Detection kit and detection method for luciola cruciata, and habitat survey method for luciola cruciata

Abstract

To provide a detection kit, a detection method, and a habitat survey method for Luciola cruciate that enable comprehensive detection of Luciola cruciata inhabiting specific regions within Japan.SOLUTION: Provided is a detection kit for Luciola cruciata, comprising a primer set for amplifying nucleic acid derived from Luciola cruciata, the primer set comprising: a first primer comprising a nucleotide sequence having a specific sequence; and a second primer comprising a nucleotide sequence having another specific sequence. Also provided are a method for detecting Luciola cruciata using the detection kit, and a habitat survey method for Luciola cruciata using the detection method.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a detection kit for fireflies, a detection method, and a method for investigating the habitat of fireflies.

Background Art

[0002] The Genji firefly (scientific name: Luciola cruciata) is a kind of aquatic firefly that inhabits various parts of Japan.

[0003] Since the Genji firefly grows in clean water quality, it has attracted attention as an indicator organism for a rich environment. However, in areas with such a rich environment, if road construction or the development of a final disposal site is carried out, it may threaten the habitat environment of organisms in the area. Preserving the environment in which the Genji firefly can grow leads to a place for local residents to relax and the creation of a habitat environment for various organisms in the surrounding environment, so the preservation of such an environment is required.

[0004] As a method for investigating the habitat of fireflies, observing the nocturnal luminescence of flying fireflies is common, but there is a problem that it is limited both in terms of time and period. In addition, since fireflies spend a long period of time as larvae in water, it is also conceivable to conduct a larval investigation. However, since such an investigation requires a capture investigation by experts, it takes time and effort to grasp the whole picture.

[0005] Therefore, environmental samples (soil, water, etc.) from areas predicted to be inhabited by fireflies are collected, nucleic acid amplification reaction (PCR) is performed using the nucleic acid extracted from the collected samples as a template, and the amplified products are analyzed to simply and efficiently investigate whether a specific type of firefly inhabits the area. A method has been studied.

[0006] As a method for detecting such fireflies, for example, Patent Document 1 discloses a primer set for amplifying nucleic acid derived from Luciola cruciata, which consists of a first primer and a second primer that can specifically amplify a predetermined region of the cytochrome oxidase subunit I (CO1) sequence in mitochondrial DNA, and a nucleic acid amplification step of performing a nucleic acid amplification reaction using the nucleic acid extracted from an arbitrary sample as a template with the primer set, and an analysis step of analyzing the amplification product by the nucleic acid amplification reaction.

[0007] In addition, in Non-Patent Document 1, the COII region of mitochondrial DNA of Luciola cruciata at 62 locations (494 individuals) in Japan was PCR-amplified, and after the amplification product was digested with six types of restriction enzymes, the cleavage patterns were compared by electrophoresis. As a result, 19 haplotype genotypes were detected from the obtained electrophoresis patterns, and from the types and frequencies of the haplotypes, it is described that they can be classified into six groups: Tohoku, Kanto, Chubu, Western Japan, Kitakyushu, and South Kyushu, and the six groups are divided into three clades: Tohoku-Kanto, Chubu-Western Japan, and Kitakyushu-South Kyushu.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Non-Patent Documents

[0009]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0010] However, as shown in Non-Patent Document 1, since the gentian living in Japan can be classified into 19 subtypes or 3 major groups of haplotypes, it is difficult to comprehensively detect and conduct a habitat survey of the gentian living throughout Japan using a primer set corresponding only to the gentian collected from a specific location as described in Patent Document 1.

[0011] The present invention has been made in view of such circumstances, and an object thereof is to provide a detection kit, a detection method, and a method for surveying the habitat of gentian that can comprehensively detect gentian living in a specific region of Japan from environmental samples.

Means for Solving the Problems

[0012] The inventor conducted intensive studies and found that by using a detection kit containing a primer set that amplifies a predetermined region in the mitochondrial DNA of gentian in a nucleic acid amplification reaction, it is possible to comprehensively detect gentian living in a specific region from environmental samples, and thus completed the present invention. That is, the present invention provides the following.

