Primer set and discrimination method

Abstract

To provide novel primer sets and discrimination methods for discriminating Spodoptera frugiperda from closely related species.SOLUTION: Provided is a primer set comprising: a primer consisting of a polynucleotide containing 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 containing 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 containing 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 containing a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 6.SELECTED DRAWING: None

Description

Technical Field

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

Background Art

[0002] The fall armyworm (Spodoptera frugiperda) is a pest of the family Noctuidae in the order Lepidoptera that mainly damages gramineous crops centered on corn, and its larvae mainly damage young leaves of crops, inhibiting the growth of the crops. In addition, compared with the pests of the family Noctuidae that have occurred in Japan so far, the fall armyworm often damages the vicinity of the plant growth point and is highly likely to cause growth inhibition. Therefore, in order to prevent damage to crops, it is necessary to quickly detect and identify them at an early stage.

[0003] However, expertise and experience are required for species identification based on the morphological characteristics of the fall armyworm, and it is very difficult to morphologically distinguish the larvae of the fall armyworm, especially young larvae, from related species. Also, in the occurrence survey, it is necessary to distinguish male adults captured by pheromone traps from related species such as the true armyworm, but morphological identification becomes difficult for male adults in a poor state captured by the trap. Therefore, there is a need for a simple and rapid identification method at the gene level that does not rely on morphology for the early and rapid detection and identification of the fall armyworm in the cultivation field.

[0004] Non-Patent Document 1 describes the identification of the fall armyworm from five related species by the LAMP method. However, there are 22 related species that damage corn in Japan, and a method for identifying the fall armyworm from more species is required.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

[0006] The present invention aims to provide a novel primer set and identification method for distinguishing the fall armyworm from closely related species. [Means for solving the problem]

[0007] The inventors have discovered a novel primer set that can amplify a base sequence specific to the fall armyworm using the LAMP method, and have completed the present invention.

[0008] In other words, the present invention provides, for example, the following inventions. [1] A primer consisting of a polynucleotide containing a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 1, A primer consisting of a polynucleotide containing a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 2, A primer consisting of a polynucleotide containing a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 5, and Primers consisting of polynucleotides containing a nucleotide sequence that has more than 90% sequence identity with the nucleotide sequence shown in Sequence ID No. 6. A primer set including... [2] A primer consisting of a polynucleotide containing a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 3, and / or The primer set according to [1] further comprises a primer consisting of a polynucleotide having a base sequence having 90% or more sequence identity with the base sequence shown in Sequence ID No. 4. [3] A fall armyworm detection kit comprising the primer set described in [1] or [2]. [4] A method for determining whether or not the test insect is the fall armyworm, The LAMP method is performed using a primer set and the nucleic acid of the test insect as a template. This includes identifying the test insect as the fall armyworm (Spodoptera litura) when nucleic acid amplification is confirmed by the LAMP method. The above primer set, A primer consisting of a polynucleotide containing a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 1, A primer consisting of a polynucleotide containing a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 2, A primer consisting of a polynucleotide containing a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 5, and A method comprising a primer consisting of a polynucleotide having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 6. [5] The above primer set consists of primers comprising polynucleotides having a sequence identity of 90% or more with the sequence shown in Sequence ID No. 3, and / or The method according to [4], further comprising a primer consisting of a polynucleotide having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 4. [Effects of the Invention]

[0009] According to the present invention, by amplifying the base sequence specific to the fall armyworm using the LAMP method, the fall armyworm can be detected with high sensitivity and rapidly, and it becomes possible to distinguish the fall armyworm from at least 17 closely related species. [Brief explanation of the drawing]

