KR101750837B1 - Primer set for multiple detection MNSV, SqMV, WMV, and CABYV and method for detecting said viruses using the same - Google Patents

Abstract

The present invention provides a primer pair comprising a pair of primers represented by SEQ ID NO: 1 and SEQ ID NO: 2, a pair of primers represented by SEQ ID NO: 3 and SEQ ID NO: 4, a pair of primers represented by SEQ ID NO: 5 and SEQ ID NO: 6, (MNSV), Squash mosaic virus (SqMV), Watermelon mosaic virus (WMV), and aphid-mediated sulfation Provided is a multi-diagnostic kit for melon virus multiple diagnosis capable of multiple diagnosis of a virus (Cucurbit aphid-borne yellows virus, CABYV) and a virus multiple diagnosis method using the primer set. The primer set of the present invention can simultaneously diagnose four viruses at a time, thereby saving time and cost for virus diagnosis, making it economical and contributing to virus-free disease production.

Description

[0002] MNSV, SqMV, WMV, and CABYV multiplex diagnostic primer sets and diagnostic methods using the same are described. [0002] Primer sets for multiple detection MNSV, SqMV, WMV, and CABYV [

The present invention relates to a primer set for multiplex diagnosis of viruses occurring in melon and a diagnostic method using the same. More particularly, the present invention relates to a primer set capable of simultaneously diagnosing four newly detected melon viruses in Korea, And a diagnostic method using the same.

Recently, the taste and price of melon have been maintained at an appropriate level and consumption has increased. In some areas, it has also been exported overseas, making it a great source of income for farmers in Korea. In Korea, most of the melons are cultivated in the facility, so they can be produced all year round. However, the damage caused by viruses began to occur in melon plantations a few years ago, and it is becoming a problem for the stable production of melons as the area and area of the melon grows exponentially (Park et al., 2011). The viruses that are mainly infected with melon are the Melon necrotic spot virus ( MNSV), the amber mosaic virus ( MNSV) virus (Squash mosaic virus, SqMV), watermelon mosaic virus have been reported, including (watermelon mosaic virus, WMV). In recent years, melon has been tested for the presence of cucurbit aphid -borne yellows virus ( CABYV).

Melon necrotic spot virus ( MNSV) is a virus belonging to the genus Tombusviride and Carmovirus , and is a spherical virus with a diameter of about 30 nm. The gene consists of about 4.3 kb of single stranded RNA. MNSV is known to be transmitted by seeds (10-40%) and fungi ( Olpidium ). In Korea, the first occurrence occurred in melon in Naju area, Chonnam province in 2001, and then it is occurred mainly in melon. In 2008, 54% of melons in Naju, Gokseong and Changwon districts were affected by the outbreak of MNSV. In addition, the occurrence was also observed in watermelons, and there was damage from 2005 to 2007 in Hwacheon, Gyeongbuk, Andong, Kangwon Yanggu and Jeonbuk Gochang.

Squash mosaic virus ( SqMV) is a 28-nm-diameter spherical virus belonging to the genus Comoviride and Comovirus . The gene consists of two single stranded RNAs of about 5.9 kb and 3.5 kb. It is known to be transmitted by beetle neck and seeds. In Korea, it first occurred in melon in Hadong region, Gyeongnam Province in 2007, and occurred in melon production area in Hadong and Changwon area in 2008.

Watermelon mosaic virus (WMV) is a dominant virus with a length of 750 nm, belonging to the genus Potyviridae and Potyvirus , and is known to be transmitted by aphids. The gene is composed of about 10 Kb single stranded RNA. WMV has been consistently produced in foliage and crops including watermelons, cucumbers, amber, and melons, including melons.

