KR101250388B1 - Specific primers for multiple detection of the viruses in soybean, and uses thereof - Google Patents

Abstract

The invention as species-specific primers that can be simultaneously diagnosed viruses occurring in soybean and on the development of multiple diagnostics, and more particularly, alfalfa mosaic virus (Alfalfa mosaic virus, AMV), soybean sulfide stain mosaic virus (Soybean yellow mottle mosaic virus (SYMMV), unreported new virus (USV3), Soybean yellow common mosaic virus (SYCMV), Peanut stunt virus (PSV), Soybean dwarf virus (SbDV) And a primer for diagnosing Soybean mosaic virus (SMV) and a combination thereof, and a use thereof. When using the primer set of the present invention, there is a diagnostic cost and effort reduction effect due to the spread of diagnostic methods for a number of viruses occurring in soybeans, thereby solving problems occurring in agricultural fields due to soybean virus disease, Can contribute to bean varieties breeding.

Description

Specific primers for multiple detection of the viruses in soybean, and uses approximately}

The invention as species-specific primers that can be simultaneously diagnosed viruses occurring in soybean and on the development of multiple diagnostics, and more particularly, alfalfa mosaic virus (Alfalfa mosaic virus, AMV), soybean sulfide stain mosaic virus (Soybean yellow mottle mosaic virus (SYMMV), unreported new virus (USV3), Soybean yellow common mosaic virus (SYCMV), Peanut stunt virus (PSV), Soybean dwarf virus (SbDV) ) and soybean mosaic virus (soybean mosaic virus, SMV) multiple diagnostic primer consisting of a primer that can be diagnosed with a combination thereof and to a use thereof.

In the past, electron microscopy and serological methods (ELISA) were mainly used for the diagnosis of viruses. Electron microscopy can confirm the presence of the virus, but its morphological features make it impossible to diagnose the species. Among the serological methods, enzyme linked immunosorbent assay (ELISA) is about 1000 times lower in sensitivity than the most commonly used diagnostic or PCR assays, and the unexpected nonspecificity of antibodies and test samples is unexpected. Often, the reaction fails to diagnose.

Therefore, in the current diagnosis of viruses, the PCR method having high detection sensitivity and convenience is most commonly used. An important point in the development of primers for the PCR diagnosis of pathogens is that they should be able to detect all lines and isolates present in the first viral species. To this end, primers should be designed for nucleotide sequences common to all strains and isolates of the viral species to be detected. Second, there must be specificity between different species. For this purpose, a primer should be designed that does not react to the nucleotide sequence of other viruses that are flexible to the virus species to be detected.

However, in the case of such primers, there is a problem in diagnosing crops infected with a large number of viruses since they are generally primers capable of only partial diagnosis of individual viruses.

Beans, and a number of viruses, as a representative of them, soybean mosaic virus (Soybean mosaic virus, SMV), alfalfa mosaic virus (Alfalfa mosaic virus, AMV), soybean atrophy virus (Soybean dwarf virus, SbDV), soybean sulfide stain Soybean yellow mottle mosaic virus (SYMMV) and Peanut stunt virus (PSV). However, no combination of primers capable of multiple diagnosis of the virus has been reported.

The inventors of the present invention while trying to study the primers for multiple diagnosis of viral diseases that occur in soybeans, representative virus, alfalfa mosaic virus generated from soybean (Alfalfa mosaic virus, AMV), soybean sulfide stain mosaic virus (Soybean yellow mottle mosaic virus (SYMMV) and primers and Peanut stunt virus (PSV), soybean dwarf virus (SbDV) for simultaneous diagnosis of unreported new virus (USV3). And the soybean mosaic virus ( Soybean mosaic virus , SMV) to complete the present invention for a primer set that can be further diagnosed at the same time.

Therefore, an object of the present invention, soybean virus disease multiple diagnostic primer set, soybean virus disease diagnostic method using the same and to provide a kit comprising the primer set.

In order to achieve the above object, the present invention provides a primer pair represented by SEQ ID NO: 1 and SEQ ID NO: 2, a primer pair represented by SEQ ID NO: 3 and SEQ ID NO: 4 and a primer pair represented by SEQ ID NO: 5 and SEQ ID NO: 6 It provides a soybean virus disease multiple diagnostic primer set capable of simultaneous diagnosis of AMV, SYMMV and USV3 containing.

In order to achieve another object of the present invention, the present invention provides a primer pair represented by SEQ ID NO: 7 and SEQ ID NO: 8, a primer pair represented by SEQ ID NO: 9 and SEQ ID NO: 10 and SEQ ID NO: 11 and SEQ ID NO: 12 Provided is a soybean virus disease multiple diagnostic primer set further comprising a primer combination capable of simultaneous diagnosis of PSV, SbDV and SMV comprising the indicated primer pairs.

In order to achieve another object of the present invention, the present invention comprises the steps of analyzing the total RNA of the virus from the soybean samples showing viral symptoms; Performing RT-PCR using the primer set of claim 1 as the RNA template; And it provides a method for diagnosing soybean virus disease by detecting the PCR product.

