JP6260986B2 - Method for detecting filaggrin gene mutation and use thereof - Google Patents

Description

  The present invention relates to a method for detecting mutations in the filaggrin gene and uses thereof. Specifically, it relates to a novel filaggrin gene mutation unique to Japanese and a method for detecting the same, a method for comprehensively detecting filaggrin gene mutation, and the like.

  Atopic dermatitis develops with various etiology and exacerbation factors, and has created its pathology, but barrier dysfunction of the stratum corneum is also a major onset factor. In 2006, it was shown in British that a mutation in the epidermis barrier-related protein, filaggrin, was an important onset factor for atopic dermatitis (Non-patent Document 1). In 2007, our research group identified two filaggrin gene mutations in Japanese for the first time outside of Europe, and showed that they were a significant onset factor for atopic dermatitis even in Japanese ( Non-patent document 2). Subsequently, Japanese filaggrin gene mutations were comprehensively identified (a total of 7), and it was clarified that bilagulin gene mutations were a causative factor in at least 27% of Japanese patients with atopic dermatitis ( Non-patent documents 3 to 5). Interestingly, filaggrin gene mutations vary greatly among races, and there is little to no difference between mutations in Japanese and Europeans.

  Filaggrin is important for the barrier function of the skin. Profilagrin, the precursor of filaggrin, is an important component of the keratohyaline granules of the epidermis. In the process of keratinization, one profilagrin is cleaved into 10 to 12 filaggrin molecules, and finally keratin and filaggrin degradation products fill the keratinocytes. This process is very important for normal keratinization and skin barrier formation. When there is a mutation in the FLG gene that encodes profilaggrin, if profilagrin is deficient or markedly reduced, keratohyaline granule formation is impaired, normal keratinization processes are impaired, and skin barrier function is reduced. It is thought that it will come (Non-Patent Document 1).

Smith FJ et al. Nat Genet. 2006; 38 (3): 337-42. Nomura et al. J Allergy Clin Immunol. 2007 Feb; 119 (2): 434-40. Nomura et al. J Invest Dermatol. 2008; 128 (6): 1436-41. Nomura et al. J Invest Dermatol. 2009; 129 (5): 1302-5. Nemoto-Hasebe et al. Br J Dermatol. 2009 Dec; 161 (6): 1387-90. Hamada T, Sandilands A, Fukuda S, et al. J Invest Dermatol 2008; 128: 1323-5. Maki Akiyama. Journal of the Japan Medical Association. 138: 2536-2537; 2010

  Eight kinds of filaggrin gene mutations in Japanese have been reported (Non-Patent Documents 2 to 6). In Japan, the prevalence of filaggrin gene mutation in patients with atopic dermatitis is about 27% in Japan, but some ethnic groups with about 50% in Europe have been reported. In view of this fact, Japanese filaggrin gene mutations may exist in addition to the eight types that are currently known. In addition, there are data that about 20% of Japanese are atopic dermatitis patients, and many samples need to be processed for clinical application. However, currently used methods (direct sequencing and PCR by the Sanger method) -RFLP (restriction fragment length polymorphism of PCR products) method requires about 10,000 yen per person and 4 days. Therefore, a method capable of processing a large number of specimens quickly and inexpensively for clinical examination is desired. In addition, since filaggrin gene mutations are significantly different among races, a method capable of accurately detecting mutations specific to Japanese is required.

  Therefore, a first object of the present invention is to identify and provide a novel filaggrin gene mutation. A second object of the present invention is to provide a filaggrin gene mutation detection method and its use that are suitable for clinical application and effective for testing of Japanese specimens.

