KR101093239B1 - Specific primers for detection of Allexivirus in garlic, and uses thereof - Google Patents

Abstract

The present invention is a genus of Alexivirus genus diagnostic primer set comprising at least one primer set selected from the primers represented by SEQ ID NOS: 1-6, Alexivirus genus diagnostic kit comprising the primer set, and genus Alexivirus using the primer set Specifically to a method of diagnosis.

The diagnostic method of the present invention diagnoses Alexivirus occurring in garlic, which has not been possible in the diagnostic methods used so far, in the most economical and convenient manner, and has the highest detection limit. This diagnostic can be used to produce garlic virus free bottles.

Garlic, Virus, Diagnostics, RT-PCR, Alexivirus, GVA, GVB, GVC, GVD, GVE, GVX, GMbMV, GMbFV, SVX

Description

Specific primers for detection of Allexivirus in garlic, and uses approximately}

The present invention relates to a genus specific primer and its use for diagnosing Alexivirus, a virus occurring in garlic, and more specifically, Alexivirus (GVA, GVB, GVC, GVD, GVE). , GVX, GMbMV, GMbFV, SVX) relates to a primer set consisting of a primer and a combination thereof that can genus-specifically diagnose.

For the diagnosis of garlic virus, electron microscopy and serological methods (ELISA) were used in the past. However, because filamentous viruses present in garlic exist in many species that are hard to distinguish from each other, species cannot be diagnosed by electron microscopy.

On the other hand, serological methods have been useful for the diagnosis of garlic virus, but garlic is naturally infected with many viruses, so it is difficult to accurately diagnose garlic virus with one or several antisera. In addition, the anti-serum production of the virus has a problem that it is very difficult to secure the pure garlic virus and garlic not infected with the virus. In addition, some viruses occurring in garlic are difficult to distinguish species by serological methods due to the non-specific reaction due to the classification similarity, and the detection limit is about 1000 times lower than that of molecular biological methods.

The most commonly used RT-PCR method among molecular biological diagnostics is the use of species specific primers. However, many of the primers published in this paper are difficult to use for the diagnosis of all Alexivirus strains because they are selected without a taxonomic system established. Genus-specific diagnosis of the virus requires detection of all of the many variants present in the genus. In general, heterogeneous primers (degenerated primers) to solve this problem, but because the genus Alexivirus (Allexivirus) the width of the mutation is very large, it is impossible to diagnose all variants with one heterogeneous primer. (Tsuneyoshi et al., 1998. Arch Virol 143: 1093-1107; Chen et al., 2001. Arch Virol 146: 1841-1853; Chen et al. 2004. Arch Virol 149: 435-445).

Korean Patent Registration No. 10-0578000 discloses a primer set for diagnosing onion yellow atrophy virus of garlic and a method for diagnosing onion yellow atrophy virus using the same, but different from the primer set of the present invention.

The present invention has been made according to the above requirements, and since most garlics in the natural state are complexly infected with filamentous viruses that are difficult to distinguish from each other, electron microscopy and serological methods can be used to diagnose all infected viruses. Is impossible. In addition, since the virus occurring in garlic has a lot of tinnitus in the taxonomy, it is necessary to clearly classify the species by molecular biological methods. On the other hand, since there are significant variations between species (isolate) within the species (isolate) to develop a diagnostic primer that can overcome these variations.

In order to solve the above problems, the present invention provides a diagnostic set of Alexivirus genus comprising one or more primer sets selected from the primers represented by SEQ ID NO: 1 to 6.

The present invention also provides a diagnostic kit for the genus Alexivirus comprising the primer set.

In addition, the present invention provides a method for genus-specific diagnosis of Alexiviruses using the primer set.

According to the present invention, the RT-PCR diagnosis using a specific primer for diagnosing the Alexivirus genus can react with all the Alexivirus strains known to date, and it has a detection limit of 1,000 times or more than the conventional method using antiserum. Have. In addition, the new garlic market could be pioneered by industrializing the plant virus RT-PCR diagnostic kit and producing differentiated quality garlic through the production of garlic-free bottles.

In order to achieve the object of the present invention, the present invention provides a diagnostic set of the genus Alexivirus comprising at least one primer set selected from the primers represented by SEQ ID NO: 1 to 6.

