Method and kit for detecting plant virus

Abstract

To provide a gene diagnostic method for detecting tomatobrownrugosefruitvirus (ToBRFV).SOLUTION: A method for detecting ToBRFV or diagnosing ToBRFV infection, comprising a step of performing an amplification reaction of a target nucleic acid region of ToBRFV by real-time RT-PCR using a primer set for real-time RT-PCR against the genome sequence of ToBRFV.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a genetic diagnostic method for detecting tomato brown rugose fruit virus (ToBRFV), an emerging pathogen of solanaceous plants such as tomatoes. [Background technology]

[0002] Plants in the Solanaceae family include many agriculturally important crops, among which tomato (Solanum lycopersicum), bell peppers (Capsicum annuum, C. frutescens, etc.), and eggplant (Solanum melongena) are important crops cultivated not only in Japan but also around the world (Non-Patent Document 1 (Quinet et al., 2019)).

[0003] In these crops, diseases caused by viruses are causing many problems in various countries, and tobamoviruses (family Virgaviridae, genus Tobamovirus) in particular cause poor growth, leaf mosaics, and abnormal fruit, resulting in significant reductions in yield and quality. Because they are highly infectious through sap and seeds, they are becoming increasingly important with the recent expansion of international seed distribution.

[0004] Tobamoviruses have a genome of approximately 6,400 bases of positive-sense single-stranded RNA, encoding four proteins: 180K and 130K replication proteins (Rep), a cell-to-cell movement protein (MP), and a coat protein (CP) (Non-Patent Document 2 (Ishibashi and Ishikawa, 2016)). Based on the Master Species List (MSL) 39 (https: / / ictv.global / msl / current?fid=16752#block-teamplus-page-title) published by the International Committee on Taxonomy of Viruses (ICTV) in 2023, there are 37 recognized species, broadly classified into those that use plants such as Solanaceae, Cucurbitaceae, and Brassicaceae as hosts (Non-Patent Document 3 (Adams et al., 2017)). In Japan, six types of tobamoviruses have been reported to occur in Solanaceae crops such as tomatoes and peppers: tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV), mainly in tomatoes, and pepper mild mottle virus (PMMoV), tobacco mild green mosaic virus (TMGMV), paprika mild mottle virus (PaMMV), and Rehmannia mosaic virus (ReMV), mainly in bell peppers and chili peppers (Non-patent document 4 (Fuji et al., 2022); Non-patent document 5 (Committee on Plant Virus Taxonomy of the Phytopathological Society of Japan, 2021)). Although there have been few reports of damage caused by tobamoviruses in eggplant, natural occurrence of these tobamoviruses has been reported overseas (Non-Patent Document 6 (Aghamohammadi et al., 2013); Non-Patent Document 7 (Tobias et al., 1982)), and there have also been reported cases of ToMV in Japan (Non-Patent Document 8 (Nagai and Tochihara, 1974)).In inoculation tests, all six tobamoviruses can cause systemic infection in many strains of eggplant (Non-Patent Document 9 (Takeyama et al., 2023)). Tomato mottle mosaic virus (ToMMV) has also been detected in imported bell pepper seeds in Japan (Non-Patent Document 10 (Kon et al., 2024)).

