JP7561521B2 - Primer set for detecting walnuts and method for detecting walnuts using the same - Google Patents

Description

The present invention relates to a primer set that can be used to detect walnuts, which are food allergens in foods and beverages, and a method for detecting walnuts using the same.

Walnuts are plants belonging to the genus Juglans in the family Juglandaceae, and the kernel is eaten as a nut.

Walnuts are one of the food allergens that cause allergies, and even ingesting a small amount can cause severe anaphylactic shock in some patients. In addition, the number of food allergy patients has been steadily increasing in recent years, and this has become a major social problem worldwide.
Non-Patent Document 1 describes that when several plant species belonging to the genus Juglans were examined, all of them contained allergen proteins.

One method for detecting walnuts in food and beverages is to amplify the DNA sequence specific to walnuts by a nucleic acid amplification method using a primer set and detect the presence or absence of the amplified product. Non-patent documents 2 to 8 are examples of documents disclosing nucleic acid amplification methods for detecting walnuts. Note that polymerase chain reaction (PCR) refers to a type of nucleic acid amplification reaction in which DNA polymerase is used to amplify nucleic acids of a target sequence in a chain-like manner.

The genus Carya is known as a plant closely related to the walnut genus that is eaten as a nut. The main commercially cultivated Carya plants are Carya illinoinensis (pecan) and Carya cathayensis (mountain peach). Pecans are generally recognized as a different food from walnuts.

On the other hand, Patent Document 1 discloses a primer set that can detect wheat, buckwheat, and peanuts, which are allergens contained in food and beverages, by real-time PCR under common PCR conditions.

JP 2016-101142 A

Comstock, S.S., McGranahan, G., Peterson, W.R., Teuber, S.S., 2004. Extensive in vitro cross-reactivity to seed storage proteins is present among walnut (Juglans) cultivars and species. Clin Exp Allergy 34, 1583-1590. Brezna, B., Hudecova., Kuchta, T., 2006. A novel real-time polymerase chain reaction (PCR) method for the detection of walnuts in food. Eur Food Res Technol 223, 373-377. Yano, T., Sakai, Y., Uchida, K., Nakao, Y., Ishihata, K., Nakano, S., Yamada, T., Sakai, S., Urisu, A., Akiyama, H., Maitani, T., 2007. Detection of Walnut Residues in Processed Foods by Polymerase Chain Reaction. Bioscience, Biotechnology, and Biochemistry 71, 1793-1796. Wang, H., Yuan, F., Wu, Y., Yang, H., Xu, B., Liu, Z., Chen, Y., 2009. Detection of Allergen Walnut Component in Food by an Improved Real-Time PCR Method. Journal of Food Protection 72, 2433-2435. Lopez-Calleja, I.M., de la Cruz, S., Gonzalez, I., Garcfa, T., Martin, R., 2015. Market analysis of food products for detection of allergenic walnut (Juglans regia) and pecan (Carya illinoinensis) by real-time PCR. Food Chemistry 177, 111-119. Linacero, R., Ballesteros, I., Sanchiz, A., Prieto, N., Iniesto, E., Martinez, Y., Pedrosa, M.M., Muzquiz, M., Cabanillas, B., Rovira, M., Burbano , C., Cuadrado, C., 2016. Detection by real time PCR of walnut allergen coding sequences in processed foods. Food Chemistry 202, 334-340. Ito, M., Mizota, T., Kitaguchi, T., Ohno, K., Ohba, T., Tanaka, M., 2018. Simultaneous detection of eight species of tree nut in foods using two tetraplex polymerase chain reaction assays. Bioscience, Biotechnology, and Biochemistry 82, 1985-1991. Kim, J.H., Hong, J.Y., Moon, J.-C., Kwon, K., Jang, C.S., 2018. Development of molecular markers for detecting almond, peanut, pine nut, and walnut in commercial food using quantitative real-time PCR. Appl Biol Chem 61, 345-354.

One or more embodiments of the present invention provide a new means for detecting walnuts and distinguishing them from Carya plants.

This specification discloses one or more of the following embodiments of the present invention.
(1) A primer set for detecting walnuts,
(A1) the base sequence A1 shown in SEQ ID NO: 1;
(A2) a base sequence A2 that is homologous to the base sequence A1;
(A3) a base sequence A3 which is a partial base sequence of 5 or more consecutive bases from the 3'-terminal base of the base sequence A1; and
(A4) Base sequence A4 homologous to base sequence A3
(B1) a primer A comprising an oligonucleotide comprising a base sequence A at the 3' end selected from the base sequence B1 shown in SEQ ID NO: 2,
(B2) a base sequence B2 that is homologous to the base sequence B1;
(B3) a base sequence B3 which is a partial base sequence of 5 or more consecutive bases from the 3'-terminal base of the base sequence B1; and
(B4) Base sequence B4 homologous to base sequence B3
Primer B comprising an oligonucleotide containing a base sequence B at its 3' end selected from
A primer set comprising:
(2) (C1) the base sequence C1 shown in SEQ ID NO: 3,
(C2) a base sequence C2 homologous to the base sequence C1;
(C3) a base sequence C3 which is a partial base sequence of 5 or more consecutive bases of the base sequence C1;
(C4) a base sequence C4 homologous to the base sequence C3;
(C5) a base sequence C5 complementary to the base sequence C1;
(C6) a base sequence C6 homologous to the base sequence C5;
(C7) a base sequence C7 which is a partial base sequence of 5 or more consecutive bases of the base sequence C5;
(C8) A base sequence C8 that is homologous to the base sequence C5
(C9) a base sequence C9 which is a partial base sequence of 5 or more consecutive bases among the base sequence complementary to positions 22 to 45 of SEQ ID NO: 7; and
(C10) A base sequence C10 complementary to the base sequence C9
The primer set according to (1), further comprising a probe C comprising an oligonucleotide comprising a base sequence C selected from the group consisting of:
(3) A method for detecting walnuts in a sample, comprising:
(1) or (2) is used to perform a nucleic acid amplification reaction using DNA derived from the sample as a template, and (3) an amplification product obtained by the nucleic acid amplification reaction is detected.
(4) The primer set is the primer set according to (2),
The detection of the amplification product is carried out by detecting binding between the amplification product and the probe C.
The method according to (3).
(5) The method according to (3) or (4), wherein the sample is a food or beverage sample.

According to one or more embodiments of the present invention, walnuts can be detected and distinguished from plants of the genus Carya.

