Gene polymorphism detection method

Abstract

To provide a novel method for detecting ApoE gene polymorphisms that is rapid and is minimally invasive to subjects.SOLUTION: The present invention can be exemplified by, for example, a method for detecting a polymorphism of apolipoprotein E present in genomic DNA collected from a subject, which comprises the following steps 1 to 3: 1. preparing a sample by isolating DNA from saliva; 2. adding and mixing (1) a PCR enzyme, (2) a PCR primer pair for amplifying a nucleic acid fragment of an apolipoprotein E gene, and (3) a fluorescently labeled probe set having an oligonucleotide for detecting a polymorphism of apolipoprotein E to the sample containing the DNA; and 3. performing PCR and measuring the fluorescence intensity corresponding to the polymorphism of apolipoprotein E from the PCR product.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention belongs to the technical field of a gene polymorphism detection method. The present invention relates to a method for detecting apolipoprotein E (hereinafter also referred to as "ApoE") gene polymorphism. More specifically, the present invention relates to a method for detecting ApoE gene polymorphism from exocrine fluid such as saliva. [Background technology]

[0002] ApoE is a protein consisting of 299 amino acids with a molecular weight of 34,200 Da. The ApoE gene has three alleles, ε2, ε3, and ε4, and each gene encodes three major isoforms, ApoE2, ApoE3, and ApoE4. These differ in two amino acid residues, 112th and 158th, with ApoE2 being Cys / Cys, ApoE3 being Cys / Arg, and ApoE4 being Arg / Arg. Of these, ApoE3, which has Cys at 112th and Arg at 158th, is the wild type and is the most frequent. ApoE2 and ApoE4 are mutant types. ApoE is a molecule involved in lipid metabolism, including cholesterol transport and lipoprotein metabolism, and is involved in the activation of lipid metabolic enzymes such as hepatic lipase, lipoprotein lipase, and lecithin-cholesterol acyltransferase.

[0003] Many risk genes related to the onset of Alzheimer's disease (AD) and cognitive impairment (MCI, etc.) have been reported, and ApoE is the most powerful risk gene. It has been reported that the frequency of ApoE4 is high and the frequency of ApoE2 is low in elderly-onset AD. Furthermore, it is known that the frequency of ApoE4 is almost the same in women in early-onset and elderly-onset types, while in men, the frequency of ApoE4 is higher in elderly-onset types than in young-onset types, ApoE4 / 4 accelerates the age of onset of AD by about 5 to 10 years, AD patients with ApoE4 / 4 have low responsiveness to AD drugs and low glucose metabolism, and brain atrophy tends to progress in the order of ApoE4 / 4, ApoE3 / 4, and ApoE3 / 3.

[0004] It is also known that the frequency of ApoE4 is high in other dementias such as vascular dementia (VD) and Lewy body disease, and that the frequency of ApoE2 is high in patients with cerebral hemorrhage due to cerebral amyloid angiopathy. In addition, it has been reported that ApoE4 is present at a high frequency in neuropsychiatric disorders such as Parkinson's disease, schizophrenia, depression, anxiety disorders, and epilepsy.

[0005] As mentioned above, ApoE gene polymorphisms are thought to be an important factor in determining the risk of developing AD and other diseases. Conventionally, when diagnosing or predicting the onset of AD, it is common to judge from images, for example, MRI (Magnetic Resonance Imaging) or CT (Computed Tomography). However, taking MRI images requires a certain amount of time, and places a heavy burden on patients. For this reason, research is being conducted into methods of diagnosing and predicting the onset of AD using information on ApoE gene polymorphisms, which allows for quicker diagnosis. Conventionally, the measurement of ApoE gene polymorphism is usually carried out by collecting blood from a subject. For example, Patent Document 1 discloses a method for detecting ApoE4 or a fragment thereof in a blood sample. Non-Patent Document 1 detects ApoE gene polymorphism using real-time PCR. Such a method for detecting ApoE gene polymorphism using blood as a sample is highly invasive, and the burden on the subject is still large, which is a problem. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 2019-536007 [Non-patent literature]

