KR102012212B1 - Primer set for detecting Panicum distortion mosaic virus and method for detecting said viruses using the same - Google Patents

Abstract

The present invention relates to a composition for diagnosing Panicum malformation mosaic virus infection, a diagnostic kit, and a diagnostic method including an agent for detecting a Panicum distortion mosaic virus gene consisting of the nucleotide sequence represented by SEQ ID NO: 1. If the panicum malformed mosaic virus infection diagnosis composition of the present invention is used, it is possible to diagnose whether the panicum malformed mosaic virus is infected or not and may be used to predict disease occurrence by monitoring or investigating the pattern of virus disease occurring in domestic crops or weeds. have. In particular, it is possible to clarify the substance of the disease occurring in the host plant, including the millet, and contribute to the dissemination and quality improvement of the host plant disease-free, including the millet.

Description

Primer set for detecting Panicum distortion mosaic virus and method for detecting said viruses using the same}

The present invention relates to a composition for diagnosing Panicum malformation mosaic virus infection, a diagnostic kit, and a diagnostic method including an agent for detecting a Panicum distortion mosaic virus gene consisting of the nucleotide sequence represented by SEQ ID NO: 1.

The emergence and risk of outbreaks and new viral diseases is increasing as ecosystems change due to climate warming, the evolution and variation of plants and viruses, and the expansion of agricultural trade. About 1,000 plant viruses reported so far are known worldwide, and new viruses are still being reported in various plants. At present, about 100 kinds of viruses have been reported in various plants in Korea, but detailed investigations on the viruses occurring have been carried out only in some crops, and most crops and natural-based weed plants have only partial surveys. Thus, it is still unknown how virus diseases are occurring in many major crops and weeds.

Plant disease is a dysfunction of host cells and tissues as a result of the constant effects of pathogens or environmental factors on the plant host, and refers to a condition in which abnormal changes in the morphology and physiology of the plant occur, including a decrease in the economic value. Sometimes. The causes of such plant diseases include fungi (Fungi), bacteria (Bacteria), nematodes (Nematode), mycoplasma (Mycoplasma), protozoa and viruses (Virus). However, since the virus is a pathogen smaller than 1 µm in size, the reality cannot be observed even under an optical microscope, and an electron microscope is required. Therefore, it is difficult to visually diagnose a virus that has occurred in a plant. Therefore, there is a need for genetic research for diagnosing such viruses, and in particular, genetic research for diagnosing viruses for domestic millet plants is needed. Millet is a plant in the genus Panicum, which is known to help inhibit lipid formation, and although research on the virus that causes disease in millet plants is still needed due to the increase in demand due to the increase in domestic cereal production Not widely done Therefore, the selection and dissemination of an uninfected panicum malaria mosaic virus is the most fundamental countermeasure. Therefore, various methods for precisely diagnosing panicum malformation mosaic virus need to be developed.

Accordingly, the present inventors identified a novel Panicum distortion mosaic virus (PDMV) gene of about 5,863bp in size, which causes disease in millet, from plant samples showing viral symptoms in major cultivated regions of domestic millet. Based on the gene, the present invention was completed by confirming that the composition for diagnosing infection, a diagnostic kit, and a diagnostic method including a primer set can be specifically diagnosed only with a panicum malformed mosaic virus.

Accordingly, an object of the present invention is to provide a panicum malformed mosaic virus infection diagnosis composition, an infection diagnosis kit, and an infection diagnosis method comprising an agent for detecting a panicum malformed mosaic virus gene consisting of the nucleotide sequence represented by SEQ ID NO: 1.

In order to achieve the above object, the present invention provides a composition for diagnosing Panicum malformation mosaic virus infection comprising an agent for detecting the Panicum distortion mosaic virus gene consisting of the nucleotide sequence represented by SEQ ID NO: 1. .

The present invention also provides a panicum malformed mosaic virus infection diagnostic kit comprising the composition for diagnosing panicum malformed mosaic virus infection.

In another aspect, the present invention comprises the steps of a) amplifying the gene using a primer specifically binding to the panicum malformed mosaic virus gene consisting of the nucleotide sequence represented by SEQ ID NO: 1; And b) detecting the amplification product obtained in step a).

If the panicum malformed mosaic virus infection diagnosis composition of the present invention is used, it is possible to diagnose whether the panicum malformed mosaic virus is infected or not and may be used to predict disease occurrence by monitoring or investigating the pattern of virus disease occurring in domestic crops or weeds. have. In particular, it is possible to clarify the substance of the disease occurring in the host plant, including the millet, and contribute to the dissemination and quality improvement of the host plant disease-free, including the millet.

1 is a diagram showing the alignment and location of the contigs for Cereal yellow dwarf virus-RPV (CYDV-RPV) showing the highest homology with the Panicum malformed mosaic virus.
FIG. 2 shows two contigs showing the highest homology among contigs of Panicum malformed mosaic virus compared with Cereal yellow dwarf virus-RPV (CYDV-RPV).
Figure 3 is a schematic diagram showing the position of the primer set on the basis of the panicum malformed mosaic virus gene.
Figure 4 is a diagram showing the results of electrophoresis after PCR using a panicum malformed mosaic virus primer set.
5 is a diagram showing the results of diagnosing a virus sample of the genus Polerovirus including a panicum malformed mosaic virus with a primer set in order to confirm the design of a species-specific primer for the panicum malformed mosaic virus.