The present inventors proceeded with investigation under the above problems. And we succeeded in identifying two novel gene mutations from the results of the complete sequence of filaggrin gene in Japanese patients with ichthyosis. On the other hand, a probe and primer pair capable of detecting a total of 10 types of Japanese filaggrin gene mutations in combination with the gene mutations reported in the past and at a low cost were designed, and their effectiveness was confirmed. That is, it succeeded in establishing the detection method which can endure practicality regarding the filaggrin gene variation peculiar to Japanese. The presence / absence and type of filaggrin gene mutation can be used to predict the onset of atopic dermatitis.
The following invention is mainly based on the above-mentioned results.
[1] For a nucleic acid sample collected from a Japanese subject, a filaggrin gene mutation comprising a step of detecting the following (1) or (2) or both of the following (1) and (2): How to detect:
(1) c.441_2delAG mutation in the filaggrin gene;
(2) c.5368C> T mutation of the filaggrin gene.
[2] The mutation detection method according to [1], wherein in addition to the mutation, one or more mutations selected from the group consisting of the following (3) to (10) are also detected:
(3) c.1501C> T mutation;
(4) c.5084C> G mutation;
(5) c.5101C> G mutation;
(6) c.7661C> G mutation;
(7) c.8666_7CC> GA mutation;
(8) c.9887C> A mutation;
(9) c.12064A> T mutation; and (10) c.3321delA mutation.
[3] The mutation detection method according to [2], wherein all of (1) to (10) are targeted for detection.
[4] The mutation detection method according to any one of [1] to [3], wherein each mutation is detected by a real-time PCR method.
[5] The mutation detection method according to any one of [1] to [4], wherein the real-time PCR method is a measurement method using a fluorescent probe using the principle of fluorescence resonance energy transfer.
[6] A risk test method for atopic dermatitis, comprising the step of determining a genetic risk of atopic dermatitis from the mutation information obtained in the step.
[7] A nucleic acid probe for detecting a mutation in the filaggrin gene, targeting a DNA sequence containing the mutation position of the c.441_2delAG mutation in the filaggrin gene.
[8] A DNA sequence that includes the mutation position of the c.441_2delAG mutation of the filaggrin gene is a target and consists of a sequence that hybridizes to the DNA region when the mutation position is a mutant type. The 3 ′ end contains a fluorescent probe to which a quencher that induces quenching of the reporter fluorescent dye is bound by the principle of fluorescence resonance energy transfer, or the mutation position of the c.441_2delAG mutation of the filaggrin gene It consists of a sequence that targets the DNA sequence and hybridizes to the DNA region when the mutation position is the wild type. A reporter fluorescent dye is bound to the 5 'end, and fluorescence resonance energy transfer to the 3' end. A fluorescent probe bound with a quencher that induces quenching of the reporter fluorescent dye according to the principle of
The nucleic acid probe for mutation detection according to [7], wherein
[9] A nucleic acid probe for detecting a mutation in the filaggrin gene, targeting a DNA sequence containing the mutation position of the c.5368C> T mutation in the filaggrin gene.
[10] Targeting a DNA sequence containing the mutation position of the filaggrin gene c.5368C> T mutation, comprising a sequence that hybridizes to the DNA region when the mutation position is a mutant type, and reporter fluorescence at the 5 ′ end A fluorescent probe to which a quencher that induces quenching of the reporter fluorescent dye by the principle of fluorescence resonance energy transfer is bound, or the c.5368C> T mutation of the filaggrin gene. It consists of a sequence that hybridizes to the DNA region when the DNA sequence containing the mutation position is targeted, and the mutation position is wild type, and a reporter fluorescent dye is bound to the 5 ′ end, and the 3 ′ end, A fluorescent probe bound with a quencher that induces quenching of the reporter fluorescent dye by the principle of fluorescence resonance energy transfer;
The nucleic acid probe for mutation detection according to [9], wherein
[11] (1) a nucleic acid set for detecting the 441_2delAG mutation of the filaggrin gene,
(2) Nucleic acid set for detecting c.5368C> T mutation of filaggrin gene,
(3) Nucleic acid set for detecting c.1501C> T mutation of filaggrin gene,
(4) Nucleic acid set for detecting c.5084C> G mutation of filaggrin gene,
(5) Nucleic acid set for detecting c.5101C> G mutation of filaggrin gene,
(6) Nucleic acid set for detecting c.7661C> G mutation of filaggrin gene,
(7) Nucleic acid set for detecting c.8666_7CC> GA mutation of filaggrin gene,
(8) Nucleic acid set for detecting c.9887C> A mutation of filaggrin gene,
(9) a nucleic acid set for detecting c.12064A> T mutation of filaggrin gene and (10) a nucleic acid set for detecting c.3321delA mutation of filaggrin gene, comprising two or more nucleic acid sets selected from the group consisting of: A filaggrin gene mutation detection kit comprising:
Each nucleic acid set is
It consists of a sequence that hybridizes to the DNA sequence when the mutation position is a mutant type, targeting the DNA sequence containing the mutation position to be detected, and the first reporter fluorescent dye is bound to the 5 ′ end, At the 3 ′ end, a first fluorescent probe to which a quencher that induces quenching of the reporter fluorescent dye by the principle of fluorescence resonance energy transfer is bound, and / or a DNA sequence including a mutation position to be detected is targeted, It consists of a sequence that hybridizes to the DNA sequence when the mutation position is wild type, and a second reporter fluorescent dye different from the first reporter fluorescent dye is bound to the 5 'end, and the 3' end A second fluorescent probe to which a quencher that induces quenching of the reporter fluorescent dye is bound by the principle of fluorescence resonance energy transfer; and
A primer pair for amplifying a DNA region containing the DNA sequence;
A mutation detection kit comprising:
[12] The mutation detection kit according to [11], comprising at least the above (1) or (2) or both (1) and (2).
[13] The mutation detection kit according to [11], including all of (1) to (10).
[14] The first fluorescent probe in the nucleic acid set of (1) includes the sequence of SEQ ID NO: 3,
The second fluorescent probe in the nucleic acid set of (1) includes the sequence of SEQ ID NO: 4, the first fluorescent probe in the nucleic acid set of (2) includes the sequence of SEQ ID NO: 5,
The second fluorescent probe in the nucleic acid set of (2) includes the sequence of SEQ ID NO: 6, the first fluorescent probe in the nucleic acid set of (3) includes the sequence of SEQ ID NO: 7,
The second fluorescent probe in the nucleic acid set of (3) includes the sequence of SEQ ID NO: 8, the first fluorescent probe in the nucleic acid set of (4) includes the sequence of SEQ ID NO: 9,
The second fluorescent probe in the nucleic acid set of (4) includes the sequence of SEQ ID NO: 10, the first fluorescent probe in the nucleic acid set of (5) includes the sequence of SEQ ID NO: 11,
The second fluorescent probe in the nucleic acid set of (5) includes the sequence of SEQ ID NO: 12, the first fluorescent probe in the nucleic acid set of (6) includes the sequence of SEQ ID NO: 13,
The second fluorescent probe in the nucleic acid set of (6) includes the sequence of SEQ ID NO: 14, the first fluorescent probe in the nucleic acid set of (7) includes the sequence of SEQ ID NO: 15,
The second fluorescent probe in the nucleic acid set of (7) includes the sequence of SEQ ID NO: 16, the first fluorescent probe in the nucleic acid set of (8) includes the sequence of SEQ ID NO: 17,
The second fluorescent probe in the nucleic acid set of (8) includes the sequence of SEQ ID NO: 18, the first fluorescent probe in the nucleic acid set of (9) includes the sequence of SEQ ID NO: 19,
The second fluorescent probe in the nucleic acid set of (9) includes the sequence of SEQ ID NO: 20, the first fluorescent probe in the nucleic acid set of (10) includes the sequence of SEQ ID NO: 21,
The second fluorescent probe in the nucleic acid set of (10) comprises the sequence of SEQ ID NO: 22;
The mutation detection kit according to any one of [11] to [13].
[15] The primer pair in the nucleic acid set of (1) comprises a forward primer consisting of the sequence of SEQ ID NO: 23 and a reverse primer consisting of the sequence of SEQ ID NO: 24,
The primer pair in the nucleic acid set of (2) comprises a forward primer comprising the sequence of SEQ ID NO: 25 and a reverse primer comprising the sequence of SEQ ID NO: 26,
The primer pair in the nucleic acid set of (3) comprises a forward primer comprising the sequence of SEQ ID NO: 27 and a reverse primer comprising the sequence of SEQ ID NO: 28,
The primer pair in the nucleic acid set of (4) comprises a forward primer comprising the sequence of SEQ ID NO: 29 and a reverse primer comprising the sequence of SEQ ID NO: 30;
The primer pair in the nucleic acid set of (5) comprises a forward primer comprising the sequence of SEQ ID NO: 31 and a reverse primer comprising the sequence of SEQ ID NO: 32,
The primer pair in the nucleic acid set of (6) comprises a forward primer comprising the sequence of SEQ ID NO: 33 and a reverse primer comprising the sequence of SEQ ID NO: 34,
The primer pair in the nucleic acid set of (7) comprises a forward primer comprising the sequence of SEQ ID NO: 35 and a reverse primer comprising the sequence of SEQ ID NO: 36,
The primer pair in the nucleic acid set of (8) comprises a forward primer comprising the sequence of SEQ ID NO: 37 and a reverse primer comprising the sequence of SEQ ID NO: 38,
The primer pair in the nucleic acid set of (9) comprises a forward primer comprising the sequence of SEQ ID NO: 39 and a reverse primer comprising the sequence of SEQ ID NO: 40;
The primer pair in the nucleic acid set of (10) comprises a forward primer comprising the sequence of SEQ ID NO: 41 and a reverse primer comprising the sequence of SEQ ID NO: 42.
The mutation detection kit according to any one of [11] to [14].

List of primer pairs and probes for TaqMan®-PCR. Analysis results of filaggrin gene mutation. The mutation retention rate is (89 + 2) /820=11.1%.

The first aspect of the present invention relates to a method for detecting a mutation in the filaggrin gene (hereinafter also referred to as the mutation detection method of the present invention), and the step of detecting a specific mutation in a nucleic acid sample collected from a Japanese subject comprises Done. In the detection method of the present invention, the filaggrin gene mutation unique to the Japanese newly identified by the present inventors is targeted for detection. Specifically, the following mutation (1), mutation (2), or both are the objects of mutation.
(1) c.441_2delAG mutation (2) c.5368C> T mutation (p.Gln1790X)

  In accordance with common practice, in the present specification, each mutation position is specified by the number of the cDNA translation initiation codon standard, that is, the position numbered with the translation initiation codon A as the first (base 1). For mutations involving amino acid substitutions in the amino acid sequence encoded by the filaggrin gene, the corresponding amino acid substitutions may be indicated together.

