KR20130032922A - Genetic markers for detecting quarantine plant viruses and methods for detecting by using thereof - Google Patents

Abstract

PURPOSE: A marker for detecting pathogenic viruses in Cucurbitaceae plants or Solanaceae plants is provided to simultaneously detect viruses in the Cucurbitaceae plants or Solanaceae plants. CONSTITUTION: A primer set for detecting viruses infecting Cucurbitaceae plants contains a sequence numbers 1 and 2, and 7 and 8. The viruses are a cucumber vein yellowing virus(CVYV) or a cucurbit yellow stunting disorder virus(CYSDV). A method for simultaneously detecting viruses infecting the Cucurbitaceae plants or Solanaceae plants comprises: a step of isolating RNA from a sample; a step of performing RT-PCR using the RNA as a template and the primer set; and a step of separating a PCR product by size.

Description

Genetic Markers for Detecting Quarantine Plant Viruses and Methods for Detecting by Using Thereof}

The present invention relates to a marker for detecting a pathogenic virus infected with a gourd or eggplant and a detection method using the same.

Due to the increase in global trade, various kinds of plants are being imported to Korea. It is therefore possible that potential or unclear pathogens, especially plant viruses, will come along with host plants such as grains, feed, hard woods, seedlings, vegetables, fruits and herbal medicines. Recently, the import of some host plants with high sensitivity to various viruses has been strictly prohibited by the National Plant Quarantine Service (NPQS). However, since the host range for many plant viruses is not well known, foreign plant viruses that are not reported in Korea can enter through unknown host plants.

In order to prevent unreported plant viruses from coming in from abroad, it is necessary to prepare specific, sensitive and rapid detection methods for quarantine. One known method is based on reverse transcription-polymerase chain reaction (RT-PCR), which is superior to other methods (Park and Kim, 2004). Therefore, the present inventors have developed a specific primer set for detecting RT-PCR of plant viruses that can be introduced into Korea through imported plants for the diagnostic detection system based on RT-PCR and completed the present invention. It was.

We selected five plant single-stranded RNA viruses from a variety of plant viruses, such as Cucumber vein yellowing virus (CVYV) (Janssen et al., 2007), cucurbit yellow stunting disorder virus; CYSDV (Yakoubi et al., 2007), Potato aucuba mosaic virus (PAMV) (Manoussopoulos, 2001), Potato yellow dwarf virus (PYDV) (Ghosh et al., 2008) ), Tomato chlorosis virus (ToCV) (Wintermantel et al., 2005). These viruses cause serious economic loss to the plant belonging to the family of the family of the family of the family of fruits of gourd or eggplant. They belong to various genus and family and show distinct biological properties as shown in Table 1. For example, CYSDV and ToCV belong to the genus Crinivirus belonging to the family of Closteroviridae, while CVYV, PAMV and PYDV belong to Potyviridae, Alphaflexiviridae and Lavdovirus ( It belongs to the genus Ipomovirus, Potexvirus and Nucleorhabdovirus belonging to the family Rhabdoviridae (ICTV, 2010). Three viruses, CVYV, CYSDV, and ToCV, are commonly transmitted by Bemisia tabaci (Berdiales et al., 1999; Martinez-Garcia et al., 2004; Morris et al., 2006). PAMV and PYDV commonly use aphids and insects, the Agalia constricta (Ghosh et al., 2008; Manoussopoulos, 2001) as vectors. CVYV and CYSDV infect a large number of host plants, especially gourds, including cucumbers, melons and pumpkins (Janssen et al., 2007; Yakoubi et al., 2007). In contrast, PAMV, PYDV and ToCV infect eggplants, including potatoes and tomatoes (Bandyopadhyay et al., 2010; de Bokx, 1975; Louro et al., 2000).

TABLE 1 List of plant viruses used in the present invention

The occurrence of all five of these viruses has been reported in Southern Europe, including Spain and Portugal (Cuadrado et al., 2001; Font et al., 2004; Louro et al., 2000; Lozano et al. , 2004). For example, since ToCV was first encountered in Malaga, Almeria and Spain, tomato infections with foliar chlorosis have become increasingly potent infectious viruses, particularly in southern and eastern Spain (Font). et al., 2004). In the last few years, ToCV has spread to new areas and plants such as paprika (Lozano et al., 2004). In addition, cucumbers grown in greenhouses in Almería (Spain) were severely damaged by CVYV in 2000 (Cuadrado et al., 2001).