[0005] Among these, tomato brown rugose fruit virus (ToBRFV) was discovered in Israel in 2014 and Jordan in 2015 (Non-Patent Document 11 (Luria et al., 2017); Non-Patent Document 12 (Salem et al., 2016)), and has since spread rapidly around the world, causing devastating damage to tomato production in many countries and regions (Non-Patent Document 13 (Salem et al., 2023)). When ToBRFV infects tomatoes, it can cause mosaics and abnormal fruit, resulting in zero yield. It also destroys the Tm-2 gene, a resistance gene used to control tobamoviruses such as ToMV. 2Because ToBRFV has complete resistance to the virus (Non-Patent Document 11 (Luria et al., 2017); Non-Patent Document 14 (Kubota et al., 2023)), countries have been on high alert for the virus's entry via seeds and seedlings. However, outbreaks have been reported in approximately 40 countries in the Middle East, Africa, Europe, America, and Asia to date (Non-Patent Document 13 (Salem et al., 2023)). Natural occurrence of ToBRFV has been reported not only in tomatoes but also in bell peppers in Italy and eggplants in Mexico (Non-Patent Document 15 (Panno et al., 2020); Non-Patent Document 16 (EPPO, 2020)), and systemic infection has also been confirmed by inoculation tests (Non-Patent Document 17 (Eldan et al., 2022); Non-Patent Document 14 (Kubota et al., 2023); Non-Patent Document 18 (Yan et al., 2021)). Furthermore, seed transmission has been confirmed in tomatoes and peppers (Non-Patent Document 19 (Davino et al., 2020); Non-Patent Document 13 (Salem et al., 2023); Non-Patent Document 20 (Matsushita et al., 2024)), and although seed transmission has not been observed in eggplant, seeds obtained from infected plants contain large amounts of infectious virus particles (Non-Patent Document 20 (Matsushita et al., 2024)).

[0006] In Japan, when tomato and bell pepper seeds are imported from overseas for cultivation, real-time RT-PCR (RT-qPCR) is used in the exporting country to confirm the absence of ToBRFV, and imported seeds are also inspected by the Plant Protection Station. However, eggplant seeds are not subject to this inspection. While ToBRFV has not been confirmed in domestic cultivation fields, given Japan's heavy reliance on imported seeds for cultivation of tomatoes and other crops, the possibility of ToBRFV escaping seed inspections and entering the country cannot be completely ruled out. The Ministry of Agriculture, Forestry and Fisheries (MAFF) will launch a nationwide "Invasion Survey Project" in April 2023 to conduct a nationwide survey of important pests, and has designated ToBRFV as one of the target pests (invasive and threatening pests) (Non-Patent Document 21 (MAFF, 2023)).

[0007] Countries around the world are on alert for the outbreak of ToBRFV, and several RT-qPCR methods have been developed for the detection and diagnosis of ToBRFV (Non-patent document 22 (ISHI-Veg, 2019) and Non-patent document 19 (Davino S, et al., 2020)).

[0008] However, these conventional RT-qPCR methods have the following problems and need to be improved when considering their use in diagnosing domestic outbreaks: (i) The primers were designed based on the sequences of only isolates from the affected country or a limited number of isolates, and it is unclear whether they can detect all ToBRFV isolates occurring worldwide; (ii) the unclear distinguishability of ToBRFV from other closely related solanaceous tobamoviruses; (iii) Although most RT-qPCRs developed for ToBRFV use TaqMan probes, there is no easy way to determine whether samples generated in the latter half of the reaction cycle are true positives or false positives (or the detection of other viruses); (vi) The applicability of this method to one-step RT-qPCR reagents commonly used in Japan has not been verified. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Quinet, M., Angosto, T., Yuste-Lisbona, FJ, Blanchard-Gros, R., Bigot, S., Martinez, JP and Lutts, S. (2019). Tomato fruit development and metabolism. Front. Plant Sci. 10: 1554. [Non-patent document 2] Ishibashi, K. and Ishikawa, M. (2016). Replication of tobamovirus RNA. Annu. Rev. Phytopathol. 54: 55-78. [Non-licensed document 3] Adams, MJ, Adkins, S., Bragard, C., Gilmer, D., Li, D., MacFarlane, SA, Wong, S.-M., Melcher, U., Ratti, C. and Ryu, KH (2017). ICTV Virus taxonomy profile: Virgaviridae. J. Gen. Virol. 98:1999-2000.