In order to achieve another object of the present invention, the present invention provides a kit for diagnosing multiple beans virus diseases comprising the primer set.

Hereinafter, the present invention will be described in detail.

The present invention relates to multiple diagnostic primer sets of AMV, SYMMV and USV3 comprising primer pairs selected from the group consisting of SEQ ID NOs: 1-12.

That is, the present invention includes AMV, SYMMV, and a primer pair represented by SEQ ID NO: 1 and SEQ ID NO: 2, primer pairs represented by SEQ ID NO: 3 and SEQ ID NO: 4 and primer pairs represented by SEQ ID NO: 5 and SEQ ID NO: 6 and Provided is a primer set (hereinafter referred to as primer combination I) capable of simultaneous diagnosis of USV3.

In addition, the present invention includes the primer set and the primer pair represented by SEQ ID NO: 7 and SEQ ID NO: 8, the primer pair represented by SEQ ID NO: 9 and SEQ ID NO: 10 and the primer pair represented by SEQ ID NO: 11 and SEQ ID NO: 12 Provided is a primer set for multiple diagnosis of soybean virus disease, further comprising a primer set (hereinafter, referred to as primer combination II) capable of simultaneous diagnosis of PSV, SbDV, and SMV.

Viruses that can be diagnosed using the primer set of the present invention are soybean mosaic virus (Soybean mosaic virus, SMV), alfalfa mosaic virus (Alfalfa mosaic virus, AMV), soybean atrophy virus (Soybean dwarf virus, SbDV), soybean sulfide stain Soybean yellow mottle mosaic virus (SYMMV), Peanut stunt virus (PSV) and unreported new virus (USV3, soybean yellow common mosaic virus, SYCMV).

The primer set of the present invention includes primer combination I for simultaneous diagnosis of AMV, SYMMV and USV3 in the virus, and may further include primer combination II for simultaneous diagnosis of PSV, SbDV and SMV.

As a primer combination constituting the simultaneous diagnostic primer set, primer combination I is a primer pair represented by SEQ ID NO: 1 and SEQ ID NO: 2 for detection of AMV virus, a primer represented by SEQ ID NO: 3 and SEQ ID NO: 4 for SYMMV virus detection Pairs, a primer pair represented by SEQ ID NO: 5 and SEQ ID NO: 6 for USV3 virus detection. In addition, primer combination II is a primer pair represented by SEQ ID NO: 7 and 8 for the detection of PSV virus, SEQ ID NO: 9 for the detection of SbDV virus and primer pair represented by SEQ ID NO: 10, SEQ ID NO: 11 for the detection of SMV virus And a primer pair represented by SEQ ID NO: 12.

The primer set of the present invention was designed for species-specific primers for the virus through the isolation and analysis of genetic information for the six viruses occurring in soybean. Since the virus diagnostic primer should act species-specifically, RT-PCR for each virus is performed using the designed primers to select species-specific sequences except for common nucleotide sequences of all strains and isolates. It was. From these species specific sequences, sequences with the optimal conditions for the primers were finally selected.

Through the above procedure, primer combination I for simultaneous diagnosis of AMV, SYMMV and USV3 of the present invention and primer combination II for simultaneous diagnosis of PSV, SbDV and SMV were selected, respectively. Finally, the selected primer combination does not react with nucleic acids other than the virus of interest, and is highly sensitive to the virus of interest.

In order to confirm the simultaneous diagnosis of the primer set of the present invention, it was confirmed whether the simultaneous diagnosis of AMV, SYMMV, USV3, AMV + SYMMV + USV3. In addition, it was confirmed whether the simultaneous diagnosis of PSV, SbDV, SMV, PSV + SbDV + SMV. As a result, it was confirmed that the primer set of the present invention can diagnose not only each virus but also simultaneously diagnose a plurality of viruses.

The present invention also relates to a method for diagnosing soybean virus disease using the primer set and soybean virus disease diagnosis kit.

More specifically, the present invention,

(a) separating the total RNA of the virus from the sample;

(b) performing RT-PCR using the primer set as the RNA template; And

(c) provides a method for diagnosing soybean virus comprising the step of separating the PCR product by size.

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

In the step (b), RT-PCR uses the isolated RNA as a template, a reverse primer among the primer pairs of the present invention as a primer, and synthesizes a complementary DNA strand through reverse transcription using a reverse transcriptase, followed by a PCR reaction. Can be performed. The PCR reaction may be carried out using a PCR reaction mixture containing various components known in the art required for the PCR reaction. The PCR reaction mixture contains an appropriate amount of DNA polymerase, dNTP, PCR buffer and water (dH ₂ O) in addition to the complementary DNA synthesized by reverse transcription and the PCR primer set provided in the present invention. The PCR buffer comprises Tris -HCl (Tris-HCl), MgCl 2, KCl and the like. The primer set of the present invention may be a primer set including the primer combination I or the primer combinations I and II simultaneously. In addition, the concentration of MgCl ₂ significantly affects the specificity and quantity of amplification. In general, when Mg ²⁺ is excessive, nonspecific PCR amplification products are increased, and when Mg ²⁺ is insufficient, the yield of PCR products is decreased. The PCR buffer may further include an appropriate amount of Triton X-100. PCR also denatured the template DNA at 94-95 ° C., followed by denaturation; Annealing; And a cycle of extension, followed by general PCR reaction conditions which are finally elongated at 72 ° C. The denaturation and amplification in the above may be carried out at 94 to 95 ℃ and 72 ℃, respectively, the temperature at the time of binding may vary depending on the type of primer, preferably 52 to 57 ℃, more preferably 55 ℃ . The time and number of cycles in each step can be determined according to the conditions generally made in the art.

