Method for detecting apple snails

Abstract

To detect apple snails from environmental DNA with a high detection rate.SOLUTION: This method amplifies and detects environmental DNA in a sample with a first primer having a specific base sequence, a second primer having another specific base sequence, and a probe having still another base sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for detecting apple snails using environmental DNA. [Background technology]

[0002] The Pomacea canaliculata is a type of freshwater snail native to South America that was introduced to Japan for aquaculture purposes for consumption and then became an invasive alien species that became feral in the environment. It inhabits rice paddies and feeds on young rice plants after transplanting, making it a target for control as a pest. The closely related species, the La Plata apple snail, which has a very similar morphology to the Pomacea canaliculata, has also invaded Japan and is hybridizing with the Pomacea canaliculata in various places. It is not easy to distinguish between the Pomacea canaliculata and the La Plata apple snail in terms of appearance or ecology.

[0003] The presence of apple snails such as Pomacea canaliculata in the environment is usually determined by the presence or absence of pink egg masses in the vicinity. However, when population densities are low, observing egg masses is not necessarily a reliable method of determination.

[0004] Environmental DNA is a general term for DNA derived from organisms present in aquatic environments such as the sea, rivers, and lakes, as well as in soil and the atmosphere. Techniques are known for estimating the types of organisms, particularly aquatic organisms, present in an environment by analyzing environmental DNA. Non-Patent Document 1 reports a method for detecting the cytochrome oxidase 1 (COI) gene of the apple snail Pomacea canaliculata and the La Plata apple snail from environmental DNA. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] P. Banerjee, et al., Hydrobiologia (2022), 849: 4241-4257 Summary of the Invention [Problem to be solved by the invention]

[0006] The method described in Non-Patent Document 1 had a detection rate of 50 to 70% for Pomacea canaliculata and La Plata apple snails from environmental DNA. The method described in Non-Patent Document 1 aims to distinguish between Pomacea canaliculata and La Plata apple snails. However, although it is meaningful to detect these apple snails while distinguishing them from native species when considering apple snail control, distinguishing between these apple snails is not particularly meaningful because the two species may hybridize. Therefore, there is a need for a method to detect Pomacea canaliculata, La Plata apple snails, and hybrids of these two species with high sensitivity without distinguishing between them.

[0007] An object of the present invention is to provide a method capable of detecting apple snails from environmental DNA with a high detection rate, and a reagent or kit for use in said method. [Means for solving the problem]

[0008] This specification provides the following inventions. [1] A first primer having any one of the following nucleotide sequences (a1) to (a4): (a1) a nucleotide sequence represented by SEQ ID NO: 1; (a2) a nucleotide sequence of 10 or more consecutive nucleotides in length in the nucleotide sequence represented by SEQ ID NO: 1; (a3) a nucleotide sequence in which 1 to 9 nucleotides are deleted, substituted, or added in the nucleotide sequence represented by SEQ ID NO: 1; (a4) a nucleotide sequence having 80% or more sequence identity with any of the nucleotide sequences (a1) to (a3); A second primer having any one of the following nucleotide sequences (b1) to (b4): (b1) a base sequence represented by SEQ ID NO: 2; (b2) a nucleotide sequence of 10 or more consecutive nucleotides in length in the nucleotide sequence represented by SEQ ID NO: 2; (b3) a nucleotide sequence in which 1 to 9 nucleotides are deleted, substituted, or added in the nucleotide sequence represented by SEQ ID NO: 2; (b4) a nucleotide sequence having 80% or more sequence identity with any of the nucleotide sequences (b1) to (b3); and a probe having a base sequence selected from the following (c1) to (c4): (c1) a nucleotide sequence represented by SEQ ID NO: 3; (c2) a contiguous nucleotide sequence of 10 or more nucleotides including the nucleotide sequence represented by SEQ ID NO: 4 in the nucleotide sequence represented by SEQ ID NO: 3; (c3) a nucleotide sequence in which 1 to 9 bases are deleted, substituted or added in the nucleotide sequence represented by SEQ ID NO: 3, and no deletion or addition occurs in the nucleotide sequence represented by SEQ ID NO: 4; (c4) a nucleotide sequence having 80% or more sequence identity with any of the nucleotide sequences of (c1) to (c3), and having no mutation in the nucleotide sequence portion represented by SEQ ID NO: 4; amplifying nucleic acid in the sample using Methods for detecting apple snails. [2] The method according to [1], wherein the sample is collected from a freshwater environment or a soil environment. [3] The method according to either [1] or [2], wherein the nucleic acid is environmental DNA. [4] The method according to any one of [1] to [3], wherein the probe comprises a fluorescent substance and a quencher substance on the 5'-end and 3'-end sides of the base sequence, respectively. [5] A first primer having any one of the following nucleotide sequences (a1) to (a4): (a1) a nucleotide sequence represented by SEQ ID NO: 1; (a2) a nucleotide sequence of 10 or more consecutive nucleotides in length in the nucleotide sequence represented by SEQ ID NO: 1; (a3) a nucleotide sequence in which 1 to 9 nucleotides are deleted, substituted, or added in the nucleotide sequence represented by SEQ ID NO: 1; (a4) a nucleotide sequence having 80% or more sequence identity with any of the nucleotide sequences (a1) to (a3); and a second primer having any one of the following nucleotide sequences (b1) to (b4): (b1) a base sequence represented by SEQ ID NO: 2; (b2) a nucleotide sequence of 10 or more consecutive nucleotides in length in the nucleotide sequence represented by SEQ ID NO: 2; (b3) a nucleotide sequence in which 1 to 9 nucleotides are deleted, substituted, or added in the nucleotide sequence represented by SEQ ID NO: 2; (b4) a nucleotide sequence having 80% or more sequence identity with any of the nucleotide sequences (b1) to (b3); and a probe having a base sequence selected from the following (c1) to (c4): (c1) a nucleotide sequence represented by SEQ ID NO: 3; (c2) a contiguous nucleotide sequence of 10 or more nucleotides including the nucleotide sequence represented by SEQ ID NO: 4 in the nucleotide sequence represented by SEQ ID NO: 3; (c3) a nucleotide sequence in which 1 to 9 bases are deleted, substituted or added in the nucleotide sequence represented by SEQ ID NO: 3, and no deletion or addition occurs in the nucleotide sequence represented by SEQ ID NO: 4; (c4) a nucleotide sequence having 80% or more sequence identity with any of the nucleotide sequences of (c1) to (c3), and having no mutation in the nucleotide sequence portion represented by SEQ ID NO: 4; A reagent or kit for detecting apple snails, comprising: [6] The reagent or kit according to [5], which is for real-time PCR. [7] A first primer having any one of the following nucleotide sequences (a1) to (a4): (a1) a nucleotide sequence represented by SEQ ID NO: 1; (a2) a nucleotide sequence of 10 or more consecutive nucleotides in length in the nucleotide sequence represented by SEQ ID NO: 1; (a3) a nucleotide sequence in which 1 to 9 nucleotides are deleted, substituted, or added in the nucleotide sequence represented by SEQ ID NO: 1; (a4) a nucleotide sequence having 80% or more sequence identity with any of the nucleotide sequences (a1) to (a3); and a second primer having any one of the following nucleotide sequences (b1) to (b4): (b1) a base sequence represented by SEQ ID NO: 2; (b2) a nucleotide sequence of 10 or more consecutive nucleotides in length in the nucleotide sequence represented by SEQ ID NO: 2; (b3) a nucleotide sequence in which 1 to 9 nucleotides are deleted, substituted, or added in the nucleotide sequence represented by SEQ ID NO: 2; (b4) a nucleotide sequence having 80% or more sequence identity with any of the nucleotide sequences (b1) to (b3); A primer set for detecting apple snails, consisting of: [8] A probe for detecting apple snails, having a base sequence selected from the following (c1) to (c4): (c1) a nucleotide sequence represented by SEQ ID NO: 3; (c2) a contiguous nucleotide sequence of 10 or more nucleotides including the nucleotide sequence represented by SEQ ID NO: 4 in the nucleotide sequence represented by SEQ ID NO: 3; (c3) a nucleotide sequence in which 1 to 9 bases are deleted, substituted or added in the nucleotide sequence represented by SEQ ID NO: 3, and no deletion or addition occurs in the nucleotide sequence represented by SEQ ID NO: 4; (c4) A base sequence having 80% or more sequence identity with any of the base sequences (c1) to (c3), which does not contain a mutation in the base sequence portion represented by SEQ ID NO:4. [Effects of the Invention]

