KR101678851B1 - Primer set for diagnosing or detecting Plantain mottle virus and uses thereof - Google Patents

Abstract

The present invention relates to a primer for diagnosis or detection of a Plantain mottle virus and a use thereof, and more particularly to a primer set of SEQ ID NO: 3 and SEQ ID NO: 4; A primer set of SEQ ID NO: 5 and SEQ ID NO: 6; A primer set of SEQ ID NO: 7 and SEQ ID NO: 8; A primer set of SEQ ID NO: 9 and SEQ ID NO: 10; And at least one primer set selected from the group consisting of primers set forth in SEQ ID NO: 11 and SEQ ID NO: 12, a primer set for specific diagnosis or detection of Plantain mottle virus, A kit for specific diagnosis or detection of Plantain mottle virus and a method for specifically diagnosing or detecting Plantain mottle virus using the above primer set.

Description

PRIMER SET FOR DIAGNOSIS OR DETECTION OF PLANTINE MOTHLE VIRUS AND USE THEREOF {

More particularly, the present invention relates to a primer set for diagnosing or detecting a Plantain mottle virus, and more particularly, to an oligonucleotide primer set of SEQ ID NO: 3 and SEQ ID NO: 4; An oligonucleotide primer set of SEQ ID NO: 5 and SEQ ID NO: 6; An oligonucleotide primer set of SEQ ID NO: 7 and SEQ ID NO: 8; An oligonucleotide primer set of SEQ ID NO: 9 and SEQ ID NO: 10; And an oligonucleotide primer set of SEQ ID NO: 11 and SEQ ID NO: 12, a plantain mottle virus-specific oligonucleotide primer set for detection or detection, A plantain mottle virus-specific diagnostic or detection kit comprising a primer set and a reagent for carrying out an amplification reaction, and a kit for detecting a Plantain mottle virus using the above-described primer set To a method for diagnosis or detection.

The emergence and risk of emergent viruses and new viral diseases are increasing gradually due to ecosystem changes due to climate warming, evolution and variation of plants and viruses, and increased trade in agricultural products. About 1,000 plant viruses have been reported so far worldwide, and new viruses are still reported in various plants. Currently, about 100 viruses have been reported in various plants in Korea. However, detailed investigations of the viruses have been carried out only in some crops, and most crops are only fragmented. Thus, it is not yet clear which viral diseases are occurring in many major crops.

Plant diseases are abnormalities of the host cells and tissues that result from the continued influences of pathogens or environmental factors on the plant host, including abnormalities in plant morphology, physiology, etc., resulting in a reduction in the resulting economic value It is also said. Causes of these plant diseases include fungi, bacteria, Nematode, Mycoplasma, protozoa and viruses. However, the virus is a pathogen having a size of less than 1 占 퐉 and its substance can not be observed even with an optical microscope, and since it requires an electron microscope, it is difficult to diagnose a virus that has developed in a plant with the naked eye.

In the past, electron microscopy and serological methods were mainly used for virus diagnosis. Electron microscopy can detect the presence of the virus, but it is impossible to diagnose it as a morphological feature. Among the serologic methods, ELISA (Enzyme-Linked Immunosorbent Assay) is the most commonly used diagnostic method, and its detection sensitivity is about 1,000 times lower than diagnosis using PCR. If the diagnosis fails due to nonspecific reaction of antibody and test sample Lt; / RTI > In the case of BPMV (bean pod mottle virus), ELISA is mainly used for virus diagnosis. However, OM is frequently diagnosed due to nonspecific reaction between plant extract and antiserum. In cases where the infection rate of BPMV is low, There is a high probability of failure. In addition, since the diagnosis of various pathogens must be performed on a single test sample at the quarantine site, many laboratories and inspection costs are required when using various diagnostic methods. Accordingly, various methods for diagnosing viruses have been developed and used. In particular, it is necessary to study viruses causing diseases in plantain plants.

On the other hand, Korean Patent No. 1093239 discloses a specific primer for diagnosis of garlic syphilis virus and its use, Korean Patent No. 1491789 discloses a primer for diagnosis of a bean pod colitis virus and its use, Likewise, a primer set for diagnosis or detection of a plantia mottle virus and its use has not been disclosed.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-described needs, and it is an object of the present invention to provide a method for diagnosing or diagnosing a Plantain mottle virus, The present invention has been accomplished by preparing five sets of primers that can be detected, and applying them to the diagnosis of a virus-infected plant sample to confirm its specificity.