In the step (c), the PCR products may be separated according to a method widely known in the art. Preferably it can be confirmed by an agarose gel (agarose gel) or polyacrylamide gel electrophoresis or fluorescence analysis (ABI prism 3100 genetic analyzer-electropherogram). At this time, in order to use the fluorescence analyzer PCR is performed in the step (b) using a primer pair attached to a fluorescent die known in the art. PCR amplification results can be preferably confirmed by agarose gel electrophoresis. After electrophoresis, the result of electrophoresis can be analyzed by ethidium bromide staining. General reverse transcription reactions, PCR performance and the resulting analysis methods are well known in the art.

In addition, the present invention relates to a bean virus disease multiple diagnostic kit comprising the primer set.

The primer set of the present invention may be a primer set including the primer combination I or the primer combinations I and II simultaneously.

In addition, but not limited thereto, the kit may further include a DNA polymerase and a buffer solution of the above-described composition in order to easily perform RT-PCR in addition to the primer set. Components necessary for performing electrophoresis capable of confirming amplification may be additionally included in the kit of the present invention.

Meanwhile, standard recombinant DNA and molecular cloning techniques used in the present invention are well known in the art and described in the following literature (Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual) , 2nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1989); by Silhavy, TJ, Bennan, ML and Enquist, LW, Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1984) and by Ausubel, FM et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-lnterscience (1987)).

Bar Reviewing the above, when using soybean multiple diagnostic primer set of the present invention, the alfalfa mosaic virus (Alfalfa mosaic virus, AMV), soybean sulfide stain mosaic virus (Soybean yellow mottle mosaic virus, SYMMV) and not reported new virus (USV3 , Tentatively: three viruses including Soybean yellow common mosaic virus (SYCMV), or Soybean mosaic virus (SMV), Soybean dwarf virus (SbDV), peanut atrophy virus ( Peanut stunt virus , PSV) can be diagnosed at the same time, including six additional viruses, thereby reducing the diagnostic cost and effort due to the spread of the diagnostic method for the virus, from which the soybean virus disease caused in the agricultural field Can solve problems and contribute to breeding high quality bean varieties.

Figure 1 shows the results of analyzing the species specific response of the SYMMV diagnostic primer.
Figure 2 shows the results of analyzing the RT-PCR response of SYMMV species specific primers and soybean-related virus.
Figure 3 shows the location of the final selected SYMMV primers.
Figure 4 shows the results of analyzing the species-specific response of the USV3 diagnostic primer.
Figure 5 shows the results of analyzing the RT-PCR reaction of USV3 species specific primers and soybean-related virus.
Figure 6 shows the location of the final selected USV3 primer.
Figure 7 shows the results of analyzing the species-specific response of the AMV diagnostic primer.
Figure 8 shows the results of analyzing the RT-PCR response of AMV species specific primers and soybean-related virus.
9 shows the location of the final selected AMV primers.
Figure 10 shows the results of analyzing the species specific response of the primer for PSV diagnostics.
Figure 11 shows the results of analyzing the RT-PCR response of PSV species specific primers and soybean-related virus.
12 shows the location of the final selected PSV primers.
Figure 13 shows the results of analyzing the species-specific response of the SMV diagnostic primers.
Figure 14 shows the results of analyzing the RT-PCR response of SMV species specific primers and soybean-related virus.
15 shows the location of the final selected SMV primers.
Figure 16 shows the results of analyzing the species specific response of the SbDV diagnostic primer.
Figure 17 shows the results of analyzing the RT-PCR reaction of SbDV species specific primers and soybean-related virus.
18 shows the location of the final selected SbDV primers.
Figure 19 shows the results of selection by RT-PCR of primer combination I for simultaneous diagnosis of AMV, SYMMV and USV3.
20 shows the results of selection by RT-PCR of primer combination I for simultaneous diagnosis of PSV, SbDV and SMV.
Figure 21 shows the packaging sample application test results using the selected primer combination.
Figure 22 shows the multi-diagnosis results of the six types of viruses occurring in domestic soybean, each lane is as follows; Lane 1: AMV, Lane 2: SYMMV, Lane 3: USV3, Lane 4: AMV + SYMMV + USV3, Lane 5: PSV, Lane 6: SbDV, Lane 7: SMV, Lane 8: PSV + SbDV + SMV.
Figure 23 shows the results of analyzing the detection limit of the multi-diagnostic primer combination, each lane is as follows; Lane 1, 7: Positive control (total RNA stock), Lane 2, 8: Negative control (distilled water), Lane 3, 9: Total RNA 10 ¹ dilution, Lane 4, 10: Total RNA 10 ² Dilution, Lane 5, 10: Total RNA 10 ³ fold Dilution, Lane 6, 12: Total RNA 10 ⁴ fold Dilution.