  The mRNA sequence of the wild-type (normal) filaggrin gene is represented by SEQ ID NO: 1 (NCBI GenBank ACCESSION NM_002016 XM_048104 XM_378901, DEFINITION Homo sapiens filaggrin (FLG), mRNA.), And the amino acid sequence of SEQ ID NO: 2 (NCBI GenPept ACCESSION NP_002007 XP_378901, DEFINITION filaggrin [Homo sapiens].)

  The c.441_2delAG mutation represents a deletion of base 441 and base 442. The c.5368C> T mutation represents substitution of the 5368th base C to T. This mutation results in the substitution of the 1790 amino acid Gln to the stop codon (nonsense mutation).

  In the present invention, a mutation is detected in order to determine whether the nucleic acid sample has a specific mutation, that is, whether it is a wild type or a mutant type with respect to the specific mutation. Therefore, detection or measurement for examining the presence or absence of mutation is expressed as “mutation detection” or “mutation detection”.

  In the mutation detection method of the present invention, first, a nucleic acid sample collected from a Japanese subject is prepared. A nucleic acid sample can be prepared from a subject's blood, saliva, lymph, urine, sweat, skin cells, mucosal cells, hair, and the like using known extraction methods and purification methods. Any genomic DNA of any length can be used as a nucleic acid sample as long as it contains the mutation position to be detected.

  The subject is not particularly limited. As will be described later, information (presence or absence of mutation) obtained by executing the mutation detection method of the present invention is useful for determining the risk (prediction of onset) of atopic dermatitis. Therefore, the present invention can be widely applied to those who need risk determination for atopic dermatitis. For example, a subject is a patient with atopic dermatitis, a person suspected of having atopic dermatitis, or a healthy person. The determination results of the former two are useful for determining a more appropriate treatment policy, and promote the improvement of the treatment effect and the improvement of the patient's quality of life (QOL). On the other hand, the determination results for healthy individuals are useful for the prevention and early diagnosis of atopic dermatitis. That is, if preventive measures and lifestyle improvements are made based on the information that the risk is high, the possibility of occurrence of atopic dermatitis (possibility of morbidity) can be reduced. In addition, information that the risk is high is an opportunity to receive a screening for atopic dermatitis, thereby enabling early diagnosis (early detection) of atopic dermatitis. If newborn infants, infants, or children are subjects, it is possible to identify those who have a high risk of developing atopic dermatitis in the pre-onset stage and actively apply externally supplementing skin barrier functions such as moisturizers. It is possible to prevent the onset of atopic dermatitis by reducing the exposure of mites and pollen.

In one aspect of the present invention, in addition to the above mutation, one or more mutations selected from the group consisting of the following (3) to (10) are also detected. The mutations (3) to (10) are Japanese filaggrin gene mutations reported so far (Non-Patent Documents 2 to 6).
(3) c.1501C> T mutation (p.Arg501X)
(4) c.5084C> G mutation (p.Ser1695X)
(5) c.5101C> G mutation (p.Gln1701X)
(6) c.7661C> G mutation (p.Ser2554X)
(7) c.8666_7CC> GA mutation (p.Ser2889X)
(8) c.9887C> A mutation (p.Ser3296X)
(9) c.12064A> T mutation (p.Lys4022X)
(10) c.3321delA mutation

  The more mutations to be detected, the higher the probability that a subject having a mutation in the filaggrin gene can be identified. In other words, people who have mutations in the filaggrin gene are often overlooked. Therefore, the number of mutations to be detected is preferably 3 to 10, more preferably 5 to 10, even more preferably 7 to 10, and most preferably 10 (all mutations). Here, among the mutations of (3) to (10), (6), (7), (9) and (10) are highly important and are particularly important mutations. Therefore, it is preferable to include these mutations in the detection target.

  The mutation detection method (measurement method) is not particularly limited. For example, using an allele-specific primer (and probe), amplification by PCR method, and detection of mutation of amplified product by fluorescence or luminescence, PCR ( PCR-RFLP (restriction fragment length polymorphism) method using polymerase chain reaction) method, PCR-SSCP (single strand conformation polymorphism) method (Orita, M. et. al., Proc. Natl. Acad. Sci., USA, 86, 2766-2770 (1989)), PCR-SSO (specific sequence oligonucleotide) method, PCR-SSO method and dot hybridization method ASO (allele specific oligonucleotide) hybridization method (Saiki, Nature, 324, 163-166 (1986), etc.), TaqMan (registered trademark, Roche Molecular Systems) -PCR method (Livak, KJ, Genet Anal, 14, 143 (1999), Morris, T. et al., J. Clin. Microbiol., 34, 2933 (1996)), Invader (registered trademark, Third Wave Technologies) method (Lyamichev V et al., Nat Biotechnol, 17, 292 (1999)), FRET (Fluorescence Resonance Energy Transfer (Heller, Academic Press Inc, pp. 245-256 (1985), Cardullo et al., Proc. Natl. Acad. Sci. USA, 85, 8790-8794 (1988), published internationally No. 99/28500 pamphlet, JP-A-2004-121232, etc.), ASP-PCR (Allele Specific Primer-PCR) method (International Publication No. 01/042498 etc.), MALDI-TOF / MS (matrix) method (Haff LA, Smirnov IP, Genome Res 7,378 (1997)), RCA (rolling cycle amplification) method (Lizardi PM et al., Nat Genet 19,225 (1998)), method using DNA chip or microarray (Wang DG et al., Science 280, 1077 (1998), etc.), primer extension method, Southern blot hybridization method, dot hybridization method (Southern, E., J. Mol. Biol. 98, 503-517 (1975)) can be used. Further, the mutation position to be detected may be directly sequenced. Note that mutation may be detected by arbitrarily combining these methods. In addition, any of the detection methods described above can be applied after a nucleic acid sample has been previously amplified (including amplification of a partial region of the nucleic acid sample) by a nucleic acid amplification method such as a PCR method or a method applying the PCR method.

  When detecting a large number of nucleic acid samples, use the allele-specific PCR method, allele-specific hybridization method, TaqMan (registered trademark) -PCR method, Invader method, FRET-based method, ASP-PCR method, and primer extension method. It is particularly preferable to use a detection method that can detect a large number of specimens in a relatively short time, such as the MALDI-TOF / MS (matrix) method, the RCA (rolling cycle amplification) method, or the method using a DNA chip or microarray. preferable.

  In the above methods, nucleic acids (also referred to as “mutation detection nucleic acids” in the present invention) such as probes and primers according to each method are used. Examples of the nucleic acid for mutation detection used as a probe include a nucleic acid that specifically hybridizes to a chromosomal region (partial chromosomal region) containing the mutation position to be detected. For example, if the mutation to be detected is c.441_2delAG, a probe may be designed targeting the chromosomal region containing positions 441 and 442. The length of the “partial chromosome region” here is, for example, 16 to 500 bases long, preferably 18 to 200 bases long, and more preferably 20 to 50 bases long. The nucleic acid preferably has a sequence complementary to a partial chromosomal region, but may have some mismatch as long as specific hybridization is not hindered. The degree of mismatch is 1 to several, preferably 1 to 3, and more preferably 1 to 2. The term “specific hybridization” as used herein refers to hybridization with a target nucleic acid (partial chromosomal region) under the hybridization conditions (preferably stringent conditions) usually employed for detection with a nucleic acid probe. It means that while it soy, it does not cause significant cross-hybridization with other nucleic acids. A person skilled in the art can easily set hybridization conditions with reference to, for example, Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York).

