Plant RNA virus detection methods

Abstract

The present invention provides a plant RNA virus detection method that is highly efficient in detection while achieving labor savings and lower costs compared to conventional techniques. [Solution] A plant RNA virus detection method comprising a step of heat-treating a mixture obtained by grinding a plant, the mixture being mixed with a primer solution, wherein the heat-treating step heats the mixture at 60°C to 98°C for 4 to 6 minutes. Alternatively, the plant RNA virus detection method is one in which the plant grinding mixture is obtained by grinding plant tissue together with a solution containing at least one selected from the group consisting of a buffer solution and RNase-free water.

Description

[Technical Field]

[0001] The present invention relates to a method for detecting plant RNA viruses. [Background technology]

[0002] RT-PCR is a conventional method for detecting viruses in plants suspected of being infected with a virus. In this method, RNA is purified from plant tissues suspected of being infected with a virus, and cDNA is synthesized using this as a template. PCR is then performed using primers containing the viral gene region to amplify the synthesized cDNA. If the plant is infected with a virus, DNA containing the viral gene region will be amplified, allowing the virus to be detected.

[0003] Improvements have been disclosed for virus detection methods using RT-PCR to improve virus detection efficiency and reduce labor. RT-PCR is divided into one-step RT-PCR, in which cDNA synthesis and PCR are carried out in one reaction, and two-step RT-PCR, in which cDNA synthesis and PCR are carried out in two reactions. One example of an improvement of two-step RT-PCR is the RT-PCR described in Non-Patent Document 1, and one example of an improvement applicable to both one-step and two-step RT-PCR is the RT-PCR described in Non-Patent Document 2.

[0004] In the RT-PCR described in Non-Patent Document 1, a purified RNA solution and a primer solution for cDNA synthesis are heat-treated at 65°C for 5 minutes before the cDNA synthesis reaction is carried out. This improves detection efficiency. In the RT-PCR described in Non-Patent Document 2, the step of purifying RNA from plant tissue is omitted, and the homogenate obtained by grinding the plant is used in RT-PCR directly or after heat treatment, thereby achieving labor savings. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Takara Bio Catalog PrimeScript II High Fidelity RT PCR Kit https: / / catalog.takara-bio.co.jp / com / tech_info_detail.php?mode=2&masterid=M100004593&unitid=U100006031 [Non-patent document 2] Hirano, Simple detection of viruses by (RT-)PCR method using crude juice, Research Report of Saitama Prefectural Agriculture and Forestry Research Center, 2005, No. 4, p61-66 Summary of the Invention [Problem to be solved by the invention]

[0006] However, the RT-PCR of Non-Patent Document 1 can be problematic in terms of the burden on workers and the cost of reagents in the process of purifying RNA from plant tissues, while the RT-PCR of Non-Patent Document 2 can achieve labor savings but can also be problematic in terms of detection efficiency.

[0007] In light of the above circumstances, the present invention aims to provide a plant RNA virus detection method that is highly efficient in detection while achieving less labor and lower costs than conventional techniques. [Means for solving the problem]

[0008] In order to solve the above problems, one aspect of the present invention includes the following aspects.

[0009] [1] A method for detecting plant RNA viruses, comprising a step of heat-treating a mixture obtained by grinding a plant and mixing the mixture with a primer solution, wherein the heat-treating step heat-treats the mixture at 60°C to 98°C for 4 to 6 minutes. [2] The plant RNA virus detection method according to [1], wherein the plant homogenate is obtained by homogenizing plant tissue together with a solution containing at least one selected from the group consisting of a buffer solution and RNase-free water. [3] The plant RNA virus detection method according to [1] or [2], wherein the heat-treated mixture is used in one-step RT-PCR. [4] The plant RNA virus detection method according to [3], wherein the DNA amplified by the one-step RT-PCR is subjected to agarose gel electrophoresis. [Effects of the Invention]

[0010] According to the present invention, a plant RNA virus detection method can be provided that is highly efficient in detection while realizing labor savings and cost reduction compared to conventional techniques. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram illustrating a plant RNA virus detection method according to this embodiment. [Figure 2] 1 shows agarose gel electrophoresis of genetic sequences derived from plant RNA viruses amplified by the plant RNA virus detection method of this embodiment and by conventional techniques. DETAILED DESCRIPTION OF THE INVENTION

[0012] An embodiment of the present invention will be described with reference to Figures 1 and 2. In all the following figures, the dimensions and proportions of the components have been changed as appropriate to make the drawings easier to understand.

