repeating substrate.

According to another aspect of the present invention, the present invention provides a method for identifying a maternal origin of Ilex x wandoensis comprising the steps of:

(a) separating DNA from a sample;

(b) performing PCR (Polymerase Chain Reaction) using the separated DNA as a template and amplifying the target sequence using the primer set; And

(c) detecting the amplification product.

According to a preferred embodiment of the present invention, the primer annealing temperature of the PCR may be 40 ° C to 60 ° C, preferably 45 ° C to 55 ° C, and most preferably 50 ° C, Do not.

According to a preferred embodiment of the present invention, the detection of the amplification product can be carried out by gel electrophoresis, gel filtration, capillary electrophoresis or fluorescence measurement.

According to the present invention, DNA extracted from a sample is extracted according to a method known in the art, and then a primer set of the present invention, a composition containing the primer set of the present invention, or a primer set of the present invention is added to an extract containing the extracted DNA Perform PCR using the included kit. Agarose gel electrophoresis analysis is performed on the obtained product (reaction solution) after PCR, and the amplification product is subjected to base sequence analysis using a template to determine the difference in the nucleotide sequence to determine whether or not the mother line is present.

Since the method of the present invention utilizes the present primer sets described above, the common description is omitted in order to avoid the excessive complexity of the present specification in accordance with repetitive descriptions

According to the present invention, it is possible to easily distinguish the mother tree of Wanthong thorn tree from the pot plants having a lot of genetic identity due to the difference in the base sequence. Therefore, it can also be used for breed protection and solving of seed conflict.

Fig. 1 shows an overall experimental flow chart for identification of the mother line of Wanthosaurus thunbergii.
Fig. 2 is a graph showing the results of experiments conducted by the inventors of the present invention on the Ilex x wandoensis, the Ixia cornuta, the Ilex integra, the Ilex rotuda, Ilex latifolia, Ilex sp., Ilex delavayi, Ilex szechwanensis, Ilex polyneura, Ilex pubescens, and Ilex wilsonii extracted from NCBI GenBank were screened by using four kinds of primers of the present invention for each species, And the genetic tree (Phylogenetic tree) (UPGMA; Unweighted Pair Group Method with Arithmetic Mean).
Figures 3 to 6 show the genetic distance by applying the conventional universal markers psbA-trnH (Figure 3), trnL-trnF IGS (Figure 4), trnL intron (Figure 5) and chloroplast rbcL (Figure 6) And a genetic tree (UPGMA).

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be obvious to you.

Example 1: Experimental Materials and Sequence Analysis Method

Ilex x wandoensis Ilex x wandoensis Ilex cornuta Ilex integra, Ilex rotuda Ix rotunda Both male and female are male and male, And collected at Wando Arboretum. Samples of the collected plants were stored at -70 ° C until use.

Genomic DNA of the plant was extracted using a known method.

In the previous step of identification of the mother line, the ITS amplification of Wando fir thorn genomic DNA as a template and the cloning into a vector were performed to analyze the sequence, which revealed similar and identical ITS nucleotide sequences to ITS,

Example 2: Preparation of primers and establishment of PCR conditions

The present inventors prepared a specific primer suitable for obtaining a sample and for the purpose of PCR for identification of the mother line of Wandosaurus thunbergii. The sequence of the primer set is shown in Table 1 below.

First, genes in the chloroplasts of plants transferred to the mother line were selected as target genes.

The target genes were selected as ycf3-rps4 IGS, rpl16-rps3 IGS, ndhA and ndhC-atpB IGS, which are known to be highly mutated in the chloroplast DNA of the potato hawthorn plant. As a known chloroplast universal marker, psbA-trnH, trnL-trnF IGS, trnL intron and chloroplast rbcL were used as a control group (Table 2).

The primer set for ycf3-rps4 IGS contained a forward primer (SEQ ID NO: 1) from 45964 to 45983 from the 5 'end of the chloroplast gene (NCBI accession number: KX426465) of Ilex latifolia, The reverse primer (SEQ ID NO: 2) from 47016 to 47035 was included.

The primer set for rpl16-rps3 IGS consists of a forward primer (SEQ ID NO: 3) from 84725 to 84745 from the 5 'end of the chloroplast gene (NCBI accession number: KX426465) And the reverse primer (SEQ ID NO: 4) up to 85607th.

The primer set for ndhA contained a forward primer (SEQ ID NO: 5) from 123367 to 123387 from the 5 'end of the chloroplast gene of the broad-leaved thornbird (NCBI accession number: KX426465) and a primer from the 124481 to 124498 Reverse primer (SEQ ID NO: 6).

The primer set for ndhC-atpB IGS contained a forward primer (SEQ ID NO: 7) from 52809 to 52828 from the 5 'end of the chloroplast gene (NCBI accession number: KX426465) And reverse primer (SEQ ID NO: 8) up to 53658th.