FIG. 1 shows an amplification curve of real-time PCR carried out under condition 1 using the primer probe set of the example. FIG. 2 shows an amplification curve of real-time PCR carried out under condition 1' using the primer probe set of the comparative example. FIG. 3 shows an amplification curve of real-time PCR carried out under condition 2 using the primer-probe set of the comparative example. FIG. 4 shows the correspondence relationship between the primer probe sets used in the Examples and Comparative Examples and the ITS-2 base sequences (SEQ ID NOs: 7 to 12) of four types of walnuts in the genus Juglans (J. regi a, J. nigra, J. mandshurica, J. airanthifolia) and two types of pecans in the genus Carya (C. cathayensis, C. illinoinensis). Single underlined bases indicate bases that are common to the genus Juglans and different from the genus Carya. Double underlined bases indicate bases that are polymorphic within the genus Juglans.

1. Purpose
The primer set and the detection method using the same according to one or more embodiments of the present invention are intended to satisfy the following requirements.

(Detection target)
According to Non-Patent Document 1, all plants belonging to the genus Juglans are considered to have allergenicity. Therefore, in order to detect walnuts as allergens, it is desirable to be able to detect them without distinguishing between species.

On the other hand, plants of the genus Carya, such as Carya illinoinensis (pecan) and Carya cathayensis (mountain peach), are generally recognized as foods different from walnuts, so it is desirable to detect only plants of the genus Juglans, distinguishing them from plants of the genus Carya.

Therefore, one or more embodiments of the present invention aim to provide a primer set that does not amplify DNA from plants of the genus Carya, but can amplify walnut DNA with similar efficiency regardless of the species, and a method for detecting walnuts using the primer set.

In this specification, walnut refers to plants of the genus Juglans. Specific examples of plants of the genus Juglans that are commercially cultivated as walnuts include Juglans regia (oak walnut, European walnut, Persian walnut, Japanese walnut), Juglans nigra (black walnut), Juglans mandshurica, and Juglans airanthifolia. Both Juglans mandshurica and Juglans airanthifolia are sometimes called long walnut, Chinese walnut, or Japanese walnut.

(sensitivity)
For the detection of allergens it is desirable to be able to detect concentrations equivalent to 1 μg walnut protein/g sample (=1 ppm).

The protein content of walnut kernels is 14.6 g/100 g (Food Composition Database), so 1 μg walnut protein/g sample is equivalent to 6.8 μg walnut/g sample.

Assuming that DNA can be recovered at the same yield from the sample and walnuts, 6.8 μg walnuts/g sample corresponds to 340 fg walnut DNA/50 ng sample DNA.

Therefore, one or more embodiments of the present invention aim to provide a primer set capable of amplifying walnut DNA present at a concentration of 340 fg walnut DNA/50 ng sample DNA, and a method for detecting walnuts using the primer set.

(Robustness)
DNA derived from raw materials contained in food and beverages may be fragmented during processing. Therefore, when amplifying walnut DNA by nucleic acid amplification reaction and detecting the presence or absence of the amplified product, the shorter the length of the amplified target DNA fragment, the more stable the detection without being affected by processing.

Therefore, one or more embodiments of the present invention aim to provide a primer set capable of amplifying a target sequence contained in walnut DNA with a fragment length of 150 bp or less, and a method for detecting walnuts using the primer set.

(Comparison with conventional technology)
Non-Patent Documents 2 to 8 describe a method for amplifying a DNA sequence specific to walnuts by a nucleic acid amplification method using a specific primer set and detecting the presence or absence of an amplified product. However, they do not disclose a method that satisfies the above requirements.

For example, the method described in Non-Patent Document 2 has a detection sensitivity of 100 μg protein concentration/g sample, which does not satisfy the above-mentioned sensitivity requirement.
The method described in Non-Patent Document 3 is a method for detecting pecans and walnuts without distinguishing between them, and does not meet the above-mentioned requirements for the detection target.
The method described in Non-Patent Document 4 has a detection sensitivity of 10 μg protein concentration/g sample, which does not satisfy the above-mentioned sensitivity requirement.
The method described in Non-Patent Document 6 has a detection sensitivity of 100 μg protein concentration/g sample, which does not satisfy the above-mentioned sensitivity requirement.
In the method described in Non-Patent Document 7, the target DNA fragment length is 501 bp, which does not satisfy the above-mentioned requirement of robustness.
The method described in Non-Patent Document 8 is a method for detecting pecans and walnuts without distinguishing between them, and does not meet the requirements for the above-mentioned detection targets.

The method described in Non-Patent Document 5 can detect walnuts separately from pecans, with a detection sensitivity of 0.1 μg protein/g sample and a target DNA fragment length of 70. However, as confirmed in the Examples described below, the detection method using the primer probe set described in Non-Patent Document 5 has been confirmed to have a large difference in detection sensitivity between oak walnuts and Japanese walnuts. For this reason, it does not meet the above requirements for the detection target.

(PCR conditions)
Patent Document 1 describes the detection of wheat, buckwheat, and peanut DNA by a real-time PCR method under common PCR conditions (specifically, condition 1 shown in the examples). The common PCR conditions for amplifying wheat, buckwheat, and peanut DNA described in Patent Document 1 have a relatively short number of cycles. If walnut DNA can be amplified under the PCR conditions described in Patent Document 1, it will be possible for users to detect the presence or absence of walnut, wheat, buckwheat, and peanut DNA with simple operations.

Therefore, one or more embodiments of the present invention more preferably have a further object to provide a primer set capable of amplifying walnut DNA under common PCR conditions for amplifying wheat, buckwheat, and peanut DNA as described in Patent Document 1, and a method for detecting walnuts using the primer set.

<2. Primer set for detecting walnuts>
According to one or more embodiments of the present invention, the primer set for detecting walnuts comprises:
(A1) the base sequence A1 shown in SEQ ID NO: 1;
(A2) a base sequence A2 that is homologous to the base sequence A1;
(A3) a base sequence A3 which is a partial base sequence of 5 or more consecutive bases from the 3'-terminal base of the base sequence A1; and
(A4) Base sequence A4 homologous to base sequence A3
(B1) a primer A comprising an oligonucleotide comprising a base sequence A at the 3' end selected from the base sequence B1 shown in SEQ ID NO: 2,
(B2) a base sequence B2 that is homologous to the base sequence B1;
(B3) a base sequence B3 which is a partial base sequence of 5 or more consecutive bases from the 3'-terminal base of the base sequence B1; and
(B4) Base sequence B4 homologous to base sequence B3
Primer B comprising an oligonucleotide containing a base sequence B at its 3' end selected from
The present invention is characterized by comprising:

With regard to the primer A, the primer B, and the primer C described below, when two base sequences are homologous to each other, that is, when the base sequence X is a base sequence Y, preferably, any one or more of the following relationships (A), (B), and (C) are satisfied:
(A) Base sequence Y is a base sequence in which one or several bases have been deleted, substituted, added and/or inserted in base sequence X.
(B) The base sequence Y has an identity of 80% or more but less than 100% with the base sequence X.
(C) Base sequence Y can bind (hybridize) to the complementary base sequence of base sequence X under stringent conditions.

