Primer set for detecting sweet potato pathogenic viruses and method for detecting sweet potato pathogenic viruses

Abstract

To provide a primer set for the detection of sweet potato pathogen virus that can be used for the detection of sweet potato pathogen virus.SOLUTION: Prepared is a primer set for detection of at least one sweet potato pathogen virus selected from an oligonucleotide group consisting of a specific base sequence. There is also provided a method including the steps of performing PCR using the primer set(s), analyzing an amplification fragment obtained by the PCR, and detecting sweet potato pathogen virus in a sample.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] The present invention relates to a primer set for detecting a sweet potato pathogenic virus, and a method for detecting a sweet potato pathogenic virus using the primer set. [Background technology]

[0002] A wide variety of plant viruses exist in nature, and they have caused severe damage to sweet potato (Ipomoea batatas (L. Lam), one of the major agricultural crops in Japan. To date, the following viruses have been identified: Sweet potato feathery mottle virus (SPFMV), Sweet potato latent virus (SPLV), Sweet potato symptomless virus (SPV), Sweet potato larvae ... Reported viruses include Sweet potato leaf curl virus (SPSMV-1), Sweet potato leaf curl virus (SPLCV), and Sweet potato virus G (SPVG). In addition to these, many other viruses have been reported in foreign countries, including Sweet potato chlorotic stunt virus (SPCSV) (Non-Patent Document 1).

[0003] In addition to the above, there are more than 20 known virus species that infect sweet potatoes. Among these many types, some are known to be highly virulent, such as the sweet potato feathery mosaic virus (SPFMV), which causes serious diseases such as zona rotunda.

[0004] So far, only methods have been proposed for detecting individual viruses that infect sweet potato. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] YAMASAKI, S. et al., Jpn. J. Phytopathol. 75: 102-108 (2009) Summary of the Invention [Problem to be solved by the invention]

[0006] However, it is believed that only a few highly virulent virus species cause serious damage. Furthermore, sweet potatoes infected with highly virulent virus species are likely to be co-infected with almost all other virus species. Therefore, if we can detect several major virus species, we may be able to comprehensively detect sweet potatoes infected with some virus strain. An object of the present invention is to provide a means for simultaneously detecting multiple sweet potato pathogenic viruses quickly and with high accuracy. [Means for solving the problem]

[0007] In order to solve the above problems, the present inventors focused on the coat protein gene, replication-related protein gene, and cell-cell movement protein gene regions of highly virulent virus species of sweet potato virus, examined multiple primers, compared the comprehensive detection rate and specificity from among them, selected a suitable primer set, and further improved the comprehensive detection and specificity. Furthermore, they attempted to design a primer set that enables detailed lineage understanding of SPFMV virus, whose effects on tuberous roots differ depending on the lineage. As a result, they succeeded in establishing a primer set that can efficiently and correctly detect multiple types of viruses known to be highly virulent viruses, and in establishing a primer set that can understand viruses of the same lineage, thereby completing the present invention.

[0008] That is, the present invention provides the following primer set for detecting a sweet potato pathogenic virus. Item 1. A primer set for detecting at least one sweet potato pathogenic virus selected from the group consisting of the following (1) to (11): (1) a primer set for detecting a sweet potato pathogenic virus, comprising an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:1 and an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:2; (2) a primer set for detecting a sweet potato pathogenic virus, comprising an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:1 and an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:3; (3) a primer set for detecting a sweet potato pathogenic virus, comprising an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:1 and an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:4; (4) a primer set for detecting a sweet potato pathogenic virus, comprising an oligonucleotide having the base sequence shown in SEQ ID NO:5 and an oligonucleotide having the base sequence shown in SEQ ID NO:6; and (5) a primer set for detecting a sweet potato pathogenic virus, comprising an oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 7 and an oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 8; (6) A primer set for detecting a sweet potato pathogenic virus, comprising an oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 1 and an oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 9; (7) A primer set for detecting a sweet potato pathogenic virus, comprising an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:10 and an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:11; (8) A primer set for detecting a sweet potato pathogenic virus, comprising an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:12 and an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:13; (9) A primer set for detecting a sweet potato pathogenic virus, comprising an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:14 and an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:15; (10) A primer set for detecting a sweet potato pathogenic virus, comprising an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:16 and an oligonucleotide having the nucleotide sequence shown in SEQ ID NO:17; (11) A primer set for detecting a sweet potato pathogenic virus, comprising an oligonucleotide having the base sequence shown in SEQ ID NO:16 and an oligonucleotide having the base sequence shown in SEQ ID NO:18.

[0009] The present invention also provides Section 2. A method for detecting a sweet potato pathogenic virus in a sample, comprising: performing PCR using the primer set according to Item 1 and amplified fragments obtained by PCR, using nucleic acid of a sweet potato-derived sample as a template; and analyzing the amplified fragments obtained by PCR; Section 3. A method for detecting the presence of the sweet potato pathogenic viruses SPFMV and SPVG in a sample by performing PCR using a primer set for detecting sweet potato pathogenic viruses, which is composed of an oligonucleotide having the base sequence shown in SEQ ID NO:1 and an oligonucleotide having the base sequence shown in SEQ ID NO:2, and analyzing the amplified fragment obtained by the PCR; Section 4. A method for detecting the presence of the sweet potato pathogenic virus SPFMV in a sample by performing PCR using a primer set for detecting a sweet potato pathogenic virus, which is composed of an oligonucleotide having the base sequence shown in SEQ ID NO: 1 and an oligonucleotide having the base sequence shown in SEQ ID NO: 3, and analyzing the amplified fragment obtained by the PCR; Section 5. A method for detecting the presence of sweet potato pathogenic viruses SPVG and SPV2 in a sample by performing PCR using a primer set for detecting sweet potato pathogenic viruses, which is composed of an oligonucleotide having the base sequence shown in SEQ ID NO:1 and an oligonucleotide having the base sequence shown in SEQ ID NO:4 in the sequence listing, and analyzing the amplified fragment obtained by the PCR; Section 6. A method for detecting the presence of sweet potato pathogenic virus SPVG in a sample by performing PCR using a primer set for detecting sweet potato pathogenic virus, which is composed of an oligonucleotide having the base sequence shown in SEQ ID NO: 1 and an oligonucleotide having the base sequence shown in SEQ ID NO: 9, and analyzing the amplified fragment obtained by the PCR; Section 7. a method for detecting the presence of sweet potato pathogenic virus SPLCV in a sample, comprising: a primer set for detecting sweet potato pathogenic virus, the primer set being composed of an oligonucleotide having the base sequence shown in SEQ ID NO:5 and an oligonucleotide having the base sequence shown in SEQ ID NO:6; or a primer set for detecting sweet potato pathogenic virus, the primer set being composed of an oligonucleotide having the base sequence shown in SEQ ID NO:7 and an oligonucleotide having the base sequence shown in SEQ ID NO:8, and performing PCR using the primer set, and analyzing the amplified fragment obtained by the PCR; Section 8. A method for detecting the presence of the sweet potato pathogenic virus SPSMV-1 in a sample, comprising carrying out PCR using a primer set for detecting a sweet potato pathogenic virus, the primer set being composed of an oligonucleotide having the base sequence shown in SEQ ID NO: 10 and an oligonucleotide having the base sequence shown in SEQ ID NO: 11, and analyzing the amplified fragment obtained by the PCR; Section 9. A method for detecting the presence of the sweet potato pathogenic virus SPFMV-RC strain in a sample by performing PCR using a primer set for detecting a sweet potato pathogenic virus, which is composed of an oligonucleotide having the base sequence shown in SEQ ID NO: 12 and an oligonucleotide having the base sequence shown in SEQ ID NO: 13 in the sequence listing, and analyzing the amplified fragment obtained by the PCR; Section 10. A method for detecting the presence of the sweet potato pathogenic virus SPFMV-O strain in a sample by performing PCR using a primer set for detecting a sweet potato pathogenic virus, which is composed of an oligonucleotide having the base sequence shown in SEQ ID NO: 14 and an oligonucleotide having the base sequence shown in SEQ ID NO: 15 in the sequence listing, and analyzing the amplified fragment obtained by the PCR; Section 11. A primer set for detecting a sweet potato pathogenic virus, which is composed of an oligonucleotide having the base sequence shown in SEQ ID NO: 16 and an oligonucleotide having the base sequence shown in SEQ ID NO: 17, or A method for detecting the presence of the sweet potato pathogenic virus SPFMV-C strain in a sample by performing PCR using a primer set for detecting a sweet potato pathogenic virus, which is composed of an oligonucleotide having the base sequence shown in SEQ ID NO: 16 and an oligonucleotide having the base sequence shown in SEQ ID NO: 18 in the sequence listing, and analyzing the amplified fragment obtained by the PCR; Regarding.