The present invention provides a composition for diagnosing Panicum malformation mosaic virus infection, comprising an agent for detecting a Panicum distortion mosaic virus gene consisting of the nucleotide sequence represented by SEQ ID NO: 1.

Hereinafter, the present invention will be described in more detail.

The Panicum distortion mosaic virus, which can be diagnosed with the primer set of the present invention, is derived from Proso millet plants. Millet, a virus-derived plant, is an annual herb belonging to the family Asteraceae. Fruits are flat, round or oval, and ripen in autumn with white, tan, reddish brown, and black. It is usually grown in a field, and is native to the equator of Africa, and is distributed in Korea, Japan, Northeast China, Southwest Asia, and Africa.

The panicum malformed mosaic virus gene derived from the millet plant may be composed of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto. The homolog of the nucleotide sequence is included within the scope of the present invention. Specifically, the gene has a base sequence having a sequence homology with at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% of the base sequence of SEQ ID NO: 1, respectively. It may include. The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).

Panicum malformed mosaic virus gene of the present invention is a genus of Polerovirus of the Luteoviridae strain, the total length of the base sequence is 5,863bp. In addition, 5 'untranslated region (5' UTR) is located at 1-117bp and 3'UTR is located at 5,623-5,808bp. The panicum malformed mosaic virus gene of the present invention has six open reading frames (ORF), and ORF0 (118-861, nt positions) encodes the P0 protein. ORF1 (251-2,140) and ORF2 (1,604-3,411) encode RNA dependent RNA polymerase P1-P2 fusion proteins due to -1 ribosomal frame variation of the 1,604 bp region. In addition, ORF5 (3,607-5,622) encodes P3-P5 translated protein by read-through mechanism in the stop codon of P3 protein, ORF3 (3,607-4,221) and ORF4 (3,638-4,093), respectively It encodes coat protein (CP) and movement protein (MP).

As used herein, the term "primer" refers to a nucleic acid sequence having a short free 3 'hydroxyl group, which may form a base pair and a base pair of complementary nucleic acids, By a short nucleic acid sequence that serves as a starting point for. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures.

In addition to the primers designed in the present invention may include DNA polymerase, reverse transcriptase, dNTP and reaction buffer known in the art for performing reverse transcription and polymerization. Furthermore, DNase, RNase inhibitor, DEPC-water, sterile water, etc. may be included if necessary.

Primer sets produced and used in the present invention are shown in Table 1 below.

TABLE 1

When designing the primer, there are various restrictions such as the A, G, C, T content ratio of the primer, prevention of primer dimer formation, and prohibition of repeating three or more times of the same nucleotide sequence. (template) amounts of DNA, the conditions of the concentration of the primer, the dNTP concentration, Mg concentration of ^{2 +,} such as reaction temperature, reaction time should be adequate.

Such primers may incorporate additional features that do not change the basic properties. That is, the nucleic acid sequence can be modified using many means known in the art. Examples of such modifications include methylation, capping, substitution of one or more homologs of nucleotides and uncharged linkages, such as phosphonates, phosphoresteres, phosphoramidates or carbamates, or phosphorothioates or phosphorodithioates. Modification of the nucleotides to charged linkers, etc. is possible. Nucleic acids may also include one or more of nucleases, toxins, antibodies, signal peptides, proteins such as poly L-lysine, insertion agents such as acridine or psoralen, chelating agents such as metals, radioactive metals, iron oxidizing metals, and alkylating agents. It may have additional covalently linked residues.

In addition, the primer sequence of the present invention can be modified using a label that can provide a detectable signal directly or indirectly. The primer can include a label that can be detected using spectroscopy, photochemistry, biochemistry, immunochemistry or chemical means. Useful labels include 32P, fluorescent dyes, electron dense reagents, enzymes (generally used in ELISA), biotin or hapten and proteins with antiserum or monoclonal antibodies available.

The primers of the present invention are suitable for cloning and restriction enzyme digestion of the appropriate sequence and phosphoester ester method such as Narang (1979, Meth, Enzymol. 68: 90-99), diethylphosphoramidie such as Beaucage, etc. And any other well known methods, including direct chemical synthesis, such as the Tetrahedron Lett. 22: 1859-1862, and the solids support method of US Pat. No. 44,580,66.

The agent for detecting a viral gene of the present invention may comprise a primer or probe that specifically binds to a Panicum malformed mosaic virus gene, but preferably the primers are SEQ ID NOs: 2 and 3; SEQ ID NOs: 4 and 5; SEQ ID NOs: 6 and 7; SEQ ID NOs: 8 and 9; And it may be one or more primer sets selected from the group consisting of primer pairs represented by SEQ ID NO: 10 and 11; Using the primer set, only panicum malformed mosaic virus can be specifically detected from a sample infected with Polerovirus belonging to the same genus as panicum malformed mosaic virus.

In addition, the primer set of the present invention may be a primer set for diagnosing panicum malformed mosaic virus infection in flower and crops. The flower and crop may be one or more selected from the group consisting of ragweed, yulmu, crude, millet, sorghum, rice and corn, preferably may be millet.

The composition for diagnosing panicum malformed mosaic virus infection of the present invention may further include a reaction amplification mixture, and the reaction amplification mixture may include reagents, thermally stable DNA polymerases, deoxynucleotides, and nucleases necessary for carrying out the amplification reaction. Sterile water and divalent metal cations, and the like, and preferably include reaction buffers, deoxynucleotides, and DNA polymerases.