[0010] [Figure 1]This photograph shows the results of the LAMP method in Example 1, using the DNA of the fall armyworm and closely related species as templates and the developed set of primers. When the DNA is amplified, the reaction solution changes from red to yellow. The arrows in the photograph indicate when the reaction solution is yellow. Each sample number indicates the results using the following DNA as a template: 21: Fall armyworm, 1-3: Allomyelia, 4-5: Pseudocorticophyta, 6-7: Rice armyworm, 8: Large red armyworm, 9-11: Helicobacter nicolai, 12: Turnip moth, 13: Cynthia japonica, 14: White-spotted armyworm, 15-16: Cabbage moth, 17: Spodoptera litura, 18-19: Spodoptera litura, 20: Armyworms: 22: White armyworm, 23-25: Sweet flag armyworm, 26: Northern sweet flag armyworm, 27-28: Black-spotted moth, 29-31: Fall armyworm, P1-P2: Positive control (identified white fall armyworm), N: Negative control (identified black fall armyworm), W: Negative control (no DNA) [Figure 2] This graph shows real-time turbidity data when the LAMP method was performed using the developed primer set with DNA from the fall armyworm and closely related species as templates in Example 1. Turbidity increases as DNA amplification progresses. Samples showing increased turbidity after 15-20 minutes are fall armyworm samples. "posi" indicates the positive control. The other sample numbers are the same as in Figure 1. [Figure 3] This graph shows real-time turbidity data when the LAMP method was performed using the developed primer set with DNA from the fall armyworm and closely related species as templates in Example 2. Turbidity increases as DNA amplification progresses. Samples showing increased turbidity between 18 and 23 minutes are the positive control (identified fall armyworm). n represents the negative control. [Figure 4]This is a graph showing the real-time turbidity data when the LAMP method was performed using a set of developed primers with the DNA of Plutella xylostella and related species as templates in Example 2. As DNA amplification progresses, the turbidity increases. The samples with increasing turbidity at 18 - 23 minutes are positive controls (identified Plutella xylostella). n indicates the negative control. [Figure 5] This is a graph showing the real-time turbidity data when the LAMP method was performed using a set of developed primers with the DNA of Plutella xylostella and related species as templates in Example 2. As DNA amplification progresses, the turbidity increases. The samples with increasing turbidity at 18 - 23 minutes are positive controls (identified Plutella xylostella). n indicates the negative control. [Figure 6] This is a graph (upper part of Figure 6) showing the real-time turbidity data when the LAMP method was performed using a set of developed primers with the DNA of 1st instar larvae (sample numbers 53 - 66) captured in the field as templates in Example 3. p indicates the positive control (identified Plutella xylostella), and n indicates the negative control. The table in the lower part of Figure 6 shows the insect species of each sample identified based on the cytochrome c oxidase subunit I (Col) gene sequence. As DNA amplification progresses, the turbidity increases. In the upper graph, it can be seen from the comparison with the table that sample numbers 56, 57, 58, 59, and 66 with increasing turbidity at 15 - 25 minutes are samples identified as Plutella xylostella based on the Col gene sequence.

Modes 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 the test insect to be used as a template in the LAMP method can be obtained. For example, the larvae, adults, pupae, molted skin (e.g., larval molt, pupal molt, and adult molt) of the test insect, insect dung, plant material damaged by the test insect, and frass can be used. The sample may be the whole or a part of the test insect. These samples may be pretreated by separation, extraction, concentration, purification, etc. For example, the LAMP method may be performed using DNA or RNA extracted and purified from the sample by a conventional method, the LAMP method may be performed using the sample directly, or the LAMP method may be performed using a crude extract with water, etc.

[0012] The test insects are not particularly limited, and for example, they may be Lepidoptera insects, may be moths, or may be selected from Spodoptera frugiperda and related species of Spodoptera frugiperda. Examples of related species of Spodoptera frugiperda include, for example, Mythimna separata, Mythimna polysticha, Sesamia inferens, Apamea lateritia, Helicoverpa armigera, Agrotis segetum, Spodoptera cilium, Spodoptera exigua, Agrotis ipsilon, Mythimna loreyi, Spodoptera litura, Mamestra brassicae, Spodoptera mauritia, Amphipoea ussuriensis, Amphipoea fucosa, Euxoa sibirica, Hydraecia amurensis, Agrotis exclamationis informis, Lateroligia ophiogramma, Helotropha leucostigma, Autographa gamma, Mesapamea concinnata, Apamea sordens basistriga, Gortyna fortis, Agrotis tokionis, Ostrinia furnacalis, etc. By using the primer set of the present embodiment, Spodoptera frugiperda can be distinguished and identified from related species of Spodoptera frugiperda. 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 isothermal amplification reaction that does not require the temperature control essential for the PCR method. Because the LAMP method has a shorter processing time than the PCR method and allows for visual inspection, it is suitable for simple and rapid identification in cultivation settings.

