JP7153436B2 - Primer and method for detecting nuts - Google Patents

Description

The present invention does not contain tree nuts that may cause allergies (almonds, beach nuts, Brazil nuts, butternuts, cashew nuts, chestnuts, tinkapins, coconuts, ginkgo nuts, hazelnuts, hickory nuts, litchi, macadamia nuts, pecan nuts, pine nuts, Pili nuts, pistachios, shea nuts, and walnuts) can be detected, and if the nuts are contained in food ingredients or products, even if the amount is very small, the detection of nuts can be performed with high sensitivity. A method and PCR primers, primer sets and kits for use in the method.

Among the foods that may cause food allergies, milk, eggs, fish, crustaceans, wheat, soybeans, peanuts, and tree nuts, which have a particularly large number of patients, are called "The Big-8." Many countries and regions around the world, such as Canada, Singapore, and the EU, commonly require labeling of foods containing these substances.

Foods that may cause allergies can be unintentionally mixed in small amounts during production, distribution, and processing. is important.

For milk and eggs, a method to detect proteins characteristic to each (ELISA method, Western blot method, and immunochromatography), and for fish, a method to detect characteristic DNA sequences (PCR method). However, for crustaceans, wheat, soybeans, and peanuts, detection methods for both have been established, and are commercially available and available as test kits. On the other hand, the term "nut" is a general term and includes a very wide range of biological species, so no method has been established to collectively detect them as nuts.

For example, in the EU and Singapore, the nuts subject to labeling are almonds, walnuts, pecan nuts, hazelnuts, macadamia nuts, Brazil nuts, cashew nuts, and pistachios. Nine species including fruit are listed. In the United States, in addition to the above nine, there are beach nuts, butternuts, chestnuts, chinkapins, coconuts, ginkgo nuts, hickory nuts, lychees, pili nuts, and shea nuts, for a total of 19 types.

Unlike other countries, it is recommended that walnuts and cashew nuts be individually labeled as "specified September 20, 2013, Shoshoku table No. 257). In recent years, with the westernization of the Japanese diet, the amount of nuts consumed is increasing.In the future, the number of patients with food allergies to nuts other than walnuts and cashew nuts will increase, and the accompanying increase in the number of people who are allergic to "specific raw materials". There are likely to be many additions.

In order to test for the presence or absence of tree nut contamination, multiple detection methods for individual tree nuts must be combined, but performing ELISA and Western blotting multiple times requires a huge amount of time and money. In the case of the PCR method, on the other hand, if the PCR conditions are the same, multiple sets of reagents can be prepared and tested all at once, so it is considered useful as a nut detection method. In addition, compared to DNA, proteins are generally less stable to various processing treatments in the food manufacturing process, so methods to detect proteins may not be applicable to highly processed test foods. high. Therefore, the PCR method targeting the base sequence of DNA, which is said to be relatively resistant to processing compared to proteins, is considered suitable for detection from food materials and products that have undergone various processing steps.

Currently, individual detection methods using the PCR method have been established for 8 types of almonds, walnuts, pecan nuts, hazelnuts, macadamia nuts, Brazil nuts, cashew nuts, and pistachios, and they are commercially available and used as test kits. It is possible. The kit ensures detection specificity by adopting a real-time PCR method using TaqMan probes. It takes In addition, as described above, there are at least 19 kinds of nuts that may cause food allergy, and many of them cannot be detected by the kit.

Almonds, hazelnuts, cashew nuts, and walnuts have also been reported to be detected by PCR (Patent Documents 1, 2, 3, and 4) as methods for detecting individual nuts (Patent Documents 1, 2, 3, and 4). It is a method of detection, and cannot detect nuts of multiple species.

In addition, a method using SSR markers has been reported for the purpose of cultivar analysis of chestnuts (Non-Patent Document 1). In the case of samples containing mixed plant species, the PCR primers used are highly likely to react with other plant DNAs, making it difficult to specifically detect chestnuts. Therefore, it cannot be used to specifically detect chestnut DNA in foods containing multiple plants.

In addition, as a detection method for coconut, a detection method using immunochromatography targeting coconut-specific proteins has been established, and it is commercially available as a test kit. There is no report on the detection method as such.

To date, no detection methods have been reported for beach nuts, butternuts, tinkapins, ginkgo nuts, hickory nuts, lychees, pili nuts, pine nuts, and shea nuts.

As noted above, tree nuts that may cause food allergies (almonds, beach nuts, brazil nuts, butternuts, cashews, chestnuts, tinkapins, coconuts, ginkgo nuts, hazelnuts, hickory nuts, litchi, macadamia nuts, pecan nuts, pine nuts) Pili nuts, pistachios, shea nuts, and walnuts) have not yet been reported. , is expected as a food quality control method.

CN 101643786A CN 101643787A CN 101792815A JP 2009-279000, Primer and method for detecting specific plant Eiichi Inoue, Toshiya Yamamoto, Shuan Ruan, Hiromi Matsuki, Hiroyuki Anzai, Hiromichi Hara, Analysis of seven chestnut varieties from the Korean peninsula using Japanese chestnut SSR markers; Sono Gakken, 7(4):475-480, 2008

The present invention does not contain tree nuts that may cause allergies (almonds, beach nuts, Brazil nuts, butternuts, cashew nuts, chestnuts, tinkapins, coconuts, ginkgo nuts, hazelnuts, hickory nuts, litchi, macadamia nuts, pecan nuts, pine nuts, Pili nuts, pistachios, shea nuts, and walnuts) were primarily aimed at providing a PCR primer set for use in a method that can specifically and simultaneously detect Pili nuts, pistachios, shea nuts, and walnuts from food raw materials and products.

In addition, as described in paragraph [0005], specific examples of nuts subject to labeling are given in each country where the labeling of nuts is obligatory. For example, 8 types in the EU and Singapore, 9 types in Canada, 19 species are described. Here, 8 types of almonds, Brazil nuts, cashew nuts, hazelnuts, macadamia nuts, pecan nuts, pistachios, and walnuts, which are commonly contained in these, are frequently used as food ingredients, and production, distribution, and processing It is thought that there is a high need for inspections for unintended minute amounts of contamination at the stage. Therefore, it was an object to provide a simultaneous PCR (multiplex PCR) method capable of simultaneously detecting specific combinations of four of these eight types in a single PCR.

In order to achieve the above objects, the present inventors focused on the commonality and specificity in the gene sequences of nuts and other organisms to be detected from a molecular biological point of view. (almonds, beach nuts, brazil nuts, butternuts, cashews, chestnuts, tinkapins, coconuts, ginkgo nuts, hazelnuts, hickory nuts, lychee nuts, macadamia nuts, pecan nuts, pine nuts, pili nuts, pistachios, shea nuts, walnuts) We have intensively studied methods that can detect As a result, almonds, beach nuts, brazil nuts, butternuts, cashews, chestnuts, tinkapins, coconuts, ginkgo nuts, hazelnuts, hickory nuts, lychees, macadamia nuts, pecans, pine nuts, pili nuts, pistachios, shea nuts, or walnuts Almonds, beach nuts, brazil nuts, butternuts, cashew nuts, chestnuts, tinkapins, coconuts, ginkgo nuts, coconuts, ginkgo nuts, etc. We found that hazelnuts, hickory nuts, litchi, macadamia nuts, pecan nuts, pine nuts, pili nuts, pistachios, shea nuts, and walnuts can be easily detected. The present invention has been completed by devising such a method that detection can be performed with That is, the present invention relates to the following PCR primer set for detecting nuts. That is, the present invention first relates to the following items.

Item 1. A PCR primer consisting of a maximum of 30 bases of DNA containing the nucleotide sequence of base numbers 9 to 23 in SEQ ID NO: 1 in the sequence listing on the 3' end side, and the bases of base numbers 5 to 19 in SEQ ID NO: 2 in the sequence listing A PCR primer set for detecting almonds, comprising a PCR primer consisting of a maximum of 30 nucleotides of DNA containing a sequence on the 3' end side.
Item 2. A PCR primer consisting of a maximum of 30 bases of DNA containing the nucleotide sequence of nucleotide numbers 7 to 21 in SEQ ID NO: 3 of the sequence listing on the 3' end side, and the nucleotides of nucleotide numbers 6 to 20 in SEQ ID NO: 4 of the sequence listing A PCR primer set for detecting beechnuts, comprising a PCR primer consisting of a maximum of 30 nucleotides of DNA containing a sequence on the 3' end side.
Item 3. A PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of nucleotide numbers 9 to 23 in SEQ ID NO: 5 of the sequence listing on the 3' end side, and the nucleotides of nucleotide numbers 5 to 19 in SEQ ID NO: 6 of the sequence listing A PCR primer set for detecting Brazil nuts, comprising a PCR primer consisting of a maximum of 30 nucleotides of DNA containing a sequence on the 3' end side.
Item 4. A PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of nucleotide numbers 6 to 20 in SEQ ID NO: 7 in the sequence listing on the 3' end side, and the nucleotides of nucleotide numbers 5 to 19 in SEQ ID NO: 8 in the sequence listing A PCR primer set for cashew nut detection, comprising a PCR primer consisting of a maximum of 30 nucleotides of DNA containing a sequence on the 3' end side.
Item 5. A PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of nucleotide numbers 6 to 20 in SEQ ID NO: 9 in the sequence listing on the 3' end side, and the nucleotides of nucleotide numbers 4 to 18 in SEQ ID NO: 10 in the sequence listing A PCR primer set for chestnut detection, comprising a PCR primer consisting of a maximum of 30 bases of DNA containing a sequence on the 3' end side.
Item 6. A PCR primer consisting of a maximum of 30 bases of DNA containing the nucleotide sequence of nucleotide numbers 4 to 18 in SEQ ID NO: 11 of the sequence listing on the 3' end side, and the nucleotides of nucleotide numbers 4 to 18 in SEQ ID NO: 12 of the sequence listing A PCR primer set for coconut detection, comprising a PCR primer consisting of a maximum of 30 nucleotides of DNA containing a sequence on the 3' end side.
Item 7. A PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of nucleotide numbers 7 to 21 in SEQ ID NO: 13 of the sequence listing on the 3' end side, and the nucleotides of nucleotide numbers 13 to 27 in SEQ ID NO: 14 of the sequence listing A PCR primer set for detecting ginkgo biloba, comprising a PCR primer consisting of DNA of up to 30 bases containing a sequence on the 3′ end side.
Item 8. A PCR primer consisting of a maximum of 30 bases of DNA containing the nucleotide sequence of nucleotide numbers 7 to 21 in SEQ ID NO: 15 of the sequence listing on the 3' end side, and the nucleotides of nucleotide numbers 11 to 25 in SEQ ID NO: 16 of the sequence listing A PCR primer set for detecting hazelnuts, comprising a PCR primer consisting of a maximum of 30 nucleotides of DNA containing a sequence on the 3' end side.
Item 9. A PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of nucleotide numbers 6 to 20 in SEQ ID NO: 17 of the sequence listing on the 3' end side and the nucleotides of nucleotide numbers 9 to 23 in SEQ ID NO: 18 of the sequence listing A PCR primer set for hickory nut detection, comprising a PCR primer consisting of a maximum of 30 nucleotides of DNA containing a sequence on the 3' end side.
Item 10. A PCR primer consisting of a maximum of 30 bases of DNA containing the nucleotide sequence of base numbers 7 to 21 in SEQ ID NO: 19 in the sequence listing on the 3' end side, and the bases of base numbers 6 to 20 in SEQ ID NO: 20 in the sequence listing A PCR primer set for detecting lychee, comprising a PCR primer consisting of a maximum of 30 bases of DNA containing a sequence on the 3' end side.
Item 11. A PCR primer consisting of a maximum of 30 bases of DNA containing the nucleotide sequence of base numbers 6 to 20 in SEQ ID NO: 21 of the sequence listing on the 3' end side, and the bases of base numbers 8 to 22 in SEQ ID NO: 22 of the sequence listing A PCR primer set for detecting macadamia nuts, comprising a PCR primer consisting of a maximum of 30 bases of DNA containing a sequence on the 3′ end side.
Item 12. A PCR primer consisting of a maximum of 30 bases of DNA containing the nucleotide sequence of nucleotide numbers 4 to 18 in SEQ ID NO: 23 of the sequence listing on the 3' end side, and the nucleotides of nucleotide numbers 6 to 20 in SEQ ID NO: 24 of the sequence listing A PCR primer set for detecting pecan nuts, comprising a PCR primer consisting of a maximum of 30 nucleotides of DNA containing a sequence on the 3' end side.
Item 13. A PCR primer consisting of a maximum of 30 bases of DNA containing the nucleotide sequence of nucleotide numbers 5 to 19 in SEQ ID NO: 25 of the sequence listing on the 3' end side, and the nucleotides of nucleotide numbers 8 to 22 in SEQ ID NO: 26 of the sequence listing A PCR primer set for detecting pine nuts, comprising a PCR primer consisting of a maximum of 30 bases of DNA containing a sequence on the 3' end side.
Item 14. A PCR primer consisting of a maximum of 30 bases of DNA containing the nucleotide sequence of base numbers 8 to 22 in SEQ ID NO: 27 in the sequence listing on the 3' end side, and the bases of base numbers 4 to 18 in SEQ ID NO: 28 in the sequence listing A PCR primer set for detecting pili nut, comprising a PCR primer consisting of a maximum of 30 bases of DNA containing a sequence on the 3' end side.
Item 15. A PCR primer consisting of a maximum of 30 bases of DNA containing the nucleotide sequence of base numbers 10 to 24 in SEQ ID NO: 29 of the sequence listing on the 3' end side, and the bases of base numbers 15 to 29 in SEQ ID NO: 30 of the sequence listing A PCR primer set for pistachio detection, comprising a PCR primer consisting of a maximum of 30 nucleotides of DNA containing a sequence on the 3' end side.
Item 16. A PCR primer consisting of a maximum of 30 bases of DNA containing the nucleotide sequence of base numbers 8 to 22 in SEQ ID NO: 31 in the sequence listing on the 3' end side, and the bases of base numbers 4 to 18 in SEQ ID NO: 32 in the sequence listing A PCR primer set for detecting shea nut, comprising a PCR primer consisting of a maximum of 30 bases of DNA containing a sequence on the 3' end side.
Item 17. A PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of nucleotide numbers 6 to 20 in SEQ ID NO: 33 of the sequence listing on the 3' end side, and the nucleotides of nucleotide numbers 6 to 20 in SEQ ID NO: 34 of the sequence listing A PCR primer set for detecting walnuts, comprising a PCR primer consisting of a maximum of 30 bases of DNA containing a sequence on the 3' end side.

Furthermore, among the nuts, almonds, brazil nuts, cashew nuts, hazelnuts, macadamia nuts, pecan nuts, pistachios, and walnuts, each of the above primer sets capable of detecting specific four combinations of these are mixed and simultaneously detected by a single PCR test (multiplex PCR) is possible. That is, the present invention also intends to relate to the following items.

Claim 18. Pecan nuts, walnuts, pistachios and Simultaneous PCR detection method for cashew nuts.
Claim 19. Almonds, hazelnuts, macadamia nuts and Simultaneous PCR detection method for Brazil nuts.
Claim 20. Pecan nuts, walnuts, hazelnuts and peanuts using a primer set comprising the primer set of claim 9, the primer set of claim 17, the primer set of claim 8 and the primer set of claim 4 Simultaneous PCR detection method for cashew nuts.
Claim 21. Almonds, pistachios, macadamia nuts and almonds using a primer set comprising the primer set according to claim 1, the primer set according to claim 15, the primer set according to claim 11 and the primer set according to claim 3 Simultaneous PCR detection method for Brazil nuts.
Item 22. Pecan nuts, walnuts and macadamia nuts using a primer set comprising the primer set according to claim 9, the primer set according to claim 17, the primer set according to claim 11 and the primer set according to claim 3 and Brazil nut simultaneous PCR detection method.
Item 23. Almonds, hazelnuts, pistachios and cashew nuts using a primer set comprising the primer set according to claim 1, the primer set according to claim 8, the primer set according to claim 15 and the primer set according to claim 4 simultaneous PCR detection method.

According to the present invention, tree nuts (almonds, beach nuts, Brazil nuts, butternuts, cashew nuts, chestnuts, tinkapins, coconuts, ginkgo nuts, hazelnuts, hickory nuts, lychees, macadamia nuts, pecan nuts , pine nuts, pili nuts, pistachios, shea nuts, and walnuts). This has the effect of making it possible to carry out management inspections. In addition, it can contribute to the prevention of allergies, investigation of causative substances when allergic symptoms occur, and the like.

For almonds, brazil nuts, cashew nuts, hazelnuts, macadamia nuts, pecan nuts, pistachios, and walnuts, simultaneous PCR (multiplex PCR) was performed by mixing primers that detect these nuts. It becomes possible to detect four species at the same time.