Marker Size (bp) primer order ycf3-rps4 IGS 1078 F
R 5'-ATGGATGTTTGTTGTCCCAA-3 '(SEQ ID NO: 1)
5'-GGCCATCTCTCCTACATACT-3 '(SEQ ID NO: 2) rpl16-rps3 IGS 883 F
R 5'-GGGCGAATATCTACTCTTTCA-3 '(SEQ ID NO: 3)
5'-TATGGGGTCTTAGGCATCAA-3 '(SEQ ID NO: 4) ndhA 1132 F
R 5'-TAAATCTAAGGACCCACTCAC-3 '(SEQ ID NO: 5)
5'-TTTAGGTGGTCTCCGAGC-3 '(SEQ ID NO: 6) ndhC-atpB IGS 849 F
R 5'-CCTTTGTTTACTGTGGGGAA-3 '(SEQ ID NO: 7)
5'-TCGTGGATAAATAAACCAACC-3 '(SEQ ID NO: 8)

5 ㎕ of commercial GOLD 10X PCR buffer, 0.5 ㎕ of 5 unit AmpliTaq Gold DNA polymerase, and 10 ㎕ of DNA template 10 extracted from tangerine plants were added to a tube containing 4 pairs of primer sets (5 ㎕ each, 20 pmole / ㎕) -100 ng, and then subjected to PCR using the amplification equipment ABI 9700 system (Applied BioSystems)

First, denatured at 95 DEG C for 11 minutes. The amplification reaction was repeated 28 times at 95 ° C for 1 minute, 50 ° C for 1 minute, and 72 ° C for 1 minute, followed by reaction at 72 ° C for 7 minutes to terminate the PCR.

For the positive control of the amplification reaction, Papaver somniferum (10 ng) gene was used as a template and distilled water was used as a negative control group.

The amplification result was confirmed by 2% (wt / vol) agarose electrophoresis. The amplified products were purified using QIAquick PCR purification kit (QIAGEN).

The purified amplified products were subjected to sequencing using ABI PRISM BigDye Terminator v3.1 Cycle Sequencing Kit (Applied BioSystems) using the same primers amplified by direct sequencing method. At this time, the fluorescent substance not participating in the nucleotide sequence reaction was purified with DyeEX ^TM 2.0 Spin Kit (QIAGEN).

Gene sequences were determined using the ABI 3500 Sequencer and DNA Sequencing Analysis Software 5.4 (Applied BioSystems).

The analyzed sequences were summarized and compared using MEGA 7 software (Kumar et al., 2016).

Ilex latifiola, Ilex sp., Ilex delavayi, Ilex szechwanensis, Ilex polyneura, Ilex pubescens, Ilex sp., Which were directly tested in NCBI GenBank using BLAST (basic local alignment search tool) Wilsonii) (Altschul et al., 1997) to complete a total of 11 genetic trees in MEGA 7 software (see Kumar S, Stecher G, and Tamura K. MEGA 7: Molecular Evolutionary Genetics Analysis version 7.0 Gap BLAST and PSI-BLAST: a new generation (1, 2, 3, 4, 5, 6, 7, of protein database search programs. Nucleic Acids Res 25: 3389-3402 (1997).

Example 3: Mother line discrimination

The present inventors carried out PCR using the primer set of the present invention in the obtained sample under the conditions established in Example 1, and confirmed the possibility of discrimination of the same mother tree among the spinach and the rhizome trees.

First, PCR reaction was performed on each of these primer pairs, and then amplification products were confirmed by agarose gel electrophoresis, and amplification products were purified from the remaining primers and dNTPs. The purified amplification products were sequenced by the Sanger method.

In addition, four marker nucleotide sequences obtained for each species (Ilex x wandoensis, Ilex cornuta, Ilex integra and Ilex rotuda, respectively) (Ilex latifolia, Ilex spp., Ilex delavayi, Ilex szechwanensis, Ilex polyneura, Ilex pubescens, and Ilex wilsonii) from the same region extracted from NCBI GenBank genetic tree, UPGMA (Unweighted Pair Group Method with Arithmetic Mean)).

This method assumes that the nucleotide or amino acid replacement rate is the same for all evolutionary lines. This method creates a single rooted tree because it assumes a constant rate of evolution.

In addition, the phylogenetic tree uses the mutation rate of biomolecules to deduce the time at which two or more life forms split into one species. The biomolecule data used in this calculation is generally the DNA base sequence or the amino acid sequence of the protein. Taking into account the commonalities and differences of living things, if we show an evolutionary relationship, we can know the system of organisms. The phylogenetic tree is created by examining morphological characteristics and DNA sequence. The closer they are to each other, the closer they are to each other.