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Non-Patent Document 12

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Non-Patent Document 15

Outdoor Content 16

Outdoor Track 17

Outdoor Tools 18

Outdoor Tools 19

[0010] In light of the above-mentioned circumstances, the present invention aims to provide a method for accurately detecting and diagnosing ToBRFV by designing new ToBRFV-specific primers using RT-qPCR, based on the following indicators: the ability to detect a wide range of ToBRFV isolates based on the base sequences of all ToBRFV isolates registered in the database to date; the ability to minimize the detection of other tobamoviruses that use Solanaceae plants as hosts; and the ability to be used in real-time RT-PCR (RT-qPCR). [Means for solving the problem]

[0011] As a result of intensive research to solve the above problems, the inventors collected and compared all sequences of ToBRFV isolates reported worldwide (a total of 114 isolates), selected regions that are highly conserved among ToBRFV isolates but less conserved with tobamoviruses other than ToBRFV (Figure 1), designed two primer sets (NARO2 and NARO5) that target these regions, and found that ToBRFV can be specifically detected using one-step RT-qPCR with the SYBR Green method with these primer sets, thereby completing the present invention.

[0012] That is, the present invention includes the following. [1] A primer set (1) or (2) below for amplifying a nucleotide sequence specific to tomato brown rugose fruit virus (ToBRFV) by real-time RT-PCR. (1) a primer set consisting of a primer containing the nucleotide sequence set forth in SEQ ID NO: 1 and a primer containing the nucleotide sequence set forth in SEQ ID NO: 2; (2) A primer set consisting of a primer containing the nucleotide sequence set forth in SEQ ID NO: 3 and a primer containing the nucleotide sequence set forth in SEQ ID NO: 4. [2] A kit for detecting ToBRFV or diagnosing ToBRFV infection by real-time RT-PCR, comprising the primer set described in [1]. [3] A method for detecting ToBRFV or diagnosing ToBRFV infection, comprising a step of carrying out an amplification reaction of a target nucleic acid region of ToBRFV by real-time RT-PCR using the primer set described in [1] or the kit described in [2]. [4] The method described in [3], wherein the template RNA used in real-time RT-PCR is RNA derived from seeds of a Solanaceae plant to be detected for the presence or absence of ToBRFV infection. [Effects of the Invention]

[0013] According to the present invention, ToBRFV infection in seeds of solanaceous crops such as tomatoes can be detected simply and quickly.

[0014] The method for detecting ToBRFV or diagnosing ToBRFV infection according to the present invention is useful as a method for testing for viral contamination of seeds in inspections such as import and export quarantine of seeds such as tomato seeds, and in quality control during the seed production and distribution process. [Brief explanation of the drawings]

[0015] [Figure 1] The target sites of the primers according to the present invention in the alignment of genome sequences between ToBRFV isolates and representative isolates of seven species of tobamoviruses that use Solanaceae plants as hosts are shown. "ToBRFV-IL" is an isolate of ToBRFV. "PaMMV-J" is an isolate of paprika mild mottle virus. "PMMoV-J" is an isolate of pepper mild mottle virus. "ReMV-J" is an isolate of Rehmannia mosaic virus. "TMGMV-J" is an isolate of tobacco mild green mosaic virus. "TMV-OM" is an isolate of tobacco mosaic virus. "ToMV-L" is an isolate of tomato mosaic virus. "ToMMV-Sh" is an isolate of tomato mottle mosaic virus. [Figure 2A]1 shows amplification plots and melting curve plots for the detection of ToBRFV and other tobamovirus species by one-step RT-qPCR using the SYBR Green method, using the primer set NARO2 (A) and NARO5 (B) according to the present invention and the known primer set CSP1325 (C). [Figure 2B] Continuation of Figure 2A. [Figure 2C] Continuation of Figure 2B. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention will be described in detail below.

[0017] The primer set of the present invention is a primer set for amplifying a nucleotide sequence specific to tomato brown rugose fruit virus (ToBRFV) by real-time RT-PCR, and includes real-time RT-PCR primers that selectively hybridize to a specific nucleotide sequence in the genomic sequence of ToBRFV.

