JP2011177113A - Method for discriminating citrus depressa fruit and processed product thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for easily discriminating in high accuracy citrus depressa and calamansi fruit and their processed products (especially, heat-processed ones). <P>SOLUTION: The method for discriminating citrus depressa fruit from calamansi fruit is provided, being characterized by including the following procedure: using a set of primers each thereof being such that the 3'-terminal is at a site corresponding to the SNP part of citrus depressa fruit and calamansi fruit present on a chloroplast DNA, and the 3'-terminal is a base identical with either one of the SNPs of citrus depressa fruit and calamansi fruit, and the third base present upstream of the 3'-terminal is a mismatched base, an allyle-specific PCR is carried out to confirm the presence/absence of the amplification of the target sequence. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention is characterized in that allele-specific PCR is carried out using the presence of a SNP site present in the trnL-trnF region and / or a SNP site present in the trnT-trnL region on the chloroplast DNA as a DNA marker. The present invention relates to a method for discriminating between sea squasher and calamancy (fruit, processed product).

Citrus depressa, a special citrus from Okinawa Prefecture, has a unique aroma and taste and is used for cooking and raw food in the Okinawa area.
Shikwasha fruit is a characteristic citrus that contains a large amount of nobiletin, tangeretin and the like, which are polymethoxyflavonoid components, compared to other citrus fruits. In recent years, nobiletin abundantly contained in fruit juice has a carcinogenic effect (see Non-Patent Document 1), and it has been clarified that ingestion of a paste containing pericarp has an effect of suppressing blood sugar and reducing body fat percentage. It was.
For this reason, the demand for sea quashers (fruits, processed products) is increasing more and more.

However, at present, there is a large shortage of raw materials for seek squash. On the other hand, calamancy from Taiwan and the Philippines is very cheap and can be imported.
Under these circumstances, there are many cases where beverages containing Calamansi juice are mislabeled and sold as ingredients for Sikhwasha juice, which has become a major social problem, such as administrative sanctions in accordance with the exclusion order of the Fair Trade Commission.
It should be noted that the form of the calamancy fruit is very similar to the sea quashsha and it is extremely difficult to discriminate in appearance. Moreover, once it is squeezed, it cannot be determined at present unless a component analysis (see Non-Patent Document 2) of flavonoid components (phloretin, polymethoxyflavonoids, etc.) is performed.

Bracke, M.E. (1994), Food Technology, 48, 104 Y., Nogata, Bioscience, Biotechnology, and Biochemistry Vol. 70 (2006), No. 1 pp.178-192

  This invention solves the said subject, and makes it a subject to provide the method of discriminate | determining the fruit and processed goods (especially heat processing goods) of a sea squash and a calamancy with high precision and easily.

  In view of such circumstances, the present inventors have partially analyzed the chloroplast DNA of Shikuwasha and Calamansea, and have found the presence of SNPs that can identify the varieties of both. Then, it was found that by performing an allele-specific PCR using a primer (DNA marker) having the SNP at the 3 ′ end, it is possible to discriminate between seekers and calamancy with extremely high accuracy and high sensitivity.

The present invention has been made based on these findings.
That is, the present invention according to claim 1 is to confirm the presence or absence of amplification of a target sequence by performing allele-specific PCR using a primer set comprising a primer having the following characteristics (A) or (B): The present invention relates to a method for distinguishing between sea quashers and calamansi.
(A): The 3 ′ end is a position corresponding to the SNP site of Sequachia and Calamansi existing in the trnL-trnF region on the chloroplast DNA, and the 3 ′ end is either Sequasher or Calamansi The same base as the SNP, the third base upstream from the 3 ′ end is a mismatch base, and the 4th to 16th bases upstream from the 3 ′ end are Sequwasha and Karamansi It is in complete agreement with the sequences of both trnL-trnF regions.
(B): The 3 ′ end is a position corresponding to the SNP site of Sequashas and Calamansi that exists in the trnT-trnL region on the chloroplast DNA, and the 3 ′ end is either Sichuasha or Karamansi A base that matches the SNP, the third base upstream from the 3 ′ end is a mismatch base, and the fourth to 16th bases upstream from the 3 ′ end are Sequwasha and Karamanshi Are completely identical to the sequences of both trnT-trnL regions.
The present invention according to claim 2 is characterized in that, in the primer set, the other primer that is not the primer having the characteristics (A) or (B) is seeking at least the 16th base upstream from the 3 ′ end. The present invention relates to a method for discriminating between a seek washer and a calamancy according to claim 1, which is designed to be completely coincident with both the washer and the calamancy sequences, and the amplified fragment is designed at a position of 400 bp or less.
Further, the present invention according to claim 3 confirms the presence or absence of amplification of the target sequence by performing allele-specific PCR using a primer set comprising the primer having the characteristics of (A) according to claim 1 or 2 And confirming the presence or absence of amplification of the target sequence by performing allele-specific PCR using a primer set comprising the primer having the characteristics of (B) according to claim 1, It is related with the discrimination method of Shikwasha and Karamanshi.
The present invention according to claim 4 is a primer set consisting of SEQ ID NOs: 5 and 6 capable of specifically amplifying the seeker trnL-trnF region, wherein the primer set comprising the primer having the characteristics of A is described above; The primer set comprising a primer having the characteristics of B is a primer set consisting of SEQ ID NOs: 7 and 8 capable of specifically amplifying the Karamansi trnT-trnL region; The present invention relates to a method for determining calamancy.

