JP4122380B2 - Analysis method of seaweed - Google Patents

Description

  The present invention relates to a method for analyzing seaweed. In particular, the present invention relates to a method for analyzing whether a test sample is a seaweed belonging to the genus Aonori and DNA used as a primer in this method.

  The seaweeds of the genus Aonori classified in the effective tariff rate table 1212.20-139 are IQ applicable items (items for which quota is determined). In contrast, seaweeds of the genus Aosa used as a substitute are AA items (items that can be freely imported). However, since the trade name is “blue paste” and the imported form is mainly powder, it was difficult to distinguish between the two in microscopic observation at customs clearance.

By the way, seaweeds of the genus Aonori and seaweeds of the genus Aosa can be identified by comparing the base sequences of the ITS (internal transscribed spacer) region. (Non-Patent Documents 1-7)
Woolcott, GW & King, RJ 1999. Ulva and Enteromorpha (Ulvales, Ulvophyceae, Chlorophyta) in eastern Australia: comparison of morphological features and analyzes of nuclear rDNA sequences data.Aust. Sts. Bot. 12: 709-25. Tan, IH, Blomster, J., Hansen, G., Leskinen, E., Maggs, CA, Mann, DG Sluiman, H. J & Stanhope, MJ 1999. Molecular phylogenetic evidence for a reversible morphogenetic switch controlling the gross morphology of two common genera of green seaweeds, Ulva and Enteromorpha. Mol. Biol. Evol. 16: 1011-8. Malta, EJ, Draisma, SGA & Kamermans, P. 1999. Free-floating Ulva in the southwest Netherlands: species or morphotypes? A morphological, molecular and ecological comparison. Eur. J. Phycol. 34: 443-54. Coat, G., Dion, P., Noailles, MC, De Reviers, B., Fontaine, JM, Bergaer-Perrot, Y. & Loiseaux-De Goer, S. 1998. Ulva armoricana (Ulvales, Chlorophyta) from the coasts of Brittany (France). II. Nuclear rDNA ITS sequences analysis. Eur. J. Phycol. 33: 81-6. Blomster, J. 2000. Molecular and morphological approaches to the evolutionary history of the Enteromorpha-Ulva species complex.W. & A. de Nottbeck Foundation Sci. Rep. 20: 1-24. Shimada, S., Hiraoka, M., Nabata, S., Iima, M. & Masuda, M. 2003. Molecular phylogenetic analyzes of the Japanese Ulva and Enteromorpha (Ulvales, Ulvophyceae), with special reference to the free-floating Ulva Phycological Research 51: 99-108. Hayden, HS, Blomster, J., Maggs, CA, Silva, PC, Stanhope, MJ & Waaland, RJ 2003 Linnaeus was right all along: Ulva and Enteromorpha are not distinct genera. Eur. J. Phycol. 38: 277-294 .

  However, although the DNA sequencer is required for the identification work by analyzing the base sequence of the ITS region, the DNA sequencer is not deployed at customs nationwide and cannot be implemented in practice. Therefore, this method is not suitable at present as an analysis method for IQ-related cargo that is requested to be analyzed with permission pending. Further, when processing a large number of specimens, analyzing the base sequence of each specimen takes time and costs for the analysis, and this is also problematic.

  Therefore, an object of the present invention is to provide a method for analyzing whether or not a specimen is a seaweed of the genus Aonori, without using a DNA sequencer, at a simple and low cost.

