Primer set, kit, and identification method

Abstract

To provide a novel primer set, a kit, and an identification method for identifying Tuta absoluta among closely related species.SOLUTION: A primer set comprises: a primer consisting of a polynucleotide comprising a base sequence having at least 90% sequence identity to the base sequence represented by SEQ ID NO:1; a primer consisting of a polynucleotide comprising a base sequence having at least 90% sequence identity to the base sequence represented by SEQ ID NO:2; a primer consisting of a polynucleotide comprising a base sequence having at least 90% sequence identity to the base sequence represented by SEQ ID NO:5; and a primer consisting of a polynucleotide comprising a base sequence having at least 90% sequence identity to the base sequence represented by SEQ ID NO:6.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a primer set, a kit, and a discrimination method.

Background Art

[0002] Tuta absoluta is a pest of the Gelechiidae family of Lepidoptera that damages Solanaceae plants centered on tomatoes. In tomatoes, the larvae bore into the leaves and fruits and damage the interior, causing significant damage to the harvest. Since the larvae of this species grow by crawling inside the leaves and fruits, it is difficult to confirm the invasion of this species until significant damage occurs to the plant after it invades the production field. Therefore, it is necessary to conduct surveillance around the field to quickly confirm the occurrence of this species.

[0003] However, expertise and experience are required for species identification based on the morphological characteristics of Tuta absoluta, and morphological discrimination from adult moths of related species captured by traps is difficult in the production field. Therefore, a simple and rapid identification method at the cultivation site is required.

[0004] Non-Patent Document 1 describes an identification method using the Real-Time PCR method for Tuta absoluta. However, the method using the Real-Time PCR method requires equipment and reagents for Real-Time PCR and is not suitable for rapid identification at the cultivation site.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide a novel primer set, kit, and discrimination method for discriminating tomato stink bugs from related species.

Means for Solving the Problems

[0007] The present inventor has found a novel primer set capable of amplifying a base sequence specific to tomato stink bugs by the LAMP method, and has completed the present invention.

[0008] That is, the present invention provides, for example, the following inventions. 〔1〕 A primer comprising a polynucleotide having a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 1, A primer comprising a polynucleotide having a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 2, A primer comprising a polynucleotide having a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 5, and A primer comprising a polynucleotide having a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 6 A primer set comprising 〔2〕 A primer comprising a polynucleotide having a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 3, and A primer comprising a polynucleotide having a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 4 The primer set according to 〔1〕, further comprising 〔3〕 A tomato stink bug detection kit comprising the primer set according to 〔1〕 or 〔2〕. 〔4〕 A method for discriminating whether a test insect is a tomato stink bug, performing the LAMP method using the primer set with the nucleic acid of the test insect as a template, When nucleic acid amplification is confirmed by the LAMP method, it includes determining that the test insect is the tomato leafminer, wherein the primer set comprises a primer consisting of a polynucleotide comprising a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 1, a primer consisting of a polynucleotide comprising a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 2, a primer consisting of a polynucleotide comprising a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 5, and a primer consisting of a polynucleotide comprising a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 6. [5] The method according to [4], wherein the primer set further comprises a primer consisting of a polynucleotide comprising a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 3, and a primer consisting of a polynucleotide comprising a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 4. [Advantages of the Invention]

[0009] According to the present invention, by amplifying a base sequence specific to the tomato leafminer by the LAMP method, the tomato leafminer can be quickly detected, and it becomes possible to distinguish the tomato leafminer from related species. [Brief Description of the Drawings]

[0010]

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Mode for Carrying Out the Invention

[0011] The sample used in the present invention may be any sample from which nucleic acid (DNA or RNA) of a test insect to be used as a template in the LAMP method can be obtained. For example, larvae, adults, pupae, post-molting skin (e.g., larval molting, pupal molting, and adult molting) of the test insect, insect feces, plants damaged by the test insect, and frass can be used. Also, for example, a part of an insect captured by a trap such as a pheromone trap can be used as a sample. The sample may be the whole or a part of the test insect. These samples may be subjected to pretreatment such as separation, extraction, concentration, and purification. For example, the LAMP method may be carried out using a sample from which DNA or RNA has been extracted and purified by a conventional method, or the LAMP method may be carried out directly using the sample, or the LAMP method may be carried out using a sample roughly extracted with water or the like. As a simple method for DNA extraction, for example, a part of the adult body is immersed in a buffer solution (such as TE), heated at 90 - 95°C for 10 - 15 minutes, and the supernatant stored at 1 - 5°C is used as the DNA extract.