[0009] According to the present invention, it is possible to detect apple snails from environmental DNA with a high detection rate. [Brief explanation of the drawings]

[0010] [Figure 1] This is a schematic diagram showing the design positions of example primers and probes on the base sequence of the cytochrome oxidase 1 (COI) gene of the apple snail. For the La Plata apple snail, only the mismatched bases in the base sequence corresponding to positions 354 to 420 are shown. For other freshwater snails, only the mismatched bases in the base sequence corresponding to positions 382 to 407 are shown. [Figure 2] FIG. 1 is a flow chart showing an example of a procedure for collecting and detecting environmental DNA. [Figure 3]3A and 3B show the results of quantitative PCR of DNA solutions derived from tissues and rearing water of various freshwater snails. The vertical axis shows fluorescence intensity, and the horizontal axis shows PCR cycle number. Figure 3A shows the results of quantitative PCR of the DNA solution derived from tissues, and Figure 3B shows the results of quantitative PCR of the DNA solution derived from rearing water. [Figure 4] 1 is a graph showing the results of quantitative PCR of environmental DNA collected from environments A and B. The vertical axis shows fluorescence intensity, and the horizontal axis shows PCR cycle number. DETAILED DESCRIPTION OF THE INVENTION

[0011] 1 Definition / Configuration In this specification, "apple snails" refers to freshwater snails belonging to the family Ampullariidae, and in particular to the Pomacea canaliculata, the La Plata apple snail (Pomacea maculata), and their hybrids, which are widespread invasive alien species in Japan. "Invasive alien species" here refers to alien species that have a significant impact on the local natural environment and pose a threat to biodiversity.

[0012] As used herein, "freshwater environment" encompasses all freshwater areas where ecosystems are established, such as rivers, lakes, marshes, rice paddies, and irrigation channels. As used herein, "soil environment" refers to the soil where ecosystems are established. Generally, soil refers to the layer that covers the Earth's surface, formed by the long-term action of organisms, topography, climate, etc. on rocks, peat, and volcanic ash.

[0013] As used herein, "nucleic acid" encompasses deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). As used herein, "environmental DNA" refers to DNA released into the environment, such as water, soil, and air. Environmental DNA is extracted from sampled water or soil and used to comprehensively understand the organisms present in the environment or to detect the presence of specific organisms.

[0014] Herein, "T" in a base sequence can be changed to "U" as necessary. Herein, "D" in a base sequence represents "A / G / T," "H" represents "A / T / C," "K" represents "G / T," "M" represents "A / C," "R" represents "A / G," and "Y" represents "C / T." Herein, when "D," "H," "K," "M," "R," and / or "Y" are present in the base sequence of a primer, this indicates that the base is any one of the bases (for example, "Y" represents C or T, and "K" represents G or T), or that the primer is a degenerate primer.

[0015] As used herein, nucleic acid amplification may be performed using any technique capable of amplifying nucleic acids, particularly DNA. Numerous nucleic acid amplification techniques are known, including PCR (Polymerase Chain Reaction), LAMP (Loop-Mediated Isothermal Amplification), SDA (Strand Displacement Amplification), NASBA (Nucleic Acid Sequence Based Amplification), RPA (Recombinase Polymerase Amplification), NEAR (Nicking Enzyme Amplification Reaction), and HDA (Helicase-Dependent Amplification). Nucleic acid amplification and post-amplified nucleic acid detection may be performed simultaneously (in real time) or sequentially. Unless otherwise specified, the following describes an embodiment in which real-time PCR is used as the nucleic acid amplification technique, but it is not intended to limit the nucleic acid amplification technique of the present invention to real-time PCR.

[0016] As used herein, a "primer" refers to an artificially prepared oligonucleotide of about 10 to 30 bases in length that serves as the starting point for nucleic acid amplification. As used herein, a "probe" refers to an artificially prepared oligonucleotide of about 10 to 50 bases in length that has a sequence that specifically binds to an amplified nucleic acid.

[0017] As used herein, the "sequence identity" of a base sequence refers to the proportion (percentage) of identical bases in the total overlapping base sequence in an optimal alignment when two base sequences are aligned with or without introducing gaps, and refers to the value calculated by formula (1). Sequence identity (%) = number of matches (ignoring gaps) / length of shorter sequence (length excluding gaps) × 100... Equation (1) Sequence identity can be easily determined using BLAST (Basic Local Alignment Search Tool) (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), an algorithm commonly used in this field.