Cucurbit aphid-borne yellows virus ( CABYV) is a 23-nm- diameter spherical virus belonging to the genus Luteoviridae and Polerovirus . It is known to be persistent (circulated) by aphids. The gene is a single stranded RNA. There have been reports of occurrences in Europe, Africa, North America and Asia since it was first reported in France (Lecoq et al., 1999). CABYV has a wide host range such as amber, watermelon, cucumber, lettuce, and especially melon which is infected with CABYV causes symptoms such as retirement spots, mosaic, and sulphate in leaf, irregular net formation in the fruit with which there is no commerciality, It is known that the infected host does not form fruit formation. In Korea, it was first confirmed in Namwon, Gumi and Cheongyang in 2013, and it was confirmed to occur in melon cultivation areas such as Goseong and Hoengseong.

Viruses are pathogens smaller than 1 ㎛ in size and can not be observed with an optical microscope. They require an electron microscope to see them with the naked eye. And yet, there has not yet been developed a drug control method that directly affects viruses. In the past, electron microscopy and serological methods have been used for virus diagnosis. Electron microscopy can detect the presence of the virus, but it is impossible to diagnose it as a morphological feature. Among the serological methods, the enzyme-linked immunosorbent assay has a detection sensitivity about 1000 times lower than that of the most commonly used diagnostic methods or PCR methods, and fails to be diagnosed due to the unexpected nonspecific reaction of the antibody and the test sample Often occurs.

For this reason, precise and rapid diagnosis is a prerequisite for the management of viral diseases. On the other hand, reverse transcription-polymerase chain reaction (RT-PCR) method, which is commonly used for precise diagnosis in plant viruses, determines the specificity and accuracy of the diagnostic primer. In general, primers are designed to clone viruses and are not species specific. And sometimes produce nonspecific products of plants or other viruses, and thus are often unsuitable for diagnostic use. In addition, in order to diagnose various kinds of viruses in the same sample, there is a problem that many diagnosis cost and labor force are required by using various types of primers and performing reverse transcription-polymerase chain reaction respectively.

To the present invention is to solve the technical problems as described above, new in the country the black melon necrotic spot virus (Melon necrotic spot virus, MNSV), pumpkin mosaic virus (Squash mosaic virus, SqMV), watermelon mosaic virus (Watermelon mosaic virus, (WMV), and a pair of primers capable of specifically detecting Cucurbit aphid-borne yellows virus ( CABYV) were designed and subjected to a reverse-transcription-polymerase chain reaction (RT-PCR) A primer set having high sensitivity to the virus is selected and provided.

The present invention also provides a virus diagnostic method and a diagnostic kit capable of detecting the viruses individually or simultaneously using a primer composition comprising the primer set.

In order to accomplish the above object, the present invention provides a primer pair comprising a pair of primers represented by SEQ ID NO: 1 and SEQ ID NO: 2, a pair of primers represented by SEQ ID NO: 3 and SEQ ID NO: 4, a pair of primers represented by SEQ ID NO: , And Melon necrotic spot virus (MNSV), Squash mosaic virus (SqMV) comprising two or more primer pairs selected from the group consisting of primers represented by SEQ ID NO: 7 and SEQ ID NO: 8, There is provided a set of at least two melanoma virus-specific multiple RT PCR primers selected from the group consisting of Watermelon mosaic virus (WMV), and Cucurbit aphid-borne yellows virus (CABYV).

The present invention also provides a primer pair represented by SEQ ID NO: 1 and SEQ ID NO: 2, a pair of primers represented by SEQ ID NO: 3 and SEQ ID NO: 4, a pair of primers represented by SEQ ID NO: 5 and SEQ ID NO: 6, (MNSV), Squash mosaic virus (SqMV), Watermelon mosaic virus (WMV), and aphid and aphid mediators containing a pair of primers represented by SEQ ID NO: 8 Provides a set of multiple reverse transcription PCR primers for melon virus diagnosis of Cucurbit aphid-borne yellows virus (CABYV).