The diagnostic set of the genus Alexivirus of the present invention comprises a primer set of SEQ ID NO: 1 and SEQ ID NO: 2 and 3; Primer sets of SEQ ID NO: 1 and SEQ ID NOs: 4 and 5; And it may be selected from the group consisting of the primer set of SEQ ID NO: 1 and SEQ ID NO: 6.

The diagnostic set of Alexivirus genus of the present invention is preferably a primer set of SEQ ID NO: 1 and SEQ ID NO: 2 and 3; Primer sets of SEQ ID NO: 1 and SEQ ID NOs: 4 and 5; And primer sets of SEQ ID NO: 1 and SEQ ID NO: 6.

Simultaneous diagnosis of the Alexivirus genus can be achieved by using three primer sets simultaneously.

Alexivirus genus that can be diagnosed using the primer set of the present invention is Garlic virus X (GVX), Garlic virus A (GVA), Garlic virus B (GVB), Garlic virus C (GVC), Garlic virus D (GVD), Garlic virus E (GVE), Shallot virus X (SHX), Garlic mite-borne mosaic virus (GMbMV) and Garlic mite-borne filamentous virus (GMbFV) can be one or more selected from the group consisting of. Preferably, the primer set of the present invention can diagnose the nine viruses simultaneously.

In the primer sets of SEQ ID NO: 1 and SEQ ID NOs: 2 and 3 of the present invention, the forward primer is the primer of SEQ ID NO: 1, and the reverse primer is a primer mixture of SEQ ID NOs: 2 and 3. In order to diagnose the genus Alexivirus, it is preferable to use a primer mixture of SEQ ID NOS: 2 and 3, rather than using primers SEQ ID NO: 2 or SEQ ID NO: 3 as reverse primers, respectively.

Likewise, in the primer sets of SEQ ID NO: 1 and SEQ ID NOs: 4 and 5 of the present invention, the forward primer is the primer of SEQ ID NO: 1, and the reverse primer is the primer mixture of SEQ ID NOs: 4 and 5. In order to diagnose the genus Alexivirus, it is preferable to use the primer mixture of SEQ ID NO: 4 and SEQ ID NO: 4 rather than using the primer of SEQ ID NO: 4 or SEQ ID NO: 5 as the reverse primer, respectively.

The primers are at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22 consecutive nucleotides within the sequence of SEQ ID NO: 1 to SEQ ID NO: 6, depending on the sequence length of each primer It may include an oligonucleotide consisting of a fragment of. For example, the primers of SEQ ID NO: 1 (23 oligonucleotides) are at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22 consecutive in the sequence of SEQ ID NO: 1 And oligonucleotides consisting of fragments of nucleotides, wherein the primers of SEQ ID NO: 2 (23 oligonucleotides) are at least 16, at least 17, at least 18, at least 19, at least 20 in the sequence of SEQ ID NO: 2 Oligonucleotides consisting of fragments of at least 21, 22 or more consecutive nucleotides.

In the present invention, all genetic information about viruses occurring in garlic was collected and the classification system was established using the nucleotide sequence of the envelope protein gene. Based on this classification system, the genus of Alexiviruses occurring in domestic garlic (9 species: GarV-A, Gar-B, GarV-C, GarV-D, GarV-E, GarMbMV, GarMbFV, ShV-X and GarV-X) Genus specific diagnostic primers were developed.

Although many studies have been conducted on plant viruses occurring in garlic, it is difficult to diagnose them because the taxonomic system of these viruses is not established. To this end, they were systematically analyzed through their coat protein sequencing (FIG. 1) to establish the classification system of major viruses occurring in garlic, and to establish a diagnosis system for these viruses. In the case of the Alexivirus genus, the genotype-specific primer was designed to diagnose the Alexivirus genus at once because the taxonomic system for the species is still unclear. In this method, the nucleotide sequences of all reported Alexivirus genus isolates are divided into two groups, and heterogeneous primers are selected and mixed at the same location for more precise diagnosis than the previously reported genus specific primers. It was made possible.