In order to achieve the above object, the present invention provides oligonucleotide primer set of SEQ ID NO: 3 and SEQ ID NO: 4; An oligonucleotide primer set of SEQ ID NO: 5 and SEQ ID NO: 6; An oligonucleotide primer set of SEQ ID NO: 7 and SEQ ID NO: 8; An oligonucleotide primer set of SEQ ID NO: 9 and SEQ ID NO: 10; And a set of oligonucleotide primers selected from the group consisting of the oligonucleotide primer set of SEQ ID NO: 11 and SEQ ID NO: 12, and a set of oligonucleotide primers specific for plantain mottle virus specific diagnosis or detection do.

In addition, the present invention provides a plant-mottle virus-specific diagnostic or detection kit comprising the primer set and the reagent for carrying out the amplification reaction.

In addition, the present invention provides a method for specifically diagnosing or detecting Plantain mottle virus using the primer set.

Using the oligonucleotide primer set for diagnosis or detection of a plantain mottle virus of the present invention, a kit for diagnosing or detecting a placenta mottle virus, and a method for diagnosing a placenta mottle virus using PCR, In addition, it can be used at the quarantine site because it can be confirmed more accurately and more accurately than the diagnosis method using the antiserum against the mottle virus. Therefore, And can contribute to improvement of quality and quality.

Figure 1 shows a primer map designed for the diagnosis of Plantain mottle virus. Primer positions are based on the Plantain motil virus genome.
FIG. 2 shows the results of PCR analysis of primer combinations for the detection of Plantain mottle virus (see Table 1).

In order to accomplish the object of the present invention, the present invention provides oligonucleotide primer set of SEQ ID NO: 3 and SEQ ID NO: 4; An oligonucleotide primer set of SEQ ID NO: 5 and SEQ ID NO: 6; An oligonucleotide primer set of SEQ ID NO: 7 and SEQ ID NO: 8; An oligonucleotide primer set of SEQ ID NO: 9 and SEQ ID NO: 10; And a set of oligonucleotide primers selected from the group consisting of the oligonucleotide primer set of SEQ ID NO: 11 and SEQ ID NO: 12, and a set of oligonucleotide primers specific for plantain mottle virus specific diagnosis or detection do.

The primer set for diagnosing or detecting a Planta strain mottle virus of the present invention is preferably one or more, two or more, three or more selected from the group consisting of the aforementioned five Planta mottle virus-specific diagnostic or detection primer sets The primer sets for four or more Planta mottle virus specific diagnostics or detection, and most preferably the five sets of primers, i.e., the primer set of SEQ ID NO: 3 and SEQ ID NO: 4; A primer set of SEQ ID NO: 5 and SEQ ID NO: 6; A primer set of SEQ ID NO: 7 and SEQ ID NO: 8; A primer set of SEQ ID NO: 9 and SEQ ID NO: 10; And an oligonucleotide primer set of SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

By using the five primer sets simultaneously, it is possible to diagnose the plantain mottle virus more accurately.

Wherein the primer comprises at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22 consecutive nucleotides in the sequence of SEQ ID NOS: 3 to 12 according to the sequence length of each primer RTI ID = 0.0 > oligonucleotides < / RTI > For example, the primer (19 oligonucleotides) of SEQ ID NO: 3 may comprise oligonucleotides consisting of fragments of 16 or more, 17 or more, 18 or more consecutive nucleotides in the sequence of SEQ ID NO: 3, The primer (23 oligonucleotides) of the oligonucleotide of SEQ ID NO: 4 comprises an oligonucleotide consisting of fragments of 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, or 22 or more consecutive nucleotides . ≪ / RTI >

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

As used herein, an oligonucleotide used as a primer may also include a nucleotide analogue, such as phosphorothioate, alkylphosphorothioate, or peptide nucleic acid, or alternatively, And may include an intercalating agent.

The present invention also provides a kit for the specific diagnosis or detection of plantain mottle virus, which comprises the oligonucleotide primer set and a reagent for carrying out the amplification reaction.

In the kit of the present invention, the reagent for carrying out the amplification reaction may include reverse transcriptase, DNA polymerase, dNTPs, buffer and the like. 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 addition,

Isolating total RNA from the plant sample;

Amplifying the target sequence by using the separated total RNA as a template and carrying out an amplification reaction using the primer set; And

And detecting the amplification product. The present invention also provides a method for specifically diagnosing or detecting Plantain mottle virus.

In the method of the present invention, a method of isolating total RNA from a plant sample can be performed by a method known in the art. For example, Tri Reagent (MRC Co.) can be used. The target sequence may be amplified by RT (reverse transcriptase) -PCR using the separated total RNA as a template and using at least one primer set according to an embodiment of the present invention as a primer. Such PCR methods are well known in the art, and commercially available kits may be used.