[0007] [Non-Patent Document 1] Clinical Chemistry 45,No.7,1999,pp.1094-1097 Summary of the Invention [Problem to be solved by the invention]

[0008] Until now, ApoE gene polymorphisms have typically been measured by taking blood samples from subjects, which is highly invasive and, like diagnostic imaging, still places a heavy burden on subjects. The present invention aims to provide a novel method for detecting or determining ApoE gene polymorphisms, which is rapid and minimally invasive to subjects and can solve the above-mentioned problems. In addition, kits and systems therefor are also provided. [Means for solving the problem]

[0009] As a result of extensive investigations, the present inventors have found that ApoE gene polymorphisms can be detected simply and quickly from exocrine fluids such as saliva, thereby solving the above-mentioned problems, and have thus completed the present invention. The present invention can include, for example, the following aspects.

[0010] [1] A method for detecting a genetic polymorphism of apolipoprotein E present in genomic DNA collected from a subject, comprising the following steps 1 to 3: 1. A step of isolating DNA from exocrine secretions or mucus containing epithelial cells collected from a subject to prepare a sample containing the DNA; 2. The sample containing the DNA is (1) PCR enzyme, (2) a PCR primer pair for amplifying a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding the 112th or 158th amino acid residue (Cys or Arg) of apolipoprotein E; and (3) A set of a fluorescently labeled probe having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene, the nucleic acid fragment containing a codon that codes for the wild-type Cys at the 112th amino acid residue of apolipoprotein E, and a set of a fluorescently labeled probe having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene, the nucleic acid fragment containing a codon that codes for the mutant Arg at the 112th amino acid residue, the fluorescently labeled probe set comprising fluorescently labeled probes having different fluorescent dyes for labeling. or a step of adding and mixing a set of a fluorescently labeled probe having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene, the nucleic acid fragment containing a codon that codes for Arg, which is a wild type, at the 158th amino acid residue of apolipoprotein E, and a set of a fluorescently labeled probe having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene, the nucleic acid fragment containing a codon that codes for Cys, which is a mutant type, at the 158th amino acid residue of the apolipoprotein E, the fluorescently labeled probe sets being different from each other in fluorescent dyes used for labeling; 3. A step of carrying out PCR on the mixture and measuring the fluorescence intensity from the PCR product corresponding to the 112th or 158th amino acid residue of the apolipoprotein E of the subject. [2] The method for detecting a genetic polymorphism according to the above [1], wherein in step 1, DNA is released using a surfactant and protease K. [3] The method for detecting a genetic polymorphism according to [2] above, wherein the surfactant is sodium dodecyl sulfate. [4] The method for detecting a genetic polymorphism according to any one of the above [1] to [3], wherein a Tris-HCl buffer containing potassium chloride, magnesium chloride, and a dNTP mix is ​​further added to the sample and mixed. [5] The method for detecting a genetic polymorphism according to any one of the above [1] to [4], further comprising adding to the sample a negatively charged substance of biological origin that adsorbs to a PCR enzyme and a positively charged substance of biological origin that adsorbs to DNA, the substance binding to a substance that inhibits PCR and neutralizing the PCR inhibitory action of the negatively charged substance and the positively charged substance, and mixing the sample. [6] The method for detecting a genetic polymorphism according to any one of the above [1] to [5], wherein the exocrine fluid or mucus is saliva. [7] The method for detecting a genetic polymorphism according to the above [6], wherein the saliva is collected using a cotton swab, absorbent cotton, spit, or a DNA collection kit. [8] The method for detecting a genetic polymorphism according to any one of the above [1] to [7], wherein the PCR primer pair is a pair of base sequences represented by the following SEQ ID NO: 1 and SEQ ID NO: 2, or SEQ ID NO: 3 and SEQ ID NO: 4, or SEQ ID NO: 5 and SEQ ID NO: 6, or SEQ ID NO: 7 and SEQ ID NO: 8.