[0018] Real-time RT-PCR (one-step real-time RT-PCR) using the primer set of the present invention was used to verify the ability to detect ToBRFV and distinguish it from other viruses in seeds of Solanaceae plants such as tomatoes and bell peppers infected with ToBRFV and various tobamoviruses that have already emerged in Japan (seeds that mimic infection), and to evaluate the detection sensitivity.The real-time RT-PCR using the primer set of the present invention demonstrated good detection and discrimination capabilities and is therefore practical as a diagnostic method for ToBRFV infection in seeds, etc.

[0019] ToBRFV is a positive-sense single-stranded RNA virus belonging to the genus Tobamovirus in the family Virgaviridae, whose genome encodes four proteins: 180K and 130K replication proteins (Rep), a cell-to-cell movement protein (MP), and a coat protein (CP).

[0020] The primer set according to the present invention is shown in Table 1 below: (1) a primer set (NARO2) consisting of a primer (NARO2-F; forward primer) containing or consisting of the nucleotide sequence set forth in SEQ ID NO: 1 and a primer (NARO2-R; reverse primer) containing or consisting of the nucleotide sequence set forth in SEQ ID NO: 2; or (2) A primer set (NARO5) consisting of a primer (NARO5-F; forward primer) containing or consisting of the nucleotide sequence set forth in SEQ ID NO: 3 and a primer (NARO5-R; reverse primer) containing or consisting of the nucleotide sequence set forth in SEQ ID NO: 4. is.

[0021] Furthermore, the primer of the present invention may be capable of specifically annealing to the nucleotide sequence of the ToBRFV genome or its complementary sequence, and may have the same or substantially the same nucleotide sequence as the nucleotide sequence of the genome or its complementary sequence. Here, "substantially the same nucleotide sequence" means a nucleotide sequence that can specifically anneal to the nucleotide sequence of the ToBRFV genome or its complementary sequence, even if it differs from the nucleotide sequences set forth in SEQ ID NOs: 1 to 4 by one or several bases (for example, 1 to 3 bases, preferably 1 or 2 bases).

[0022] Alternatively, the primers may alternatively include primers having a base sequence in which one or several (e.g., 1 to 10, 1 to 5, 1 to 3, preferably 1 or 2) bases have been deleted, substituted, inserted or added in the base sequence shown by the SEQ ID NO of each primer, and which have the respective primer functions in real-time RT-PCR.

[0023] The present invention also relates to a kit for detecting ToBRFV or diagnosing ToBRFV infection by real-time RT-PCR, which includes the primer set of the present invention. The kit may further include, for example, reverse transcriptase and DNA polymerase used in RT-PCR; nucleic acid synthesis substrates (dNTPs), buffer solutions, salts, containers, etc. used in RT-PCR; reagents necessary for detecting the real-time RT-PCR amplification product (e.g., a staining reagent (intercalator) capable of detecting nucleic acids); instructions for use, etc.

[0024] Furthermore, the present invention relates to a method for detecting ToBRFV or diagnosing ToBRFV infection (hereinafter referred to as "the method"), which includes a step of carrying out a real-time RT-PCR amplification reaction of a target nucleic acid region of ToBRFV using the primer set or kit according to the present invention described above. The method can also be said to be a method for identifying Solanaceae plants infected with ToBRFV. The method includes a step of carrying out a nucleic acid amplification reaction using the primer set according to the present invention, and specifically utilizes real-time (quantitative) RT-PCR. In particular, one-step real-time RT-PCR can be preferably used in the method. The presence or absence of ToBRFV infection can be tested or evaluated by the method.

[0025] In this method, RNA is first extracted and purified from a sample derived from a plant of the Solanaceae family (e.g., tomato (Solanum lycopersicum), bell peppers (Capsicum annuum, C. frutescens, etc.), or eggplant (Solanum melongena)) to be tested for ToBRFV infection. Examples of samples derived from Solanaceae plants include whole plants, plant organs (e.g., leaves, pedicels, petioles, petals, stems, roots, seeds, and fruits), plant tissues (e.g., epidermis, phloem, parenchyma, xylem, and vascular bundles), and cultured plant cells. In particular, seed samples can be used to test for viral contamination in Solanaceae seeds during import and export quarantine inspections and quality control during seed production and distribution. These samples may exhibit typical symptoms, such as mosaicism, or may be asymptomatic. For example, the detection rate can be increased by using a leaf section affected by mosaicism.