[0014] The LAMP method includes at least four types of primers (i) to iv) below, which recognize the base sequences of a total of six regions in the target nucleic acid template: the F3c, F2c, and F1c regions from the 3' end and the B3, B2, and B1 regions from the 5' end (the complementary strand of the target double-stranded DNA has the F, F2, and F1 regions from the 3' end and the B3c, B2c, and B1c regions from the 5' end). i) FIP (Forward Inner Primer): Designed to have an F2 region complementary to the F2c region of the target DNA at its 3' end, and the same sequence as the F1c region of the target DNA at its 5' end. ii) F3 primers: Designed to have an F3 region complementary to the F3c region of the target DNA. iii) BIP (Backward Inner Primer): Designed to have a B2 region complementary to the B2c region of the target DNA at its 3' end and the same sequence as the B1c region of the target DNA at its 5' end. iv) B3 primers: 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 specific DNA in the fall armyworm using the LAMP method. It includes a FIP primer consisting of a polynucleotide containing a nucleotide sequence with 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 5, a BIP primer consisting of a polynucleotide containing a nucleotide sequence with 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 6, an F3 primer consisting of a polynucleotide containing a nucleotide sequence with 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 1, and a B3 primer consisting of a polynucleotide containing a nucleotide sequence with 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 2. The fall armyworm DNA amplified using these primers includes the sequence shown in SEQ ID NO: 7 in the variable region of the mitochondrial COI (cytochrome c oxidase subunit I) gene. By using this primer set, the fall armyworm can be detected with sufficient sensitivity.

[0016] The primer set may also further include the loop primers shown in v) and vi) below. v) LF (Loop Primer F): Designed to have a complementary sequence in the region between the F1 and F2 regions. vi) LB (Loop Primer B): Designed to have a complementary sequence in the area between the B1 and B2 regions. The primer set of this embodiment may further include, as LF, a primer consisting of a polynucleotide containing a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 3, and / or as LB, a primer consisting of a polynucleotide containing a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 4. This allows for more sensitive identification of the fall armyworm.

[0017] The relationship between the target DNA and the F1, F2, F3, F1c, F2c, F3c, B1, B2, B3, B1c, B2c, and B3c regions, as well as details of the LAMP method, can be confirmed, for example, by referring to the principle of the LAMP method explained on Eiken Chemical's website (http: / / www.eiken.co.jp / ) or to Japanese Patent Publication No. 2007-236392.

[0018] The primer set of this embodiment includes a primer made 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 made 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 made 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 made of a polynucleotide containing a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 6.

[0019] The primer set may include a primer consisting of a polynucleotide containing a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 3, and / or a primer consisting of a polynucleotide containing a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 4.

[0020] The primer set may include, for example, a primer made of a polynucleotide consisting of a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 1, a primer made of a polynucleotide consisting of a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 2, a primer made of a polynucleotide consisting of a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 3, a primer made of a polynucleotide consisting of a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 4, a primer made of a polynucleotide consisting of a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 5, and a primer made of a polynucleotide consisting of a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 6.

[0021] Here, the sequence identity with the base sequences shown in Sequence IDs 1-6 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%.

[0022] Furthermore, the primers may contain sequences in which 0 to 5, 0 to 3, 0 to 2, 0 to 1, 1 to 3, or 1 or 2 mutations have been introduced into the nucleotide sequences shown in SEQ ID NOs: 1 to 6. The mutations may be identical or different, selected from substitutions, deletions, insertions, and non-substitutions, and may be selected from single nucleotide substitutions, single nucleotide deletions, single nucleotide insertions, and single nucleotide additions.

[0023] The length of oligonucleotides used as primers is not particularly limited, but may be, for example, 10 to 80 bases, 15 to 70 bases, or 18 to 55 bases, or 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, or 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, F3 primers and B3 primers may be 15 to 30 bases or 18 to 27 bases, respectively, forward loop primers and backward loop primers may be 20 to 35 bases or 25 to 31 bases, respectively, and FIP primers and BIP primers may be 45 to 60 bases or 50 to 55 bases, respectively.

[0024] Furthermore, the primer may include a specific base sequence, or a base sequence that includes the addition or deletion of 0 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 nucleotide at its 3' or 5' end.

[0025] Primers can be designed based on the sequence of the target sequence and can be synthesized by conventional methods in the art.

[0026] In this specification, A, C, G, and T in the base sequence of nucleic acids represent the adenine base, cytosine base, guanine base, and thymine base in deoxyribonucleotides, respectively. Primers may consist of bases A, G, C, T or their analogues according to the base notation defined by the International Union of Pure and Applied Chemistry (IUPAC), or degenerate bases (M, R, W, S, Y, K).

[0027] As one embodiment, the present invention also provides a method for determining whether or not a test insect is the fall armyworm using the above-mentioned primer set.