The present invention for solving the above problems is as follows.
[Claim 1] A method for analyzing whether a test sample is a seaweed of Aonori,
Using DNA contained in the test sample as a template,
One kind of gene sequence common to the above three kinds of seaweeds, using one of the three kinds of sequences complementary to the species-specific sequences in each of the genes of the seaweeds of the genus Aonori, Usbaaonori, Hiiraaoori and Bouaoonori As the other primer
Perform PCR,
The said method of determining whether a test sample is a seaweed of Aonori genus by the presence or absence of a PCR product.
[Claim 2] The method according to claim 1, which is used to distinguish seaweeds of the genus Aonori and seaweeds of the genus Aosa.
[Claim 3] The method according to claim 1 or 2, wherein the one primer is a forward primer and the other primer is a reverse primer.
[4] The method according to any one of [1] to [3], wherein the one primer has the base sequence of SEQ ID NOs: 1 to 3 described in the sequence listing.
[5] The method according to any one of [1] to [4], wherein the other primer has the base sequence of SEQ ID NO: 4 described in the sequence listing.
[6] The method according to any one of [1] to [5], wherein the presence or absence of the PCR product is confirmed by electrophoresis.
[7] The method according to any one of [1] to [6], wherein DNA contained in the test sample is extracted, and the extracted DNA is used as a PCR template.
[8] The method according to any one of [1] to [7], wherein the PCR is carried out after confirming that the DNA contained in the test sample has been extracted.
[9] The method according to [8], wherein the DNA is confirmed to be extracted by PCR using a pair of primers having a base sequence common to a seaweed of Aonori and a seaweed of Aosa.
[10] The method according to [9], wherein the pair of primers has the nucleotide sequences of SEQ ID NOs: 5 and 6.
[Claim 11] PCR is performed using DNA contained in the test sample as a template, and it is determined whether or not the test sample is a seaweed of the genus Aonori, based on the presence or absence of a PCR product. DNA used as a primer in the method for analyzing whether the DNA has any one of the nucleotide sequences of SEQ ID NOS: 1 to 4 described in the sequence listing, or the DNA of SEQ ID NOS: 1-4 described in the sequence listing DNA having a partial base sequence of any one of the base sequences and having 15 or more bases.
[Claim 12] PCR is carried out using DNA contained in the test sample as a template, and it is determined whether the test sample is a seaweed of the genus Aonori, based on the presence or absence of a PCR product. 4 types of DNA having any one of the nucleotide sequences of SEQ ID NOS: 1 to 4 described in the Sequence Listing, which are used as primers in the method for analyzing whether at least one of the four types of DNAs is used Is a primer set which is a DNA having a partial base sequence of any one of SEQ ID NOS: 1 to 4 described in the Sequence Listing and having 15 or more bases.

  According to the present invention, it is possible to analyze whether or not a specimen is a seaweed belonging to the genus Aonori, without using a DNA sequencer, simply and at low cost.

  The method of the present invention is a method for analyzing whether a test sample is a seaweed of the genus Aonori. In the present invention, three primers that are complementary to the species-specific sequences of each of the genes of Usubaaonori, Hieraonoori, and Boaonori, which are seaweeds of the genus Aonori, are used as a template with the DNA contained in the test sample as a template. In addition, PCR is carried out using one gene sequence common to the above three types of seaweeds as the other primer. Then, whether or not the test sample is a seaweed of the genus Aonori is determined based on the presence or absence of the PCR product.

  The four species of Usubaaonori, Hiraonori and Bouaoonori plus Susioonori are the main species of the seaweed of the genus Aonori, and almost 100% of the customary Aonori seaweed belongs to one of these four types. However, although Usubaaonori and Susiaoonori are different species, the ITS region is common, and Susioonori can also be detected by using a species-specific sequence of Usbaaonori. Therefore, by examining whether or not a PCR product can be obtained for three kinds of seaweeds of Usubaaoori, Hiiraonoori and Bouaooriori, it can be determined substantially whether or not the test sample is seaweed of the genus Aonori.

  Therefore, in the present invention, species-specific regions are searched for in these three types of seaweed genes, for example, ITS sequences, and three types of sequences complementary to these species-specific sequences are used as one primer. Also, PCR is performed using one gene sequence common to these three types of seaweeds as the other primer. If the test sample is a seaweed belonging to the genus Aonori, that is, any one of Usubaaonori, Susiaoonori, Hiiraonori and Bouaoonori, a PCR product can be obtained, and it can be determined that the test sample is a seaweed belonging to the genus Aonori. If no PCR product is obtained, it can be determined that the test sample is not a seaweed of the genus Aonori.