Within virus species, there are lines or isolates with slightly different sequences. Thus, if isolate-specific primers are used for diagnosis, there is a risk of failure. In other words, the virus diagnostic primer should be species specific for each virus. Primers in the present invention are designed to work specifically genus. In other words, the nucleotide sequences known to date are collected in the genus to search for common nucleotide sequences of all strains and isolates. Of the common sequences, genus specific sequences were selected by excluding the sequences of other viruses with a flexible relationship from the design of the primers. From the genus specific base sequence, a sequence having optimal conditions as a primer was designed as a genus specific primer. The designed primers were subjected to the actual reverse transcriptase-polymerase chain reaction (RT-PCR) for each combination and did not have a nonspecific reaction with nucleic acids other than the target virus, and finally selected a combination having excellent sensitivity to the target virus.

Garlic is cultivated in Korea, four kinds of genus 1 virus (GLV, LYSV, GCLV, OYDV and Allexivirus) cause most of the disease. Among them, genus specific diagnostic primers were developed for Alexivirus.

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

As used herein, oligonucleotides used as primers may also include nucleotide analogues such as phosphorothioate, alkylphosphothioate or peptide nucleic acid, or It may comprise an intercalating agent.

In order to achieve another object of the present invention, the present invention

At least one primer set according to the present invention; And it provides a diagnostic kit for the genus Alexivirus comprising a reagent required for RT-PCR. In the kit of the present invention, the reagents required for the RT-PCR may be, for example, but not limited to, a forward primer and a reverse primer, a reverse transcriptase, a ribonuclease inhibitor, a heat resistant polymerase, a reaction buffer, and the like, and a reaction buffer Silver includes both reverse transcriptase and PCR buffer, and heat resistant polymerase may be EF Taq polymerase and the like, and reverse transcriptase is a reverse transcriptase originating from various sources, for example, avian myeloblastosis virus-derived reverse transcriptase. (avian myeloblastosis virus-derived virus reverse transcriptase (AMV RTase)), mouse leukemia virus-derived virus reverse transcriptase (MuLV RTase) and Rouse-associated virus 2 reverse transcriptase 2 reverse transcriptase (RAV-2 RTase).

The kit may also include instructions. The guide is a printed document that explains how to use the kit, such as how to prepare reverse transcription buffer and PCR buffer, and the reaction conditions presented. The instructions include brochures in the form of pamphlets or leaflets, labels affixed to the kit, and instructions on the surface of the package containing the kit. In addition, the guide includes information that is disclosed or provided through electronic media such as the Internet.

In order to achieve another object of the present invention, the present invention

Separating total RNA from garlic samples;

Amplifying a target sequence by using the isolated whole RNA as a template and performing RT-PCR using at least one primer set according to the present invention; And

It provides a method for genus specific diagnosis of Alexivirus, comprising detecting the amplification product.

As a method for separating total RNA from garlic samples, a method known in the art may be used, for example, a phenol extraction method may be used. Using the isolated whole RNA as a template, one or more primer sets according to an embodiment of the present invention may be used as a primer to amplify a target sequence by performing RT-PCR. 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 may be, but is not limited to, a material that emits fluorescence, phosphorescence, or radioactivity. Preferably, the labeling substance is Cy-5 or Cy-3. When amplifying the target sequence, PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of the primer to allow the target sequence to be labeled with a detectable fluorescent labeling substance. In addition, the label using the radioactive material may add radioactive isotopes such as ³² P or ³⁵ S to the PCR reaction solution when PCR is performed, and the amplification product may be incorporated into the amplification product while the amplification product is synthesized. One or more sets of oligonucleotide primers used to amplify the target sequence are as described above.