[0012] The test insects are not particularly limited, and for example, they may be Lepidoptera insects, may be moths, or may be selected from Tuta absoluta and related insect species of Tuta absoluta. Examples of related insect species of Tuta absoluta include, for example, Scrobipalpa ergasima, Phthorimaea operculella, Keiferia lycopersicella, Plutella xylostella, Tyrolimnas anthraconesa, Batrachedra sp., Tortricidae sp., Batrachedra sp., Coleophora sp., Blastobasis sp., etc. By using the primer set of the present embodiment, Tuta absoluta can be distinguished and identified from related insect species of Tuta absoluta. As described above, the sample is not limited to larvae, and adults, pupae, etc. can also be used.

[0013] The LAMP (Loop-mediated Isothermal Amplification) method is an amplification reaction under isothermal conditions that does not require temperature control, which is indispensable for the PCR method. Since the LAMP method has a shorter processing time than the PCR method and can also be assayed visually, it is suitable for simple and rapid identification at the cultivation site.

[0014] In the LAMP method, at least four types of primers of the following i) to iv) are included, which recognize the base sequences of a total of six regions in the target nucleic acid serving as a template, namely, the regions F3c, F2c, and F1c from the 3'-terminal side and the regions B3, B2, and B1 from the 5'-terminal side (the complementary strand of the target double-stranded DNA has the regions F, F2, and F1 from the 3'-terminal side and the regions B3c, B2c, and B1c from the 5'-terminal side). i) FIP (Forward Inner Primer): It is designed to have an F2 region complementary to the F2c region of the target DNA at the 3'-end side and the same sequence as the F1c region of the target DNA at the 5'-end side. ii) F3 primer: It is designed to have an F3 region complementary to the F3c region of the target DNA. iii) BIP (Backward Inner Primer): It is designed to have a B2 region complementary to the B2c region of the target DNA at the 3'-end side and the same sequence as the B1c region of the target DNA at the 5'-end side. iv) B3 primer: It is designed to have a B3 region complementary to the B3c region of the target DNA.

[0015] The primer set of this embodiment is a primer set for amplifying a specific DNA in tomato leafminer by the LAMP method. As the FIP primer, it consists of a polynucleotide containing a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 5. As the BIP primer, it consists of a polynucleotide containing a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 6. Also, as the F3 primer, it consists of a polynucleotide containing a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 1. As the B3 primer, it consists of a polynucleotide containing a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 2. The DNA of tomato leafminer amplified using these primers contains the sequence shown in SEQ ID NO: 7 (amplified by F3 - B3) in the variable region of the mitochondrial COI (cytochrome c oxidase subunit I) gene. Also, the region of the dumbbell structure between F2 - B2 is the sequence shown in SEQ ID NO: 8. By using this primer set, tomato leafminer can be detected with sufficient sensitivity.

[0016] In addition, the primer set may further include loop primers shown in the following v) and vi). v) LF (Loop Primer F): It is designed to have a sequence complementary to the portion between the F1 region and the F2 region. vi) LB (Loop Primer B): It is designed to have a sequence complementary to the portion between the B1 region and the B2 region. The primer set of this embodiment may further include a primer consisting of a polynucleotide having a nucleotide sequence with 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 3 as LF, and / or a primer consisting of a polynucleotide having a nucleotide sequence with 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 4 as LB. Thereby, tomato powdery mildew can be identified with higher sensitivity.

[0017] Regarding the relationship between the target DNA and the F1, F2, F3, F1c, F2c, F3c, B1, B2, B3, B1c, B2c, B3c regions, and the details of the LAMP method, for example, it can be confirmed by referring to the principle of the LAMP method described on the homepage of Eiken Chemical Co., Ltd. (http: / / www.eiken.co.jp / ) and Japanese Patent Application Laid-Open No. 2007-236392.

[0018] The primer set of this embodiment includes a primer consisting of a polynucleotide having a nucleotide sequence with 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 1, a primer consisting of a polynucleotide having a nucleotide sequence with 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 2, a primer consisting of a polynucleotide having a nucleotide sequence with 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 5, and a primer consisting of a polynucleotide having a nucleotide sequence with 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 6.