These viruses are often infected with two or more kinds of viruses, and the appearance of the virus-infected plants is very diverse and complex depending on cultivars, types, and environmental factors, making it difficult to accurately diagnose the virus with the naked eye. On the other hand, the virus diagnostic antibody reagent that is generally used can only diagnose one virus, so when it is necessary to diagnose a complex infected sample, there are problems of using several reagents or diagnosing it several times.

On the other hand, Korean Patent Registration No. 10-496015 "Specific primer for diagnosing Curinnovan mosaic virus", Korean Patent Registration No. 10-0703599 "Set of primers for detecting begomovirus, detection method and kit using the same", Korean Patent Registration No. 10 -496017 discloses a primer for detecting plant viruses, such as "a combination of five crop virus-producing primers for simultaneous diagnosis". The Korean Patent No. 10-496017 is commonly used in gourd crops and is a primer combination for diagnosing a virus already reported in Korea. However, there was no detection primer set specific to virus which has not been reported in Korea. The present inventors have completed the present invention by recognizing that a primer set for detection specific to a virus has not been reported in Korea.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

It is an object of the present invention to provide a primer set as a marker for the simultaneous detection of pathogenic viruses against gourds or eggplants.

The present invention is to provide a method and a detection kit for the simultaneous detection of viruses infected with fruit or eggplant using the primer set.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

The first aspect of the present invention is a primer set for the simultaneous detection of viruses infected with gourd plants, characterized in that consisting of a primer set of SEQ ID NO: 1 and SEQ ID NO: 2, and a primer set of SEQ ID NO: 7 and SEQ ID NO: 8 To provide.

In more detail, the virus is Cucumber vein yellowing virus (CVYV) or Cucurbit yellow stunting disorder virus (CYSDV). Provide a set of primers for.

The second aspect of the present invention is a branched fruit comprising a primer set of SEQ ID NO: 9 and SEQ ID NO: 10, a primer set of SEQ ID NO: 15 and SEQ ID NO: 16, and a primer set of SEQ ID NO: 19 and SEQ ID NO: 20 Provided is a primer set for simultaneous detection of virus infected with plants.

More specifically, the virus may be a potato macular mosaic virus Potato aucuba mosaic virus; PAMV), Potato yellow dwarf virus (PYDV) and Tomato chlorosis virus (ToCV), which provide a primer set for simultaneous detection of viruses infected with eggplant plants, characterized in that selected from the group consisting of. do.

The primer used in the present invention is hybridized or annealed at one site of the template to form a double-stranded structure. Suitable nucleic acid hybridization conditions for forming such a double-chain structure include Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization , A Practical Approach, IRL Press, Washington, DC (1985).

Various DNA polymerases can be used for amplification of the present invention and include the "Klenow fragment" of E. coli DNA polymerase I, thermostable DNA polymerase and bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtainable from various bacterial species, which is Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus , Thermococcus literalis , and Pyrococcus furiosus (Pfu).

As used herein, the term "primer" refers to a single-strand that can act as an initiation point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) at a suitable temperature. Oligonucleotides. Suitable lengths of primers are typically 15-30 nucleotides, although varying depending on various factors, such as temperature and the use of the primer. Short primer molecules generally require lower temperatures to form a hybrid complex that is sufficiently stable with the template.

Primers of the invention can be suitably labeled to provide signals capable of analyzing amplification products of DNA markers. Suitable labels include fluorophores (eg, fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia), chromophores, chemilumines, magnetic particles, Radioisotopes (P ³² and S ³⁵ ) and the like, but are not limited thereto. The labeling may be carried out in various manners conventionally practiced in the art, such as nick translation method, random priming method (Multiprime DNA labeling systems booklet, "Amersham" (1989)) and chination method (Maxam & Gilbert, Methods in Enzymology, 65: 499 (1986)). The label provides a signal that can be detected by methods such as fluorescence, radioactivity, colorimetry, weight measurement, and the like.