[0010] The present invention also relates to a kit for detecting a pathogenic virus in sweet potato, comprising the primer set according to item 1. Effect of the Invention

[0011] The primer set of the present invention also makes it possible to simultaneously detect multiple types of sweet potato pathogenic viruses. [Brief description of the drawings]

[0012] [Figure 1] 1 shows an electrophoresis pattern of PCR amplification products of a gene of a sweet potato pathogenic virus using a primer set according to an embodiment. [Diagram 2] 1 shows an electrophoresis pattern of PCR amplification products of a gene of a sweet potato pathogenic virus using a primer set according to an embodiment. [Diagram 3] 1 shows an electrophoresis pattern of PCR amplification products of a gene of a sweet potato pathogenic virus using a primer set according to an embodiment. [Figure 4] Electrophoretograms of PCR amplification products of genes of four kinds of sweet potato pathogenic viruses using the primer set of the example are shown. [Diagram 5] Electrophoretograms of PCR amplification products of genes of four kinds of sweet potato pathogenic viruses using the primer set of the example are shown. [Figure 6]Electrophoretograms of PCR amplified products of genes of sweet potato pathogenic virus strains SPFMV-RC, SPFMV-O, and SPFMV-C using the primer set of the example are shown. [Figure 7] Electrophoretograms of PCR amplified products of genes of sweet potato pathogenic virus strains SPFMV-RC, SPFMV-O, and SPFMV-C using the primer set of the example are shown. [Figure 8] Electrophoretograms of PCR amplified products of genes of sweet potato pathogenic virus strains SPFMV-RC, SPFMV-O, and SPFMV-C using the primer set of the example are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] [Primer set for detecting sweet potato pathogenic viruses] The primer set of the present invention is a primer set consisting of a pair of oligonucleotides capable of efficiently amplifying a gene of a sweet potato pathogenic virus, and is capable of detecting the sweet potato pathogenic virus.

[0014] Each primer constituting the primer set for detecting sweet potato pathogenic viruses of the present invention is an oligonucleotide, and is a sequence of 20 to 25 consecutive nucleotides designed as a forward primer and a reverse primer in the viral coat protein gene, replication-associated protein gene, and cell-to-cell movement protein gene regions.

[0015] In designing the primers, the virus gene sequence information infecting sweet potato was obtained by virus type from the NCBI site (https: / / www.ncbi.nlm.nih.gov / nuccore / ). The base sequence information can also be obtained from publications. The inventors used the coat protein (CP) gene, the replication-related protein gene, and the cell-cell movement protein gene region, which have the largest number of registered data. Then, a comparison table of the nucleic acid sequences of both gene regions was created by virus type. At this time, in consideration of the current situation of agricultural product import and export, in addition to virus isolates from neighboring Asian countries such as China and Korea, mainly Japan, virus isolates from North and South America and African countries were also compared. Alignment software (e.g., GENETYX ver.14) can be used to align the base sequences.

[0016] Primers are designed based on the identified base sequence. In designing the primers, the complementarity between oligonucleotides is taken into consideration in order to eliminate the drawbacks of conventional degenerate primers, which were not able to adequately accommodate base substitutions between multiple strains of the same species.

[0017] Specifically, we focused on SPFMV, SPLCV, SPVG, SPV2 and SPSMV-1, and paid special attention to creating degenerate primers compatible with base substitutions.

[0018] Based on these points of view, the following primer set was created as a primer set for detecting a sweet potato pathogenic virus of the present invention. (Primer set: for simultaneous detection of SPFMV and SPVG) Forward: 5'-DTGGTGYATHGARAATGGMACATC-3' (SEQ ID NO: 1 in the sequence listing) Reverse: 5'-TGCACACCCCTCATWCCYAAGAG-3' (SEQ ID NO: 2 in the sequence listing)

[0019] (Primer set: for identifying SPFMV) Forward: 5'-DTGGTGYATHGARAATGGMACATC-3' (SEQ ID NO: 1 in the sequence listing) Reverse: 5'-TACAGTGCARAGKWTSWCCTAYTGC-3' (SEQ ID NO: 3 in the sequence listing)

[0020] (Primer set: for simultaneous detection of SPVG and SPV2) Forward: 5'-DTGGTGYATHGARAATGGMACATC-3' (Sequence No. 1 in the sequence listing) Reverse: 5'-CATATGYGCYTCYCTRGCBCGAAC-3' (SEQ ID NO: 4 in the sequence listing)

[0021] (Primer set: for SPLCV detection) Forward: 5'-CTACACTKGGAATGCYGTCCCAATTG-3' (SEQ ID NO: 5 in the sequence listing) Reverse: 5'-AGTCCTGKAYCTTACAGGGACCVACRC-3' (SEQ ID NO: 6 in the sequence listing)

[0022] (Primer set: for SPLCV detection) Forward: 5'-CTCTCCTGGATTGCAGARGAAGAYWGTG-3' (SEQ ID NO: 7 in the sequence listing) Reverse: 5'-AYAGYAAYAGTGCYTGGTAYAACGTC-3' (SEQ ID NO: 8 in the sequence listing)

[0023] (Primer set: for identifying SPVG) Forward: 5'-DTGGTGYATHGARAATGGMACATC-3' (Sequence No. 1 in the sequence listing) Reverse: 5'-TCYCTRGCSCGAACWGGAGTGTTC-3' (SEQ ID NO: 9 in the sequence listing)

[0024] (Primer set: for identifying SPSMV-1) Forward: 5'-ACGGAGATAGGGTTCGGWCAGAC-3' (SEQ ID NO: 10 in the sequence listing) Reverse: 5'-GAGCGCAGATCTGACCMGGTTTAC-3' (SEQ ID NO: 11 in the sequence listing)

[0025] (Primer set: for detecting SPFMV-RC) Forward: 5'-AAGATGCAYYAAYTGGTARCATCC-3' (SEQ ID NO: 12 in the sequence listing) Reverse: 5'-CTGTAKAGYCCCCAYCTCCATTTYAC-3' (SEQ ID NO: 13 in the sequence listing)