[0018] In this specification, a "reagent" refers to a single element of a solution, component, or other element contained in a container (however, packaging may be provided separately). On the other hand, a "kit" refers to an item containing two or more elements of a solution, component, or other element contained in a container. In this specification, there is no particular limitation on the number of elements between a reagent and a kit, as long as the elements necessary for the purpose are provided. In this specification, a reagent and a kit are also collectively referred to as "reagents, etc."

[0019] 2. How to detect apple snails A first embodiment of the present invention is a method for detecting apple snails. The method of this embodiment comprises using a first primer having any one of the following base sequences (a1) to (a4): (a1) the base sequence represented by SEQ ID NO: 1 (Table 1); (a2) a nucleotide sequence of 10 or more consecutive nucleotides in length in the nucleotide sequence represented by SEQ ID NO: 1; (a3) a base sequence in which 1 to 9 bases, preferably 8 bases, 7 bases, 6 bases, 5 bases, 4 bases, 3 bases, 2 bases, or 1 base is deleted, substituted, or added in the base sequence represented by SEQ ID NO: 1; (a4) a nucleotide sequence having 80% or more, preferably 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to any of the nucleotide sequences of (a1) to (a3); A second primer having any one of the following nucleotide sequences (b1) to (b4): (b1) the base sequence represented by SEQ ID NO: 2 (Table 1); (b2) a nucleotide sequence of 10 or more consecutive nucleotides in length in the nucleotide sequence represented by SEQ ID NO: 2; (b3) a base sequence in which 1 to 9 bases, preferably 8 bases, 7 bases, 6 bases, 5 bases, 4 bases, 3 bases, 2 bases, or 1 base is deleted, substituted, or added in the base sequence represented by SEQ ID NO: 2; (b4) a nucleotide sequence having 80% or more, preferably 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to any of the nucleotide sequences of (b1) to (b3); and a probe having a base sequence selected from the following (c1) to (c4): (c1) the nucleotide sequence represented by SEQ ID NO: 3 (Table 1); (c2) a contiguous nucleotide sequence of 10 or more nucleotides containing the nucleotide sequence represented by SEQ ID NO: 4 (Table 1) on the nucleotide sequence represented by SEQ ID NO: 3; (c3) a nucleotide sequence in which 1 to 9 bases, preferably 8 bases, 7 bases, 6 bases, 5 bases, 4 bases, 3 bases, 2 bases, or 1 base are deleted, substituted, or added in the nucleotide sequence represented by SEQ ID NO: 3, and no deletion or addition occurs in the nucleotide sequence represented by SEQ ID NO: 4; (c4) a nucleotide sequence having 80% or more, preferably 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to any of the nucleotide sequences of (c1) to (c3), and having no mutation in the nucleotide sequence portion represented by SEQ ID NO: 4; and amplifying the nucleic acid in the sample using the method. The method of this embodiment has the above-mentioned features, and thus has the advantage of being able to detect apple snails with high sensitivity without cross-reaction with native species.

[0020] 2-1 Primers and probes The first primer is selected from the base sequence represented by SEQ ID NO: 1, a partial sequence of the base sequence represented by SEQ ID NO: 1, a base sequence having 1 to 9 base mutations in the base sequence represented by SEQ ID NO: 1, and a base sequence having 80% or more sequence identity with these base sequences. Most preferably, the first primer has the base sequence represented by SEQ ID NO: 1.

[0021] The second primer is selected from the base sequence represented by SEQ ID NO: 2, a partial sequence of the base sequence represented by SEQ ID NO: 2, a base sequence having 1 to 9 base mutations in the base sequence represented by SEQ ID NO: 2, and a base sequence having 80% or more sequence identity with these base sequences. Most preferably, the second primer has the base sequence represented by SEQ ID NO: 2.

[0022] The probe is selected from the base sequence represented by SEQ ID NO: 3, a partial sequence of the base sequence represented by SEQ ID NO: 3, a base sequence having 1 to 9 base mutations in the base sequence represented by SEQ ID NO: 3, and a base sequence having 80% or more sequence identity to any of these base sequences, but in any case, it is required to contain the base sequence represented by SEQ ID NO: 4. Most preferably, the probe has the base sequence represented by SEQ ID NO: 3.

[0023] Table 1 shows the nucleotide sequence (SEQ ID NO: 5) of cytochrome oxidase 1 (COI) from the Pomacea canaliculata. Figure 1 shows an alignment of the partial sequence of the COI gene from the Pomacea canaliculata with the corresponding sequences from the La Plata apple snail and other freshwater snails (Pomacanthus spp., P ...). For the La Plata apple snail nucleotide sequence, only bases that differ from SEQ ID NO: 5 are shown. For species other than the Pomacea canaliculata and the La Plata apple snail, only bases that differ from positions 382 to 407 of the nucleotide sequence of SEQ ID NO: 5 are shown. The nucleotide sequence of SEQ ID NO: 5 is the nucleotide sequence from positions 1 to 1536, which corresponds to the open reading frame of COI, in the Pomacea canaliculata mitochondrial complete genome sequence (NCBI Reference Sequence: NC_024586.1).

[0024] The first primer, particularly the primer having the nucleotide sequence of SEQ ID NO: 1, specifically binds to the complementary strand of positions 353 to 377 of the nucleotide sequence of the COI gene represented by SEQ ID NO: 5. The second primer, particularly the primer having the nucleotide sequence of SEQ ID NO: 2, specifically binds to positions 401 to 419 of the nucleotide sequence of the COI gene represented by SEQ ID NO: 5. In the figure, the first primer binds to the 5' end and the second primer binds to the 3' end, so hereinafter the first primer is also referred to as the forward primer and the second primer is also referred to as the reverse primer. The forward primer and reverse primer described above are capable of amplifying a partial sequence of positions 353 to 419 of the nucleotide sequence represented by SEQ ID NO: 5.

[0025] The probe, particularly the probe having the nucleotide sequence of SEQ ID NO: 3, specifically binds to the complementary strand of positions 382 to 399 of the nucleotide sequence of the COI gene represented by SEQ ID NO: 5. The probe has the nucleotide sequence of SEQ ID NO: 4 as at least a partial sequence. The nucleotide sequence of SEQ ID NO: 4 specifically binds to the complementary strand of positions 388 to 397 of the nucleotide sequence of the COI gene represented by SEQ ID NO: 5. Since differences in the nucleotide sequences between apple snails and other freshwater snails are particularly observed in this region, the use of the probe makes it possible to specifically detect apple snails.