In addition, the base sequence of the primer includes a sequence to which a label that binds to a molecule of the base sequence and / or a site capable of binding to a solid phase carrier is introduced.

In the case of the modified sequence, it means a sequence in which some bases are deleted, inserted, added, and substituted within a range that substantially maintains the hybridization ability with the genome of the virus. In addition, individual modification sequences should be modified to the extent that they are not hybridized with other viral genomes. When used as a primer, the number of modified bases does not require any particular limitation as long as it substantially maintains the hybridization ability, but it is generally preferred that the total number of bases of the base sequences obtained as a result of the variation is about 10 to 50.

The position of the mutation is not particularly limited as long as it does not interfere with the hybridization, but preferably, the 3 'terminal portion that affects the elongation step of the polymerase chain reaction is excluded or only minimal mutation is allowed. Should be.

A third aspect of the invention comprises the steps of (a) isolating RNA from the sample; (b) using the isolated RNA as a template and performing RT-PCR using a primer set according to claim 5; And (c) provides a method for the simultaneous detection of the virus infected by the fruit or eggplant plant, characterized in that it comprises the step of separating the PCR product by size.

A fourth aspect of the invention provides a kit for the simultaneous detection of viruses infected with nectarine or eggplant plants.

The detection kit includes PCR buffer, dNTP, Taq polymerase and primers. The primer included in the tracking kit may vary depending on the target virus to be detected, and PCR reaction conditions may vary depending on the primer. The primer is preferably adjusted to the PCR reaction conditions according to the GC%, and Tm of the primer, it is preferable to include a primer that can be PCR in the most optimal reaction conditions.

Virus specific RT-PCR and serological approaches are widely used to detect viruses. Compared to serological approaches that require many experimental steps and take a long time to produce antibodies, the RT-PCR used in the present invention is simple, fast, reproducible and accurate. Western blots and enzyme-linked immunosorbent assays (ELISAs) require large amounts of samples, while RT-PCR is sufficient for small samples, such as small pieces of virus-infected leaves. Biological samples cannot exist for a long time at airports or seaports, and many samples must be tested at the same time, which is fast for quarantine and requires accurate and specific results. Therefore, the RT-PCR based detection system of the present invention has a superior effect on plant virus quarantine than other methods.

1 shows the results of RT-PCR amplification of virus-specific dsDNA fragments using the newly developed primer set and isolation of total RNAs. (A) Agarose gel electrophoresis results of total RNAs isolated from virus infected plant leaves. M: λ DNA / HindIII size marker (600 ng / 5 μl), lane 1: CVYV, lane 2: CYSDV, lane 3: PAMV, lane 4: PYDV, lane 5: ToCV (B) RT of virus specific dsDNA fragment -PCR amplification results are shown. M: λ DNA / HindIII size marker (600 ng / 5 μl), lane 1: CVYV-1 primer set, lane 2: CYSDV-1 primer set, lane 3: PAMV-1 primer set, lane 4: ToCV-1 primer Set, lane 5: PYDV-1 primer set, lane 6: PYDV-2 primer set, lane 7: PYDV-3 primer set
2 shows Gradient RT-PCR results to improve the specificity and sensitivity of primer sets. M1: λ DNA / HindIII marker (600 ng / 5 μl), M2: 100 bp DNA ladder marker (NEB, UK), lanes 1, 4, 7, 10, 13, 16 and 19: annealing temperature 50 ° C .; lanes 2, 5, 8, 11, 14, 17, and 20: annealing temperature 53.2 ° C .; lanes 3, 6, 9, 12, 15, 18 and 21: annealing temperature 56.7 ° C.
3 shows RT-PCR results for virus detection specificities of newly developed primers. Amplified dsDNA fragments were isolated on 1% agarose gel and stained on EtBr. CDNAs prepared from healthy N. benthamiana (lanes 1, 4, 7, 10 and 13) and tomatoes (lanes 2, 5, 8, 11 and 14) and virus infected leaves (lanes 3, 6, 9, 12 and 15). Each primer set was used for PCR reaction. M: 100 bp DNA ladder marker (NEB, UK)
4 shows the results of optimization of the annealing temperature for RT-PCR. PCR was performed with each primer set at (A) annealing temperature 53 ° C. and (B) annealing temperature 56 ° C. to show amplified dsDNA fragments. lane 1: CVYV-2 primer set, lane 2: CYSDV-2 primer set, lane 3: PAMV-2 primer set, lane 4: ToCV-2 primer set, M1: λ DNA / HindIII marker (600 ng / 5 μl) , M2: 100 bp DNA ladder marker (NEB, UK)