  Thus, according to the method of the present invention, it can be determined whether or not the test sample is a seaweed of the genus Aonori. For example, in customs, it can be used to identify seaweeds of the genus Aonori and seaweeds of the genus Aosa that are subjected to customs clearance procedures in a state where identification is difficult.

  The “three types of sequences complementary to the species-specific sequence” used as one primer in the PCR and the “one type of gene sequence common to three types of seaweed” used as the other primer are, for example, in the ITS sequence. It can be selected by exploring species-specific regions and regions common to three types of seaweed. The ITS sequences of four kinds of seaweeds obtained by adding Sujionori to the above three kinds of seaweeds of the genus Aonori are known (http://www.ncbi.nlm.nih.gov/).

  In addition, the analysis method of the present invention mainly aims to distinguish seaweeds of the genus Aonori from seaweeds of the genus Aosa. It means a species-specific region.

  Such a species-specific region for the seaweed of the genus Aonori can be determined by comparing the ITS sequences of each of the three types of seaweed with the ITS sequences of the seaweed of the genus Aosa. Specifically, among the seaweeds of the genus Aosa, for example, nine kinds of seaweeds of the genus Aosa, which are often traded as “blue seaweed”, Anaaaosa, Oobaaosa, Nagaaosa, Ribbonaosa, Amiaosa, Kotsubuosa, Minamiosa, U. rigida It can be determined by comparing with the ITS sequence of U. armoricana. Of course, ITS sequences of other seaweeds belonging to the genus Aosa can be used as comparison targets, and the species specificity can be increased as the number of comparison targets increases. ITS sequences of seaweeds of the genus Aosa are also known (http://www.ncbi.nlm.nih.gov/).

  The “three types of sequences complementary to the species-specific sequence” used as one primer can be appropriately determined from the species-specific region that can be determined by the above-described method in consideration of the use as a primer. From the viewpoint of specifically hybridizing with a template as a primer, the number of bases is suitably at least 15, preferably 15 to 30 bases, more preferably 20 to 30 bases. This is because a primer of this length is sufficient for specific annealing with the template DNA.

  Specifically, the three types of sequences complementary to the species-specific sequence can have the base sequences of SEQ ID NOs: 1 to 3 described in the sequence listing. None of the nucleotide sequences of SEQ ID NOs: 1 to 3 include a sequence complementary to a species-specific sequence in the seaweed of Aonori, which does not include a complementary sequence in the gene of seaweed of Aosa. Therefore, these sequences are species-specific regions, and some of these sequences can be used as primers. That is, a sequence obtained by removing a partial base on the 5 ′ and / or 3 ′ side from these sequences can also be used as a primer. In that case, the number of bases in the sequence can be appropriately determined in consideration of the numerical range shown above. That is, a sequence having at least 15 bases among the base sequences of SEQ ID NOs: 1 to 3 described in the sequence listing can also be used as a primer.

  In addition, one type of gene sequence common to the three types of seaweed can be appropriately determined from the region common to the ITS sequences of the three types of seaweeds of the genus Aonori. In this case, there may be a region common to the ITS sequence of the seaweed belonging to the genus Aosa. The three sequences complementary to the above species-specific sequences differ from the ITS sequence of Aosa seaweed, so one gene sequence common to the three kinds of seaweed is the ITS sequence of Aosa seaweed. This is because even if there is a common part, the gene of seaweed belonging to the genus Aosa is not amplified during PCR. In addition, one kind of gene sequence common to three kinds of seaweeds is considered on the ITS sequence with a species-specific region for seaweeds of the genus Aonori, considering the number of DNA bases to be amplified by PCR. The distance can be determined as appropriate. The distance between the species-specific region (sequence) of the seaweed of the genus Aonori and one type of gene sequence common to the three types of seaweed can be in the range of, for example, 100 to 2000. If the distance is short, the molecular weight of the amplified DNA is small, which may hinder the confirmation of the presence or absence of PCR products. If the distance is long, even if there is a sequence corresponding to the DNA contained in the test sample, amplification by PCR does not proceed well, which may cause analysis errors. From the viewpoint that the presence or absence of the PCR product can be confirmed satisfactorily, the distance is preferably the number of bases, for example, in the range of 200 to 1000, and more preferably in the range of 300 to 600.