FIG. 2 is a photograph after electrophoresis showing DNA detection sensitivity when using the primer set of the present invention. FIG. (A) Photographs after electrophoresis showing almond DNA detection sensitivity in PCR using SEQ ID NO: 1 and SEQ ID NO: 2. (B) A photograph after electrophoresis showing the detection sensitivity of beach nut DNA in PCR using SEQ ID NO:3 and SEQ ID NO:4. (C) A photograph after electrophoresis showing the detection sensitivity of Brazil nut DNA in PCR using SEQ ID NO: 5 and SEQ ID NO: 6. (D) A photograph after electrophoresis showing the detection sensitivity of cashew nut DNA in PCR using SEQ ID NO: 7 and SEQ ID NO: 8. (E) is a photograph after electrophoresis showing detection sensitivity of chestnut DNA in PCR using SEQ ID NO:9 and SEQ ID NO:10. (F) A photograph after electrophoresis showing detection sensitivity of coconut DNA in PCR using SEQ ID NO: 11 and SEQ ID NO: 12. (G) A photograph after electrophoresis showing the detection sensitivity of ginkgo biloba DNA in PCR using SEQ ID NO: 13 and SEQ ID NO: 14. (H) A photograph after electrophoresis showing the detection sensitivity of hazelnut DNA in PCR using SEQ ID NO: 15 and SEQ ID NO: 16. (I) A photograph after electrophoresis showing the detection sensitivity of hickory nut DNA in PCR using SEQ ID NO: 17 and SEQ ID NO: 18. (J) A photograph after electrophoresis showing the detection sensitivity of lychee DNA in PCR using SEQ ID NO: 19 and SEQ ID NO: 20. (K) is a photograph after electrophoresis showing detection sensitivity of macadamia nut DNA in PCR using SEQ ID NO: 21 and SEQ ID NO: 22. (L) A photograph after electrophoresis showing the detection sensitivity of pecan nut DNA in PCR using SEQ ID NO: 23 and SEQ ID NO: 24. (M) is a photograph after electrophoresis showing the detection sensitivity of pine nut DNA in PCR using SEQ ID NO:25 and SEQ ID NO:26. (N) A photograph after electrophoresis showing the detection sensitivity of Pili nut DNA in PCR using SEQ ID NO: 27 and SEQ ID NO: 28. (O) is a photograph after electrophoresis showing the detection sensitivity of pistachio DNA in PCR using SEQ ID NO:29 and SEQ ID NO:30. (P) A photograph after electrophoresis showing the detection sensitivity of shea nut DNA in PCR using SEQ ID NO: 31 and SEQ ID NO: 32. (Q) is a photograph after electrophoresis showing detection sensitivity of walnut DNA in PCR using SEQ ID NO:33 and SEQ ID NO:34. (A) Photographs after electrophoresis showing the results of multiplex PCR for pecan nuts, walnuts, pistachios, and cashew nuts. (B) Photographs after electrophoresis showing the results of multiplex PCR for 4 types of almonds, hazelnuts, macadamia nuts and Brazil nuts. (C) Photographs after electrophoresis showing the results of multiplex PCR for pecan nuts, walnuts, hazelnuts and cashew nuts. (D) Photographs after electrophoresis showing the results of multiplex PCR for 4 types of almonds, pistachios, macadamia nuts, and Brazil nuts. (E) Photographs after electrophoresis showing the results of multiplex PCR for pecan nuts, walnuts, macadamia nuts, and Brazil nuts. (F) A photograph after electrophoresis showing the results of multiplex PCR for 4 types of almonds, hazelnuts, pistachios, and cashew nuts.

The present invention will be described in detail below.
PCR primer set for tree nut detection
The present inventors have developed the following 17 kinds of PCR primer sets for detecting nuts in accordance with the above purpose. The PCR primer set for detecting nuts of the present invention includes one having a base sequence characteristic of almonds, one having a base sequence characteristic of beach nuts, one having a base sequence characteristic of Brazil nuts, and one having a base sequence characteristic of cashew nuts. base sequence characteristic of chestnut, base sequence characteristic of coconut, base sequence characteristic of hazelnut, base sequence characteristic of ginkgo biloba having a base sequence characteristic of hickory nuts, having a base sequence characteristic of lychee, having a base sequence characteristic of macadamia nuts, having a base sequence characteristic of pecan nuts, pili nuts Those having a base sequence characteristic of pine nuts, those having a base sequence characteristic of pistachios, those having a base sequence characteristic of shea nuts, and those having a base sequence characteristic of walnuts Some have base sequences.

Each primer set was designed as follows. From the viewpoint of improving sensitivity, it is desirable that the target nucleotide sequence has a large number of copies per cell. maturase-encoding gene) gene sequence, rRNA gene internal transcribed spacer region 1 (ITS1) nucleotide sequence, and internal transcribed spacer region 2 (ITS2) nucleotide sequence were selected as candidates. We obtain the sequences of various plants from a known DNA database (GenBank nucleotide sequence database), or obtain some plants by our own analysis, and create a multiple parallel sequence map of those enormous base sequences. did. Among them, the ITS region is suitable as a target sequence for nut-specific primers because it evolves at a high rate and enables identification of closely related species.

When developing a primer set for detecting almonds, the species to be detected was set to be almonds (Prunus dulcis), and almonds and peach (Prunus percica), which is a related species of the same genus, were selected. ), prunus domestica, plum (Prunus mume), plum (Prunus salicina), apricot (Prunus armeniaca), and cherry (Prunus avium). Nucleotide sequences in the ITS region are highly homologous to related species, and it was difficult to select almond-specific nucleotide sequences. A base sequence region could be selected, and a PCR primer set was newly designed from the base sequence region. At the time of design, it is designed to hybridize under stringent conditions with the relevant nucleotide sequence region of the biological species targeted for detection, but not to hybridize with the biological species not targeted for detection. did.

When developing a primer set for detecting beach nuts, we set beach nuts (Fagaceae Fagus spp.) as the target organism for detection, Castanea spp., Castanea spp., Castanopsis spp., Quercus spp., Quercus spp., Carya spp., Pecan nuts (Carya illinoinensis) , walnut (Juglans spp.) of the walnut family Walnut genus, and hazelnut (Corylus spp.) of the birch family Corylus spp. A base sequence region specific to beech nuts was selected, and a new PCR primer set was designed from the base sequence region. At that time, it was designed so that it hybridizes under stringent conditions with the base sequence region of the biological species targeted for detection, but does not hybridize with the biological species not targeted for detection.

When developing a PCR primer set for detecting Brazil nuts, the species to be detected was set to be Brazil nuts (Bertholletia excelsa, genus Brazil nuts of the family Barringtoniaceae, of the order Ericales). Shea nuts (Vitellaria paradoxa) of the same order as the family Sapotaceae, the genus Fulcuricum of the family Sapotaceae (Synsepalum dulcificum), persimmons of the genus Persimmon (Diospyros kaki), and teas of the genus Camellia of the family Persimmon (Camellia sinensis) , a kiwifruit (Actinidia deliciosa) belonging to the genus Actinidia of the family Actinidia, and a blueberry (Vaccinium spp.) belonging to the genus Vaccinium of the family Ericaceae. A base sequence region specific to Brazil nuts was selected, and a new PCR primer set was designed from the base sequence region. At that time, it was designed so that it hybridizes under stringent conditions with the base sequence region of the biological species targeted for detection, but does not hybridize with the biological species not targeted for detection.

In addition, when developing a PCR primer set for detecting cashew nuts, the species to be detected was set to be cashew nuts (Anacardium occidentale). The nucleotide sequences of the ITS regions of Pistacia vera of the genus Mangifera indica and Pink pepper of the genus Schinus molle were compared. A base sequence region specific to cashew nuts was selected, and a new PCR primer set was designed from the base sequence region. At that time, it was designed so that it hybridizes under stringent conditions with the base sequence region of the biological species targeted for detection, but does not hybridize with the biological species not targeted for detection.

In addition, when developing a PCR primer set for detecting chestnuts, chestnuts (Castanea spp., family Fagaceae, genus Castanea) were selected as the species to be detected. Since Chincapin (Castanea pumila) is also a species of the genus Castanea, it was also included in the detection target. Chestnuts and their related species Fagus spp., Fagus spp., Castanopsis spp., Quercus spp., Quercus spp., Hickory nuts (Carya spp.), pecan nuts (Carya illinoinensis), walnuts of the genus Walnut (Jugrans spp.), and hazelnuts (Corylus spp.) of the family Betulaceae were compared. The nucleotide sequence of the ITS region is highly homologous to closely related species, and it was difficult to select a chestnut-specific nucleotide sequence. , Selected from regions with high homology to pecan nuts, and selected regions for antisense primers from regions with low homology to hickory nuts and pecan nuts, but high homology to acorns. By combining a sense primer exhibiting cross-reactivity with respect to a species (hickory nuts, pecan nuts) and an antisense primer exhibiting cross-reactivity with another species (acorn) A PCR primer set that hybridizes under stringent conditions with the base sequence region of , but that does not hybridize with organisms not targeted for detection was newly designed by ingeniously devising a PCR primer set.

In addition, when developing a PCR primer set for coconut detection, the target organism species for detection was set to coconut (Cocos nucifera). The base sequences of the ITS regions of Phoenix dactylifera of the genus and Euterpe oleracea of the genus Euterpe were compared. A nucleotide sequence region specific to coconut was selected, and a new PCR primer set was designed from this nucleotide sequence region. At that time, it was designed so that it hybridizes under stringent conditions with the base sequence region of the biological species targeted for detection, but does not hybridize with the biological species not targeted for detection.

In addition, when developing a ginkgo detection PCR primer set, the species to be detected was set to Ginkgo biloba (Ginko biloba), and the chloroplast matK gene sequence of Ginkgo and other gymnosperms was used. compared. A gene sequence region specific to Ginkgo biloba was selected, and a new PCR primer set was designed from the gene sequence region. At that time, it was designed so that it hybridizes under stringent conditions with the relevant gene sequence region of the target organism species, but does not hybridize with the target organism species.

In addition, when developing a PCR primer set for detecting hazelnuts, the species to be detected was set to be hazelnuts (Corylus spp.). Hickory nuts (Carya spp.), pecan nuts (Carya illinoinensis), walnuts (Jugrans spp.), chestnuts (Castanea spp.), beach nuts of the family Fagaceae (Fagus spp.), Castanopsis spp. (Fagaceae), and Quercus spp. (Acorn). A base sequence region specific to hazelnuts was selected, and a new PCR primer set was designed from the base sequence region. At that time, it was designed so that it hybridizes under stringent conditions with the base sequence region of the biological species targeted for detection, but does not hybridize with the biological species not targeted for detection.

In addition, when developing a PCR primer set for detecting hickory nuts, the species to be detected is set to hickory nuts (Carya spp.), and hickory nuts and related species Juglans spp., Castanea spp., Castanea spp., Fagus spp., Castanopsis spp. , acorn (Quercus spp.) of the Fagaceae family, and hazelnut (Corylus spp.) of the Betulaceae family. A base sequence region specific to hickory nuts was selected, and a new PCR primer set was designed from the base sequence region. At that time, it was designed so that it hybridizes under stringent conditions with the base sequence region of the biological species targeted for detection, but does not hybridize with the biological species not targeted for detection.

In addition, when developing a PCR primer set for detecting lychee, the species to be detected was set to lychee (Litchi chinensis). The nucleotide sequences of the ITS regions of the genus Rambutan (Nephelium lappaceum) were compared. A base sequence region specific to lychee was selected, and a new PCR primer set was designed from the base sequence region. At that time, it was designed so that it hybridizes under stringent conditions with the base sequence region of the biological species targeted for detection, but does not hybridize with the biological species not targeted for detection.

In addition, when developing a PCR primer set for detecting macadamia nuts, the biological species to be detected is set to macadamia nuts (Macadamia spp.), Macadamia nuts and related species. Nucleotide sequences of the ITS region of lotus root (Nelumbo nucifera) belonging to the genus Nelumbo of the same order as the Lotus family were compared. A base sequence region specific to macadamia nuts was selected, and a new PCR primer set was designed from the base sequence region. At that time, it was designed so that it hybridizes under stringent conditions with the base sequence region of the biological species targeted for detection, but does not hybridize with the biological species not targeted for detection.

In the development of the PCR primer set for detecting pecan nuts, the species to be detected was set to pecan nuts (Carya illinoinensis, belonging to the family Fagaceae, walnut family, genus Pecan (Carya illinoinensis)). Pecan nuts are a kind of hickory nuts (pecan genus) mentioned above, but since they are the most distributed hickory nuts in the market, we thought it necessary to distinguish pecan nuts from other hickory nuts. Pecan nuts and their related species, hickory nuts (Carya ovata, Carya laciniosa), walnuts (Juglans spp.), chestnuts (Castanea spp.), Fagaceae Fagus spp., Castanopsis spp., Castanopsis spp., Quercus spp., Quercus spp., Corylus spp. Base sequences were compared. Nucleotide sequences in the ITS region are highly homologous to related species, and it was difficult to select a pecan nut-specific nucleotide sequence. A base sequence region could be selected, and a PCR primer set was newly designed from the base sequence region. At that time, it was designed so that it hybridizes under stringent conditions with the base sequence region of the biological species targeted for detection, but does not hybridize with the biological species not targeted for detection.

In addition, when developing a PCR primer set for detecting pine nuts, the species to be detected was set to pine nuts (Pinus spp.), and pine nuts and other gymnosperms were selected. We compared the nucleotide sequences of the ITS regions of A nucleotide sequence region specific to pine nuts was selected, and a new PCR primer set was designed from the nucleotide sequence region. At that time, it was designed so that it hybridizes under stringent conditions with the base sequence region of the biological species targeted for detection, but does not hybridize with the biological species not targeted for detection.

In addition, when developing a PCR primer set for detecting pili nuts, the species to be detected was set to Pili nuts (Canarium ovatum). Anacardium occidentale of the family Anacardium occidentale, Pistacia vera of the Anacardiaceae family, Mangifera indica of the Anacardiaceae family, Schinus molle of the Anacardiaceae family, Ganoderma spp. The nucleotide sequences of the ITS regions of litchi (Litchi chinensis), rambutan (Nephelium lappaceum) of the family Sapindaceae, genus Rambutan, and orange (Citrus sinensis) of the family Rutaceae, genus Citrus were compared. A base sequence region specific to pili nuts was selected, and a new PCR primer set was designed from the base sequence region. At that time, it was designed so that it hybridizes under stringent conditions with the base sequence region of the biological species targeted for detection, but does not hybridize with the biological species not targeted for detection.

In addition, when developing a PCR primer set for detecting pistachios, we set the target species to be pistachios (Pistacia vera, genus Pistachio, genus Sapindaceae, Anacardiaceae, Order Sapindaceae). The base sequences of the ITS regions of cashew nuts (Anacardium occidentale) of the genus Mangifera indica and pink pepper (Schinus molle) of the genus Mangifera were compared. A base sequence region specific to pistachio was selected, and a new PCR primer set was designed from the base sequence region. At that time, it was designed so that it hybridizes under stringent conditions with the base sequence region of the biological species targeted for detection, but does not hybridize with the biological species not targeted for detection.

In addition, when developing a PCR primer set for detecting shea nuts, the species to be detected was set to the species of shea nuts (Vitellaria paradoxa), which is a related species of the same family. Miracle fruit of the genus (Synsepalum dulcificum), Brazil nut of the same order Barringtoniaceae Brazilnut genus (Bertholletia excelsa), Persimmon persimmon of the Persimmon family Persimmon genus (Diospyros kaki), Persimmon family Camellia genus tea (Camellia sinensis), Actinidia Actinidia Actinidia The nucleotide sequences of the ITS regions of kiwifruit (Actinidia deliciosa) of the genus Vaccinium and blueberry (Vaccinium spp.) of the Ericaceae family were compared. A base sequence region specific to shea nuts was selected, and a new PCR primer set was designed from the base sequence region. At that time, it was designed so that it hybridizes under stringent conditions with the base sequence region of the biological species targeted for detection, but does not hybridize with the biological species not targeted for detection.

In developing a PCR primer set for detecting walnuts, walnuts (Juglans spp.) were selected as the species to be detected. Butternuts (Juglans cinerea) are also included in the detection target because they belong to the walnut genus. Walnuts and their related species, hickory nuts (Carya ovata, Carya laciniosa), pecan nuts (Carya illinoinensis), castanea spp. The nucleotide sequences of the ITS regions of beach nuts (Fagus spp.), castanopsis spp. (castanopsis spp.), acorns (Quercus spp.) of the fagaceae family, and hazelnuts (Corylus spp.) of the birch family compared. A base sequence region specific to walnut was selected, and a new PCR primer set was designed from the base sequence region. At that time, it was designed so that it hybridizes under stringent conditions with the base sequence region of the biological species targeted for detection, but does not hybridize with the biological species not targeted for detection.

Each primer was designed so as not to cause hybridization between the sense primer and the antisense primer, or hybridization by the individual primers themselves, that is, to avoid the formation of so-called primer dimers as much as possible. . For example, in order to avoid the formation of primer dimers, primers are subjected to base substitutions that do not lead to lower detection specificity and detection sensitivity of the primers. The bases of base numbers 3 and 4 in SEQ ID NO: 1, which is the sense primer of the almond detection primer, are originally A, but in order to avoid the formation of primer dimers by the sense primer itself, the detection specificity and detection of the primer T is adopted instead of A as a base substitution that does not lead to a decrease in sensitivity. Similarly, the base of base number 8 in SEQ ID NO: 10 is changed from C to A, the base of base number 6 in SEQ ID NO: 15 is changed from G to A, the base of base number 3 in SEQ ID NO: 21 is changed from C to A, and SEQ ID NO: The base number 4 in 24 is replaced with A from C, and the base number 7 with A to T.

Furthermore, in order to make the PCR reaction conditions for the above 17 types of primer sets the same, each PCR primer was designed so that the annealing conditions (annealing temperature, annealing time) that most affect the reaction conditions in the PCR reaction would be the same. It was very difficult to make the annealing conditions of multiple types of PCR primers identical, but by examining the number of bases of the PCR primers and the base sequences of the primers in detail, we were able to , were designed so that the annealing conditions were identical.

Although the nucleotide sequences of the primers provided by the present invention have been exhaustively described, even if the primers are theoretically designed successfully, primers that do not possess the intended performance (detection specificity, etc.) are designed. may occur. For example, in the research stage of the present invention, in PCR using a pecan nut detection PCR primer set consisting of SEQ ID NO: 69 and SEQ ID NO: 70, pecan nut DNA as well as hickory nut (overta hickory) DNA, which did not show the intended detection specificity. Thus, simply logically designing primers does not always result in the intended performance (detection specificity, etc.), and may not possess the required performance. , not only the logical design but also further devising and performance evaluation tests are important. The primers provided by the present invention are designed by applying further ingenuity to the logical design, and finally cleared the performance evaluation test.

Therefore, using a nucleic acid analysis method such as PCR using the PCR primer set for detecting nuts of the present invention, nuts contained in various foods (almonds, beach nuts, Brazil nuts, butter nuts, cashew nuts, chestnuts, tinkapins) , coconut, ginkgo, hazelnut, hickory nut, lychee, macadamia nut, pecan nut, pine nut, pili nut, pistachio, shea nut, or walnut) can be detected simultaneously with high sensitivity and specificity.