[0026] The first primer, second primer, and probe are used for nucleic acid amplification and detection. Any known nucleic acid amplification method can be used, but real-time PCR is preferred. It is particularly preferred to use a TaqMan (registered trademark) probe, which has a fluorescent substance (reporter dye) attached to the 5' end of the probe and a quencher substance attached to the 3' end. The combination of the fluorescent substance and the quencher substance is not particularly limited, as long as it is a combination typically used in TaqMan probes. The quencher substance can be either a fluorescent quencher or a non-fluorescent quencher (NFQ). Examples of combinations of fluorescent substances and quenchers include FAM-TAMRA (fluorescence wavelength: 520 nm), HEX-TAMRA (fluorescence wavelength: 556 nm), TET-TAMRA (fluorescence wavelength: 536 nm), FAM-BHQ®-1 (fluorescence wavelength: 520 nm), HEX-BHQ-1 (fluorescence wavelength: 556 nm), TET-BHQ-1 (fluorescence wavelength: 536 nm), TAMRA-BHQ-2 (fluorescence wavelength: 583 nm), ROX-BHQ-2 (fluorescence wavelength: 610 nm), and Cy5-BHQ-3 (fluorescence wavelength: 669 nm). Alternatively, any combination of fluorescent substance and NFQ may be used. The TaqMan probe may further contain a minor groove binder (MGB) bound to the 3' end. Real-time PCR using TaqMan probes can be performed using an instrument such as the LightCycler 96 (Roche Diagnostics).

[0027] The probe does not necessarily have to be labeled with a fluorescent substance, etc. In this case, in the PCR method, the first primer and / or the second primer may be labeled with a fluorescent substance, digoxigenin (DIG), biotin, etc., and the target DNA may be amplified, and then the amplified DNA may be bound to a probe immobilized on a membrane, microplate, etc., for detection. In this case, the target DNA can be detected from the amplified DNA even if other nucleic acid amplification techniques that do not use a probe during amplification are used.

[0028] [Table 1]

[0029] 2-2 Collection and detection of environmental DNA In this embodiment, the sample is not particularly limited as long as it is a sample suspected of containing apple snail DNA. For example, it may be a biological sample (e.g., muscle tissue) collected directly from a freshwater snail that needs to be identified as an apple snail, but a sample collected from a freshwater or soil environment is preferred. For the purpose of preventing damage to apple snails in freshwater environments, a sample collected from a freshwater environment is more preferred. In other words, the DNA amplified and detected from the sample is preferably environmental DNA, particularly environmental DNA derived from a freshwater environment. Below, a method for detecting environmental DNA from a sample collected from a freshwater environment will be described as an example, but this is not intended to limit the sample used in this embodiment.

[0030] For procedures for collecting and detecting environmental DNA, see, for example, the Environmental DNA Survey and Experiment Manual Ver. 2.2 (published April 3, 2020) compiled by the Environmental DNA Society and the Environmental DNA Technology Standardization Committee. The procedures described in the manual may be modified as needed. Figure 2 shows an example of the procedure for collecting and detecting environmental DNA. The steps shown in the figure include collecting and detecting environmental DNA from a freshwater environment, filtering the sample, preserving the filter in a preservative solution, recovering DNA from the filter, purifying the DNA, and amplifying and detecting apple snail DNA. Each step is described below as an example, but the conditions for each step are not intended to be limiting.

[0031] 2-2-1 Water sampling process Environmental samples are collected from rivers, lakes, marshes, rice paddies, irrigation channels, etc. First, the sampling point is determined and the water is collected. Containers (paper cups, etc.) used for collecting the water should be unused, or if they are reused, the inside of the container should be sprayed with a sodium hypochlorite solution (approximately 0.1%) and wiped down before sampling to remove any nucleic acids adhering to the inside of the container.

[0032] 2-2-2 Filter filtration process Environmental DNA is concentrated from collected water samples as quickly as possible by filtration, before DNA degradation occurs. Filters such as Sterivex filter cartridges (pore size 0.45 μm, Merck Millipore, SVHV010RS) and glass fiber filters can be used. When using a Sterivex filter cartridge, the sample is aspirated into a 50 mL syringe, then the filter cartridge is attached to the syringe and water is passed through. By repeating this process, approximately 200 mL to 1 L of sample can usually be filtered with one filter cartridge.

[0033] 2-2-3 Preservation process After filtration, the filter cartridge is preferably filled with a preservation solution to prevent degradation of the internal DNA. Air is passed through the filter cartridge with a syringe to remove any remaining water. The outlet port of the filter cartridge is then sealed with parafilm, a luer fitting (e.g., VRSP6, manufactured by Isis Co., Ltd.), or the like, and the preservation solution is then injected into the filter cartridge with a syringe. Examples of preservation solutions that can be used include RNAlater™ stabilization solution (Thermo Fisher Scientific), ethanol, and aqueous benzalkonium chloride solution. After injecting the preservation solution, the injection port of the filter cartridge is sealed with parafilm, a luer fitting (e.g., VRMP6, manufactured by Isis Co., Ltd.), or the like. The filter cartridge is then preferably immediately stored in a cool, dark place, such as in a cooler box. It is then preferably frozen (e.g., at -20°C or below) as soon as possible.

[0034] If it is difficult to carry out the above-mentioned filter filtration and storage steps at the water collection site, it is preferable to place the collected water in an unused sealed container or one that has been decontaminated with a sodium hypochlorite aqueous solution or the like, add a preservative such as benzalkonium chloride as necessary, and immediately place the water in a cooler box or the like for transportation, after which filter filtration is carried out as soon as possible.

[0035] 2-2-4 DNA extraction process Next, the DNA attached to the filter is recovered. If the filter cartridge was frozen and filled with preservative solution, it is first thawed at room temperature, and the preservative solution in the filter cartridge is removed by aspiration, centrifugation, or other methods. When aspiration is used, the filter cartridge can be placed in a QIAvac (Qiagen) connected to an aspirator, and the preservative solution can be removed by aspiration. When centrifugation is used, the preservative solution can be removed by, for example, placing a 5 mL tube in a 50 mL conical tube, placing the filter cartridge with both open ends in the 5 mL tube with the injection hole facing downwards, closing the lid of the 50 mL conical tube, and centrifuging.

[0036] After removing the storage solution, the filter cartridge's outlet hole is sealed and filled with an elution solution. The elution solution is not particularly limited, but may be, for example, a solution containing a chaotropic protein denaturant (e.g., guanidine hydrochloride) and a protease (e.g., proteinase K). The elution solution can be prepared, for example, by diluting Buffer AL and proteinase K from the DNeasy Blood and Tissue kit (Qiagen, 69504) with PBS(-).