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 only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example  One: RNA  extraction

The five plant viruses used in the present invention were obtained from DSMZ GmbH (Braunschweig, Germany), a plant virus collection, in the form of virus infected dry plant tissue or total plant nucleic acid extracts stored at -20 ° C. All viruses were imported with approval from NPQS, and all experiments were performed in close cooperation with NPQS staff.

Extraction of total RNA from healthy leaves and virus-infected plant tissues of Nicotiana ventamiana ( N. benthamiana ) and tomatoes was performed by Trizol Reagent? (Molecular Research Center, Inc., USA), Phenol (phenol): Chloroform (chloroform): Isoamylalcohol (25: 24: 1) extraction, ethanol (ethanol) precipitation method was used (Park and Kim, 2004). Quantification of total RNA was performed using NanoPhotometer ^™ (Implen GmbH, Germany) and electrophoresis on 1% agarose gel.

Example  2: primer  making

For RT-PCR, primers were prepared to enable separation and identification of various viruses due to different molecular sizes of the final PCR products based on the nucleotide sequence of each virus. CVYV [5'-GAATGGCACAAGTGATGAGAGG-3 '(SEQ ID NO: 1) / 5'-ATTAGCGGCAAGTGTCTGGTG-3' (SEQ ID NO: 2) or 5'-GGCACAAGTGATGAGAGGAAAAC-3 '(SEQ ID NO: 3) / 5'-CATTAGCGGCAAGTGTCTGGTG-3' Number 4)], CYSDV [5'-GGACGGCTGACTTTATCAATTATG-3 '(SEQ ID NO: 5) / 5'-TGTGACACGTTATAATATTTCTTTTC-3' (SEQ ID NO: 6) or 5'-CATGCACGGTGACCAAAAGAAG-3 '(SEQ ID NO: 7) / 5'-CAAAGCCTGGCATTTCATCAAC -3 '(SEQ ID NO: 8)] and ToCV [5'-GAGGTTAGACCCAAAATGTCCG-3' (SEQ ID NO: 19) / 5'-CTGAGATATTCATCAACGAACCAT-3 '(SEQ ID NO: 20)] for well-preserved coat protein regions RT-PCR primers were prepared using the nucleotide sequence. To detect PAMV [5'-GCTGTGGGCTGTGTGTAAAG-3 '(SEQ ID NO: 9) / 5'-TCCAATTGTCTTTAATGAACGC-3' (SEQ ID NO: 10) consisting of 6 ORFs, the sequence of the putative polymerase was subjected to RT-PCR amplification. Used. PYDV [5'-CGCTCAGTTCGTGCAGTTGT-3 '(SEQ ID NO: 13) / 5'-TCTGATTCTAGTCCACGCTGC-3' (SEQ ID NO: 14) or 5'-GATGCAGTCAGCGGAAGTCAC-3 '(SEQ ID NO: 15) / 5'-GGTAATGGCTGGCAGTCCA-3' (SEQ ID NO: 13) No. 16), specific primers for RT-PCR were prepared using a nucleocapsid protein portion based on an RNA sequence transcribed from a negative strand. PCR product predicted by RT-PCR ranges from 192 to 354 bp. In the case of PYDV, three different primer pairs were produced (Table 2). All primers were made in Bioneer (Korea).