Here, nucleic acid analysis refers to the existence of characteristic base sequences for individual species, genera, or groups in organism classification, and by analyzing the presence or absence of the characteristic base sequences, It is an effective means for ascertaining the species, genus, or group of the organism, and is a method useful for detecting specific microorganisms, identifying organism species, and the like.

The base sequences of the primers provided by the present invention are as follows.
Base number 12345678901234567890123456789
SEQ ID NO: 1 (almond S) AGTTCTAGTTTCAAAGCGGGGGC
SEQ ID NO: 2 (almond AS) ACGACGGGCAACCGAGGTC
SEQ ID NO: 3 (Beach Nut S) GCCTTTCGTCCCCAAACGGTC
SEQ ID NO: 4 (Beach Nut AS) TGCCACGGTCGCTTCGAATG
SEQ ID NO: 5 (Brazil Nut S) GACGAGTGGTGGATCACGACACG
SEQ ID NO: 6 (Brazil nut AS) TCGATGCCTTGCCGCTTCG
SEQ ID NO: 7 (cashew nut S) TGGCGTTCGGAACGAACCCG
SEQ ID NO: 8 (cashew AS) GCGATGCGGGCGGGCATAG
SEQ ID NO: 9 (KuriS) GGAACGCGCCAAGGAATCA
SEQ ID NO: 10 (Clear AS) TCCGCCCAGCCCCGAACC
SEQ ID NO: 11 (Coconut S) GACGCTCCGCCCCCTCGA
SEQ ID NO: 12 (Coconut AS) GTGCGGCCACGCACCTGGG
SEQ ID NO: 13 (Gingko S) GGTGCTCACTCCCCAGTCAGG
SEQ ID NO: 14 (Ginkgo biloba AS) CATCTAGCATTTTGATCCTAACCACGG
SEQ ID NO: 15 (Hazelnut S) ATGCCAGGGGCGAATCTTGTG
SEQ ID NO: 16 (Hazelnut AS) GCTACAGGGTCACAGAGCACAAGAC
SEQ ID NO: 17 (Hickory Nut S) TGGGAGGGCACGATGAAAGC
SEQ ID NO: 18 (Hickory Nut AS) CGACGCAATAGGGTCGAGGAGAG
SEQ ID NO: 19 (Litchi S) GAGGCGTGGGGATGCGTTATC
SEQ ID NO: 20 (Litchi AS) CGAGAGCCTTACGCCGACCG
SEQ ID NO: 21 (Macadamia Nut S) GCACCCCGTGTCTCTGTTCG
SEQ ID NO: 22 (Macadamia Nut AS) CGATGTCCGAACAATGGCAAAG
SEQ ID NO: 23 (pecan nut S) ACGGTTGGGAGGGCACGA
SEQ ID NO: 24 (Pecan Nut AS) TGTAGTTGCGCTCGTCGCCC
SEQ ID NO: 25 (pine nut S) CAGGCGATGTTTCGTGGCG
SEQ ID NO: 26 (pine nut AS) CCCTACCCAATGGGAGGACAAG
SEQ ID NO: 27 (Pilinut S) TGGTCGTAACGAACCTCGGTGC
SEQ ID NO: 28 (Pilinut AS) CGTCACGGCGGCGCTTTC
SEQ ID NO: 29 (pistachio S) GGGTGTCGGTCGTATGCTTCTGCAT
SEQ ID NO: 30 (Pistachio AS) GTCGTTATTGATAATGAAAGAAGGCTACC
SEQ ID NO: 31 (Shea Nut S) GCGTTGCCTCGGCTAAAAACTC
SEQ ID NO: 32 (Shea Nut AS) GCGGTCAAGGCTCCCCAA
SEQ ID NO: 33 (Walnut S) GGTTGGGAGGGCACGTTGAG
SEQ ID NO: 34 (Walnut AS) CGACGGGTCACGAGGGTTTC
In addition, as the primer consisting of 30-base DNA provided by the present invention, for example, those having the following base sequences can be mentioned.
SEQ ID NO: 35 (almond S) cgacccgAGTTCTAGTTTCAAAGCGGGGGC
SEQ ID NO: 36 (almond AS) gacgaacgcacACGACGGGCAACCGAGGTC
SEQ ID NO: 37 (Beach Nut S) ggcttcgcgGCCTTTCGTCCCCAAACGGTC
SEQ ID NO: 38 (Beach Nut AS) cggacgaggtTGCCACGGTCGCTTCGAATG
SEQ ID NO: 39 (Brazil Nut S) acggcacGACGAGTGGTGGATCACGACACG
SEQ ID NO: 40 (Brazil Nut AS) tggggtcgcggTCGATGCCTTGCCCGCTTCG
SEQ ID NO: 41 (cashew nut S) cgacggtcggTGGCGTTCGGAACGAACCCG
SEQ ID NO: 42 (cashew AS) ggggtcgcgacGCGATGCGGGCGGGCATAG
SEQ ID NO: 43 (Kuri S) accccggcgcGGAACGGCCCAAGGAAATCA
SEQ ID NO: 44 (Kuri AS) gggaggccaactTCCGGCCCAGCCCCGAACC
SEQ ID NO: 45 (Coconut S) atcatgccaagcGACGCTCCGCCCCCTCGA
SEQ ID NO: 46 (Coconut AS) ccgatggctgggGTGCGGCACGCACCTGGG
SEQ ID NO: 47 (Gingko S) gctctgaagGGTGCTCACTCCCCAGTCAGG
SEQ ID NO: 48 (Ginkgo biloba AS) aatCATCTAGCATTTTGATCCTAACCACGG
SEQ ID NO: 49 (Hazelnut S) acggcgtgcATGCCAGGGGGCGAATCTTGTG
SEQ ID NO: 50 (Hazelnut AS) gcgcaGCTACAGGGTCACAGAGCACAAGAC
SEQ ID NO: 51 (Hickory Nut S) caaaaacggtTGGGAGGGCACGATGAAAGC
SEQ ID NO: 52 (Hickory Nut AS) gcaagaaCGACGCAATAGGGTCGAGGAGAG
SEQ ID NO: 53 (Litchi S) gcctcggacGAGGCGTGGGGATGCGTTATC
SEQ ID NO: 54 (Litchi AS) gcttcagggtCGAGAGCCTTACGCCGACCG
SEQ ID NO: 55 (Macadamia Nut S) gcgaagattgGCACCCCGTGTCTCTGTTCG
SEQ ID NO: 56 (Macadamia Nut AS) cacgcacaCGATGTCCGAACAATGGCAAAG
SEQ ID NO: 57 (pecan nut S) cccctcccaaaaACGGTTGGGAGGGCACGA
SEQ ID NO: 58 (Pecan Nut AS) cgggaggccaTGTAGTTGCGCTCGTCGCCC
SEQ ID NO: 59 (pine nut S) ctgaaatgtggCAGGCGATGTTTCGTGGCG
SEQ ID NO: 60 (pine nut AS) taaatccgCCCTACCCAATGGGAGGACAAG
SEQ ID NO: 61 (Pilinut S) accttcggTGGTCGTAACGAACCTCGGTGC
SEQ ID NO: 62 (Pilinut AS) gtgcgggcgccgCGTCACGGCGGCGCTTTC
SEQ ID NO: 63 (Pistachio S) tcgtcgGGTGTCGGTCGTATGCTTCTGCAT
SEQ ID NO: 64 (pistachio AS) aGTCGTTATTGATAATGAAAGAAGGCTACC
SEQ ID NO: 65 (Sheanut S) cctgaattGCGTTGCCTCGGCTAAAAACTC
SEQ ID NO: 66 (Shea Nut AS) tgacctggggtcGCGGTCAAGGCTCCGCAA
SEQ ID NO: 67 (Walnut S) cccaaaaaaacGGTTGGGAGGGCACGTTGAG
SEQ ID NO: 68 (Walnut AS) gggcaacacaCGACGGGTCACGAGGGTTTC

The base sequences of primers that did not exhibit the intended detection specificity in the research stage of the present invention are as follows.
SEQ ID NO: 69 (pecan nut S) CCCCAACACCTCGTATGATGTGTA
SEQ ID NO: 70 (Pecan Nut AS) TGTAGTTGCGCTCGTCGCCC
(S stands for sense primer and AS stands for antisense primer.)

According to the present invention, as the first primer set, PCR primers consisting of DNA of a minimum of 15 bases and a maximum of 30 bases containing the base sequences of base numbers 9 to 23 in SEQ ID NO: 1 of the sequence listing on the 3' end side are used as sense primers. A primer set is proposed in which a PCR primer consisting of a minimum of 15 bases and a maximum of 30 bases of DNA containing the nucleotide sequence of nucleotide numbers 5 to 19 in SEQ ID NO: 2 in the sequence list is used as an antisense primer. This primer set can be suitably used for almond detection.

In the present invention, as the second primer set, PCR primers consisting of DNA of minimum 15 bases and maximum 30 bases containing the nucleotide sequence of base numbers 7 to 21 in SEQ ID NO: 3 of the sequence listing on the 3' end side are used as sense primers. A primer set is proposed in which a PCR primer consisting of a minimum of 15 bases and a maximum of 30 bases of DNA containing the nucleotide sequence of base numbers 6 to 20 in SEQ ID NO: 4 in the sequence list is used as an antisense primer. This primer set can be suitably used for detecting beach nuts.

According to the present invention, as a third primer set, PCR primers consisting of a minimum of 15 and a maximum of 30 bases of DNA containing the base sequences of base numbers 9 to 23 in SEQ ID NO: 5 of the sequence listing on the 3' end side are used as sense primers. A primer set is proposed in which a PCR primer consisting of a minimum of 15 bases and a maximum of 30 bases of DNA containing the nucleotide sequence of nucleotide numbers 5 to 19 in SEQ ID NO: 6 in the sequence list is used as an antisense primer. This primer set can be suitably used for Brazil nut detection.

In the present invention, as a fourth primer set, PCR primers consisting of DNA of minimum 15 bases and maximum 30 bases containing base sequences of base numbers 6 to 20 in SEQ ID NO: 7 of the sequence listing on the 3' end side are used as sense primers. A primer set is proposed in which a PCR primer consisting of a minimum of 15 bases and a maximum of 30 bases of DNA containing the nucleotide sequence of base numbers 5 to 19 in SEQ ID NO: 8 in the sequence list is used as an antisense primer. This primer set can be suitably used for cashew nut detection.

According to the present invention, as a fifth primer set, PCR primers consisting of DNA of minimum 15 bases and maximum 30 bases containing base sequences of base numbers 6 to 20 in SEQ ID NO: 9 of the sequence listing on the 3' end side are used as sense primers. A primer set is proposed in which a PCR primer consisting of a minimum of 15 bases and a maximum of 30 bases of DNA containing the nucleotide sequence of base numbers 4 to 18 in SEQ ID NO: 10 in the sequence list on the 3' end side is used as an antisense primer. This primer set can be suitably used for chestnut detection.

In the present invention, as a sixth primer set, PCR primers consisting of DNA of minimum 15 bases and maximum 30 bases containing base sequences of base numbers 4 to 18 in SEQ ID NO: 11 of the sequence listing on the 3' end side are used as sense primers. A primer set is proposed in which a PCR primer consisting of a minimum of 15 bases and a maximum of 30 bases of DNA containing the nucleotide sequence of base numbers 4 to 18 in SEQ ID NO: 12 in the sequence list is used as an antisense primer. This primer set can be suitably used for coconut detection.

According to the present invention, as a seventh primer set, PCR primers consisting of DNA of a minimum of 15 bases and a maximum of 30 bases containing the nucleotide sequence of base numbers 7 to 21 in SEQ ID NO: 13 of the sequence listing on the 3' end side are used as sense primers. A primer set is proposed in which a PCR primer consisting of a minimum of 15 bases and a maximum of 30 bases of DNA containing the base sequence of base numbers 13 to 27 in SEQ ID NO: 14 in the sequence list is used as an antisense primer. This primer set can be suitably used for detecting ginkgo biloba.

In the present invention, as the eighth primer set, PCR primers consisting of DNA of minimum 15 bases and maximum 30 bases containing the base sequence of base numbers 7 to 21 in SEQ ID NO: 15 of the sequence listing on the 3' end side are used as sense primers. A primer set is proposed in which PCR primers consisting of DNA of a minimum of 15 bases and a maximum of 30 bases containing the nucleotide sequence of base numbers 11 to 25 in SEQ ID NO: 16 in the sequence list are used as antisense primers. This primer set can be suitably used for hazelnut detection.

According to the present invention, as a ninth primer set, PCR primers consisting of DNA of a minimum of 15 bases and a maximum of 30 bases containing the nucleotide sequence of base numbers 6 to 20 in SEQ ID NO: 17 of the sequence listing on the 3' end side are used as sense primers. A primer set is proposed in which a PCR primer consisting of a minimum of 15 bases and a maximum of 30 bases of DNA containing the nucleotide sequence of base numbers 9 to 23 in SEQ ID NO: 18 in the sequence list is used as an antisense primer. This primer set can be suitably used for hickory nut detection.

According to the present invention, as a tenth primer set, PCR primers consisting of DNA of a minimum of 15 bases and a maximum of 30 bases containing the nucleotide sequence of base numbers 7 to 21 in SEQ ID NO: 19 of the sequence listing on the 3' end side are used as sense primers. A primer set is proposed in which PCR primers consisting of a minimum of 15 bases and a maximum of 30 bases of DNA containing the nucleotide sequence of nucleotide numbers 6 to 20 in SEQ ID NO: 20 in the sequence list are used as antisense primers. This primer set can be suitably used for litchi detection.

According to the present invention, as an eleventh primer set, PCR primers consisting of DNA of a minimum of 15 bases and a maximum of 30 bases containing the nucleotide sequence of base numbers 6 to 20 in SEQ ID NO: 21 of the sequence listing on the 3' end side are used as sense primers. A primer set is proposed in which PCR primers consisting of a minimum of 15 bases and a maximum of 30 bases of DNA containing the nucleotide sequence of nucleotide numbers 8 to 22 in SEQ ID NO: 22 in the sequence list are used as antisense primers. This primer set can be suitably used for macadamia nut detection.

In the present invention, as a twelfth primer set, PCR primers consisting of DNA of a minimum of 15 bases and a maximum of 30 bases containing the base sequences of base numbers 4 to 18 in SEQ ID NO: 23 of the sequence listing on the 3' end side are used as sense primers. A primer set is proposed in which a PCR primer consisting of a minimum of 15 bases and a maximum of 30 bases of DNA containing the base sequence of base numbers 6 to 20 in SEQ ID NO: 24 in the sequence list is used as an antisense primer. This primer set can be suitably used for pecan nut detection.

In the present invention, as a thirteenth primer set, PCR primers consisting of a minimum of 15 and a maximum of 30 bases of DNA containing the base sequences of base numbers 5 to 19 in SEQ ID NO: 25 of the sequence listing on the 3' end side are used as sense primers. A primer set is proposed in which a PCR primer consisting of a minimum of 15 bases and a maximum of 30 bases of DNA containing the base sequence of base numbers 8 to 22 in SEQ ID NO: 26 in the sequence list is used as an antisense primer. This primer set can be suitably used for detecting pine nuts.

In the present invention, as the 14th primer set, PCR primers consisting of DNA of minimum 15 and maximum 30 bases containing the nucleotide sequence of base numbers 8 to 22 in SEQ ID NO: 27 of the sequence listing on the 3' end side are used as sense primers. A primer set is proposed in which PCR primers consisting of DNA of a minimum of 15 bases and a maximum of 30 bases containing the base sequences of base numbers 4 to 18 in SEQ ID NO: 28 in the sequence list are used as antisense primers. This primer set can be suitably used for Pili nuts detection.

According to the present invention, as a fifteenth primer set, PCR primers consisting of DNA of a minimum of 15 bases and a maximum of 30 bases containing the base sequences of base numbers 10 to 24 in SEQ ID NO: 29 of the sequence listing on the 3' end side are used as sense primers. A primer set is proposed in which PCR primers consisting of a minimum of 15 bases and a maximum of 30 bases of DNA containing the base sequences of base numbers 15 to 29 in SEQ ID NO: 30 in the sequence list are used as antisense primers. This primer set can be suitably used for pistachio detection.

According to the present invention, as the 16th primer set, PCR primers consisting of DNA of minimum 15 bases and maximum 30 bases containing base sequences of base numbers 8 to 22 in SEQ ID NO: 31 of the sequence listing on the 3' end side are used as sense primers. A primer set is proposed in which a PCR primer consisting of a minimum of 15 bases and a maximum of 30 bases of DNA containing the nucleotide sequence of nucleotide numbers 4 to 18 in SEQ ID NO: 32 in the sequence list is used as an antisense primer. This primer set can be suitably used for shea nut detection.

According to the present invention, as the 17th primer set, PCR primers consisting of DNA of minimum 15 bases and maximum 30 bases containing base sequences of base numbers 6 to 20 in SEQ ID NO: 33 of the sequence listing on the 3' end side are used as sense primers. A primer set is proposed in which a PCR primer consisting of a minimum of 15 bases and a maximum of 30 bases of DNA containing the base sequence of base numbers 6 to 20 in SEQ ID NO: 34 in the sequence list on the 3' end side is used as an antisense primer. This primer set can be suitably used for walnut detection.

In each of the above primers, the length of the base sequence is 15 to 30 because the appropriate length of the base sequence as a PCR primer is about 15 to 30, and the 15 bases on the 3' end side About 15 nucleotides on the 3' end side are important for the specific amplification reaction of PCR to specify the sequence, and the nucleotide sequence on the 5' end side is slightly different, and the length of the sequence is slightly different. This is because, in most cases, the adverse effect on the PCR reaction itself is small.