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 1 >

Design and Selection of Primer for Soybean Virus Diagnosis

Through the following experimental method, diagnostic primers for six soybean viruses were designed. Total RNA isolation and RT-PCR methods used in the present invention are as follows.

(1) Total RNA Isolation

Total RNA was isolated from 50 mg of virus infected leaf tissues using Solgent's RNA Mini prep kit (Cat.No. SRP31-C100) and finally dissolved in 50 μl of distilled water and used for RT-PCR analysis.

(2) RT-PCR

Reverse transcription reaction was performed with 1 μl total RNA isolated, 10 pmole downstream primer, 5-fold reverse transcription buffer (250 mM Tris-HCl, 150 mM KCl, 40 mM MgCl ₂ , 5 mM DTT, pH 8.5). ) 5 μl of the reaction solution containing 1 μl, 10 mM dNTP 0.5 μl, 10 U RNase inhibitor, and 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 transcriptase and 12.5 pmole upstream primer, 1.25 U Taq DNA polymerase, 10-fold polymerase chain reaction buffer (100 mM Tris-HCl, 500 mM KCl, 15). 2 μl of 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 35 times at 94 ° C 45 seconds, 55 ° C 60 seconds, 72 ° C 60 seconds and finally treated at 72 ° C for 5 minutes. RT-PCR products were electrophoresed using 0.5 × TBE buffer and 1.5% agarose gel, stained with ethidium bromide, and confirmed by ultraviolet lamp.

<1-1> Design and Selection of SYMMV Diagnostic Primer

SYMMV sequence search designed 45 primer combinations based on the entire sequence. As shown in Figure 1, using the selected primer combination to perform the target virus and RT-PCR primarily to select 38 primer combinations, and secondly with five related viruses except for the target virus as shown in FIG. RT-PCR was performed. Of these species-specific and nonspecific sequences, 15 diagnostic primers satisfying primer conditions were designed as shown in Table 1 below. The position of the SYMMV primer is shown in FIG. 3.

Finalized SYMMV Diagnostic Primer primer Base sequence Length location ^a SYMM-F01 TATCCACCATTTAATCTTTCAGG 23 17-39 SYMM-F16 GAGGATGCAGAATGAGTGGTGACA 23 1574-1597 SYMM-F19 TAAGCCTACAGTCAGTCTATCAAA 24 1863-1886 SYMM-F23 CCAAAGGGGACGTAGTAGAAGC 22 2294 ~ 2315 SYMM-F GCCAGAGAACAAGCAGATTCA 21 2351 ~ 2371 SYMM-F29 ACAAAACAAACCGGTCATTCAGC 23 2876 ~ 2898 SYMM-F34 CAACCCTCAGCCACATTCAACTAT 24 3365 ~ 3388 SYMM-R05 TAACGGGTCATTCATCAACTTCAT 24 544-567 SYMM-R09 AACACCCTCTCGATCAAACCTCTC 24 897-920 SYMM-R GATCACCGCCAGGTGGTTTA 20 2405 ~ 2424 SYMM-C30 CAGCGGGGACTCTTCGTGTTGAC 23 2807 ~ 2829 SYMM-R30 CCTGTGGAGAGCGGTTGCA 19 2999 ~ 3016 SYMM-C20 CGCCAAGACAATCGATCCCGTAGT 24 3704-3101 SYMM-R35 GCCAGCAGTCGAAAATGTGAGAAC 24 3480 ~ 3503 SYMM-C10 ATCGCGCACAACATAAACATCC 22 3680 ~ 3701

^a Primer position based on FJ457015

<1-2> Design and Selection of USV3 Diagnostic Primers

USV3 is a virus that has not been reported worldwide. USV3, which was used for species-specific primer selection, was used as RT-PCR template by separating total RNA from leaves showing mosaic symptoms from naturally infected soybeans. Total sequencing and RT-PCR were used in the same manner as in <1-1>.

As a result, as shown in FIG. 4, 31 primer pairs were designed based on the entire nucleotide sequence. Thirty primer pairs were selected by first performing RT-PCR with the target virus using the selected primer pairs.

In addition, RT-PCR was performed with five related viruses except for the target virus.

As a result, as shown in Figure 6, among these species-specific and non-specific nucleotide sequences 19 diagnostic primers of Table 2 to satisfy the primer conditions were designed.

