KR101139802B1 - Primers for detecting orchid infecting virusinfect and screening method of virus using same - Google Patents

Abstract

The present invention relates to a primer for egg plant virus assay and a virus detection method using the same. In particular, the present invention is ORSV-420F primer of SEQ ID NO: 1, ORSV-420R primer of SEQ ID NO: 2, CyMV-620F primer of SEQ ID NO: 3, which can be easily tested for ORSV, CyMV, OFV infection of the eggplant It relates to a primer for egg plant virus assay selected from the group consisting of CyMV-620R primer of No. 4, OFV-873F primer of SEQ ID NO: 5, and OFV-873R primer of SEQ ID NO: 6.

Description

Primer for egg plant virus assay and virus detection method using same {{PRIMERS FOR DETECTING ORCHID INFECTING VIRUSINFECT AND SCREENING METHOD OF VIRUS USING SAME}

The present invention I and plant viruses black primer, and relates to a detecting method using the same, and more particularly, ORSV (Odontoglossum infecting eggs and plants ring spot virus ), CyMV ( Cymbidium mosaic virus ) and OFV ( Orchid fleck The present invention relates to a PCR primer that specifically amplifies virus ) and three virus detection methods using the primer.

Since eggs are nutrient propagated, if a virus-infected plant is continuously grown and grown without knowing whether the virus is infected, Lee Byeong-ju accumulates the virus, and the disease spreads to surrounding crops, causing the virus to spread. Ovarian viruses deteriorate egg quality due to symptoms such as leaf specks, leaf epidermis dents, leaf defoliation, poor growth, early flowering and discoloration of flowers.

Representative egg virus is ORSV ( Odontoglossum ring spot virus ), CyMV ( Cymbidium mosaic virus ) and OFV ( Orchid fleck virus ).

The diagnostic techniques of oocyte viruses include bioassays using indicator plants, antiserum assays and RT-PCR assays.

Bioassay using indicator plants has a disadvantage in that the sensitivity of the reaction is inferior when the disease expression is poor or the virus concentration is low, depending on the method using the herbal indicator plants or susceptibility to the virus.

The antiserum assay is an ELISA (Enzyme-linked Immune Antibody) method, and when it is necessary to test a plant with a low virus concentration, it is difficult to obtain an accurate reaction result because the sensitivity of the reaction is low. There are disadvantages.

The RT-PCR assay is a method of assaying using a primer that specifically amplifies a specific viral RNA fragment. It is difficult to establish primers specific to each virus and RT-PCR reaction conditions.

 It is an object of the present invention to provide specific RT-PCR primers capable of amplifying egg plant viruses.

It is also an object of the present invention to provide a method for detecting whether or not the virus of the eggplant virus.

Another object of the present invention is to provide a method for detecting three types of egg plant viruses at once.

In order to achieve the above object, the present invention provides an ORSV-420F primer of SEQ ID NO: 1, an ORSV-420R primer of SEQ ID NO: 2, a CyMV-620F primer of SEQ ID NO: 3, a CyMV-620R primer of SEQ ID NO: 4, an OFV of SEQ ID NO: 5 Primer for oval plant virus assay for simultaneously diagnosing Odontoglossum ring spot virus (ORSV), Cymbidium mosaic virus (CyMV) and Orchid fleck virus (OFV) consisting of -873F primer and OFV-873R primer of SEQ ID NO: 6 is provided.

In addition, the present invention (a) the RNA extracted from the sample as a template ORSV-420F primer of SEQ ID NO: 1, ORSV-420R primer of SEQ ID NO: 2, CyMV-620F primer of SEQ ID NO: 3, CyMV-620R primer of SEQ ID NO: 4, Performing reverse transcription polymerase chain reaction (RT-PCR) with a primer set for an oval plant virus assay consisting of OFV-873F primer of SEQ ID NO: 5 and OFV-873R primer of SEQ ID NO: 6, and (b) amplified PCR product A method for detecting an oval plant virus that simultaneously diagnoses an ORSV ( Odontoglossum ring spot virus ), a CyMV ( Cymbidium mosaic virus ), and an OFV (Orange fleck virus ), including determining the infection of an oval plant virus by checking its size To provide.