In the present invention, a preferred embodiment of the first primer set is a PCR primer (1') consisting of a maximum of 30 bases of DNA containing the base sequence of SEQ ID NO: 1 in the sequence listing on the 3' end side (most preferably is a primer consisting of the DNA of the nucleotide sequence of SEQ ID NO: 1) as a sense primer, and a PCR primer (2') (most preferably proposes a primer set in which the primer consisting of the DNA of the nucleotide sequence of SEQ ID NO: 2 is used as an antisense primer. This primer set can be suitably used for detecting almonds, and it is most preferable to use the primer of SEQ ID NO: 1 and the primer of SEQ ID NO: 2 as a set. A specific example of (1′) is the primer of SEQ ID NO: 35, and a specific example of (2′) is the primer of SEQ ID NO: 36.

In the present invention, a preferred embodiment of the second primer set is a PCR primer (3') consisting of DNA of up to 30 bases containing the nucleotide sequence of SEQ ID NO: 3 in the sequence listing on the 3' end side (most preferably is a primer consisting of DNA of the nucleotide sequence of SEQ ID NO: 3) as a sense primer, and a PCR primer (4') (most preferably proposes a primer set in which the primer consisting of the DNA of the base sequence of SEQ ID NO: 4 is used as an antisense primer. This primer set can be suitably used for detecting beach nuts, and it is most preferable to use the primer of SEQ ID NO: 3 and the primer of SEQ ID NO: 4 as a set. A specific example of (3') is the primer of SEQ ID NO: 37, and a specific example of (4') is the primer of SEQ ID NO: 38.

In the present invention, a preferred embodiment of the third primer set is a PCR primer (5') consisting of DNA of up to 30 bases containing the nucleotide sequence of SEQ ID NO: 5 in the sequence listing on the 3' end side (most preferably is a primer consisting of DNA of the nucleotide sequence of SEQ ID NO: 5) as a sense primer, and a PCR primer (6') (most preferably proposes a primer set in which the primer consisting of the DNA of the base sequence of SEQ ID NO: 6) is used as an antisense primer. This primer set can be suitably used for detecting Brazil nuts, and it is most preferable to use the primer of SEQ ID NO: 5 and the primer of SEQ ID NO: 6 as a set. A specific example of (5') is the primer of SEQ ID NO: 39, and a specific example of (6') is the primer of SEQ ID NO: 40.

In the present invention, a preferred embodiment of the fourth primer set is a PCR primer (7') consisting of a maximum of 30 bases of DNA containing the nucleotide sequence of SEQ ID NO: 7 in the sequence listing on the 3' end side (most preferably is a primer consisting of DNA of the nucleotide sequence of SEQ ID NO: 7) as a sense primer, and a PCR primer (8') (most preferably proposes a primer set in which the primer consisting of the DNA of the nucleotide sequence of SEQ ID NO: 8 is used as an antisense primer. This primer set can be suitably used for cashew nut detection, and it is most preferable to use the primer of SEQ ID NO: 7 and the primer of SEQ ID NO: 8 as a set. A specific example of (7') is the primer of SEQ ID NO: 41, and a specific example of (8') is the primer of SEQ ID NO: 42.

In the present invention, a preferred embodiment of the fifth primer set is a PCR primer (9') consisting of a maximum of 30 nucleotides of DNA containing the nucleotide sequence of SEQ ID NO: 9 in the sequence listing on the 3' end side (most preferably is a primer consisting of the DNA of the nucleotide sequence of SEQ ID NO: 9) as a sense primer, and a PCR primer (10') (most preferably proposes a primer set in which the primer consisting of the DNA of the base sequence of SEQ ID NO: 10) is used as an antisense primer. This primer set can be suitably used for chestnut detection, and it is most preferable to use the primer of SEQ ID NO: 9 and the primer of SEQ ID NO: 10 as a set. A specific example of (9′) is the primer of SEQ ID NO: 43, and a specific example of (10′) is the primer of SEQ ID NO: 44.

In the present invention, a preferred embodiment of the sixth primer set is a PCR primer (11') consisting of a maximum of 30 bases of DNA containing the nucleotide sequence of SEQ ID NO: 11 in the sequence listing on the 3' end side (most preferably is a primer consisting of the DNA of the nucleotide sequence of SEQ ID NO: 11) as a sense primer, and a PCR primer (12') consisting of DNA of up to 30 nucleotides containing the nucleotide sequence of SEQ ID NO: 12 in the sequence listing on the 3' end side (most preferably proposes a primer set in which the primer consisting of the DNA of the base sequence of SEQ ID NO: 12) is used as an antisense primer. This primer set can be suitably used for coconut detection, and it is most preferable to use the primer of SEQ ID NO: 11 and the primer of SEQ ID NO: 12 as a set. A specific example of (11′) is the primer of SEQ ID NO: 45, and a specific example of (12′) is the primer of SEQ ID NO: 46.

In the present invention, a preferred embodiment of the seventh primer set is a PCR primer (13') consisting of DNA of up to 30 bases containing the nucleotide sequence of SEQ ID NO: 13 in the sequence listing on the 3' end side (most preferably is a primer consisting of the DNA of the nucleotide sequence of SEQ ID NO: 13) as a sense primer, and a PCR primer (14') consisting of DNA of up to 30 nucleotides containing the nucleotide sequence of SEQ ID NO: 14 in the sequence listing on the 3' end side (most preferably proposes a primer set in which the primer consisting of the DNA of the nucleotide sequence of SEQ ID NO: 14 is used as an antisense primer. This primer set can be suitably used for detecting ginkgo biloba, and it is most preferable to use the primer of SEQ ID NO: 13 and the primer of SEQ ID NO: 14 as a set. A specific example of (13') is the primer of SEQ ID NO: 47, and a specific example of (14') is the primer of SEQ ID NO: 48.

In the present invention, a preferred embodiment of the eighth primer set is a PCR primer (15') consisting of a maximum of 30 bases of DNA containing the nucleotide sequence of SEQ ID NO: 15 in the sequence listing at the 3' end (most preferably is a primer consisting of DNA of the nucleotide sequence of SEQ ID NO: 15) as a sense primer, and a PCR primer (16') (most preferably proposes a primer set in which the primer consisting of the DNA of the base sequence of SEQ ID NO: 16) is used as an antisense primer. This primer set can be suitably used for detecting hazelnuts, and it is most preferable to use the primer of SEQ ID NO: 15 and the primer of SEQ ID NO: 16 as a set. A specific example of (15') is the primer of SEQ ID NO: 49, and a specific example of (16') is the primer of SEQ ID NO: 50.

In the present invention, a further preferred embodiment of the ninth primer set is a PCR primer (17') consisting of a maximum of 30 bases of DNA containing the base sequence of SEQ ID NO: 17 in the sequence listing on the 3' end side (most preferably is a primer consisting of DNA of the nucleotide sequence of SEQ ID NO: 17) as a sense primer, and a PCR primer (18') (most preferably proposes a primer set in which the primer consisting of the DNA of the nucleotide sequence of SEQ ID NO: 18 is used as an antisense primer. This primer set can be suitably used for hickory nut detection, and it is most preferable to use the primer of SEQ ID NO: 17 and the primer of SEQ ID NO: 18 as a set. A specific example of (17') is the primer of SEQ ID NO: 51, and a specific example of (18') is the primer of SEQ ID NO: 52.

In the present invention, a preferred embodiment of the tenth primer set is a PCR primer (19') (most preferably is a primer consisting of the DNA of the base sequence of SEQ ID NO: 19) as a sense primer, and a PCR primer (20') consisting of a maximum of 30 bases of DNA containing the base sequence of SEQ ID NO: 20 in the sequence listing at the 3' end (most preferably proposes a primer set in which the primer consisting of the DNA of the base sequence of SEQ ID NO: 20) is used as an antisense primer. This primer set can be suitably used for litchi detection, and it is most preferable to use the primer of SEQ ID NO: 19 and the primer of SEQ ID NO: 20 as a set. A specific example of (19') is the primer of SEQ ID NO: 53, and a specific example of (20') is the primer of SEQ ID NO: 54.

In the present invention, a further preferred embodiment of the eleventh primer set is a PCR primer (21') consisting of DNA of up to 30 bases containing the nucleotide sequence of SEQ ID NO: 21 in the sequence listing on the 3' end side (most preferably is a primer consisting of DNA of the nucleotide sequence of SEQ ID NO: 21) as a sense primer, and a PCR primer (22') (most preferably proposes a primer set in which the primer consisting of the DNA of the base sequence of SEQ ID NO: 22) is used as an antisense primer. This primer set can be suitably used for detecting macadamia nuts, and it is most preferable to use the primer of SEQ ID NO: 21 and the primer of SEQ ID NO: 22 as a set. A specific example of (21′) is the primer of SEQ ID NO:55, and a specific example of (22′) is the primer of SEQ ID NO:56.

In the present invention, a preferred embodiment of the twelfth primer set is a PCR primer (23') consisting of DNA of up to 30 bases containing the nucleotide sequence of SEQ ID NO: 23 in the sequence listing on the 3' end side (most preferably is a primer consisting of DNA of the nucleotide sequence of SEQ ID NO: 23) as a sense primer, and a PCR primer (24') (most preferably proposes a primer set in which the primer consisting of the DNA of the base sequence of SEQ ID NO: 24 is used as an antisense primer. This primer set can be suitably used for detecting pecan nuts, and it is most preferable to use the primer of SEQ ID NO: 23 and the primer of SEQ ID NO: 24 as a set. A specific example of (23') is the primer of SEQ ID NO: 57, and a specific example of (24') is the primer of SEQ ID NO: 58.

In the present invention, a preferred embodiment of the thirteenth primer set is a PCR primer (25') consisting of a maximum of 30 bases of DNA containing the base sequence of SEQ ID NO: 25 in the sequence listing on the 3' end side (most preferably is a primer consisting of DNA of the nucleotide sequence of SEQ ID NO: 25) as a sense primer, and a PCR primer (26') (most preferably proposes a primer set in which the primer consisting of the DNA of the base sequence of SEQ ID NO: 26) is used as an antisense primer. This primer set can be suitably used for detecting pine nuts, and it is most preferable to use the primer of SEQ ID NO: 25 and the primer of SEQ ID NO: 26 as a set. A specific example of (25') is the primer of SEQ ID NO: 59, and a specific example of (26') is the primer of SEQ ID NO: 60.

In the present invention, a further preferred embodiment of the fourteenth primer set is a PCR primer (27') consisting of a maximum of 30 bases of DNA containing the base sequence of SEQ ID NO: 27 in the sequence listing on the 3' end side (most preferably is a primer consisting of DNA of the nucleotide sequence of SEQ ID NO: 27) as a sense primer, and a PCR primer (28') (most preferably proposes a primer set in which the primer consisting of the DNA of the base sequence of SEQ ID NO: 28 is used as an antisense primer. This primer set can be suitably used for detecting Pili nuts, and it is most preferable to use the primer of SEQ ID NO: 27 and the primer of SEQ ID NO: 28 as a set. A specific example of (27') is the primer of SEQ ID NO: 61, and a specific example of (28') is the primer of SEQ ID NO: 62.

In the present invention, a further preferred embodiment of the fifteenth primer set is a PCR primer (29') consisting of a maximum of 30 nucleotides of DNA containing the nucleotide sequence of SEQ ID NO: 29 in the sequence listing on the 3' end side (most preferably is a primer consisting of DNA of the nucleotide sequence of SEQ ID NO: 29) as a sense primer, and a PCR primer (30') (most preferably proposes a primer set in which the primer consisting of the DNA of the base sequence of SEQ ID NO: 30) is used as an antisense primer. This primer set can be suitably used for detecting pistachio, and it is most preferable to use the primer of SEQ ID NO: 29 and the primer of SEQ ID NO: 30 as a set. A specific example of (29') is the primer of SEQ ID NO: 63, and a specific example of (30') is the primer of SEQ ID NO: 64.

In the present invention, a further preferred embodiment of the 16th primer set is a PCR primer (31') consisting of DNA of up to 30 nucleotides containing the nucleotide sequence of SEQ ID NO: 31 in the sequence listing on the 3' end side (most preferably is a primer consisting of the DNA of the base sequence of SEQ ID NO: 31) as a sense primer, and a PCR primer (32') consisting of a maximum of 30 bases of DNA containing the base sequence of SEQ ID NO: 32 in the sequence listing at the 3' end (most preferably proposes a primer set in which the primer consisting of the DNA of the base sequence of SEQ ID NO: 32) is used as an antisense primer. This primer set can be suitably used for detecting shea nut, and it is most preferable to use the primer of SEQ ID NO: 31 and the primer of SEQ ID NO: 32 as a set. A specific example of (31′) is the primer of SEQ ID NO: 65, and a specific example of (32′) is the primer of SEQ ID NO: 66.

In the present invention, a further preferred embodiment of the 17th primer set is a PCR primer (33') consisting of a maximum of 30 nucleotides of DNA containing the nucleotide sequence of SEQ ID NO: 33 in the sequence listing on the 3' end side (most preferably is a primer consisting of DNA of the nucleotide sequence of SEQ ID NO: 33) as a sense primer, and a PCR primer (34') (most preferably proposes a primer set in which the primer consisting of the DNA of the base sequence of SEQ ID NO: 34) is used as an antisense primer. This primer set can be suitably used for detecting walnuts, and it is most preferable to use the primer of SEQ ID NO: 33 and the primer of SEQ ID NO: 34 as a set. A specific example of (33') is the primer of SEQ ID NO: 67, and a specific example of (34') is the primer of SEQ ID NO: 68.

Nut detection method The nut detection method of the present invention includes the steps of extracting DNA from a sample, performing PCR using the DNA as a template and the primer set of the present invention, and detecting the amplified DNA. Nuts (almonds, beach nuts, Brazil nuts, cashew nuts, chestnuts, coconuts, ginkgo nuts, hazelnuts, hickory nuts, lychees, macadamia nuts, pecan nuts, pine nuts, pili nuts, pistachios, shea nuts, walnuts) and detecting whether it is present.

That is, it is possible to specifically detect nuts in a sample by nucleic acid analysis of DNA in the sample by PCR or the like using the PCR primer set for detecting nuts of the present invention. At this time, when detecting almonds, a PCR primer set for almond detection is used, and beach nuts, Brazil nuts, cashew nuts, chestnuts, coconuts, ginkgo nuts, hazelnuts, hickory nuts, lychees, macadamia nuts, pecan nuts, When detecting pine nuts, pili nuts, pistachios, shea nuts, and walnuts, use a PCR primer set for beach nut detection, a PCR primer set for Brazil nut detection, a PCR primer set for cashew nut detection, a PCR primer set for chestnut detection, and a coconut. PCR primer set for detection, PCR primer set for detection of ginkgo nuts, PCR primer set for detection of hazelnuts, PCR primer set for detection of hickory nuts, PCR primer set for detection of lychee, PCR primer set for detection of macadamia nuts, PCR primer set for detection of pecan nuts , a set of PCR primers for detecting pine nuts, a set of PCR primers for detecting pili nuts, a set of PCR primers for detecting pistachios, a set of PCR primers for detecting shea nuts, and a set of PCR primers for detecting walnuts.

The type of detection method (nucleic acid analysis) is not particularly limited, and known methods can be used as appropriate. For example, a method of PCR amplification using PCR primers, a method of detecting a PCR amplification product with a probe, and the like can be exemplified.

As a method for PCR amplification using PCR primers, a target base sequence in DNA in a sample is selectively amplified using the above-described primer set for detecting nuts of the present invention selected according to the purpose of detection, and a PCR amplification product is obtained. A method of measuring the presence or absence of The presence or absence of PCR amplification products can usually be confirmed by separating the PCR amplification products by agarose gel electrophoresis and staining them with nucleic acid such as ethidium bromide or SYBR Green I. When PCR is performed using a real-time PCR device, the presence or absence of PCR amplification products can be automatically confirmed by the detection system of the device. For example, when Cybergreen I is added to the PCR reaction solution, the amount of PCR amplification product depending on the amount of fluorescence emitted when Cybergreen I binds to double-stranded DNA can be monitored in each cycle. When PCR amplification is observed by the amount of fluorescence, the presence and size of the amplified product can be confirmed by the aforementioned agarose gel electrophoresis.

Specifically, the size of the PCR amplification product using the primers of SEQ ID NO: 1 and SEQ ID NO: 2 is about 515 bp. Detection of an amplification product of this size in agarose electrophoresis suggests the presence of almonds in the test sample.

Specifically, the size of the PCR amplification product using the primers of SEQ ID NO: 3 and SEQ ID NO: 4 is about 75 bp. Detection of an amplification product of this size in agarose electrophoresis suggests the presence of beach nuts in the test sample.

Specifically, the size of the PCR amplification product using the primers of SEQ ID NO: 5 and SEQ ID NO: 6 is about 91 bp. Detection of an amplification product of this size in agarose electrophoresis suggests the presence of Brazil nuts in the test sample.

Specifically, the size of the PCR amplification product using the primers of SEQ ID NO: 7 and SEQ ID NO: 8 is about 102 bp. Detection of an amplification product of this size in agarose electrophoresis suggests the presence of cashew nuts in the test sample.

Specifically, the size of the PCR amplification product using the primers of SEQ ID NO: 9 and SEQ ID NO: 10 is about 293 bp. Detection of an amplification product of this size in agarose electrophoresis suggests the presence of chestnuts in the test sample.

Specifically, the size of the PCR amplification product using the primers of SEQ ID NO: 11 and SEQ ID NO: 12 is about 224 bp. Detection of an amplification product of this size in agarose electrophoresis suggests the presence of coconut in the test sample.

Specifically, the size of the PCR amplification product using the primers of SEQ ID NO: 13 and SEQ ID NO: 14 is about 186 bp. Detection of an amplification product of this size in agarose electrophoresis suggests the presence of ginkgo in the test sample.

Specifically, the size of the PCR amplification product using the primers of SEQ ID NO: 15 and SEQ ID NO: 16 is about 361 bp. Detection of an amplification product of this size in agarose electrophoresis suggests the presence of hazelnuts in the test sample.