The present invention also provides a panicum malformed mosaic virus infection diagnostic kit comprising a composition for diagnosing panicum malformed mosaic virus infection.

Kits of the present invention may be prepared by placing the composition in a plastic tube in a freeze-dried form. The kit may further comprise, in addition to the composition, suitable reagents and tools for use in the assay, such reagents and tools including suitable carriers, labels capable of producing detectable signals, solubilizers, cleaning agents and the like.

Such suitable carriers include, but are not limited to, soluble carriers such as physiologically acceptable buffers known in the art such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, polyester , Polyacrylonitrile, fluorine resin, crosslinked dextran, polysaccharides, polymers such as magnetic fine particles plated with latex metal, other paper, glass, metal, agarose and combinations thereof.

In another aspect, the present invention comprises the steps of a) amplifying the gene using a primer specifically binding to the panicum malformed mosaic virus gene consisting of the nucleotide sequence represented by SEQ ID NO: 1; And b) detecting the amplification product obtained in step a).

Primers used in step a) of the present invention are SEQ ID NOs: 2 and 3; SEQ ID NOs: 4 and 5; SEQ ID NOs: 6 and 7; SEQ ID NOs: 8 and 9; And it may be one or more primer sets selected from the group consisting of primer pairs represented by SEQ ID NO: 10 and 11;

In addition, the a) step amplification of the present invention is a polymerase chain reaction (Polymerase Chain Reaction), duplex polymerase chain reaction (duplex PCR), multiplex polymerase chain reaction (multiplex Polymerase Chain Reaction, multiplex PCR) , Competitive polymerase chain reaction, real-time polymerase chain reaction, realtime quantitative polymerase chain reaction, DNA chip, isothermal amplification (Loop-mediated isothermal amplification) and a combination thereof may be any one selected from the group consisting of.

Detection of the amplification product of step b) of the present invention may use any one method selected from the group consisting of capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement, phosphorescence measurement and combinations thereof.

Terms not defined otherwise in this specification are intended to have a meaning commonly used in the art to which the present invention pertains.

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

Example  One. Panicum  Identification of Malformed Mosaic Virus

1.1 Obtaining and Panicum  Total RNA Extraction of Malformed Mosaic Virus

Sixty millet samples were collected from eight regions nationwide (Gangwon-do Hoengseong, Gochang, Gyeongbuk Uiseong, Miryang, Jeonbuk Gochang, Jeonnam Naju, Yeosu, Jeju Island). Next generation sequencing (NGS) was commissioned to identify millet-infected viruses, and total RNA was extracted using each plant as a TRI Reagent (MRC, Cincinnati, OH, USA).

1.2 RT - PCR  Conduct of diagnosis

The extracted total RNA was synthesized cDNA using RevertAid H Minus Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) according to the manual of the sales company, as follows.

Contig specific primers were designed to identify the contigs of the samples and find the entire genome sequence of the virus, which is shown in Table 2.

TABLE 2

The sequence similarity test of the virus-related database was analyzed by NCBI BLAST in order to determine the sequence of the entire gene of the virus with the contigro, and the step of identifying the virus with the highest homology was performed. The alignment of the contigs is shown in FIG. 1, and the two contigs with the highest homology were identified, and the two contigs were compared with Cereal yellow dwarf virus-RPV (CYDV-RPV). Shown in As confirmed in FIG. 2, it was confirmed that the contig having the highest homology had 72% homology with Cereal yellow dwarf virus-RPV (CYDV-RPV).

Later, GeNetBio SuPrimeScript RT-PCR Premix (2X) Cat. No. RT-PCR diagnosis was performed by SR-8000 (GenetBio, Daejeon, Korea). 1 μl RNA template, 10 pmol of 1 μl forward primer and 10 pmol of 1 μl reverse primer were added to 10 μl of 10 μl premix and adjusted to 20 μl with ddH ₂ O. RT-PCR was reacted at 50 ° C. for 30 minutes in the cDNA synthesis step, and was performed by setting conditions for 10 minutes at 95 ° C. in the initial denaturation step. Thereafter, three stages of a denaturation step at 95 ° C. for 30 seconds, an annealing step at 55 ° C. for 30 seconds, an extension step at 72 ° C. for 1 minute, and a final expansion step at 72 ° C. for 5 minutes were performed. RT-PCR was performed at 35 cycles. PCR products were analyzed by electrophoresis on a 1.5% agarose gel using 0.5 TAE (tris base, acetic acid and EDTA) buffer containing Ethidium Bromide (EtBr).

As a result, it was confirmed that all six sets of primers designed on the basis of contig showed positive reaction. As a result of sequencing by direct sequencing, it was confirmed that it is consistent with the result analyzed by next generation sequencing (NGS). Therefore, the virus was finally identified as a new Polerovirus species.