  One type of gene sequence common to the three types of seaweed can have, for example, the base sequence of SEQ ID NO: 4 described in the sequence listing. The base sequence of SEQ ID NO: 4 does not contain a complementary sequence in the gene for seaweed belonging to the genus Aosa, and is a sequence complementary to a species-specific sequence in seaweed belonging to the genus Aonori. Therefore, this sequence is a species-specific region, and a part of these sequences can be used as a primer. That is, a sequence obtained by removing a partial base on the 5 ′ and / or 3 ′ side from these sequences can also be used as a primer. In that case, the number of bases in the sequence can be appropriately determined from a range of 15 or more.

  The three sequences complementary to the species-specific sequence can be either forward primers or reverse primers, and if the three sequences complementary to the species-specific sequence are forward primers, 3 One kind of gene sequence common to various kinds of seaweeds can be used as a reverse primer. In addition, when three types of sequences complementary to a species-specific sequence are reverse primers, one type of gene sequence common to three types of seaweeds can be used as a forward primer. The base sequences of SEQ ID NOs: 1 to 3 are forward primers, and the base sequence of SEQ ID NO: 4 is a reverse primer.

  The present invention performs PCR using DNA contained in the test sample as a template, and determines whether the test sample is a seaweed belonging to the genus Aonori, based on the presence or absence of a PCR product. The method for analyzing whether it is a seaweed includes DNA used as a primer, which DNA has any one of the nucleotide sequences of SEQ ID NOS: 1 to 4 described in the Sequence Listing, and the sequence described in the Sequence Listing DNA having a part of the base sequence of any one of Nos. 1 to 4 and having 15 or more bases. If the number of bases is 15 or more, it functions as a primer.

  Furthermore, the present invention performs PCR using DNA contained in the test sample as a template, and determines whether or not the test sample is a seaweed belonging to the genus Aonori, based on the presence or absence of a PCR product. The primer set used as a primer is included in the method for analyzing whether or not, and this primer set includes four types of DNAs having any one of the base sequences of SEQ ID NOS: 1 to 4 described in the sequence listing. Alternatively, in the primer set of the present invention, at least one of the four types of DNA has a partial base sequence of any one of the base sequences of SEQ ID NOS: 1 to 4 described in the sequence listing, and the number of bases is 15 It can also be DNA which is the above.

  PCR in the present invention can be performed by a conventional method. Specifically, PCR can be performed in 35 cycles of 94 ° C. for 45 seconds, 50 ° C. for 45 seconds, and 68 ° C. for 60 seconds. The number of cycles can be appropriately increased or decreased in consideration of the amount of mold in the sample. Commercially available products can be used as PCR reagents, for example, ex Taq (manufactured by TAKARA) can be used. The sample content of one sample can be, for example, as follows (Buffer 3 μL, dNTP mix 2.5 μL, DMSO 1.5 μL, Taq 0.2 μL, DW 22.3 μL, total 30 μL).

  In the PCR method, DNA contained in a test sample is used as a template. Specifically, it is appropriate to extract DNA contained in the test sample and use the extracted DNA as a PCR template. Extraction of DNA contained in the test sample can be performed, for example, by the following method. Imported dried Aosa and Aonori are crushed to about 1 square meter and powdered. This is first soaked in seawater and separated into individual pieces, which are then cleaned with a paintbrush or tweezers under a stereomicroscope. Place the cleaned piece in a 1.5ml tube and dry under reduced pressure for 10 minutes using a pump. Place liquid nitrogen in a 1.5 ml tube containing the sample and crush it with a pestle. DNA can be extracted from the crushed sample using, for example, DNeasy Plant Mini Kit (manufactured by Qiagen).