Finalized USV3 Diagnostic Primer primer Base sequence Length location USV3-N01 NACAAAATATAAGAACAAAGAGTC 24 1-23 USV3-N04 GGAGTTGGTGCCTGGGTAT 19 386-404 USV3-N16 GTTGAGATCGTAGGGAGAGGTAAGG 25 1574 ~ 1598 USV3-N23 CAGTTTGTTGCTCTTTGTGTTA 22 2259-2280 USV3-N28 AGCAGCCTGGAATAATGAAGTC 22 2795 ~ 2816 USV3-N33 TAGCCAAGGCCATAGCGAACACT 23 3255 ~ 3277 USV3-C03 ACACCGGGCAGTCTATCAATCTAA 24 268-291 USV3-C04 CTCGGCAGTTGATGGATAGC 20 332-351 USV3-C05 GACCAAGAGCGTGTACGCAA 20 542-561 USV3-C08 TGGGCTACATAATACTTCCGTTCCT 25 744-768 USV3-C12 AGCCACGAAATCGAGCATCTG 21 1160-1180 USV3-C24 TCGACCATTTCAACGGGAGTAA 22 2337-2358 USV3-C27 CTCCAAAGCCGCCCAGAACTATT 23 2685-2707 USV3-C31 AGTACTTGAACAGGGTCTTAGG 22 3073-3094 USV3-C35 ACGGCTCACGAAGACGCACCATAGT 25 3398 ~ 3422 USV3-C38 TGATGAAACACAAGCCGGACGAACC 25 3737 ~ 3761 USV3-C42 CATTTGGATTACGCTCCATTTCTA 24 4128 ~ 4151

<1-3> Design and Selection of AMV Diagnostic Primer

The AMV used for the species-specific primer selection was used as RT-PCR template by separating the whole RNA by extracting leaves showing mosaic symptoms from naturally infected soybeans. AMV sequence search designed 16 primer pairs based on the entire sequence.

Using the primer pair thus selected, 13 primer pairs were selected by performing RT-PCR with the target virus as shown in FIG. 7. In addition, RT-PCR was performed with five related viruses except for the target virus as shown in FIG. 8. Among these species-specific and nonspecific sequences, four diagnostic primers having the position of FIG. 9 and satisfying the primer conditions as shown in Table 3 were designed.

Final Selected AMV Diagnostic Primer primer Base sequence Length location ^a AM-N10 TCGTGAGTAAGTTGCAAATGGAGA 24 241-264 AM-N20 AGGAAATGTTACTAGCTGATGAAG 24 331-354 AM-C50 GGTAGCATCATGGTCGGTGTCAAC 24 627-650 AM-C40 CTTTCGAGGATCTGCTTTCTGCT 23 804-826

<1-4> Design and Selection of PSV Diagnostic Primer

PSV used for species-specific primer selection was used as RT-PCR template by separating the total RNA from the leaves showing mosaic symptoms from the naturally infected soybean. PSV sequence search designed 20 primer pairs based on the entire sequence.

Using the primer pair thus selected, 12 primer pairs were selected by performing RT-PCR with the target virus as shown in FIG. 10. In addition, RT-PCR was performed with five related viruses except for the target virus as shown in FIG. 11. Among these species-specific and nonspecific sequences, seven primers for satisfying the primer conditions and having the position of FIG. 12 were designed as shown in Table 4.

PSV Diagnostic Primer primer Base sequence Length location ^a PSV-N20 CGTGTTGTCGTGTTGGTGA 19 84-102 PSV-N30 CCTCCCTGCTCTGGTATCGTTC 22 512 ~ 533 PSV-N50 CTGGCGTGTTGGAAGGCTAAGG 22 1051-1072 PSV-C50 GAGCAGGGAGGTCTCGGTTA 20 503-522 PSV-C40 AGGTTGCTTTCTATTGCGATTTA 23 871 ~ 893 PSV-C30 CGCCAGCATCATCGTAACAC 20 1037-1056 PSV-C20 AAACACAATACAAACAAATACGAT 24 1185-1208

<1-5> Design and Selection of SMV Diagnostic Primer

The SMV used for species-specific primer selection was used as RT-PCR template by separating the whole RNA by extracting leaves showing mosaic symptoms from naturally infected soybeans. SMV sequence search designed 9 primer pairs based on the entire sequence.

Using the primer pairs selected as described above, nine primer pairs were selected by performing RT-PCR with the target virus as shown in FIG. 13, and with five related viruses except the target virus as shown in FIG. RT-PCR was performed.

Of these species-specific and non-specific nucleotide sequences having the position of Figure 15, nine diagnostic primers that meet the primer conditions were designed as shown in Table 9.

Final selected SMV diagnostic primer primer Base sequence Length location ^a SMV-N10 ACTACATTTCTACAAGCAACC 21 59-79 SMV-N20 TAAATAAGCATAGTTCCATACAAT 24 373-396 SMV-N30 TTTGCTTCTAATGACCTTC 19 2382-2400 SMV-N40 CATATCAGTTTGTTGGGCA 19 5014-5032 SMV-C60 TCTTTAGTCTTCTTTGGAGTTGGT 24 599-622 SMV-C50 AATTCAACCAGCTTACCACTC 21 692-712 SMV-C40 TTTTGGGAGTAGTGCTGAATA 21 1046-1066 SMV-C30 AATTGGTTGTTGGATGCT 18 2816 ~ 2833 SMV-C20 TGCCTATACCCTCAACAT 18 5488 ~ 5505

<1-6> Design and Selection of SbDV Diagnostic Primer

SbDV, which was used for species-specific primer selection, was used as RT-PCR template by separating the whole RNA by extracting mosaic leaves from naturally infected beans. SbDV sequence search designed seven primer pairs based on the entire sequence.