[0026] Examples of RNA extraction methods include a method using a commercially available RNA extraction reagent, and a method in which a sample is triturated with Tris buffer or sterilized water and diluted to obtain crudely purified RNA.

[0027] Next, in this method, RT-PCR, preferably one-step RT-PCR, is performed. For example, purified RNA is used as a template and subjected to one-step RT-PCR using reverse transcriptase, a heat-stable DNA polymerase, and the primer set of the present invention. The RT-PCR reaction solution is prepared so that, per 25 μL of reaction solution, each primer included in the primer set of the present invention is present at a final concentration of 50 to 500 nM (preferably 100 to 300 nM), 0.05 to 5 μL (preferably 0.2 to 2 μL) of RNA solution, 100 to 1000 U (preferably 400 to 600 U) of reverse transcriptase, 0.2 to 5 U (preferably 1 to 2 U) of DNA polymerase, and dNTPs at final concentrations of 100 to 800 μM (preferably 300 to 500 μM). Thermal cycling conditions for RT-PCR include, for example, reverse transcription: 30 to 80°C (preferably 50 to 70°C) for 5 to 60 minutes (preferably 20 to 40 minutes), reverse transcriptase inactivation: 90 to 99°C (preferably 94 to 98°C) for 0.5 to 30 minutes (preferably 1 to 5 minutes), followed by 15 to 45 cycles (e.g., 30 to 40 cycles) of denaturation: 90 to 99°C (preferably 94 to 98°C) for 5 to 60 seconds (preferably 10 to 20 seconds), annealing: 40 to 75°C (preferably 60 to 70°C) for 5 to 60 seconds (preferably 10 to 20 seconds), and extension: 60 to 80°C (preferably 70 to 75°C) for 5 to 60 seconds (preferably 40 to 50 seconds). Melting curve analysis after the PCR reaction involves, for example, denaturation treatment at 90 to 99°C (preferably 93 to 96°C) for 5 to 30 seconds (preferably 10 to 20 seconds), annealing at 50 to 70°C (preferably 55 to 65°C) for 0.5 to 5 minutes (preferably 0.5 to 2 minutes), and then increasing the temperature at, for example, 0.01 to 2°C / second, preferably 0.05 to 0.2°C / second, and particularly preferably 0.1°C / second, and measuring the fluorescent signal every 0.05 to 0.5°C (preferably every 0.2 to 0.3°C).

[0028] In this method, real-time RT-PCR is used. For example, PCR can be performed using, in addition to the reagents used in the RT-PCR method, a staining reagent capable of detecting nucleic acids (an intercalator such as SYBR Green) or a fluorescent probe that complementarily binds to the amplified region (for example, a fluorescent probe that has a base sequence that can specifically anneal to a sequence present between the regions to which the primers of the present invention anneal, and is labeled with a reporter at the 5' end and a quencher at the 3' end), and the amplified product can be quantified using the fluorescent signal as an indicator.

[0029] For example, real-time RT-PCR is performed using serially diluted, known amounts of ToBRFV-derived RNA as a standard, and a calibration curve is created by plotting the threshold cycle (Ct value) at which a certain amount of amplification product is obtained on the vertical axis against the amount of added RNA on the horizontal axis. Real-time RT-PCR is also performed on a sample to be detected to determine the Ct value, and the amount of RNA in the sample to be detected can be determined from the resulting calibration curve.

[0030] Specificity can also be determined by melting temperature (Tm) values. In melting curve analysis after PCR, if the Tm value of the positive control (e.g., 81.0 to 81.6°C (preferably 81.2°C) for the NARO2 primer set (FIG. 2A), and 81.5 to 82.0°C (preferably 81.7°C) for the NARO5 primer set (FIG. 2B)) matches the Tm value of the sample to be detected, it is determined that the target amplification product has been obtained. If the Tm values ​​differ, it can be determined that nonspecific amplification has occurred. [Example]

[0031] The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to these examples.