[0037] The filter cartridge filled with the elution solution is sealed at both ends and heated at approximately 50 to 60°C (e.g., 56°C). For heating, a dry heat sterilizer can be used, for example. The heating time can be approximately 20 to 30 minutes. During heating, the filter cartridge may be rotated at a speed of approximately 10 rpm using a rotator to ensure that the elution solution is evenly distributed inside the filter cartridge. Alternatively, the amount of elution solution used may be increased (see, for example, Wong et al., Nature Scientific Reports, (2020) 10:21531).

[0038] After heating, the eluted solution is recovered from the filter cartridge. For example, the filter cartridge with both open ends is placed with the injection port facing downwards in a 5 mL tube placed inside a 50 mL conical tube, and the 50 mL conical tube is then closed with its lid and centrifuged to recover the eluted solution in the 5 mL tube.

[0039] 2-2-5 DNA purification process The DNA in the recovered elution solution is purified. The DNA purification method is not particularly limited, and any known purification method can be used. For example, the DNeasy Blood and Tissue kit can be used. Alternatively, known methods such as the phenol / chloroform method or ethanol precipitation can be used.

[0040] 2-2-6 DNA amplification and detection process The DNA in the solution containing the purified DNA (hereinafter referred to as the DNA solution) is amplified to detect the apple snail-derived DNA. Any known nucleic acid amplification method can be used for DNA amplification, but real-time PCR, particularly TaqMan® PCR, which allows simultaneous nucleic acid amplification and probe detection, is particularly suitable.

[0041] In real-time PCR, a reaction solution for DNA amplification is usually mixed with a DNA solution containing template DNA, and then the mixture is reacted. The reaction solution for DNA amplification in real-time PCR usually contains at least the following components: -DNA polymerase; -First primer; - second primer; -probe; -deoxyribonucleotide triphosphates (dNTPs); - divalent metal ions, preferably magnesium ions (Mg 2+ ) - pH buffering agent. The pH of the reaction solution for DNA amplification is usually about 7.5 to 10.5, particularly about 8.0 to 9.0.

[0042] The DNA polymerase is not particularly limited, and examples thereof include Taq DNA polymerase, Tth DNA polymerase, KOD DNA polymerase, Bst DNA polymerase, and Bsu DNA polymerase. The pH buffer may be any known pH buffer commonly used for real-time PCR, such as Tris, phosphate buffer, or Good's buffer such as HEPES. The divalent metal ion may be Mg 2+ In this case, the concentration can be, for example, about 2 mM. The probe can be a fluorescently labeled probe, particularly a TaqMan (registered trademark) probe.

[0043] Using a probe not bound to a fluorescent substance, PCR (amplification) and detection may be performed separately instead of real-time PCR. In this case, by using the first primer and / or the second primer labeled with a fluorescent substance, DIG, biotin, etc., the amplified DNA can be detected using a probe-immobilized microplate (microplate hybridization), membrane (Southern hybridization), etc.

[0044] The DNA solution can be mixed with the above-mentioned DNA amplification reagents and reacted under a predetermined temperature condition to amplify the DNA. DNA amplification can be performed using a real-time PCR or PCR thermal cycler, such as the LightCycler® 96 (Roche Diagnostics).

[0045] To reduce the risk of errors due to atmospheric DNA contamination, particularly carryover contamination, the steps from the DNA recovery step to just before loading into the thermal cycler are preferably performed in a laboratory separate from the laboratory where post-DNA amplification steps are performed. Alternatively, uracil-DNA glycosylase or the like is preferably used to reduce the risk of carryover contamination.

[0046] Whether or not apple snail DNA is present in the DNA solution can be determined from the signal obtained by real-time PCR, etc. If the presence of apple snail DNA is detected in the DNA solution, it can be determined that apple snails live in the environment from which the original sample of the DNA solution was collected.

[0047] 3. Reagents or kits for detecting apple snails A second embodiment of the present invention is a reagent or kit for detecting apple snails. The reagent or kit (hereinafter also referred to as "reagent, etc.") of this embodiment comprises a first primer having any of the following base sequences (a1) to (a4): (a1) a nucleotide sequence represented by SEQ ID NO: 1; (a2) a nucleotide sequence of 10 or more consecutive nucleotides in length in the nucleotide sequence represented by SEQ ID NO: 1; (a3) a base sequence in which 1 to 9 bases, preferably 8 bases, 7 bases, 6 bases, 5 bases, 4 bases, 3 bases, 2 bases, or 1 base is deleted, substituted, or added in the base sequence represented by SEQ ID NO: 1; (a4) a nucleotide sequence having 80% or more, preferably 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to any of the nucleotide sequences of (a1) to (a3); A second primer having any one of the following nucleotide sequences (b1) to (b4): (b1) a base sequence represented by SEQ ID NO: 2; (b2) a nucleotide sequence of 10 or more consecutive nucleotides in length in the nucleotide sequence represented by SEQ ID NO: 2; (b3) a base sequence in which 1 to 9 bases, preferably 8 bases, 7 bases, 6 bases, 5 bases, 4 bases, 3 bases, 2 bases, or 1 base is deleted, substituted, or added in the base sequence represented by SEQ ID NO: 2; (b4) a nucleotide sequence having 80% or more, preferably 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to any of the nucleotide sequences of (b1) to (b3); and a probe having a base sequence selected from the following (c1) to (c4): (c1) a nucleotide sequence represented by SEQ ID NO: 3; (c2) a contiguous nucleotide sequence of 10 or more nucleotides including the nucleotide sequence represented by SEQ ID NO: 4 in the nucleotide sequence represented by SEQ ID NO: 3; (c3) a nucleotide sequence in which 1 to 9 bases, preferably 8 bases, 7 bases, 6 bases, 5 bases, 4 bases, 3 bases, 2 bases, or 1 base are deleted, substituted, or added in the nucleotide sequence represented by SEQ ID NO: 3, and no deletion or addition occurs in the nucleotide sequence represented by SEQ ID NO: 4; (c4) a nucleotide sequence having 80% or more, preferably 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to any of the nucleotide sequences of (c1) to (c3), and having no mutation in the nucleotide sequence portion represented by SEQ ID NO: 4; The present invention is characterized in that it includes:

[0048] The reagents etc. of this embodiment have the above-mentioned characteristics and can be used to detect apple snails with high sensitivity without cross-reaction with native species.

[0049] The reagent etc. of this embodiment is preferably a reagent etc. for real-time PCR. In the case of a reagent etc. for real-time PCR, the probe can be, for example, a TaqMan probe.

[0050] Specifically, the reagents and the like of this embodiment are the reagents and the like used in the method described in Section 2. "Method for detecting apple snails." Unless otherwise specified, the sample to be detected, the conditions for detection, and the like are as described in Section 2. "Method for detecting apple snails."