Hereinafter, the present invention will be described in detail.

The present invention relates to a primer for egg virus assay, ORSV ( Odontoglossum ring spot virus ), CyMV ( Cymbidium mosaic virus ) and OFV ( Orchid fleck virus ) to provide a primer for testing.

ORSV is a 6618 bp ssRNA (+) strand virus (NC_001728), belonging to Tobamovirus. The primers for ORSV assay are ORSV-420F primer of SEQ ID NO: 1 and ORSV-420R primer of SEQ ID NO: 2. The ORSV-420F / ORSV-420R primer set amplifies ORSV's Virus Replicase 420 bp (FIG. 1).

CyMV is an ssRNA (+) strand virus (NC_001812) consisting of 6227 bp and belongs to the Potexvirus. CyMV assay primers are CyMV-620F primers of SEQ ID NO: 3 and CyMV-620R primers of SEQ ID NO: 4. The CyMV-620F / CyMV-620R primer set amplifies 620 bp of viral replication enzyme (RDRP) of CyMV (FIG. 2).

OFV is a ssRNA (-) strand virus consisting of two segmental genomes of RNA1 (NC009608) consisting of 6413 bp and RNA 2 (NC009609) consisting of 6001 bp and belongs to Rhabdovirus. The primer for OFV assay amplifies nucleocapsid protein 873 bp (FIG. 3).

The primer for the oocyte virus assay of the present invention can detect an oocyte virus by extracting RNA from an oocyte leaf, root or flower and performing RT-PCR with one primer set or one or more primer sets. have. The eggplant virus may be any kind of eggplant to which ORSV or CyMV may be infected. For example, the egg yolk, western egg symbidium, phalaenopsis egg, dendrobium, onsidium, cattleya, denpare and the like.

Primer for oocyte virus assay of the present invention can be easily and simply assayed for the infection by ORSV, CyMV and OFV, as well as can accurately identify the complex infection by ORSV and CyMV most common in oocytes. Therefore, it is possible to diagnose whether or not the virus infection on the oocytes using the egg yolk virus assay primer of the present invention to prevent the spread of virus pathogens by aliquotation and virus spread to the surrounding individuals due to neglected pathogens in the cultivation greenhouse Damage can be prevented in advance.

In addition, the present invention (a) the RNA extracted from the sample as a template ORSV-420F primer of SEQ ID NO: 1, ORSV-420R primer of SEQ ID NO: 2, CyMV-620F primer of SEQ ID NO: 3, CyMV-620R primer of SEQ ID NO: 4, Performing reverse transcriptase polymerase chain reaction (RT-PCR) with one or more primers for eggplant virus assay selected from the group consisting of the OFV-873F primer of SEQ ID NO: 5 and the OFV-873R primer of SEQ ID NO: 6, and (b The present invention provides an oocyte virus detection method comprising the step of determining the size of an amplified PCR product to determine whether an oocyte virus is infected.

The present invention provides a primer that specifically amplifies the oocyte virus, and can be easily and simply assayed for infection by ORSV, CyMV, OFV, as well as to accurately identify the complex infection of the viruses. Therefore, using the ophthalmic plant virus assay primer of the present invention, it is possible to trace the path of infection of the oocyte plant virus or diagnose the virus infection of the parent strain for proliferation, and to prevent and control the oocyte virus infection in advance Can be.

Example 1 Preparation of Primer for Egg Plant Virus Assay

1-1. Primer for ORSV Assay

An ORSV-420F of SEQ ID NO: 1 and an ORSV-420R primer set of SEQ ID NO: 2 were prepared to amplify a portion of the RNA viral replica gene (NC_001728, 1121-1542). The ORSV-420F / ORSV-420R primer set amplifies DNA fragments of 420 bp.