[0013] The following embodiments are merely examples of configurations for embodying the technical concept of the present invention, and the present invention is not limited to these embodiments. Various modifications can be made to the technical concept of the present invention within the technical scope defined by the claims.

[0014] In one embodiment, the present invention provides a plant RNA virus detection method, comprising a step of heat-treating a mixture obtained by grinding a plant and mixing the mixture with a primer solution, wherein the heat-treating step heat-treats the mixture at 60°C to 98°C for 4 to 6 minutes. An outline of the plant RNA virus detection method of this embodiment is shown in Figure 1.

[0015] The detection target in the present invention is an RNA virus that infects plants, but is not particularly limited thereto. Examples of viruses to be detected include, but are not limited to, one or more viruses selected from the group consisting of potato leaf curl virus (PLRV), potato virus S (PVS), potato virus X (PVX), potato virus Y (PVY), alfalfa mosaic virus (AMV), cucumber mosaic virus (CMV), potato yellow mosaic virus (PAMV), potato mop top virus (PMTV), potato virus A (PVA), potato virus M (PVM), tomato ringspot virus (ToRSV), tomato spotted wilt virus (TSWV), beet yellow leaf virus (BLYV), wheat dwarf virus (SBWMV), and wheat yellow dwarf virus (WYMV).

[0016] (Plant grinding liquid) The plant homogenate is obtained by grinding the whole plant or a part of the plant. The plant used as the source of the plant homogenate is not particularly limited. Examples of plants include, for example, the Solanaceae family. Examples of Solanaceae plants include, but are not limited to, potato and Nicotiana benthamiana. The plant parts that can be used as the source of the plant homogenate are not particularly limited. Examples of plant parts include, but are not limited to, one or more parts selected from the group consisting of leaves, petioles, stems, roots, rhizomes, petals, fruits, and seeds. The plant used for the plant homogenate may be one or more plant tissues selected from the group consisting of the epidermis, phloem, parenchyma, xylem, and vascular bundles of the plant, or may be plant-derived cultured cells, etc.

[0017] Any known method can be used to grind plants and obtain a plant homogenate, as long as it does not destroy plant viral RNA or cause contamination of the plant homogenate. Methods for grinding plants and obtaining a plant homogenate include, for example, placing the plants and a buffer solution in a plastic bag and grinding them using a test tube placed over the bag, grinding the plants using a mortar and pestle, or adding the plants and a buffer solution to a grinding tube containing zirconia beads and grinding them using a homogenizer. Alternatively, a plant homogenate can be obtained using a conventional method for obtaining RNA from plant samples.

[0018] (Buffer solution and RNase-free water) At least one selected from the group consisting of a buffer solution and RNase-free water may be added to the ground plant. A known buffer solution can be used as the buffer solution added to the ground plant. Examples of buffer solutions that can be added to the ground plant include, but are not limited to, phosphate-buffered saline. A known surfactant or the like may be added to the at least one selected from the group consisting of a buffer solution and RNase-free water. It is recommended to confirm the amount of the at least one selected from the group consisting of a buffer solution and RNase-free water to be added to the ground plant by conducting a preliminary experiment in advance. When detecting viruses, it is recommended to determine the amount of the at least one selected from the group consisting of a buffer solution and RNase-free water to be added to the ground plant based on the results of the preliminary experiment.

[0019] The plant homogenate of the present invention can be used for cDNA synthesis and PCR without undergoing an RNA extraction step or adding an RNA extraction reagent, which is expected to reduce labor by omitting the RNA extraction step and reduce costs by not using an RNA extraction reagent.

[0020] (primer solution) The primer solution is a solution containing single-stranded DNA (hereinafter referred to as a primer) that contains a sequence complementary to at least one end of the sequence to be amplified in the gene of the plant RNA virus to be detected. The primer may be one type or two or more types depending on the number of species of RNA virus to be detected. The primer can be prepared by a known method. The primer may be diluted with a known buffer solution. An example of a buffer solution for diluting the primer is Tris buffer, but it is not limited to this.