1.3. Determination of Whole Virus Genome Sequence

1.3.1 Analysis of Virus Whole Genome Sequence

One virus positive sample was selected for total sequencing and cDNA was synthesized using enzynomics SuperiorScript III Reverse Transcriptase (enzynomics, Daejeon, South Korea). PCR 4 μl of mixed 5X first strand buffer, 1 μl of SuperiorScript III reverse transcriptase, 2 μl of 2 mM dNTP mixture, 2 μl of 0.1 M DTT, 2 μl of RNA template and 0.2 μl of 10 pmol random primer (25 mer) The tube was filled with up to 20 μl of RNase-free water. Synthesis conditions were set to an annealing step at 25 ° C. for 10 minutes, a first incubation step at 50 ° C. for 60 minutes, and a second incubation step at 70 ° C. for 15 minutes. PCR was performed using Solg ^™ EF-Taq DNA Polymerase (Solgent, Daejeon, South Korea). 0.5 μl of mixed Solg ^™ EF-Taq DNA polymerase, 0.5 μl of each 10 mM dNTP, 1.5 μl of cDNA template, 1 μl of forward primer, 1 μl of reverse primer, 2.5 μl of 10X EF-Taq reaction buffer into 0.2 μl PCR tube And filled with 20 μl of ddH ₂ Ofh. PCR conditions were set at an initial denaturation step at 95 ° C. for 2 minutes. Thereafter, three steps of a denaturation step at 95 ° C. for 20 seconds, an annealing step at 55 ° C. for 40 seconds, an extension step at 72 ° C. for 1 minute, and a final expansion step at 72 ° C. for 5 minutes RT-PCR was performed at 35 cycles. PCR products were analyzed by electrophoresis on a 1.5% agarose gel using 0.5 TAE (tris base, acetic acid and EDTA) buffer containing Ethidium Bromide (EtBr).

Using GeneAll ^® Expin SV ^TM PCR Kit (GeneAll, Seoul, Republic of Korea) and purified PCR product as follows:

1. Binding step: 5 volumes of buffer PB was added to 1 volume of sample and mixed.

The mixture was transferred to an SV column and centrifuged at 13,000 rpm for 1 minute at room temperature. The emulsion was then discarded and the SV column replaced.

2. Wash step: 700 μl of buffer NW was added and centrifuged at 13,000 rpm for 1 min at room temperature. The emulsion was then discarded and the SV column replaced. Centrifuge at 13,000 rpm for 2 minutes at room temperature to remove residual wash buffer.

3. Elution step: 50 μl of double distilled water (ddH ₂ O) was added to the filter in a fresh 1.5 ml tube on top of the SV column. After incubation for 1 minute at room temperature, and centrifuged at 13,000 rpm for 1 minute at 4 ℃.

1.3.2 5 'and 3' ends Nucleotides  Sequencing

Rapid amplification PCR method of cDNA (RACE-PCR) was used to analyze the genomic sequence. 5 'RACE PCR was performed using Invitrogen ^™ 5' RACE System for cDNA Ends (Thermo Fisher SCIENTIFIC, California, USA) as follows.

1. First strand cDNA synthesis step: 2.5 pmol 1 μl reverse primer, 3 μl RNA template was added and filled with ddH ₂ O up to 15.5 μl. The mixture was incubated at 70 ° C. for 10 minutes and then cooled on ice for 1 minute. 2.5 μl 25 mM MgCl, 10 mM 1 μl dNTP, 2.5 μl 0.1 M DTT and 2.5 μl 10X PCR buffer were added to the mixture, mixed gently and incubated at 42 ° C. for 1 minute. 1 μl of SuperScript ^TM II Reverse Transcriptase was added to the final mixture and incubated at 42 ° C. for 50 minutes.

2. Purification step: ddH ₂ O at 65 ° C. was prepared for elution. The reaction of the first strand synthesis reaction was added to 120 μl of binding solution and mixed. The mixture was transferred to a SNAP column, centrifuged at 13,000 rpm for 1 minute at room temperature, the flow-through was removed and the SNAP column was returned to the centrifuge. 400 μl of 1 × Wash Buffer was added to the SNAP and centrifuged at 13,000 rpm for 1 minute at room temperature. After removing the flow-through and reinserting the SNAP column, the step was repeated three times. 400 μl of 70% ethanol was added to the SNAP and centrifuged at 13,000 rpm for 1 minute at room temperature. The emulsion was removed and the SNAP column was placed back into the centrifuge and this step was repeated twice. The column filter was centrifuged at 13,000 rpm for 1 minute 30 seconds at room temperature to dry. Place a new 1.5 ml tube on top of the SNAP column and add 50 μl of 65 ° C. ddH ₂ O. Centrifuged at room temperature for 1 minute and centrifuged at 13,000 rpm for 1 minute at room temperature.

3. C-tailing step of cDNA step: 10 μl of purified cDNA, 2.5 μl of 2 mM dCTP, 5 μl of 5 × tailing buffer was added, and 24 μl of ddH ₂ O was added. After incubating at 94 ° C. for 3 minutes and cooling on ice, 1 μl of Terminal deoxynucleotidyl transferase (TdT) was added to the mixture and mixed gently. Incubated at 37 ° C for 10 minutes and at 65 ° C for 10 minutes.

4. PCR step: PCR was performed using C-tailed cDNA synthesized using Solg ^™ EF-Taq DNA Polymerase (Solgent, Daejeon, Korea). 1 μl of Oligo dG Anchor primer designed by Invitrogen (5'-GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GGG IIG -3 ') and reverse primer (5'-GCT GCT GGG CAC TTA GCC 3') were used. . 3'RACE PCR was performed using Epicenter Poly (A) tailing kit (Epicentre ^® , Wisconsin, USA).

5. A-tailing step of RNA step: 3 μl of RNA template, 2 μl of 10 × reaction buffer, 2 μl of 10 mM ATP, 1 μl of poly (A) Polymerase were added and filled with ddH ₂ O up to 20 μl. . Incubated at 37 ° C. for 20 minutes and then cooled on ice.