TABLE 2 Base sequences of oligonucleotide primers used in RT-PCR

Example  3: cDNA synthesis

Total RNA isolated was quantified on 1% agarose gel stained with ethidium bromide (EtBr). All total RNA extracted showed very good quality (FIG. 1A). Using the isolated total RNA as a template, we synthesized cDNA. For each virus, 2 μg total RNA (5 μl), 10 pmol reverse primer (1 μl), 3 μl 2.5 mM dNTPs and 6 μl of DEPC treated dH2O were added and denatured at 65 ° C for 5 minutes. Do this. And 100 units of M-MLV reverse transcriptase (RT), M-MLV 5X buffer (5 μl) and 4.5 μl of DEPC treated dH2O in a mixture of RNA / nucleotide / primer (Promega, USA). give. The RT reaction proceeds at 42 ° C. for 1 hour, and 1 hour later at 80 ° C. for 3 minutes to terminate the RT reaction.

Example  4: polymerase chain reaction ( PCR ) And sequencing

To amplify PCR fragments from each cDNA, 2 μl cDNA mixture, 5 μl 10X buffer, 3 μl 2.5 mM dNTPs, 2 μl primer pairs (Table 2), 37.5 μl dH 2 O and 2.5 units of Taq DNA Prepare 50μl PCR mixture containing polymerase (Takara, Japan). PCR product was 94 ° C./3 min (once); 92 ° C./20 sec, 53 ° C./1 min, 72 ° C./1 min (30 times); The reaction was carried out using Peltier Thermal Cycler (MJ Research, USA) according to the condition of 72 ° C / 10 min (once). PCR products were separated on 1% agarose gel, QIAquick ^? Extraction was performed using gelextractionkit (Qiagen). The DNA sequence was confirmed by ABI Prism 3730 XL DNA Analyzer (Applied Biosystems) of the National Instrumentation Center for Environmental Management at Seoul National University. The sequence was analyzed by BLAST and Clustal W.

Using the synthesized cDNA as a template, PCR was performed at annealing temperature of 53 ° C. for 30 seconds. As a result, all primer pairs except PYDV-3 amplified with two PCR products were amplified with specific fragments (FIG. 1B).

Example  5: Gradient RT - PCR

To increase the specificity of the primer binding gradient PCR was performed to change the annealing temperature. PCR product was 94 ° C./3 min (once); 92 ° C./20 sec, 50 ° C./1 min, 72 ° C./1 min (30 times); 72 ° C./10 min (once), 94 ° C./3 min (once); 92 ° C./20 sec, 53.2 ° C./1 min, 72 ° C./1 min (30 times); 72 ° C./10 min (once), 94 ° C./3 min (once); 92 ° C./20 sec, 56.7 ° C./1 min, 72 ° C./1 min (30 times); The reaction was carried out using Peltier Thermal Cycler (MJ Research, USA) according to the conditions of 72 ° C / 10 min (once). PCR products were separated on 1% agarose gel (FIG. 2). As a result, it was found that increasing the annealing temperature amplified a lot of PCR products, but the PYDV-3 primer pair failed to obtain a single specific amplification fragment (FIG. 2).

To test the virus specificity of the newly prepared primer pairs, total RNA was isolated from the leaves of healthy N. benthamiana and tomatoes. Virus specific primer pairs were detected only in each virus of the plant leaves infected with the virus and not from healthy N. benthamiana and tomatoes (FIG. 3). In addition, the inventors further prepared primer pairs in four viruses except PYDV. CVYV, CYSDV and ToCV were used for the other CP moieties, and PAMV for the polymerase moiety for primer preparation. The annealing temperature for Tm of the newly prepared primers is in the range of 53.5 ° C. to 59.4 ° C. (Table 2). At the annealing temperature of 53 ° C., only the CYSDV-2 primer set was amplified into the band predicted, while the other primer sets were amplified into several nonspecific bands. This indicates that the annealing temperature is critical for amplifying a band specific for each virus (FIG. 4A). PCR reactions were performed at 56 ° C. to find a suitable annealing temperature for each primer pair. As a result, CVYV-2 and CYSDV-2 were each amplified into specific bands as predicted (FIG. 4B). However, PAMV and ToCV primer pairs showed strong nonspecific bands and failed to obtain a single band. The present inventors have developed two effective pairs of primers for detecting CVYV, CYSDV and PYDV and one primer pair for PAMV and ToCV detection. In addition, the optimized annealing temperature of all primer sets was determined to be 56 ° C.