Specifically, the size of the PCR amplification product using the primers of SEQ ID NO: 17 and SEQ ID NO: 18 is about 543 bp. Detection of an amplification product of this size in agarose electrophoresis suggests the presence of hickory nuts in the test sample.

Specifically, the size of the PCR amplification product using the primers of SEQ ID NO: 19 and SEQ ID NO: 20 is about 162 bp. Detection of an amplification product of this size in agarose electrophoresis suggests the presence of lychee in the test sample.

Specifically, the size of the PCR amplification product using the primers of SEQ ID NO: 21 and SEQ ID NO: 22 is about 111 bp. Detection of an amplification product of this size in agarose electrophoresis suggests the presence of macadamia nuts in the test sample.

Specifically, the size of the PCR amplification product using the primers of SEQ ID NO: 23 and SEQ ID NO: 24 is about 468 bp. Detection of an amplification product of this size in agarose electrophoresis suggests the presence of pecan nuts in the test sample.
Since pecan nuts are a type of hickory nut, pecan nuts can also be detected using the aforementioned detection primer for hickory nuts.

Specifically, the size of the PCR amplification product using the primers of SEQ ID NO: 25 and SEQ ID NO: 26 is about 139 bp. Detection of an amplification product of this size in agarose electrophoresis suggests the presence of pine nuts in the test sample.

Specifically, the size of the PCR amplification product using the primers of SEQ ID NO: 27 and SEQ ID NO: 28 is about 83 bp. Detection of an amplification product of this size in agarose electrophoresis suggests the presence of pili nuts in the test sample.

Specifically, the size of the PCR amplification product using the primers of SEQ ID NO: 29 and SEQ ID NO: 30 is about 163 bp. Detection of an amplification product of this size in agarose electrophoresis suggests the presence of pistachios in the test sample.

Specifically, the size of the PCR amplification product using the primers of SEQ ID NO: 31 and SEQ ID NO: 32 is about 518 bp. Detection of an amplification product of this size in agarose electrophoresis suggests the presence of shea nuts in the test sample.

Specifically, the size of the PCR amplification product using the primers of SEQ ID NO: 33 and SEQ ID NO: 34 is about 501 bp. Detection of an amplification product of this size in agarose electrophoresis suggests the presence of walnuts in the test sample.

Furthermore, among the above primer sets, specific combinations of four of the eight main types (almonds, brazil nuts, cashew nuts, hazelnuts, macadamia nuts, pecan nuts, pistachios, and walnuts) can be detected by the multiplex method. be. Specific combinations are as follows.
Multiplex PCR refers to a method of simultaneously amplifying multiple gene regions by simultaneously using multiple primers in one PCR reaction system. By performing multiplex PCR, it is possible to save PCR reagents, avoid the complexity of PCR experimental work by simultaneous detection, and have the advantage of saving and effectively using template DNA to be detected.

(1) For the four types of pecan nuts, walnuts, pistachios, and cashew nuts, the primer described in SEQ ID NO: 17 and the primer described in SEQ ID NO: 18 (for hickory nuts), the primer described in SEQ ID NO: 33 and the primer described in SEQ ID NO: 34 ( walnut), the primer of SEQ ID NO: 29 and the primer of SEQ ID NO: 30 (for pistachios), and the primer of SEQ ID NO: 7 and the primer of SEQ ID NO: 8 (for cashew nuts) were all mixed. By subjecting to the test (multiplex), PCR amplification product of pecan nuts (approximately 543 bp), PCR amplification product of walnuts (approximately 501 bp), PCR amplification product of pistachio (approximately 163 bp) and PCR amplification product of cashew nuts (approximately 102 bp) can be detected at the same time, and the detection of an amplification product of either of these sizes suggests the presence of the nuts in the test sample.

Since pecan nuts are a type of hickory nut, pecan nuts can also be detected using the aforementioned detection primer for hickory nuts. In this multiplex PCR, detection primers for hickory nuts are used for detection of pecan nuts.
Also, for example, detection of amplification products of all these sizes suggests that both nuts were present in the test sample.
In addition, when an amplification product of a nut to be detected is detected by multiplexing, it is preferable to perform a confirmation test again using individual primers as a secondary test.

In the case of PCR in this multiplex, the ratio of the primer concentration of each primer for pecan nuts, walnuts, pistachios, and cashew nuts is not particularly limited, but preferably, pecan nuts Regarding the detection primers, it is preferable to use the other three types (walnut, pistachio, and cashew nut) at a ratio of 0.9 or less to 1, respectively. More preferably, it is used at a ratio of 0.1 to 0.7. Furthermore, it is most preferably used in a ratio of 0.2 to 0.4.
By setting such a ratio, it is possible to suitably detect any of the four types.

(2) For four types of almonds, hazelnuts, macadamia nuts, and Brazil nuts, the primers described in SEQ ID NO: 1 and the primers described in SEQ ID NO: 2 (for almonds), the primers described in SEQ ID NO: 15 and the primers described in SEQ ID NO: 16 ( hazelnut), the primer of SEQ ID NO: 21 and the primer of SEQ ID NO: 22 (for macadamia nuts), and the primer of SEQ ID NO: 5 and the primer of SEQ ID NO: 6 (for Brazil nuts) are all mixed By subjecting to PCR test (multiplex), almond PCR amplification product (about 515bp), hazelnut PCR amplification product (about 361bp), macadamia nut PCR amplification product (about 111bp) and Brazil nut PCR amplification product ( 91 bp) can be detected at the same time, and the detection of amplification products of either of these sizes suggests the presence of the nut in the test sample.

Also, for example, detection of amplification products of all these sizes suggests that both nuts were present in the test sample.
In addition, when an amplification product of a nut to be detected is detected by multiplexing, it is preferable to perform a confirmation test again using individual primers as a secondary test.

In the case of PCR in this multiplex, the ratio of the primer concentration of each primer for almond, hazelnut, macadamia nut and Brazil nut is not particularly limited, but preferably almond Regarding the detection primers, it is preferable to use two or more of the other three types (hazelnuts, macadamia nuts, and brazil nuts) at a ratio of one to one. More preferably, it is used at a ratio of 4 to 8. Furthermore, it is most preferred to use a ratio of 5 to 7.

By setting such a ratio, it is possible to suitably detect any of the four types. Furthermore, by conducting both tests (1) and (2), it is possible to detect all eight major species (almonds, brazil nuts, cashew nuts, hazelnuts, macadamia nuts, pecan nuts, pistachios, and walnuts). is.
In this way, it is also possible to provide a method that enables comprehensive detection of all of the above eight types by performing (1) and (2) four times twice.

(3) For the four types of pecan nuts, walnuts, hazelnuts, and cashew nuts, the primer described in SEQ ID NO: 17 and the primer described in SEQ ID NO: 18 (for hickory nuts), the primer described in SEQ ID NO: 33 and the primer described in SEQ ID NO: 34 ( walnuts), the primer of SEQ ID NO: 15 and the primer of SEQ ID NO: 16 (for hazelnuts), and the primer of SEQ ID NO: 7 and the primer of SEQ ID NO: 8 (for cashew nuts) were mixed. By subjecting it to the test (multiplex), the pecan nut PCR amplification product (approximately 543bp), walnut PCR amplification product (approximately 501bp), hazelnut PCR amplification product (approximately 361bp) and cashew nut amplification product (approximately 102bp) were obtained. Detection of amplification products of either of these sizes, which can be detected simultaneously, suggests the presence of the nut in the test sample.

Since pecan nuts are a type of hickory nut, pecan nuts can also be detected with the above-mentioned detection primer for hickory nuts. use.

Also, for example, detection of amplification products of all these sizes suggests that both nuts were present in the test sample.
In addition, when an amplification product of a nut to be detected is detected by multiplexing, it is preferable to perform a confirmation test again using individual primers as a secondary test.

In the case of PCR in this multiplex, the primer concentration ratio of each primer for pecan nuts, walnuts, hazelnuts, and cashew nuts is not particularly limited, but preferably, pecan nuts Regarding the detection primers, it is preferable to use the other three species (walnut, hazelnut, cashew nut) at a ratio of 0.9 or less to 1 each. More preferably, it is used at a ratio of 0.1 to 0.7. Furthermore, it is most preferably used in a ratio of 0.2 to 0.4.
By setting such a ratio, it is possible to suitably detect any of the four types.

(4) For four types of almonds, pistachios, macadamia nuts, and Brazil nuts, the primers described in SEQ ID NO: 1 and the primers described in SEQ ID NO: 2 (for almonds), the primers described in SEQ ID NO: 29 and the primers described in SEQ ID NO: 30 ( pistachio), the primer of SEQ ID NO: 21 and the primer of SEQ ID NO: 22 (for macadamia nuts), and the primer of SEQ ID NO: 5 and the primer of SEQ ID NO: 6 (for Brazil nuts) are mixed. By subjecting to PCR test (multiplex), almond PCR amplification product (about 515bp), pistachio PCR amplification product (about 163bp), macadamia nut PCR amplification product (about 111bp) and Brazil nut PCR amplification product ( 91 bp) can be detected at the same time, and the detection of amplification products of either of these sizes suggests the presence of the nut in the test sample.

Also, for example, detection of amplification products of all these sizes suggests that both nuts were present in the test sample. In addition, when an amplification product of a nut to be detected is detected by multiplexing, it is preferable to perform a confirmation test again using individual primers as a secondary test.

In the case of PCR in this multiplex, the ratio of the primer concentration of each primer for almond, pistachio, macadamia nut and Brazil nut is not particularly limited, but preferably almond As for the detection primers, it is preferable to use two or more of the other three types (pistachio, macadamia nut, and brazil nut) at a ratio of one to one. More preferably, it is used at a ratio of 4 to 8. Furthermore, it is most preferred to use a ratio of 5 to 7.
By setting such a ratio, it is possible to suitably detect any of the four types.

Furthermore, by conducting both tests (3) and (4), it is possible to detect all eight major species (almonds, brazil nuts, cashew nuts, hazelnuts, macadamia nuts, pecan nuts, pistachios, and walnuts). is.
In this way, it is also possible to provide a method that makes it possible to exhaustively detect all of the above eight types by carrying out (3) and (4) four times twice.

(5) For the four types of pecan nuts, walnuts, macadamia nuts, and Brazil nuts, the primer described in SEQ ID NO: 17 and the primer described in SEQ ID NO: 18 (for hickory nuts), the primer described in SEQ ID NO: 33 and the primer described in SEQ ID NO: 34 All the primers of the primer (for walnuts), the primer set forth in SEQ ID NO: 21 and the primer set forth in SEQ ID NO: 22 (for macadamia nuts), and the primer set forth in SEQ ID NO: 5 and the primer set forth in SEQ ID NO: 6 (for Brazil nuts) were mixed. By subjecting it to a PCR test (multiplex), the PCR amplification product of pecan nuts (about 543 bp), the PCR amplification product of walnuts (about 501 bp), the PCR amplification product of macadamia nuts (about 111 bp) and the PCR of Brazil nuts Amplification products (approximately 91 bp) can be detected simultaneously, and detection of amplification products of either of these sizes suggests the presence of the nut in the test sample.

Since pecan nuts are a type of hickory nut, pecan nuts can also be detected with the above-mentioned detection primer for hickory nuts. use. Also, for example, detection of amplification products of all these sizes suggests that both nuts were present in the test sample.

In addition, when an amplification product of a nut to be detected is detected by multiplexing, it is preferable to perform a confirmation test again using individual primers as a secondary test.
In the case of PCR in this multiplex, the primer concentration ratio of each primer for pecan nuts, walnuts, macadamia nuts, and Brazil nuts is not particularly limited, but preferably, As for the primers for detecting pecan nuts, it is preferable to use a ratio of 0.9 or less for each of the other three types (walnuts, macadamia nuts, and Brazil nuts) at a ratio of 1. More preferably, it is used at a ratio of 0.1 to 0.7. Furthermore, it is most preferably used in a ratio of 0.2 to 0.4.
By setting such a ratio, it is possible to suitably detect any of the four types.

(6) For the four types of almonds, hazelnuts, pistachios, and cashew nuts, the primer described in SEQ ID NO: 1 and the primer described in SEQ ID NO: 2 (for almonds), the primer described in SEQ ID NO: 15 and the primer described in SEQ ID NO: 16 (for hazelnuts ), the primer described in SEQ ID NO: 29 and the primer described in SEQ ID NO: 30 (for pistachios), and the primer described in SEQ ID NO: 7 and the primer described in SEQ ID NO: 8 (for cashew nuts) were all mixed in the PCR test. By providing (multiplex), the almond PCR amplification product (about 515 bp), the hazelnut PCR amplification product (about 361 bp), the pistachio PCR amplification product (about 163 bp) and the cashew nut amplification product (about 102 bp) are simultaneously detected. Detecting an amplification product of either of these sizes suggests the presence of the nut in the test sample.

Also, for example, detection of amplification products of all these sizes suggests that both nuts were present in the test sample.
In addition, when an amplification product of a nut to be detected is detected by multiplexing, it is preferable to perform a confirmation test again using individual primers as a secondary test.
In the case of PCR in this multiplex, the primer concentration ratio of each primer for almond, hazelnut, pistachio and cashew nut is not particularly limited, but preferably for almond detection It is preferable to use 2 or more of the other three primers (hazelnut, pistachio, and cashew nut) at a ratio of 1 to 1, respectively. More preferably, it is used at a ratio of 4 to 8. Furthermore, it is most preferred to use a ratio of 5 to 7.

By setting such a ratio, it is possible to suitably detect any of the four types. Furthermore, by conducting both tests (5) and (6), it is possible to detect all eight major species (almonds, brazil nuts, cashew nuts, hazelnuts, macadamia nuts, pecan nuts, pistachios, and walnuts). is.
In this way, it is also possible to provide a method that enables exhaustive detection of all of the above eight types by carrying out (5) and (6) four times twice.

As a method for detecting a PCR amplification product with a probe, as typified by the TaqMan method, a fluorescent substance-labeled probe that hybridizes with the internal nucleotide sequence of the PCR amplification product is added to the reaction solution, and the probe is detected by PCR. A method of confirming the presence or absence of a PCR amplification product by automatically detecting the amount of fluorescence generated when the probe is degraded after hybridizing to the amplification product with a real-time PCR device is included. Other methods for detecting PCR amplification products with probes include the Molecular Beacon method, the CycleavePCR method, and methods using hybrid probes. Any of the PCR primer sets for detecting nuts of the present invention may be used as the PCR primers used in these methods. A common nucleotide sequence region may be separately selected and probes based on that region may be used.

In the method for detecting nuts of the present invention, the type of target test sample is not particularly limited. For example, test samples include food raw materials and processed foods. Examples of food raw materials include food raw materials that do not intentionally contain nuts that are separately produced in a food raw material production factory that handles nuts. Processed foods include confectionery, noodles, powdered soups, liquid soups, hot-air dried or freeze-dried ingredients, and various cooked foods containing these processed foods. Processed foods that do not intentionally contain nuts that are separately produced in food manufacturing factories that handle nuts are also included. In addition, after manufacturing processed food containing nuts, when manufacturing processed food not containing nuts, careful cleaning of food manufacturing equipment is essential, taking into consideration the removal of nut residues. From the viewpoint of confirming the effectiveness of this cleaning work method and the presence or absence of nut residues in the food manufacturing equipment, a wiping sample of the manufacturing equipment is also included as a test sample.

In addition, the form of the sample is not particularly limited, and a general known DNA extraction method (Notification of the Deputy Director-General of the Consumer Affairs Agency, Inspection methods for foods containing allergens, March 26, 2014, Shoshoku table No. 36) or various commercially available DNA extraction kits [for example, Nucleon PhytoPure, plant and fungal DNA extraction kits (GE Healthcare Biosciences Corp., USA), DNA Extraction IsoplantII kit (Nippon Gene Co. Ltd., Japan), DNeasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany)], any sample from which DNA can be recovered can be applied to the above detection method. Genomic DNA and organelle-derived DNA (mitochondrial DNA and chloroplast DNA) can usually be extracted from a sample by the DNA extraction method described above.