[0013] (1) A first aspect of the present invention is a detection kit for gentian, comprising a primer set for amplifying nucleic acid derived from gentian, the primer set consisting of a first primer containing the nucleotide sequence of SEQ ID NO: 1 and a second primer containing the nucleotide sequence of SEQ ID NO: 2.

[0014] (2) A second aspect of the present invention is the detection kit according to (1), wherein the gentian includes gentian living in the Tohoku and Kanto regions.

[0015] (3) The third aspect of the present invention is a method for detecting Luciola cruciata, comprising: a nucleic acid amplification step of performing a nucleic acid amplification reaction using the nucleic acid extracted from an environmental sample as a template with the detection kit described in (1); and an analysis step of analyzing the amplification product obtained by the nucleic acid amplification reaction.

[0016] (4) The fourth aspect of the present invention is a method for investigating the habitat of Luciola cruciata, characterized by using the detection method described in (3).

Advantages of the Invention

[0017] According to the present invention, it is possible to provide a detection kit, a detection method, and a method for investigating the habitat of Luciola cruciata that can comprehensively detect Luciola cruciata inhabiting specific regions in Japan.

Brief Description of the Drawings

[0018]

Figure 1

Figure 2

Embodiments for Carrying Out the Invention

[0019] Hereinafter, embodiments for carrying out the present invention (hereinafter simply referred to as "the present embodiment") will be described in detail. The following present embodiment is an exemplification for explaining the present invention and is not intended to limit the present invention to the following content. The present invention can be appropriately modified and implemented within the scope of its gist.

[0020] <Detection Kit for Luciola cruciata> The detection kit for Luciola cruciata according to the present embodiment is a detection kit containing a primer set for amplifying nucleic acid derived from Luciola cruciata (scientific name: Luciola cruciata). Non-Patent Document 1 describes that fireflies inhabiting Japan can be divided into 19 fine and 3 large groups of haplotypes. However, the firefly detection kit of the present invention can be preferably used to detect fireflies mainly inhabiting the Tohoku and Kanto regions.

[0021] In addition, the firefly detection kit may include, together with the primer set of the present invention, reagents for DNA extraction, reagents for nucleic acid amplification reaction (such as PCR), reagents for detecting nucleic acid amplification reaction products, and the like. Hereinafter, the primer set will be described in detail.

[0022] (Primer Set) The primer set of the present invention is a primer set for amplifying nucleic acid derived from firefly (scientific name: Luciola cruciata), and consists of the following two primers. (1) The first primer containing the nucleotide sequence of SEQ ID NO: 1 (5’- CGGGCTTACTTCACCTAGC -3’) (2) The second primer containing the nucleotide sequence of SEQ ID NO: 2 (5’- TGCAAATACTGCYCCTATTGA -3’)

[0023] The primer set of the present invention is used in a nucleic acid amplification reaction and can specifically amplify a predetermined region of the cytochrome oxidase subunit I (CO1) sequence in the mitochondrial DNA of fireflies. The base sequence length of the amplification product obtained by the nucleic acid amplification reaction using the primer set of the present invention is usually about 261 bp. However, this value may vary due to non-specific base insertions or deletions that occur during the nucleic acid amplification reaction.

[0024] The nucleic acid amplified using the primer set of the present invention is DNA. In addition, the primers constituting the primer set of the present invention can be synthesized by any method known as a method for synthesizing oligonucleotides.

[0025] (First primer) The first primer containing the nucleotide sequence of SEQ ID NO: 1 (hereinafter, also simply referred to as the "first primer") corresponds to the forward primer (sense primer) in the nucleic acid amplification reaction. The first primer may consist of the nucleotide sequence of SEQ ID NO: 1, or 1 to 30 bases may be added to the 5'-end and / or 3'-end of the nucleotide sequence of SEQ ID NO: 1, but preferably it consists of the nucleotide sequence of SEQ ID NO: 1. The bases added to the nucleotide sequence of SEQ ID NO: 1 can be appropriately designed based on the nucleotide sequence of CO1 of Geomyces destructans or the predicted Tm value of the resulting primer, etc.