In the above (A), "one or several" preferably refers to one to five, more preferably one to four, more preferably one to three, particularly preferably one or two, and most preferably one. Furthermore, "deletion, substitution, addition, and/or insertion" more preferably refers to substitution.

In (B), the identity value indicates a value calculated with default settings using software (e.g., FASTA, DANASYS, and BLAST) that calculates identity between multiple base sequences. The identity value of base sequences is calculated by calculating the number of matching bases when a pair of base sequences are aligned to maximize the degree of identity, and is calculated as the ratio of the number of matching bases to the total number of bases in the compared base sequences. Here, when there are gaps, the total number of bases mentioned above is the number of bases counted with one gap as one base. For details on the method of determining identity, see, for example, Altschul et al., Nuc. Acids. Res. 25, 3389-3402, 1977 and Altschul et al., J. Mol. Biol. 215, 403-410, 1990.

In (B) above, the identity is more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more.

As used herein, including the above (C), "binding (hybridizing) under stringent conditions" means that two nucleic acid fragments hybridize to each other under standard hybridization conditions (Expression of cloned genes in E. coli (Molecular Cloning: A laboratory manual (1989)) Cold Springharbor Laboratory Press, New York, USA, pp. 9.47-9.62 and 11.45-11.61). More specifically, this means that hybridization (e.g., about 3.0×SSC or 2.0×SSC, 30° C. or 37° C.) is performed based on the Tm value calculated by the following formula, followed by washing under conditions with higher stringency than the hybridization conditions (e.g., about 2.0×SSC, 30° C., 37° C., 40° C., 44° C. or 48° C. or higher, or 1.0×SSC or 0.5×SSC, 37° C. or higher). It is well known in the art to select "stringent conditions" appropriate for hybridization and washing depending on the base sequence to be hybridized, etc.
Tm = 81.5 + 16.6 (log ₁₀ [Na ⁺ ]) + 0.41 (fraction G + C) - (600/N)
[Na ⁺ ]: Molar concentration of Na ⁺ (mol / L)
Fraction G+C: The percentage of G and C in the oligonucleotide
N: length of oligonucleotide (number of bases)

The phrase "bind (hybridize) under stringent conditions" will be used in the following with the same meaning unless otherwise specified.

The primer set is designed to amplify DNA derived from walnuts, more specifically, a region in the ITS (Internal Transcribed Spacer)-2 of the ribosomal RNA gene.

Primer A can bind (hybridize) under stringent conditions with the complementary sequence of base sequence A1 shown in SEQ ID NO:1 on one strand of ITS-2 of double-stranded DNA derived from walnut. Primer B can bind (hybridize) under stringent conditions with the complementary sequence of base sequence B1 shown in SEQ ID NO:2 on the other strand of ITS-2 of DNA derived from walnut. When a nucleic acid amplification reaction is carried out using a primer set including primers A and B and walnut-derived DNA as a template, a DNA fragment including a region of 150 bp or less included in ITS-2 of walnut-derived DNA is generated as an amplification product.

With reference to the sequence table, FIG. 4, and Table 1 in the Examples, we explain why the primer set containing primers A and B can amplify walnut DNA with the same efficiency regardless of the species, but does not amplify the DNA of plants in the genus Carya.

A specific example of primer A is the "JI2 forward primer" described in the Examples, which is composed of an oligonucleotide consisting of the base sequence A1 shown in SEQ ID NO: 1. A specific example of primer B is the "JI2 reverse primer" described in the Examples, which is composed of an oligonucleotide consisting of the base sequence B1 shown in SEQ ID NO: 2. A part of the base sequence (AF303809.1) of ITS-2 of the DNA of Juglans regia is shown in SEQ ID NO: 7. As shown in FIG. 4, the JI2 forward primer matches positions 1 to 21 of SEQ ID NO: 7, and the JI2 reverse primer matches the reverse complementary strand of positions 56 to 74 of SEQ ID NO: 7. By performing a nucleic acid amplification reaction using the JI2 forward primer and the JI2 reverse primer and the DNA of Juglans regia as a template, a DNA fragment of the target region consisting of 74 bp of ITS-2 can be amplified. Because the target region to be amplified is shorter than 150 bp, it is highly likely that the DNA of Juglans regi a will remain even if the sample is subjected to processing such as heating.

Figure 4 also shows the alignment results between the target region shown in sequence number 7 of ITS-2 of Juglans regia and the corresponding regions of Juglans nigra, Juglans mandshurica, and Juglans airanthifolia belonging to the genus Juglans, and Carya cathayensis and Carya illinoinensis belonging to the genus Carya (shown in sequence numbers 8, 9, 10, 11, and 12, respectively). In the target region shown in SEQ ID NO:7 of ITS-2 for Juglans regia, the 21st, 25th, 33rd, 43rd, 52nd, 56th, 64th, and 65th positions are bases (single underlined) that are common to the genus Juglans but different in the genus Carya. In addition, in the target region shown in SEQ ID NO:7 of ITS-2 for Juglans regia, the 46th and 49th positions are bases (double underlined) that are polymorphic within the genus Juglans.

The JI2 forward primer (consisting of the base sequence shown in SEQ ID NO:1) contains a base common to Juglans but different to Caria at position 21 of the 3' end, which has a large effect on specificity. The JI2 reverse primer (consisting of the base sequence shown in SEQ ID NO:2) contains a base common to Juglans but different to Caria at position 19 of the 3' end, which has a large effect on specificity, and also at positions 10 and 11. Therefore, when a nucleic acid amplification reaction is performed using walnut DNA as a template with the JI2 forward primer and the JI2 reverse primer, the walnut DNA can be amplified with the same efficiency regardless of the species, and the DNA of Caria plants is not amplified. Similarly, when using the primer set according to this embodiment, which includes primer A capable of hybridizing to the ITS-2 region of walnut DNA to which the JI2 forward primer hybridizes, and primer B capable of hybridizing to the ITS-2 region of walnut DNA to which the JI2 reverse primer hybridizes, it is possible to amplify the amplification product of walnut DNA with the same efficiency regardless of the species, without amplifying the DNA of Caria plants.