The inventors of the present invention collect known nucleotide sequences in the above-mentioned four species viruses and search common sequences possessed by all strains and isolates of the species. Then, Specific sequences were selected by excluding sequences from the primer design, and then sequences having optimal conditions as primers from these species-specific base sequences were designed as species-specific primers, and these primers were designed as combinations of actual reverse transcription-polymerase The present invention has been accomplished based on the final selection of a combination which is free of nonspecific reaction with a nucleic acid other than a target virus through a chain reaction (RT-PCR), and which is excellent in sensitivity to a target virus.

The primers of the present invention were developed to allow RT-PCR products to be synthesized species-specifically for the virus. In the present invention, the term "species-specific" means that the primer can synthesize a specific RT-PCR product from the genomic sequence of the virus to be diagnosed, and it is common for all the strains in the virus species to be diagnosed And nonspecific reactions with nucleic acids derived from other similar species or plants should not occur. Generally used primers are designed to amplify specific genes of viruses and are not species specific, and are sometimes unsuitable for diagnostic use because they may produce nonspecific products by the nucleic acid of plants or other viruses .

Specifically, the primer of the present invention was designed in the following manner. (a) selecting the target genome of the target virus; (b) search for common sequences in known strains of viruses and isolate genomic sequences to date; (C) excluding the same or similar sequence as the genome of a similar virus; (d) A primer was designed by selecting only a base sequence capable of functioning as a primer. A pair of primers that can be subjected to species-specific amplification by RT-PCR among the designed primers and which does not cause nonspecific reaction was selected.

In the present invention, a 'primer' is a nucleic acid sequence having a short free 3 'hydroxyl group and can form base pairs with a template of a complementary nucleic acid. Quot; means a short nucleic acid sequence which functions as a starting point. The primers can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures.

In order to distinguish the four viruses from each other by RT-PCR electrophoresis, it was necessary to distinguish the sizes of the viruses. Therefore, considering the size of amplified product, a pair of primers for detecting each virus was selected.

(152 bp) represented by SEQ ID NOS: 1 and 2 for MNSV detection, a primer pair (491 bp) represented by SEQ ID NOS: 3 and 4 for SqMV detection, and SEQ ID NOS: 5 and 6 for WMV detection (392 bp), and a pair of primers (271 bp) represented by SEQ ID NOS: 7 and 8 were used for CABYV detection.

RT-PCR was performed on a sample in which the selected primer pairs were infected with MNSV, SqMV, WMV, and CABYV alone, two kinds of compound infections, three kinds of compound infections, and four kinds of compound infections and the products were subjected to electrophoresis, Four different viruses could be distinguished by size. Therefore, it can be seen that the above primer pair can be useful for diagnosing the four viruses simultaneously or individually.

According to another embodiment of the present invention, the present invention provides at least one melon virus diagnostic kit selected from the group consisting of MNSV, SqMV, WMV, and CABYV comprising the primer set.

The present invention also provides MNSV, SqMV, WMV, and CABYV multi-diagnostic compositions comprising the primer set and a kit comprising the same.

The composition means a reagent, a thermostable DNA polymerase, a dNTP and a solution containing a divalent metal cation necessary for performing an RT-PCR amplification reaction.

The kit includes a primer pair shown in SEQ ID NO: 1 and 2, a primer pair shown in SEQ ID NO: 3 and 4, a primer pair shown in SEQ ID NO: 5 and 6, and SEQ ID NO: 7 and 8 One or more primer pairs selected from the group consisting of a pair of primers and a reagent for performing a reverse reaction and an amplification reaction. The reagent for carrying out the amplification reaction in the kit may include reverse transcriptase, DNA polymerase, dNTPs, buffer, and the like. In addition, components necessary for conducting electrophoresis to confirm whether the RT-PCR product is amplified can be further included in the kit of the present invention. Further, it may contain an RNase inhibitor, DEPC-water, sterilized water and the like if necessary.

In the present invention, 'RT-PCR kit' can be prepared by putting the RT-PCR composition into a plastic tube in a lyophilized form, and the kit further includes appropriate reagents and tools used for analysis in addition to the RT-PCR composition Such reagents and tools may include suitable carriers, labels capable of generating detectable signals, solubilizers, detergents, 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, polysaccharide, polymer such as magnetic fine particles plated with metal on latex, other paper, glass, metal, agarose, and combinations thereof.