The method of the present invention includes detecting the amplification product. Detection of the amplification product may be performed through DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto. As one of the methods for detecting amplification products, gel electrophoresis can be performed. Gel electrophoresis may use agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product. In addition, in the fluorescence measurement method, PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of a primer, and a target sequence is labeled with a detectable fluorescent labeling substance, and the labeled fluorescence is measured using a fluorimeter. can do. In addition, radioactivity measuring method is to add a radioactive isotope such as ³² P or ³⁵ S to the PCR reaction solution to label the amplification product when performing PCR, and then radioactive measuring apparatus, for example, Geiger counter or liquid flash The radioactivity can be measured using a liquid scintillation counter.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example

Example 1 Design, Selection and Specificity of the Diagnostic Primer for Alexivirus Genus

9 viruses belonging to the genus Alexiviruses ( Garlic virus X (GVX), Garlic virus A (GVA), Garlic virus B (GVB), Garlic virus C (GVC), Garlic virus D (GVD), Garlic virus E (GVE), Shallot virus X (SHX), Garlic mite-borne mosaic virus (GMbMV), and Garlic mite-borne filamentous virus (GMbFV) ) were genotype-specific primers designed for simultaneous diagnosis.

The Alexivirus consensus sequence search was made by multiple comparisons of base sequences (Table 1) of 81 Alexivirus genus isolates known to date.

Table 1.Sequences of isolates of the genus Alexivirus used for common sequencing

Separation Gene source gene
Size (bp) GVA-CFH AJ551467 755 GVA AB010300 8660 GVA AF478197 1573 GVA-LA AJ551473 755 GVA = BT AJ551468 755 GVA-CFL AJ551469 755 GVA-CS1 AJ551470 755 GVA-CS2 AJ551471 755 GVA-HK AJ551472 755 GVA NC003375 8660 GVA-LZ AJ551474 755 GVA-SS AJ551475 755 GVA-WH AJ551476 755 GVA-YY1 AJ551477 755 GVA-YY2 AJ551478 755 GVB-YC AJ551482 772 GVB AB010301 5077 GVB-EN-373 AJ543829 272 GVB-TS1-1 AJ551479 772 GVB-TS1-2 AJ551480 772 GVB-TS2 AJ551481 772 GVC AB010302 8405 GVC AY170322 591 GVC NC003376 8405 GVD-AB AJ551483 755 GVD AB010303 4497 GVD AF519572 1544 GVD-LZ AJ551489 755 GVD-CFH AJ551484 755 GVD-CFL AJ551485 755 GVD-DL AJ551486 755 GVD-HK AJ551487 755 GVD-LA AJ551488 755 GVD-YY1 AJ551495 755 GVD-SS AJ551490 755 GVD-TM AJ551491 755 GVD-WH AJ551492 752 GVD-XA AJ551493 755 GVD-XX AJ551494 755 GVD-YY2 AJ551496 755 GVD-ZHaZ AJ551497 755

Separation Gene source gene
Size (bp) GVE-TS1 AJ551500 752 GVE-YH AJ292230 8451 GVE-CFH AJ551498 755 GVE-CS2 AJ551499 752 GVE-TS2 AJ551501 752 GVE-YC AJ551502 752 GVE NC004012 8451 GVX-AB2 AJ551504 765 GVX-YH AJ292229 8319 GVX-AB1 AJ551503 767 GVX-CS2-1 AJ551510 741 GVX--BD AJ551505 767 GVX-BT AJ551506 766 GVX-CQ AJ551507 767 GVX-CS1-1 AJ551508 765 GVX-CS1-2 AJ551509 769 GVX-LZ AJ551516 765 GVX-CS2-2 AJ551511 769 GVX-DL AJ551512 767 GVX-GY AJ551513 767 GVX-JX AJ551514 765 GVX-LA AJ551515 767 GVX-XA AJ551522 766 GVX-NJ AJ551517 767 GVX-TM AJ551518 766 GVX-TP AJ551519 767 GVX-TS1 AJ551520 766 GVX-TS2 AJ551521 766 GVX U89243 8106 GVX-XX AJ551523 767 GVX-YC AJ551524 766 GVX-ZHaZ AJ551525 767 GVX-ZHaZ AJ551526 767 GVX NC001800 8106 SVX NC003795 8832 SVX L76292 2452 SVX M97264 8832 GMbFV AY390254 723 GMbFV X98991 762 GMbMV D49443 2518

Of these genera-specific nucleotide sequences, 13 Alexivirus genus diagnostic primers were designed in the region satisfying the primer conditions (Table 2).