[0051] 4 Primer sets for detecting apple snails A third embodiment of the present invention is a primer set for detecting apple snails. The primer set of this embodiment comprises a first primer having any of the following base sequences (a1) to (a4): (a1) a nucleotide sequence represented by SEQ ID NO: 1; (a2) a nucleotide sequence of 10 or more consecutive nucleotides in length in the nucleotide sequence represented by SEQ ID NO: 1; (a3) a base sequence in which 1 to 9 bases, preferably 8 bases, 7 bases, 6 bases, 5 bases, 4 bases, 3 bases, 2 bases, or 1 base is deleted, substituted, or added in the base sequence represented by SEQ ID NO: 1; (a4) a nucleotide sequence having 80% or more, preferably 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to any of the nucleotide sequences of (a1) to (a3); and a second primer having any one of the following nucleotide sequences (b1) to (b4): (b1) a base sequence represented by SEQ ID NO: 2; (b2) a nucleotide sequence of 10 or more consecutive nucleotides in length in the nucleotide sequence represented by SEQ ID NO: 2; (b3) a base sequence in which 1 to 9 bases, preferably 8 bases, 7 bases, 6 bases, 5 bases, 4 bases, 3 bases, 2 bases, or 1 base is deleted, substituted, or added in the base sequence represented by SEQ ID NO: 2; (b4) a nucleotide sequence having 80% or more, preferably 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to any of the nucleotide sequences of (b1) to (b3); The present invention is characterized in that it comprises:

[0052] Specifically, the primer set of this embodiment is a primer set for use in the method described in Section "2. Method for detecting apple snails." It is also a primer set used to produce the reagent or kit described in Section "3. Reagent or kit for detecting apple snails." The primer set of this embodiment can be used in combination with the probe described in Section "5. Probe for detecting apple snails," which will be described later.

[0053] 5. Probes for detecting apple snails A fourth embodiment of the present invention is a probe for detecting apple snails. The probe of this embodiment is characterized by having a base sequence selected from the following (c1) to (c4): (c1) a nucleotide sequence represented by SEQ ID NO: 3; (c2) a contiguous nucleotide sequence of 10 or more nucleotides including the nucleotide sequence represented by SEQ ID NO: 4 in the nucleotide sequence represented by SEQ ID NO: 3; (c3) a nucleotide sequence in which 1 to 9 bases, preferably 8 bases, 7 bases, 6 bases, 5 bases, 4 bases, 3 bases, 2 bases, or 1 base are deleted, substituted, or added in the nucleotide sequence represented by SEQ ID NO: 3, and no deletion or addition occurs in the nucleotide sequence represented by SEQ ID NO: 4; (c4) A base sequence having 80% or more, preferably 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to any of the base sequences of (c1) to (c3), and having no mutation in the base sequence portion represented by SEQ ID NO: 4.

[0054] Specifically, the probe of this embodiment is a probe for use in the method described in Section "2. Method for detecting apple snails." It is also a probe used to produce the reagent or kit described in Section "3. Reagent or kit for detecting apple snails." The primer set of this embodiment can be used in combination with the primer set described in Section "4. Primer set for detecting apple snails."

[0055] The probe of this embodiment is not particularly limited, but is preferably a TaqMan (registered trademark) probe. The fluorescent substance and quencher substance used in the TaqMan probe may be any of those typically used in TaqMan probes. Furthermore, the TaqMan probe may have an MGB (Minor Groove Binder) bound to its 3' end. [Example]

[0056] [Example 1] DNA detection from breeding water and tissue samples of various freshwater snails (1) DNA collection from breeding water Pomacea canaliculata, La Plata apple snail, scorpion snail, semi-snail snail, and scorpion snail were reared for two weeks. The rearing density was 2–3 individuals per 5 L for Pomacea canaliculata and La Plata apple snail, and 20 individuals per 0.5 L for the other snails. After rearing, water was collected in a paper cup, aspirated using a 50 mL syringe, and a Sterivex filter cartridge (pore size 0.45 μm, Merck Millipore, SVHV010RS) was attached to the syringe suction port and pressure-filtered. By repeating this process, 200 mL–1 L of rearing water was filtered. The syringe filled with air was reattached to the filter cartridge after filtration, and pressure was applied to expel the water from the cartridge. This procedure was repeated several times until the cartridge was completely depleted. A nucleic acid storage solution (RNAlater solution (Applied Biosystems, AM7021)) was injected into the cartridge using a micropipette to fill it. Luer fittings (VRSP6 and VRMP6, manufactured by Isis) were attached to the outlet and inlet holes of the filter cartridge, which was then sealed. The sealed filter cartridge was stored under refrigerated conditions (below -20°C or below -30°C).

[0057] (2) Collection of DNA from breeding water The filter cartridge containing the frozen DNA was returned to room temperature and the RNALater solution was thawed. A 5 mL tube with its lid open was placed in a 50 mL conical tube with its lid facing up, and the filter cartridge, with the Luer fitting removed, was placed with the injection hole facing downward and inside the opening of the 5 mL tube. The 50 mL conical tube was centrifuged at 4000 rpm for 2 minutes, and the RNALater solution was collected in the 5 mL tube.

[0058] Proteinase K (600 mAU / ml, QIAGEN 19133), Buffer AL (QIAGEN 19075), and PBS(-) were mixed in a volume ratio of 10:91:99 to prepare an elution solution. After attaching a Luer fitting to the outlet port of the filter cartridge, 2 mL of the elution solution was injected into the injection port using a micropipette. After attaching a Luer fitting to the injection port and sealing it, the cartridge was heated in a dry heat sterilizer at 56°C for 30 minutes.

[0059] An open 5 mL tube was placed in an open 50 mL conical tube with the opening facing up. The luer fitting of the heated filter cartridge was removed, and the injection hole was placed facing downwards so that it was inside the opening of the 5 mL tube. The 50 mL conical tube was centrifuged at 4,000 rpm for 10 minutes, and the liquid in the cartridge was collected in a 5 mL tube. The liquid in the 5 mL tube was transferred to the same 50 mL conical tube and centrifuged at 10,000 rpm for 10 minutes. The supernatant was then collected and used as the DNA recovery solution.