6. cDNA Synthesis Step: A-tailed RNA was synthesized by Enzynomics SuperiorScript III reverse transcriptase (Enzynomics, Daejeon, Korea). 1 μl of Oligo dT Anchor primer (5′-GGC CAC GCG TCG ACT AGT ACT TTT TTT TTT TTT TTT T-3 ′) designed by Invitrogen was used.

7. PCR step: C-tailed cDNA was synthesized by PCR using Solg ^TM EF-Taq DNA Polymerase (Solgent, Daejeon, Korea). 1 μl of forward primer (5′-CAT TGA AAC TAC AGG GGA GG-3 ′) and 1 μl of Invitrogen designed Anchor primer (5′-GGC CAC GCG TCG ACT AGT AC-3 ′) were used.

8. As a result of electrophoresis after PCR reaction, the positive reaction of the expected size of about 1200 bp and 1000 bp at the 5 'end and 1000 bp was confirmed, and the base sequence was finally determined by direct sequencing.

1.3.3 Whole Genome of New Virus Sequence  analysis

The viral sequences finally identified in Examples 1.1 to 1.3.2 were identified and shown in SEQ ID NO: 1. The newly identified sequence of viruses differs by more than 10% in identity from previously known Poleroviruses ORFs and whole genome sequences, so the new virus is classified according to the species classification criteria of the International Committee on Taxonomy of Viruses (ICTV). It was identified as. The virus was named Panicum distortion mosaic virus (PDMV) and it was confirmed that PDMV is a new species of the genus Polerovirus of the Luteoviridae strain.

The total length of the newly identified virus was 5,863bp, the 5 'untranslated region (5' UTR) was 1-117bp, and the 3'UTR was 5,623-5,808bp. The virus has six open reading frames (ORFs), ORF0 (118-861) encodes P0 protein, ORF1 (251-2,140) and ORF2 (1,604-3,411) are -1 ribosomal frame mutations in the 1,604 bp region. It was confirmed that it encodes an RNA dependent RNA polymerase P1-P2 fusion protein. ORF5 (3,607-5,622) encodes P3-P5 translated protein by read-through mechanism in the stop codon of P3 protein, ORF3 (3,607-4,221) and ORF4 (3,638-4,093) are envelope proteins, respectively (coat protein, CP) and the movement protein (movement protein, MP) was confirmed to encode.

Example  2. Panicum  Diagnosis of Malformed Mosaic Virus Of primer  making

Species specific primers were designed for diagnosing the virus based on the new virus Panicum malformed mosaic virus nucleotide sequence obtained in Example 1. Specifically, Viral contigu sequences obtained by NGS analysis were secured to design primers. In addition, Polerovirus belonging to Panicum malformed mosaic virus for species-specific design The genome sequence information of 18 viruses belonging to the genus was downloaded from NCBI GenBank, alignment was performed using DNAMAN ver 7.0 software, and there was a homology difference between the panicum malformation mosaic virus nucleotide sequence and the nucleotide sequence of 18 viruses of the same genus. Five pairs of primer sets were designed for many non-conserved regions. 18 virus sequence information belonging to the genus is shown in Table 3, and the panicum malformed mosaic virus primer position is schematically shown in FIG. In addition, the designed primer sequence is shown in Table 1.

TABLE 3

TABLE 1

Example  3. Species Specific Primers  Use of diagnostics

3.1 primer  Through set Panicum  Diagnosis of Malformed Mosaic Virus

In order to determine whether panicum malformed mosaic virus can be diagnosed using the primer set designed in Example 2, electrophoresis was performed after PCR amplification of the virus sample obtained in Example 1. Polymerization chain reaction (Poymerase chain reaction, PCR) reaction was performed using the cDNA synthesized in Example 1 as a template strand with the primer designed by Neoprobe (Daejeon, Korea). PCR reactions were carried out using Prime Taq ™ Premix (2 ×) (Genet Bio, Cheonan, Korea) with 1 μL cDNA template, 10 pmol / μL primer set 1 μL, Prime Taq Premix (2 ×) and 10 μL PCR was performed with 7 μL of ionized water as the composition.

PCR was performed under the following conditions. An initial denaturation step of 5 minutes was performed at 94 ° C. Thereafter, the reaction was performed at 94 ° C. for 5 minutes, followed by amplification at 72 ° C. for 1 minute after reaction at 55 ° C. for 30 minutes, followed by 35 cycles and finally extending at 72 ° C. for 5 minutes.

After the PCR experiment, 1% agarose gel was added to the etidium bromide (EtBr) and electrophoresis was performed for 40 minutes at 120V, to confirm the amplification product, the results are shown in FIG.

As shown in FIG. 4, it was confirmed that all five primer sets were able to diagnose panicum malformed mosaic virus through high sensitivity.

3.2 Panicum  Malformed mosaic virus primer  Specificity of the set

PCR was performed to confirm whether the panicum malformed mosaic virus primer set can specifically detect only panicum malformed mosaic virus from a sample infected with the genus Polerovirus . Polymerization chain reaction (Poymerase chain reaction, PCR) reaction was performed using the cDNA synthesized in Example 1 as a template strand with the primer designed by Neoprobe (Daejeon, Korea). PCR reactions were carried out using Prime Taq ™ Premix (2 ×) (Genet Bio, Cheonan, Korea) with 1 μL cDNA template, 10 pmol / μL primer set 1 μL, Prime Taq Premix (2 ×) and 10 μL PCR was performed with 7 μL of ionized water as the composition.