  As an example of a nucleic acid for mutation detection used as a primer, it has a sequence complementary to a chromosomal region (partial chromosomal region) containing a mutation position to be detected, and can specifically amplify a DNA fragment containing the mutated portion. And nucleic acids designed as such. As another example of a nucleic acid for mutation detection used as a primer, a primer pair designed to specifically amplify a DNA fragment containing the mutation position only when the mutation position to be detected is any base Can be mentioned. More specifically, it is a primer pair designed to specifically amplify a DNA fragment containing the mutation position to be detected, including the mutation position of the antisense strand whose mutation position is any base A primer pair consisting of a sense primer that specifically hybridizes to a chromosomal region (partial chromosomal region) and an antisense primer that specifically hybridizes to a partial region of the sense strand (region near the mutation position) Can be illustrated. Here, the length of the DNA fragment to be amplified is appropriately set within a range suitable for the detection, and is, for example, 15 to 1000 bases long, preferably 20 to 500 bases long, and more preferably 30 to 200 bases long. As in the case of the probe, the mutation detection nucleic acid used as a primer also has a template sequence as long as it can specifically hybridize to the amplification target (template) and amplify the target DNA fragment. There may be some mismatch. The degree of mismatch is 1 to several, preferably 1 to 3, and more preferably 1 to 2.

  For the nucleic acid for detection of mutation (probe, primer), DNA, RNA, peptide nucleic acid (PNA: Peptide nucleic acid :) or the like is used as appropriate according to the detection method. The nucleotide length of the nucleic acid for mutation detection may be any length that allows its function to be exhibited. Examples of the base length when used as a probe are 16 to 500 base length, preferably 18 to 200 base length, more preferably It is 20-50 bases long. On the other hand, examples of the base length when used as a primer are 10 to 50 bases, preferably 15 to 40 bases, and more preferably 15 to 30 bases.

  Nucleic acid for detection of mutation (probe, primer) can be synthesized by a known method such as a phosphodiester method. Regarding the design, synthesis, etc. of nucleic acids for detecting mutations (eg, Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, New York, Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987)) ) Can be helpful.

  The nucleic acid for mutation detection in the present invention can be labeled with a labeling substance in advance. By using such a labeled nucleic acid, for example, mutation can be detected using the labeling amount of the amplified product as an index. In addition, if the probe specific to the partial DNA region in the mutant type gene and the specific name probe in the partial DNA region in the wild type gene are labeled with different labeling substances, the labeling substance detected from the amplified product and The presence or absence of mutation can be determined by the amount of labeling.

Labeling substances used for labeling nucleic acids for mutation detection include FAM ^™ (6-carboxyfluorescein), VIC ^™ , TET ^™ (tetrachlorofluorescein), NED ^™ , HEX ^™ , CAL Fluor Gold 540, CAL Fluor Orange 560 (CFO), CAL Fluor Red 590, CAL Fluor Red 610, CAL Fluor Red 635, Quasar 570, Quasar 670, Quasar 705, T (JOE ^™ ), 7-AAD, Alexa Fluor (R) 488, Alexa Fluor (R) 350, Alexa Fluor (registered trademark) 546, Alexa Fluor (registered trademark) 555, Alexa Fluor (registered trademark) 568, Alexa Fluor (registered trademark) 594, Alexa Fluor (registered trademark) 633, Alexa Fluor (registered trademark) 647, Cy ^TM 2 , DsRED, EGFP, EYFP, FITC, PerCP ^TM , R-Phycoerythrin, Propidium Iodide, AMCA, DAPI, ECFP, MethylCoumarin, Allophycocyanin (APC), Cy ^TM 3, Cy ^TM 5, Rhodamine-123, Tetramethylrhodamine, Texas Red (Texas Red (R)), PE, PE-Cy TM 5, PE-Cy TM 5.5, PE-Cy TM 7, APC-Cy TM 7, Oregon Green (Oregon Green , Carboxyfluorescein, carboxyfluorescein diacetate, fluorescent dyes such as quantum dots, radioisotopes such as ^{32 P, 131 I, 125 I} , can be exemplified biotin, 5 as labeling methods using alkaline phosphatase and T4 polynucleotide kinase Examples include 'end labeling method, 3' end labeling method using T4 DNA polymerase and Klenow fragment, nick translation method, random primer method (Molecular Cloning, Third Edition, Chapter 9, Cold Spring Harbor Laboratory Press, New York) it can.

  It can also be used in the state where the nucleic acid for mutation detection is immobilized on an insoluble support. If the insoluble support used for immobilization is processed into a chip shape, a bead shape, etc., mutation can be detected more easily using these immobilized nucleic acids.

  As described above, various detection means can be employed, but it is particularly preferable to use a real-time PCR method. Real-time PCR has the advantage of being quick and sensitive, and excellent in quantification. A specific example of the method using real-time PCR is TaqMan (registered trademark, Roche Molecular Systems) -PCR method. In this method, a fluorescent probe using the principle of fluorescence resonance energy transfer (FRET) is used.

  Information (presence or absence of mutation) obtained by executing the mutation detection method of the present invention described above can be used for determination of risk of atopic dermatitis (prediction of onset). That is, the present invention also provides a risk test method for atopic dermatitis, comprising the step of determining a genetic risk of atopic dermatitis from mutation information obtained by the mutation detection method. In the present invention, “risk of atopic dermatitis” refers to the degree of risk (susceptibility) of causing atopic dermatitis. Therefore, according to the risk test method of the present invention, it is possible to determine and evaluate the susceptibility of atopic dermatitis.

  In determining the risk of atopic dermatitis, in the present invention, for example, a criterion for determining that the risk of atopic dermatitis is high when any of the above mutations (1) to (10) is detected. (Criteria 1) is adopted. Thus, in the present invention, the risk is determined based on the result of gene analysis. On the other hand, a criterion (determination criterion 2) that the risk of atopic dermatitis is particularly high when two or more mutations are detected may be adopted. Determination criteria 1 and 2 can also be used in combination. Note that the determination here can be automatically / mechanically performed without depending on the determination of a person having specialized knowledge such as a doctor or a clinical laboratory technician, as is apparent from the determination criteria.

(Mutation detection probe)
The second aspect of the present invention provides a probe that can be used in the mutation detection method of the present invention. Specifically, detection of a newly identified mutation (c.441_2delAG mutation), detection of a filaggrin gene mutation targeting a DNA sequence containing the mutation position of the filaggrin gene c.441_2delAG mutation Nucleic acid probes are provided. In addition, it is for detecting another novel mutation (c.5368C> T mutation). Detection of a filaggrin gene mutation targeting a DNA sequence containing the mutation position of the filaggrin gene c.5368C> T mutation. Nucleic acid probes are also provided. The arrangement of these probes is designed according to the detection means employed.