Prediction example  One : RT - PCR through Plant virus  Simultaneous detection

In order to simultaneously detect viruses infected with nectarine plants, the prepared sets of CVYV-1_F (SEQ ID NO: 1) and CVYV-1_R (SEQ ID NO: 2) and CYSDV-2_F (SEQ ID NO: 7) and CYSDV-2_R (SEQ ID NO: No. 8) Simultaneous RT-PCR reaction using the primer set yields PCR products of 334bp and 289bp sizes. It is expected to detect Cucumber vein yellowing virus (CVYV) or Cucurbit yellow stunting disorder virus (CYSDV) infected with gourd plants respectively or simultaneously.

On the other hand, PAMV-1_F (SEQ ID NO: 9) and PAMV-1_R (SEQ ID NO: 10) primer sets, PYDV-2_F (SEQ ID NO: 15), and PYDV-2_R (SEQ ID NO: 16) for simultaneously detecting viruses infected with branched plants ) PCR products of 338 bp, 253 bp and 193 bp can be obtained by simultaneously performing RT-PCR reaction using the primer set and the ToCV-1_F (SEQ ID NO: 19) and ToCV-1_R (SEQ ID NO: 20) primer sets. It can detect Potato aucuba mosaic virus (PAMV), Potato yellow dwarf virus (PYDV) or Tomato chlorosis virus (ToCV) infected with eggplant plants. It is expected to be.

<References>

1.Bandyopadhyay, A., Kopperud, K., Anderson, G., Martin, K. and Goodin, M. M. 2010. An integrated protein localization and interaction map for Potato yellow dwarf virus, type species of the genus Nucleorhabdovirus. Virology 402: 61-71.

Berdiales, B., Bernal, JJ, Saez, E., Woudt, B., Beitia, F. and Rodriguez-Cerezo, E. 1999.Occurrence of Cucurbit yellow stunting disorder virus (CYSDV) and Beet pseudo-yellows virus in cucurbit crops in spain and transmission of CYSDV by two biotypes of Bemisia tabaci. Eur. J. Plant Pathol. 105: 211-215.

Cuadrado, I. M., Janssen, D., Velasco, L., Ruiz, L. and Segundo, E. 2001. First report of Cucumber vein yellowing virus in Spain. Plant Dis. 85: 336.

De Bokx, J. A., 1975. Reactions of various plant species to inoculation with Potato aucuba mosaic virus. Potato Res. 18: 397-409.

5.Font, M. I., Juarez, M., Martinez, O. and Jorda, C. 2004. Current status and newly discovered natural hosts of Tomato infectious chlorosis virus and Tomato chlorosis virus in Spain. Plant Dis. 88:82.

6.Ghosh, D., Brooks, R. E., Wang, R., Lesnaw, J. and Goodin, M. M. 2008. Cloning and subcellular localization of the phosphoprotein and nucleocapsid proteins of Potato yellow dwarf virus, type species of the genus Nucleorhabdovirus. Virus Res. 135: 26-35.

7. International Committee on Taxonomy of Viruses (ICTV). 2010. Virus Taxonomy: 2009 Release. http://www.ictvonline.org/ virusTaxonomy.asp? version = 2009.

8. Janssen, D., Velasco, L., Martin, G., Segundo E. and Cuadrado, I. M. 2007. Low genetic diversity among Cucumber vein yellowing virus isolates from spain. Virus Genes 34: 367-371.

9. Louro, D., Accotto, G. P. and Vaira, A. M. 2000. Occurrence and diagnosis of Tomato chlorosis virus in portugal. Eur. J. Plant Pathol. 106: 589-592.

10.Lozano, G., Moriones, E. and Navas-Castillo, J. 2004. First report of sweet pepper (Capsicum annuum) as a natural host plant for Tomato chlorosis virus. Plant Dis. 88: 224.

11.Manoussopoulos, I. N. 2001. Acquisition and retention of Potato virus y helper component in the transmission of Potato aucuba mosaic virus by aphids. J. Phytopathol. 149: 103-106.

12. Martinez-Garcia, B., Marco, CF, Goytia, E., Lopez-Abella, D., Serra, MT, Aranda MA and Lopez-Moya, JJ 2004. Development and use of detection methods specific for Cucumber vein yellowing virus (CVYV). Eur. J. Plant Pathol. 110: 811-821.