[0060] (3) Collection of DNA (genomic DNA) from tissue DNA was extracted from tissues of the apple snail, the apple snail, the stag beetle, the semi-sulphur snail, and the stag beetle, using the DNeasy Blood and Tissue kit (Qiagen, 69504). 200 mg of tissue from each freshwater snail was collected, placed in a solution containing 180 μL of Buffer ATL and 20 μL of proteinase K, and disrupted using a homogenizer. After disruption, the tissue was heated at 56°C for 30 minutes, and then collected. 200 μL of Buffer AL was added to obtain the DNA recovery solution.

[0061] (4) DNA purification Approximately 2 mL and 0.4 mL of the DNA recovery solution obtained in (2) and (3) were placed in 5 mL tubes, respectively, and 1 mL and 0.2 mL of ethanol were added, followed by mixing with a pipette. DNA was then extracted using the DNeasy Blood and Tissue kit (Qiagen, 69504). 650 μL of the mixed solution was placed in the spin column of the DNeasy Blood and Tissue kit and centrifuged at 8000 rpm for 1 minute. If any mixed solution remained, the discharged liquid was removed, and 650 μL of the remaining solution was added to the same spin column and centrifuged in the same manner. This procedure was repeated until the solution in the tube was depleted.

[0062] The spin column was placed in a new 2 mL collection tube, and 800 μL of Buffer AW1 was added to the spin column and centrifuged at 8,000 rpm for 1 minute. 800 μL of Buffer AW2 was then added to the spin column and centrifuged at 13,000 rpm for 4 minutes. The spin column was then transferred to a new 2 mL collection tube and centrifuged again at 13,000 rpm for 3 minutes. A new 1.5 mL tube was prepared, and the centrifuged spin column was placed in it. 75 μL of Buffer AE preheated to 56°C was poured onto the column membrane. After leaving the tube at room temperature for 1 minute, the DNA solution was eluted into the 1.5 mL tube by centrifugation at 8,000 rpm for 1 minute.

[0063] (5) Quantitative PCR Quantitative PCR of each DNA solution was performed using TaqMan Environmental Master Mix 2.0 (Thermo Fisher Scientific, 4398021). An amplification detection solution with the composition shown in Table 2 was prepared and dispensed into eight tubes for quantitative PCR.

[0064] [Table 2]

[0065] The forward primer, reverse primer, and probe used had the following base sequences: All of the primers used were degenerate primers. Forward primer: 5'-GTGCYGGAACKGGATGRACAGTATA-3' (SEQ ID NO: 1) Reverse primer: 5'- ACAGAMCCACCYGCATGAG -3' (SEQ ID NO: 2) Probe: 5'- FAM-CCTTTAGCTGGTAATTTA -NFQ-MGB-3' (SEQ ID NO: 3)

[0066] As controls, an amplification detection solution containing no template DNA and a solution in which the template was diluted with distilled water were dispensed into wells at both ends of the 8-tube strip.

[0067] The eight tubes containing the amplification and detection solution were placed in a LightCycler 96 (Roche Diagnostics) and PCR was performed under the following conditions: 95°C for 10 minutes, followed by 50 cycles of (95°C for 5 seconds + 60°C for 1 minute).

[0068] Figure 3 shows the results of quantitative PCR for DNA solutions derived from tissues and rearing water. The vertical axis represents fluorescence intensity, and the horizontal axis represents PCR cycle number. Figure 3A shows the results of quantitative PCR for DNA solutions derived from tissues, and Figure 3B shows the results for DNA solutions derived from rearing water. DNA amplification was confirmed in DNA solutions derived from tissues of Pomacea canaliculata and La Plata apple snails, but not in DNA solutions derived from tissues of S. sieboldii, S. chirimensis, and S. sieboldii. Similarly, DNA amplification was confirmed in DNA solutions derived from rearing waters of Pomacea canaliculata and S. sieboldii, but not in DNA solutions derived from tissues of S. sieboldii, S. chirimensis, and S. sieboldii. This demonstrates that quantitative PCR can specifically detect apple snails and S. sieboldii.

[0069] [Example 2] Quantitative PCR from environmental DNA (1) Environmental DNA sampling We performed quantitative PCR to detect apple snails using environmental DNA from an irrigation channel in Omitama City (hereafter, Environment A), where large numbers of apple snails have been confirmed, and a pond in Tsukuba City (hereafter, Environment B), where no apple snails have been confirmed to inhabit. Environmental water samples from Environments A and B were collected into paper cups. Sampling was performed at two locations in Environment A and four locations in Environment B. The environmental water was aspirated on-site using a 50 mL syringe, and a Sterivex filter cartridge (pore size 0.45 μm, Merck Millipore, SVHV010RS) was attached to the syringe suction port, allowing pressure filtration. By repeating this process, 200 mL to 1 L of environmental water was filtered. After filtration, the syringe filled with air was reattached to the filter cartridge, and pressure was applied to expel the water from the cartridge. This procedure was repeated several times until the water in the cartridge was depleted. A nucleic acid preservation solution (RNAlater solution (Applied Biosystems, AM7021)) was injected into the cartridge using a micropipette to fill the inside. Luer fittings (Isis VRSP6 and VRMP6) were attached to the outlet and inlet holes of the filter cartridge and sealed. After sealing, the filter cartridge was placed in a plastic bag with a zipper and temporarily stored in a cooler box containing ice packs. The cartridge was transferred to and stored under frozen conditions (below -20°C or below -30°C) as soon as possible.

[0070] (2) Collection of environmental DNA The filter cartridge containing the frozen environmental DNA was returned to room temperature and the RNAlater solution was thawed. A 5 mL tube with its lid open was placed in a 50 mL conical tube with its lid facing up, and the filter cartridge, with the Luer fitting removed, was placed with the injection hole facing downward and inside the 5 mL tube. The 50 mL conical tube was centrifuged at 4000 rpm for 2 minutes, and the RNAlater solution was collected in the 5 mL tube.

[0071] Proteinase K (600 mAU / ml, Qiagen, 19133), Buffer AL (Qiagen, 19075), and PBS(-) were mixed in a ratio of 10:91:99 to prepare an elution solution. After attaching a Luer fitting to the outlet port of the filter cartridge, 2 mL of the elution solution was injected into the injection port using a micropipette. After attaching a Luer fitting to the injection port and sealing it, the cartridge was heated in a dry oven at 56°C for 30 minutes.

[0072] The 5 mL tube with its lid open was placed in an open 50 mL conical tube with the opening facing up. The Luer fitting of the heated filter cartridge was removed, and the injection hole was placed facing downward so that it was inside the opening of the 5 mL tube. The 50 mL conical tube was then closed and centrifuged at 4,000 rpm for 10 minutes, and the eluate was collected in a 5 mL tube. The eluate in the 5 mL tube was transferred to the same 50 mL conical tube and centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected.