Since the present invention is a PCR-based inspection method using a region on the chloroplast as a primer, without performing complicated analysis such as analysis of the polyphenol component, it is possible to remove the squeaker and the calamancy by an extremely simple operation. It becomes possible to discriminate with high accuracy.
In addition, since the PCR method targets a short region on the chloroplast, a highly sensitive test can be performed.
As a result, the present invention is highly accurate even when the raw material is a mixed raw material or a processed product with heat treatment, which has been difficult with the conventional simple detection method of polyphenol components (phloretin and the like). Can be determined.

It is the photographic image figure which compared the external appearance of the fruit of Siquasha and Karamanshi. It is a schematic diagram of trnL-trnF region and trnT-trnL region on chloroplast DNA. 2 is an electrophoretogram showing the PCR amplification result in Example 1. FIG. 2 is an electrophoretogram showing the PCR amplification result in Example 1. FIG. In Example 2, it is a figure which shows the alignment result by 5 'side of a trnL-trnF area | region. In Example 2, it is a figure which shows the alignment result by 5 'side of a trnT-trnL area | region. In Example 3, it is a figure which shows the preparation example of the primer whose 3 'side terminal corresponds to a SNP site | part. In Example 3, it is a figure which shows the preparation example of the primer whose 3 'side terminal corresponds to a SNP site | part. 6 is an electrophoretogram showing the PCR amplification results in Example 4. FIG. FIG. 6 is an electrophoretogram showing the PCR amplification result in Example 5. 10 is an electrophoretogram showing the PCR amplification result in Example 6. FIG. FIG. 10 is an electrophoretogram showing the PCR amplification result in Example 7. It is a figure which shows the raw material display which can be discriminate | determined by the test | inspection of this invention.

  In the present invention, allele-specific PCR is performed using the presence of SNP sites in trnL-trnF region and trnT-trnL region on chloroplast DNA as a DNA marker. (Fruit, processed product).

[Target]
As the siqua washer discriminable in the present invention, any one can be used as long as it belongs to Citrus depressa of the Citrus mandarin orange section. For example, Izu-flavored Kugani from Okinawa, Ogimi Kuganee, Katsuyama Kuganee, Toyama Hayao, Carachi, B-41, B-41-H, A-29, Stone Kunibu, B-7, B-20, B-25, Fusbuter , Kirby, Mayagher, C-22, Taiwanese sea quashers made in Taiwan, and the like.
As the calamancy that can be discriminated by the present invention, any calamancy can be used as long as it belongs to Citrus madurensis of the citrus genus Cultivation. For example, it is possible to discriminate Taiwanese calamancy; Okinawa throbbing;
In addition, as shown in FIG. 1, the form of a siquasha and a calamancy fruit is very similar to a siquashah, and it is very difficult to discriminate in appearance. Moreover, once it has been squeezed, it cannot be determined at present unless a component analysis such as a flavonoid component is performed.

[SNP]
The SNP (Single Nucleotide Polymorphisms: Single Nucleotide Polymorphisms) site present in the trnL-trnF region and trnT-trnL region on the chloroplast DNA used in the present invention is, as shown in the schematic diagram of FIG. This refers to those present in the trnF region and trnT-trnL region, and refers to a mutation site capable of distinguishing cultivars of Sikwasha and Karamanshi.
Note that these regions are spacer regions (IGS regions) between transfer RNA (trn) genes, and have a relatively high molecular evolution rate.