[0011] [ka]

[0012] [9] The method for detecting a genetic polymorphism according to any one of the above [1] to [8], wherein, in the fluorescently labeled probe, the base sequence of the oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding wild-type Cys for the 112th amino acid residue of apolipoprotein E, and the base sequence of the oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding mutant Arg for the 112th amino acid residue, are the base sequences represented by the following SEQ ID NOs: 9 and 10, respectively; and the base sequence of the oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding wild-type Arg for the 158th amino acid residue of apolipoprotein E, and the base sequence of the oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding mutant Cys for the 158th amino acid residue, are the base sequences represented by the following SEQ ID NOs: 11 and 12, respectively.

[0013] [ka]

[0014]

[10] A method for determining a genetic polymorphism of apolipoprotein E present in genomic DNA collected from a subject, comprising the following steps 1 and 2: 1. A step of obtaining an amplification curve based on the 112th amino acid residue and the 158th amino acid residue by the method for detecting a genetic polymorphism according to any one of the above-mentioned [1] to [9]; 2. (a) In the amplification curve based on the 112th amino acid residue obtained in the above step, A substantial increase in fluorescence intensity was observed only in a fluorescently labeled probe (112th Cys probe) having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding a wild-type Cys, and no substantial increase in fluorescence intensity was observed in a fluorescently labeled probe (112th Arg probe) having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding a mutant Arg, and In the amplification curve based on the 158th amino acid residue obtained in the above step, determining that the sample is apolipoprotein E3 / E3 when a substantial increase in fluorescence intensity is observed only from a fluorescently labeled probe (158th Arg probe) having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon that codes for wild-type Arg, and no substantial increase in fluorescence intensity is observed from a fluorescently labeled probe (158th Cys probe) having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon that codes for mutant Cys; (b) In the amplification curve based on the 112th amino acid residue obtained in the above step, A substantial increase in the fluorescence intensity derived from the 112th Cys probe was observed only, and no substantial increase in the fluorescence intensity derived from the 112th Arg probe was observed; and In the amplification curve based on the 158th amino acid residue obtained in the above step, determining that the sample is alipoprotein E2 / E2 when no substantial increase in the fluorescence intensity derived from the 158th Arg probe is observed and only a substantial increase in the fluorescence intensity derived from the 158th Cys probe is observed; (c) In the amplification curve based on the 112th amino acid residue obtained in the above step, A substantial increase in the fluorescence intensity derived from the 112th Cys probe was observed only, and no substantial increase in the fluorescence intensity derived from the 112th Arg probe was observed; and In the amplification curve based on the 158th amino acid residue obtained in the above step, determining that the sample is alipoprotein E2 / E3 when a substantial increase in the fluorescence intensity derived from the 158th Arg probe and a substantial increase in the fluorescence intensity derived from the 158th Cys probe are both observed; (d) In the amplification curve based on the 112th amino acid residue obtained in the above step, No substantial increase in the fluorescence intensity derived from the 112th Cys probe was observed, and only a significant increase in the fluorescence intensity derived from the 112th Arg probe was observed; and In the amplification curve based on the 158th amino acid residue obtained in the above step, determining that the sample is alipoprotein E4 / E4 when a substantial increase in the fluorescence intensity derived from the 158th Arg probe is observed but no substantial increase in the fluorescence intensity derived from the 158th Cys probe is observed; (e) In the amplification curve based on the 112th amino acid residue obtained in the above step, A substantial increase in the fluorescence intensity derived from the 112th Cys probe and the 112th Arg probe was observed, and In the amplification curve based on the 158th amino acid residue obtained in the above step, determining that the sample is alipoprotein E3 / E4 when a substantial increase in the fluorescence intensity derived from the 158th Arg probe is observed and no substantial increase in the fluorescence intensity derived from the 158th Cys probe is observed; or (f) In the amplification curve based on the 112th amino acid residue obtained in the above step, A substantial increase in the fluorescence intensity derived from the 112th Cys probe and the 112th Arg probe was observed, and In the amplification curve based on the 158th amino acid residue obtained in the above step, a step of determining that the sample is alipoprotein E2 / E4 when a substantial increase in the fluorescence intensity derived from both the 158th Arg probe and the 158th Cys probe is observed;