Table 2. Designed primers for diagnosis of genus Alexivirus

primer Sequence ^a Length location ^b Al-N10  TGGRCNTGCTACCACAAYGG (SEQ ID NO: 7) 20 337-356 Al-N20  TTYKCNGSHTTYGAYTTCTT (SEQ ID NO: 8) 20 553-572 Al-N30  CAYTCHATGAAYGCBAARATGTC (SEQ ID NO: 1) 23 652-674 Al-C40  GGYTGYTCATGKATYTGTTG (SEQ ID NO: 9) 20 736-755  GGHGSTTCRTGRATYTGYTG (SEQ ID NO: 10) 20 Al-C30  GGCTTATTYTGWCTAGYYTTACG (SEQ ID NO: 2) 23 910-932  GGYTTATTKTSTTRTRATYTACG (SEQ ID NO: 3) 23 Al-C21  TCRAARCAYCTRTTGTARCCYTT (SEQ ID NO: 11) 23 973-995  TCAAARCAYCTGYYATARCGTTT (SEQ ID NO: 12) 23 Al-C20 (2)  CAKTCRAARCAYCTRTTGTARCG (SEQ ID NO: 4) 23 976-998  CARTCAAARCAYCTGYYATARCG (SEQ ID NO: 5) 23 Al-C20 (1)  CARTCRAARCAYCKRTYRTARCG (SEQ ID NO: 6) 23 976-998 Al-C10  CCYTTCAGCATATAGCTTAGC (SEQ ID NO: 13) 21 1087-1107

^a D: Degenerated primer

^b Primer Positions Based on GMbMV (D49443)

The designed primer is shown in FIG. 2. Designed Alexivirus genus primers were combined in 17 (Table 3), RT-PCR was performed (Fig. 3).

Table 3. Alexivirus Primer Combinations and RT-PCR Products

Combination primer Product size (bp) One Al-N10 Al-C40 419 2 Al-N10 Al-C30 596 3 Al-N10 Al-C21 659 4 Al-N10 Al-C20 (2) 662 5 Al-N10 Al-C20 (1) 662 6 Al-N10 Al-C10 771 7 Al-N20 Al-C40 203 8 Al-N20 Al-C30 380 9 Al-N20 Al-C21 443 10 Al-N20 Al-C20 (2) 446 11 Al-N20 Al-C20 (1) 446 12 Al-N20 Al-C10 555 13 Al-N30 Al-C30 281 14 Al-N30 Al-C21 344 15 Al-N30 Al-C20 (2) 347 16 Al-N30 Al-C20 (1) 347 17 Al-N30 Al-C10 456

The Alexivirus used for selection of genus specific primers was used as RT-PCR template by separating the whole RNA by extracting leaves showing mosaic symptoms from naturally infected garlic. Total RNA isolation and RT-PCR methods used in the present invention are as follows.

Total RNA Isolation: Total RNA was isolated from 50 mg of virus infected leaf tissue using phenol. The isolated total RNA was finally washed three times with 75% ethanol, dried, dissolved in 50 μl of distilled water and used for RT-PCR analysis.

RT-PCR: Reverse transcription reaction consists of 0.5 μl total RNA isolated, 12.5 pmole reverse primer, 5-fold reverse transcription buffer (250 mM Tris-HCl, 150 mM KCl, 40 mM MgCl ₂ , 5 mM DTT) , pH 8.5) 1 μl, 0.5 μl of 10 mM dNTP, 10 U RNase inhibitor, and 5 μl of the reaction solution containing 2 U AMV reverse transcriptase were incubated at 42 ° C. for 1 hour and then denatured at 94 ° C. for 10 minutes. The polymerase chain reaction was carried out in 5 μl of reverse transcription reaction with 12.5 pmole forward primer, 1.25 U Taq DNA polymerase, 10-fold polymerase chain reaction buffer (100 mM Tris-HCl, 500 mM KCl, 2 μl of 15 mM MgCl ₂ , pH 8.3) was added and distilled water was added to make 25 μl of the reaction solution. The reaction solution was amplified 40 times at 95 ° C 45 seconds, 50 ° C 60 seconds, 72 ° C 60 seconds, and finally treated at 72 ° C for 7 minutes. RT-PCR products were identified by electrophoresis using 0.5X TBE buffer and 1.5% agarose gel followed by staining with ethidium bromide.