[0028] This identification method involves performing the LAMP method using a primer set with the nucleic acid (DNA or RNA) of the test insect as a template, and identifying the test insect as the fall armyworm (Spodoptera litura) if nucleic acid amplification is confirmed by the LAMP method. The test insect, primer set, etc., are as described above.

[0029] The LAMP method using nucleic acids from the test insect as a template may be performed, for example, by using nucleic acids extracted and / or purified from the sample described above as a template, by directly using a sample containing nucleic acids as a template, or by using a crude extract obtained from the sample with water or the like as a template.

[0030] The LAMP method may include, for example, allowing a reaction mixture containing a sample or a template obtained from a sample, a strand-displacing DNA polymerase, dNTPs (dATP, dTTP, dGTP, and dCTP), and a buffer to stand at an isothermal temperature. If the RT-LAMP method is performed, reverse transcriptase may be added to the above reaction mixture.

[0031] The temperature at which the LAMP method is performed may be, for example, 50°C to 75°C, 60°C to 65°C, or 60°C to 63°C. The standing time may be, for example, 15 minutes or more, 20 minutes or more, 15 minutes to 120 minutes, 20 minutes to 90 minutes, or 30 minutes to 60 minutes.

[0032] For strand-displacement DNA polymerase, dNTPs, and buffer solutions, commercially available LAMP DNA amplification reagent kits can be used, such as Loopamp DNA Amplification Kit (Eiken Chemical Co., Ltd.), WarmStart Colorimetric LAMP 2X Master Mix (DNA & RNA) (New England Biolabs Co., Ltd.), and LAMP MASTER for Turbidity (Nippon Gene Co., Ltd.).

[0033] By performing the LAMP method using the primer set described above, it is possible to easily and quickly determine whether or not the test insect is a fall armyworm. Specifically, whether or not the test insect is a fall armyworm can be determined by the presence or absence of nucleic acid amplification by the LAMP method. That is, if nucleic acid amplification is confirmed by the LAMP method, the test insect can be determined to be a fall armyworm, and if nucleic acid amplification is not confirmed, the test insect can be determined to be a fall armyworm. Therefore, this determination method may further include a step of checking for the presence or absence of amplification products after performing the LAMP method. Alternatively, the generation of amplification products may indicate that the test insect is a fall armyworm. For example, if nucleic acid amplification is confirmed after the start of the enzymatic reaction in the LAMP method, for instance, at 10 minutes, 12 minutes, 15 minutes, within 30 minutes, within 28 minutes, within 25 minutes, 10 to 30 minutes, 12 to 25 minutes, or 15 to 20 minutes, it may indicate that the test insect is the fall armyworm.

[0034] Nucleic acid amplification using the LAMP method can be confirmed by observing changes in the reaction solution after the amplification reaction. For example, the presence or absence of amplification products can be confirmed by a change in the color of the reaction solution, the amplification of nucleic acids can be confirmed by the turbidity (whitening) of the reaction solution, and the amplification of nucleic acids can be confirmed by the fluorescence intensity. For a reagent set to confirm the presence or absence of amplification products by a change in the color of the reaction solution, for example, WarmStart Colorimetric LAMP 2X Master Mix (DNA & RNA) (New England Biolabs) can be used. For a reagent set to confirm the amplification of nucleic acids by turbidity (whitening) of the reaction solution, for example, LAMP MASTER for Turbidity (Nippon Gene Co., Ltd.) can be used. For a reagent set to confirm the amplification of nucleic acids by fluorescence intensity, for example, LAMP MASTER for Fluorescence (Nippon Gene Co., Ltd.) can be used. The turbidity of the reaction solution is due to magnesium pyrophosphate formed as a byproduct of the amplification reaction, so additional Mg should be added when performing the LAMP method. 2+ It is preferable to include in the reaction solution. Changes in the color or turbidity of the reaction solution may be confirmed visually, or by optical measurement using equipment such as a turbidity measuring device. Fluorescence intensity can be detected using a real-time PCR device or the like. The presence or absence of nucleic acid amplification may also be confirmed using agarose gel electrophoresis or the like. The presence or absence of nucleic acid amplification can be determined by conventional methods of confirmation used, such as comparison with negative and positive controls or comparison with pre-set reference values.

[0035] As one embodiment, the present invention also provides a fall armyworm detection kit including the primer set described above. The primer set is as described above.