[0026] (Second primer) The second primer containing the nucleotide sequence of SEQ ID NO: 2 (hereinafter, also simply referred to as the "second primer") corresponds to the reverse primer (antisense primer) in the nucleic acid amplification reaction. The second primer may consist of the nucleotide sequence of SEQ ID NO: 2, or 1 to 30 bases may be added to the 5'-end and / or 3'-end of the nucleotide sequence of SEQ ID NO: 2, but preferably it consists of the nucleotide sequence of SEQ ID NO: 2. The bases added to the nucleotide sequence of SEQ ID NO: 2 can be appropriately designed based on the nucleotide sequence of CO1 of Geomyces destructans or the predicted Tm value of the resulting primer, etc.

[0027] (Detection method of Geomyces destructans) The detection method of the present invention includes a nucleic acid amplification step of performing a nucleic acid amplification reaction using the nucleic acid extracted from an environmental sample as a template with the primer set of the present invention, and an analysis step of analyzing the amplification product obtained by the nucleic acid amplification reaction.

[0028] (Nucleic acid amplification step) The nucleic acid amplification step is a step of performing a nucleic acid amplification reaction using the nucleic acid extracted from an environmental sample as a template with the primer set of the present invention to obtain an amplification product of a predetermined region of CO1 of Geomyces destructans. The nucleic acid serving as the template is usually DNA.

[0029] In the present invention, the "environmental sample" is not particularly limited, and a sample collected from any environment (such as a waterside area, a breeding tank, etc.) by any method can be used. Examples of such samples include water, soil, sediment, excrement, carcasses (adult insects, larvae, etc.), molted exoskeletons, pupal cases, eggs, and the like.

[0030] The method for extracting nucleic acid from a sample is not particularly limited, and a conventionally known nucleic acid extraction method can be employed. For example, nucleic acid extraction may be performed using a phenol / chloroform / isoamyl alcohol (e.g., phenol:chloroform:isoamyl alcohol (mass ratio) = 25:24:1) solution or a commercially available kit. Also, the extracted nucleic acid may be appropriately concentrated by filtration or the like.

[0031] As the nucleic acid amplification reaction, a conventionally known nucleic acid amplification method can be adopted, and examples include polymerase chain reaction (PCR). In the nucleic acid amplification reaction, a buffer, four types of bases (dNTP), an enzyme (such as DNA polymerase), a probe, etc. can be used together with the primer set of the present invention and the nucleic acid serving as a template.

[0032] The conditions of the nucleic acid amplification reaction can be appropriately set according to the type of enzyme used, etc. Usually, the temperature and time are appropriately set, and a cycle consisting of denaturation, annealing, and extension is repeated. Also, the amplification product obtained by the nucleic acid amplification reaction may be appropriately purified.

[0033] (Analysis step) By the nucleic acid amplification reaction, a predetermined region of the CO1 of the Genji firefly can be specifically amplified. For example, even when using nucleic acid derived from organisms other than the Genji firefly (such as the Heike firefly), the target amplification product (usually about 261 bp) cannot be obtained by the nucleic acid amplification reaction. Utilizing this point, the detection of the Genji firefly can be performed by analyzing whether the amplification product obtained in the nucleic acid amplification step is an amplification product of a predetermined region of the CO1 of the Genji firefly.

[0034] In the present invention, "detecting Gentiana scabra" means determining whether Gentiana scabra exists in the area where the sample to be analyzed was collected. For example, in the analysis process, if the target amplification product (usually about 261 bp) is obtained, it can be determined that Gentiana scabra exists in the place where the sample was collected. Furthermore, by quantifying the amplification product in the analysis process, the number of Gentiana scabra inhabiting the area where the sample was collected can be estimated.

[0035] As a method for analyzing the amplification product, any method that can determine the presence or absence of the target amplification product, identify the sequence of the amplification product, quantify the nucleic acid (such as DNA) in the amplification product, etc. can be adopted. Examples of such methods include electrophoresis, sequence analysis of the base sequence, quantitative PCR, etc.