In the method of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. In one embodiment, the labeling material can be, but is not limited to, a fluorescent, phosphorescent or radioactive substance. Preferably, the labeling substance is Cy-5 or Cy-3. When the target sequence is amplified, PCR is carried out by labeling the 5'-end of the primer with Cy-5 or Cy-3, and the target sequence may be labeled with a detectable fluorescent labeling substance. 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.

The method of the present invention comprises detecting said amplification product. The detection of the amplification product can be performed through capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto. As a method of detecting the amplification product, gel electrophoresis can be performed, and gel electrophoresis can be performed using acrylamide gel electrophoresis or agarose gel electrophoresis according to the size of the amplification product. Also, in the fluorescence measurement method, Cy-5 or Cy-3 is labeled at the 5'-end of the primer. When PCR is carried out, the target is labeled with a fluorescent label capable of detecting the target sequence. The labeled fluorescence is measured using a fluorescence meter can do. In addition, 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 present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited thereto.

Experimental Method

One. Sample collection and total RNA  extraction

138 plant samples suspected of being infected with plant viruses were collected and all samples were each ground using liquid nitrogen. All samples were stored at cryogenic temperatures. Total RNA was extracted after mixing the same amount of each sample with the same amount. Total RNA extraction was performed using Tri Reagent (MRC) according to the manufacturer's instructions.

2. Ribosome RNA  Removal, double strand cDNA  Synthesis, library creation and sequencing

Ribosomal RNA was removed from the extracted total RNA using Ribo-Zero ™ Magnetic Kit (plant leaf) (Epicenter). The prepared RNA was disrupted with short fragments and synthesized with double-stranded cDNA. The synthesized double-stranded cDNA was end-repaired and an adapter was attached for the cloning required for library production. This series of procedures was performed using the TrueqRNA sample prep kit (Illumina). Inserted DNA (insert DNA) of appropriate size purified using BluePippin ™ 2% agarose gel cassette (Sage Science) was selected for PCR amplification. Finally, the size and quantity / quality of the inserted DNA suitable for library production were measured using an Agilent 2100 BioAnalyzer 2100 (Agilent Technologies). The library production was completed, and the size of the library segment was confirmed to be approximately 350-450bp. The library was sequenced using Illumina HiSeq 2500 and raw data was generated at about 40 Gbp using a paired-end sequencing method.

3. Sequenced low  data( raw data ) And plant virus-related contig ( contig ) Production

After selecting high-quality data from low-level data, we obtained plant virus-related readings and created contig. This process was carried out using de novo transcriptome analysis (SG-de novo assembler). Approximately 7,000 contigs with plant virus-related sequences were obtained. When the length of Conti was too short (<500bp), data with low coverage was removed when compared with the virus database (DataBase), and the final about 700 contigs were obtained.

4. primer  Fabrication and identification of whole genome

When compared with the virus-related database (DataBase), about 150 to 50 contigues were obtained by removing the contigus that showed about 85-90% or more homology. Each of them was confirmed the most homologous virus through database, They were grouped into two groups, each of which had high homology to the same virus. The contigs belonging to each group aligned their plant viral genome as a reference sequence and confirmed their position.

Based on the nucleotide sequences of these contigs, primers were prepared and PCR was performed on 138 plant samples using the primers. The PCR results were confirmed by agarose gel electrophoresis or by sequencing of the banded PCR products. Based on the nucleotide sequence obtained from the PCR result of the primer set based on the contig, the total nucleotide sequence of the Plantain mottle virus (SEQ ID NO: 13) was obtained.

5. Plantain Motley  For virus diagnosis Primer  design

In order to prepare a primer capable of specifically diagnosing Plantaia mottle virus, which is expected to be a new virus, the genome of Plantaia mottle virus isolated from plantain samples was BLAST in NCBI, ) And similar viruses with somewhat higher identity were selected. The Plantain mottle virus of the present invention can be obtained by using the Habenaria mosaic virus mosaic virus (99% coverage and identity 81%). Thus, after aligning the entire genome of the Plantaia mottle virus and the Habenaria mosaic virus of the present invention, six primer sets were produced at a site that can specifically function to the Plantaia mottle virus One).