Using the primer pairs selected in this way, as shown in FIG. 16, the first virus and RT-PCR were first performed to select seven primer pairs, and as shown in FIG. RT-PCR was performed.

Of these species-specific and nonspecific nucleotide sequences, six diagnostic primers satisfying primer conditions were designed as shown in Table 11.

Final selected SbDV diagnostic primer primer Base sequence Length location ^a SbDV-N10 CGTTGGGAAAGGAAATGAGGTAGT 24 1432-1455 SbDV-N20 TGGGGGTAGAAGCACTCC 18 1929-1946 SbDV-N30 TCTGTGTCGCTACCAATACTT 21 2798 ~ 2818 SbDV-C30 TTCAATCTGCATGTCAAACC 20 2265-2284 SbDV-C20 TTTGAAAGTATTGGTAGCGACACA 24 2800 ~ 2823 SbDV-C10 ATTCCATCTTTCTCGACTACTTG 23 4154 ~ 4176

<Example 2>

Development of Soybean 6 Virus Simultaneous Diagnosis

In order to develop co-diagnosis methods for SYMMV, USV3, AMV, PSV, SMV, and SbDV virus diseases in soybeans, we designed a combination of co-diagnostic primers by combining species-specific primers for individual viruses.

SYMMV, USV3, AMV, PSV, SMV, SbDV primers were used in each species specific primers selected in Example 1 above. That is, from the primers for each virus selected in Example 1, the following SYMMV (Table 7), USV3 (Table 8), AMV (Table 9), SMV (Table 10), PSV (Table 11) and SbDV Primer combinations of Table 12 were selected.

Secondary Selected SYMMV Species Specific Primer Combination Combination Primer (F / R) Product size (bp) One F01 R05 551 2 F01 R09 904 3 F16 R 851 4 F19 R 562 5 F23 C30 536 6 F23 R30 723 7 F23 C20 808 8 F C30 479 9 F29 R35 628 10 F29 C10 826 11 F34 C10 337

Secondary Selected USV3 Species Specific Primer Combination Combination Primer (F / R) Product size (bp) One N01 C03 291 2 N01 C04 351 3 N01 C05 561 4 N01 C08 768 5 N04 C05 176 6 N04 C12 795 7 N16 C24 785 8 N23 C27 449 9 N23 C31 835 10 N28 C35 628 11 N28 C38 967 12 N33 C38 507 13 N39 C42 269

Secondary Selected AMV Species Specific Primer Combination Combination Primer (F / R) Product size (bp) One N10 C50 410 2 N10 C40 586 3 N20 C50 320 4 N20 C40 496

Secondary Selected PSV Species Specific Primer Combination Combination Primer (F / R) Product size (bp) One N20 C50 439 2 N20 C40 810 3 N30 C30 545 4 N30 C20 697 5 N50 C20 158

Secondary Selected SMV Species-Specific Primer Combinations Combination Primer (F / R) Product size (bp) One N10 C60 564 2 N10 C50 654 3 N20 C60 250 4 N20 C50 340 5 N20 C40 694 6 N30 C30 452 7 N40 C20 492

Secondary Selected SbDV Species-Specific Primer Combinations Combination Primer (F / R) Product size (bp) One N10 C30 853 2 N20 C30 356

Using each of the selected primer combinations, 27 primer combinations (combination I) for simultaneous diagnosis of SYMMV, USV3, and AMV, and 16 PSV, SMV, SbDV primer combinations (combination II) were designed as shown in Tables 13 and 14 below. It was.