Nut detection kit The present invention can also be used as a nut detection kit. The configuration of the kit is not particularly limited as long as it comprises at least one set of PCR primer sets for detecting nuts of the present invention.
Specifically, a PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of nucleotide numbers 9 to 23 in SEQ ID NO: 1 on the 3' end side, and the nucleotide sequence of nucleotide numbers 5 to 19 in SEQ ID NO: 2 at 3' A set with a PCR primer consisting of DNA of up to 30 bases including the terminal side;
A PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of base numbers 7 to 21 in SEQ ID NO: 3 on the 3' end side and a maximum of DNA containing the base sequence of base numbers 6 to 20 in SEQ ID NO: 4 on the 3' end side A set with a PCR primer consisting of 30 bases of DNA;
A PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of nucleotide numbers 9 to 23 in SEQ ID NO: 5 on the 3' end side and a maximum of DNA containing the nucleotide sequence of nucleotide numbers 5 to 19 in SEQ ID NO: 6 on the 3' end side A set with a PCR primer consisting of 30 bases of DNA;
A PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of nucleotide numbers 6 to 20 in SEQ ID NO: 7 on the 3' end side and a maximum of DNA containing the nucleotide sequence of nucleotide numbers 5 to 19 in SEQ ID NO: 8 on the 3' end side A set with a PCR primer consisting of 30 bases of DNA;
A PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of nucleotide numbers 6 to 20 in SEQ ID NO: 9 on the 3' end side and a maximum of DNA containing the nucleotide sequence of nucleotide numbers 4 to 18 in SEQ ID NO: 10 on the 3' end side A set with a PCR primer consisting of 30 bases of DNA;
A PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of base numbers 4 to 18 in SEQ ID NO: 11 on the 3' end side and a maximum of DNA containing the base sequence of base numbers 4 to 18 in SEQ ID NO: 12 on the 3' end side A set with a PCR primer consisting of 30 bases of DNA;
A PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of nucleotide numbers 7 to 21 in SEQ ID NO: 13 on the 3' end side and a maximum of DNA containing the nucleotide sequence of nucleotide numbers 13 to 27 in SEQ ID NO: 14 on the 3' end side A set with a PCR primer consisting of 30 bases of DNA;
A PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of nucleotide numbers 7 to 21 in SEQ ID NO: 15 on the 3' end side and a maximum of DNA containing the nucleotide sequence of nucleotide numbers 11 to 25 in SEQ ID NO: 16 on the 3' end side A set with a PCR primer consisting of 30 bases of DNA;
A PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of nucleotide numbers 6 to 20 in SEQ ID NO: 17 on the 3' end side and a maximum of DNA containing the nucleotide sequence of nucleotide numbers 9 to 23 in SEQ ID NO: 18 on the 3' end side A set with a PCR primer consisting of 30 bases of DNA;
A PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of base numbers 7 to 21 in SEQ ID NO: 19 on the 3' end side and a maximum of DNA containing the base sequence of base numbers 6 to 20 in SEQ ID NO: 20 on the 3' end side A set with a PCR primer consisting of 30 bases of DNA;
A PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of nucleotide numbers 6 to 20 in SEQ ID NO: 21 on the 3' end side and a maximum of DNA containing the nucleotide sequence of nucleotide numbers 8 to 22 in SEQ ID NO: 22 on the 3' end side A set with a PCR primer consisting of 30 bases of DNA;
A PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of nucleotide numbers 4 to 18 in SEQ ID NO: 23 in the sequence listing on the 3' end side and the nucleotide sequence of nucleotide numbers 6 to 20 in SEQ ID NO: 24 on the 3' end side A set with PCR primers consisting of up to 30 bases of DNA contained in;
A PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of base numbers 5 to 19 in SEQ ID NO: 25 on the 3' end side and a maximum of DNA containing the base sequence of base numbers 8 to 22 in SEQ ID NO: 26 on the 3' end side A set with a PCR primer consisting of 30 bases of DNA;
A PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of base numbers 8 to 22 in SEQ ID NO: 27 on the 3' end side and a maximum of DNA containing the base sequence of base numbers 4 to 18 in SEQ ID NO: 28 on the 3' end side A set with a PCR primer consisting of 30 bases of DNA;
A PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of nucleotide numbers 10 to 24 in SEQ ID NO: 29 on the 3' end side and a maximum of DNA containing the nucleotide sequence of nucleotide numbers 15 to 29 in SEQ ID NO: 30 on the 3' end side A set with a PCR primer consisting of 30 bases of DNA;
A PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of base numbers 8 to 22 in SEQ ID NO: 31 on the 3' end side and a maximum of DNA containing the base sequence of base numbers 4 to 18 in SEQ ID NO: 32 on the 3' end side A set with a PCR primer consisting of 30 bases of DNA;
A PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of nucleotide numbers 6 to 20 in SEQ ID NO: 33 on the 3' end side and a maximum of DNA containing the nucleotide sequence of nucleotide numbers 6 to 20 in SEQ ID NO: 34 on the 3' end side A set of PCR primers consisting of 30 bases of DNA;

Preferably, a PCR primer consisting of a DNA of up to 30 bases containing the nucleotide sequence of SEQ ID NO: 1 on the 3' end side and a DNA of up to 30 bases containing the base sequence of SEQ ID NO: 2 on the 3' end side. set with PCR primers;
A set of a PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of SEQ ID NO: 3 on the 3′ end side and a PCR primer consisting of DNA of up to 30 bases containing the base sequence of SEQ ID NO: 4 on the 3′ end side;
A set of a PCR primer consisting of a maximum of 30 bases of DNA containing the nucleotide sequence of SEQ ID NO: 5 on the 3′ end side and a PCR primer consisting of a maximum of 30 bases of DNA containing the base sequence of SEQ ID NO: 6 on the 3′ end side;
A set of a PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of SEQ ID NO: 7 on the 3′ end side and a PCR primer consisting of DNA of up to 30 bases containing the base sequence of SEQ ID NO: 8 on the 3′ end side;
A set of a PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of SEQ ID NO: 9 on the 3′ end side and a PCR primer consisting of DNA of up to 30 bases containing the base sequence of SEQ ID NO: 10 on the 3′ end side;
A set of a PCR primer consisting of a maximum of 30 nucleotides of DNA containing the nucleotide sequence of SEQ ID NO: 11 on the 3′ end side and a PCR primer consisting of a maximum of 30 nucleotides of DNA containing the nucleotide sequence of SEQ ID NO: 12 on the 3′ end side;
A set of a PCR primer consisting of a maximum of 30 bases of DNA containing the nucleotide sequence of SEQ ID NO: 13 on the 3′ end side and a PCR primer consisting of a maximum of 30 bases of DNA containing the nucleotide sequence of SEQ ID NO: 14 on the 3′ end side;
A set of a PCR primer consisting of a maximum of 30 bases of DNA containing the nucleotide sequence of SEQ ID NO: 15 on the 3′ end side and a PCR primer consisting of a maximum of 30 bases of DNA containing the base sequence of SEQ ID NO: 16 on the 3′ end side;
A set of a PCR primer consisting of a maximum of 30 nucleotides of DNA containing the nucleotide sequence of SEQ ID NO: 17 on the 3′ end side and a PCR primer consisting of a maximum of 30 nucleotides of DNA containing the nucleotide sequence of SEQ ID NO: 18 on the 3′ end side;
A set of a PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of SEQ ID NO: 19 on the 3′ end side and a PCR primer consisting of DNA of up to 30 bases containing the base sequence of SEQ ID NO: 20 on the 3′ end side;
A set of a PCR primer consisting of a maximum of 30 nucleotides of DNA containing the nucleotide sequence of SEQ ID NO: 21 on the 3′ end side and a PCR primer consisting of a maximum of 30 nucleotides of DNA containing the nucleotide sequence of SEQ ID NO: 22 on the 3′ end side;
A PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of SEQ ID NO: 23 in the sequence listing on the 3' end side and a PCR primer consisting of DNA of up to 30 bases containing the base sequence of SEQ ID NO: 24 on the 3' end side a set of;
A set of a PCR primer consisting of a maximum of 30 nucleotides of DNA containing the nucleotide sequence of SEQ ID NO: 25 on the 3′ end side and a PCR primer consisting of a maximum of 30 nucleotides of DNA containing the nucleotide sequence of SEQ ID NO: 26 on the 3′ end side;
A set of a PCR primer consisting of a maximum of 30 nucleotides of DNA containing the nucleotide sequence of SEQ ID NO: 27 on the 3′ end side and a PCR primer consisting of a maximum of 30 nucleotides of DNA containing the nucleotide sequence of SEQ ID NO: 28 on the 3′ end side;
A set of a PCR primer consisting of a maximum of 30 bases of DNA containing the nucleotide sequence of SEQ ID NO: 29 on the 3′ end side and a PCR primer consisting of a maximum of 30 bases of DNA containing the base sequence of SEQ ID NO: 30 on the 3′ end side;
A set of a PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of SEQ ID NO: 31 on the 3′ end side and a PCR primer consisting of DNA of up to 30 bases containing the base sequence of SEQ ID NO: 32 on the 3′ end side;
A set of a PCR primer consisting of DNA of up to 30 bases containing the nucleotide sequence of SEQ ID NO: 33 on the 3′ end side and a PCR primer consisting of DNA of up to 30 bases containing the base sequence of SEQ ID NO: 34 on the 3′ end side Included are nut detection kits comprising one or more sets.

Among them, a set of a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 1 and a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 2 is particularly preferable;
A set of a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 3 and a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 4;
A set of a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 5 and a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 6;
A set of a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 7 and a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 8;
A set of a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 9 and a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 10;
A set of a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 11 and a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 12;
A set of a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 13 and a PCR primer consisting of SEQ ID NO: 14;
A set of a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 15 and a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 16;
A set of a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 17 and a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 18;
A set of a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 19 and a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 20;
A set of a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 21 and a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 22;
A set of a PCR primer consisting of the base sequence of SEQ ID NO: 23 in the sequence listing and a PCR primer consisting of the base sequence of SEQ ID NO: 24;
A set of a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 25 and a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 26;
A set of a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 27 and a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 28;
A set of a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 29 and a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 30;
A set of a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 31 and a PCR primer consisting of the nucleotide sequence of SEQ ID NO: 32;
Examples include a nut detection kit containing one or more sets of a PCR primer consisting of the nucleotide sequence of SEQ ID NO:33 and a PCR primer consisting of the nucleotide sequence of SEQ ID NO:34.
Also included are a nut detection kit containing each of the above primer sets and probes for detecting PCR amplification products amplified by each of the primer sets.

The kit can contain appropriate reagents, various containers, and the like, depending on the purpose of use. Specifically, polymerization reagents for synthesizing PCR primer extension products, e.g., thermostable DNA polymerase, deoxyribonucleotides, magnesium and PCR reaction buffers, and storage capable of appropriately maintaining their quality Containers and the like can be included.

EXAMPLES The present invention will be described in more detail below using examples, but the present invention should not be construed as being limited to these examples. Moreover, it is possible to make appropriate modifications without departing from the gist of the present invention.

Example 1
Confirmation of detection specificity of the PCR primer set for detecting nuts against DNA derived from various animals, plants, fungi, and bacteria In order to confirm the effectiveness of the PCR analysis method using the PCR primer set for detecting nuts of the present invention, various organisms Experiments to examine the detection specificity of PCR for derived DNA were performed as follows.

Purified human DNA was purchased from CeMines, LLC, USA.

Purified DNA of bovine, swine, chicken, white shrimp, king crab, cuttlefish, sockeye salmon, and chub mackerel was purchased from a store. After freezing the (muscle) fleshy part of each sample, it was crushed with a multi-bead shocker (Yasui Kikai, Osaka). Prepared from samples using Nucleon PhytoPure, plant and fungal DNA extraction kits (GE Healthcare Biosciences Corp., USA), DNA Extraction IsoplantII kit (Nippon Gene Co. Ltd., Japan), or DNeasy Blood&Tissue kit (Qiagen GmbH, Germany) I used what I had.

Pepper, Chinese yam, green onion, coconut, dates, acai, banana, macadamia nuts (Australia, Kenya), lotus root, peanuts, soybeans, almonds (US, Spain), peaches, prunes, plums, plums, apricots, cherries , apples, loquats, strawberries, pumpkins, pecan nuts, walnuts (from America and France), hazelnuts, pili nuts, cashew nuts (from India and Vietnam), pistachios (from Iran and America), mangoes, pink pepper, lychees, Rambutan, cabbage, Brazil nut, shea nut, miracle fruit, oyster, tea, kiwi fruit, blueberry, sesame, potato, carrot, ginkgo, and pine nut were purchased from a commercial store, and rice was obtained from a domestic grower. A portion of each sample (seed, pericarp, leaf, tuber) was washed with 70% ethanol and then quickly washed with TE buffer, and then washed with a multi-bead shocker (Yasui Kikai, Osaka). ) using Genomic-tip 20/G (Qiagen GmbH, Germany), DNeasy plant Mini kits (Qiagen GmbH, Germany), Nucleon PhytoPure, plant and fungal DNA extraction kits, or DNA Extraction IsoplantII kit I used what I had.

Buckwheat and wheat purified DNA was prepared from buckwheat flour and wheat flour purchased from a store using Genomic-tip 20/G.

Maize purified DNA was purchased from BioChain Institute, Inc., USA.

Purified DNA of hickory nuts (overta hickory) was prepared from leaves provided by the Botanical Garden, Graduate School of Science, The University of Tokyo using DNeasy Plant Mini Kit.

Purified DNA of chestnuts (Japanese chestnut, Chinese chestnut, European chestnut) was prepared from leaves provided by National Agriculture and Food Research Organization Fruit Tree Research Institute using DNeasy Plant Mini Kit.

Purified DNA of chestnut (chinkapin) was prepared using DNeasy Plant Mini Kit from leaves distributed from Tama Forest Science Garden, Forestry and Forest Products Research Institute.

Purified DNAs of beach nuts (beech, beech), sea bream, and acorn (white oak) were prepared from leaves obtained from nature using the DNeasy Plant Mini Kit.

Purified DNA of Pichia anomala IFO 0144 and Bacillus cereus ATCC11950 was obtained by culturing each strain in an appropriate medium, and then using the Puregene Yeast and Gram-Positive DNA Isolation kit ( Gentra Systems Inc., USA) was used.
All purified DNAs were subjected to removal of RNA by RNase.

SEQ. Each PCR primer described in 25, 26, 27, 28, 29, 30, 31, 32, 33, and 34 was synthesized by Eurofins Genomics and used in the following PCR.

After measuring the amounts of various animal, plant, fungal and bacterial DNA prepared as described above, the DNA concentration was adjusted to 10 ng/μL using sterilized water. A 20 μL volume reaction solution containing 5 μL of the prepared DNA sample solution was prepared using Takara Bio Inc.'s SYBR Premix Ex Taq (Tli RNaseH Plus) (Takara Bio Inc., Japan). This reagent contains TaKaRa Ex Taq HS (hot start type), dNTP Mixture, Mg ²⁺ and SYBR Green I in solution. A reaction solution was prepared based on SYBR Premix Ex Taq with a composition containing 250 nM sense primer and 250 nM antisense primer. The PCR reaction was performed with LightCycler (Roche Diagnostics GmbH, Germany), and the amplification reaction conditions were as follows.

After one cycle of 95°C for 1 minute, the temperature was raised to 95°C at a rate of 20°C/second, then maintained at the same temperature for 5 seconds, then cooled to 68°C at a rate of 20°C/second, and then the temperature was lowered to 68°C for 10 seconds. 35 amplification cycles consisting of 3 steps of maintaining the same temperature for 1 second, increasing the temperature to 72°C at a rate of 10°C/second, and maintaining the same temperature for 30 seconds were performed 35 times. PCR amplification products were recorded at the final step of each cycle as SYBR Green I dependent fluorescence levels. If amplification is confirmed by the amount of fluorescence, separate the PCR amplification product (5 μL of the reaction mixture) by 2% (wt/vol) agarose gel electrophoresis and extract the amplification product using Gel DOC EZ (Bio Rad). The presence or absence of amplification products was confirmed by visualization. OneMARK 100 (GeneDireX) was used as a molecular weight marker. A PCR reaction was determined to be positive when an amplified product of the target size was detected by electrophoresis. The results of these PCRs are listed in Table 1. + (plus) in the table indicates a positive PCR reaction, and - (minus) indicates a negative PCR reaction.

When using almond detection PCR primers (PCR primer set consisting of SEQ ID NOs: 1 and 2), an amplification product of around 515 bp was confirmed only when using almond (produced in the United States and Spain) DNA, and was determined to be positive. (table 1). Negative results were obtained when using the closely related peach DNA, prune DNA, plum DNA, plum DNA, apricot DNA, cherry DNA, and other species DNA, including other plants (Table 1).

In addition, when beach nut detection PCR primers (PCR primer set consisting of SEQ ID NOs: 3 and 4) were used, an amplified product of around 75 bp was confirmed only when beach nut (beech, dog beech) DNA was used, and was determined to be positive. (Table 1). Chestnut (Japanese chestnut, Chinese chestnut, European chestnut, Chinkapin) DNA, Shii DNA, acorn (white oak) DNA, pecan nut DNA, hickory nut DNA, walnut (American, French) DNA, hazelnut DNA, and Negative results were obtained when DNA from other species, including other plants, was used (Table 1).

In addition, when using the PCR primers for detecting Brazil nuts (PCR primer set consisting of SEQ ID NOS: 5 and 6), an amplification product of around 91 bp was confirmed only when using Brazil nut DNA, and was determined to be positive (Table 1 ). Negative results were obtained when using closely related shea nut DNA, miracle fruit DNA, persimmon DNA, tea DNA, kiwifruit DNA, blueberry DNA, and other species DNA, including other plants (Table 1). ).

In addition, when using cashew nut detection PCR primers (PCR primer set consisting of SEQ ID NOs: 7 and 8), an amplified product of around 102 bp was confirmed only when cashew nut (from India and Vietnam) DNA was used, and was determined to be positive. (Table 1). Negative results were obtained when DNA from closely related pistachio (Iranian, American), mango, pink pepper, and other species, including other plants, was used (Table 1).

In addition, when the chestnut detection PCR primers (PCR primer set consisting of SEQ ID NOs: 9 and 10) were used, an amplified product of around 293 bp was confirmed only when chestnut (Japanese chestnut, Chinese chestnut, European chestnut, Chinkapin) DNA was used. , were determined to be positive (Table 1). Contains closely related species: beach nut (beech, beech) DNA, sea bream DNA, acorn (white oak) DNA, pecan nut DNA, hickory nut DNA, walnut (American, French) DNA, hazelnut DNA, and other plant species It was negative when other species DNA was used (Table 1).

In addition, when coconut detection PCR primers (PCR primer set consisting of SEQ ID NOS: 11 and 12) were used, an amplified product of around 224 bp was confirmed only when coconut DNA was used, and was determined to be positive (Table 1). Negative results were obtained when the closely related date palm DNA, acai DNA, and other species DNA, including other plants, were used (Table 1).

In addition, when ginkgo detection PCR primers (PCR primer set consisting of SEQ ID NOS: 13 and 14) were used, an amplified product of around 186 bp was confirmed only when ginkgo biloba DNA was used, and was determined to be positive (Table 1). Negative results were obtained when DNA from other species, including other plants, was used (Table 1).

In addition, when the PCR primers for detecting hazelnuts (PCR primer set consisting of SEQ ID NOS: 15 and 16) were used, an amplified product of around 361 bp was confirmed only when hazelnut DNA was used, and was determined to be positive (Table 1). Pecan nut DNA, hickory nut DNA, walnut (American, French) DNA, chestnut (Japanese chestnut, Chinese gourd, European chestnut, Chinkapin) DNA, beach nut (beech, Japanese beech) DNA, sea bream DNA, acorn (Oak oak) DNA and other species DNA, including other plants, were negative (Table 1).