[0026] (Primer set: for detecting SPFMV-O) Forward: 5'-CAATCTGARAGGGARTAYTTCARRCCTC-3' (SEQ ID NO: 14 in the sequence listing) Reverse: 5'-GRTGRYYTTCTAYATCRTGDTCACTC-3' (SEQ ID NO: 15 in the sequence listing)

[0027] (Primer set: for detecting SPFMV-C) Forward: 5'-GCACCAAARCYRAAAGGGSCATWTRC-3' (SEQ ID NO: 16 in the sequence listing) Reverse: 5'-CACYCTTTGTGKTGAAAYAGARCGCA-3' (SEQ ID NO: 17 in the sequence listing)

[0028] (Primer set: for detecting SPFMV-C) Forward: 5'-GCACCAAARCYRAAAGGGSCATWTRC-3' (SEQ ID NO: 16 in the sequence listing) Reverse: 5'-YAGARCGCATAGRTTTCTCACYTG-3' (SEQ ID NO: 18 in the sequence listing)

[0029] Here, in the base sequences shown in SEQ ID NOs: 1 to 18 in the sequence listing, B represents either C, G, or T; D represents either A, G, or T; K represents either G or T; M represents either A or C; R represents either A or G; V represents either A, C, or G; W represents either A or T; Y represents either C or T; and S represents either C or G. (All are shown in lower case in the sequence listing.)

[0030] The GC contents and Tm values ​​of the sequences shown in SEQ ID NOs: 1 to 18 in the sequence listing are as follows: SEQ ID NO: 1: GC content 42%, Tm value 57 SEQ ID NO:2: GC content 57%, Tm value 62 SEQ ID NO:3: GC content 44%, Tm value 59 SEQ ID NO: 4: GC content 58%, Tm value 63 SEQ ID NO:5: GC content 46%, Tm value 60 SEQ ID NO: 6: GC content 56%, Tm value 65 SEQ ID NO: 7: GC content 50%, Tm value 62 SEQ ID NO: 8: GC content 46%, Tm value 61 SEQ ID NO: 9: GC content 50%, Tm value 60 SEQ ID NO: 10: GC content 57%, Tm value 61 SEQ ID NO: 11: GC content 54%, Tm value 61 SEQ ID NO: 12: GC content 46%, Tm value 59 SEQ ID NO: 13: GC content 50%, Tm value 60 SEQ ID NO: 14: GC content 43%, Tm value 59 SEQ ID NO: 15: GC content 50%, Tm value 60 SEQ ID NO: 16: GC content 50%, Tm value 62 SEQ ID NO: 17: GC content 54%, Tm value 65 Sequence number 18: GC content 50%, Tm value 59.

[0031] The above 11 types of primer sets can be used individually to detect the desired sweet potato pathogenic virus. Furthermore, by mixing two or more types and using them in a multiplex PCR method, it is possible to simultaneously and specifically detect five pathogenic viruses, SPFMV, SPVG, SPV2, SPLCV, and SPSMV-1, in multiple combinations. Furthermore, it is possible to distinguish and detect the SPFMV strains SPFMV-RC, SPFMV-O, and SPFMV-C, and to investigate in detail the effects on different tuberous roots. In particular, it is significant to be able to distinguish the SPFMV-RC strain, which may have a serious effect.

[0032] Specifically, each of the forward primers and reverse primers constituting the above primer set is a degenerate primer that simultaneously contains two or three types of bases specified at the B, D, K, M, R, V, W, Y, and S positions of the base sequence, and the ratio of each primer in the degenerate primer may be any ratio, but it is preferable that they are equal in amount.

[0033] Each primer of the primer set can be prepared by a method for synthesizing oligonucleotides known to those skilled in the art, for example, by using an automatic DNA synthesizer (such as Model 394 manufactured by Applied Biosystems).

[0034] A method for detecting a sweet potato pathogenic virus in a sample using the primer set of the present invention includes the steps of extracting RNA from a sweet potato sample, using cDNA synthesized by reverse transcription as a template, and performing a polymerase chain reaction (PCR) using any one or more or all of the above-mentioned primer sets to amplify the gene, and analyzing the amplified fragment obtained by the PCR.

[0035] Sweet potato samples may be prepared from any part of the sweet potato plant, such as leaves, stems, or tuberous roots. When the sweet potato sample is a leaf, it is possible to hollow it out with a cork poacher, and sample several tens of mg (e.g., about 50 mg) from one plant. In this case, the sampling site is the mesophyll part between the veins, and if there are any parts that show symptoms such as spots or cigar marks, it is preferable to select such parts. When the sweet potato sample is a tuberous root, it is possible to prepare the sample by cutting off several tens of mg (e.g., about 50 mg) of only the epidermis using a cutter blade or the like.

[0036] The detection of sweet potato pathogenic viruses using the primer set of the present invention can target both DNA viruses and RNA viruses. Therefore, nucleic acid extraction may involve simultaneous extraction of DNA and RNA. In such a case, simultaneous extraction of DNA and RNA is performed by modifying the standard protocol.

[0037] After preparing the sweet potato sample, nucleic acid is extracted. Nucleic acid extraction can be performed by a method known to those skilled in the art, or a commercially available kit for extracting and purifying RNA from plant tissue can be used. A method of extracting RNA from a sample and synthesizing cDNA using the RNA as a template can also be adopted. For nucleic acid extraction, a kit such as ISOSPIN Plant RNA (manufactured by NIPPON GENE Co., Ltd.) can be used, but is not limited thereto. Alternatively, first, RNA is extracted using an isothiocyanate, phenol, and chloroform, or a commercially available RNA extraction reagent (for example, TRIZOL Reagent kit (manufactured by Thermo Fisher SCIENTIFIC Co., Ltd., 15596-018)), and then cDNA is synthesized using RNA as a template. Here, commercially available random primers are used as primers, and cDNA is synthesized using reverse transcriptase in the presence of dNTPs. For reverse transcriptase, for example, SuperScriptII (manufactured by Invitrogen Co., Ltd.), Thermoscript RT, AMV reverse transcriptase, MMLV reverse transcriptase, etc. can be used.

[0038] Next, a PCR amplification reaction is carried out using one or more of the primer sets of the present invention as a template using the synthesized cDNA. The PCR amplification can be carried out using any or all of the primer sets of the present invention or a combination thereof. Other points can be carried out according to a conventional method. PCR is a PCR method or a real-time PCR method (qPCR). Specifically, in PCR, a fragment containing the base sequence of each coat protein gene region is amplified by repeating a cycle including denaturation of template DNA, annealing of primers, and extension reaction of primers using a heat-resistant enzyme. The composition of the PCR reaction solution and the PCR reaction conditions can be appropriately selected and set from those known to those skilled in the art. A method for selecting optimal PCR reaction conditions based on the Tm of primers is known.

[0039] The DNA polymerase used in the nucleic acid amplification step of the PCR method is not particularly limited, and examples thereof include α-type DNA polymerases such as KOD DNA polymerase and Pfu DNA polymerase, and Pol I-type DNA polymerases such as Taq DNA polymerase and Tth DNA polymerase. Although not particularly limited, commercially available KOD DNA polymerase (manufactured by Toyobo) and Pfu DNA polymerase (manufactured by Takara Bio Inc.) can also be used.