PCR was performed under the following conditions. An initial denaturation step of 5 minutes was performed at 94 ° C. Thereafter, the reaction was performed at 94 ° C. for 5 minutes, followed by amplification at 72 ° C. for 1 minute after reaction at 55 ° C. for 30 minutes, followed by 35 cycles and finally extending at 72 ° C. for 5 minutes.

After the PCR experiment, 1% agarose gel was added to the ethidium bromide (EtBr) and subjected to electrophoresis for 40 minutes at 120V, to confirm the amplification, and the results are shown in FIG.

M is a marker, BVG lane is Barley virus G, BWYV lane is Beet western yellow virus , CABYV lane is Cucurbit aphid-borne yellow virus , PLRV lane is Potato leafroll virus and PDMV lane is Panicum distortion mosaic virus. Indicated.

As shown in FIG. 5, it was confirmed that all five primer sets specifically reacted only to samples infected with Panicum malformed mosaic virus. Therefore, it was confirmed that only a panicum malformed mosaic virus can be diagnosed with high specificity from a sample infected with Polerovirus belonging to the same genus as the panicum malformed mosaic virus using the diagnostic method through the primer set of the present invention.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Primer set for detecting Panicum distortion mosaic virus and          method for detecting said viruses using the same <130> 1-303P <160> 11 <170> KoPatentIn 3.0 <210> 1 <211> 5863 <212> DNA <213> Unknown <220> <223> Panicum distortion mosaic virus complete genome sequnece <400> 1 tctcagcaga gttcctgcat ggcagtaaaa agatagggag gggactcacc tgtcttttga 60 agcactcctc tgctctcgca cgaagttgat caagatcctt catgcgagct attcttggca 120 caaccagaat agtcttccac ggtcgtaacg ctcccgaaac ggtcgaagag ctcgcttctt 180 gcttgacgct catcgaattt caggctagaa ggtattacgc cctcgcccga agcgaccccc 240 tctcagcaga gttcctgcat ggcgctaact tttctaatgt tggtgcttat gttagccacc 300 ttaggcgcct cttgtgcgcc tacatccctg tgctactcct taacggaacc agccttacaa 360 gcacagggct catcaccgta cgactcgact tcctggtacc agtcctacga tggttacaat 420 gtcaccggct ttacccgtac attaccatcc gtagtagaag gttcaggatt gaccaaaatc 480 gaacggtcgg accaacgctt tatcgatctc tcatatggag atatgatttt ctggttcggc 540 ttcaagatac gagtcgactt gctaacatta gcaggagagg tccagagtat ttcaacaccc 600 aaatggcagt cattctgtca gggattgaaa gaaggattct cgagattcct gaacagcact 660 ttgtggatga tatacaacta ctatctttgg tgtgttttat acacgatcaa attgatagca 720 tcattcttgt tgatgacctg gaagccgatg tcagccattg ggctggctct ggccatcaca 780 tcatccatga tttgggccat caagcggata tactcgtgca ttccggtctt ttggatcgga 840 atacctttca ggcttttgca caaagccctc cgctggctgt tcaggaagaa cggagatgaa 900 aaagctgttc aaggctttaa cagctactcc gttaagatgg cccctccagg aaagtcggtt 960 ctggaaatcc tcttcgcaga caactcacac tgtggttacg cttcgtgcgt acgccttcag 1020 accggagagg acgccctgtt aacttcacta cactgttgcg atccaacata caaagtgagg 1080 tctttcagga ctggctcaat gatcccgttg agccaattcc agatcatttt cccttccgag 1140 aagcttgatt tggtgattct tcgaggtcca accgaatgga aatcgcttct cggcgccaaa 1200 gcctcccaca tgaagacagc agaccgcctt tcaaaacaag gtgtgtcctt cttcaccttt 1260 aatggacagt gggaaatgca taacgcaaaa gtggttggct cggacggact cttcgtgagc 1320 gtcctgagca acaccactaa aggctacagc ggaaccccct acttcgttgg caaagacatt 1380 attggggtgc acaagggata tgacgggaat gactcgagca agaactacaa tctgatggct 1440 ccgatccctc ccatcgaggg attgacgaag cctcagtacg tttacgagtc ttctacgatt 1500 gacggagacg tatacagcga agaagaaatt gaagaattaa cgaaatacgc tgtagacacc 1560 tttgaaaagg ttaagaaggt gtcgtcctcc tggagaccta gccacaatga agaatggtgg 1620 ctagatagcc tcgctgatga agcagcatca cccccaaaaa ctccggcaac cgttgagttt 1680 cagggaaacg aggcgcgtgg caccgaccac caggaaaaac cggtatccac ccttgccaag 1740 cccgccgact ctaccgacga catgctgcgc gaaatcgtag cacagatagt gggcaggatc 1800 gacctgagtg ctctcactca gagggtggag gggaagctgc tgaaccaagt caaatctgca 1860 cagaacaagc aggttcggca gccggaggtc aagaggacac gatcacgaaa acgaaatacc 1920 tcgaagagtt cctcaagtcc gccttcaaat gggatgacga acccaccagt ggtgaaatcc 1980 cagggttcag gaaaacagga cgcctccccg cgctatatca tccccaaaaa ccgaaagaca 2040 caaccttcgg ggatggtatc atcagcgccc acccagaaat ggcggccaaa atcgccgggt 2100 tcgggtggcc ccagttcggc gctgccgccg aaaagacaag tctgaaactt caggctgatc 2160 gctggctctc tcgagctcag tcagcggtta aaccatcggc agcaaaacga gagctcgtga 2220 tccaacatgc ggtagaaagg tacaaactct gccaaacaca aatgccgcat actagtgccc 2280 ctggacagct taaatgggga aatttcttga aagatttcaa ggaagctgtc cactcacttg 2340 agctggatgc aggaatcggc ctcccataca aagtcctgaa gatgcaaacc cacagagata 2400 tggttgagga ccccgattac ctgcccttat tgactcgcct cgtctggaac cgcctatcga 2460 agatgtccca ggcgaagttt gaggatatga gcccagaaca gctcgtgaga gagggattgt 2520 gcgacccgat acgcttgttc atcaaaggag agccgcacaa acaatcgaaa cttgatgaag 2580 gccgctaccg cctcatcatg tcagtgtcac tagtggatca actggtagcc cgggttttat 2640 ttcaaagcca aaacaaacgc gagatatcct tgtggaggga tatcccgtca aaacccggtt 2700 tcggcttatc cactgacagc gaggcgaaag agtttatcag aacgctgtca aaggttgtgg 2760 gttgtcctcc acaaacgctc attcatgagt ggaaggacaa agtcgtaccc accgactgtt 2820 