The primer of the present invention division SEQ ID NO: Base sequence Length Tm (占 폚) location Combination number 1 Plantain Mottle Virus - Forward 89 One CTC TAT TAA AGT TTC ATA TAC GTC 24 56.7 89-112 Plantain mottle virus - reverse 686 2 CTC ATG TTC GGC ACG TCA ATG 21 61.3 666-686 Combination number 2 Plantain Mottle Virus - Forward 402 3 GGT TGA AAA GGA TGT GGA C 19 55.2 402-420 Plantain mottle virus - reverse 883 4 AAC TTT GTG CAT CCA TCA GCA AG 23 61.1 861-883 Combination number 3 Plantain Mottle Virus - Forward 2311 5 ACT GAT TTT CAT GCA AGA AGC C 22 58.4 2311-2332 Plantain Mottle Virus - Reverse 2968 6 AAC ACC CTA CTA GTC TAC TCG 21 59.4 2948-2968 Combination number 4 Plantain Mottle Virus - Forward 2942 7 ACC AAC GAG TAG ACT AGT AGG 21 59.4 2942-2962 Plantain Mottle Virus - Reverse 3312 8 ATT ACC TTC GAG TTG CTT CAA C 22 58.4 3291-3312 Combination number 5 Plantain Mottle Virus - Forward 4997 9 TAT TGG AGA ATT TGT GGA GCG 21 57.4 4997-5017 Plantain mottle virus - reverse 5394 10 ATC CAT AAC CTT GTT GAT AGT C 22 56.6 5373-5394 Combination number 6 Plantain Mottle Virus - Forward 5646 11 CCG CAT TAT AGA TTC TGA TGG C 22 60.3 5646-5667 Plantain mottle virus - reverse 6151 12 ATT CGT GTT CAA CCT CAT GCC 21 59.4 6131-6151

All the primer sets of the present invention were located as shown in FIG. 1 based on the genome of the Plantain mottle virus. PCR was performed by combining the six primers thus prepared. The PCR method is as follows.

PCR: (1) pre-denaturation at 94 ° C for 5 minutes, (2) denaturation at 94 ° C for 30 seconds, (3) annealing at 56 ° C for 30 seconds, (4) extension) The PCR was carried out at 72 ° C for 30 seconds under the condition of temperature and time, (2) to (4) 30 times, and the cDNA prepared from the plant sample was used as a template. The PCR product was subjected to electrophoresis for 30 minutes in an amount of 5 μl in each well of 1% agarose gel.

Example  One. Plantain Motley  For virus diagnosis Primer  Identification of specificity

The PCR product of Experimental Method 5 was electrophoresed and then the band of the PCR product was confirmed by EtBr (Ethidium bromide) staining. As shown in FIG. 2, each PCR product had the same size as expected at the time of primer production (Primer combination 1: 598 bp, Combination 2: 482 bp, Combination 3: 658 bp, Combination 4: 371 bp, Combination 5: 398 bp, Combination 6: 506 bp).