In one embodiment, each probe is designed to be suitable for a measurement means using the FRET principle. Specifically, the following configuration is provided. Note that (i) and (iii) are configurations for specifically detecting the mutant type, and (ii) and (iv) are configurations for specifically detecting the wild type.
(i) Configuration of probe for detecting c.441_2delAG mutation 1
It consists of a sequence that targets a DNA sequence containing the mutation position of the c.441_2delAG mutation of the filaggrin gene and hybridizes to the DNA region when the mutation position is a mutant type (ie, complementary to the target DNA region), A reporter fluorescent dye is bound to the 5 ′ end, and a quencher that induces quenching of the reporter fluorescent dye by the principle of fluorescence resonance energy transfer is bound to the 3 ′ end.
(ii) Configuration 2 of probe for detecting c.441_2delAG mutation 2
It consists of a sequence that targets the DNA sequence containing the mutation position of the c.441_2delAG mutation of the filaggrin gene and hybridizes to the DNA region when the mutation position is wild type (ie, complementary to the target DNA region), A reporter fluorescent dye is bound to the 5 ′ end, and a quencher that induces quenching of the reporter fluorescent dye by the principle of fluorescence resonance energy transfer is bound to the 3 ′ end.
(iii) Configuration of probe for detecting c.5368C> T mutation 1
It consists of a sequence that targets the DNA sequence containing the mutation position of the c.5368C> T mutation of the filaggrin gene and hybridizes to the DNA region when the mutation position is a mutant type (ie, complementary to the target DNA region) ), And a reporter fluorescent dye is bound to the 5 ′ end, and a quencher that induces quenching of the reporter fluorescent dye by the principle of fluorescence resonance energy transfer is bound to the 3 ′ end.
(iv) c.5368C> T Configuration of Probe for Detection of T Mutation 2
Targeting a DNA sequence containing the mutation position of the c.5368C> T mutation of the filaggrin gene, and consisting of a sequence that hybridizes to the DNA region when the mutation position is wild type (ie, complementary to the target DNA region) ), And a reporter fluorescent dye is bound to the 5 ′ end, and a quencher that induces quenching of the reporter fluorescent dye by the principle of fluorescence resonance energy transfer is bound to the 3 ′ end.

  If both (i) and (ii) are used in combination, the mutant type and the wild type can be detected simultaneously for the c.441_2delAG mutation. In this case, two types of reporter fluorescent dyes that can distinguish fluorescence are prepared so that the mutant type and the wild type can be distinguished. One is used as the reporter fluorescent dye in (i), and the other is used as the reporter fluorescent dye in (ii). Let it be a pigment. The relationship between (iii) and (iv) is similar to the relationship between (i) and (ii), and their combination allows simultaneous detection of mutant and wild type for the c.5368C> T mutation.

Typically, the probe of the present invention is used for real-time PCR (for example, TaqMan (registered trademark) -PCR method) using the principle of FRET. Thus, preferably, the probe sequence is designed to suit the method, and the reporter fluorescent dye and quencher are selected. The probe sequence should be designed so that the DNA sequence (PCR product) amplified by the primer pair is targeted at the approximate center. The Tm of the probe should be 60 to 65 ° C. Reporter fluorescent dyes bound to the 5 'end include FAM ^™ (6-carboxyfluorescein), VIC ^™ , TET ^™ (tetrachlorofluorescein), NED ^™ , CAL Fluor Orange 560 (CFO), HEX ^™ , CAL Fluor Gold 540, and CAL Fluor. Orange 560, CAL Fluor Red 590, CAL Fluor Red 610, CAL Fluor Red 635, Quasar 570, Quasar 670, Quasar 705, T (JOE ^™ ) and the like can be used. On the other hand, TAMRA (tetramethylrhodamine), MGB (dihydrocyclopyrroloindole tripeptide minor groove binder), BHQ (registered trademark) -1 (Black Hole Quencher 1), BHQ (registered trademark) -2 (Black Hole) Quencher 2), BHQ (registered trademark) -3 (Black Hole Quencher 3), Pulsar 650, Spacer C3, and the like can be used. Hereinafter, specific examples of the sequences constituting the probes (i) to (iv) are shown. In accordance with common practice, the 5 ′ end side is on the left side and the 3 ′ end side is on the right side.

(i) 5'-CAAGAGAAACGGGGGAAAAGGCA-3 '(SEQ ID NO: 3)
(ii) 5'-CAAGAGAAACAGGGGGGAAAAGGC-3 '(SEQ ID NO: 4)
(iii) 5'-AGGAAGATAAAGATCCCGCCACGAG-3 '(SEQ ID NO: 5)
(iv) 5'-AGGAAGACAAAGATCCCGCCACG-3 '(SEQ ID NO: 6)

  The above sequence is an example, and a part of the base sequence may be modified as long as specific hybridization can be performed without hindrance. Here, “partial modification” means that a part of the base is deleted, substituted, inserted and / or added. The number of bases involved in the modification is, for example, 1 to 3, preferably 1 to 2, and more preferably 1.

(Mutation detection kit)
The present invention further provides a kit (kit for detecting mutations in the fibrillin gene) that can be used in the mutation detection method of the present invention. The mutation detection kit of the present invention includes two or more nucleic acid sets selected from the group consisting of the following (1) to (10).
(1) Nucleic acid set for detecting 441_2delAG mutation of filaggrin gene (2) Nucleic acid set for detecting c.5368C> T (p.Gln1790X) filaggrin gene (3) c.1501C> T (p.Arg501X of filaggrin gene) ) Nucleic acid set for mutation detection (4) Nucleic acid set for c.5084C> G (p.Ser1695X) mutation of filaggrin gene (5) Nucleic acid for c.5101C> G (p.Gln1701X) mutation detection of filaggrin gene Set (6) Nucleic acid set for detecting c.7661C> G (p.Ser2554X) mutation of filaggrin gene (7) Nucleic acid set for detecting c.8666_7CC> GA (p.Ser2889X) mutation of filaggrin gene (8) Filaggrin gene Nucleic acid set for detecting c.9887C> A (p.Ser3296X) mutation in (9) Nucleic acid set for detecting c.12064A> T (p.Lys4022X) mutation in filaggrin gene (10) Detection of c.3321delA mutation in filaggrin gene Nucleic acid set

  Each nucleic acid set is roughly divided into a probe and a primer pair. As the probe, a first fluorescent probe specific to the mutant type, a second fluorescent probe specific to the wild type, or both are used. The first fluorescent probe here comprises a sequence that targets a DNA sequence containing the mutation position to be detected and hybridizes to the DNA sequence when the mutation position is a mutant type. A reporter fluorescent dye is bound, and a quencher that induces quenching of the reporter fluorescent dye by the principle of fluorescence resonance energy transfer is bound to the 3 ′ end. On the other hand, the second probe targets a DNA sequence including the mutation position to be detected, and comprises a sequence that hybridizes to the DNA sequence when the mutation position is a wild type, and the first reporter fluorescence is present at the 5 ′ end. A second reporter fluorescent dye different from the dye is bonded, and a quencher that induces quenching of the reporter fluorescent dye is bonded to the 3 ′ end by the principle of fluorescence resonance energy transfer.