For comparison, Figure 4 also shows the corresponding positions on the ITS-2 base sequence of the WalITS forward primer (SEQ ID NO: 4) and WalITS reverse primer (SEQ ID NO: 5) described in Non-Patent Document 5. The base at the 3' end of these primers is a common base between the Juglans and Caria genera. For this reason, nucleic acid amplification reactions using these primers may erroneously amplify DNA from Caria plants, which is considered undesirable for the purpose of detecting walnuts.

Next, more preferred embodiments of the primer A and the primer B will be described.
The oligonucleotide in primer A may contain a base sequence A selected from base sequence A1, base sequence A2, base sequence A3, and base sequence A4 at the 3' end, and may contain other base sequences added to the 5' end side of base sequence A, or may consist of only base sequence A. The length (number of bases) of the oligonucleotide in primer A is not particularly limited, but is usually 35 bases or less, preferably 30 bases or less, more preferably 28 bases or less, more preferably 26 bases or less, and more preferably 25 bases or less. Primer A may consist of the oligonucleotide alone, or may be the oligonucleotide to which other elements such as a labeling substance or a tag are linked. The other elements may be linked to any position of the oligonucleotide, but it is particularly preferred that they are linked to the 5' end of the oligonucleotide.
The base sequence A1 refers to the base sequence shown in SEQ ID NO:1.

The base sequence A2 is a base sequence homologous to the base sequence A1. Preferably, the base sequence A2 is identical to the base sequence A1 at least in the base at position 21 of the base sequence A1 (SEQ ID NO: 1). More preferably, the partial base sequence A2-3' of 5 or more consecutive bases, preferably 8 or more consecutive bases, more preferably 10 or more consecutive bases, more preferably 15 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases from the 3'-terminal base of the base sequence A2 is identical to the partial base sequence A1-3' of the same number of consecutive bases from the 3'-terminal base (the 21st base of SEQ ID NO: 1) of the base sequence A1 (SEQ ID NO: 1), and the remaining partial base sequence A2-5' of the base sequence A2 on the 5'-terminal side of the partial base sequence A2-3' is homologous to the remaining partial base sequence A1-5' of the base sequence A1 on the 5'-terminal side of the partial base sequence A1-3'.

The base sequence A3 is a partial base sequence of the base sequence A1 (SEQ ID NO: 1) consisting of 5 or more consecutive bases, preferably 8 or more consecutive bases, more preferably 10 or more consecutive bases, more preferably 15 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases, starting from the 3'-terminal base (i.e., the 21st base of SEQ ID NO: 1).

The base sequence A4 is a base sequence homologous to the base sequence A3. Preferably, the base sequence A4 is identical to the base sequence A3 at least in the base corresponding to the 21st position of the base sequence A1 (SEQ ID NO: 1). More preferably, the partial base sequence A4-3' of 5 or more consecutive bases, preferably 8 or more consecutive bases, more preferably 10 or more consecutive bases, more preferably 15 or more consecutive bases, more preferably 17 or more consecutive bases, more preferably 20 or more consecutive bases from the 3'-terminal base of the base sequence A4 is identical to the partial base sequence A3-3' of the same number of consecutive bases from the 3'-terminal base of the base sequence A3 (corresponding to the 21st base of SEQ ID NO: 1), and the remaining partial base sequence A4-5' of the base sequence A4 on the 5'-terminal side of the partial base sequence A4-3' is homologous to the remaining partial base sequence A3-5' of the base sequence A3 on the 5'-terminal side of the partial base sequence A3-3'.

The oligonucleotide in primer B may contain a base sequence B selected from base sequence B1, base sequence B2, base sequence B3, and base sequence B4 at the 3' end, and may contain other base sequences added to the 5' end side of base sequence B, or may consist of only base sequence B. The length (number of bases) of the oligonucleotide in primer B is not particularly limited, but is usually 30 bases or less, preferably 28 bases or less, more preferably 25 bases or less, more preferably 23 bases or less, and more preferably 20 bases or less. Primer B may consist of the oligonucleotide alone, or may be an oligonucleotide to which other elements such as a labeling substance or a tag are linked. The other elements may be linked to any position of the oligonucleotide, but it is particularly preferred that they are linked to the 5' end of the oligonucleotide.
The base sequence B1 refers to the base sequence shown in SEQ ID NO:2.

The base sequence B2 is a base sequence homologous to the base sequence B1. Preferably, the bases of the base sequence B2 at least at positions 19, 11, and 10 of the base sequence B1 (SEQ ID NO: 2) are identical to those of the base sequence B1. More preferably, the partial base sequence B2-3' of 5 or more consecutive bases, preferably 8 or more consecutive bases, more preferably 10 or more consecutive bases, more preferably 11 or more consecutive bases, more preferably 15 or more consecutive bases, more preferably 17 or more consecutive bases from the 3'-terminal base of the base sequence B2 is identical to the partial base sequence B1-3' of the same number of consecutive bases from the 3'-terminal base (the 19th base of SEQ ID NO: 2) of the base sequence B1 (SEQ ID NO: 2), and the remaining partial base sequence B2-5' of the base sequence B2 on the 5'-terminal side of the partial base sequence B2-3' is homologous to the remaining partial base sequence B1-5' of the base sequence B1 on the 5'-terminal side of the partial base sequence B1-3'.

Base sequence B3 is a partial base sequence of base sequence B1 (SEQ ID NO:2) consisting of 5 or more consecutive bases, preferably 8 or more consecutive bases, more preferably 10 or more consecutive bases, more preferably 11 or more consecutive bases, more preferably 15 or more consecutive bases, more preferably 17 or more consecutive bases, starting from the 3'-terminal base (i.e., the 19th base of SEQ ID NO:2).

The base sequence B4 is a base sequence homologous to the base sequence B3. Preferably, the bases of the base sequence B4 are identical to those of the base sequence B3 at least in the bases corresponding to the 19th, 11th, and 10th positions of the base sequence B1 (SEQ ID NO: 2). More preferably, the partial base sequence B4-3' of 5 or more consecutive bases, preferably 8 or more consecutive bases, more preferably 10 or more consecutive bases, more preferably 11 or more consecutive bases, more preferably 15 or more consecutive bases, more preferably 17 or more consecutive bases from the 3'-terminal base of the base sequence B4 is identical to the partial base sequence B3-3' of the same number of consecutive bases from the 3'-terminal base of the base sequence B3 (the base corresponding to the 19th base of SEQ ID NO: 2), and the remaining partial base sequence B4-5' of the base sequence B4 on the 5'-terminal side of the partial base sequence B4-3' is homologous to the remaining partial base sequence B3-5' of the base sequence B3 on the 5'-terminal side of the partial base sequence B3-3'.