ccggtttcga ctggtcggtc gccgattgga tgctcgagga tgatatggag gtgagaaatc 2880 gcctcacatt taactgcaac gagctcacca ggcgcctccg cgcatgctgg ttgaagtgca 2940 taacaaattc tgtgttgtgc acatccgacg gaaccttgta cgcccagatt catcctggtg 3000 tgcagaaatc cggctcgtac aatacgagct cgtcgaattc caggattaga gttatggccg 3060 catatcactg cggcgccaaa tgggcgatag ccatggggga tgacgcactt gaatccccag 3120 aaaccgacct taacgcctac aagaagctcg gtttcaaagt cgaggtatcc tcacagctag 3180 aattttgctc acacgtcttt gagcaggagg acctcgccag gccgttgaac gtcgcgaaaa 3240 cattatatag gttgatctac ggatacaacc cggcctgtgg caacccggag gtgttagcta 3300 actacctcca agcagtggcg agcgtattga acatactccg tcatgaccct gctgaggttg 3360 ccaagctccg taactggctc cttcctggcg ggggccagta aaaagatacg gagggaaaag 3420 tagccaccat cttcgttgca agagtggagc tttagtcgaa gaacgcagcc gattctggat 3480 tataaattgc tagctggtgt gctcattgga gtaatcatca caactccgct agtagtagtg 3540 gctgcgtacc tggcatttct caaaatcagt gcacatatca gagcgatagt taatgaatac 3600 gggcgctgct agacgctcac gcagaaatgt cagaaggcgc tctaaccgtc gtcgatcgac 3660 tcggccagtg gtcatggtca ggactacccc aagatctcga cgagtacgac gacgtggagc 3720 acgtgtcgga ggaaacgctg tgcgaggatt acgaggagga agcaaccggg atgttctcac 3780 tttcacggtt gacgatctca aagccaacag ctccgggatc ctcaagttcg gaccgagttt 3840 atctcagtac ccagcgttca acaatggctt actcaaagcc taccatgagt ataaaatcac 3900 aagtctcaca gtacagtata actcatgctc ctccgacgcc acttcaggtg caatcgcact 3960 tgaagtggat acgtcctgct cccaaacagc aacaggctcc aaaatcgtta gcttccccgt 4020 caagaggaac gccacgaaag tcttccccac ccagtacatt aagggtcaaa acttcatgac 4080 tacatcagct gatcaatttt ggctgttgta caaagggaat ggcgatggaa gcctagcagg 4140 acaattcgtc tgccgatttg aatgtcaatt tcagaatcca aaataggtag gcgacgctcc 4200 cccaacaccc acacccaccc ctacaccaac accgactccc actccgaccc ctactccggc 4260 accagcaccc aaattctttg gatatcaagg tgtgccgacg agtatcgtca aaaccagagg 4320 aaacaatgag ttttgcgatg tgggttcact caacaatgtc tccatgtatt tctggaaaga 4380 tgaagcttgg agtgttgaga cactgtcagc gggctacagc atcaacaatc gcgacagagc 4440 cacccccttt ttcctggttc cagttgagaa agggagatac tccgtgtaca tacaatgtga 4500 gggctttaag gcagttaaag ccaaaggtgg ttccaatgat ggccgcatga gtggtttcat 4560 cacagataat ccgagtcagg cagggtggag agcgtacaat tacgatgggt gtgtaatatc 4620 gaactacaaa gctagtaacg acagggtgca aggacatccc gacctcaagg tcaacggatg 4680 ttctttctca gaccagcttg tagagaccga tttttactgt tcgtttcacc tcgaggtggc 4740 cactgaagga tactttggtg tccaggcacc tcccatagag aaggatgatc attacaacta 4800 tgtggtgtcc tacgcaaact tcactgagaa agtgatggaa tggggctcag tctccatcgc 4860 gatggacgag gtaaacgagg gagcgtatcg cgcgtcgaaa tgggataaga ccgcaccagt 4920 gaatgctcga ttgtcgggtc agatcagcgc ctcatcggag gtttatactc cggtggaatc 4980 tgacacaaaa ccgcaatcag aacggacgga gagtacgcct ccagcccgga ccctccaggt 5040 acaaggggtt aaaaccgaac cacccatcaa aatcgctgat aaaccggatc ctaaagtcat 5100 gagctggtta catgaccaac cggttgtcga agctgtcgta aagggggatt tcactcacga 5160 aactgtgtat ccaccaccac ccccgcctct gacggcagat gacacttctc gccaagccgc 5220 aaaagtcgtt cgaactttga tggccaacgc tgatgaagcg cctgcccaaa cacctggcca 5280 agatgtgcaa acgtcgatgc cggtttccaa gacagaggag cacagcgata gcgatgagga 5340 tgcggcgtcc atcacaccct cgagagcggg aacgctctct ggagggtctc ttcgaggagg 5400 atctcttcga ggagggttaa agccaaaacc atacgcagct gaacaagcag tggatgatga 5460 attggcttca atcagcggac gttccggagg tggatccctt ggcgggggat ctctccgggg 5520 aggaaccttg cgttccacac ctagtctggt atctcgagaa atgactcaag ccgaacgaaa 5580 catgtatcgg gatatcttga acagcagagg taaaactgct gcagcagagt gggctgcagc 5640 catgggaaaa gacatacctc aggctaggag accgcgcctc tttgggtgat ttaaccacta 5700 cgccaccgtc cagctctcga cattaaatgg agcgcctgcc gaggcaaaac gagacgggct 5760 aaccacccgg ccgccctcca gctaagacca cttagactgg agtggtttct cacggtcaat 5820 gagaagacat tcctgggagc actgcttcca ggttttgttg gta 5863 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> PDMV-F1 primer sequence of primer set 1 <400> 2 gattcaatcg cccgaccgt 19 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> PDMV-R1 primer sequence of primer set 1 <400> 3 taagagcggc ttctgagcg 19 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> PDMV-F1 primer sequence of primer set 2 <400> 4 gattcaatcg cccgaccgt 19 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> PDMV-R2 primer sequence of primer set 2 <400> 5 atgaatgtcg aggcggggt 19 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> PDMV-F2 primer sequence of primer set 3 <400> 6 agagcattgc agaccgcct 19 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> PDMV-R3 primer sequence of primer set 3 <400> 7 ccacctgatg gatcacttga 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> PDMV-F3 primer sequence of primer set 4 <400> 8 cttagtacct ccggagaatc 20 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> PDMV-R4 primer sequence of primer set 4 <400> 9 ttcctacccg aggcctaga 19 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> PDMV-F3 primer sequence of primer set 5 <400> 10 cttagtacct ccggagaatc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> PDMV-R5 primer sequence of primer set 5 <400> 11 taccccaatg catccctaac 20