[0021] (mixed liquid) The mixed solution is a solution obtained by mixing the plant homogenate and the primer solution. A known method may be used to mix the plant homogenate and the primer solution. The mixing ratio of the plant homogenate and the primer solution may be based on a known method, but it is preferable to confirm the ratio by conducting a preliminary experiment in advance. When detecting viruses, it is preferable to determine the mixing ratio of the plant homogenate and the primer solution based on the results of the preliminary experiment.

[0022] (Heat treatment of the mixed liquid) The mixture is heat-treated before being used in a PCR reaction. The heat treatment temperature is preferably 50°C to 99°C, more preferably 55°C to 99°C, and even more preferably 60°C to 98°C. The temperature at which the mixture is heat-treated should be confirmed in advance by a preliminary experiment. When detecting viruses, the temperature at which the mixture is heat-treated should be determined based on the results of the preliminary experiment.

[0023] The time for heat treatment is preferably 2 to 10 minutes, more preferably 4 to 7 minutes, and even more preferably 5 to 6 minutes. The time for heat treatment of the mixture should be confirmed in advance by a preliminary experiment. When detecting viruses, the time for heat treatment of the mixture should be determined based on the results of the preliminary experiment.

[0024] Heat treatment should be applied to the mixture after mixing the two, not to the plant homogenate or the primer solution before mixing. Heat treatment of the mixture is thought to promote binding between the RNA molecules of the RNA virus contained in the plant homogenate and the DNA molecules of the primer, improving the efficiency of the RT-PCR reaction.

[0025] (One-step RT-PCR) The mixture prepared by the above method can be used for RT-PCR. For RT-PCR, one-step RT-PCR, in which cDNA synthesis and PCR are performed in the same vessel, is preferred. One-step RT-PCR allows for efficient cDNA synthesis of the RNA sequence of the RNA virus to be detected and DNA amplification by PCR, which is expected to simplify the process and reduce the risk of contamination.

[0026] (Detection of amplified DNA) The DNA amplified by PCR can be detected by known methods, including, but not limited to, agarose gel electrophoresis. Detection of the DNA allows the detection of RNA viruses infecting the plants used.

[0027] (Effects of the present invention) In conventional technology, when a solution containing plant-derived RNA was mixed with a primer solution and heated, a step of extracting RNA from the plant homogenate was required prior to heating. However, by mixing the plant homogenate with a primer solution and heat-treating it without going through the RNA extraction step, the process has been simplified and labor-saving. Furthermore, compared to conventional technology in which plant homogenate is used for RT-PCR with or without heat treatment, mixing it with a primer solution and applying heat treatment can improve detection sensitivity.

[0028] In this embodiment, the solution containing plant-derived RNA is heat-treated at a higher temperature than in conventional techniques. The inventors discovered that performing heat treatment at different temperatures depending on the target virus improves virus detection sensitivity compared to heat treatment at temperatures considered preferable in conventional techniques, and thus completed the present invention. [Example]

[0029] The effects of the present invention will be made clearer by the following examples. Note that the present invention is not limited to the following examples, and can be practiced by making appropriate modifications within the scope of the present invention.

[0030] Example 1 Potato plants were infected with potato leaf curl virus (PLRV), potato virus S (PVS), potato virus X (PVX), and potato virus Y (PVY). Infected potato leaf pieces were placed in a 0.1-mm-thick plastic bag and 5 μl of 0.05% TWEEN phosphate-buffered saline (pH 7.4) per mg leaf piece. The pieces were then ground using the corner of a test tube and diluted 100-fold with RNase-free water to obtain plant homogenates. Each plant homogenate was mixed with a primer solution containing the corresponding viral primer and then heat-treated; the plant homogenate and the corresponding primer solution were then mixed; the plant homogenate alone was heat-treated and then mixed with the primer solution; or the plant homogenate and primer solution were mixed without heat treatment. Heat treatment was performed at 70°C for 5 minutes. The composition of the primer solution was as follows:

[0031] The primer solution used was an unpublished primer set for detecting PLRV, PVS, PVX, and PVY.

[0032] Each mixture prepared as described above was used in one-step RT-PCR to amplify DNA. The amplified DNA was detected by agarose gel electrophoresis, and the band position and clarity were compared for each condition. The results are shown in Figure 2.