1-2. Primer for CyMV Assay

A set of CyMV-620F of SEQ ID NO: 3 and a CyMV-620R primer of SEQ ID NO: 4 were able to amplify a portion of the RNA viral replica gene (NC_001812, 167-786). The CyMV-620F / CyMV-620R primer set amplifies 620 bp DNA fragments.

1-3. Primer for OFV Assay

OFV-873F of SEQ ID NO: 5 and OFV-873R primer set of SEQ ID NO: 6 were prepared to amplify a portion of the RNA virus replication gene (NC_009609, 3781-4653). The OFV-873F / OFV-873R primer set amplifies 873 bp DNA fragments.

EXAMPLE 2 Eggplant Virus Detection by Using Primer for Eggplant Virus Assay

ORSV, CyMV, OFV, respectively, were identified and RNA was purified. First, virus-infected strains were selected by ELISA. Antibodies used in the ELISA were used as antibodies from the United States Agdia, and the selected virus infected strain was used as a test material while isolated in the greenhouse. Viral nucleic acid extraction was performed by grinding the plant with 0.3 ml of buffer, then mixing 0.3 ml of a solution of phenol (Sigma, pH5) and chloroform (Sigma) in the same ratio (Petol: chloroform = 1: 1) at room temperature. The reaction was shaken vigorously for 5 minutes. Thereafter, the supernatant was recovered by centrifugation at 13,000 rpm for 10 minutes, and 0.3 ml of phenol: chloroform (1: 1) solution was added thereto, followed by vigorous shaking, followed by centrifugation at 13,000 rpm for 10 minutes to recover the supernatant. 1.5 times of ethanol was added to the recovered supernatant, and the pellet was recovered by centrifugation at 13,000 rpm for 20 minutes after 1 hour at -70 ° C. 600 μl of 70% ethanol was added to the pellet, and the recovered pellet was dried by centrifugation at 13,000 rpm for 10 minutes and dissolved in 30 μl of sterilized distilled water.

2-1 ORSV

Reverse transcription of the purified ORSV RNA with the ORSV-420R primer was performed (Promega Improm-II reverse transcriptase, GoTaq). PCR was performed using the ORSV-420F / ORSV-420R primer set as a template of the cDNA prepared after the reverse transcription reaction (Promega GoTaq).

Reverse transcription ( cDNA  Synthetic composition

RNA -------------------------- 1 μl (μg / μl)

5 × Reverse Transcription Buffer -------- 4 μl

MgCl ₂ ------------------------ 2 μl

10 mM dNTP ------------------- 2 μl

Reverse transcriptase -------- 1 μl

ORSV-420R -------------------- 1 μl (10 pM)

Nuclease free water ---------- 9 μl

PCR  Composition

cDNA ------------------------- 20 μl

10 × PCR reaction buffer ---------- 5 μl

MgCl ₂ ----------------------- 2 μl

10 mM dNTP ------------------ 2 μl

ORSV-420F ------------------- 1 μl (10 pM)

ORSV-420R ------------------- 1 μl (10 pM)

Nuclease free water --------- 9 μl

Reverse transcription  Condition

70 ℃ 10min → 25 ℃ 5min → 42 ℃ 60min

PCR  Condition

92 ℃ 2min → 92 ℃ 30sec → 51 ℃ 30sec → 70 ℃ 1min (29 cycles) → 72 ℃ 10min

After PCR reaction, the agarose gel was electrophoresed to confirm the size of the PCR product. Figure 1 is an electrophoresis picture showing the ORSV PCR product amplified by the primer for ORSV assay, PCR product was confirmed at 420 bp. Therefore, it can be seen that the ORSV-420F / ORSV-420R primer set prepared in Example 1 amplifies ORSV.