[0036] In addition to the primer set described above, this kit may include one or more selected from the group consisting of strand-displacing DNA polymerase, dNTPs (dATP, dTTP, dGTP, and dCTP), buffers, and combinations thereof. A combination of strand-displacing DNA polymerase and dNTPs, a combination of strand-displacing DNA polymerase and buffers, a combination of dNTPs and buffers, or a combination of strand-displacing DNA polymerase, dNTPs and buffers may be contained in a single container (for example, as a solution).

[0037] The LAMP method using this kit is carried out as described above, for example, by allowing a reaction solution containing a sample or a template obtained from a sample, strand-displacing DNA polymerase, dNTPs, and buffer solution to stand at an isothermal temperature. This kit may further contain reagents for confirming the formation of amplification products. For example, if the presence or absence of amplification products is to be confirmed by a change in the color of the reaction solution, it is preferable to further include a dye (e.g., a pH indicator), and if the presence or absence of amplification products is to be confirmed by turbidity of the reaction solution, it is preferable to further include Mg 2+ It is preferable to include a fluorescent dye that binds to double-stranded DNA (e.g., a cyanine dye) if the presence or absence of amplification products is to be confirmed by fluorescence intensity. The kit may also include a positive control (e.g., fall armyworm DNA) and / or a negative control. The kit may further include instructions on how to detect fall armyworms by performing the LAMP method using the kit. The kit may contain each reagent in isolation in individual containers, or two or more reagents may be pre-mixed in the same container.

[0038] By performing the LAMP method using this kit, if amplification products are confirmed, it can be determined that fall armyworms have been detected. If amplification products are not confirmed, it can be determined that fall armyworms have not been detected. [Examples]

[0039] [Preparation of primer set] For captured fall armyworm specimens, the nucleotide sequence of the variable region of the mitochondrial COI (cytochrome c oxidase subunit I) gene was determined. Furthermore, the nucleotide sequences of 15 species were obtained from databases of 22 closely related species that damage maize, and these sequences were compared using multiple sequence sorting in Clustal W. The results of the sequence comparison narrowed down the region in the fall armyworm's nucleotide sequence that differed from that of closely related species. Primers were designed using Primer Explorer (https: / / primerexplorer.jp / ), a primer design support software from Eiken Chemical Co., Ltd., targeting this region, and a set of primers with partially modified sequences was created. The sequences of the created primer sets are shown in Table 1. Note that while a LAMP method primer set for identifying the fall armyworm is described in Non-Patent Literature 1, the novel primer set in Table 1 targets a different gene region on the mitochondria, and therefore has a different sequence.

[0040] [Table 1]

[0041] [Example 1: Identification of the fall armyworm using a novel primer set (1)] DNA was purified from the legs of adult fall armyworms and related species using the DNeasy Blood & Tissue Kit (QIAGEN).

[0042] Using the primer sets in Table 1, the purified DNA described above was used as a template, and the LAMP method was performed using WarmStart Colorimetric LAMP 2X Master Mix (DNA & RNA) (New England Biolabs) or LAMP MASTER for Turbidity (Nippon Gene). The primer sets in Table 1 amplified the DNA sequence of the fall armyworm shown in Sequence ID No. 7.

[0043] 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 visual confirmation of DNA / RNA amplification by the LAMP method through a color change (change from red to yellow when amplification occurs). The LAMP method was performed using 25 μL of reaction solution, and the reaction was stopped after 30 minutes of incubation at 60°C to confirm whether or not a reaction occurred. The results are shown in Figure 1. In the photograph in Figure 1, the arrows indicate that the reaction solution was yellow. Each sample number indicates the results using the following DNA as a template. 21: White-tipped armyworm, 1-3: Round armyworm, 4-5: False white armyworm, 6-7: Rice armyworm, 8: Large red armyworm, 9-11: Tobacco budworm, 12: Turnip moth, 13: Cactus spp., 14: White-striped armyworm, 15-16: Cabbage moth, 17: Grass white armyworm, 18-19: Beet armyworm, 20: Armyworms: 22: White armyworm, 23-25: Sweet flag armyworm, 26: Northern sweet flag armyworm, 27-28: Black-spotted moth, 29-31: Fall armyworm, P1-P2: Positive control (identified White fall armyworm), N: Negative control (identified Black fall armyworm), W: Negative control (no DNA).

[0044] Only 21: Fall armyworm and P1-P2: Positive control (identified fall armyworm) turned yellow, confirming that their DNA was amplified.