As the SNP existing in the trnL-trnF region, specifically, the SNP shown by the alignment in FIG. 5 can be used. In addition, the said site | part is a site | part corresponded to the 51401th base in the chloroplast DNA sequence (Accession No: DQ864733) of sweet orange (Citrus sinensis) registered in NCBI.
As the SNP existing in the trnT-trnL region, specifically, the SNP shown by the alignment in FIG. 6 can be used. In addition, the said site | part is a site | part corresponded to the 50268th base in the chloroplast DNA sequence (Accession No: DQ864733) of sweet orange (Citrus sinensis) registered in NCBI.

[Primer set]
The present invention utilizes the SNP site as a DNA marker by designing the 3 ′ end of the primer to be the SNP site.

As the primer set used in the present invention, one in which the 3 ′ end of one of the two primers is designed at a position corresponding to the SNP can be used.
In addition, by designing the base at the 3 ′ end to be the same base as that of the seek washer, the seek squash can be specifically detected.
Further, by designing to the same base as calamancy, it becomes possible to specifically detect calamancy.

In addition, as a primer to be designed at the SNP site, a PCR background can be obtained by using a primer in which the third base is replaced with a mismatched base (a base that is different from both Shikuwasha and Karamanshi) upstream from the 3 ′ end. Can be significantly reduced.
For example, when the site is T (thymine), a base other than T (thymine) such as A (adenine), C (cytosine), G (guanine), or a specific base such as I (inosine) It can be replaced with a non-bonded base.

  The primer designed in the SNP site may include a modified base sequence or the like (sequence that does not match the template) at the 5 ′ end, but at least from the 3 ′ end to the 4th to 16th upstream, Preferably, the 4th to 18th bases, and more preferably the 4th to 20th bases, are perfectly matched with the target DNA (both the sequence of both Seekwasher and Karamancy) as a template.

The other primer in the primer set is a position where a fragment with a length (for example, 1.5 kbp or less) that can be easily amplified by normal PCR is obtained when used in combination with the primer designed in the SNP site. , Can be designed in any position.
From the viewpoint of PCR detection sensitivity, it is desirable to design at a position where a short amplified fragment can be obtained. Specifically, for example, by performing the measurement at 400 bp or less, preferably 300 bp or less, more preferably 200 bp or less, more preferably 110 bp or less, it is possible to cope with discrimination of a processed product for which high sensitivity detection is required.
The other primer may include a modified base sequence or the like (sequence that does not match the template) at the 5 ′ end, but at least the 16th upstream from the 3 ′ end, preferably 18 It is desirable that the bases up to the eye, and more preferably the base up to the 20th, are completely identical with the template target DNA (both the Sequwasha and Karamanshi sequences).

The length of the primer used in the primer set may be in the range of a normal PCR primer. For example, any primer having a length of about 16 to 50 bp which is a normal primer length range can be used, but the lower limit is preferably 18 bp or more, more preferably 20 bp or more. The upper limit is 40 bp or less, preferably 35 bp or less.
In order to reduce the background, it is desirable to design the Tm values of the two primers to be approximately the same.
Further, a detection modifying substance (for example, a fluorescent substance such as biotin, DIG, Cy5, or Cy3) or a modified base sequence (for example, a sequence including a restriction enzyme site) may be added to the 5 ′ end.

Among the primer sets that satisfy the above conditions, those that can specifically detect seek squash are "CiDeLF-F (SEQ ID NO: 5)" and "CiDeLF-R (SEQ ID NO: 5)" that can specifically amplify the trnL-trnF region. 6) ”can be mentioned (amplified fragment 76 bp).
As a specific example of a set that can specifically detect calamancy, it consists of “CiMaTL-F (SEQ ID NO: 7)” and “CiMaTL-R (SEQ ID NO: 8)” that can specifically amplify the calamancy trnT-trnL region. A primer set (amplified fragment 105 bp) can be mentioned.

[Allyl-specific PCR]
Allyl-specific PCR using the above primer set can be performed by a normal PCR reaction. Preferably, the reaction is performed using an amplification enzyme and a reaction solution having high sensitivity and low background. For example, KOD, Ex-Taq, AmpliTaq-Gold, Pfu, etc. can be mentioned.
In addition, the annealing temperature varies depending on the Tm value of the primer and the composition of the PCR reaction solution. Therefore, the annealing temperature should be appropriately set at a temperature at which specific amplification of the target region can be clearly distinguished between the seeker and the calamancy. Can do. For example, it can be set at about 46 to 68 ° C, preferably about 50 to 65 ° C.