(A) separating the viral RNA from the sample; (b) a primer pair shown in SEQ ID NO: 1 and SEQ ID NO: 2, a primer pair shown in SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 And a primer pair shown in SEQ ID NO: 7 and SEQ ID NO: 8; And (c) isolating the RT-PCR product of step (b) by size. The method comprises the steps of: (a) isolating the RT-PCR product of step (b)

Preferably, the present invention provides a method of detecting a virus, comprising: (a) separating viral RNA from a sample; (b) a primer pair shown in SEQ ID NO: 1 and SEQ ID NO: 2, a primer pair shown in SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 And a primer pair shown in SEQ ID NO: 7 and SEQ ID NO: 8; And (c) isolating the RT-PCR products of step (b) by size. The method comprises the steps of: (a) isolating the MNSV, SqMV, WMV, and CABYV.

The RNA extraction in the step (a) may be performed according to a method commonly used in the art, and may be carried out using a commercially available RNA extraction kit.

In addition, in the step (b), the cDNA strands were synthesized by reverse transcription using reverse transcriptase using the RNA isolated as the template in step (a) as the template and the reverse primer of the primer pair of the present invention as the primer Next, the PCR reaction mixture may be used. The PCR reaction may be performed using a PCR reaction mixture containing various components known in the art necessary for the PCR reaction. The PCR reaction mixture may contain a suitable amount of DNA polymerase, dNTP, PCR buffer solution and water in addition to the cDNA synthesized by the reverse transcription reaction and the primer pair provided in the present invention. The PCR buffer solution may include Tris-HCl, MgCl ₂ , KCl, and the like. In this case, MgCl ₂ concentration greatly affects the specificity and yield of amplification. In general, when Mg ²⁺ is excessive, nonspecific PCR amplification product is increased, and when Mg ²⁺ is insufficient, the yield of PCR product is decreased . The PCR buffer solution may further contain an appropriate amount of Triton X-100 (Triton X-100). Also, PCR was performed by denaturing the template DNA at 94-95 ° C, followed by denaturation; Annealing; Followed by a cycle of amplification and extension followed by final elongation at 72 ° C. Modification and amplification may be performed at 94-95 ° C and 72 ° C, respectively, and the temperature at the time of binding may vary depending on the type of the primer, but is preferably 50-57 ° C, more preferably 55 ° C . The time and number of cycles in each step can be determined according to the conditions commonly practiced in the art.

In the step (c), the DNA of the PCR product can be isolated by size according to a method well known in the art. Preferably by agarose gel or polyacrylamide gel electrophoresis or an ABI prism 3100 genetic analyzer-electropherogram. In this case, in order to use the fluorescence analyzer, PCR is performed in step (b) using a pair of primers having a fluorescent dye well known in the art.

The PCR amplification result can preferably be confirmed by agarose gel electrophoresis. After electrophoresis, electrophoresis results can be analyzed by ethidium bromide staining. Methods for performing general reverse transcription, PCR and analysis of the results are well known in the art.

The melon virus diagnostic primer set according to the present invention can diagnose MNSV, SqMV, WMV, and CABYV, as well as simultaneously diagnose four kinds of viruses at a time.

Through the primer set, it is possible to diagnose melon virus infection early, thereby preventing economic loss of the farm due to the infection.

The virus can be accurately and specifically diagnosed using the primer set according to the present invention.

The multi-diagnosis method of the present invention can reduce the cost and time required for the diagnosis of the virus, which is economical and can be effectively used for development of disease-free production of melon virus.