[0019] The primer set may further include a primer consisting of a polynucleotide having a nucleotide sequence with 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 3, and / or a primer consisting of a polynucleotide having a nucleotide sequence with 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 4. Thereby, tomato powdery mildew can be identified with higher sensitivity.

[0020] Here, the sequence identity with the base sequences shown in SEQ ID NOs: 1 to 8 may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0021] Also, the primer may include a sequence in which 0 to 5, 0 to 3, 0 to 2, 0 to 1, 1 to 3, or 1 or 2 mutations are introduced into the base sequences shown in SEQ ID NOs: 1 to 6. The mutations may be the same or different mutations selected from substitution, deletion, insertion, and inversion, and may be selected from single-base substitution, single-base deletion, single-base insertion, and single-base addition.

[0022] The length of the oligonucleotide used as the primer is not particularly limited, and may be, for example, 10 bases to 80 bases, 15 bases to 70 bases, or 18 bases to 55 bases, may be 10 bases or more, 15 bases or more, 20 bases or more, 25 bases or more, 30 bases or more, or 35 bases or more, and may also be 80 bases or less, 70 bases or less, 60 bases or less, 55 bases or less, 50 bases or less, 40 bases or less, 35 bases or less, or 30 bases or less. Also, for example, the F3 primer and the B3 primer may each be 15 bases to 30 bases or 18 bases to 28 bases, the forward loop primer and the backward loop primer may each be 20 bases to 35 bases or 25 bases to 31 bases, and the FIP primer and the BIP primer may each be 30 bases to 60 bases or 35 bases to 55 bases.

[0023] Also, the primer may include a base sequence that is a specific sequence, or a base sequence including 0 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 nucleotide addition or deletion at its 3'-end or 5'-end.

[0024] The primer can be designed based on the sequence of the target sequence and can be synthesized by conventional methods in the art.

[0025] In this specification, A, C, G, and T in the nucleotide sequence of nucleic acid respectively represent the adenine base, cytosine base, guanine base, and thymine base in deoxyribonucleotide. The primer may consist of the bases A, G, C, T or analogs thereof, or degenerate bases (M, R, W, S, Y, K) according to the base notation defined by the International Union of Pure and Applied Chemistry (IUPAC).

[0026] As one embodiment, the present invention also provides a method for discriminating whether a test insect is a tomato leafminer using the above primer set.

[0027] This identification method includes performing the LAMP method using the nucleic acid (DNA or RNA) of the test insect as a template with the primer set, and discriminating that the test insect is a tomato leafminer when nucleic acid amplification is confirmed by the LAMP method. The test insect, primer set, etc. are as described above.

[0028] The implementation of the LAMP method using the nucleic acid of the test insect as a template may, for example, be to perform the LAMP method using the nucleic acid extracted and / or purified from the above-mentioned sample as a template, or to directly perform the LAMP method using a sample containing nucleic acid, or to perform the LAMP method using a crude extract from the sample with water or the like as a template.

[0029] The step of performing the LAMP method may include, for example, allowing a reaction solution containing a sample or a template obtained from the sample, a strand displacement DNA synthase, dNTPs (dATP, dTTP, dGTP, and dCTP), and a buffer solution to stand isothermally. When performing the RT-LAMP method, a reverse transcriptase may be further added to the above reaction solution.

[0030] The temperature for performing the LAMP method may be, for example, 50°C to 75°C, may be 60°C to 65°C, or may be 60°C to 63°C. The standing time may be, for example, 15 minutes or more, may be 20 minutes or more, may be 15 minutes to 120 minutes, may be 20 minutes to 90 minutes, or may be 30 minutes to 70 minutes.

[0031] As the strand displacement type DNA synthase, dNTPs, and buffer, for example, commercially available DNA amplification reagent sets for the LAMP method or the like can be used. Specific examples include Loopamp DNA Amplification Kit (Eiken Chemical Co., Ltd.), WarmStart Colorimetric LAMP 2X Master Mix (DNA&RNA) (New England Biolabs), and LAMP MASTER for Turbidity (Nippon Gene Co., Ltd.).