No. Virus Forward p. Reverse p. Size No. Virus Forward p. Reverse p. Size One USV3 NO4 C05 176 15 AMV N20 C50 320 AMV N20 C50 320 SYMMV F19 R 562 SYMMV F01 R05 551 USV3 N16 C24 785 2 USV3 NO4 C05 176 16 AMV N20 C50 320 AMV N20 C50 320 SYMMV F23 C30 536 SYMMV F19 R 562 USV3 N01 C08 768 3 USV3 NO4 C05 176 17 AMV N20 C50 320 AMV N20 C50 320 SYMMV F23 C30 536 SYMMV F23 C30 536 USV3 N04 C12 795 4 USV3 N04 C05 176 18 AMV N20 C50 320 AMV N10 C50 410 SYMMV F23 C30 536 SYMMV F01 R05 551 USV3 N16 C24 785 5 USV3 N04 C05 176 19 AMV N10 C50 410 AMV N10 C50 410 SYMMV F01 R05 551 SYMMV F19 R 562 USV3 N01 C08 768 6 USV3 N04 C05 176 20 AMV N10 C50 410 AMV N10 C50 410 SYMMV F01 R05 551 SYMMV F23 C30 536 USV3 N04 C12 795 7 USV3 N01 C03 291 21 AMV N10 C50 410 AMV N10 C50 410 SYMMV F01 R05 551 SYMMV F01 R05 551 USV3 N16 C24 785 8 USV3 N01 C03 291 22 AMV N10 C50 410 AMV N10 C50 410 SYMMV F19 R05 562 SYMMV F19 R 562 USV3 N01 C08 768 9 USV3 N01 C03 291 23 AMV N10 C50 410 AMV N10 C50 410 SYMMV F19 R05 562 SYMMV F23 C30 536 USV3 N04 C12 795 10 AMV N20 C50 320 24 AMV N10 C50 410 SYMMV F01 R05 551 SYMMV F19 R05 562 USV3 N01 C08 768 USV3 N16 C24 785 11 AMV N20 C50 320 25 AMV N10 C50 410 SYMMV F01 R05 551 SYMMV F23 C30 536 USV3 N04 C12 795 USV3 N01 C08 768 12 AMV N20 C50 320 26 AMV N10 C50 410 SYMMV F01 R05 551 SYMMV F23 C30 536 USV3 N16 C24 785 USV3 N04 C12 795 13 AMV N20 C50 320 27 AMV N10 C50 410 SYMMV F19 R 562 SYMMV F23 C30 536 USV3 N01 C08 768 USV3 N16 C24 785 14 AMV N20 C50 320 SYMMV F19 R 562 USV3 N04 C12 795

No. Virus Forward p. Reverse p. Size One SMV N30 C30 452 PSV N30 C20 697 SbDV N10 C30 853 2 SMV N10 C60 564 PSV N30 C20 697 SbDV N10 C30 853 3 PSV N50 C20 158 SbDV N20 C30 356 SMV N30 C30 452 4 PSV N50 C20 158 SbDV N20 C30 356 SMV N40 C20 492 5 PSV N50 C20 158 SbDV N20 C30 356 SMV N10 C60 564 6 PSV N50 C20 158 SbDV N20 C30 356 SMV N10 C50 654 7 PSV N50 C20 158 SbDV N20 C30 356 SMV N20 C40 694 8 PSV N50 C20 158 SMV N20 C60 250 SbDV N20 C30 356 9 PSV N50 C20 158 SMV N20 C60 250 SbDV N10 C30 853 10 PSV N50 C20 158 SMV N20 C50 340 SbDV N10 C30 853 11 PSV N50 C20 158 SMV N30 C30 452 SbDV N10 C30 853 12 PSV N50 C20 158 SMV N40 C20 492 SbDV N10 C30 853 13 PSV N50 C20 158 SMV N10 C60 564 SbDV N10 C30 853 14 PSV N50 C20 158 SMV N10 C50 654 SbDV N10 C30 853 15 PSV N50 C20 158 SMV N20 C40 694 SbDV N10 C30 853 16 PSV N20 C50 439 SMV N20 C40 694 SbDV N10 C30 853

Of the above combinations, RT-PCR was performed using the designed primer pairs in order to select a pair of primers capable of simultaneous diagnosis.

As a result, as shown in FIGS. 19 and 20, eight combinations of primers for combination diagnosis were selected for combination I and four combinations for combination II, which are shown in Tables 15 and 16.

No. Virus Forward p. Reverse p. Size No. Virus Forward p. Reverse p. Size 2 USV3 NO4 C05 176 11 PSV N50 C20 158 AMV N20 C50 320 SMV N30 C30 452 SYMMV F19 R 562 SbDV N10 C30 853 5 USV3 N04 C05 176 13 PSV N50 C20 158 AMV N10 C50 410 SMV N10 C60 564 SYMMV F19 R 562 SbDV N10 C30 853 7 PSV N50 C20 158 14 PSV N50 C20 158 SbDV N20 C30 356 SMV N10 C50 654 SMV N20 C40 694 SbDV N10 C30 853 8 PSV N50 C20 158 15 PSV N50 C20 158 SMV N20 C60 250 SMV N20 C40 694 SbDV N20 C30 356 SbDV N10 C30 853

No. Virus Forward p. Reverse p. Size No. Virus Forward p. Reverse p. Size 4 PSV N50 C20 158 6 PSV N50 C20 158 SbDV N20 C30 356 SbDV N20 C30 356 SMV N40 C20 492 SMV N10 C50 654 5 PSV N50 C20 158 8 PSV N50 C20 158 SbDV N20 C30 356 SMV N20 C60 250 SMV N10 C60 564 SbDV N20 C30 356

As shown in Table 15 and Table 16 it was confirmed with the samples taken from the package using a total of 12 selected combinations. As a result, as shown in Fig. 21, two combinations of design I and one combination of design II were obtained.

Among the above combinations, the soybean virus multiple diagnostic primer combinations finally showing the best diagnostic effect were selected as shown in Tables 17 and 18. As a result of diagnosing six viruses using the selected primers, as shown in FIG. 22, it was confirmed that simultaneous diagnosis of all six viruses was possible.