[0033] Figure 2 shows agarose gel electrophoresis of genetic sequences derived from plant RNA viruses amplified by the plant RNA virus detection method of this embodiment and by conventional techniques. PLRV stands for potato leaf curl virus, PVS for potato virus S, PVX for potato virus X, and PVY for potato virus Y. The base length following each virus abbreviation indicates the length of the PCR product.

[0034] The numbers next to each lane under each virus indicate the RT-PCR conditions described above. Lane 1 shows the results of mixing the plant homogenate and primer solution, followed by heat treatment, followed by one-step RT-PCR. Lane 2 shows the results of mixing the plant homogenate and primer solution, each of which was heat-treated, followed by one-step RT-PCR. Lane 3 shows the results of heat-treating only the plant homogenate, mixing it with the primer solution, followed by one-step RT-PCR. Lane 4 shows the results of mixing the plant homogenate and primer solution without heat treatment, followed by one-step RT-PCR, followed by agarose gel electrophoresis.

[0035] For all viruses, PLRV, PVS, PVX, and PVY, the PCR product band in lane 1 showed the strongest fluorescence, demonstrating that the plant RNA virus detection method of this embodiment has higher detection sensitivity than other detection methods.

[0036] Example 2 Potato plants were infected with PLRV, PVS, PVX, PVY, alfalfa mosaic virus (AMV), cucumber mosaic virus (CMV), potato yellow mosaic virus (PAMV), potato A virus (PVA), potato M virus (PVM), tomato ringspot virus (ToRSV), and tomato spotted wilt virus (TSMV). Nicotiana benthamiana was infected with potato mop top virus (PMTV). Sugar beet was infected with beet yellow leaf virus (BLYV). Wheat was infected with wheat dwarf virus (SBWMV) and wheat yellow mosaic virus (WYMV). Leaves or tubers (stolon bases) from the infected plants were collected as samples and ground in the same manner as in Example 1 to obtain plant homogenates.

[0037] The plant homogenate from each plant infected with each virus was mixed with one of the following primer sets depending on the infected virus: a primer set for detecting PLRV, PVS, PVX, and PVY; a primer set for detecting AMV, PMTV, PVA, and PVM; a primer set for detecting PAMV, CMV, ToRSV, and TSWV; or a primer set for detecting BLYV, SBWMV, and WYMV. The primer sets and heat treatment conditions were as follows:

[0038] The primer sets used to detect PLRV, PVS, PVX, and PVY were unpublished. The primer sets used to detect AMV, PMTV, PVA, and PVM, as well as PAMV, CMV, ToRSV, and TSWV, were published in "Development of a one-step multiplex RT-PCR method for detecting eight potato viruses reported in Japan" (Northern Japan Pest Research Association Bulletin, 2023).

[0039] The mixture of plant homogenate and primer set from each virus-infected plant was heat-treated for 5 min at at least two temperatures selected from the group consisting of 60°C, 70°C, 80°C, 90°C, and 98°C. For PLRV, PVS, PVX, and PVY, a non-heat-treated mixture was also prepared as a control.

[0040] The mixture prepared as described above was used for one-step RT-PCR to amplify the viral RNA cDNA. Primescript One Step RT-PCR Kit Ver. 2 (TaKaRa) was used for one-step RT-PCR. The obtained DNA was subjected to agarose gel electrophoresis to compare whether bands corresponding to each virus could be detected.

[0041] The relationship between the RNA virus, plant, sample part, and heat treatment temperature and the band detection for each virus is shown in Tables 1 and 2. A indicates band detection, B indicates band detection but is slightly unclear, D indicates band detection not possible, and - indicates not performed.

[0042] [Table 1]

[0043] [Table 2]

[0044] The above results demonstrate that this embodiment can detect various plant RNA viruses.

Claims

1. a step of heat-treating a mixture obtained by grinding a plant and mixing the ground plant solution with a primer solution, the step of heat-treating the mixture comprises heat-treating the mixture at 60 to 98°C for 4 to 6 minutes; The heat-treated mixture is used in one-step RT-PCR.

2. The plant RNA virus detection method according to claim 1, wherein the plant homogenate is prepared by homogenizing plant tissue together with a solution containing at least one selected from the group consisting of a buffer solution and RNase-free water.

3. 3. The plant RNA virus detection method according to claim 1, wherein the DNA amplified by the one-step RT-PCR is used for agarose gel electrophoresis.

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