[0032] By performing the LAMP method using the above primer set, it becomes possible to simply and quickly determine whether the test insect is a tomato leafminer. Specifically, it can be determined whether the test insect is a tomato leafminer based on the presence or absence of nucleic acid amplification by the LAMP method. That is, when nucleic acid amplification is confirmed by the LAMP method, it can be determined that the test insect is a tomato leafminer, and when nucleic acid amplification is not confirmed, it can be determined that the test insect is not a tomato leafminer. Therefore, this determination method may further include a step of confirming the presence or absence of an amplification product after performing the LAMP method. Also, the generation of the amplification product may indicate that the test insect is a tomato leafminer. For example, when nucleic acid amplification is confirmed, for example, 10 minutes later, 12 minutes later, 15 minutes later, within 30 minutes, within 28 minutes, within 25 minutes, 10 to 30 minutes, 12 to 25 minutes, or 15 to 20 minutes after the start of the enzyme reaction in the LAMP method, it may indicate that the test insect is a tomato leafminer.

[0033] The amplification of nucleic acid by the LAMP method can be confirmed by observing the change in the reaction solution after the amplification reaction. For example, the presence or absence of the amplification product can be confirmed by the change in the color of the reaction solution, the amplification of nucleic acid can be confirmed by the turbidity (cloudiness) of the reaction solution, and the amplification of nucleic acid can be confirmed by the fluorescence intensity. As a reagent set for confirming the presence or absence of the amplification product by the change in the color of the reaction solution, for example, WarmStart Colorimetric LAMP 2X Master Mix (DNA&RNA) (New England Biolabs) can be used. As a reagent set for confirming the amplification of nucleic acid by the turbidity (cloudiness) of the reaction solution, for example, LAMP MASTER for Turbidity (Nippon Gene) can be used. As a reagent set for confirming the amplification of nucleic acid by the fluorescence intensity, for example, LAMP MASTER for Fluorescence (Nippon Gene) can be used. Since the turbidity of the reaction solution is due to magnesium pyrophosphate formed as a by-product of the amplification reaction, it is preferable to include Mg 2+ in the reaction solution. The change in the color or turbidity of the reaction solution may be visually confirmed, or may be optically measured using an instrument such as a turbidity measuring device. The fluorescence intensity can be detected by a real-time PCR device or the like. Also, the presence or absence of nucleic acid amplification may be confirmed using agarose gel electrophoresis or the like. The presence or absence of nucleic acid amplification can be determined by a conventional method in the confirmation method used, such as comparison with a negative control and a positive control or comparison with a preset reference value.

[0034] In one embodiment, the present invention also provides a tomato brown rugose fruit virus detection kit containing the above primer set. The primer set is as described above.

[0035] In addition to the above primer set, the kit may also contain one or more selected from the group consisting of a strand displacement DNA synthase, dNTPs (dATP, dTTP, dGTP, and dCTP), a buffer, and combinations thereof. Combinations of a strand displacement DNA synthase and dNTPs, combinations of a strand displacement DNA synthase and a buffer, combinations of dNTPs and a buffer, or combinations of a strand displacement DNA synthase, dNTPs, and a buffer may be contained in one container (e.g., as a solution).

[0036] The implementation of the LAMP method using this kit is as described above. For example, it can be implemented by allowing a reaction solution containing a sample or a template obtained from the sample, a strand displacement DNA synthase, dNTPs, and a buffer to stand isothermally. The kit may further contain reagents for confirming the generation of amplification products. For example, when confirming the presence or absence of amplification products by the change in the color of the reaction solution, it is preferably further contained a dye (e.g., a pH indicator). When confirming the presence or absence of amplification products by the turbidity of the reaction solution, it is preferably further contained Mg 2+ When confirming the presence or absence of amplification products by fluorescence intensity, it is preferably further contained a fluorescent dye (e.g., a cyanine-based dye) that binds to double-stranded DNA. The kit may contain a positive control (e.g., the DNA of tomato ringspot virus) and / or a negative control. The kit may further contain an instruction manual regarding a method for detecting tomato ringspot virus by implementing the LAMP method using this kit. The kit may contain each reagent, etc. in a form isolated in individual containers, or two or more reagents may be premixed in the same container.

[0037] When an amplification product is confirmed by implementing the LAMP method using this kit, it can be determined that tomato ringspot virus has been detected. When no amplification product is confirmed, it can be determined that tomato ringspot virus has not been detected.