Figure 1 is a photograph showing the main symptoms of the MNSV virus.
Figure 2 is a photograph showing the main symptoms of SqMV virus.
Figure 3 is a photograph showing the main symptoms of WMV virus.
4 is a photograph showing the main symptoms of CABYV virus.
Fig. 5 shows the results of selecting MNSV diagnostic primers.
Fig. 6 shows the result of selecting a primer for SqMV diagnosis.
Fig. 7 shows the result of selecting a primer for WMV diagnosis.
Fig. 8 shows the result of selecting a CABYV diagnostic primer.
FIG. 9 is an electrophoresis photograph of a melon virus multiplex RT-PCR result (M: 100 bp ladder, 1: SqMV, 2: WMV, 3: CABYV, 4: MNSV, 5: SqMV + WMV, 6: SqMV + CABYV, 7: SqMV + MNSV, 8: WMV + CABYV, 9: WMV + MNSV, 10: CABYV + MNSV, 11: SqMV + WMV + CABYV, 12: SqMV + WMV + MNSV, 13: SqMV + CABYV + WMV + CABYV + MNSV, 15: SqMV + WMV + CABYV + MNSV).

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Example 1: Establishment of melon virus assay system

The diagnostic primers for MNSV, SqMV, WMV, and CABYV, newly identified viruses in domestic melons, were designed with reference to previously reported nucleotide sequences. Only the primers that specifically reacted with the target virus were selected .

1-1. Selection of MNSV diagnostic primer

MNSV diagnostic primers were designed with four pairs of size 138 bp, 217 bp, 172 bp and 152 bp for RNA-dependent RNA polymerase (RdRp), movement protein (MP) and coat protein Respectively. Among them, 3 pairs except 138 bp were selected as specific primers for MNSV diagnosis. The primers used in the following Table 2 are shown, and RT-PCR results are shown in FIG.

Name of the primer Locus The sequence (5 '- > 3') Expected size MNS-N55 2,987-3,010 GAGGCAACATTTCGTACACTCAGG 138 bp MNS-C06 3,124-3,104 TAAACTCCCTATGAACTATCT MNS-N53 2,908-2,930 GCTATAGATGTGGTTCCTTTGGT 217 bp MNS-C06 3,124-3,104 TAAACTCCCTATGAACTATCT MNS-N40 2,505-2,524 AGCGGGGGAAAACAGAAGAA 172 bp MNS-C20 2,676-2,659 CCCCGGGGCTGGAGTCAC MNS-N30 1,607-1,628 CCGACGCTCTTAAATGGGAACA 152 bp MNS-C40 1,758-1,736 CATCCGGCACCCATGAACCTTAT

Lane Sample No. Primer used One One MNS-N55 MNS-C06 2 MNS-N53 MNS-C06 3 MNS-N40 MNS-C20 4 MNS-N30 MNS-C40

1-2. SqMV diagnostic primer selection

SqMV diagnostic primers were designed for two pairs of size 628 bp and 491 bp for coat protein (CP) and are shown in Table 3. As a result of RT-PCR, SqMV was selected as a specific primer for SqMV diagnosis. The results are shown in Table 4 and FIG. 6.

Name of the primer location The sequence (5 '- > 3') Expected size SqMV 1F-10 120-138 ACGGCCCTGTGGTAGATTG 628 bp SqMV 1R-10 748-727 TAATAATACTCTCCACTGTCCA SqMV 1F 2388-2405 TGGAGAGTTTYCCCCACAAG 491 bp SqMV 1R 2878-2859 TTCTTCCAAGCAGCCACTTT

Lane Sample No. Primer used One One SqMV 1F-10 SqMV 1R-10 2 SqMV 1F SqMV 1R

1-3. WMV diagnostic primer selection

The WMV diagnostic primer was designed to be a universal primer capable of diagnosing both WMV 2 strains reported in Korea. Nuclear inclusion protein b (NIb) and coat protein (CP) were measured in 392 bp, 717 bp and 580 bp 3 pairs were designed, which are shown in Table 5. As a result of the RT-PCR test, the specimens infected with each of the WMV strains were selected as specific primers for WMV diagnosis. The results are shown in Table 6 and FIG.