  Furthermore, when extracting DNA contained in the test sample, it is preferable to confirm that the DNA contained in the test sample is reliably extracted before performing PCR in the method of the present invention. Confirmation that DNA is extracted can be performed, for example, by PCR using a pair of primers having a base sequence common to seaweeds of the genus Aonori and seaweeds of the genus Aosa. Such a pair of primers can have, for example, the nucleotide sequences of SEQ ID NOs: 5 and 6. By performing PCR using the above-mentioned pair of primers and confirming the presence or absence of the PCR product, it can be confirmed whether or not the DNA contained in the test sample is reliably extracted. A DNA pair having a partial sequence of the nucleotide sequences of SEQ ID NOs: 5 and 6 can also be used as a primer. PCR can be performed in the same manner as described above.

  The confirmation of the presence or absence of a PCR product in order to confirm the presence or absence of a PCR product in the method of the present invention and to confirm whether or not the DNA contained in the test sample has been reliably extracted can be performed by a conventional method. it can. For example, the PCR product can be subjected to electrophoresis, and the presence or absence of a PCR product can be determined from the presence or absence of a band. Electrophoresis can be performed, for example, on a 1% agarose gel (1 × TAE buffer). Specifically, 4 μL of one sample is loaded, and electrophoresed for about 15 minutes. After electrophoresis, for example, it can be stained with ethidium bromide and the presence or absence of a band can be confirmed with a transilluminator.

  According to the present invention, whether or not a test sample is a seaweed of the genus Aonori can be determined based on the presence or absence of a PCR product. If necessary, depending on the molecular weight of the PCR product, depending on the result of electrophoresis (band position), if the test sample is a seaweed belonging to the genus Aonori, any one of Usubaaonori (or Sueaonori), Hiraaoori and Boaonori It is also possible to determine whether it is a seaweed. In addition, by attaching a marker such as a fluorescent marker to any one of the three types of sequences (primers) complementary to the species-specific sequence or each of them, the above three types (four types) of seaweed can be identified. It can also be made easier.

  Hereinafter, the present invention will be described in detail with reference to examples.

Example 1
[Generation of species-specific primers for the three major blueberry species]
From the DNA sequence of the ITS region (Internal Transcribed Spacer region) of about 300 Aosa-Aonori species around the world, we searched for specific regions in each of the four major Aonori species. Species-specific primers for the three major Aonori species on the Forward side (Usuba aonori / Sujiaonori: 5'-CGCGTGCGCTCCCCTCGGGGGGCG-3 '(SEQ ID NO: 1), Hira Aonori: 5'-TGAGGTGCGCTCCCCCGGGGGCGCGCCCCT-3' (SEQ ID NO: 2), Bow Aonori: 5 '-TGGGAGGGGGTGGTGGTGCTCACG-3' (SEQ ID NO: 3)) was prepared, and 5'-TGATAGGTTAAGTTCAGC-3 '(SEQ ID NO: 4) was prepared on the reverse side.

In addition, although Usubaaonori and Susiaoonori are now considered as separate species, research on ITS has reported that the Susioonori of Japan is likely to be the same species as Usubaaonori ( Masahira Hiraoka, Satoshi Hamada : Marine and Biological 2004, Shimanto Biology of river special product Susioonori "26 (6) 508-515. Therefore, the same primer is used for both species. The reverse primer has already been reported ( Satoshi Shimada , Masanori Hiraoka, Shinichi Nabata, Masafumi Iima & Michio Masuda. 2003. Molecular phylogenetic analyzes of the Japanese Ulva and Enteromorpha (Ulvales, Ulvophyceae), with special reference to the free-floating Ulva. Phycological Research 51: 99-108.).