[0040] The PCR procedure can be carried out using a commercially available PCR kit and PCR device, following the operating instructions. For example, the PCR reaction solution can be SYBR Green Real-Time PCR Master Mixes (Thermo Fisher Scientific, #A25742), and the PCR device can be the 7500 Fast Real-Time PCR System (Applied Biosystems).

[0041] PCR reaction can be carried out by performing multiple cycles, with template thermal denaturation, primer annealing, and extension reaction as one cycle. Any condition known to ordinary skilled artisans can be used, and is not limited thereto. For example, it is preferable to perform about 30 to 40 cycles, with one cycle consisting of thermal denaturation at 90°C to 96°C for 20 seconds to 1 minute, annealing at a temperature range of about 50°C to 58°C for 20 seconds to 1 minute, and extension reaction at 70°C to 73°C for 20 seconds to 1 minute. In particular, the annealing temperature is more preferably about 54°C to 58°C, and even more preferably about 55°C to 56°C. By setting the annealing temperature relatively high, the risk of non-specific amplification can be further reduced.

[0042] In addition, the detection of the amplified product obtained by PCR is not particularly limited, and examples thereof include agarose gel electrophoresis, capillary electrophoresis, RFLP, a method for detecting the amplified product using an intercalator, and a method for detecting the amplified product using a nucleic acid probe that specifically binds to the base sequence of the amplified product. More preferred is a method for detecting the amplified product using a nucleic acid probe, and examples of the nucleic acid probe include TaqMqn probe, quenching probe (QProbe), and MolecularBeacon. Other conditions for amplification and detection can be appropriately selected in consideration of known nucleic acid amplification and nucleic acid detection techniques.

[0043] [Methods for detecting sweet potato pathogenic viruses] The present invention also relates to a method for detecting a sweet potato pathogenic virus in a sample, comprising carrying out PCR using one or more types of the primer set of the present invention and analyzing the amplified fragment obtained by the PCR.

[0044] In the present invention, PCR is performed using a primer set for detecting sweet potato pathogenic viruses consisting of an oligonucleotide having the base sequence shown in SEQ ID NO: 1 in the sequence listing and an oligonucleotide having the base sequence shown in SEQ ID NO: 2, and the amplified fragment obtained by the PCR is analyzed, thereby enabling the sweet potato pathogenic viruses SPFMV and SPVG to be specifically detected in a sample.

[0045] In the present invention, PCR is performed using a primer set for detecting sweet potato pathogenic viruses, which is composed of an oligonucleotide having the base sequence shown in SEQ ID NO: 1 in the sequence listing and an oligonucleotide having the base sequence shown in SEQ ID NO: 3, and the amplified fragment obtained by the PCR is analyzed, thereby making it possible to specifically identify the sweet potato pathogenic virus SPFMV in a sample.

[0046] In the present invention, PCR is performed using a primer set for detecting sweet potato pathogenic viruses, which is composed of an oligonucleotide having the base sequence shown in SEQ ID NO: 1 in the sequence listing and an oligonucleotide having the base sequence shown in SEQ ID NO: 4, and the amplified fragment obtained by the PCR is analyzed, thereby making it possible to specifically identify the sweet potato pathogenic viruses SPVG and SPV2 in a sample.

[0047] In the present invention, PCR is performed using a primer set for detecting sweet potato pathogenic viruses, which is composed of an oligonucleotide having the base sequence shown in SEQ ID NO: 1 in the sequence listing and an oligonucleotide having the base sequence shown in SEQ ID NO: 9, and the amplified fragment obtained by the PCR is analyzed, thereby making it possible to specifically identify sweet potato pathogenic virus SPVG in a sample.

[0048] In the present invention, the presence of the sweet potato pathogenic virus SPLCV in a sample can be detected by performing PCR using a primer set for detecting sweet potato pathogenic viruses, which is composed of an oligonucleotide having the base sequence shown in SEQ ID NO: 5 and an oligonucleotide having the base sequence shown in SEQ ID NO: 6, and analyzing the amplified fragment obtained by the PCR. Similarly, in the present invention, the presence of the sweet potato pathogenic virus SPLCV in a sample can be detected by performing PCR using a primer set for detecting sweet potato pathogenic viruses, which is composed of an oligonucleotide having the base sequence shown in SEQ ID NO: 7 and an oligonucleotide having the base sequence shown in SEQ ID NO: 8, and analyzing the amplified fragment obtained by the PCR.

[0049] In the present invention, oligonucleotides consisting of the base sequences shown in SEQ ID NOs: 1, 3, 4, 7, and 8 are used as primers for detecting sweet potato pathogenic viruses to perform PCR, and the amplified fragments obtained by the PCR are analyzed, thereby making it possible to detect the presence of sweet potato pathogenic viruses SPFMV, SPVG, SPV2, and SPLCV in a sample collectively and simultaneously or separately.

[0050] These primer sets of the present invention can also be used for multiplex RT-PCR. As previously reported primers use the common sequences of SPFMV-S, O, Bungo, and SPVG, it was necessary to cleave the gene fragments of SPFMV-S, O, Bungo, and SPVG, which are simultaneously amplified and detected, at specific restriction enzyme recognition sites to identify the viruses. However, by performing multiplex RT-PCR (reverse transcriptase PCR) using the primer set of the present invention, it is possible to directly identify the viruses, and it is also possible to perform labor-saving and rapid testing.

[0051] In the present invention, the presence of sweet potato pathogenic virus SPSMV-1 in a sample can be detected by performing PCR using a primer set for detecting sweet potato pathogenic viruses consisting of an oligonucleotide having the base sequence shown in SEQ ID NO: 10 and an oligonucleotide having the base sequence shown in SEQ ID NO: 11, and analyzing the amplified fragment obtained by the PCR.

[0052] In the present invention, oligonucleotides consisting of the base sequences shown in SEQ ID NOs: 1, 3, 7, 8, 9, 10 and 11 are used as primers for detecting sweet potato pathogenic viruses to perform PCR, and the amplified fragments obtained by the PCR are analyzed, thereby making it possible to simultaneously and differentially detect the presence of four types of sweet potato pathogenic viruses, SPFMV, SPVG, SPLCV and SPSMV-1, in a sample.

[0053] On the other hand, in the present invention, by performing PCR using oligonucleotides consisting of the base sequences shown in SEQ ID NOs: 12 to 18 as primers for detecting sweet potato pathogenic viruses and analyzing the amplified fragments obtained by the PCR, it is possible to distinguish and detect the strains of sweet potato pathogenic virus SPFMV in a sample as SPFMV-RC strain, SPFMV-O strain, and SPFMV-C strain.

[0054] When SPFMV and SPVG are detected simultaneously, the primers of the set of oligonucleotides shown in SEQ ID NOs: 1 and 2 are preferably present at a molar ratio of 1:1 or close thereto. For detecting SPFMV, the primers of the set of oligonucleotides shown in SEQ ID NOs: 1 and 3 are preferably present at a molar ratio of 1:1 or close thereto. For simultaneously distinguishing SPVG and SPV2, the primers of the set of oligonucleotides shown in SEQ ID NOs: 1 and 4 are preferably present at a molar ratio of 1:1 or close thereto. For detecting SPLCV, it is preferable that all types of primers of the oligonucleotides shown in SEQ ID NOs: 5 and 6 are present at a molar ratio of 1:1 or close thereto. Alternatively, for detecting SPLCV, it is preferable that the primers of the oligonucleotides shown in SEQ ID NOs: 7 and 8 are present at a molar ratio of 1:1 or close thereto.