Name of the primer location The sequence (5 '- > 3') Expected size WMV-UNI-1F 9246-9268 CAGTTTGAATCATGGTACAGCGC 392 bp WMV-UNI-1R 9637-9614 TGTGCTATTGCTTCTCTTGCCC WMV-UNI-2F 8859-8881 ACACAACMAAGTGAATTGCAAAR 717 bp WMV-UNI-2R 9575-9554 TAGCGTGCTAATTCCCTGTCTC WMV-UNI-3F 7822-7844 GAGCGCYTGTTGTAYCTTAGCTG 580 bp WMV-UNI-3R 8401-8381 ACCAACCTTGTTTACAGCACG

Lane Sample No. Primer used One 1 (K1 system) WMV-UNI-1F WMV-UNI-1R 2 2 (K2 system) 3 1 (K1 system) WMV-UNI-2F WMV-UNI-2R 4 2 (K2 system) 5 1 (K1 system) WMV-UNI-3F WMV-UNI-3R 6 2 (K2 system)

1-4. Selection of CABYV diagnostic primer

The CABYV diagnostic primers were designed for 12 pairs of coat proteins (CP), 272 to 746 bp in size, as shown in Table 7. RT-PCR was performed on CABYV single infected specimens. As a result, a specific primer for CABYV diagnosis was selected and the results are shown in Table 8 and FIG.

Name of the primer location The sequence (5 '- > 3')  Forward primer CABYV-u1 3060-3085 GTCGAGGAGTCCTCAAAACTGGAATT CABYV-u2 3180-3127 GAGGACCTCGCCATTCCGGT CABYV-u3 3312-3338 GGGGAGTATAAAGAACACTAGCCAAGC CABYV-u4 3341-3364 ACACGAGTTGCAAGCATTGGAAGT CABYV-F 3405-3432 TTTCTCGCAGGGTTTTCWAGCGGTCTAT CABYV u3535 3535-3560 GCTAGAAATCAAAATGCAGGGAGGCG  Reverse primer CABYV-R 3732-3708 ACTGCCCGTGAGATTGTCCTTTGAA CABYV d3806 3806-3783 AGTATTCCAGAGCTGAATGCTGGG

Lane Primer used Expected size One CABYV-u1 CABYV-R
673 bp 2 CABYV-u2 553 bp 3 CABYV-u3 421 bp 4 CABYV-u4 392 bp 5 CABYV-F 328 bp 6 CABYV u3535 197 bp 7 CABYV-u1 CABYV d3806 746 bp 8 CABYV-u2 626 bp 9 CABYV-u3 495 bp 10 CABYV-u4 466 bp 11 CABYV-F 402 bp 12 CABYV u3535 272 bp

Example 2: Development of multiple diagnostic methods for four viruses in melon

Among the specific diagnostic primers designed for melon viruses MNSV, SqMV, WMV, and CABYV distributed in Korea, multiple diagnostic primers as shown in Table 9 below, which can simultaneously diagnose four viruses by one RT-PCR, We have developed a multiplex RT-PCR method that can diagnose melon virus MNSV, SqMV, WMV, and CABYV at once.

virus Name of the primer The sequence (5 '- > 3') location Expected size density SqMV SqMV 1F TGGAGAGTTTYCCCCACAAG 2388-2405 491bp 40 pmol SqMV 1R TTCTTCCAAGCAGCCACTTT 2878-2859 WMV WMV-UNI-1F CAGTTTGAATCATGGTACAGCGC 9246-9268 392 bp 40 pmol WMV-UNI-1R TGTGCTATTGCTTCTCTTGCCC 9637-9614 CABYV CABYV u3535 GCTAGAAATCAAAATGCAGGGAGGCG 3535-3560 271 bp 60 pmol CABYV d3806 AGTATTCCAGAGCTGAATGCTGGG 3806-3783 MNSV MNS N30 CCGACGCTCTTAAATGGGAACA 1607-1628 152bp 40 pmol MNS C40 CATCCGGCACCCATGAACCTTAT 1758-1736

For the diagnosis of MNSV, SqMV, WMV, and CABYV, primers selected from the above were used. Melon samples were obtained by crushing diseased leaves with liquid nitrogen and then extracting whole genomes using a total RNA extraction kit (Intron Co.).