As a combination of the first reporter fluorescent dye and the second reporter fluorescent dye, FAM ^TM and CFO (any of them may be the first reporter fluorescent dye, and the same applies to the following combinations), FAM ^TM and VIC ^™ , FAM ^™ and HEX ^™ , FAM ^™ and TET ^™ , HEX ^™ and Joe ^™ , HEX ^™ and TET ^{™ and the} like can be mentioned.

  In a preferred embodiment of the mutation detection kit of the present invention, the kit is configured to include at least the nucleic acid set (1) or the nucleic acid set (2), or both the nucleic acid set (1) and the nucleic acid set (2). . According to this aspect, the newly identified mutation can be detected.

  As already mentioned, the greater the number of mutations to be detected, the higher the probability that a subject having a mutation in the filaggrin gene can be identified, and the more often a person having a mutation is overlooked. Therefore, in order to detect more mutations, the number of nucleic acid sets included in the mutation detection kit is 3 to 10, more preferably 5 to 10, still more preferably 7 to 10, and most preferably. Is 10 (all mutations). Here, the c.7661C> G mutation, the c.8666_7CC> GA mutation, the c.12064A> T mutation, and the c.3321delA mutation are particularly important mutations with a high retention rate. Therefore, the nucleic acid set of (6), (7), (9) and (10) is preferably included in the mutation detection kit so that these mutations can be detected.

  Typically, the mutation detection kit of the present invention is used for real-time PCR (for example, TaqMan (registered trademark) -PCR method) using the principle of FRET. Therefore, preferably, the probe and primer pair are configured so as to be compatible with the method. For the design of the probe, the description in the column of “Mutation detection probe” is used, and a duplicate description is omitted. The primer pair is preferably designed so that a region of 100 to 200 bp including the mutation position is amplified and Tm is 68 to 70 ° C.

Specific examples of the sequences of probes and primer pairs constituting the nucleic acid sets of (1) to (10) above are shown. In accordance with common practice, the 5 ′ end side is on the left side and the 3 ′ end side is on the right side.
<Nucleic acid set (1)>
First fluorescent probe: 5′-CFO-CAAGAGAAACGGGGGAAAAGGCA-BHQ1-3 ′ (SEQ ID NO: 3)
Second fluorescent probe: 5'-FAM-CAAGAGAAACAGGGGGGAAAAGGC-BHQ1-3 '(SEQ ID NO: 4)
Forward primer: 5'-AAGGGAATAAGGGAAGATCCAAGA-3 '(SEQ ID NO: 23)
Reverse primer: 5'-CTTAGCCTCTTCCTATTGTCTCCTAATCT-3 '(SEQ ID NO: 24)
<Nucleic acid set (2)>
First fluorescent probe: 5'-CFO-AGGAAGATAAAGATCCCGCCACGAG-BHQ1-3 '(SEQ ID NO: 5)
Second fluorescent probe: 5'-FAM-AGGAAGACAAAGATCCCGCCACG-BHQ1-3 '(SEQ ID NO: 6)
Forward primer: 5'-CACAGGGCCCAGCACTG-3 '(SEQ ID NO: 25)
Reverse primer: 5'-ACCGATTGCTCATAGTGGGATC-3 '(SEQ ID NO: 26)
<Nucleic acid set (3)>
First fluorescent probe: 5'-CFO-CAGGCATGAGACAGCTCCAGGC-BHQ1-3 '(SEQ ID NO: 7)
Second fluorescent probe: 5′-FAM-AGGCACGAGACAGCTCCAGGC-BHQ1-3 ′ (SEQ ID NO: 8)
Forward primer: 5'-GCACTGGAGGAAGACAAGGATC-3 '(SEQ ID NO: 27)
Reverse primer: 5'-TCCTCATTTCGTGTTTGTCTGC-3 '(SEQ ID NO: 28)
<Nucleic acid set (4)>
First fluorescent probe: 5′-CFO-CCACAGACTGAGGCACTGGGC-BHQ1-3 ′ (SEQ ID NO: 9)
Second fluorescent probe: 5′-FAM-CCACAGACTCAGGCACTGGGC-BHQ1-3 ′ (SEQ ID NO: 10)
Forward primer: 5'-ACCAGCAGTCAGCAGACAGC-3 '(SEQ ID NO: 29)
Reverse primer: 5'-CTGACTCTTCTGAGTGTCCCTCG-3 '(SEQ ID NO: 30)
<Nucleic acid set (5)>
First fluorescent probe: 5′-CFO-CACTGGGYGCAGATAAGATTCATCTG-BHQ1-3 ′ (SEQ ID NO: 11)
Second fluorescent probe: 5′-FAM-TGGGYGCAGACAAGATTCATCTG-BHQ1-3 ′ (SEQ ID NO: 12)
Forward primer: 5'-GTCAGCAGACAGCTCCACAGAC-3 '(SEQ ID NO: 31)
Reverse primer: 5'-CTGACTCTTCTGAGTGTCCCTCG-3 '(SEQ ID NO: 32)
<Nucleic acid set (6)>
First fluorescent probe: 5'-CFO-CCCTCTCATTGTCCCTGGMCC-BHQ1-3 '(SEQ ID NO: 13)
Second fluorescent probe: 5'-FAM-CCCTCTGATTGTCCCTGGMCC-BHQ1-3 '(SEQ ID NO: 14)
Forward primer: 5'-GCTCCAGGCACTCAGGGTC-3 '(SEQ ID NO: 33)
Reverse primer: 5'-TGGATCCCCAGTTCCTGCT-3 '(SEQ ID NO: 34)
<Nucleic acid set of (7)>
First fluorescent probe: 5′-CFO-TCCTGGCTMACACTTCATCCCTG-BHQ1-3 ′ (SEQ ID NO: 15)
Second fluorescent probe: 5′-FAM-CCTGGCTMACACTGGATCCCTG-BHQ1-3 ′ (SEQ ID NO: 16)
Forward primer: 5'-CTCAGGGTCGCGTCACC-3 '(SEQ ID NO: 35)
Reverse primer: 5'-CTCTCAGAGTCTTCTGAATGTCCCT-3 '(SEQ ID NO: 36)
<Nucleic acid set of (8)>
First fluorescent probe: 5′-CFO-TGAACAGGCAAGATAAAGTCCAGGAGAG-BHQ1-3 ′ (SEQ ID NO: 17)
Second fluorescent probe: 5'-FAM-ACAGGCAAGATCAAGTCCAGGAGAGA-BHQ1-3 '(SEQ ID NO: 18)
Forward primer: 5'-TGGTGGACAGGCTGCATC-3 '(SEQ ID NO: 37)
Reverse primer: 5'-AAGCYTGTCCACGCGGA-3 '(SEQ ID NO: 38)
<Nucleic acid set (9)>
First fluorescent probe: 5'-CFO-CCTTGGATGATCTTAACCAAACGCACT-BHQ1-3 '(SEQ ID NO: 19)
Second fluorescent probe: 5′-FAM-CCTTGGATGATCTTTACCAAACGCACT-BHQ1-3 ′ (SEQ ID NO: 20)
Forward primer: 5'-AGACACTCTCAGCACGGAAGTGT-3 '(SEQ ID NO: 39)
Reverse primer: 5'-GCCACATAAACCTGGGTCCTTA-3 '(SEQ ID NO: 40)
<Nucleic acid set of (10)>
First fluorescent probe: 5′-CFO-ACGTGACTGGTCGGGGGAAGGT-BHQ1-3 ′ (SEQ ID NO: 21)
Second fluorescent probe: 5′-FAM-CGTGACTGGTCAGGGGGAAGGTC-BHQ1-3 ′ (SEQ ID NO: 22)
Forward primer: 5'-AGGCCATGGACAGGATGG-3 '(SEQ ID NO: 41)
Reverse primer: 5'-CTCACCTGGTAGATGAAAGACCCT-3 '(SEQ ID NO: 42)