[0055] On the other hand, when used in multiplex PCR, the set includes two or more kinds of primers, preferably all kinds of oligonucleotides of SEQ ID NOs: 1, 3, 4, 7, and 8. In this case, it is also preferable to have equal amounts of oligonucleotides of SEQ ID NOs: 3, 4, 7, and 8 in molar ratio, and 1 to 3 times, preferably about 2 times, the oligonucleotide of SEQ ID NO: 1. Alternatively, all oligonucleotides can be used with the molar ratio being within the range of 1:1 to 3.

[0056] Furthermore, when used in multiplex PCR, it preferably contains all kinds of primers of the oligonucleotides of SEQ ID NOs: 1, 3, 7, 8, 9, 10, and 11. In this case, it is also preferable to have the oligonucleotides of SEQ ID NOs: 3, 7, 8, 9, 10, and 11 present in equal amounts at a molar concentration ratio, and the oligonucleotide of SEQ ID NO: 1 present in a two- or three-fold amount. Alternatively, for the purpose of PCR efficiency, the molar ratios may be, for example, 1 for the oligonucleotide of SEQ ID NO: 1, preferably 0.1 to 1, more preferably 0.3 to 0.8, and even more preferably about 0.5 to 0.7 for the oligonucleotide of SEQ ID NO: 3, preferably 0.1 to 1, more preferably 0.1 to 0.3, and even more preferably about 0.2 for the oligonucleotide of SEQ ID NO: 9, preferably 0.1 to 1, more preferably 0.1 to 0.3, and even more preferably about 0.2 for the oligonucleotides of SEQ ID NO: 7 and 8, preferably 0.1 to 1, more preferably 0.2 to 0.6, and even more preferably about 0.5 for the oligonucleotides of SEQ ID NO: 10 and 11, preferably 0.1 to 1, more preferably 0.2 to 0.4, and even more preferably about 0.3. In this way, it is also possible to simultaneously and discriminately detect SPFMV, SPVG, SPLCV, and SPSMV-1.

[0057] Alternatively, the primers of SEQ ID NO: 12 and SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, and SEQ ID NO: 16 and SEQ ID NO: 18 may be used in pairs or all types of primers may be included in a multiplex PCR method. In multiplex PCR, although not limited thereto, for the sake of PCR efficiency, the molar ratio may be, for example, 1 for the oligonucleotide of SEQ ID NO: 12, 1 for the oligonucleotide of SEQ ID NO: 13, preferably 0.5 to 1.5, more preferably about 1; 0.5 to 1.5 for the oligonucleotides of SEQ ID NO: 14 and 15, respectively, more preferably about 1; and 0.1 to 1 for the oligonucleotides of SEQ ID NO: 16 and 18, preferably 0.1 to 1, more preferably 0.2 to 0.6, even more preferably about 0.3. In this way, it is also possible to distinguish and detect the strains of SPFMV as SPFMV-RC strain, SPFMV-O strain, and SPFMV-C strain.

[0058] Although not limited thereto, in the present invention, each of these oligonucleotides can be used in a range of preferably 0.1 μM to 50 μM, more preferably 1 μM to 15 μM.

[0059] [kit] The primer set of the present invention can be made into a kit. The kit of the present invention includes at least one or more of the primer sets described in the section [Primer set for detecting sweet potato pathogenic viruses], and can be appropriately changed depending on the target virus to be detected. In addition, various reagents required for detecting nucleic acid amplification can be optionally included together. Specifically, the primer of the present invention, four types of dNTPs that serve as substrates for nucleic acid synthesis, template-dependent nucleic acid synthetase, buffer solutions and salts, or instructions can be provided as a kit. Such a kit can be conveniently used, for example, to test whether sweet potato seedlings are infected with a virus. EXAMPLES

[0060] The present invention will be described in more detail below with reference to the following examples. However, the present invention is not limited to these examples. In the examples, the ratio of each primer in the same sequence number to each degenerate primer represented by each sequence number was equal.

[0061] (Example 1) Primer set design We collected information on the nucleotide sequences of viral genes from the NCBI website (https: / / www.ncbi.nlm.nih.gov / nuccore / ). We used GENETYX ver.14 to create multiple alignments of these nucleotide sequences. This comparison showed that the sequences of the coat protein gene and replication-related protein gene regions were conserved. We therefore considered that this conservation could be used to simultaneously detect different strains. In particular, we focused on the viruses SPFMV, SPVG, and SPLCV, and designed multiple forward and reverse primers that are complementary to parts of the coat protein gene region and the replication-related protein gene as candidate primers.

[0062] The above candidate primers were used to detect the presence of the previously known sweet potato pathogenic viruses SPFMV-S, SPFMV-O, and SPFMV-Bungo, and the primers were initially selected. Furthermore, the primers were further improved using a strain infected with the three target viruses.

[0063] The sweet potato pathogenic virus strains mentioned above consisted of the Japanese SPFMV-S strain, the Japanese SPFMV-O strain, and the Japanese SPFMV-Bungo strain.

[0064] Sweet potato samples used were leaves and tuberous roots. When the sweet potato samples were leaves, they were hollowed out with a cork power and 50 mg was sampled from each individual. The sampling site was the mesophyll between the veins, where symptoms such as spots or curling were present. When the sweet potato samples were tuberous roots, 50 mg of only the epidermis was cut off using a cutter blade.

[0065] The procedures and conditions for nucleic acid extraction from virus strains and RT-PCR amplification were as follows.

[0066] ISOSPIN Plant RNA (NIPPON GENE) was used to extract nucleic acids from virus strains. The primer set targeted both DNA and RNA viruses to extract both nucleic acids. Therefore, both nucleic acids were extracted simultaneously by omitting the standard protocol's PT Wash 1 Buffer and DNase I solution treatment. Samples were disrupted using Micro SmashTM MS-100R (TOMY SEIKO) at 4200 rpm for 160 seconds, using one stainless steel bead φ4.8 per sample.

[0067] Reverse transcription and PCR amplification were performed using PrimeScriptTM One Step RT-PCR Kit Ver.2 (Dye Plus) (Takara, Japan). The PCR reaction solution composition and conditions are as follows. After the reaction, 3 μl of the PCR amplification product was analyzed by 2.0% agarose gel electrophoresis.

[0068] (PCR reaction mixture composition: 10 μl per reaction) Nucleic acid 1μl 1.0 μl of each primer (5 pmol each) PrimeScript 1 step Enzyme Mix 2μl 2×1 step Buffer (Dye Plus) 25μl RNase Free dH2O up to 50μl

[0069] (RT-PCR conditions) cDNA synthesis was performed by incubating at 50°C for 30 minutes and at 94°C for 2 minutes, and the subsequent PCR reaction was performed for a total of 35 cycles, each cycle consisting of heat denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 30 seconds.

[0070] From the results of PCR analysis, primer sets having the sequences shown in Table 1 below (the upper sequence in each column is the forward primer, and the lower sequence is the reverse primer) were finally determined, and primer sets composed of these primer sets were designed.

[0071] [Table 1]

[0072] (Example 2) Detection of sweet potato pathogenic viruses using primer sets Detection of sweet potato pathogenic viruses using the primer set established in Example 1 was examined using the sweet potato pathogenic viruses SPFMV, SPVG, and SPLCV diseased strains. Viral nucleic acid extraction was performed in the same manner as in Example 1. The PCR reaction solution composition and conditions were as follows.