(RT), 0.5 microliters of a mixture of 40 pmol each of the genomic template and 3'-primer, 0.5 microliters of 2.5 mM dNTPs, 0.1 microliter of 10 mg / ml BSA, 0.2 microliters of 40 U / ul RNase inhibitor, 5x RT buffer, AMV reverse transcriptase, and reacted with the final 5 μl reaction solution at 42 ° C for 30 minutes. Using the cDNA produced in the process, the 5'-primer 40 pmol each of a mixture of 0.5 microliters, 10mg / ml BSA 0.1 microliter, 2.5 mM dNTPs 1.5 _{microliters, 25mM MgCl 2 2.5ul, 5X PCR} buffer, Taq polymerase into the A final 25 microliter reaction mixture was prepared and PCR was performed.

The DNA was subjected to initial denaturation at 95 ° C for 3 minutes, followed by 35 cycles of 95 ° C for 30 seconds, denaturation at 55 ° C for 30 seconds for DNA primer annealing, 1 minute at 72 ° C for DNA chain extension, , And the products amplified by MNSV, SqMV, WMV, and CABYV sequences were subjected to 1.2% agarose gel electrophoresis at 100 mV for 1 hour 30 minutes under conditions of 72 ° C for 10 minutes for DNA chain extension.

The results are shown in Fig. Both the single and complex infections showed the correct response and the size of the amplified sequences was also markedly different.

As a result of the nucleotide sequence analysis of the RT-PCR product, the nucleotide sequence was 95 to 99% homologous with the nucleotide sequence registered in NCBI, and the results are shown in Table 10 below.

object
virus primer Product size The base sequence (5 '- > 3') Comparable / homologous MNSV MNS N30 / MNS C40 152bp CCGACT AB250687 / 99% SqMV SqMV 1F / SqMV 1R 491bp TGGAGAGTTTTCCCCACAAGCTCGTGCAATTCGCTGAAATTCAGGGGCGTACCACTATAACGTTCACGCAAGGCGAGTTTTTGACGGCATGGTCCACACAaGTATTAAGCACTGTTAATCCTCAGAAAGATGGGTGTCCCCACTTGTATGCACTTTTGCATGACTCTGCTACTTCAACCATTGAAGGAAATTTTGTCATTGGTGTTAAATTGCTGGACATTAGAAATTATCGTGCTTATGGTCACAACCCCGGTTTTGAGGGAGCTCGCTTGCTAGGAATTTCTGGGCAGAGTACCATGGTACAGCAGCTTGGAACTTATAATCCAATTTGGATGGTTCGTACGCCTCTAGAAAGCACAGCCCAACAAAATTTTGCGAGTTTCACTGCTGATTTGATGGAATCCACGATAAGTGGGGACTCTACTGGGAACTGGAATATCACAGTTTATCCTAGCCCTATAGCTAATTTGCTGAAAGTGGCTGCTTGGAAGAA AB054689 / 95% WMV WMV-UNI-1F
/ WMV-UNI-1R
392 bp
GTTTGAAATCATGGTACAGCGCAGTTAAAGTTGAtATgATGAtCTTaATGATGAGCaAATGGGTGTGATTATGAATGGTTTTATGGTTTGGTGCATCGATAACGGTACATCTCCAGATGTTAATGGAGTGTGGGTGATGATGGATGGGGAAGAGCAAGTTGAGTACCCACTAAAGCCAATTGTTGAAAATGCAAAGCCAACTTTGAGACAAATCATGCACCATTTCTCAGACGCAGCAGAAGCGTATATTGAAATGAGAAACTCTGAAAGTCCGTATATGCCTAGATACGGATTACTAAGAAATTTGAGAGACAGGGAATTAGCACGCTATGCTTtCGACTTCTaTGAAGTTACTTCCAAAACACCAAATAGGGCAAGAGAACAAATAGCACA JF273469 / 98% CABYV CABYV u3535 / CABYV d3806 271 bp CTAGAAATCCAAAATGCAGGGAGGCGGAGGCGAAGAAATCAGCGCTCTACACGGCGCGACCGCGTGGTTGTGGTCAACCCCTCTGGGGGACCATCGcCGCGGAAGACGACAACGAAGAAACCGCCGACGCCCTAATAGAGGAGGCAGAGCTAGAGGAAGAAGCTCAGGCGAAACATTTGTATTTTCAAAGGACAATCTCACGGGCAGTtCCTCAGGAAGTATCACcTTCggGccGtCtCTTTCAgAGaGCCCAGCATTCAGCTC KF427704 / 99%