Example

[0038] [Preparation of Primer Set] For the captured individuals of the tomato leafminer, the nucleotide sequence of the variable region of the mitochondrial COI (cytochrome c oxidase subunit I) gene was determined. Also, the nucleotide sequences of this region registered in the database for a total of four species, namely the closely related species of the tomato leafminer and small Lepidoptera pests, were searched, and these nucleotide sequences were subjected to sequence comparison by multiple sequence alignment using Clustal W. As a result of the sequence comparison, regions where the nucleotide sequence of the tomato leafminer differed from these species were narrowed down. A primer set was designed for that region. The design diagram of the primer is shown in Figure 1, and the sequences of the prepared primer set are shown in Table 1. Although the primer set for the Real-Time PCR method for identifying the tomato leafminer is described in Non-Patent Document 1, the novel primer set in Table 1 targets different gene regions on the mitochondrion and is a primer set for the LAMP method.

[0039]

Table 1

[0040] [Method for identifying the tomato leafminer using a novel primer set] DNA was purified from a part of the body (mainly legs) of adult tomato leafminers and related species using a simple extraction method with TE (pH 8.0). Specifically, a part of the adult body was immersed in TE (pH 8.0), heated at 95°C for 15 minutes, and the supernatant stored at 4°C was used as the DNA extract. Using the primer set in Table 1, with the above-purified DNA as a template, the LAMP method was carried out using WarmStart Colorimetric LAMP 2X Master Mix (DNA&RNA) (New England Biolabs) or LAMP MASTER for Turbidity (Nippon Gene). The DNA sequence of the tomato leafminer shown in SEQ ID NO: 7 is amplified by the primer set in Table 1.

[0041] The WarmStart Colorimetric LAMP 2X Master Mix contains Bst 2.0 WarmStart DNA polymerase, WarmStart RTx (reverse transcriptase), dyes, etc., and is a master mix reagent that allows the amplification of DNA / RNA by the LAMP method to be confirmed by a color change (changing from red to yellow upon amplification), enabling visual confirmation of the reaction solution changes. LAMP MASTER for Turbidity (Nippon Gene) contains a heat-resistant strand-displacing DNA polymerase, Mg 2+ , dNTPs, an optimized buffer, etc., and is a master mix reagent that allows the amplification of DNA / RNA by the LAMP method to be confirmed by detecting turbidity.

[0042] In the WarmStart Colorimetric LAMP 2X Master Mix (DNA&RNA), for the LAMP method, 25 μL of the reaction solution was used, the reaction (incubation) was carried out at 65°C for 40 minutes, and then the reaction was stopped to confirm the presence or absence of the reaction. In LAMP MASTER for Turbidity (Nippon Gene), for the LAMP method, 25 μL of the reaction solution was used, and the turbidity was continuously measured in real-time at 65°C for 50 - 60 minutes to confirm the presence or absence of the reaction.

[0043] [Example 1] The pheromone trap-caught insects of the tomato leafminer and the DNA extracted from the tomato leafminer were purified as described above. Using the primer sets in Table 1, with the purified DNA as a template, the LAMP method was carried out using LAMP MASTER for Turbidity (Nippon Gene) as described above. A graph showing the real-time turbidity data is shown in Figure 2. Each sample number shows the results using the following DNA as a template. 1 - 4: Tuta absoluta, Sp·p: tomato leafminer (positive control), Sn·n: negative control (distilled water). As DNA amplification progresses, the turbidity increases.

[0044] Only the samples of the positive control (identified tomato leafminer) showed an increase in turbidity within 15 to 20 minutes, and it was confirmed that DNA of other species did not amplify during the DNA amplification time of the tomato leafminer.

[0045] [Example 2] The pheromone trap-caught insects of the tomato leafminer and the DNA extracted from the tomato leafminer were purified as described above. Using the primer sets in Table 1, with the purified DNA as a template, the LAMP method was carried out by LAMP MASTER for Turbidity (Nippon Gene) as described above. A graph showing the real-time turbidity data is shown in Figure 3. Each sample number shows the result with the following DNA as a template. KC24·KW2·KW5·KC53: potato tuberworm, KC54·KC55: tomato leafminer, n: negative control (distilled water), p: positive control (tomato leafminer). Turbidity increases as DNA amplification progresses. Only the samples of KC54, KC55 and the positive control (identified tomato leafminer) showed an increase in turbidity within 15 to 20 minutes, and it was confirmed that DNA of other species did not amplify during the DNA amplification time of the tomato leafminer.