After the PCR reaction, it is possible to distinguish the cultivar very easily by performing electrophoresis on an agarose gel or the like and detecting the presence or absence of an amplified fragment having a size corresponding to the target sequence.
Nucleic acid detection can be easily performed by ethidium bromide staining and UV irradiation, but in the case of detection that requires more sensitivity, real-time PCR, Southern hybridization, surface plasmon resonance (SPR) method, quartz crystal (QCM) It is also possible to perform detection with higher accuracy by performing a method or the like.

[Distinguishing inspection]
In the allyl-specific PCR in the present invention, as described above, the target region as a template is on chloroplast DNA (which is much easier to amplify than the nuclear DNA), and a relatively short sequence is targeted. To do. Therefore, it is possible to perform inspection with high accuracy even for an inspection object in which degradation or deterioration of DNA such as a processed product with heat treatment is recognized.
Further, the sensitivity can be further increased by setting the size of the amplified fragment (primer position) to be shorter.

The inspection that can be discriminated by the present invention can be carried out on processed products such as fruit juice, juice, jam, ice confectionery, ice cream, and jelly, in addition to direct discrimination on fresh fruit. In particular, it can also be performed after processing such as heat treatment.
It is desirable in terms of sensitivity that PCR for these test objects is performed after DNA is extracted from the test objects. Depending on the subject, it may be possible to perform direct PCR by direct PCR.

As a discrimination test, either a test using a primer set that can specifically detect seek squash or a test using a primer set that can specifically detect calamancy can be performed, but more detailed inspection can be performed by performing both tests. Inspection can be performed.
For example, as shown in FIG. 13, when a result of positive detection of seikwasher (+) / negative detection of calamancy (+) is obtained, a result that “Karamanshi is mixed in the raw material as a raw material” can be obtained. it can.
In addition, when it is desired to know the mixing rate in detail, it is possible to estimate the mixing rate by quantifying the amount of template DNA by a real-time PCR method or the like.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, the scope of the present invention is not limited by these.

[Example 1] Preparation of primate discrimination primer set for Siquasha and Karamancy (1) Amplification of trnL-trnF region and trnT-trnL region The fruit samples shown in Table 1 were lyophilized and DNeasy Plant Mini kits manufactured by QIAGEN were used. Total DNA was extracted.
Next, for the samples 1 to 9 out of the extracted total DNA, a combination of primers shown in Table 2 (Taberlet P. et al. Plant Mol. Biol. 17, 1105-1109, (1991)) was used. PCR was performed under the conditions shown in 3, and the trnL-trnF region and trnT-trnL region were amplified.
Each obtained PCR product was electrophoresed on a 2% agarose gel. FIG. 3 shows the result of amplification of the trnL-trnF region, and FIG. 4 shows the result of the trnT-trnL region.
In the drawing, lanes 1 to 7 show the amplification results for the Siquasha sample, and lanes 8 to 9 show the amplification results for the Calamancy sample. Lane NC represents negative control (no template), and lane M represents a 100 bp marker.

  As a result, the amplification recognized as the trnL-trnF region (437 bp) and the trnT-trnL region (1121 bp) was observed from all of the used siquasha and calamancy (samples 1 to 9).

[Example 2] Determination of base sequence For each amplified fragment obtained above, the base sequence was determined by the direct sequencing method using the same primer as each amplification primer. Then, alignment was performed for each of the obtained sequences and a sequence of Valencia (Citrus sinensis) (Accession No: DQ864733). FIG. 5 shows the alignment result on the 5 ′ side in the trnL-trnF region, and FIG. 6 shows the alignment result on the 5 ′ side in the trnT-trnL region.

As a result, it was revealed that SNP specific to calamancy (samples 8 and 9) exists in both the trnL-trnF region and the trnT-trnL region. In addition, each homologous site in Shikwasha was the same base as Valencia.