[0073] The primers were used in the amounts (pmol / μL) shown in Table 2 below, at 0.2 μL per 10 μL of reaction solution.

[0074] [Table 2]

[0075] The virus strains used were as follows:

[0076] [Table 3]

[0077] MC2: SPFMV, SPVG, SPLCV affected strains MC4: SPLCV disease strain 1-36: SPFMV, SPVG, SPLCV affected strains

[0078] Template nucleic acid concentration MC2: 366.8 (ng / μl) MC4: 177.9 (ng / μl) 1-36:403.5(ng / μl)

[0079] The PCR reaction was carried out for a total of 35 cycles, each cycle consisting of heat denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 30 seconds.

[0080] The PCR amplification products were verified by electrophoresis using agarose gel. The results are shown in Figure 1. Although all of the products were at a level that could be verified, the results were particularly favorable when the primers of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 8 were used simultaneously, allowing clear identification of all three types of viruses.

[0081] (Example 3) Redetection of sweet potato pathogenic viruses using primer sets Based on the results of Example 2, the primer concentrations were adjusted again, and the sweet potato pathogenic viruses SPFMV, SPVG, and SPLCV diseased strains were used for the study. Viral nucleic acid extraction was performed in the same manner as in Example 1. The PCR reaction solution composition and conditions were as follows.

[0082] The primers were used in the amounts (pmol / μL) shown in Table 4 below, at 0.2 μL per 10 μL of reaction solution.

[0083] [Table 4]

[0084] The virus strains used were as follows:

[0085] [Table 5] Potatoes 1-3, 4-3, 11, 12, 15, 16, 17, 18, and 19 are tuberous root samples suspected of being infected with the virus.

[0086] MC2: SPFMV, SPVG, SPLCV affected strains 1-36: SPFMV, SPVG, SPLCV affected strains Template nucleic acid concentration (undiluted) MC2: 366.8 (ng / μl) 1-36:403.5(ng / μl) Potato 1-3: 155.0 (ng / μl) Potato 4-3: 126.2 (ng / μl) Potato 11: 121.2 (ng / μl) Potato 12: 86.0 (ng / μl) Potato 15: 50.3 (ng / μl) Potato 16: 26.1 (ng / μl) Potato 17: 59.3 (ng / μl) Potato 18: 69.9 (ng / μl) Potato 19: 96.1 (ng / μl)

[0087] The PCR reaction was carried out for a total of 40 cycles, each cycle consisting of heat denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 30 seconds.

[0088] The PCR amplification products were verified by electrophoresis on an agarose gel. The results are shown in Figure 2. Although all of the results were verifiable, the three viruses were clearly identified under the conditions in which the primers of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, and SEQ ID NO:8 were used simultaneously, and good results were obtained. For the tuberous root samples suspected of virus infection, potatoes 1-3, 4-3, 11, 12, 15, 17, 18, and 19 were shown to be co-infected with SPFMV and SPVG, and potato 16 was shown to be infected with SPFMV.

[0089] (Example 4) Detection sensitivity of sweet potato pathogenic viruses using primer sets The detection sensitivity of the sweet potato pathogenic viruses using the primer set established in Example 3 was examined by serial dilution of the template nucleic acid concentration using the sweet potato pathogenic viruses SPFMV, SPVG, and SPLCV diseased strains. The viral nucleic acid was extracted in the same manner as in Example 1. The PCR reaction solution composition and conditions are as follows.

[0090] The primers were used in the amounts (pmol / μL) shown in Table 6 below, at 0.2 μL per 10 μL of reaction solution.

[0091] [Table 6]

[0092] The virus strains used were as follows:

[0093] 1-36: SPFMV, SPVG, SPLCV affected strains Template nucleic acid concentration 10 0 : 403.5 (ng / μl) 10 -1 : 40.35 (ng / μl) 10 -2 : 4.35 (ng / μl) 10 -3 : 403.5 (pg / μl) 10 -4 : 40.35 (pg / μl)

[0094] The PCR reaction was carried out for a total of 40 cycles, each cycle consisting of heat denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 30 seconds.

[0095] The PCR amplification products were verified by electrophoresis on an agarose gel, and the results are shown in Figure 3. Under the condition where the primers of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 8 were used simultaneously, the three types of viruses could be clearly identified, and the results were similar to those of PCR performed separately using the primer set of SEQ ID NO: 1 and SEQ ID NO: 3, the primer set of SEQ ID NO: 1 and SEQ ID NO: 4, and the primer set of SEQ ID NO: 7 and SEQ ID NO: 8.

[0096] (Reference Example 1) Comparison of properties of primer set with previously reported primers in Japan and overseas The nucleotide sequence information of the three viral genes was collected for isolates from around the world on the NCBI website. For those nucleotide sequences, multiple alignments including primer sequences were created using GENETYX ver.14. According to this comparison, among the virus isolates with 100% homology to the primer set of the present invention, there were occasional differences in the bases on the viral genome between the primers and those in the primers previously reported in Japan and those previously reported in China and Korea.

[0097] The previously reported primers used for comparison with the primer set of the present invention are shown in Table 7.

[0098] [Table 7]

[0099] The comparison results of primers for detecting SPFMV are shown in Table 8. The + in the table indicates 100% homology, and the numbers in the table indicate the number of different bases between the primer sequence and the viral genome. Additionally, ND in the table indicates that viral genome information for the primer recognition site is not registered in the database.

[0100] [Table 8]

[0101] The results of a comparison of the properties of primers for detecting SPFMV are shown in Table 9.

[0102] [Table 9]

[0103] The comparison results of primers for SPVG detection are shown in Table 10. The + in the table indicates 100% homology, and the numbers in the table indicate the number of different bases between the primer sequence and the viral genome. Additionally, the ND in the table indicates that the viral genome information of the primer recognition site is not registered in the database.

[0104] [Table 10]

[0105] The results of a comparison of the properties of primers for detecting SPVG are shown in Table 11.

[0106] [Table 11]

[0107] The comparison results of primers for detecting SPLCV are shown in Tables 12 and 13. The "+" in the table indicates 100% homology, and the numbers in the table indicate the number of different bases between the primer sequence and the viral genome. Additionally, the "ND" in the table indicates that the viral genome information of the primer recognition site is not registered in the database.

[0108] [Table 12] [Table 13]

[0109] The results of a comparison of the properties of primers for detecting SPLCV are shown in Table 14.

[0110] [Table 14]

[0111] As shown in Tables 8 to 14, it was found that by using the primer sets shown in SEQ ID NOs: 1 to 8 in the sequence listing, no non-specific amplification was observed and the target virus could be specifically detected.

[0112] (Example 5) Primer set design We collected information on the nucleotide sequences of viral genes from the NCBI website (https: / / www.ncbi.nlm.nih.gov / nuccore / ). We used GENETYX ver.14 to create multiple alignments of these nucleotide sequences. This comparison showed that the sequences of the coat protein gene and the cell-to-cell movement protein gene regions were conserved. Therefore, we thought that this conservation could be used to simultaneously detect different strains. We focused in particular on the SPVG and SPSMV-1 viruses, and designed multiple forward and reverse primers that are complementary to parts of the coat protein gene region and the cell-to-cell movement protein gene as candidate primers.