The primer set for detecting walnuts, which includes the above-mentioned primer A and primer B, may further include a probe. The probe may include an oligonucleotide that can bind (hybridize) under stringent conditions to at least a partial region of the target sequence in the amplification product amplified by the nucleic acid amplification reaction using primer A and primer B.

The probe may consist of only an oligonucleotide, or the oligonucleotide may be labeled with a fluorescent substance (e.g., FAM ^™ , TET ^™ , VIC ^™ , HEX ^™ , NED ^™ , PET, etc.) and/or a quencher (e.g., TAMRA ^™ , ROX ^™ , MGB, BHQ (Black Hole Quencher) ^™ , BBQ (Blackberry Quencher) ^™ , etc.). A probe labeled in this manner is also called a TaqMan ^™ probe. Preferably, a fluorescent substance is linked to one end of the oligonucleotide and a quencher is linked to the other end.

The probe is more preferably
(C1) the base sequence C1 shown in SEQ ID NO: 3,
(C2) a base sequence C2 homologous to the base sequence C1;
(C3) a base sequence C3 which is a partial base sequence of 5 or more consecutive bases of the base sequence C1;
(C4) a base sequence C4 homologous to the base sequence C3;
(C5) a base sequence C5 complementary to the base sequence C1;
(C6) a base sequence C6 homologous to the base sequence C5;
(C7) a base sequence C7 which is a partial base sequence of 5 or more consecutive bases of the base sequence C5;
(C8) A base sequence C8 that is homologous to the base sequence C5
(C9) a base sequence C9 which is a partial base sequence of 5 or more consecutive bases among the base sequence complementary to positions 22 to 45 of SEQ ID NO: 7; and
(C10) A base sequence C10 complementary to the base sequence C9
and probe C comprising an oligonucleotide comprising a base sequence C selected from the group consisting of:

With reference to the sequence table, Figure 4, and Table 1 in the Examples, we explain why probe C can bind to the amplification products of walnut DNA using primers A and B with similar efficiency regardless of the walnut species.

A specific example of probe C is the "JI2 probe" described in the Examples, which is composed of an oligonucleotide consisting of the base sequence C1 shown in SEQ ID NO: 3. As shown in FIG. 4, the JI2 probe is the reverse complementary sequence of the base sequence from positions 26 to 45 of SEQ ID NO: 7, which is part of the base sequence (AF303809.1) of ITS-2 DNA of Juglans regia. The JI2 probe can hybridize to the base sequence corresponding to positions 26 to 45 of SEQ ID NO: 7 in the amplification product of walnut DNA using primers A and B.

As shown in Figure 4, the 33rd and 43rd positions in the target region shown in SEQ ID NO:7 of ITS-2 of Juglans regia are bases that are common to the genus Juglans but different in the genus Carya (indicated by single underlining), and the 46th and 49th positions are bases that are polymorphic within the genus Juglans (indicated by double underlining).

The JI2 probe (consisting of the base sequence shown in SEQ ID NO:3) hybridizes to the base sequence corresponding to positions 26 to 45 of the base sequence shown in SEQ ID NO:7 of ITS-2 of Juglans regia, and can therefore hybridize with the same degree of efficiency to the amplification products of walnut DNA using primers A and B, regardless of the species of walnut. Similarly, another primer C that can hybridize to the region of ITS-2 of walnut DNA to which the JI2 probe hybridizes, or its complementary strand, can also hybridize with the same degree of efficiency to the amplification products of walnut DNA using primers A and B, regardless of the species of walnut.

For comparison, Figure 4 also shows the corresponding position on the ITS-2 base sequence of the WalITS probe (SEQ ID NO: 6) described in Non-Patent Document 5. The WalITS probe contains a portion that hybridizes to bases at positions 46 and 49 of SEQ ID NO: 7, which are polymorphic within the genus Juglans. For this reason, when using the WalITS probe to detect amplification products by nucleic acid amplification reaction, there is a possibility that the detection sensitivity will differ between species of the genus Juglans, and this is considered undesirable.

Next, a more preferred embodiment of the probe C will be described.
The oligonucleotide in the probe C may contain at least a part of the base sequence C selected from the base sequence C1, base sequence C2, base sequence C3, base sequence C4, base sequence C5, base sequence C6, base sequence C7, and base sequence C8, and may contain other base sequences added to the 3'-end and/or 5'-end (preferably only the 5'-end) of the base sequence C, or may consist of only the base sequence C. The length (number of bases) of the oligonucleotide in the probe C is not particularly limited, but is usually 30 bases or less, preferably 28 bases or less, and more preferably 25 bases or less. The probe C may consist of only the oligonucleotide, but preferably the fluorescent substance and/or the quencher is linked to the oligonucleotide.
The base sequence C1 refers to the base sequence shown in SEQ ID NO:3.

The base sequence C2 is a base sequence homologous to the base sequence C1. Preferably, the bases of the base sequence C2 are identical to those of the base sequence C1 at least in the third and thirteenth positions of the base sequence C1 (SEQ ID NO: 3). More preferably, the base sequence C2 has a partial base sequence C2-1 of 5 or more consecutive bases, preferably 8 or more consecutive bases, more preferably 10 or more consecutive bases, more preferably 15 or more consecutive bases, more preferably 17 or more consecutive bases, which is identical to the corresponding partial base sequence C1-1 of the base sequence C1 (SEQ ID NO: 3), and the remaining partial base sequence of the base sequence C2 other than the partial base sequence C2-1 is homologous to the remaining partial base sequence of the base sequence C1 other than the partial base sequence C1-1.

The base sequence C3 is a partial base sequence of the base sequence C1 (SEQ ID NO: 3) that is 5 or more consecutive bases, preferably 8 or more consecutive bases, more preferably 10 or more consecutive bases, more preferably 15 or more consecutive bases, more preferably 17 or more consecutive bases. It is preferable that the base sequence C3 is a partial base sequence that includes at least the bases at positions 3 and 13 of the base sequence C1 (SEQ ID NO: 3).

The base sequence C4 is a base sequence homologous to the base sequence C3. Preferably, the base sequence C4 is identical to the base sequence C3 at least in the bases corresponding to the 3rd and 13th positions of the base sequence C1 (SEQ ID NO: 3). More preferably, the base sequence C4 has a partial base sequence C4-1 of 5 or more consecutive bases, preferably 8 or more consecutive bases, more preferably 10 or more consecutive bases, more preferably 15 or more consecutive bases, more preferably 17 or more consecutive bases, which is identical to the corresponding partial base sequence C3-1 of the base sequence C3, and the remaining partial base sequence of the base sequence C4 other than the partial base sequence C4-1 is homologous to the remaining partial base sequence of the base sequence C3 other than the partial base sequence C3-1.
The base sequence C5 is a base sequence complementary to the base sequence C1 shown in SEQ ID NO:3.