[0045] LAMP MASTER for Turbidity (Nippon Gene Co., Ltd.) is a heat-resistant strand-displacement DNA polymerase, Mg 2+This is a master mix reagent containing dNTPs, optimized buffer, etc., which allows for confirmation of DNA / RNA amplification by LAMP method by detecting turbidity. In the LAMP method, 25 μL of reaction solution was used, and turbidity was continuously measured in real time for 90 minutes at 60°C to confirm whether or not a reaction occurred. The results are shown in Figure 2. "posi" indicates the positive control. The other sample numbers are the same as in Figure 1. The sample numbers are the same as in Figure 1. Only the samples of 21: Fall armyworm and P1-P2: Positive control (identified Fall armyworm) showed an increase in turbidity within 15-20 minutes.

[0046] Table 1 demonstrates that the primer set can be used to specifically amplify the DNA of the fall armyworm, making it possible to identify only the fall armyworm from closely related species.

[0047] [Example 2: Identification of the fall armyworm using a novel primer set (2)] DNA was purified from the legs of adult fall armyworms and related species using the DNeasy Blood & Tissue Kit (QIAGEN). Using the primer set shown in Table 1, the purified DNA was used as a template, and the LAMP method was performed using LAMP MASTER for Turbidity (Nippon Gene Co., Ltd.) in the same manner as in Example 1.

[0048] The LAMP method involved using 25 μL of reaction solution and continuously measuring turbidity in real time for 90 minutes at 60°C to confirm the presence or absence of reaction. The results are shown in Figures 3 to 5. n represents the negative control (no DNA).

[0049] Only the positive control (identified fall armyworm) sample showed an increase in turbidity within 18-23 minutes, confirming that the DNA of other species did not amplify during the same time as the fall armyworm DNA amplification period.

[0050] Table 1 demonstrates that the primer set can be used to specifically amplify the DNA of the fall armyworm, making it possible to identify only the fall armyworm from closely related species.

[0051] [Example 3: Identification of the fall armyworm using a novel primer set (3)] Fourteen first-instar caterpillars were captured in the field from corn leaves and assigned sample numbers 53-66. DNA was purified from each first-instar larva using the DNeasy Blood & Tissue Kit (QIAGEN). Using the primer set shown in Table 1, the purified DNA was used as a template, and the LAMP method was performed using LAMP MASTER for Turbidity (Nippon Gene Co., Ltd.) in the same manner as in Example 1.

[0052] The LAMP method used 25 μL of reaction solution, and turbidity was continuously measured in real time for 90 minutes at 60°C to confirm the presence or absence of reaction. The results are shown in Figure 6. p represents the positive control (identified fall armyworm), and n represents the negative control (no DNA). The table below Figure 6 shows the insect species of each sample identified based on the cytochrome c oxidase subunit I (Col) gene sequence. Only samples 56, 57, 58, 59, 66, and the positive control, which were identified as fall armyworms based on the Col gene sequence, showed an increase in turbidity within 15-25 minutes, and it was confirmed that the DNA of other species was not amplified.

[0053] Table 1 demonstrates that using the primer set makes it possible to identify only the fall armyworm (Spodoptera litura) from closely related species in first-instar larvae, which are difficult to identify with the naked eye.

Claims

1. A primer consisting of a polynucleotide containing a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 1, A primer consisting of a polynucleotide containing a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 2, A primer consisting of a polynucleotide containing a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 5, and Primers consisting of polynucleotides containing a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No.

6. A primer set for specifically amplifying the DNA of the fall armyworm, including [specific primers].

2. A primer consisting of a polynucleotide containing a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 3, and Primers consisting of polynucleotides containing a base sequence having 90% or more sequence identity with the base sequence shown in Sequence ID No.

4. The primer set according to claim 1, further comprising:

3. A fall armyworm detection kit comprising the primer set described in claim 1 or 2.

4. A method for determining whether or not the test insect is the fall armyworm, The LAMP method is performed using nucleic acids from the test insect as a template, with a primer set specifically designed to amplify the DNA of the fall armyworm. This includes identifying the test insect as the fall armyworm (Spodoptera litura) when nucleic acid amplification is confirmed by the LAMP method. The aforementioned primer set A primer consisting of a polynucleotide containing a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 1, A primer consisting of a polynucleotide containing a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 2, A primer consisting of a polynucleotide containing a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 5, and A method comprising a primer consisting of a polynucleotide having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No.

6.

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

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