<110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Primer set for diagnosing or detecting Plantain mottle virus and          uses thereof <130> PN15105 <160> 13 <170> Kopatentin 2.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 1 ctctattaaa gtttcatata cgtc 24 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 ctcatgttcg gcacgtcaat g 21 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 ggttgaaaag gatgtggac 19 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 aactttgtgc atccatcagc aag 23 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 actgattttc atgcaagaag cc 22 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 aacaccctac tagtctactc g 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 accaacgagt agactagtag g 21 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 attaccttcg agttgcttca ac 22 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 9 tattggagaa tttgtggagc g 21 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 10 atccataacc ttgttgatag tc 22 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 11 ccgcattata gattctgatg gc 22 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 12 attcgtgttc aacctcatgc c 21 <210> 13 <211> 9515 <212> DNA <213> Plantain mottle virus <400> 13 aaaatataaa aactcataca aacatacaga aaacaaacga attcaagcaa acaagcactc 60 aaagcattca caaagcattc aaaacaatct ctattaaagt ttcatatacg tcttaatact 120 ctctagcaat cagaaatggc aattcaattt ggaacaatta ctagtgaagt actggctgca 180 tgcaacgtga aaccgcagtt gacacacagc gaaatacttg atcgtgctat gatcgaaaag 240 tgcggagttg caaaaatatc tgtactaccg tacattaaga gtctgaatgt gcaagccccg 300 acaccacgca tccgtaaagc atggcgagtg gagaacaaag gttttgggga aacccaaata 360 gtggaaacaa ttgtagatac ctttgataat catattgtca aggttgaaaa ggatgtggac 420 acgaagggta aagttgtagc cacttcgttc aggacacgct ctgccaaggt tgcacgacca 480 attgtgcaat cattgggcga aaagaactcg ctactgcata aagtgtgtgc actagcgttt 540 aagcgaaaca taccagtgac attcattggt aagcgaactg agcgtgttcg tgcagcacgc 600 cacgtgacat cgaacttctc ttgcatggca atagtaacac accaccatgc tgggaagaca 660 cgcaacattg acgtgccgaa catgagctca ttgcgtgata tggttgttag tgtcgcgcac 720 gcaacgtgga aaggtaacaa aatacacgaa cggaatatca aagttggtga tagtggttgc 780 attataccac gtgaaatgat tgaaggcact gtccactgtg acaaggatga tgtttttatt 840 gtgcgtggac gctacagtaa cttgctgatg gatgcacaaa gttacttacc gatgagttat 900 tgcaccaagg tagttccgta ctctacagca gagcaatact ggaaaggttt tgacgtggca 960 tttcgtgcga ataggagcaa ccaactaata cacgaacctg gggagaagct ggatgttgag 1020 caatgtggtt ctatcgcagc aatactgcac cagtcattgc taccctgttg taagattacg 1080 tgcaccactt gcagtaaaat tctcgaggag agcagtgctg aggaaacacg acaacgaatc 1140 gggcaaaccg caaggaaagg ggcgcagctc attaggcgta attatcgtgg cttcgaacat 1200 gtctatcaac tgctaatgaa tcatgcgaat atgctagatt ctgttaacag caatcgagaa 1260 gcatgcggta aggtgcaata tataatcgga gagcgcacag atgcaccgtt cagtcatgta 1320 ctacgcatca atgaaacact cataaaaggt aatcaagcca ctgcaagtga gttatcagca 1380 gcatctaatc atctcctgga aattgcgcgt tatttgaaaa accgcacaga aaacattcag 1440 aaggggtcac tgcgctcgtt ccgaaataaa gtttcagcaa aagctcatct aaatccacaa 1500 ttgctttgtg ataaccagtt ggatgcagat ggaaatttcg tgtggggtga tcgagcatac 1560 catgccaaac gcttcttctc gaacttcttt gaggagataa atcctgaaca tgggtatgat 1620 aagtacataa tacgcaagtt tccgaatggt agtagaaaac tagcaatagg gcagttaata 1680 ctgtcaacaa atttagatcg gttacgtgaa cagttagtag gggaacccat aaaacccgaa 1740 ccgttaactg acgcatgtgt tagtaaaatt catgaaacat ttatataccc atgctcatgt 1800 gtaacgtatg acgacgggac accagttcta tccgagatga aggcacccac gagcaatcac 1860 ctagtccttg taacgcaggg gattcaaagt acttggactt accgacagga agaggagatc 1920 gcatgttcat ttgcaaaaga aggttattgc tacatgaaca tctttttggc tatgcttgtt 1980 aatgttgata aagataaagc caaagatttt actaagtggg ttcgggatac aattgtgacg 2040 cagctaggcc aatggcctac aataacagat gtcgcactcg catgtttcca gctatccatt 2100 atgttccctt cagtgcgtaa tgcagaatta ccaagaatac tcgttgatca ccatacgaaa 2160 acgttgcatg tcttggactc gtatggatca ttaactacag gttttcacat acttaagatg 2220 aacacggttg atcagctcat taaaatagct aatgaaactt tagagtctga gattaagcat 2280 tatagagttg gaggaactga ttatgctggc actgattttc atgcaagaag cctaaaacaa 2340 gtaatccgcg gagtataccg tccgagcgaa cttcgaagca tcctcaatca tgacccgtac 2400 atactgacga tggcactcct atcaccagca attctgacag gcttatttac aacaggatcc 2460 ctctaccaag ccactttgtc