The base sequence C6 is a base sequence homologous to the base sequence C5. Preferably, the bases of the base sequence C6 are identical to those of the base sequence C5 at least in the bases corresponding to the bases at positions 3 and 13 of the base sequence C1 (SEQ ID NO: 3). More preferably, the base sequence C6 has a partial base sequence C6-1 of 5 or more consecutive bases, preferably 8 or more consecutive bases, more preferably 10 or more consecutive bases, more preferably 15 or more consecutive bases, more preferably 17 or more consecutive bases, which is identical to the corresponding partial base sequence C5-1 of the base sequence C5, and the remaining partial base sequence of the base sequence C6 other than the partial base sequence C6-1 is homologous to the remaining partial base sequence of the base sequence C5 other than the partial base sequence C5-1.

The base sequence C7 is a partial base sequence of the base sequence C5, which is 5 or more consecutive bases, preferably 8 or more consecutive bases, more preferably 10 or more consecutive bases, more preferably 15 or more consecutive bases, more preferably 17 or more consecutive bases. It is preferable that the base sequence C7 is a partial base sequence of the base sequence C5, which includes bases corresponding to the bases at the 3rd and 13th positions of the base sequence C1 (SEQ ID NO: 3).

The base sequence C8 is a base sequence homologous to the base sequence C7. Preferably, the bases of the base sequence C8 are identical to those of the base sequence C7 at least in the bases corresponding to the third and thirteenth positions of the base sequence C1 (SEQ ID NO: 3). More preferably, the base sequence C8 has a partial base sequence C8-1 of 5 or more consecutive bases, preferably 8 or more consecutive bases, more preferably 10 or more consecutive bases, more preferably 15 or more consecutive bases, more preferably 17 or more consecutive bases, which is identical to the corresponding partial base sequence C7-1 of the base sequence C7, and the remaining partial base sequence of the base sequence C8 other than the partial base sequence C8-1 is homologous to the remaining partial base sequence of the base sequence C7 other than the partial base sequence C7-1.

The base sequence C9 is a partial base sequence of 5 or more consecutive bases, preferably 8 or more consecutive bases, more preferably 10 or more consecutive bases, more preferably 15 or more consecutive bases, more preferably 17 or more consecutive bases, of the base sequence complementary to positions 22 to 45 of SEQ ID NO: 7. The base sequence C9 is preferably a partial base sequence of the base sequence complementary to positions 22 to 45 of SEQ ID NO: 7, including at least the 3rd and 13th bases from the 5' end.

The base sequence C10 is a base sequence complementary to the base sequence C9. Preferably, the base sequence C10 is identical to the base sequence C9 in at least the bases corresponding to the third and thirteenth positions from the 5' end of the base sequence complementary to the 22nd to 45th positions of SEQ ID NO:7. More preferably, the base sequence C10 has a partial base sequence C10-1 of 5 or more consecutive bases, preferably 8 or more bases, more preferably 10 or more bases, more preferably 15 or more bases, more preferably 17 or more bases, which is identical to the corresponding partial base sequence C9-1 of the base sequence C9, and the remaining partial base sequence of the base sequence C10 other than the partial base sequence C10-1 is homologous to the remaining partial base sequence of the base sequence C9 other than the partial base sequence C9-1.

The oligonucleotides contained in the above primer A, primer B or probe can be synthesized by a method known in the art for synthesizing oligonucleotides, such as the phosphotriethyl method or the phosphodiester method, using a commonly used automated DNA synthesizer.

In the primer set according to one or more embodiments of the present invention, each of the above elements may be contained in a separate container, or each element may be contained in a container for a single use and the amount to be used. Alternatively, an amount sufficient for multiple uses may be contained in one container (a user can take out and use the amount required for one use). Each of the above elements may be contained in a container in a dry form, or in a form dissolved in an appropriate solvent.

The primer set according to one or more embodiments of the present invention can include one or more elements selected from a dNTP mix, a DNA polymerase, ^SYBR® Green I, magnesium chloride, and instructions for use.

The primer set according to one or more embodiments of the present invention may be combined with one or more other primer sets for detecting food allergens to form a kit for detecting food allergens.

The one or more other primer sets for detecting food allergens include one or more primer sets for detecting food allergens such as wheat, buckwheat, and peanuts. As the primer set for detecting wheat, the primer set for detecting buckwheat, and/or the primer set for detecting peanuts, the primer set described in Patent Document 1 (JP Patent Publication No. 2016-101142) is preferred. The primer set according to one or more embodiments of the present invention is capable of amplifying and detecting walnut DNA by a real-time PCR method under common PCR conditions for detecting wheat, buckwheat, and peanuts as described in Patent Document 1. Therefore, by using the primer set according to one or more embodiments of the present invention, it is possible to detect walnuts under PCR conditions for detecting wheat, buckwheat, and/or peanuts. Therefore, the detection of wheat, buckwheat, and/or peanuts, and walnuts can be carried out simultaneously under the same conditions, which reduces the effort required for analysis.

3. How to detect walnuts in a sample
According to one or more embodiments of the present invention, a method for detecting walnuts in a sample comprises the steps of:
The method includes: performing a nucleic acid amplification reaction using DNA derived from the sample as a template and the primer set for detecting walnuts; and detecting an amplification product obtained by the nucleic acid amplification reaction.

The sample is preferably a food or drink sample. The food or drink sample may be a sample containing a food or drink composition, a raw material for a food or drink composition, a raw material for a food or drink in the middle of processing, or a processed product thereof (e.g., an extract, a crushed product, a dissolved product), etc. The results obtained by this walnut detection method can be used to confirm the accuracy of allergy labeling and to determine whether or not allergy labeling should be made.
The range of walnuts to be detected is as described above.

DNA can be extracted from a sample using any method known to those skilled in the art as a nucleic acid extraction method, such as the phenol/chloroform method, cell lysis with a surfactant or protease enzyme, physical destruction using glass beads, repeated freezing and thawing, or a combination of these. In addition, various commercially available DNA extraction kits such as DNeasy Plant mini Kit, Genomic-tip 20/G, DNeasy mericon Food Kit (all from QIAGEN), and GM quicker 4 (Nippon Gene) can also be used.