Claims (10)

A composition for diagnosing Panicum malformation mosaic virus infection, comprising an agent detecting a Panicum distortion mosaic virus (PDMV) gene consisting of the nucleotide sequence represented by SEQ ID NO: 1.
The method of claim 1, wherein the agent for detecting the virus gene is characterized in that the primer or probe specifically binding to the panicum malformation mosaic virus gene, panicum malformation mosaic virus infection diagnostic composition.
The method of claim 2, wherein the primers comprise SEQ ID NOs: 2 and 3; SEQ ID NOs: 4 and 5; SEQ ID NOs: 6 and 7; SEQ ID NOs: 8 and 9; And at least one selected from the group consisting of primer pairs represented by SEQ ID NOs: 10 and 11; panicum malformation mosaic virus infection diagnostic composition.
The composition for diagnosing Panicum malformed mosaic virus infection according to claim 1, wherein the diagnostic composition is for diagnosing Panicum malformed mosaic virus infection in plants and crops.
The method of claim 4, wherein the flower and crop is one or more selected from the group consisting of ragweed, yulmu, crude, millet, sorghum, rice and corn, composition for diagnosing panicum malformation mosaic virus infection.
A panicum malformed mosaic virus infection diagnostic kit comprising a composition for diagnosing panicum malformed mosaic virus infection according to claim 1.
a) amplifying the gene using a primer that specifically binds to a Panicum malformed mosaic virus gene consisting of the nucleotide sequence represented by SEQ ID NO: 1; And
b) detecting the amplification product obtained in step a); panicum malformation mosaic virus infection diagnostic method comprising a.
The method of claim 7, wherein
Primers used in step a) are SEQ ID NOs: 2 and 3; SEQ ID NOs: 4 and 5; SEQ ID NOs: 6 and 7; SEQ ID NOs: 8 and 9; And at least one selected from the group consisting of primer pairs represented by SEQ ID NOs: 10 and 11; panicum malformation mosaic virus infection diagnosis method.
The method of claim 7, wherein
The a) amplification step a) polymerase chain reaction (Polymerase Chain Reaction), duplex polymerase chain reaction (duplex PCR), multiplex polymerase chain reaction (multiplex Polymerase Chain Reaction, multiplex PCR), competitive polymerase Competitive Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Realtime Quantitative Polymerase Chain Reaction, DNA Chip, Loop-mediated isothermal amplification) and a combination thereof. A method for diagnosing panicum malformed mosaic virus infection, characterized in that using any one method selected from the group consisting of.
The method of claim 7, wherein
The detection of the amplification product of step b) is characterized in that using any one method selected from the group consisting of capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement, phosphorescence measurement and combinations thereof. Method for diagnosing a Kum malformed mosaic virus infection.

Images