[0032] [Real-time RT-PCR (SYBR Green method) using the primer set (NARO2, NARO5) according to the present invention] We investigated the specificity (discrimination ability) of real-time RT-PCR (SYBR Green method) using the primer set (NARO2, NARO5) of the present invention for the target ToBRFV and six other closely related tobamoviruses that infect Solanaceae, which are not the target.

[0033] 1. Materials and Methods The sample was an RNA solution prepared by adding 360 pg (equivalent to 10^8 copies) of each tobamovirus genomic RNA to RNA (150 ng) extracted from healthy tomato or bell pepper seeds.

[0034] The evaluation items were the threshold cycle (Ct) value and the melting temperature (Tm) value. The greater the difference between ToBRFV and other viruses, the higher the discrimination ability.

[0035] The primer sets tested are shown in Table 1 below. The CSP1325 primer set is a known primer set (ISHI-Veg, 2019). "Position" is the position in the ToBRFV genome sequence to which the primer corresponds, and "Product size (bp)" is the size of the amplified product by RT-PCR using each primer set.

[0036] [Table 1]

[0037] The virus strains tested are shown in Table 2. A series of tests were conducted using the ToBRFV DSMZ PV-1241 strain (GenBank Accession No. MZ202349), which was isolated from a tomato in Israel, under the import permit conditions of the Minister of Agriculture, Forestry and Fisheries (Ministry of Agriculture, Forestry and Fisheries Directive Yokoseki No. 503).

[0038] [Table 2]

[0039] The composition of the real-time RT-PCR reaction solution is shown in Table 3 below. "Primer Fw" is a forward primer (F or Fw), and "primer Rv" is a reverse primer (R or Rv).

[0040] [Table 3]

[0041] The combinations of healthy seed RNA and viral RNA used in sample preparation are shown in Table 4 below.

[0042] [Table 4]

[0043] Finally, the temperature conditions for real-time RT-PCR and melting curve analysis are shown in Table 5 below.

[0044] [Table 5]

[0045] 2. Results and Discussion The results are shown in Figure 2. Ct value: For both primers, the Ct value for ToBRFV was around 12, while for other viruses it was 30-35, with a difference in Ct value of around 20, and the detection sensitivity and specificity (discrimination) were comparable. Tm value: For both primers, a sharp peak was formed around 80°C for ToBRFV, whereas a broad peak was formed for the other viruses. The difference in Tm value between the two viruses was the largest for NARO2, at more than 3°C.

[0046] In addition, for CSP1325, the peak Tm value of ToBRFV was 79.8°C, while that of the other viruses was approximately 79°C, and the difference in Tm values ​​between the two viruses was small, making it difficult to clearly distinguish between them.