The concentration and purity of the extracted DNA can be evaluated by measuring the absorbance at wavelengths of 230, 260, and 280 nm using a spectrophotometer.
For example, the concentration of extracted DNA can be calculated using the following formula:
DNA concentration (ng/μL) = absorbance at a wavelength of 260 nm × 50

The degree of purification of DNA is preferably such that the absorbance ratio at 260/230 nm is 2.0 or more, and the absorbance ratio at 260/280 nm is 1.8 to 2.0, so that the nucleic acid amplification reaction can proceed smoothly.

Furthermore, the extracted DNA may be subjected to a nucleic acid amplification reaction using a primer set for an endogenous gene shared by plants or animals, and it may be confirmed that an amplified product is obtained.

The concentration of DNA extracted from the sample can be adjusted appropriately based on the DNA preparation method described in the information provided by the Consumer Affairs Agency, "About testing methods for foods containing allergens." The DNA with the adjusted concentration can be used as template DNA as appropriate. It is preferably used as template DNA in an amount of 5 to 500 ng, more preferably 50 ng (this amount is specified in the information provided by the Consumer Affairs Agency, "About testing methods for foods containing allergens").

The nucleic acid amplification reaction may be a nucleic acid amplification reaction using a heat-stable polymerase or a nucleic acid amplification reaction using a strand-displacing polymerase. The polymerase is preferably a DNA polymerase.

An example of a nucleic acid amplification reaction method that uses DNA polymerase is the polymerase chain reaction (PCR).

The PCR method can be carried out based on the usual method described in, for example, Saiki RK, et al., Science, 230: 1350-1354 (1985) or Plant Cell Engineering Special Issue, Plant PCR Experimental Protocol, edited by Shimamoto Isao and Sasaki Takuji (1995). The PCR conditions (temperature and time for each step of denaturation, annealing, and extension, as well as the number of cycles, etc.) and the composition of the PCR reaction solution (type of buffer, primer concentration, type and concentration of DNA polymerase, dNTP concentration, magnesium chloride concentration, etc.) can be appropriately selected and set by a person skilled in the art through preliminary experiments, etc., so that a PCR amplification product can be obtained with high sensitivity in PCR using the primer set. PCR master mixes in which the DNA polymerase, dNTP concentration, magnesium chloride concentration, etc. are almost optimized are commercially available, and these may be used.

Qualitative PCR can be used as the PCR method. In qualitative PCR, walnut DNA contained in DNA extracted from a sample is amplified by PCR using the primer set including primer A and primer B, and then separated and stained by electrophoresis to detect it.

When the primer set including primer A and primer B is used, the presence or absence of an amplification product, preferably an amplification product of about 74 bp in size, can be used as an indicator to detect the presence or absence of walnut contamination. If an amplification product containing at least a portion of the walnut ITS-2 sequence, preferably of the above size, is detected, it can be determined that the sample is contaminated with walnuts, and if the amplification product is not detected, it can be determined that the sample is not contaminated with walnuts.

Using the above primer set allows for highly sensitive detection of walnuts. As detailed in the Examples below, 50 fg of walnut DNA can be detected in a 25 μL PCR reaction system. This detection sensitivity is comparable between walnut species.

The primer set preferably further includes a probe, and more preferably includes the probe C. The amplification product can be detected by detecting binding between the amplification product and the probe. An example of a detection method using a probe is a real-time PCR method using the TaqMan ^™ method described below.

The PCR method can be a real-time PCR method.

For the real-time PCR method, either the intercalator method or the TaqMan ^™ method can be used, but it is preferable to use the TaqMan ^™ method.

In the intercalator method, a fluorescent compound (e.g., SYBR ^{(registered trademark)} Green I, etc.) is added to the PCR reaction solution, and this binds to the amplified DNA. When this is irradiated with excitation light, it emits fluorescence, and by measuring the intensity of this fluorescence, the amount of the amplified product produced can be measured.

On the other hand, in the TaqMan ^TM method, a probe (TaqMan ^TM probe) labeled with a fluorescent substance is included in the PCR reaction solution, and this binds (hybridizes) to the target sequence in the DNA amplified in the annealing step. In the subsequent extension reaction step, the probe bound (hybridized) to the DNA is decomposed by the 5'→3' exonuclease activity of Taq DNA polymerase, at which time the fluorescent substance is released from the probe and emits fluorescence. By measuring this fluorescence intensity, the amount of amplified product produced can be measured. The device for performing real-time PCR is not particularly limited, and various models that enable real-time PCR, such as ABI PRISM 7900HT (Life Technologies), LightCycler 96 (Roche Diagnostics), etc., can be used, and is not particularly limited.

In the real-time PCR method, the walnut DNA contained in the DNA extracted from the sample is amplified by PCR using the primer set, and the amount of PCR amplified product is measured by measuring the fluorescence intensity as described above to obtain an amplification curve. Next, the cycle number at which the fluorescence signal significantly increases (Cq value) is used as an index, or an appropriate threshold (Threshold) of the increase in the amount of fluorescence (ΔRn) is set in the region where the fluorescence signal is exponentially related to the cycle number, and the cycle number (Ct value (Threshold Cycle)) at which the amplification curve intersects with the threshold (Threshold) under appropriate cycle number conditions is used as an index to detect the presence or absence of walnut contamination. In other words, if a significant increase in the fluorescence signal is observed or the amplification curve intersects with the threshold under appropriate cycle number conditions, it indicates that walnuts are present in the sample, while if no significant increase in the fluorescence signal is observed or the amplification curve does not intersect with the threshold, it indicates that walnuts are not present in the sample.

The qualitative PCR method or real-time PCR method using the above primer set and the qualitative PCR method or real-time PCR method using the wheat detection primer set, buckwheat detection primer set, and/or peanut detection primer set described in Patent Document 1 can obtain target amplification products only in the desired sequence while maintaining the desired sensitivity, even under the same conditions. Therefore, the walnut detection method of the present invention can be performed simultaneously under the same conditions as the wheat detection method, buckwheat detection method, and/or peanut detection method described in Patent Document 1, and food allergens in a sample can be detected quickly and efficiently.

Specific embodiments of the present invention will be described in detail below based on examples, but the technical scope of the present invention is not limited to the following examples.

1. Goals
The objectives of the walnut qualitative real-time PCR method were to enable analysis under the same PCR temperature conditions as the wheat, buckwheat, and peanut qualitative real-time PCR method, to specifically detect walnuts belonging to the genus Juglans, and to ensure that the sensitivity does not differ between species of the genus Juglans.