tctaattcct gaagatactt cagcgagaca tttagtgtgc 2520 ttattgactt cgctcgcggg tcgtgtttca cgattgaagg atttacatga tcaagtcaat 2580 atcattgagg aaaatctagg agcttttcta gaaattttgt ctgttggtga tcgttgtagt 2640 tatgctcgag catttatgca gcgcacgata gaggcacggt tagaatcgat tagtgcggat 2700 gaagaactcg atgctagtgg gttcagaaca cttcgttgga aatccgtcca agtgctggaa 2760 aaaatttaca cagaggattt ggaggcttca tggcaagatt tgcagtttgt ggaaaagtgc 2820 tatataatgt tgcaaaagtt gagatggcga aggcgtatta tagtagaact cagccaagaa 2880 agtgccatat ctttcaagaa ggtctacgag cattgcagca caggcttgca cctcgctatg 2940 aaaccaacga gtagactagt agggtgttgt acaaataaat ttggtaacgt agctaagagt 3000 gtgcataata gattgttgag tggatttgtc tatggtttca ggtgtgtatt ccgcgatcta 3060 tttcattttg ttcaagtttt agctatttgt aatatatttc ttatgatttt agattcgtta 3120 ctacgtctta ggagtgcata tatagctaat gcacgccaag ttcattatat gcgtgaacaa 3180 cagaataggg ataaactaga aaaattgtat agtgtcctca aacacaaact cggaatggag 3240 ccaacgttcg atgaattcaa agagtttgtt agtggagtga atcctgaatt gttgaagcaa 3300 ctcgaaggta atgatgagtt agaagtggag caccaggccg ttaagcgaga gagtgaagct 3360 cgactcgaac aaatcgtggc attcattgca ctggttctta tggtattcga taatgaacgt 3420 agtgactgtg tctatcgagt tatgaataaa cttaagaatg tagtgggtgt cgccgaccaa 3480 gggggaacc accagagcat ggacgatgag aatgaagtat ttgatgataa tgcaacaatc 3540 agctttgagc tggagtgtga agacccagtc cgtgcatacc cgagttcatc tacactcgag 3600 cagtggtggg ataaccaact cgctctcaac agaacaatcc ctcattaccg aactgagggc 3660 tatttcatgg agttcacaag agctaactgc gcgcaagtta taaatgagat tgtgcacaac 3720 gagcacaagg atattctatt gagaggtgcg gttggctctg gtaaatcaac tggtttacca 3780 gctggattgt cgacccgcgg aaaagttcta cttctggaat caactaagcc actcagcaga 3840 aacgtgttta atcaactgag acaggatccc ttccatcttt caccatctct catgatgcgt 3900 gactccacta catttggttc aacaccgatc actatcatga cgagtggtta tgctttccac 3960 tactttgcaa acaatgcagg gaaacttcga gattatcaat tcgttatgat agatgaatgt 4020 cacgtacttg acgcaaatgc catggcattc agatgtctac ttgaagaaca tgactaccaa 4080 gggaagatta ttaaagtgtc agcgacacca ccaggacggg aagtggaatt taccacgcaa 4140 cacaaagttg aaatacgaat cgaggattcg ttatcattcc aacaatttgt cgccacactt 4200 ggcacaaaag gaaacgcaga cgtgactgat aaggccgaca acatccttgt atatgtagcg 4260 agctacaatg aagttgataa attatcaaag ctgctacaag agaagggtta tctggtgaca 4320 aaagttgacg ggaggactat gaagaatggt gttagcgacg ttgtaacaaa aggaacaccc 4380 acaaagaagc atttcatagt tgcaacgaac ataattgaga atggtgtcac tctggatatt 4440 gaagcagttg tagattttgg caccaaagta gtaccaactc tcgacacgga ctctcgaagg 4500 atactatact ctcacacatc cattagttat ggggagcgca tacaacgatt gggtcgtgta 4560 ggaagattca aacctggtgt agcactaagg attgggcata cgcagaaagg gatttgcgct 4620 attccctcaa taatcgcgac agaggctgcg ttcttgtgtt ttatttacgg tctaccagtt 4680 atgacatctc aggtaagtac cagcctgctg cgaaagtgca cagtgcaaca agcaagggtc 4740 atgaaactat tcgaattgcc cacatacttc atgcttgatt tagttaggca tgatggcaca 4800 atgcacccag atgttcacag attgctcact aaatataagt tgcgtgagag tgaaatcgta 4860 ttgaacagaa tggctatacc acatgcgcga acatatcatt ggatggacgt gcggacatat 4920 aacgcttgtg gtaccaactt aacacttgca ccagacataa agataccgtt ctactgcaag 4980 gacttgcctg agcaattatt ggagaatttg tggagcgtca tccaaagaaa taagggtgat 5040 gcaggtttca gaactcttaa aagtcatgat gctgcaaaaa ttgcatacaa gctgcacact 5100 gatgagcact ccattcaacg aacagttgcg atcattgatg cacttatcat tgaggaacag 5160 actaagaaag cacactttga ttcgctagtt gtgaacacat gctcgagcgc ttctttctct 5220 ttgcaatcag ttagtaacgc gctgcgtgca agatataagc gaaacaacac aactgagaat 5280 ataagtgtgt taatggcagc gaaggcgcag ttacttgact ttcaacactc ttgctatgag 5340 gaaagcattg ttataaaccc aggttcgcaa cggactatca acaaggttat ggataatgga 5400 gcccttgaaa ctgtcttgca tcaaagccgg gaagggataa tcaagagctt aaatctgcaa 5460 ggtaagtgga agggaacgtt actcacaaga gatctattgg tttgcgctgg agttgcttgt 5520 ggtggtgtgt ggttgttata ccaatatgtt cagaatttca tgaatgaacc agtagagcac 5580 caagctaaaa acaagcgaca gaagcagaag cttaaatttc gcgatgcgcg cgatagaaaa 5640 gttggccgca ttatagattc tgatggctgt ggtgaagctg ttgaatggct ctttggtgat 5700 gcatacacga agaaaggcaa gcgcggtgga aaaacacgag gcatgggaac gaaaacacga 5760 aggtttgtga atatgtatgg ctttgatgaa gctgaattta catacatacg cttcgtagat 5820 ccggtgacag gagaaatctt ggatgagagt gtgatgaccg acatctcgat agtgcaggat 5880 cattttggtg atctgaggag agagtacatt ggggaggata aaatttcacc acaggcactg 5940 tatagcaacc cgggaatcaa agcctatctt gttaagaata aaacatcgcc agtgcttgag 6000 