  The above probe and primer sequences are only examples, and the probe is modified to a part of the base sequence to the extent that specific hybridization can be performed without hindrance, and to the extent that the target amplification reaction can be performed without hindrance if it is a primer. May be given. Here, “partial modification” means that a part of the base is deleted, substituted, inserted and / or added. The number of bases involved in the modification is, for example, 1 to 3, preferably 1 to 2, and more preferably 1.

  The mutation detection kit of the present invention includes a nucleic acid set, but may include other reagents (DNA polymerase, reaction solution, etc.), containers, instruments, and the like necessary for detecting the mutation. Usually, an instruction manual is attached to the mutation detection kit of the present invention.

1. Detection of a novel mutation of filaggrin gene (FLG) (1) Purpose The following experiment was conducted for the purpose of finding a novel filaggrin gene mutation unique to Japanese.

(2) Subjects Samples collected from patients with wheat-dependent exercise-induced anaphylaxis (WDEIA) were used for experiments.

(3) Method
By the method of Sandilands et al. (Sandilands A, Terron-Kwiatkowski A, Hull PR et al. Comprehensive analysis of the gene encoding filaggrin uncovers prevalent and rare mutations in ichthyosis vulgaris and atopic dermatitis. Nat Genet 2007; 39: 650-4.) The exon region of the filaggrin gene (FLG) was directly sequenced to detect a novel mutation.

(4) Results
Two novel mutations of FLG, p.Gln1790X and c.441_442delAG, were identified. In addition to the 8 mutations reported so far, 2 mutations can be targeted for detection, so that individuals with FLG mutations can be found more accurately and comprehensively, with clinical significance. It has risen.

2. Simplified screening method for filaggrin gene (FLG) mutation (1) Purpose The following experiment was conducted with the aim of establishing a method for easily and rapidly detecting a filaggrin gene (FLG) mutation.

(2) Method For detection of each mutation, a primer pair and a probe for TaqMan (registered trademark) -PCR were designed (FIG. 1). Two types of probes for wild type detection (-wild) and mutation type detection (-mut) were prepared for each mutation. FAM ^™ as a reporter fluorescent dye is bound to the 5 ′ end of the wild type detection probe. On the other hand, CFO as a reporter fluorescent dye is bound to the 5 ′ end of the mutant detection probe. By using two types of fluorescent dyes having different fluorescence characteristics in this way, simultaneous detection of wild type and mutant type is possible. A quencher BHQ-1 was bound to the 3 ′ end of each probe.

If the wild-type detection probe and the mutant-type detection probe are assayed together, and the fluorescence intensity from FAM ^TM and the fluorescence intensity from CFO are measured (for example, two fluorescence intensities are plotted in two dimensions for evaluation) ), Whether the specimen falls under (i) a homozygote of a wild type allele, (ii) a heterozygote of a wild type allele and a mutant allele, or (iii) a homozygote of a mutant allele I understand.

The operation procedure is as follows. First, genomic DNA is extracted from the blood, saliva, etc. of the subject. A genotyping assay by a real-time PCR method using the prepared TaqMan probe (wild-type detection probe and mutant-type detection probe) and a primer pair (FIG. 1) was performed. LightCycler 480 system II 384 plate (Roche Diagnostics) was used as a real-time PCR apparatus. 2x LightCycler 480 Probes Master, wild-type detection probe, mutation-type detection probe and primer pair (forward and reverse), and genomic DNA were added to form a reaction solution, and a PCR reaction was performed. The PCR reaction was treated at 95 ° C. for 10 minutes, followed by 40 temperature cycles of 95 ° C. for 10 seconds, 60 ° C. for 1 minute, 72 ° C. for 1 second, and finally 40 ° C. for 30 seconds. During the reaction, fluorescence was detected over time. The obtained data was analyzed with LightCycler 480 software. The above operation was performed for each mutation (10 in total), and the genotype of each subject was determined.
(3) Results With this method, FLG mutations could be screened quickly and inexpensively compared to the conventional direct sequencing method. When FLG mutations were detected in the 820 Japanese population, 91 mutations were detected (FIG. 2). These were confirmed by the direct sequencing method and all agreed.

3. Summary According to this method, although it varies depending on the device, reagent, and environment to be used, when using currently available devices and reagents, detection results can be obtained in about several hundred yen per person for about 2 hours. This is possible and provides dramatic improvements in both cost and time. The two mutations added this time are thought to account for about 5% of all Japanese people with the filaggrin gene mutation. It has clinical significance to be able to recognize that these Japanese people, which had not been mutated so far, can possess mutations.

  Information obtained by carrying out the mutation detection method of the present invention is useful for risk determination (prediction of onset) of atopic dermatitis. That is, the present invention provides clinically useful risk information. The risk information can be used for early diagnosis (early detection) of atopic dermatitis, prevention of onset, selection of treatment method, and the like. It is expected to contribute to the realization of tailor-made treatment / prevention of atopic dermatitis.

  The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

SEQ ID NOs: 3-21: Description of artificial sequence: probe SEQ ID NOs: 22-42: Description of artificial sequence: primer

Claims (13)