[0113] Based on the results of PCR analysis similar to that in Example 1, primer sets having the sequences shown in Table 15 below (the upper sequence in each column is the forward primer, and the lower sequence is the reverse primer) were finally determined, and primer sets composed of these primer sets were designed.

[0114] [Table 15]

[0115] (Example 6) Detection of sweet potato pathogenic viruses using primer sets and comparison of sensitivity Detection of sweet potato pathogenic viruses using the primer sets established in Examples 1 and 5 was examined using diseased strains of sweet potato pathogenic viruses SPFMV, SPVG, SPLCV, and SPSMV-1. Viral nucleic acid extraction was performed in the same manner as in Example 1. The PCR reaction solution composition and conditions were as follows.

[0116] The oligonucleotide primers shown in SEQ ID NOs: 1, 3, 7, 8, 9, 10, and 11 were used separately or together in the amounts shown below per 10 μL of reaction solution. The oligonucleotide primers shown in SEQ ID NOs: 1, 3, 7, 8, 9, 10, and 11 were, respectively, SEQ ID NO: 1: 10 μM, SEQ ID NO: 3: 5 μM, SEQ ID NO: 9: 2 μM, SEQ ID NOs: 7 and 8: 5 μM, and SEQ ID NOs: 10 and 11: 3 μM. The virus disease strains in FIG. 4 corresponded to the following template concentrations and types of primers used: Template nucleic acid concentration 10 0 : 403.5 (ng / μl) 10 -1 : 40.35 (ng / μl) 10 -2 : 4.35 (ng / μl) 10 -3 : 403.5 (pg / μl) 10 -4 : 40.35 (pg / μl)

[0117] The PCR reaction was carried out for a total of 40 cycles, each cycle consisting of heat denaturation at 94°C for 30 seconds, annealing at 52 to 56°C for 30 seconds, and extension at 72°C for 30 seconds.

[0118] The PCR amplification products were verified by agarose gel electrophoresis. The results are shown in Figure 4. Figure 4(a) shows the detection limit when the primer sets were used separately, while Figure 4(b) shows the detection sensitivity under multiplex RT-PCR conditions. Both were at a level that could be verified, demonstrating that highly sensitive detection is possible even under multiplex PCR conditions.

[0119] Example 7 Detection of sweet potato pathogenic viruses using primer sets Detection of sweet potato pathogenic viruses using the primer sets established in Examples 1 and 5 was examined using diseased strains of sweet potato pathogenic viruses SPFMV, SPVG, SPLCV, and SPSMV-1. Viral nucleic acid extraction was performed in the same manner as in Example 1. The PCR reaction solution composition and conditions were as follows.

[0120] The oligonucleotide primers shown in SEQ ID NOs: 1, 3, 7, 8, 9, 10, and 11 were used in the amounts shown below (μM) per 10 μL of reaction solution. The oligonucleotide primers shown in SEQ ID NOs: 1, 3, 7, 8, 9, 10, and 11 were, respectively, SEQ ID NO: 1: 10 μM, SEQ ID NO: 3: 5 μM, SEQ ID NO: 9: 2 μM, SEQ ID NOs: 7 and 8: 5 μM, and SEQ ID NOs: 10 and 11: 3 μM. The virus disease strains in FIG. 5 were adjusted to a template nucleic acid concentration of 300 to 500 ng per reaction.

[0121] The PCR reaction was carried out for a total of 35 cycles, each cycle consisting of heat denaturation at 94°C for 30 seconds, annealing at 52 to 56°C for 30 seconds, and extension at 72°C for 30 seconds.

[0122] The PCR amplification products were verified by agarose gel electrophoresis. The results are shown in Figure 5. In Figure 5, the leftmost lane shows the DNA marker, and the following four columns on the left show the amplification products of each virus-single infected strain. The seven lanes on the right show the verification results of sweet potatoes infected with the viruses in nature. Various infection patterns were confirmed by multiplex RT-PCR, demonstrating that it is a useful technique for simultaneously identifying four types of viruses.

[0123] Example 8 Primer set design Separately, information on the nucleotide sequence of the viral gene was collected from the NCBI website (https: / / www.ncbi.nlm.nih.gov / nuccore / ). A multiple alignment was created for those nucleotide sequences using GENETYX ver.14. This comparison revealed that even if the virus was the same, different lineages had different sequence regions, and multiple forward and reverse primers were designed as candidate primers.

[0124] Based on the results of PCR similar to those in Example 1, the primer sets having the sequences shown in Table 16 below (the upper sequences in each column are forward primers, and the lower sequences are reverse primers) were finally determined, and primer sets composed of these primer sets were designed.

[0125] [Table 16]

[0126] Example 9 Detection of sweet potato pathogenic viruses using primer sets Detection of sweet potato pathogenic viruses using the primer set established in Example 8 was examined using diseased strains of sweet potato pathogenic viruses SPFMV-RC, SPFMV-O, and SPFMV-C. Viral nucleic acid extraction was performed in the same manner as in Example 1. The composition and conditions of the PCR reaction solution are as follows.

[0127] The primers were used in the amounts (μM) shown below per 10 μL of reaction solution. The virus disease strains in FIG. 6 corresponded to the following types of primers used: Lanes 1 and 13: DNA marker Lanes 2-4: RC, O, C series primers (5 μM, 5 μM, 3 μM) Lanes 6-8: RC, O, C series primers (5 μM, 5 μM, 2.5 μM), Lanes 10-12: RC, O, C series primers (5 μM, 5 μM, 2 μM)

[0128] The template nucleic acid concentrations were adjusted to 300 ng / μl for the RC, O, and C strains.

[0129] The PCR reaction was carried out for a total of 35 cycles, each cycle consisting of heat denaturation at 94°C for 30 seconds, annealing at 52 to 56°C for 30 seconds, and extension at 72°C for 30 seconds.

[0130] The PCR amplification products were verified by agarose gel electrophoresis. The results are shown in Figure 6. Although all of the results were at a level where they could be verified, the RC, O, and C lineage viruses could be clearly identified under the conditions where the primers of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17 were used simultaneously, and good results were obtained.

[0131] Example 10 Efficiency verification of uniplex and multiplex PCR by primer set Detection of sweet potato pathogenic viruses using the primer set established in Example 8 was examined using diseased strains of sweet potato pathogenic viruses SPFMV-RC, SPFMV-O, and SPFMV-C. Viral nucleic acid extraction was performed in the same manner as in Example 1. The composition and conditions of the PCR reaction solution are as follows.

[0132] The primers for SPFMV-RC were an oligonucleotide having the base sequence shown in SEQ ID NO:12 and an oligonucleotide having the base sequence shown in SEQ ID NO:13, each at 5 μM. For SPFMV-O, an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 14 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 15 were each: 5 μM For SPFMV-C, an oligonucleotide consisting of the base sequence shown in SEQ ID NO:16 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO:17 were used at 3 μM each per 10 μL of reaction solution.

[0133] The results are shown in Figure 7. The virus disease strains in FIG. 7 corresponded to the following primers used: Lanes 1, 7, 13, 20: DNA marker; Lanes 2-6: detection limit of uniplex RT-PCR (RC lineage); Lanes 8-12: detection limit of uniplex RT-PCR (O lineage); Lanes 14-18: detection limit of uniplex RT-PCR (C lineage); Lanes 21-25: detection limit in multiplex RT-PCR.