gttgatctta cactccatga accactcaag ctatgtgaca actcatcaac cattgcaggg 6060 ttcccagaga aggaaggaat tctaaggcag acaggtccag ccagacaaat caagtatgag 6120 gacatgcctg ggcatgaggt tgaacacgaa tctaaatcac tgaaccgtgg gcttcgcgat 6180 tacacgccaa tctcaaaatc catttgtttg ctgcaaaaca catcagatgg gtgcagcacc 6240 acaatacatg gcgttggata tggttcactc attgtttcaa atgcgcattt gttgaagcga 6300 aataacggca ccttgacaat aaagtccatg catggggagt tcaaaattca gaatacaacg 6360 gcaattagaa tagcaccagt gcccaattgt gatttaattg tcttaagatt gccaaaggac 6420 ttcccaccat tttccacgaa gttaaagttc agagtgccag agtcaaatga gcaagtgtgt 6480 atggttggca ccaattttca ggagaagtgg atgtcgagta ccgtttcaag cacaagctac 6540 atacagcata tacctgggac acaatttgtg aaacactgga ttgatacaaa ggatggacac 6600 tgcggtcttc caatggtggc atcgaaagat ggagcaattc ttggtcttca tagtttgact 6660 aatacaaaac aagaatataa ctgctttgcc tccgccacga gtgtgttaat ggaaatacta 6720 gatgccccag agcatgctga ttggcgtaaa ggttggatgt acaaccctaa tgacatcagt 6780 tggggtttca tgcgcttgaa agagagtaca ccatctggtt tgtttaagcc tgctaaatcc 6840 attagggatt tagagctcga cattgttaat gagcaggcac aagtgcagga acgttggttt 6900 aaaaaccagt tgcattgtaa ccttaaagct gttggacata gcgacagtca gcttgtgacg 6960 aagcacgtga taaaaggaaa gtgtccactc tttgaacggt atttatgtga aactccgagc 7020 gctgcaaaat atttcagacc attaatgggt gcttaccaga aaagccgact aaatcgcgta 7080 gcatatgcga aagatgcact caagtacgca acaatcattg aatgcggttt agtcgagcca 7140 gaatcgtttg aacaggccat cgtgaatgtc atacaaattc tcaagaaggc tggcttctca 7200 gagtgtgcat acatcactga tccagaggaa atatttgcta atcttaacat gaaagcagca 7260 gtgggggcct tgtatgctgg gaagaagaag gattattttt ccgagtacac acaagagcag 7320 agagaagaaa tcctacaaca aagtgcggag cgtttgtata aaggtttcaa gggcgtgtgg 7380 aatggagcaa ttaaagcaga gctgcgaact cgtgagaaag tggaagcaga taaaacaagg 7440 actttcacgg cggcaccaat agacacatta ctcgcaggca aaatatgtgt tgatgatttc 7500 aacctccagt tctatagctt acacactaaa gccccatgga gcgttggtat ttctaaattc 7560 tcgtgtggat gggatgcgtt attacgtaag ctaccagatg ggtggatata ttgtgatgct 7620 gatggaagtc gcttcgacag ctcgttgact ccatacatta tcaatgcgat acctattatt 7680 cggcttgctt tcatggaaaa gtgggacatt ggtgaggtta tgatgcggaa cttatacaca 7740 gagattgtgt acaccccgat ccttactgca gatggcacga ttgtaaagaa gtttaaaggc 7800 aataatagtg gacagccttc tacagttgtg gataatacac ttatggttct actggctatg 7860 caatatgcac tcgagcgact cagtatagat tttacagtgc aggaacaagt gtgcgtatat 7920 ttcgccaatg gtgatgatct tgttgtagct gtagcacctg gatacgaaca cattctggac 7980 gcattgcagg gctacttctc tgagttgggt ttgaactacg atttatcaag tagacacaaa 8040 gaccgcgcaa atctctggtt tatgtcacac aaaggtgtaa taagagatgg tctatacata 8100 cccaaacttg agagtgaaag gattgtatca atacttgaat ggacacgtgc taatgagccg 8160 gctcatagat tggaggccat ttgtgcagct atggttgaag cgtggggtta taatgaactc 8220 ctgcatgaaa tacgtttgtt ctacaactgg attctccagc aacaaccata tgctacacta 8280 gctcaggaag gtaaagcacc ttacatttcg gagtgtgcat tacggagatt atacatggac 8340 aagttgatag agccgcacga atacgcaacg tatcttgaaa agttagtggc atccgtgcaa 8400 acttttgatg atagcgcgaa tttcatgatg catcaagcca gtgagcaaag agtggatgct 8460 actaacccat ttggaaaaca ggcacaaaca aaagggtctg attctgaaag aagcagctca 8520 cgtgatgaac aacagaggaa cgaagataat cagcgcaaag attcagcacc actagcaccg 8580 gtaccagatc gggatataaa tgctggcaca acagggacat ttactgttcc aaagctcaag 8640 ggtatgagta caaagctcac aatacctaaa gtgaaaggaa aagtcgttgt gaatttacaa 8700 cacttgctgc aatatacgcc agaccaggag aaactgtcaa acactttcgc aactgatgaa 8760 caatttgcca tatggtataa tggtgtcaaa ggtgattatg aagtgtctga cgatgagatg 8820 caaataatat tgaatggtct aatggtgtgg tgtattgaga atggcacctc accaaactta 8880 tctggagtgt gggttatgat ggatggggaa gaacaagtga catacccaat taaaccacta 8940 cttgatcatg cacaaccaac ttttaggcag atcatgcacc acttcagcaa cttggcagaa 9000 gcgtatatag aaaagcgtaa ttacattagt ccgtatatgc caagatatgg tcgtaatcgc 9060 aatcttaccg atatgtccct cgcacgctat gcattcgatt tttacgcagt aacttcgcgc 9120 acaccagagc gagcaaaaga ggcacatatg cagatgaagg ccgcagccct tcgtaatacc 9180 agtacacgaa tgtttggact ggacggtaag gtcggcacac aagcagagga cacggaaagg 9240 cacacagcag aggacgttaa ccgaaacatg cacaacctac taggtgtgcg tggcgtttaa 9300 agtgtgctat tagactggca tcgaacttat ataatatatt atgtaagagt ttaatcgtta 9360 gtatcctcct tgccgtacgt atttttataa gcaaagagtg gttcgtccac gtacttatac 9420 tacatccgta catcgctttc tatacttatg tcagacaaag tgagatttta tctcggtctg 9480 gggttttata gagagagaga aaaaaaaaaaaaaa 9515