A method for detecting a filaggrin gene mutation comprising a step of detecting the following (1) or (2) or both of the following (1) and (2) for a nucleic acid sample collected from a Japanese subject: :
(1) c.441_2delAG mutation in the filaggrin gene;
(2) c.5368C> T mutation of the filaggrin gene. The mutation detection method according to claim 1, wherein one or more mutations selected from the group consisting of the following (3) to (10) are also detected in addition to the mutation:
(3) c.1501C> T mutation;
(4) c.5084C> G mutation;
(5) c.5101C> G mutation;
(6) c.7661C> G mutation;
(7) c.8666_7CC> GA mutation;
(8) c.9887C> A mutation;
(9) c.12064A> T mutation; and (10) c.3321delA mutation.   The mutation detection method according to claim 2, wherein all of (1) to (10) are targeted for detection.   Each mutation is detected by real-time PCR method, The mutation detection method as described in any one of Claims 1-3 characterized by the above-mentioned.   The mutation detection method according to any one of claims 1 to 4, wherein the real-time PCR method is a measurement method using a fluorescent probe utilizing the principle of fluorescence resonance energy transfer.   A nucleic acid probe for detecting mutations in the filaggrin gene, targeting a DNA sequence containing the mutation position of the c.441_2delAG mutation in the filaggrin gene. Targeting a DNA sequence containing the mutation position of the filaggrin gene c.441_2delAG mutation, it consists of a sequence that hybridizes to the DNA region when the mutation position is a mutant type, and a reporter fluorescent dye is bound to the 5 'end. cage, 3 'end, a fluorescent probe quencher to induce quenching of the reporter fluorescent dye on the principle of fluorescence resonance energy transfer is attached,
The nucleic acid probe for mutation detection according to claim 6, wherein A nucleic acid probe for detecting mutations in the filaggrin gene , targeting a DNA sequence containing the mutation position of the filaggrin gene c.5368C> T mutation and targeting a Japanese subject . Targeting a DNA sequence containing the mutation position of the filaggrin gene c.5368C> T mutation, it consists of a sequence that hybridizes to the DNA region when the mutation position is mutant, and a reporter fluorescent dye is bound to the 5 'end and is, 3 'end, a fluorescent probe quencher to induce quenching of the reporter fluorescent dye on the principle of fluorescence resonance energy transfer is attached,
The nucleic acid probe for mutation detection according to claim 8, wherein (1) Nucleic acid set for detecting 441_2delAG mutation of filaggrin gene,
(2) Nucleic acid set for detecting c.5368C> T mutation of filaggrin gene,
(3) Nucleic acid set for detecting c.1501C> T mutation of filaggrin gene,
(4) Nucleic acid set for detecting c.5084C> G mutation of filaggrin gene,
(5) Nucleic acid set for detecting c.5101C> G mutation of filaggrin gene,
(6) Nucleic acid set for detecting c.7661C> G mutation of filaggrin gene,
(7) Nucleic acid set for detecting c.8666_7CC> GA mutation of filaggrin gene,
(8) Nucleic acid set for detecting c.9887C> A mutation of filaggrin gene,
(9) a nucleic acid set for detecting c.12064A> T mutation of filaggrin gene and (10) a nucleic acid set for detecting c.3321delA mutation of filaggrin gene, comprising two or more nucleic acid sets selected from the group consisting of: A filaggrin gene mutation detection kit comprising:
Each nucleic acid set is
It consists of a sequence that hybridizes to the DNA sequence when the mutation position is a mutant type, targeting the DNA sequence containing the mutation position to be detected, and the first reporter fluorescent dye is bound to the 5 ′ end, The 3 ′ end includes a first fluorescent probe to which a quencher that induces quenching of the reporter fluorescent dye is bound by the principle of fluorescence resonance energy transfer, or the first fluorescent probe and a mutation position to be detected It consists of a sequence that targets the DNA sequence and hybridizes to the DNA sequence when the mutation position is wild type, and a second reporter fluorescent dye different from the first reporter fluorescent dye is bound to the 5 ′ end. and which, 3 'end, a fluorescent probe and a second fluorophore quencher to induce quenching of the reporter fluorescent dye on the principle of fluorescence resonance energy transfer is attached And Tsu door,
A primer pair for amplifying a DNA region containing the DNA sequence;
Consists of
(1) or (2), or the (1) and both (2), at least viewed including the Japanese is the subject, the mutation detection kit.   The mutation detection kit according to claim 10, comprising all of (1) to (10). The first fluorescent probe in the nucleic acid set of (1) includes the sequence of SEQ ID NO: 3,
The second fluorescent probe in the nucleic acid set of (1) includes the sequence of SEQ ID NO: 4, the first fluorescent probe in the nucleic acid set of (2) includes the sequence of SEQ ID NO: 5,
The second fluorescent probe in the nucleic acid set of (2) includes the sequence of SEQ ID NO: 6, the first fluorescent probe in the nucleic acid set of (3) includes the sequence of SEQ ID NO: 7,
The second fluorescent probe in the nucleic acid set of (3) includes the sequence of SEQ ID NO: 8, the first fluorescent probe in the nucleic acid set of (4) includes the sequence of SEQ ID NO: 9,
The second fluorescent probe in the nucleic acid set of (4) includes the sequence of SEQ ID NO: 10, the first fluorescent probe in the nucleic acid set of (5) includes the sequence of SEQ ID NO: 11,
The second fluorescent probe in the nucleic acid set of (5) includes the sequence of SEQ ID NO: 12, the first fluorescent probe in the nucleic acid set of (6) includes the sequence of SEQ ID NO: 13,
The second fluorescent probe in the nucleic acid set of (6) includes the sequence of SEQ ID NO: 14, the first fluorescent probe in the nucleic acid set of (7) includes the sequence of SEQ ID NO: 15,
The second fluorescent probe in the nucleic acid set of (7) includes the sequence of SEQ ID NO: 16, the first fluorescent probe in the nucleic acid set of (8) includes the sequence of SEQ ID NO: 17,
The second fluorescent probe in the nucleic acid set of (8) includes the sequence of SEQ ID NO: 18, the first fluorescent probe in the nucleic acid set of (9) includes the sequence of SEQ ID NO: 19,
The second fluorescent probe in the nucleic acid set of (9) includes the sequence of SEQ ID NO: 20, the first fluorescent probe in the nucleic acid set of (10) includes the sequence of SEQ ID NO: 21,
The second fluorescent probe in the nucleic acid set of (10) comprises the sequence of SEQ ID NO: 22;
The mutation detection kit according to claim 10 or 11. The primer pair in the nucleic acid set of (1) comprises a forward primer comprising the sequence of SEQ ID NO: 23 and a reverse primer comprising the sequence of SEQ ID NO: 24,
The primer pair in the nucleic acid set of (2) comprises a forward primer comprising the sequence of SEQ ID NO: 25 and a reverse primer comprising the sequence of SEQ ID NO: 26,
The primer pair in the nucleic acid set of (3) comprises a forward primer comprising the sequence of SEQ ID NO: 27 and a reverse primer comprising the sequence of SEQ ID NO: 28,
The primer pair in the nucleic acid set of (4) comprises a forward primer comprising the sequence of SEQ ID NO: 29 and a reverse primer comprising the sequence of SEQ ID NO: 30;
The primer pair in the nucleic acid set of (5) comprises a forward primer comprising the sequence of SEQ ID NO: 31 and a reverse primer comprising the sequence of SEQ ID NO: 32,
The primer pair in the nucleic acid set of (6) comprises a forward primer comprising the sequence of SEQ ID NO: 33 and a reverse primer comprising the sequence of SEQ ID NO: 34,
The primer pair in the nucleic acid set of (7) comprises a forward primer comprising the sequence of SEQ ID NO: 35 and a reverse primer comprising the sequence of SEQ ID NO: 36,
The primer pair in the nucleic acid set of (8) comprises a forward primer comprising the sequence of SEQ ID NO: 37 and a reverse primer comprising the sequence of SEQ ID NO: 38,
The primer pair in the nucleic acid set of (9) comprises a forward primer comprising the sequence of SEQ ID NO: 39 and a reverse primer comprising the sequence of SEQ ID NO: 40;
The primer pair in the nucleic acid set of (10) comprises a forward primer comprising the sequence of SEQ ID NO: 41 and a reverse primer comprising the sequence of SEQ ID NO: 42.
The mutation detection kit according to any one of claims 10 to 12.

Images