2. Method
2.1. Sample
The following four types of samples were obtained from the market. All of them were roasted.
Shelled oak walnut (Juglans regia)
Shelled Japanese walnut (Juglans mandshurica, or Juglans airanthifolia)
Shelled pecans (Carya illinoinensis)
Carya cathayensis kernels

2.2. DNA Extraction
The shells of Japanese walnuts were cracked and the kernels (0.1 g) were extracted with Genomic-tip 20/G according to the kit's instructions to obtain an extracted DNA solution.

For walnuts and pecans, 0.1 g and 0.5 g of kernels were cracked and removed from the shells, respectively, and DNA solutions were extracted using a GM Quicker 4 according to the kit's instructions.

1.0 g of Yamanaka peach was obtained by scraping off the kernel, and an extracted DNA solution was obtained using a GM Quicker 4 according to the kit's instructions.

The DNA concentration of the extracted DNA solution was quantified using a microspectrophotometer and diluted to 20 ng/μL with TE (pH 8.0).

The serial dilutions of the extracted DNA from Japanese walnut and oak walnut were made by diluting the DNA ten-fold with 20 ng/μL of salmon sperm DNA. The extracted DNA solutions with final concentrations of 20 pg/μL, 2 pg/μL, 200 fg/μL, and 20 fg/μL, respectively, were used for real-time PCR.

<2.3. Primers and probes>

<2.4. Real-time PCR>
The PCR reaction solution contained 1x QuantiTect Probe PCR Master mix, 0.2 μM forward primer and reverse primer, and 0.1 M probe, and was prepared by adding 2.5 μL of template DNA sample solution to make the total volume 25 μL. The LightCycler 96 was used as the apparatus.

There are three types of PCR temperature conditions:

Condition 1 is a common PCR condition for detecting wheat, buckwheat, and peanuts, as described in Patent Document 1 (JP Patent Publication No. 2016-101142).

Condition 1' is a condition in which the number of cycles in condition 1 is extended from 42 cycles to 50 cycles.

Condition 2 is the optimized condition for PCR using the comparative primer probe set (WalITS).

The analysis conditions used to calculate the Cq value were the default in LightCycler 96.

The extracted DNA solution was used as the template DNA sample solution. The amount of DNA in the measurement sample was as follows:
Oak walnut DNA, Japanese walnut DNA: 50 pg, 5 pg, 500 fg, 50 fg respectively
Pecan DNA, Japanese peach DNA: 50 ng (= 5 x ¹⁰⁷ fg)

2.5. Calculation of DNA amount
A calibration curve was created with the common logarithm of the nominal DNA amount of the walnut DNA sample on a fg basis on the x-axis and the obtained Cq value on the y-axis. The DNA amount was calculated from the slope and intercept of the calibration curve.
DNA amount (fg) = 10^((Cq value of sample - intercept) / slope)

3. Results
The amounts of DNA calculated from the PCR results are shown in the following table.

Figure 1 shows the amplification curve of real-time PCR performed under condition 1 using the primer probe set of the example.

Figure 2 shows the amplification curve of real-time PCR performed under condition 1' using the primer probe set of the comparative example.

Figure 3 shows the amplification curve of real-time PCR performed under condition 2 using the primer probe set of the comparative example.

Using the primer probe set of the embodiment, real-time PCR was performed under condition 1, which is a common PCR condition for detecting wheat, buckwheat, and peanuts as described in Patent Document 1 (JP Patent Publication 2016-101142 A), and was able to detect Japanese walnut and oak walnut without mistakenly detecting pecan and Japanese walnut. The detection sensitivity of Japanese walnut was within 1/2 to 2 times that of oak walnut.

When real-time PCR was performed under condition 1 using the primer probe set of the comparative example, there were samples in which the amount of amplified product did not reach the threshold value at the 42nd cycle. Therefore, real-time PCR was performed under condition 1', which was the same as condition 1, except that the number of cycles was extended to 50, using the primer probe set of the comparative example. As a result, although pecan was not erroneously detected, the detection sensitivity of Japanese walnut was approximately 1/100 of that of Japanese walnut.

When real-time PCR was performed using the comparative primer-probe set under condition 2, which was optimized according to the sequence of the comparative primer-probe set, the detection sensitivity of Japanese walnut was improved to about half that of Japanese walnut, but the shapes of the amplification curves differed between Japanese walnut and Japanese walnut, and Japanese walnut was mistakenly detected (Figure 3).

<4. Discussion>
The primer-probe set of the embodiment was able to specifically detect walnuts belonging to the genus Juglans by real-time PCR under common PCR conditions for detecting wheat, buckwheat, and peanuts, as described in Patent Document 1 (JP Patent Publication No. 2016-101142), and the sensitivity did not differ between species of the genus Juglans. In other words, the above-mentioned goal was achieved.

On the other hand, the primer probe set of the comparative example showed different detection sensitivities between Juglans species, both under common PCR conditions for detecting wheat, buckwheat, and peanuts, and under optimized PCR conditions, and the above-mentioned goal could not be achieved.

The present invention can be used for the purpose of detecting allergens in the food and beverage industry.

Claims (5)

A primer set for detecting walnuts,
Primer A comprising an oligonucleotide containing the base sequence A1 shown in SEQ ID NO: 1 at its 3'end; and
Primer B containing an oligonucleotide containing the base sequence B1 shown in SEQ ID NO:2 at its 3' end
A primer set comprising: (C1) the base sequence C1 shown in SEQ ID NO: 3,
(C2) a base sequence C2 homologous to the base sequence C1;
(C3) a base sequence C3 which is a partial base sequence of 5 or more consecutive bases of the base sequence C1;
(C4) a base sequence C4 homologous to the base sequence C3;
(C5) a base sequence C5 complementary to the base sequence C1;
(C6) a base sequence C6 homologous to the base sequence C5;
(C7) a base sequence C7 which is a partial base sequence of 5 or more consecutive bases of the base sequence C5;
(C8) A base sequence C8 that is homologous to the base sequence C5
(C9) a base sequence C9 which is a partial base sequence of 5 or more consecutive bases among the base sequence complementary to positions 22 to 45 of SEQ ID NO: 7; and
(C10) A base sequence C10 complementary to the base sequence C9
The primer set according to claim 1 , further comprising a probe C comprising an oligonucleotide comprising a base sequence C selected from the group consisting of: 1. A method for detecting walnuts in a sample, comprising:
A method comprising: carrying out a nucleic acid amplification reaction using DNA derived from the sample as a template and the primer set according to claim 1 or 2; and detecting an amplification product by the nucleic acid amplification reaction. The primer set is the primer set according to claim 2,
The detection of the amplification product is carried out by detecting binding between the amplification product and the probe C.
The method according to claim 3. The method according to claim 3 or 4, wherein the sample is a food or drink sample.