Forward and reverse primers were synthesized as degenerated primers.

RT-PCR was performed on the first selected primer pairs with viruses occurring in garlic (FIG. 4) to finally select four diagnostic primers and three primer pairs using the same (Table 4). All of the naturally infected garlic samples were infected with Alexivirus, which confirmed the nonspecific reaction with Garlic latent virus (GLV), Like yellow stripe virus (LYSV), Garlic common latent virus (GCLV), and Onion yellow dwarf virus (OYDV). This was impossible.

Table 4. Specific primer combinations of the final selected Alexivirus genus

Combination Forward primer Reverse primer Product size (bp) 13 Al-N30 Al-C30 281 15 Al-N30 Al-C20 (2) 347 16 Al-N30 Al-C20 (1) 347

As can be seen in Figure 4, it can be seen that the three primer sets of the present invention can specifically detect or diagnose the genus Alexivirus.

When the diagnostic method of the present invention is carried out, industrialization of the RT-PCR diagnostic kit for garlic virus is possible first, and by using this diagnostic method, it is possible to produce disease-free garlic that is not infected with the virus. It is expected to be very large.

Figure 1 shows the establishment of the taxonomic system of viruses occurring in garlic using the envelope protein gene sequence. Isolators shown in red are domestic isolates.

Figure 2 shows the designed Alexivirus primer position.

3 shows the RT-PCR results of 17 primer pairs in combination with Alexivirus. Lanes 1 to 17 represent Alexivirus primer combinations 1 to 17 described in Table 3, respectively.

4 shows the RT-PCR results with garlic generating virus of the final selected Alexivirus primer combination.

PepMOV (Pepper mottle virus)

TBRV (Tomato black ring virus)

PVY (Potato virus Y)

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Specific primers for detection of Allexivirus in garlic, and uses          the <130> PN08151E <160> 13 <170> KopatentIn 1.71 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Al-N30 primer <400> 1 caytchatga aygcbaarat gtc 23 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Al-C30 primer <400> 2 ggcttattyt gwctagyytt acg 23 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Al-C30 primer <400> 3 ggyttattkt sttrtratyt acg 23 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Al-C20 (2) primer <400> 4 caktcraarc ayctrttgta rcg 23 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Al-C20 (2) primer <400> 5 cartcaaarc ayctgyyata rcg 23 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Al-C20 (1) primer <400> 6 cartcraarc ayckrtyrta rcg 23 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Al-N10 primer <400> 7 tggrcntgct accacaaygg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Al-N20 primer <400> 8 ttykcngsht tygayttctt 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Al-C40 primer <400> 9 ggytgytcat gkatytgttg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Al-C40 primer <400> 10 gghgsttcrt gratytgytg 20 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Al-C21 primer <400> 11 tcraarcayc trttgtarcc ytt 23 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Al-C21 primer <400> 12 tcaaarcayc tgyyatarcg ttt 23 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Al-C10 primer <400> 13 ccyttcagca tatagcttag c 21  

Claims (6)

delete Primer sets of SEQ ID NO: 1 and SEQ ID NOs: 2 and 3; Primer sets of SEQ ID NO: 1 and SEQ ID NOs: 4 and 5; And a set of primers for diagnosing a genus of Alexyvirus genus specific comprising a set of primers SEQ ID NO: 1 and SEQ ID NO: 6. According to claim 2, wherein the genus Alexivirus is Garlic virus X, Garlic virus A, Garlic virus B, Garlic virus C, Garlic virus D, Garlic virus E, Shallot virus X, Garlic mite-borne mosaic virus and Garlic mite-borne filamentous A specific diagnostic primer set for the genus Alexivirus, characterized in that at least one selected from the group consisting of virus . A primer set according to claim 2 or 3; And Alexivirus genus specific diagnostic kit comprising a reagent required for RT-PCR. Separating total RNA from garlic samples; Amplifying a target sequence by using the isolated whole RNA as a template and performing RT-PCR using the primer set according to claim 2 or 3; And A method for diagnosing genus specific virus, comprising detecting the amplification product. The method of claim 5, wherein the detection of the amplification product is carried out by DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement.

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