[0047] 〔Cited Literature〕 Aghamohammadi, V., Rakhshandehroo, F., Shams-Bakhsh, M. and Palukaitis, P. (2013). Distribution and genetic diversity of tomato mosaic virus isolates in Iran. J. Plant. Pathol. 95: 339-347. Davino S, Caruso AG, Bertacca S, Barone S, Panno S (2020) Tomato Brown Rugose Fruit Virus: Seed Transmission Rate and Efficacy of Different Seed Disinfection Treatments. Plants (Basel). 9:1615. doi: 10.3390 / plants9111615. Eldan, O., Ofir, A., Luria, N., Klap, C., Lachman, O., Bakelman, E., Beausov, E., Smith, E. and Dombrovsky, A. (2022). Pepper plants harboring L resistance alleles showed tolerance toward manifestations of tomato brown rugose fruit virus disease. Plants 11: 2378. https: / / doi.org / 10.3390 / plants11182378 EPPO (2020). Tomato brown rugose fruit virus. EPPO Bulletin 50: 529-534. Fuji, S., Mochizuki, T., Okuda, M., Tsuda, S., Kagiwada, S., Sekine, K.-T., Ugaki, M., Natsuaki, K.T., Isogai, M., Maoka, T., Takeshita, M., Yoshikawa, N., Mise, K., Sasaya, T., Kondo, H., Kubota, K., Yamaji, Y., Iwanami, T., Ohshima, K., Kobayashi, K., Hataya, T., Sano, T. and Suzuki, N. (2022). Plant viruses and viroids in Japan. J. Gen. Plant Pathol. 88: 105-127. Hamada, H., Takeuchi, S., Morita, Y., Sawada, H., Kiba, A. and Hikichi, Y. (2003). Characterization of Paprika mild mottle virus first isolated in Japan. J. Gen. Plant Pathol. 69: 199-204. Ishibashi, K. and Ishikawa, M. (2016). Replication of tobamovirus RNA. Annu. Rev. Phytopathol. 54: 55-78. ISHI-Veg (2019). Detection of infectious Tomato brown rugose fruit virus (ToBRFV) in tomato and pepper seed. International Seed Federation. https: / / worldseed.org / wp-content / uploads / 2019 / 09 / Tomato-ToBRFV_2019.09.pdf Kirita, M., Akutsu, K., Watanabe, Y. and Tsuda, S. (1997). Nucleotide sequence of the Japanese isolate of pepper mild mottle tobamovirus (TMV-P) RNA. Ann. Phytopathol. Soc. Jpn. 63: 373-376. Kon, T., Sato, C. and Fuji, S. (2024). Molecular characterization and host reaction to tomato mottle mosaic virus isolated from sweet pepper seeds in Japan. Arch. Virol. 169: 113 Kubota K, Takeyama S, Ishibashi K, Matsushita Y, Tomitaka Y, Matsuyama M, Shinosaka H, ​​and Osaki K (2023). Host range and pathogenicity of Tomato brown rugose fruit virus. Japanese Journal of Plant Pathology 89: 225-234. Kubota, K., Usugi, T., Tomitaka, Y., Matsushita, Y., Higashiyama, M., Kosaka, Y. and Tsuda, S. (2012). Characterization of Rehmannia mosaic virus isolated from chili pepper (Capsicum annuum) in Japan. J. Gen. Plant Pathol. 78:43-48. Kubota, K., Takeyama, S., Matsushita, Y. and Ishibashi, K. (2024). Isolation of spontaneous mutants of tomato brown rugose fruit virus that efficiently infect Tm-1 homozygote tomato plants. J. Gen. Plant Pathol. 90: 187-195. Luria, N., Smith, E., Reingold, V., Bekelman, I., Lapidot, M., Levin, I., Elad, N., Tam, Y., Sela, N., Abu-Ras, A., Ezra, N., Haberman, A., Yitzhak, L., Lachman, P. and Dombrovsly, A. (2017). A new Israeli tobamovirus isolate infects tomato plants harboring Tm-2 2 resistance genes. PLoS One 12: e01704. Matsushita, Y., Takeyama, S., Tomitaka, Y., Matsuyama, M., Ishibashi, K., Shinosaka, H., Osaki, K. and Kubota, K. (2024). Elucidating the nature of seed-borne transmission of tomato brown rugose fruit virus in tomato, bell pepper, and eggplant. J. Gen. Plant Pathol. 90: 23-34. Adams, M.J., Adkins, S., Bragard, C., Gilmer, D., Li, D., MacFarlane, S.A., Wong, S.-M., Melcher, U., Ratti, C. and Ryu, K.H. (2017). ICTV Virus taxonomy profile: Virgaviridae. J. Gen. Virol. 