  Verification was made as to whether or not the species-specific primer prepared above was effective.

[sample]
For comparison, DNA of 9 major Aosa (anaanaosa, Oobaaosa, Nagaaosa, Ribbonaosa, Amiaosa, Kotobuaosa, Minamiosa, U. rigida, U. armoricana) and 4 major Aonori DNAs were prepared.

[First PCR]
The first PCR was performed using the extracted DNA of the prepared sample as a template. This is a control experiment to confirm whether the extracted DNA is DNA that can be PCR. Seaweed is rich in polysaccharides and polyphenols, and PCR may be inhibited. Therefore, even if the extracted DNA is directly confirmed by electrophoresis, it does not prove that the sample is capable of PCR. Ex Taq (manufactured by TAKARA) was used as a PCR reagent. The contents of one sample are as follows (Buffer 3 μL, dNTP mix 2.5 μL, DMSO 1.5 μL, Taq 0.2 μL, DW 22.3 μL, total 30 μL). The primer used the primer set (F: 5'-CTCTCAACAACGGATATCT-3 '(sequence number 5), R: 5'-TGATAGGTTAAGTTCAGC-3' (sequence number 6)) which amplifies the ITS2 area | region of Aosa-Aonori. This has already been reported ( Satoshi Shimada , Masanori Hiraoka, Shinichi Nabata, Masafumi Iima & Michio Masuda. 2003. Molecular phylogenetic analyzes of the Japanese Ulva and Enteromorpha (Ulvales, Ulvophyceae), with special reference to the free-floating Ulva. Phycological Research 51: 99-108.).

  In order to check DNA contamination such as reagents, a sample without DNA was also prepared. PCR reaction conditions are as follows (35 cycles of 94 ° C 45 seconds, 50 ° C 45 seconds, 68 ° C 60 seconds). The presence or absence of a band was confirmed by electrophoresis on a 1% agarose gel. As a result, all the bands other than the sample without DNA appeared, and it was found that there was no problem as DNA (Figure 1).

[Second PCR]
PCR was performed using a forward primer (SEQ ID NO: 1 to 3) specific to each of the three major blueberry species, and a common primer (SEQ ID NO: 4) on the reverse side. Reagents, programs, etc. were the same conditions as the first time. The presence or absence of a band was confirmed by electrophoresis on a 1% agarose gel. As a result, in each species-specific primer set, a band appeared only for that species (FIGS. 2 (A) to (C)).

[Sequence for verification]
A sequence was performed to see if the band that appeared in the second round of PCR really belongs to that kind of ITS domain. The PCR product was purified, and a cycle sequence was performed using a dye terminator sequencing kit (BIG DYE: Applied Biosystems). The reaction conditions were 30 cycles of 96 ° C. for 30 seconds, 50 ° C. for 15 seconds, and 60 ° C. for 240 seconds. As a result of decoding the base sequence of the cycle sequence reaction product using a DNA automatic sequencer, it was confirmed that the bands that appeared in the second PCR belong to the ITS region of each species. From the above, it can be seen that the prepared primer has a species-specific sequence capable of species-specific band amplification.

Example 2
[Sample cleaning]
Imported dried Aosa and Aonori are crushed to about 1 square meter and powdered. This was first soaked in seawater and separated into individual pieces, which were then cleaned with a paintbrush and tweezers under a stereomicroscope. The cleaned piece was placed in a 1.5 ml tube and dried under reduced pressure for 10 minutes using a pump. Ten samples were prepared.

[DNA extraction]
Liquid nitrogen was placed in a 1.5 ml tube containing the sample and crushed with a pestle. DNA was extracted with DNeasy Plant Mini Kit (Qiagen).