Claims (7)

An oligonucleotide primer set of SEQ ID NO: 3 and SEQ ID NO: 4; An oligonucleotide primer set of SEQ ID NO: 5 and SEQ ID NO: 6; An oligonucleotide primer set of SEQ ID NO: 7 and SEQ ID NO: 8; An oligonucleotide primer set of SEQ ID NO: 9 and SEQ ID NO: 10; And a set of oligonucleotide primers selected from the group consisting of the oligonucleotide primers set forth in SEQ ID NO: 11 and SEQ ID NO: 12, and a set of oligonucleotide primers specific for plantain mottle virus. 2. The oligonucleotide primer set of claim 1, wherein the oligonucleotide primer set of SEQ ID NO: 3 and SEQ ID NO: 4; An oligonucleotide primer set of SEQ ID NO: 5 and SEQ ID NO: 6; An oligonucleotide primer set of SEQ ID NO: 7 and SEQ ID NO: 8; An oligonucleotide primer set of SEQ ID NO: 9 and SEQ ID NO: 10; And a set of oligonucleotide primers of SEQ ID NO: 11 and SEQ ID NO: 12 for the detection of a plant mottle virus specific diagnostic or detection oligonucleotide primer set. A kit for the diagnosis or detection of plantain mottle virus, comprising an oligonucleotide primer set according to claim 1 or 2 and a reagent for carrying out an amplification reaction. 4. The kit for the diagnosis or detection of plantain mottle virus according to claim 3, wherein the reagent for carrying out the amplification reaction comprises DNA polymerase, dNTPs and a buffer. Isolating total RNA from the plant sample;
Amplifying the target sequence by performing amplification reaction using the separated total RNA as a template and using the primer set according to claim 1 or 2; And
And detecting the amplification product. A method for specifically diagnosing or detecting Plantain mottle virus. 6. The method of claim 5, wherein the amplified target sequence is labeled with a substance that emits fluorescence, phosphorescence, or radioactive measurement. 6. The method of claim 5, wherein the detection of the amplification product is performed by DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement.

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