[Example 3] Design of DNA marker (primer set) Based on alignment results of Example 2 and allele-specific PCR method (Okayama et al. J. Lab. Clin. Med., 114, 105-113 (1989)) Thus, a primer set was designed that can be used to discriminate between Shikuwasha and Karamanshi.
Specifically, as shown in Table 4, CiDeLF-F: SEQ ID NO: 5 and CiDeLF-R: SEQ ID NO: 6 were designed as primer sets for detection of the seeker trnL-trnF region. In addition, CiMaTL-F: SEQ ID NO: 7 and CiMaTL-R: SEQ ID NO: 8 were designed as primer sets for detecting the calamancy trnT-trnL region.
In addition, the forward primer (CiDeLF-F: SEQ ID NO: 5, CiMaTL-F: SEQ ID NO: 7) of each primer set is designed at a position where the 3 ′ end becomes a SNP site to reduce non-specific amplification. 'A mismatch base was introduced at the third position from the end. FIG. 7 and FIG. 8 show examples of preparation of the primer. Moreover, the forward primer preparation site is shown under each alignment of FIG. 5 and FIG.

[Example 4] Examination of primer set conditions The annealing temperature in the allele-specific PCR was examined for the primer set for detection of the siquashr trnL-trnF region designed in Example 3.
“CiDeLF-F (SEQ ID NO: 5)”, “CiDeLF-R (SEQ ID NO: 6)” using the DNA of sample 2 (SEIKWASHA) or sample 9 (KARAMANCY) among the total DNA extracted in Example 1 above as a template. The PCR was performed under the conditions shown in Table 5. The annealing temperature was 58 ° C, 59 ° C, 60 ° C, 61 ° C, 62 ° C, 63 ° C, 64 ° C, or 65 ° C.
Each obtained PCR product was electrophoresed on a 2% agarose gel. The results are shown in FIG.
In the figure, lanes 1 to 8 are annealing temperatures of 58 ° C. (lane 1), 59 ° C. (lane 2), 60 ° C. (lane 3), 61 ° C. (lane 4), 62 ° C. (lane 5), 63, respectively. The amplification results at each temperature of ° C. (lane 6), 64 ° C. (lane 7), and 65 ° C. (lane 8) are shown. In addition, the left lane shows the amplification results for Siqua Wash (sample 2), and the right lane shows the results for calamancy (sample 9) calamancy. Lane M shows a 100 bp marker.

As a result, amplification (amplification recognized as 76 bp) was confirmed in the Siqwasher under the conditions of annealing temperatures of 58 ° C. to 63 ° C. (lanes 1 to 6), and the clearest band was confirmed at 61 ° C. (lane 4). On the other hand, no amplification of calamancy was observed in this temperature range.

[Example 5] Examination of primer set conditions The annealing temperature in allele-specific PCR was examined for the primer set for detecting the Calamancy trnT-trnL region designed in Example 3.
In the PCR reaction, “CiMaTL-F (SEQ ID NO: 7)” and “CiMaTL-R (SEQ ID NO: 8)” were used as primer sets, and annealing temperatures were 54.9 ° C., 55.8 ° C., 56.7 ° C., 57.6 ° C., The same procedure as in Example 4 was performed except that the test was performed at 58.5 ° C, 59.4 ° C, 60.3 ° C, or 61.2 ° C. The results are shown in FIG.
In the figure, lanes 1 to 8 have annealing temperatures of 54.9 ° C. (lane 1), 55.8 ° C. (lane 2), 56.7 ° C. (lane 3), 57.6 ° C. (lane 4), 58.5 ° C. (lane 5), and 59.4, respectively. The amplification results at each temperature of ° C (lane 6), 60.3 ° C (lane 7), and 61.2 ° C (lane 8) are shown. Further, the left lane shows the amplification result for Siquasha (sample 2), and the right lane shows the calamancy (sample 9). Lane M shows a 100 bp marker.

  As a result, amplification (amplification recognized as 105 bp) was confirmed at an annealing temperature of 54.9 ° C to 59.4 ° C (lanes 1 to 6), and the clearest band was confirmed at 54.9 ° C to 58.5 ° C (lanes 1 to 5). It was. On the other hand, in this temperature range, almost no amplification was observed in Shikuwasha, but there was a possibility of amplification slightly at 54.9 ° C (lane 1).

[Example 6] Variety discrimination example "CiDeLF-F (SEQ ID NO: 5)" and "CiDeLF-R (SEQ ID NO: 6)" which are primer sets for detecting the siquashr trnL-trnF region designed in Example 3 were used. Allyl-specific PCR was performed to discriminate between Sikwasher and Karamanshi.
The PCR reaction was performed on samples 1 to 10 of the total DNA extracted in Example 1 at an annealing temperature of 61 ° C. (conditions where specific amplification was clearly obtained in Example 4). This was carried out in the same manner as in Example 4. The results are shown in FIG.
In the figure, lanes 1 to 7 and 10 show the amplification results for Siquasha, and lanes 8 and 9 show the amplification results for Karamansi. Lane M shows a 100 bp marker.