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Primer set for multiple detection MNSV, SqMV, WMV, and CABYV and          method for detecting said viruses using the same <130> 2014-0421-10-A, P14-349 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MNS N30 <400> 1 ccgacgctct taaatgggaa ca 22 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> MNS C40 <400> 2 catccggcac ccatgaacct tat 23 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SqMV 1F <400> 3 tggagagttt yccccacaag 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SqMV 1R <400> 4 ttcttccaag cagccacttt 20 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> WMV-UNI-1F <400> 5 cagtttgaat catggtacaga cgc 23 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> WMV-UNI-1R <400> 6 tgtgctattg cttctcttgc cc 22 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> CABYV u3535 <400> 7 gctagaaatc aaaatgcagg gaggcg 26 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CABYV d3806 <400> 8 agtattccag agctgaatgc tggg 24

Claims (6)

A pair of primers represented by SEQ ID NO: 1 and SEQ ID NO: 2, a pair of primers represented by SEQ ID NO: 3 and SEQ ID NO: 4, a pair of primers represented by SEQ ID NO: 5 and SEQ ID NO: 6, and a pair of SEQ ID NO: (MNSV), Squash mosaic virus (SqMV), Watermelon mosaic virus (WMV), and the like, which comprise two or more primer pairs selected from the group consisting of primers And a set of multiple RT PCR primers for the detection of two or more melon viruses selected from the group consisting of Cucurbit aphid-borne yellows virus (CABYV). A pair of primers represented by SEQ ID NO: 1 and SEQ ID NO: 2, a pair of primers represented by SEQ ID NO: 3 and SEQ ID NO: 4, a pair of primers represented by SEQ ID NO: 5 and SEQ ID NO: 6, and a pair of SEQ ID NO: (MNSV), Squash mosaic virus (SqMV), Watermelon mosaic virus (WMV), and aphid mediated sulphidation virus (Cucurbit aphid-borne yellows virus, CABYV) for melanoma virus diagnosis. At least one melanoma virus diagnostic kit selected from the group consisting of MNSV, SqMV, WMV, and CABYV comprising the primer set of claim 1 or 2. (MNSV), Squash mosaic virus (SqMV), Watermelon mosaic virus (WMV), and the like, including primers represented by the nucleotide sequences of SEQ ID NOS: 1 to 8, And a diagnostic composition for the diagnosis of Cucurbit aphid-borne yellows virus (CABYV). A kit comprising the composition of claim 4. (a) separating the viral RNA from the sample;
(b) using the RNA of step (a) as a template,
A pair of primers represented by SEQ ID NO: 1 and SEQ ID NO: 2, a pair of primers represented by SEQ ID NO: 3 and SEQ ID NO: 4, a pair of primers represented by SEQ ID NO: 5 and SEQ ID NO: 6, and a pair of SEQ ID NO: Performing RT-PCR using a primer pair that is a primer pair; And
(c) a step of isolating the RT-PCR products of step (b) by size, the method comprising the steps of: (a) isolating the RTV-PCR product of step (b)

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