Primer Combination for Final Soybean Design I Virus Diagnosis Target Virus Product size primer Base sequence AMV 320 bp N20 AGGAAATGTTACTAGCTGATGAAG C50 GGTAGCATCATGGTCGGTGTCAAC SYMMV 551 bp F01 TATCCACCATTTAATCTTTCAGG R05 TAACGGGTCATTCATCAACTTCAT USV3 795 bp N04 GGAGTTGGTGCCTGGGTAT C12 AGCCACGAAATCGAGCATCTG

Primer Combination for Final Soybean Design II Virus Diagnosis Target Virus Product size primer Base sequence PSV 158 N50 CTGGCGTGTTGGAAGGCTAAGG C20 AAACACAATACAAACAAATACGAT SbDV 356 N20 TGGGGGTAGAAGCACTCC C30 TTCAATCTGCATGTCAAACC SMV 492 N40 CATATCAGTTTGTTGGGCA C20 TGCCTATACCCTCAACAT

< Example  3>

Detection Limit Analysis of Multiple Diagnostic Primer Combinations

To find out the detection limit of the virus of the soybean virus multi-diagnostic primer combination of the present invention selected from Example 2, the virus is isolated from the plant infected with soybean virus, and the total RNA of the virus is separated by concentration (1/10, 1/100, 1/1000, 1 / 10,000).

As a result, as shown in Figure 23, the primer combination of the present invention had a detection effect until the RNA concentration of the virus 1/100, the effect began to decrease somewhat at a concentration of 1/1000

Multiple diagnostic primer set of the present invention is soybean virus ( Soybean mosaic virus (SMV), Alfalfa mosaic virus ( Alfalfa) mosaic virus , AMV), Soybean dwarf virus , SbDV), Soybean yellow mottle mosaic virus (SYMMV), Peanut stunt virus (PSV), and unreported new virus (USV3, soybean yellow common mosaic, Soybean yellow common mosaic) virus, SYCMV) can be diagnosed at the same time, and the cost and effort of the virus can be reduced due to the spread of diagnostic methods for the virus, and soybean virus diseases can be solved in the agricultural field. In addition, it can contribute to the breeding of high quality soybean varieties, and also to the expansion of the market for soybean virus pathogen diagnostic kits.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Specific primers for multiple detection of the viruses in          soybean, and uses apparent <130> NP11-0013 <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 24 <212> DNA <213> Alfalfa mosaic virus <400> 1 aggaaatgtt actagctgat gaag 24 <210> 2 <211> 24 <212> DNA <213> Alfalfa mosaic virus <400> 2 ggtagcatca tggtcggtgt caac 24 <210> 3 <211> 23 <212> DNA <213> Soybean yellow mottle mosaic virus <400> 3 tatccaccat ttaatctttc agg 23 <210> 4 <211> 24 <212> DNA <213> Soybean yellow mottle mosaic virus <400> 4 taacgggtca ttcatcaact tcat 24 <210> 5 <211> 19 <212> DNA <213> Soybean yellow common mosaic virus <400> 5 ggagttggtg cctgggtat 19 <210> 6 <211> 21 <212> DNA <213> Soybean yellow common mosaic virus <400> 6 agccacgaaa tcgagcatct g 21 <210> 7 <211> 22 <212> DNA <213> Peanut stunt virus <400> 7 ctggcgtgtt ggaaggctaa gg 22 <210> 8 <211> 24 <212> DNA <213> Peanut stunt virus <400> 8 aaacacaata caaacaaata cgat 24 <210> 9 <211> 18 <212> DNA <213> Soybean dwarf viru <400> 9 tgggggtaga agcactcc 18 <210> 10 <211> 20 <212> DNA <213> Soybean dwarf viru <400> 10 ttcaatctgc atgtcaaacc 20 <210> 11 <211> 19 <212> DNA <213> Soybean mosaic virus <400> 11 catatcagtt tgttgggca 19 <210> 12 <211> 18 <212> DNA <213> Soybean mosaic virus <400> 12 tgcctatacc ctcaacat 18

Claims (4)

Simultaneous diagnosis of AMV, SYMMV and USV3 comprising a primer pair represented by SEQ ID NO: 1 and SEQ ID NO: 2, a primer pair represented by SEQ ID NO: 3 and SEQ ID NO: 4, and a primer pair represented by SEQ ID NO: 5 and SEQ ID NO: 6 Primer for soybean virus disease multiple diagnosis.
According to claim 1, PSV, SbDV comprising a primer pair represented by SEQ ID NO: 7 and SEQ ID NO: 8, a primer pair represented by SEQ ID NO: 9 and SEQ ID NO: 10 and a primer pair represented by SEQ ID NO: 11 and SEQ ID NO: 12 And primer sets for diagnosing soybean disease multiplex further comprising primer combinations capable of diagnosing SMV.
(a) isolating viral RNA from the sample;
(b) performing RT-PCR using the primer set of claim 1 or 2 as a template of the RNA of step (a); And
(C) method for diagnosing soybean virus disease comprising the step of separating the PCR product of step (b) by size.
A kit for diagnosing multiple beans virus diseases comprising the primer set of claim 1.

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