As a result, a clear amplification (amplification recognized as 76 bp) was observed for the used Shiqwasha (samples 1 to 7 and 10). On the other hand, no amplification was observed for Calamancy (samples 8 and 9).
From this, it was shown that DNA of Shiquashar and Karamanshi can be clearly distinguished by allele-specific PCR using CiDeLF-F (SEQ ID NO: 5) and CiDeLF-R (SEQ ID NO: 6).

[Example 7] Variety discrimination example Using "CiMaTL-F (SEQ ID NO: 7)" and "CiMaTL-R (SEQ ID NO: 8)" which are primer sets for detecting the Calamancie trnT-trnL region designed in Example 3 Allyl-specific PCR was performed to discriminate between Sikwasher and Karamanshi.
The PCR reaction was performed on samples 1 to 10 of the total DNA extracted in Example 1 at an annealing temperature of 58 ° C. (conditions where specific amplification was clearly obtained in Example 5). This was carried out in the same manner as in Example 5. The results are shown in FIG.
In the figure, lanes 1 to 7 and 10 show the amplification results for Siquasha, and lanes 8 and 9 show the amplification results for Karamansi. Lane M shows a 100 bp marker.

As a result, clear amplification (amplification recognized as 105 bp) was observed for the used calamancy (samples 8 and 9). On the other hand, no amplification was seen for Seek washer (Samples 1-7, 10).
From this, it was shown that DNA of Siquashar and Karamanshi can be clearly distinguished by allele specific PCR using CiMaTL-F (SEQ ID NO: 7) and CiMaTL-R (SEQ ID NO: 8).

ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to discriminate | determine easily and with high precision the mixing | mixing of the calamancy in the heat processing product of a sea washer which was difficult conventionally. As a result, it is expected that it can contribute to preventing illegal mixing of the calamancy raw material in the processed sea quashsha product.
Further, the principle of the present invention is not limited to seek squash and calamancy, and is expected to be applied to discrimination of other mixed fruit juice products.

Claims (4)

A method for discriminating between seek washer and calamancy, comprising performing allele-specific PCR using a primer set comprising a primer having the following characteristics (A) or (B) and confirming the presence or absence of amplification of the target sequence .
(A): The 3 ′ end is a position corresponding to the SNP site of Sequachia and Calamansi existing in the trnL-trnF region on the chloroplast DNA, and the 3 ′ end is either Sequasher or Calamansi The same base as the SNP, the third base upstream from the 3 ′ end is a mismatch base, and the 4th to 16th bases upstream from the 3 ′ end are Sequwasha and Karamansi It is in complete agreement with the sequences of both trnL-trnF regions.
(B): The 3 ′ end is a position corresponding to the SNP site of Sequashas and Calamansi that exists in the trnT-trnL region on the chloroplast DNA, and the 3 ′ end is either Sichuasha or Karamansi A base that matches the SNP, the third base upstream from the 3 ′ end is a mismatch base, and the fourth to 16th bases upstream from the 3 ′ end are Sequwasha and Karamanshi Are completely identical to the sequences of both trnT-trnL regions. In the primer set, the other primer that is not the primer having the characteristics of (A) or (B) is such that at least the 16th base upstream from the 3 ′ end is completely identical to the sequences of both Sikwasher and Karamancy. The method for discriminating between a seek washer and a calamancy according to claim 1, which is designed at a position where the amplified fragment is 400 bp or less. Performing allele-specific PCR using a primer set comprising the primer having the characteristics of (A) according to claim 1 or 2, and confirming the presence or absence of amplification of the target sequence; and A method for discriminating between seek washer and calamancy, comprising performing allele-specific PCR using a primer set comprising a primer having the characteristics of (B) above and confirming the presence or absence of amplification of the target sequence. The primer set including the primer having the characteristics of A is a primer set consisting of SEQ ID NOs: 5 and 6 capable of specifically amplifying the Sequwacher trnL-trnF region; and the primer set including the primer having the characteristics of B The primer set consisting of SEQ ID NOs: 7 and 8 capable of specifically amplifying the calamancy trnT-trnL region;