98:1999-2000. Morishima, N., Ido, T., Hamada, H., Yoshimoto, E., Mizumoto, H., Takeuchi, S., Kiba, A., Hikichi, Y. and Okuno, T. (2003). Infectious in vitro transcripts from a cDNA clone of Tobacco mild green mosaic tobamovirus and its biological activity in host and nonhost plants and in their protoplasts. J. Gen. Plant Pathol. 69: 335-338. Committee on Plant Virus Taxonomy, Japanese Phytopathological Society (2021). Plant viruses and viroids occurring in Japan. https: / / www.ppsj.org / pdf / mokuroku-viroid_2021.pdf?1005 Nagai, Y. and Tochihara, H. (1974). Mosaic disease of eggplant caused by TMV. Jpn. J. Plant Pathology 40: 212. (Abstract) Ministry of Agriculture, Forestry and Fisheries (2023). About the Invasion Survey Project. https: / / www.maff.go.jp / j / syouan / syokubo / keneki / k_kokunai / shinnyuuchousa / shinnyuuchousa.html. Nozu, Y. and Okada, Y. (1968). Amino acid sequence of a common Japanese strain of tobacco mosaic virus. J. Mol. Biol. 35:643-646. Ohno, T., Aoyagi, M., Yamanashi, Y., Saito, H., Ikawa, S., Meshi, T. and Okada, Y. (1984). Nucleotide sequence of the tobacco mosaic virus (tomato strain) genome and comparison with the common strain genome. J. Biochem. 96: 1915-1923. Panno, S., Caruso, A.G., Blanco, G., and Davino, S. (2020). First report of Tomato brown rugose fruit virus infecting sweet pepper in Italy. New Dis. Rep. 41: 20. doi:10.5197 / j.2044-0588.2020.041.020. Quinet, M., Angosto, T., Yuste-Lisbona, F.J., Blanchard-Gros, R., Bigot, S., Martinez, J.P. and Lutts, S. (2019). Tomato fruit development and metabolism. Front. Plant Sci. 10: 1554. Salem, N., Mansour, A., Ciuffo, M., Falk, B.W. and Turina, M. (2016). A new tobamovirus infecting tomato crops in Jordan. Arch. Virol. 161: 503-506. Salem, NM, Jewehan, A., Aranda, MA and Fox, A. (2023). Tomato brown rugose fruit virus pandemic. Annu. Rev. Phytopathol. 61: 14.1-14.18. https: / / doi.org / 10.1146 / annurev-phyto-021622-120703 Tobias, I., Rast, A.Th.B. and Maat, DZ (1982). Tobamoviruses of pepper, eggplant and tobacco: comparative host reactions and serological relationships, Neth. J. Pl. 88: 257-268. Takeyama, Sawana, Miyatake, K., and Kubota, K. (2023). Evaluation of resistance differences among tobamovirus-resistant lines selected from eggplant genetic resources. Program Abstracts of the 70th Annual Meeting of the Japanese Society for Virology. p. 341. (Abstract) Yan, Z., Zhao, M., Ma, H., Liu, L., Yang, G., Geng, C., Tian, ​​Y. and Li, X. (2021). Biological and molecular characterization of tomato brown rugose fruit virus and development of quadruplex RT-PCR detection. J. Integr. Agric. 20: 1871-1879. https: / / doi.org / 10.1016 / S2095-3119(20)63275-0

Claims

1. A primer set (1) or (2) below for amplifying a nucleotide sequence specific to tomato brown rugose fruit virus (ToBRFV) by real-time RT-PCR. (1) a primer set consisting of a primer containing the nucleotide sequence set forth in SEQ ID NO: 1 and a primer containing the nucleotide sequence set forth in SEQ ID NO: 2; (2) A primer set consisting of a primer containing the base sequence set forth in SEQ ID NO: 3 and a primer containing the base sequence set forth in SEQ ID NO:

4.

2. A kit for detecting ToBRFV or diagnosing ToBRFV infection by real-time RT-PCR, comprising the primer set according to claim 1.

3. A method for detecting ToBRFV or diagnosing ToBRFV infection, comprising a step of carrying out an amplification reaction of a target nucleic acid region of ToBRFV by real-time RT-PCR using the primer set according to claim 1 or the kit according to claim 2.

4. The method according to claim 3, wherein the template RNA used in the real-time RT-PCR is RNA derived from seeds of a solanaceous plant to be detected for the presence or absence of ToBRFV infection.

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