2-2 CyMV

Reverse transcription was carried out with CyMV-620R primers using the purified CyMV RNA as a template (Promega Improm-II reverse transcriptase, GoTaq). CDNA prepared after reverse transcription was subjected to PCR using CyMV-620F / CyMV-620R primer set as a template (Promega GoTaq).

Reverse transcription ( cDNA  Synthetic composition

RNA --------------------------- 1 μl (μg / μl)

5 × Reverse Transcription Buffer --------- 4 μl

MgCl ₂ -------------------------- 2 μl

10 mM dNTP-------------------- 2 μl

Reverse transcriptase ---------- 1 μl

CyMV-620R ---------------------- 1 μl (10 pM)

Nuclease free water ------------ 9 μl

PCR  Composition

cDNA --------------------------- 20 μl

10 × PCR reaction buffer ------------ 5 μl

MgCl ₂ -------------------------- 2 μl

10 mM dNTP -------------------- 2 μl

CyMV-620F --------------------- 1 μl (10 pM)

CyMV-620R --------------------- 1 μl (10 pM)

Nuclease free water ----------- 19 μl

Reverse transcription  Condition

70 ℃ 10min → 25 ℃ 5min → 42 ℃ 60min

PCR  Condition

92 ° C 2 minutes → 92 ° C 30 seconds → 50 ° C 30 seconds → 70 ° C 1 minute 20 seconds (29 cycles) → 72 ° C 10 minutes

After PCR reaction, the agarose gel was electrophoresed to confirm the size of the PCR product. Figure 2 is an electrophoresis picture showing the CyMV PCR product amplified with a primer for CyMV assay, PCR product was confirmed at 620 bp. Thus, it was confirmed that the CyMV-620F / CyMV-620R primer set prepared in Example 1 amplifies CyMV.

2-3 OFV

Reverse transcription was carried out using OFV-873R primer as a template of purified OFV RNA (Promega Improm-II reverse transcriptase, GoTaq). CDNA prepared after reverse transcription was subjected to PCR reaction using OFV-873F / OFV-873R primer set as a template (Promega GoTaq).

Reverse transcription ( cDNA  Synthetic composition

RNA ------------------------ 1 μl (μg / μl)

5 × Reverse Transcription Buffer ------ 4 μl

MgCl ₂ ---------------------- 2 μl

10 mM dNTP ---------------- 2 μl

Reverse transcriptase ----- 1 μl

OFV-873R ------------------ 1 μl (10 pM)

Nuclease free water ------- 9 μl

PCR  Composition

cDNA ---------------------- 20 μl

10 × PCR Reaction Buffer ------- 5 μl

MgCl ₂ ---------------------- 2 μl

10 mM dNTP ----------------- 2 μl

OFV-873F ------------------- 1 μl (10 pM)

OFV-873R ------------------- 1 μl (10 pM)

Nuclease free water -------- 19 μl

Reverse transcription  Condition

70 ℃ 10min → 25 ℃ 5min → 42 ℃ 60min

PCR  Condition

92 ° C 2 minutes → 92 ° C 30 seconds → 50 ° C 30 seconds → 70 ° C 1 minute 30 seconds (29 cycles) → 72 ° C 10 minutes

After PCR reaction, the agarose gel was electrophoresed to confirm the size of the PCR product. Figure 3 is an electrophoresis picture showing the OFV PCR product amplified with a primer for OFV assay, PCR product was confirmed at 873 bp. Therefore, it was confirmed that OFV-873F / OFV-873R primer set prepared in Example 1 amplifies OFV.

Example 3 Multiple Detection of Egg Plant Virus

RNA was isolated from ORSV and CyMV infected symbidium (QIAGEN, RNeasy Plant RNA extraction kit), and subjected to reverse transcription and PCR reactions using the primers of Example 1.

PCR results are shown in FIG. 4.