In addition, when hickory nut detection PCR primers (PCR primer set consisting of SEQ ID NOS: 17 and 18) were used, an amplification product of around 543 bp was confirmed only when hickory nut (overta hickory) DNA and pecan nut DNA were used. , were determined to be positive (Table 1). Walnut (American, French) DNA, chestnut (Japanese chestnut, Chinese nut, European chestnut, Chinkapin) DNA, beach nut (beech, Japanese beech) DNA, hazelnut DNA, sea bream DNA, acorn (white oak) DNA, and , was negative when DNA from other species including other plants was used (Table 1).

In addition, when PCR primers for detecting lychee (PCR primer set consisting of SEQ ID NOS: 19 and 20) were used, an amplified product of around 162 bp was confirmed only when lychee DNA was used, and was determined to be positive (Table 1). Negative results were obtained when DNA from closely related species, rambutan, and DNA from other species, including other plants, were used (Table 1).

In addition, when using PCR primers for detecting macadamia nuts (PCR primer set consisting of SEQ ID NOS: 21 and 22), an amplification product of around 111 bp was confirmed only when macadamia nut (Australian and Kenyan) DNA was used, and it was positive. (Table 1). It was negative when DNA from lotus root, a related species, and DNA from other species, including other plants, were used (Table 1).

In addition, when using pecan nut detection PCR primers (PCR primer set consisting of SEQ ID NOS: 23 and 24), an amplification product of around 468 bp was confirmed only when pecan nut DNA was used, and was determined to be positive (Table 1 ). Hickory nut (overta hickory) DNA, walnut (American, French) DNA, chestnut (Japanese chestnut, Chinese gourd, European chestnut, Chinkapin) DNA, beach nut (beech, Japanese beech) DNA, hazelnut DNA, Negative results were obtained when using shii DNA, acorn (white oak) DNA, and other species DNA, including other plants (Table 1).

In addition, when using PCR primers for detecting pine nuts (PCR primer set consisting of SEQ ID NOS: 25 and 26), an amplification product of around 139 bp was confirmed only when pine nut DNA was used, and was determined to be positive ( table 1). Negative results were obtained when DNA from other species, including other plants, was used (Table 1).

In addition, when PCR primers for detecting Pili nuts (PCR primer set consisting of SEQ ID NOS: 27 and 28) were used, an amplification product of around 83 bp was confirmed only when Pili nuts DNA was used, and was determined to be positive (Table 1). Relative cashew (India, Vietnam) DNA, pistachio (Iran, USA) DNA, mango DNA, pink pepper DNA, lychee DNA, rambutan DNA, orange DNA and others including other plants It was negative when species DNA was used (Table 1).

In addition, when PCR primers for detecting pistachios (PCR primer set consisting of SEQ ID NOs: 29 and 30) were used, an amplification product of around 163 bp was confirmed only when pistachio (Iranian and American) DNA was used, and the results were positive. (Table 1). Negative results were obtained using closely related species cashew (India, Vietnam) DNA, mango DNA, pink pepper DNA, and DNA from other species, including other plants (Table 1).

In addition, when shea nut detection PCR primers (PCR primer set consisting of SEQ ID NOs: 31 and 32) were used, an amplification product of around 518 bp was confirmed only when shea nut DNA was used, and was determined to be positive (Table 1). Negative results were obtained when using the closely related miracle fruit DNA, Brazil nut DNA, persimmon DNA, tea DNA, kiwifruit DNA, blueberry DNA, and DNA from other species, including other plants (Table 1). 1).

In addition, when PCR primers for walnut detection (PCR primer set consisting of SEQ ID NOs: 33 and 34) were used, an amplified product of around 501 bp was confirmed only when walnut (produced in the United States and France) DNA was used, and was determined to be positive. (Table 1). Related species pecan nut DNA, hickory nut (overta hickory) DNA, chestnut (Japanese chestnut, Chinese gourd, European chestnut, chinkapin) DNA, beach nut (beech, Japanese beech) DNA, hazelnut DNA, seaweed DNA, acorn (white oak) ) and other species, including other plants, were negative (Table 1).

Example 2
Confirmation of PCR Detection Sensitivity Using PCR Primer Set for Nut Detection In order to examine the detection sensitivity of PCR using the PCR primer set for nut detection used in Example 1, the following experiment was carried out.
Almonds (produced in the United States), beach nuts (beech), Brazil nuts, cashew nuts (produced in India), chestnuts, coconuts, hazelnuts, ginkgo nuts, litchi, macadamia nuts (produced in Australia), pecan nuts prepared in Example 1 , pine nut, pili nut, pistachio (US), shea nut, and walnut (US) DNA were diluted with sterile water to 1 fg/μl, 10 fg/μl, 100 fg/μl, 1 pg/μl , 10 pg/μl, and 100 pg/μl, and 1 μL of these diluted test DNA solutions were subjected to PCR under the PCR reaction conditions described in Example 1. After the PCR reaction, the presence or absence of amplification products was confirmed by agarose gel electrophoresis.

As shown in Figure 1, the detection sensitivity when using the almond detection PCR primer (PCR primer set consisting of SEQ ID NOS: 1 and 2) was 100 fg DNA/analysis, and the beach nut detection PCR primer (SEQ ID NO: The detection sensitivity when using the PCR primer set consisting of 3 and 4) is 10 fg DNA / analysis, and the detection when using the Brazil nut detection PCR primer (PCR primer set consisting of SEQ ID NOS: 5 and 6) The sensitivity is 100 fg DNA/analysis, and the detection sensitivity when using cashew nut detection PCR primers (PCR primer set consisting of SEQ ID NOs: 7 and 8) is 1 pg DNA/analysis. The detection sensitivity when using (PCR primer set consisting of SEQ ID NOS: 9 and 10) is 1 pg DNA/analysis, and when using the coconut detection PCR primer (PCR primer set consisting of SEQ ID NOS: 11 and 12) The detection sensitivity is 100 pg DNA / analysis, and the detection sensitivity when using the ginkgo detection PCR primer (PCR primer set consisting of SEQ ID NOS: 13 and 14) is 100 fg DNA / analysis, for hazelnut detection The detection sensitivity when using PCR primers (PCR primer set consisting of SEQ ID NOS: 15 and 16) is 100 fg DNA/analysis, and PCR primers for hickory nut detection (PCR primer set consisting of SEQ ID NOS: 17 and 18) The detection sensitivity when used is 100 fg DNA/analysis, and the detection sensitivity when using PCR primers for lychee detection (PCR primer set consisting of SEQ ID NOs: 19 and 20) is 100 fg DNA/analysis. The detection sensitivity when using the PCR primers for detecting macadamia nuts (PCR primer set consisting of SEQ ID NOS: 21 and 22) is 100 fg DNA/analysis, and the PCR primers for detecting pecan nuts (PCR primer set consisting of SEQ ID NOS: 23 and 24 The detection sensitivity when using the primer set) is 10 fg DNA / analysis, and the detection sensitivity when using the PCR primers for detecting pine nuts (PCR primer set consisting of SEQ ID NOS: 25 and 26) is 100 fg DNA/analytical PCR primers for detection of pili nuts (PCR primers consisting of SEQ ID NOs: 27 and 28) set) is 100 fg DNA/analysis, and the detection sensitivity is 100 fg DNA/analysis when using PCR primers for pistachio detection (PCR primer set consisting of SEQ ID NOs: 29 and 30). and the detection sensitivity when using the shea nut detection PCR primers (PCR primer set consisting of SEQ ID NOS: 31 and 32) is 10 fg DNA / analysis, and the walnut detection PCR primers (SEQ ID NOS: 33 and 34 consist of The detection sensitivity when using the PCR primer set) was 1 fg DNA/analysis.

Example 3
Detection of nut-derived DNA in nut-containing food products by PCR using the nut-detecting PCR primer set and investigated the practicality of the present invention.

A commercially available food product (Table 2) was prepared, and 10 to 100 g of the food product was pulverized using a multi-bead shocker. Each DNA was extracted from 2 g of the mixed homogenate using the Genomic-tip 20/G kit. At the time of extraction, RNA was removed by RNase. After measuring the amount of the extracted food sample DNA, sterilized water was used to adjust the concentration of the food sample DNA to 10 ng/μL. Since it was difficult to obtain commercial foods containing beach nuts, hickory nuts, and shea nuts as raw materials, 10 pg of each DNA was added to a coconut sable (Nissin Cisco) DNA solution (10 ng/μL) as an alternative sample. /μL concentration was used. 5 μL of these prepared DNA solutions were subjected to PCR under the PCR reaction conditions described in Example 1 using the PCR primer set for nut detection used in Example 1. If amplification is confirmed by the amount of fluorescence, separate the PCR amplification product (5 μL of the reaction mixture) by 2% (wt/vol) agarose gel electrophoresis and extract the amplification product using Gel DOC EZ (Bio Rad). The presence or absence of amplification products was confirmed by visualization. OneMARK 100 (GeneDireX) was used as a molecular weight marker. A PCR reaction was determined to be positive when an amplified product of the target size was detected by electrophoresis. The results of these PCRs are listed in Table 2. + (plus) in the table indicates a positive PCR reaction, and - (minus) indicates a negative PCR reaction.
Furthermore, the PCR amplification product amplified from the food sample DNA was directly sequenced from both directions using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems), followed by nucleotide sequencing using the Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems). was analyzed. The analyzed nucleotide sequence data is compared with the GenBank nucleotide sequence database (BLAST search) or the DNA database owned by the inventors, and based on the similarity of the nucleotide sequences, the biological species of the DNA derived from the PCR amplification product is assigned/analyzed. identified.

As a result, when using PCR primers for detecting almonds (PCR primer set consisting of SEQ ID NOS: 1 and 2), DNA derived from almond-containing foods, rearatie muesli (Dorset cereal) and petit chocolate leaf pie (El Madron) Only in , an amplified product of around 515 bp was confirmed and determined to be positive. It was negative when DNA from other food samples was used (Table 2). As a result of nucleotide sequence analysis of the PCR amplification products, the nucleotide sequences of the PCR amplification products amplified from DNA derived from rearnutty muesli (Dorset cereal) and petit chocolate leaf pie (El Madron) match the ITS nucleotide sequences of almonds. did. In other words, PCR using the PCR primers does not detect almond-derived DNA or other DNA from almond-free food samples in multiple commercial foods, but can specifically detect almond-derived DNA from almond-containing food samples. confirmed.

In addition, when using beach nut detection PCR primers (PCR primer set consisting of SEQ ID NOs: 3 and 4), alternative samples of beach nut-containing food [coconut sable (Nisshin Cisco) added with 50 pg beach nut DNA] Only in DNA, an amplified product of around 75 bp was confirmed and determined as positive. It was negative when DNA from other food samples was used (Table 2). As a result of nucleotide sequence analysis of the PCR amplification product, the nucleotide sequence of the PCR amplification product amplified from the DNA derived from the alternative sample of the beach nut-containing food matched the ITS nucleotide sequence of the beach nut. That is, PCR using the PCR primers did not detect beech nut-derived DNA or other DNA from beech nut-free food samples in multiple commercial foods, but trace amounts of beech nut added to beech nut-free foods were detected. It was confirmed that nut-derived DNA can be specifically detected.

In addition, when using a PCR primer for detecting Brazil nuts (PCR primer set consisting of SEQ ID NOS: 5 and 6), an amplification product of around 91 bp was obtained only in DNA derived from rearnut tea muesli (Dorset cereal), which is a food containing Brazil nuts. was confirmed and tested positive. It was negative when DNA from other food samples was used (Table 2). As a result of nucleotide sequence analysis of the PCR amplification product, the nucleotide sequence of the PCR amplification product amplified from DNA derived from rearnutty muesli (Dorset cereal) matched the ITS nucleotide sequence of Brazil nuts. In other words, PCR using the PCR primers does not detect Brazil nut-derived DNA or other DNA from Brazil nut-free food samples in multiple commercial foods, but does not detect Brazil nut-derived DNA from Brazil nut-containing food samples. It was confirmed that it can be detected at

In addition, when cashew nut detection PCR primers (PCR primer set consisting of SEQ ID NOS: 7 and 8) are used, cashew nut-containing foods, Real Nutty Muesli (Dorset Cereal) and Triple Nut Chocolate (Merry Chocolate Company)-derived DNA Only in , an amplified product of around 102 bp was confirmed and determined as positive. It was negative when DNA from other food samples was used (Table 2). As a result of nucleotide sequence analysis of the PCR amplification products, the nucleotide sequences of the PCR amplification products amplified from DNA derived from Rear Nutty Muesli (Dorset Cereal) and Triple Nuts Chocolate (Mary Chocolate Company) match the ITS nucleotide sequences of cashew nuts. did. That is, PCR using the PCR primers does not detect cashew nut-derived DNA or other DNA from cashew nut-free food samples, but can specifically detect cashew nut-derived DNA from cashew nut-containing food samples. confirmed.

In addition, when PCR primers for chestnut detection (PCR primer set consisting of SEQ ID NOS: 9 and 10) were used, an amplification product of around 293 bp was confirmed only in DNA derived from chestnut-containing food, chestnut bun (Yamakyu YK). , was determined to be positive. It was negative when DNA from other food samples was used (Table 2). As a result of nucleotide sequence analysis of the PCR amplified product, the nucleotide sequence of the PCR amplified product amplified from the chestnut manju (Yamakyu YK)-derived DNA matched the ITS nucleotide sequence of chestnut. In other words, it was confirmed that PCR using these PCR primers can specifically detect chestnut-derived DNA from food samples containing chestnuts, although it does not detect chestnut-derived DNA or other DNA from food samples that do not contain chestnuts. did.

In addition, when using coconut detection PCR primers (PCR primer set consisting of SEQ ID NOS: 11 and 12), in coconut-containing foods, triple nut chocolate (Mary Chocolate Company) and coconut sable (Nissin Cisco)-derived DNA Only, an amplified product of around 224 bp was confirmed and determined as positive. It was negative when DNA from other food samples was used (Table 2). As a result of nucleotide sequence analysis of the PCR amplification products, the nucleotide sequences of the PCR amplification products amplified from DNA derived from Triple Nuts Chocolate (Mary Chocolate Company) and Coconut Sable (Nissin Cisco) matched the ITS nucleotide sequence of coconut. . That is, PCR using the PCR primers does not detect coconut-derived DNA or other DNA from coconut-free food samples in multiple commercial foods, but can specifically detect coconut-derived DNA from coconut-containing food samples. confirmed.

In addition, when PCR primers for detecting ginkgo nuts (PCR primer set consisting of SEQ ID NOs: 13 and 14) were used, an amplification product of around 186 bp was confirmed only in the DNA derived from ginkgo nut-containing food, Ginnan Yokan (Yamagishi Motors). , was determined to be positive. It was negative when DNA from other food samples was used (Table 2). As a result of nucleotide sequence analysis of the PCR amplification product, the nucleotide sequence of the PCR amplification product amplified from the DNA derived from Ginnan Yokan (Yamagishi Motors) was consistent with the ginkgo chloroplast matK gene sequence. That is, PCR using the PCR primers does not detect ginkgo-derived DNA or other DNA from food samples that do not contain ginkgo biloba in multiple commercial foods, but can specifically detect ginkgo-derived DNA from food samples containing ginkgo. confirmed.

In addition, when using PCR primers for detecting hazelnuts (PCR primer set consisting of SEQ ID NOS: 15 and 16), an amplification product of around 361 bp was detected only in DNA derived from rearnut muesli (Dorset cereal), a hazelnut-containing food. Confirmed and tested positive. It was negative when DNA from other food samples was used (Table 2). As a result of nucleotide sequence analysis of the PCR amplification product, the nucleotide sequence of the PCR amplification product amplified from DNA derived from rearnut muesli (Dorset cereal) matched the ITS nucleotide sequence of hazelnuts. That is, PCR using the PCR primers does not detect hazelnut-derived DNA or other DNA from hazelnut-free food samples in multiple commercial foods, but can specifically detect hazelnut-derived DNA from hazelnut-containing food samples. confirmed.

In addition, when hickory nut detection PCR primers (PCR primer set consisting of SEQ ID NOs: 17 and 18) are used, alternative samples of hickory nut-containing food [coconut sable with 50 pg hickory nut DNA added (Nissin Cisco)] An amplification product of around 543 bp was confirmed only in DNA and triple nuts chocolate (Mary Chocolate Company), which is a pecan nut-containing food, and was determined to be positive. It was negative when DNA from other food samples was used (Table 2). As a result of nucleotide sequence analysis of the PCR amplification products, the nucleotide sequences of the PCR amplification products amplified from the DNA derived from the substitute sample of hickory nut-containing food and the DNA derived from triple nuts chocolate (Mary Chocolate Company) were the ITS nucleotide sequences of hickory nuts. coincided with That is, PCR using the PCR primers does not detect hickory-derived DNA or other DNA from hickory nut-free food samples in multiple commercial foods, but in pecan nut-containing food samples and hickory nut-free foods It was confirmed that a small amount of added hickory nut-derived DNA could be specifically detected.

In addition, when PCR primers for lychee detection (PCR primer set consisting of SEQ ID NOS: 19 and 20) were used, an amplification product of around 162 bp was confirmed only in the DNA derived from Fruit Therapy White Peach (Fujicco), which is a lychee-containing food. and tested positive. It was negative when DNA from other food samples was used (Table 2). As a result of nucleotide sequence analysis of the PCR amplification product, the nucleotide sequence of the PCR amplification product amplified from DNA derived from Fruit Therapy White Peach (Fujicco) matched the ITS nucleotide sequence of lychee. In other words, PCR using the PCR primers does not detect lychee-derived DNA or other DNA from food samples that do not contain lychee, but can specifically detect lychee-derived DNA from food samples that contain lychee. confirmed.