[0036] Electrophoresis is a method of determining the presence or absence of the target amplification product (usually about 261 bp) by subjecting the amplification product to electrophoresis on an agarose gel or the like and then staining the gel. Sequence analysis is a method of identifying the base sequence of the amplification product. Examples of sequence analysis methods include the Sanger method and methods using next-generation sequencers. The base sequence thus identified is queried using a base sequence database (for example, BLAST of NCBI). If the sequence identity with the base sequence of Gentiana scabra is high (for example, the sequence identity is preferably 95 - 100%, more preferably 97 - 100%), it can be known that the target amplification product has been obtained.

[0037] Quantitative PCR (such as real-time PCR) is a method of measuring the fluorescence intensity simultaneously with the nucleic acid amplification reaction using a fluorescent probe or SYBR Green to quantify the amplification product. Such a method corresponds to a method in which the nucleic acid amplification step and the analysis step are performed simultaneously. Preferred fluorescent probes used in quantitative PCR include those modified by the Hypercool method (Japan Gene Research Institute).

[0038] In quantitative PCR, preferable probes that can be used together with the primer set of the present invention include those containing the nucleotide sequence (5'-CCACTATCCACGGARCCAA-3') described in SEQ ID NO: 3. For this probe, a fluorescent substance may be added to the 5'-terminal side and a quencher may be added to the 3'-terminal side. Examples of such a fluorescent probe include "5'-FAM-CCACTATCCACGGARCCAA-BHQ-3'" ("FAM" means fluorescein, which is a fluorescent substance, and "BHQ" means a black hole quencher).

[0039] In the nucleotide sequence described in SEQ ID NO: 3, it is preferable that the 8th nucleotide residue and the 17th nucleotide residue are each an LNA-modified base. By being an LNA-modified base, the Tm value of the probe can be increased, and thereby the binding affinity to DNA can also be increased.

[0040] <Method for investigating the habitat of Genji fireflies> In addition, as a method for investigating the habitat of Genji fireflies, by detecting Genji fireflies by the detection method of the present invention, it is possible to estimate the presence or absence of fireflies in the area where the sample was collected, the location where the fireflies pupated, and the presence or absence of firefly egg laying. Furthermore, it is also possible to evaluate whether the area where the sample was collected is suitable for the growth of fireflies.

[0041] As described above, the present invention has been described using embodiments, but it goes without saying that the technical scope of the present invention is not limited to the scope described in the above embodiments. It is obvious to those skilled in the art that various changes or improvements can be made to the above embodiments. Also, it is clear from the description of the claims that forms to which such changes or improvements are added can also be included in the technical scope of the present invention.

Examples

[0042] Hereinafter, the present invention will be described in detail with reference to examples. Note that the present invention is not limited to the examples shown below.

[0043] (Template DNA) For the DNA of Amanita muscaria in the Kanto region, a synthetic one was used. Also, as a control experiment, a synthetic DNA of an insect in the family Staphylinidae (scientific name: Astenus pulchellus), which is likely to inhabit the same area as Amanita muscaria, was similarly used.

[0044] (Design of primer set) As the primer set, a forward primer, a reverse primer, and a (forward) probe designed according to the sequences shown in Table 1 were used. In the "Forward probe" described in Table 1, "FAM" means fluorescein, a fluorescent substance, and "BHQ" means a black hole quencher, respectively.

Table 1

[0045] (Quantitative PCR) Using the above-extracted DNA and primer set, quantitative PCR was performed under the reaction solution composition shown in Table 2 and the reaction conditions shown in Table 3. For the PCR reagent, TaqMan (registered trademark) Environmental Master Mix 2.0 (ThermoFisher) described in the official manual of the Environmental DNA Society was used. Also, for LightCycler (registered trademark) Uracil-DNA Glycosylase, a reagent from Roche was used, and the PCR reaction conditions shown in Table 3 were referred to the procedure manuals of various reagents.

Table 2

[0046]

Table 3

[0047] Table 4 shows the Cq values at each concentration as the results of quantitative PCR for DNA derived from *Lycoris radiata*. Table 5 similarly shows the results of quantitative PCR for DNA derived from *Cicadella viridis*. **[Table 4]**

[0048] **[Table 5]**

[0049] In addition, Fig. 1 shows a graph of the quantitative results in Table 4.