PCR  Condition

92 ° C 2 minutes → 92 ° C 30 seconds → 50 ° C 30 seconds → 70 ° C 1 minute 30 seconds (29 cycles) → 72 ° C 10 minutes

Figure 4 shows the results of multiple RT-PCR with ORSV-420F / ORSV-420R primer set and CyMV-620F / CyMV-620R primer set.

4, "1" is used for ORSV infected symbidium RNA, "2" is used for CyMV infected eggs, and "3" is used for ORSV / CyMV infected symbidium RNA.

RNA was isolated from ORSV, CyMV, OFV infected symbidium (QIAGEN, RNeasy Plant RNA extraction kit), and subjected to reverse transcription and PCR reactions using the primers of Example 1.

PCR results are shown in FIG. 5.

PCR  Condition

92 ° C 2 minutes → 92 ° C 30 seconds → 50 ° C 30 seconds → 70 ° C 2 minutes (29 cycles) → 72 ° C 10 minutes

Figure 5 shows the results of multiple RT-PCR with ORSV-420F / ORSV-420R primer set, CyMV-620F / CyMV-620R primer set and OFV-873F / OFV-873R.

In Fig. 5, “1” is used for ORSV infected symbidium RNA, “2” for 5 is used for CyMV infected eggs, “3” for 5 is used for OFV infected symbidium RNA, and “4” for 5 is ORSV / CyMV. / OFV infected symbidium RNA.

Figure 1 is an electrophoresis picture showing the ORSV PCR product amplified with a primer for ORSV assay.

Figure 2 is an electrophoresis picture showing the CyMV PCR product amplified with a primer for CyMV assay.

Figure 3 is an electrophoresis picture showing the OFV PCR product amplified with a primer for OFV assay.

Figure 4 shows the results of multiple RT-PCR with ORSV-420F / ORSV-420R primer set and CyMV-620F / CyMV-620R primer set.

Figure 5 shows the results of multiple RT-PCR with ORSV-420F / ORSV-420R primer set, CyMV-620F / CyMV-620R primer set and OFV-873F / OFV-873R.

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Claims (5)

ORSV-420F primer represented by SEQ ID NO: 1, ORSV-420R primer represented by SEQ ID NO: 2, CyMV-620F primer represented by SEQ ID NO: 3, CyMV-620R primer represented by SEQ ID NO: 4, represented by SEQ ID NO: 5 Comprised of OFV-873F primer and OFV-873R primer represented by SEQ ID NO: 6 for oocyte plant virus assay for simultaneous diagnosis of ORSV ( Odontoglossum ring spot virus ), CyMV ( Cymbidium mosaic virus ) and OFV ( Orchid fleck virus ) Primer set. The method of claim 1, The ORSV-420F primer and ORSV-420R primer are ORSV ( Odontoglossum ring spot virus ) A primer set for eggplant virus assay, which is a primer for assay. The method of claim 1, The CyMV-620F primer and CyMV-620R primer are CyMV ( Cymbidium mosaic virus ) A primer set for eggplant virus assay, which is a primer for assay. The method of claim 1, The OFV-873F primer and OFV-873R primer are OFV ( Orchid fleck virus ) A primer set for eggplant virus assay, which is a primer for assay. a) ORSV-420F primer represented by SEQ ID NO: 1, ORSV-420R primer represented by SEQ ID NO: 2, CyMV-620F primer represented by SEQ ID NO: 3, and CyMV- represented by SEQ ID NO: 4 A primer set for eggplant virus assay consisting of a 620R primer, OFV-873F primer represented by SEQ ID NO: 5, and OFV-873R primer represented by SEQ ID NO: 6, reverse transcription polymerase chain reaction (RT-PCR) and polymerase chain reaction (PCR); And (b) Simultaneously diagnose ORSV ( Odontoglossum ring spot virus ), CyMV ( Cymbidium mosaic virus ), and OFV (Orange fleck virus ), which include determining the size of the amplified PCR product to determine whether an oocyte virus is infected. Egg plant virus detection method.

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