In addition, when using PCR primers for detecting macadamia nuts (PCR primer set consisting of SEQ ID NOS: 21 and 22), only in DNA derived from petit chocolate leaf pie (El Madron), which is a macadamia nut-containing food, an amplification product of around 111 bp was confirmed and tested positive. It was negative when DNA from other food samples was used (Table 2). As a result of nucleotide sequence analysis of the PCR amplified product, the nucleotide sequence of the PCR amplified product amplified from DNA derived from petit chocolate leaf pie (El Madron) matched the ITS nucleotide sequence of macadamia nuts. That is, PCR using the PCR primers does not detect macadamia nut-derived DNA or other DNA from macadamia nut-free food samples in multiple commercial foods, but specifically macadamia nut-derived DNA from macadamia nut-containing food samples. It was confirmed that it can be detected at

In addition, when PCR primers for detecting pecan nuts (PCR primer set consisting of SEQ ID NOs: 23 and 24) were used, an amplification product of around 468 bp was generated only in triple nuts chocolate (Mary Chocolate Company), which is a pecan nut-containing food. Confirmed and tested positive. It was negative when DNA from other food samples was used (Table 2). As a result of nucleotide sequence analysis of the PCR amplified product, the nucleotide sequence of the PCR amplified product amplified from DNA derived from Triple Nuts Chocolate (Mary Chocolate Company) matched the ITS nucleotide sequence of pecan nuts. That is, PCR using the PCR primers does not detect pecan nut-derived DNA or other DNA from pecan nut-free food samples in multiple commercial foods, but specifically pecan nut-derived DNA from pecan nut-containing food samples. It was confirmed that it can be detected at

In addition, when using a PCR primer for detecting pine nuts (a PCR primer set consisting of SEQ ID NOS: 25 and 26), only in the spaghetti sauce basil (SB food) that is a food containing pine nuts, it is around 139 bp. of amplification products were confirmed and determined to be positive. It was negative when DNA from other food samples was used (Table 2). As a result of nucleotide sequence analysis of the PCR amplification product, the nucleotide sequence of the PCR amplification product amplified from DNA derived from Spaghetti Sauce Basil (SB Foods), which is just mixed, matched the ITS nucleotide sequence of pine nuts. That is, PCR using the PCR primers does not detect pine nut-derived DNA or other DNA from pine nut-free food samples in multiple commercial foods, but pine nut-derived DNA from pine nut-containing food samples. It was confirmed that DNA can be specifically detected.

In addition, when PCR primers for detecting pili nuts (PCR primer set consisting of SEQ ID NOS: 27 and 28) were used, an amplification product of around 83 bp was confirmed only in LA2PU CRISPY PILINUT with Garlic (Royal Concern), a food containing pili nuts. and tested positive. It was negative when DNA from other food samples was used (Table 2). As a result of nucleotide sequence analysis of the PCR amplified product, the nucleotide sequence of the PCR amplified product amplified from DNA derived from LA2PU CRISPY PILINUT with Garlic (Royal Concern) matched the ITS nucleotide sequence of pili nuts. That is, PCR using the PCR primers does not detect pili nut-derived DNA or other DNA from food samples that do not contain pili nuts, but can specifically detect pili nut-derived DNA from food samples that contain pili nuts. confirmed.

In addition, when using PCR primers for detecting pistachios (PCR primer set consisting of SEQ ID NOS: 29 and 30), an amplification product of around 163 bp was confirmed only in DNA derived from petit chocolate leaf pie (El Madron), which is a pistachio-containing food. and tested positive. It was negative when DNA from other food samples was used (Table 2). As a result of nucleotide sequence analysis of the PCR amplification product, the nucleotide sequence of the PCR amplification product amplified from DNA derived from petit chocolate leaf pie (El Madron) matched the ITS nucleotide sequence of pistachio. That is, PCR using the PCR primers does not detect pistachio-derived DNA or other DNA from pistachio-free food samples in multiple commercial foods, but can specifically detect pistachio-derived DNA from pistachio-containing food samples. confirmed.

In addition, when using PCR primers for detecting shea nuts (PCR primer set consisting of SEQ ID NOS: 31 and 32), only DNA derived from alternative samples of shea nut-containing food [coconut sable with 50 pg shea nut DNA added (Nisshin Cisco)] , an amplified product of around 518 bp was confirmed and determined to be positive. It was negative when DNA from other food samples was used (Table 2). As a result of nucleotide sequence analysis of the PCR amplification product, the nucleotide sequence of the PCR amplification product amplified from the DNA derived from the alternative sample of the shea nut-containing food matched the ITS nucleotide sequence of shea nut. That is, PCR using the PCR primers does not detect shea nut-derived DNA or other DNA from shea nut-free food samples in multiple commercial foods, but a trace amount of shea nut-derived DNA added to shea nut-free foods It was confirmed that it can be specifically detected.

In addition, when PCR primers for walnut detection (PCR primer set consisting of SEQ ID NOS: 33 and 34) were used, an amplification product of around 501 bp was confirmed only in the DNA derived from Azuki Walnut Danish (Shikishima Bakery), which is a walnut-containing food. and tested positive. It was negative when DNA from other food samples was used (Table 2). As a result of nucleotide sequence analysis of the PCR amplified product, the nucleotide sequence of the PCR amplified product amplified from DNA derived from Azuki Kurumi Danish (Shikishima Bread Co., Ltd.) matched the ITS nucleotide sequence of walnut. That is, PCR using the PCR primers does not detect walnut-derived DNA or other DNA from walnut-free food samples, but can specifically detect walnut-derived DNA from walnut-containing food samples. confirmed.

Example 4
Detection of 8 types of almonds, brazil nuts, cashew nuts, hazelnuts, macadamia nuts, pecan nuts, pistachios, and walnuts by multiplex method with specific combinations of 4 types

DNAs of almonds (USA), Brazil nuts, cashew nuts (India), hazelnuts, macadamia nuts (Australia), pecan nuts, pistachios (USA) and walnuts (USA) prepared in Example 1 were dissolved in sterilized water. to prepare a sample solution.

Each PCR primer set forth in SEQ ID NOs: 1, 2, 5, 6, 7, 8, 15, 16, 17, 18, 21, 22, 29, 30, 33, and 34 was synthesized by Eurofins Genomics. were used in the following PCR.

The PCR reaction solution was composed of Takara Bio's SYBR Premix Ex Taq (Tli RNaseH Plus), 1 μL of each DNA solution adjusted to 1 pg/μL, and four types of sense primers and antisense primers in a volume of 20 μL. was prepared to The concentration of each primer was 100 nM for the pecan nut detection primer and 250 nM for the other three primers used in the same reaction system. The almond detection primer was 500 nM, and the other three primers used in the same reaction system were 100 nM. The PCR reaction was performed with an Applied Biosystems 7500 real-time PCR system (Thermo Fisher Scientific), and the amplification reaction conditions are as follows.

After one cycle at 95°C for 30 seconds, the temperature was raised to 95°C, maintained at the same temperature for 5 seconds, then cooled to 70°C and maintained at the same temperature for 40 seconds. 35 times. PCR amplification products (5 μL of reaction solution) were separated by 4% (wt/vol) agarose gel electrophoresis, and after ethidium bromide staining, the presence or absence of amplification products was confirmed using Chemi DOC Touch (Bio Rad). OneMARK 100 (GeneDireX) was used as a molecular weight marker.

In the case of multiplex (simultaneous detection) of four species of pecan nuts, walnuts, pistachios, and cashew nuts, as shown in FIG.
In FIG. 2 (A), each lane number from 1 to 5 is a PCR reaction with the addition of only pecan nut DNA (lane 1), a PCR reaction with the addition of only walnut DNA (lane 2 ), PCR reaction with the addition of pistachio DNA only (lane 3), PCR reaction with the addition of cashew nut DNA only (lane 4), all (pecan nuts, walnuts, pistachios, cashew nuts) It shows what was added and PCR-reacted (lane 5).

In the case of multiplex (simultaneous detection) for four species of almonds, hazelnuts, macadamia nuts, and Brazil nuts, as shown in FIG.

In FIG. 2 (B), each lane number from 1 to 5 indicates the PCR reaction with the addition of almond DNA alone (lane 1), the PCR reaction with the addition of hazelnut DNA alone (lane 2). , PCR reaction by adding only macadamia nut DNA (lane 3), PCR reaction by adding only Brazil nut DNA (lane 4), all (almonds, hazelnuts, macadamia nuts, Brazil nuts) A PCR reaction with the addition of DNA is shown (lane 5).

In the case of multiplexing (simultaneous detection) of pecan nuts, walnuts, hazelnuts, and cashew nuts, all of the nuts to be detected could be detected, as shown in FIG. 2(C).
In FIG. 2(C), each lane number from 1 to 5 is a PCR reaction with the addition of only pecan nut DNA (lane 1), a PCR reaction with the addition of only walnut DNA (lane 2 ), PCR reaction with the addition of hazelnut DNA only (lane 3), PCR reaction with the addition of cashew nut DNA only (lane 4), all (pecan nuts, walnuts, hazelnuts, cashew nuts) It shows what was added and PCR-reacted (lane 5).

In the case of multiplex (simultaneous detection) of four species of almond, pistachio, macadamia nut, and brazil nut, as shown in FIG.
In FIG. 2(D), each lane number from 1 to 5 indicates a PCR reaction with the addition of almond DNA alone (lane 1), a PCR reaction with the addition of pistachio DNA alone (lane 2). , PCR reaction by adding only macadamia nut DNA (lane 3), PCR reaction by adding only Brazil nut DNA (lane 4), all (almonds, pistachios, macadamia nuts, Brazil nuts) A PCR reaction with the addition of DNA is shown (lane 5).

In the case of multiplexing (simultaneous detection) of four species of pecan nuts, walnuts, macadamia nuts, and Brazil nuts, as shown in FIG.
In FIG. 2 (E), each lane number from 1 to 5 is a PCR reaction with the addition of pecan nut DNA only (lane 1), a PCR reaction with the addition of walnut DNA only (lane 2 ), PCR reaction with the addition of macadamia nut DNA only (lane 3), PCR reaction with the addition of Brazil nut DNA only (lane 4), all (pecan nuts, walnuts, macadamia nuts, Brazil nuts ) was added and subjected to PCR reaction (lane 5).

In the case of multiplex (simultaneous detection) for four species of almond, hazelnut, pistachio, and cashew nut, as shown in FIG.
In FIG. 2 (F), each lane number from 1 to 5 indicates the PCR reaction with the addition of almond DNA alone (lane 1), the PCR reaction with the addition of hazelnut DNA alone (lane 2). , PCR reaction with the addition of pistachio DNA only (lane 3), PCR reaction with the addition of cashew nut DNA only (lane 4), all (almond, hazelnut, pistachio, cashew nut) DNA was added. (lane 5).

Claims (6)

a hickory nut detection PCR primer set comprising a PCR primer for the nucleotide sequence of SEQ ID NO: 17 in the sequence listing and a PCR primer for the nucleotide sequence of SEQ ID NO: 18 in the sequence listing;
a PCR primer set for detecting walnuts, comprising a PCR primer having the nucleotide sequence of SEQ ID NO: 33 in the sequence listing and a PCR primer having the nucleotide sequence of SEQ ID NO: 34 in the sequence listing;
a PCR primer set for detecting pistachios, comprising a PCR primer for the nucleotide sequence of SEQ ID NO: 29 in the sequence listing and a PCR primer for the nucleotide sequence of SEQ ID NO: 30 in the sequence listing;
a cashew nut detection PCR primer set comprising a PCR primer for the nucleotide sequence of SEQ ID NO: 7 in the sequence listing and a PCR primer for the nucleotide sequence of SEQ ID NO: 8 in the sequence listing;
A simultaneous PCR detection method for pecan nuts, walnuts, pistachios and cashew nuts using a primer set containing
Simultaneous PCR detection method for pecan nuts, walnuts, pistachios and cashew nuts with a concentration ratio of 2:5 between the PCR primer set for detecting hickory nuts and the other three primer sets in the PCR reaction solution. A PCR primer set for detecting almonds, which consists of a PCR primer having the nucleotide sequence of SEQ ID NO: 1 in the sequence listing and a PCR primer having the nucleotide sequence of SEQ ID NO: 2 in the sequence listing;
a PCR primer set for detecting hazelnuts, comprising a PCR primer for the nucleotide sequence of SEQ ID NO: 15 in the sequence listing and a PCR primer for the nucleotide sequence of SEQ ID NO: 16 in the sequence listing;
A PCR primer set for detecting macadamia nuts, comprising a PCR primer for the nucleotide sequence of SEQ ID NO: 21 in the sequence listing and a PCR primer for the nucleotide sequence of SEQ ID NO: 22 in the sequence listing;
A PCR primer set for detecting Brazil nuts, comprising a PCR primer for the nucleotide sequence of SEQ ID NO: 5 in the sequence listing and a PCR primer for the nucleotide sequence of SEQ ID NO: 6 in the sequence listing;
A simultaneous PCR detection method for almonds, hazelnuts, macadamia nuts and Brazil nuts using a primer set containing
A simultaneous PCR detection method for almonds, hazelnuts, macadamia nuts and brazil nuts in which the concentration ratio of the PCR primer set for detecting almonds in the PCR reaction solution and each of the other three primer sets is 5:1. a hickory nut detection PCR primer set comprising a PCR primer for the nucleotide sequence of SEQ ID NO: 17 in the sequence listing and a PCR primer for the nucleotide sequence of SEQ ID NO: 18 in the sequence listing;
a PCR primer set for detecting walnuts, comprising a PCR primer having the nucleotide sequence of SEQ ID NO: 33 in the sequence listing and a PCR primer having the nucleotide sequence of SEQ ID NO: 34 in the sequence listing;
a PCR primer set for detecting hazelnuts, comprising a PCR primer for the nucleotide sequence of SEQ ID NO: 15 in the sequence listing and a PCR primer for the nucleotide sequence of SEQ ID NO: 16 in the sequence listing;
a cashew nut detection PCR primer set comprising a PCR primer for the nucleotide sequence of SEQ ID NO: 7 in the sequence listing and a PCR primer for the nucleotide sequence of SEQ ID NO: 8 in the sequence listing;
A simultaneous PCR detection method for pecan nuts, walnuts, hazelnuts and cashew nuts using a primer set containing
Simultaneous PCR detection method for pecan nuts, walnuts, hazelnuts and cashew nuts in which the concentration ratio of the hickory nut detection PCR primer set and the other three primer sets in the PCR reaction solution is 2:5. a PCR primer set for detecting almonds, comprising a PCR primer for the nucleotide sequence of SEQ ID NO: 1 in the sequence listing and a PCR primer for the nucleotide sequence of SEQ ID NO: 2 in the sequence listing;
a PCR primer set for detecting pistachios, comprising a PCR primer for the nucleotide sequence of SEQ ID NO: 29 in the sequence listing and a PCR primer for the nucleotide sequence of SEQ ID NO: 30 in the sequence listing;
A PCR primer set for detecting macadamia nuts, comprising a PCR primer for the nucleotide sequence of SEQ ID NO: 21 in the sequence listing and a PCR primer for the nucleotide sequence of SEQ ID NO: 22 in the sequence listing;
A PCR primer set for detecting Brazil nuts, comprising a PCR primer for the nucleotide sequence of SEQ ID NO: 5 in the sequence listing and a PCR primer for the nucleotide sequence of SEQ ID NO: 6 in the sequence listing;
A simultaneous PCR detection method for almonds , pistachios, macadamia nuts and Brazil nuts using a primer set containing
A simultaneous PCR detection method for almonds, pistachios, macadamia nuts, and Brazil nuts in which the concentration ratio of the PCR primer set for detecting almonds in the PCR reaction solution and each of the other three primer sets is 5:1. a hickory nut detection PCR primer set comprising a PCR primer for the nucleotide sequence of SEQ ID NO: 17 in the sequence listing and a PCR primer for the nucleotide sequence of SEQ ID NO: 18 in the sequence listing;
a PCR primer set for detecting walnuts, comprising a PCR primer having the nucleotide sequence of SEQ ID NO: 33 in the sequence listing and a PCR primer having the nucleotide sequence of SEQ ID NO: 34 in the sequence listing;
A PCR primer set for detecting macadamia nuts, comprising a PCR primer for the nucleotide sequence of SEQ ID NO: 21 in the sequence listing and a PCR primer for the nucleotide sequence of SEQ ID NO: 22 in the sequence listing;
A PCR primer set for detecting Brazil nuts, comprising a PCR primer for the nucleotide sequence of SEQ ID NO: 5 in the sequence listing and a PCR primer for the nucleotide sequence of SEQ ID NO: 6 in the sequence listing;
A simultaneous PCR detection method for pecan nuts, walnuts, macadamia nuts and Brazil nuts using a primer set containing
Simultaneous PCR detection method for pecan nuts, walnuts, macadamia nuts and brazil nuts with a concentration ratio of 2:5 between the hickory nut detection PCR primer set and the other three primer sets in the PCR reaction solution. a PCR primer set for detecting almonds, comprising a PCR primer for the nucleotide sequence of SEQ ID NO: 1 in the sequence listing and a PCR primer for the nucleotide sequence of SEQ ID NO: 2 in the sequence listing;
a PCR primer set for detecting hazelnuts, comprising a PCR primer for the nucleotide sequence of SEQ ID NO: 15 in the sequence listing and a PCR primer for the nucleotide sequence of SEQ ID NO: 16 in the sequence listing;
a PCR primer set for detecting pistachios, comprising a PCR primer for the nucleotide sequence of SEQ ID NO: 29 in the sequence listing and a PCR primer for the nucleotide sequence of SEQ ID NO: 30 in the sequence listing;
a cashew nut detection PCR primer set comprising a PCR primer for the nucleotide sequence of SEQ ID NO: 7 in the sequence listing and a PCR primer for the nucleotide sequence of SEQ ID NO: 8 in the sequence listing;
A simultaneous PCR detection method for almonds, hazelnuts, pistachios and cashews using a primer set comprising
A simultaneous PCR detection method for almonds, hazelnuts, pistachios and cashew nuts, wherein the concentration ratio of the PCR primer set for detecting almonds in the PCR reaction solution and the other three primer sets is 5:1.

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