[First PCR]
The first PCR was performed using the extracted DNA as a template. This is a control experiment for confirming whether or not the above-mentioned DNA extraction sample has extracted DNA that can be PCR. Seaweed is rich in polysaccharides and polyphenols, and PCR may be inhibited. Therefore, even if the extracted DNA is directly confirmed by electrophoresis, it does not prove that the sample is capable of PCR. Ex Taq (manufactured by TAKARA) was used as a PCR reagent. The contents of one sample are as follows (Buffer 3 μL, dNTP mix 2.5 μL, DMSO 1.5 μL, Taq 0.2 μL, DW 22.3 μL, total 30 μL). As a primer, a primer set (SEQ ID NOs: 5 and 6)) for amplifying the ITS2 region of Aosa aonoris was used. In order to check DNA contamination such as reagents, a sample without DNA was also prepared. PCR reaction conditions are as follows (35 cycles of 94 ° C 45 seconds, 50 ° C 45 seconds, 68 ° C 60 seconds). The presence or absence of a band was confirmed by electrophoresis on a 1% agarose gel. The second sample was different from a normal bunt and several bands were detected (Fig. 3). Although DNA extraction has been successful, it is probably not Aosa or Aonori. All samples were subjected to the second PCR as they were.

[Second PCR]
PCR was performed using a forward primer specific to each of the three major blueberry species (SEQ ID NOs: 1 to 3) and a common primer (SEQ ID NO: 4) on the reverse side. Reagents, programs, etc. are the same as the first time. The presence or absence of a band was confirmed by electrophoresis on a 1% agarose gel (FIGS. 4 (A) and (B)). Then, only when the Usbaaonori / Sujiaoori species-specific primer was used, clear bands could be confirmed in the 1st, 4th, 5th, 7th and 10th samples, and 5 out of 10 samples were positive. That is, 5 samples were seaweeds of the genus Aonori.

  The present invention can be used for identification of seaweed species.

FIG. 2 is an electrophoresis photograph of the first PCR product of Example 1. FIG. 2 is an electrophoresis photograph of the second PCR product of Example 1. FIG. FIG. 3 is an electrophoresis photograph of the first PCR product of Example 2. FIG. 2 is an electrophoresis photograph of the second PCR product of Example 2. FIG.

Claims (9)

A method for analyzing whether a test sample is a seaweed of Aonori,
Using DNA contained in the test sample as a template,
A DNA comprising the nucleotide sequences of SEQ ID NOs: 1 to 3 described in the Sequence Listing, which are three types of sequences complementary to the species-specific sequences in the genes of each species of the seaweeds of the genus Aonori, Usubaaonori, Hiraonori and Bouaoonori PCR is carried out using one of the primers and a DNA consisting of the base sequence of SEQ ID NO: 4 described in the Sequence Listing, which is one kind of sequence common to the three seaweeds, as the other primer,
The said method of determining whether a test sample is a seaweed of Aonori genus by the presence or absence of a PCR product. The method according to claim 1, which is used for discriminating between seaweeds of the genus Aonori and seaweeds of the genus Aosa. The method according to claim 1 or 2, wherein the one primer is a forward primer and the other primer is a reverse primer. The method according to any one of claims 1 to 3, wherein the presence or absence of a PCR product is confirmed by electrophoresis. The method according to any one of claims 1 to 4, wherein DNA contained in a test sample is extracted, and the extracted DNA is used as a PCR template. The method according to any one of claims 1 to 5, wherein the PCR is performed after confirming that DNA contained in the test sample has been extracted. The method according to claim 6, wherein the DNA is confirmed to be extracted by PCR using a pair of primers having a base sequence common to the seaweed of the genus Aonori and the seaweed of the genus Aosa. The method according to claim 7, wherein the pair of primers has the nucleotide sequences of SEQ ID NOs: 5 and 6. Perform PCR using the DNA contained in the test sample as a template, and determine whether the test sample is a seaweed of the genus Aonori, based on the presence or absence of the PCR product. Analyze whether the test sample is a seaweed of the genus Aonori A primer set comprising four types of DNAs consisting of any one of the nucleotide sequences of SEQ ID NOs: 1 to 4 described in the Sequence Listing, which are used as primers in the method.