[0073] (3) Purification of environmental DNA DNA was purified from the supernatant in the same manner as in Example 1(4).

[0074] (4) Quantitative PCR Quantitative PCR of the environmental DNA solution was carried out in the same manner as in Example 1(5).

[0075] Figure 4 shows the results of quantitative PCR of environmental DNA from environments A and B. The vertical axis shows fluorescence intensity, and the horizontal axis shows the number of PCR cycles. Table 3 shows the Ct values ​​of quantitative PCR of environmental DNA from environments A and B. DNA amplification was confirmed in environment A, but not in environment B. This demonstrates that quantitative PCR can specifically detect apple snails in the environment.

[0076] [Table 3]

[0077] [Example 3] Quantitative PCR from environmental DNA from various locations Quantitative PCR was used to detect apple snails in environmental DNA collected from various locations in Japan where apple snails have been confirmed to exist. Environmental DNA was collected from the aquatic environments of Iwata City, Shizuoka Prefecture (3 locations), Yokkaichi City, Mie Prefecture (3 locations), Matsusaka City, Mie Prefecture (2 locations), Yasu City, Shiga Prefecture (2 locations), Nara City, Nara Prefecture (2 locations), and Sasebo City, Nagasaki Prefecture (2 locations). The procedures for sampling, collection, purification, and quantitative PCR of environmental DNA were all the same as those in Example 2.

[0078] Table 4 shows the Ct values ​​of quantitative PCR for environmental DNA collected from various locations. DNA amplification was confirmed in all samples. These results demonstrate that it is possible to detect apple snails from environmental DNA throughout Japan, regardless of regional differences.

[0079] [Table 4] [Industrial Applicability]

[0080] The method, reagent or kit of the present invention is useful as a countermeasure against pest damage in rice farming and the like, and can be used in industrial fields such as agriculture.

Claims

1. A first primer having any one of the following base sequences (a1) to (a4): (a1) a base sequence represented by SEQ ID NO: 1; (a2) a contiguous base sequence of 10 or more bases in the base sequence represented by SEQ ID NO: 1; (a3) a base sequence in which 1 to 9 bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 1; (a4) a nucleotide sequence having 80% or more sequence identity with any one of the nucleotide sequences (a1) to (a3); A second primer having any one of the following base sequences (b1) to (b4): (b1) a base sequence represented by SEQ ID NO: 2; (b2) a contiguous base sequence of 10 or more bases in the base sequence represented by SEQ ID NO: 2; (b3) a base sequence in which 1 to 9 bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 2; (b4) a base sequence having 80% or more sequence identity with any of the base sequences of (b1) to (b3); and a probe having a base sequence selected from the following (c1) to (c4): (c1) a base sequence represented by SEQ ID NO: 3; (c2) a contiguous base sequence of 10 or more bases including the base sequence represented by SEQ ID NO: 4 on the base sequence represented by SEQ ID NO: 3; (c3) a base sequence in which 1 to 9 bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 3, and there is no deletion or addition in the base sequence portion represented by SEQ ID NO: 4; (c4) a nucleotide sequence having 80% or more sequence identity with any of the nucleotide sequences of (c1) to (c3), and having no mutation in the nucleotide sequence portion represented by SEQ ID NO: 4; amplifying nucleic acid in the sample using Methods for detecting apple snails.

2. 10. The method of claim 1, wherein the sample is a sample taken from a freshwater environment or a soil environment.

3. The method of claim 1 , wherein the nucleic acid is environmental DNA.

4. The method according to claim 1 , wherein the probe comprises a fluorescent substance and a quencher substance at the 5′-end and 3′-end of the base sequence, respectively.

5. A first primer having any one of the following base sequences (a1) to (a4): (a1) a base sequence represented by SEQ ID NO: 1; (a2) a contiguous base sequence of 10 or more bases in the base sequence represented by SEQ ID NO: 1; (a3) a base sequence in which 1 to 9 bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 1; (a4) a nucleotide sequence having 80% or more sequence identity with any one of the nucleotide sequences (a1) to (a3); A second primer having any one of the following base sequences (b1) to (b4): (b1) a base sequence represented by SEQ ID NO: 2; (b2) a contiguous base sequence of 10 or more bases in the base sequence represented by SEQ ID NO: 2; (b3) a base sequence in which 1 to 9 bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 2; (b4) a base sequence having 80% or more sequence identity with any of the base sequences of (b1) to (b3); and a probe having a base sequence selected from the following (c1) to (c4): (c1) a base sequence represented by SEQ ID NO: 3; (c2) a contiguous base sequence of 10 or more bases including the base sequence represented by SEQ ID NO: 4 on the base sequence represented by SEQ ID NO: 3; (c3) a base sequence in which 1 to 9 bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 3, and there is no deletion or addition in the base sequence portion represented by SEQ ID NO: 4; (c4) a nucleotide sequence having 80% or more sequence identity with any of the nucleotide sequences of (c1) to (c3), and having no mutation in the nucleotide sequence portion represented by SEQ ID NO: 4; A reagent or kit for detecting apple snails, comprising:

6. The reagent or kit according to claim 5, which is for real-time PCR.

7. A first primer having any one of the following base sequences (a1) to (a4): (a1) a base sequence represented by SEQ ID NO: 1; (a2) a contiguous base sequence of 10 or more bases in the base sequence represented by SEQ ID NO: 1; (a3) a base sequence in which 1 to 9 bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 1; (a4) a nucleotide sequence having 80% or more sequence identity with any one of the nucleotide sequences (a1) to (a3); and a second primer having any one of the following base sequences (b1) to (b4): (b1) a base sequence represented by SEQ ID NO: 2; (b2) a contiguous base sequence of 10 or more bases in the base sequence represented by SEQ ID NO: 2; (b3) a base sequence in which 1 to 9 bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 2; (b4) a base sequence having 80% or more sequence identity with any of the base sequences of (b1) to (b3); A primer set for detecting apple snails, consisting of:

8. A probe for detecting apple snails, having a base sequence selected from the following (c1) to (c4): (c1) a base sequence represented by SEQ ID NO: 3; (c2) a contiguous base sequence of 10 or more bases including the base sequence represented by SEQ ID NO: 4 on the base sequence represented by SEQ ID NO: 3; (c3) a base sequence in which 1 to 9 bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 3, and there is no deletion or addition in the base sequence portion represented by SEQ ID NO: 4; (c4) A base sequence having 80% or more sequence identity with any of the base sequences of (c1) to (c3), which does not have a mutation in the base sequence portion represented by SEQ ID NO: 4.

Images