[0046] [Example 3] The DNA extracted from the pheromone trap-caught insects of the tomato leafminer and the tomato leafminer was purified as described above. Using the primer sets in Table 1, with the above-purified DNA as a template, the LAMP method was carried out using LAMP MASTER for Turbidity (Nippon Gene Co., Ltd.) as described above. A graph showing the real-time turbidity data is shown in Figure 4. Each sample number shows the result using the following DNA as a template. KC29, KC30, KC31, KC32, KC39, KC40: tomato leafminer; KC33: Tyrolimnas anthraconesa; KC34: a species of Batrachedra; KC35: a species of Gelechiidae; KC36: a species of Batrachedra; KC37: a species of Coleophora; KC38: a species of Blastobasis; n: negative control; p: positive control (tomato leafminer). As DNA amplification proceeds, the turbidity increases. It was confirmed that only the samples of KC29, KC30, KC31, KC32, KC39, KC40 and the positive control (identified tomato leafminer) had an increase in turbidity within 15 to 20 minutes, and the DNA of other species did not amplify within the DNA amplification time of the tomato leafminer.

[0047] [Example 4] The DNA extracted from the pheromone trap-caught insects of the tomato leafminer and the tomato leafminer was purified as described above. Using the primer sets in Table 1, with the above-purified DNA as a template, the LAMP method was carried out using WarmStart Colorimetric LAMP 2X Master Mix (DNA&RNA) (New England Biolabs) as described above. The change in the hue of the reaction solution is shown in Figure 5. Each sample number shows the result using the following DNA as a template. T: tomato leafminer; P: potato tuberworm; E: onion maggot; N: negative control (distilled water). Only the reaction solution of the tomato leafminer with sample number T changed from red to yellow.

[0048] [Example 5] The pheromone trap-caught insects of the tomato stink bug and the DNA extracted from the tomato stink bug were purified as described above. Using the primer sets in Table 1, with the purified DNA as a template, the LAMP method was carried out using LAMP MASTER for Turbidity (Nippon Gene Co., Ltd.) as described above. Graphs showing real-time turbidity data are shown in FIGS. 6 to 8. n indicates a negative control, and p indicates a positive control (tomato stink bug). A species of the family Pyrrhocoridae refers to Phaeoses sp. As DNA amplification progresses, the turbidity increases. Only the samples of the positive control (identified tomato stink bug) showed an increase in turbidity within 15 to 20 minutes, and the samples of other insect species did not show an increase in turbidity even after reacting for 60 minutes.

[0049] From the above results, it was shown that by using the primer sets in Table 1, the DNA of the tomato stink bug can be specifically amplified, and it is possible to identify only the tomato stink bug from related species.

Claims

1. A primer consisting of a polynucleotide containing a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO:1; A primer consisting of a polynucleotide containing a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 2; A primer consisting of a polynucleotide containing a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO:5; and A primer consisting of a polynucleotide containing a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO:

6. A primer set comprising:

2. A primer consisting of a polynucleotide containing a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO:3, and A primer consisting of a polynucleotide containing a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO:

4. The primer set of claim 1 , further comprising:

3. A tomato budworm detection kit comprising the primer set according to claim 1 or 2.

4. A method for determining whether a test insect is a tomato tussock moth, comprising: carrying out the LAMP method using the primer set and the nucleic acid of the test insect as a template; and when amplification of nucleic acid is confirmed by the LAMP method, determining that the test insect is a tomato tussock moth; The primer set is A primer consisting of a polynucleotide containing a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO:1; A primer consisting of a polynucleotide containing a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 2; A primer consisting of a polynucleotide containing a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO:5; and A method comprising a primer consisting of a polynucleotide containing a base sequence having 90% or more sequence identity to the base sequence shown in SEQ ID NO:

6.

5. the primer set comprises a primer consisting of a polynucleotide comprising a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO:3; and The method according to claim 4, further comprising a primer consisting of a polynucleotide comprising a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO:4.

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