[0134] The template nucleic acid concentration was 10 -0 : 300ng / μl, 10 -1 : 30ng / μl, 10 -2 : 3ng / μl, 10 -3 : 300pg / μl, 10 -4 :Adjusted to 30pg / μl.

[0135] The PCR reaction was carried out for a total of 35 cycles, each cycle consisting of heat denaturation at 94°C for 30 seconds, annealing at 54°C for 30 seconds, and extension at 72°C for 30 seconds.

[0136] The PCR amplification products were verified by agarose gel electrophoresis, and the results are shown in Figure 7. When the primers of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17 were used simultaneously, the RC, O, and C lineages could be clearly identified with the same sensitivity as when they were used separately, and good results were obtained.

[0137] Example 11 Detection of sweet potato pathogenic viruses using primer sets The infection status of various sweet potatoes was examined by detecting the sweet potato pathogenic virus using the primer set established in Example 8. The viral nucleic acid was extracted in the same manner as in Example 1. The PCR reaction solution composition and conditions were as follows.

[0138] The primers were used in the amounts (μM) shown below per 10 μL of reaction solution. The primers used in FIG. 8 were an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 14 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 15, each of which was 5 μM for SPFMV-O. For SPFMV-C, an oligonucleotide consisting of the base sequence shown in SEQ ID NO:16 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO:17 were used at 3 μM each per 10 μL of reaction solution.

[0139] The template nucleic acid concentration was adjusted to 100 to 500 ng / μl.

[0140] The PCR reaction was carried out for a total of 35 cycles, each cycle consisting of heat denaturation at 94°C for 30 seconds, annealing at 54°C for 30 seconds, and extension at 72°C for 30 seconds.

[0141] The PCR amplification products were verified by agarose gel electrophoresis. The results are shown in Figure 8. Various infection patterns were observed, demonstrating that this method is useful for identifying SPFMV strains in actual cultivated strains.

[0142] As shown in Figures 6 to 8, by using the primer set shown in SEQ ID NOs: 12 to 18 in the sequence listing, no non-specific amplification was observed and the target virus could be detected specifically and efficiently.

Claims

1. A primer set for detecting at least one sweet potato pathogenic virus selected from the group consisting of (2) to (10) below: (2) A primer set for detecting a sweet potato pathogenic virus, which is composed of an oligonucleotide having a base sequence shown in SEQ ID NO: 1 and an oligonucleotide having a base sequence shown in SEQ ID NO: 3; (3) A primer set for detecting a sweet potato pathogenic virus, which is composed of an oligonucleotide having a base sequence shown in SEQ ID NO: 1 and an oligonucleotide having a base sequence shown in SEQ ID NO: 4; (4) A primer set for detecting a sweet potato pathogenic virus, which is composed of an oligonucleotide having a base sequence shown in SEQ ID NO:5 and an oligonucleotide having a base sequence shown in SEQ ID NO:6; (5) A primer set for detecting a sweet potato pathogenic virus, which is composed of an oligonucleotide having a base sequence shown in SEQ ID NO: 7 and an oligonucleotide having a base sequence shown in SEQ ID NO: 8; (6) A primer set for detecting a sweet potato pathogenic virus, which is composed of an oligonucleotide having a base sequence shown in SEQ ID NO: 1 and an oligonucleotide having a base sequence shown in SEQ ID NO: 9; (7) A primer set for detecting a sweet potato pathogenic virus, comprising an oligonucleotide having the base sequence shown in SEQ ID NO: 10 and an oligonucleotide having the base sequence shown in SEQ ID NO: 11; (8) A primer set for detecting a sweet potato pathogenic virus, comprising an oligonucleotide having the base sequence shown in SEQ ID NO: 12 and an oligonucleotide having the base sequence shown in SEQ ID NO: 13; (9) A primer set for detecting a sweet potato pathogenic virus, comprising an oligonucleotide having the base sequence shown in SEQ ID NO: 14 and an oligonucleotide having the base sequence shown in SEQ ID NO: 15; (10) A primer set for detecting a sweet potato pathogenic virus, comprising an oligonucleotide having the base sequence shown in SEQ ID NO: 16 and an oligonucleotide having the base sequence shown in SEQ ID NO:

17.

2. A method for detecting a sweet potato pathogenic virus in a sample, comprising carrying out PCR using one or more of the primer sets according to claim 1 and analyzing the amplified fragments obtained by the PCR.

3. A method for detecting the presence of sweet potato pathogenic virus SPFMV in a sample by performing PCR using a primer set for detecting sweet potato pathogenic viruses, which is composed of an oligonucleotide having the base sequence shown in SEQ ID NO: 1 of the sequence listing and an oligonucleotide having the base sequence shown in SEQ ID NO: 3, and analyzing the amplified fragment obtained by the PCR.

4. A method for detecting the presence of sweet potato pathogenic viruses SPVG and SPV2 in a sample by performing PCR using a primer set for detecting sweet potato pathogenic viruses, which is composed of an oligonucleotide having the base sequence shown in SEQ ID NO: 1 of the sequence listing and an oligonucleotide having the base sequence shown in SEQ ID NO: 4, and analyzing the amplified fragment obtained by the PCR.

5. A method for detecting the presence of sweet potato pathogenic virus SPVG in a sample by performing PCR using a primer set for detecting sweet potato pathogenic virus, which is composed of an oligonucleotide having the base sequence shown in SEQ ID NO: 1 in the sequence listing and an oligonucleotide having the base sequence shown in SEQ ID NO: 9, and analyzing the amplified fragment obtained by the PCR.

6. A method for detecting the presence of sweet potato pathogenic virus SPLCV in a sample, comprising: a primer set for detecting sweet potato pathogenic virus, the primer set being composed of an oligonucleotide having the base sequence shown in SEQ ID NO:5 in the sequence listing and an oligonucleotide having the base sequence shown in SEQ ID NO:6; or a primer set for detecting sweet potato pathogenic virus, the primer set being composed of an oligonucleotide having the base sequence shown in SEQ ID NO:7 and an oligonucleotide having the base sequence shown in SEQ ID NO:8, and analyzing the amplified fragment obtained by the PCR.

7. A method for detecting the presence of sweet potato pathogenic virus SPSMV-1 in a sample by performing PCR using a primer set for detecting sweet potato pathogenic viruses, the primer set being composed of an oligonucleotide having the base sequence shown in SEQ ID NO:10 in the sequence listing and an oligonucleotide having the base sequence shown in SEQ ID NO:11, and analyzing the amplified fragment obtained by the PCR.

8. A method for detecting the presence of sweet potato pathogenic viruses SPFMV-RC, SPFMV-O, and SPFMV-C in a sample, comprising: a primer set for detecting sweet potato pathogenic viruses comprising an oligonucleotide having the base sequence shown in SEQ ID NO:12 of the sequence listing and an oligonucleotide having the base sequence shown in SEQ ID NO:13; a primer set for detecting sweet potato pathogenic viruses comprising an oligonucleotide having the base sequence shown in SEQ ID NO:14 and an oligonucleotide having the base sequence shown in SEQ ID NO:15; and / or an oligonucleotide having the base sequence shown in SEQ ID NO:16 and SEQ ID NO:17, performing PCR and analyzing the amplified fragment obtained by the PCR.

9. A kit for detecting a sweet potato pathogenic virus, comprising the primer set according to claim 1.

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