13. Park, M.-R. and Kim, K.-H. 2004. RT-PCR detection of three non-reported fruit tree viruses useful for quarantine purpose in Korea. Plant Pathol. J. 20: 147-154.

Morris, J., Steel, E., Smith, P., Boonham, N., Spence, N. and Barker, I. 2006.Host range studies for Tomato chlorosis virus and Cucumber vein yellowing virus transmitted by Bemisia tabaci ( Gennadius). Eur. J. Plant Pathol. 114: 265-273.

15. Wintermantel, W. M., Wisler, G. C., Anchieta, A. G., Liu, H.-Y., Karasev, A. V. and Tzanetakis, I. E. 2005. The complete nucleotide sequence and genome organization of tomato chlorosis virus. Arch. Virol. 150: 2287-2298.

Yakoubi, S., Desbiez, C., Fakhfakh, H., Wipf-Scheibel, C., Marrakchi, M. and Lecoq, H. 2007.Occurrence of Cucurbit yellow stunting disorder virus and Cucumber vein yellowing virus in Tunisia. J. Plant Pathol. 89: 417-420.

<110> SNU R & DB Foundation <120> Genetic Markers for Detecting Quarantine Plant Viruses and          Methods for Detecting by Using Thereof <130> PN110025 <160> 22 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gaatggcaca agtgatgaga gg 22 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 attagcggca agtgtctggt g 21 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ggcacaagtg atgagaggaa aac 23 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 cattagcggc aagtgtctgg tg 22 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ggacggctga ctttatcaat tatg 24 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 tgtgacacgt tataatattt cttttc 26 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 catgcacggt gaccaaaaga ag 22 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 caaagcctgg catttcatca ac 22 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 gctgtgggct gtgtgtaaag 20 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 tccaattgtc tttaatgaac gc 22 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 gcagtgggtg aagaaagtgg ag 22 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 gaacgcccca aagtcctcag 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 cgctcagttc gtgcagttgt 20 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 tctgattcta gtccacgctg c 21 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 gatgcagtca gcggaagtca c 21 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 ggtaatggct ggcagtcca 19 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 gtatagcaat gcagatggga g 21 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 ctccggattg tagagcagca t 21 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 gaggttagac ccaaaatgtc cg 22 <210> 20 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 ctgagatatt catcaacgaa ccat 24 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 cagaacttgg gcaggaggga c 21 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 ccacttaaat ctcgcaccat c 21

Claims (8)

A primer set for simultaneous detection of a virus infected with a nectarine plant, comprising a primer set of SEQ ID NO: 1 and SEQ ID NO: 2, and a primer set of SEQ ID NO: 7 and SEQ ID NO: 8. The virus of claim 1, wherein the virus is Cucumber vein yellowing virus (CVYV) or Cucurbit yellow stunting disorder virus (CYSDV). Primer set for simultaneous detection. (a) isolating RNA from the sample;
(b) using the isolated RNA as a template and performing RT-PCR using the primer set according to claim 1; And
(C) a method for the simultaneous detection of viruses infected with the fruit plant, characterized in that it comprises the step of separating the PCR product by size. A kit for the simultaneous detection of viruses infected with nectarine plants using the primer set according to claim 1. For the simultaneous detection of viruses infected with branched plants, comprising a primer set of SEQ ID NO: 9 and SEQ ID NO: 10, a primer set of SEQ ID NO: 15 and SEQ ID NO: 16, and a primer set of SEQ ID NO: 19 and SEQ ID NO: 20 Primer set. The method according to claim 5, wherein the virus is in the group consisting of Potato aucuba mosaic virus (PAMV), Potato yellow dwarf virus (PYDV), Tomato chlorosis virus (ToCV) A primer set for simultaneous detection of a virus infected with an eggplant plant, characterized in that it is selected. (a) isolating RNA from the sample;
(b) using the isolated RNA as a template and performing RT-PCR using a primer set according to claim 5; And
(C) a method for the simultaneous detection of viruses infected with eggplant plants, comprising the step of separating the PCR product by size. A kit for simultaneous detection of a virus infected with an eggplant plant using the primer set according to claim 5.

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