[0050] As shown in Table 4 and Fig. 1, DNA derived from *Lycoris radiata* could be successfully amplified by quantitative PCR using the primer set included in the detection kit of the present invention.

[0051] On the other hand, as shown in Table 5, DNA derived from *Cicadella viridis* was not amplified by quantitative PCR using the primer set included in the detection kit of the present invention at the concentrations used in normal PCR. From this, it was found that DNA derived from *Lycoris radiata* could be specifically amplified by quantitative PCR using the primer set included in the detection kit of the present invention.

[0052] <Detection of *Lycoris radiata* produced in Chiba Prefecture using primers in prior art documents> Compared with the detection kit for *Lycoris radiata* according to the present invention, it was verified whether DNA of *Lycoris radiata* inhabiting Chiba Prefecture could be detected among the Tohoku and Kanto regions using the primer set and detection kit described in the prior art document (Japanese Unexamined Patent Application Publication No. 2020-195346).

[0053] (Template DNA) As the DNA of *Lycoris radiata* produced in Chiba Prefecture, a synthesized 389 bp base sequence (SEQ ID NO: 7) shown in Table 6, which is a part of the COI gene sequence of *Lycoris geniculata* produced in Chiba Prefecture that has been analyzed in the past, was used. In addition, as a primer set, a forward primer (SEQ ID NO: 4), a reverse primer (SEQ ID NO: 5), and a (forward) probe (SEQ ID NO: 6) designed according to the sequences shown in Table 6 were used. [Table 6]

[0054] (Quantitative PCR) The PCR reagents and reaction conditions were based on the information described in the prior art documents. In addition, to prevent carry-over contamination, the addition of Uracil-DNA-Glycosylase (UNG) to the reaction system and the activation reaction were added. The PCR composition is shown in Table 7, and the PCR reaction conditions are shown in Table 8, respectively. [Table 7]

[0055] [Table 8]

[0056] Then, as shown in Table 9, DNA solutions (6.6×10 0 copies / μL to 6.6×10 6 copies / μL) obtained by serially diluting each synthetic gene by a factor of 10 were used as templates, and each was analyzed in triplicate (n = 3) (in the table, "copies" means the same as "cp" in Table 4, and "reaction" means the same as "reaction" in Table 4). [Table 9]

[0057] Figure 2 shows the fluorescence quantification waveforms at each concentration as the result of quantitative PCR for the DNA of Gentiana triflora produced in Chiba Prefecture using the primer set of the prior art document.

[0058] As shown in Figure 2, in the DNA sequence of Gentiana triflora produced in Chiba Prefecture, 1.3×101 copies / reaction solution from 1.3×10 7 For any sample between copies / reaction solution up to 1.3×10, no quantitative fluorescence was obtained. According to sequence analysis, this is because there is a one-base mismatch in the probe sequence, which is considered to be the reason why it could not be detected.

[0059] From the above examples, according to the present invention, it was confirmed that a detection kit, a detection method, and a method for investigating the habitat of Ganoderma lucidum that can comprehensively detect Ganoderma lucidum inhabiting a specific region in Japan can be provided.

Claims

1. A detection kit for Ganoderma tsugae, comprising a primer set for amplifying a nucleic acid derived from Ganoderma tsugae, the primer set consisting of a first primer containing the nucleotide sequence of SEQ ID NO: 1 and a second primer containing the nucleotide sequence of SEQ ID NO:

2.

2. The detection kit according to claim 1, wherein the Ganoderma tsugae includes Ganoderma tsugae inhabiting the Tohoku and Kanto regions.

3. A method for detecting Ganoderma tsugae, comprising: a nucleic acid amplification step of performing a nucleic acid amplification reaction using, as a template, a nucleic acid extracted from an environmental sample, with the detection kit according to claim 1; and an analysis step of analyzing an amplification product obtained by the nucleic acid amplification reaction.

4. A method for investigating the habitat of Ganoderma tsugae, characterized by using the detection method according to claim 3.

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