For example, the DNA sequences of Ilex integra and Ilex wandoenesis are the same as those of Ilex wandoenesis. That is, they are close to each other in the phylogenetic tree because of high homology, which is close to that of chloroplast DNA.

As a result, as shown in Fig. 2, it was found that the potted tree was the same as the wandering tree of Wando.

In other words, it could be concluded that the mutant tree that has the same base sequence as Wando fir thorn tree from the pot plants is the same mother tree.

On the other hand, among the conventional chloroplast universal markers listed in Table 2, psbA-trnH (FIG. 3) can judge that the same parent tree of the wandering rhizome is a sparrow tree, but the rhizomes have a difference of 2 bp from the Wando rhizome It is difficult to accurately determine whether or not the mother is the same. When the trnL-trnF IGS (FIG. 4), trnL intron (FIG. 5) and rbcL (FIG. 6) among the universal markers shown in Table 2 were applied, I could not figure out whether it was the mother line.

In the case of psbA-trnH (492 bp), there was only a difference of 2 bp between the spruce tree and the vernal spruce tree.

In addition, the universal markers (trnL-trnF IGS (413 bp), trnL intron (526 bp), and rbcL (446 bp) showed no difference between the three species.

The length of the base sequence obtained with the primer for the marker of the present invention is as follows:

In the case of ycf3-rps4 IGS, 973 bp in 973 bp of the waxy and thornbirds, 973 bp in the molasses and sum of the spruce trees, 979 bp in the molasses of the vernacular tree, 978 bp of the vernacellus, We obtained 971 bp from the series.

In the case of rpl16-rps3 IGS, 883bp was obtained in both of the waxy and prickly rhizomes and the sumi and the sumi and irises of the rosewood trees, and 893bp in the females.

In the case of ndhA, 1087 bp in the waxy and thornbirds of the Wanshore and thornbirds, 1087 bp in the ammonium and sum of the spruce trees, 1089 bp in the whites of the rhizomes, 1088 bp in the fractions of the rhizomes, 1087 bp.

In the case of ndhC-atpB IGS, 849 bp was obtained in all of the waxy rhizomes, in the sumi and the sumi of the rhizomes, in the sumi and sumuga of the twigs, and in 853 bp.

From the above results, it can be confirmed that the primer set for the markers of the present invention can distinguish the same parental system of Wanthorrhoeae.

<110> Republic of Korea (National Forensic Service Director Ministry of Interior and Safety) <120> Primer Set for Identifying Maternal origin of Ilex x wandoensis          and Uses Thereof <130> NFSDM1-8p <160> 8 <170> KoPatentin 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ycf3-rps4 IGS primer F <400> 1 atggatgttt gttgtcccaa 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ycf3-rps4 IGS primer R <400> 2 ggccatctct cctacatact 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> rpl16-rps3 IGS primer F <400> 3 gggcgaatat ctactctttc a 21 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rpl16-rps3 IGS primer R <400> 4 tatggggtct taggcatcaa 20 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ndHA primer F <400> 5 taaatctaag gacccactca c 21 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> ndHA primer R <400> 6 tttaggtggt ctccgagc 18 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> NdhC-atpB IGS primer F <400> 7 cctttgttta ctgtggggaa 20 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> NdhC-aTPB IGS primer R <400> 8 tcgtggataa ataaaccaac c 21

Claims (6)

A primer set consisting of a primer represented by the nucleotide sequence of SEQ ID NO: 1 and a primer represented by the nucleotide sequence of SEQ ID NO: 2; A primer set consisting of a primer represented by the nucleotide sequence of SEQ ID NO: 3 and a primer represented by the nucleotide sequence of SEQ ID NO: 4; A primer set consisting of a primer represented by the nucleotide sequence of SEQ ID NO: 5 and a nucleotide sequence represented by SEQ ID NO: 6; And a primer set consisting of a primer represented by the nucleotide sequence of SEQ ID NO: 7 and a primer represented by the nucleotide sequence of SEQ ID NO: 8, and a primer set for identifying the maternal origin of Ilex x wandoensis . The primer set according to claim 1, wherein the primer set is for PCR (Polymerase Chain Reaction). A composition for identification of the maternal origin of Ilex x wandoensis comprising the primer set of claim 1. A kit for identifying a maternal origin of Ilex x wandoensis comprising the primer set of claim 1. Identify the maternal origin of Ilex x wandoensis , including the following steps:
(a) separating DNA from a sample;
(b) performing PCR (Polymerase Chain Reaction) using the separated DNA as a template and using the primer set of claim 1 to amplify the target sequence; And
(c) detecting the amplification product. 6. The method according to claim 5, wherein the primer annealing temperature of the PCR in step (b) is 45 ° C to 55 ° C.

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