[0015]

[11] A kit for detecting or determining a genetic polymorphism of apolipoprotein E present in genomic DNA collected from a subject, the kit comprising the following (1) to (3): (1) PCR enzyme, (2) a PCR primer pair for amplifying a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding the 112th or 158th amino acid residue (Cys or Arg) of apolipoprotein E; and (3) A set of a fluorescently labeled probe having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene, the nucleic acid fragment containing a codon that codes for the wild-type Cys at the 112th amino acid residue of apolipoprotein E, and a set of a fluorescently labeled probe having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene, the nucleic acid fragment containing a codon that codes for the mutant Arg at the 112th amino acid residue, the fluorescently labeled probe set comprising fluorescently labeled probes having different fluorescent dyes for labeling. or a set of a fluorescently labeled probe having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene, the nucleic acid fragment containing a codon encoding the wild-type Arg at the 158th amino acid residue of apolipoprotein E, and a fluorescently labeled probe having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene, the nucleic acid fragment containing a codon encoding the mutant Cys at the 158th amino acid residue, the fluorescently labeled probe set using different fluorescent dyes for labeling.

[12] The kit according to

[11] above, further comprising a surfactant and protease K.

[13] The kit according to

[12] , wherein the surfactant is sodium dodecyl sulfate.

[14] The kit according to any one of the above

[11] to

[13] , further comprising a Tris-HCl buffer solution containing potassium chloride, magnesium chloride, and a dNTP mix.

[15] The kit according to any one of the above

[11] to

[14] , further comprising a negatively charged substance of biological origin that adsorbs to a PCR enzyme and a positively charged substance of biological origin that adsorbs to DNA, which binds to a substance that inhibits PCR and neutralizes the PCR inhibitory effect of the negatively charged substance and the positively charged substance.

[16] The kit according to any one of

[11] to

[14] above, further comprising a cotton swab, absorbent cotton, a saliva collection tool, and a DNA collection kit.

[17] The kit according to any one of the above

[11] to

[16] , wherein the PCR primer pair is a pair of base sequences represented by the following SEQ ID NO: 1 and SEQ ID NO: 2, or SEQ ID NO: 3 and SEQ ID NO: 4.

[0016] [ka]

[0017]

[18] The kit according to any one of the above

[11] to

[17] , wherein in the fluorescently labeled probe, the base sequence of the oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding wild-type Cys for the 112th amino acid residue of apolipoprotein E, and the base sequence of the oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding mutant Arg for the 112th amino acid residue, are the base sequences represented by the following SEQ ID NO:9 and SEQ ID NO:10, respectively; and the base sequence of the oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding wild-type Arg for the 158th amino acid residue of apolipoprotein E, and the base sequence of the oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding mutant Cys for the 158th amino acid residue, are the base sequences represented by the following SEQ ID NO:11 and SEQ ID NO:12, respectively.

[0018] [ka]

[0019]

[19] A dementia diagnosis system comprising the kit according to any one of

[11] to

[18] above and MRI or CT.

[20] The dementia diagnostic system described in

[19] above, wherein the dementia is Alzheimer's disease. Effect of the Invention

[0020] According to the present invention, gene polymorphism of apolipoprotein E can be detected and determined relatively simply and quickly with little burden on the subject. [Brief description of the drawings]

[0021] [Figure 1]These are PCR amplification curves for each gene polymorphism of apolipoprotein E. The upper figures show the amplification curves corresponding to the 112th amino acid residue, and the lower figures show the amplification curves corresponding to the 158th amino acid residue. The vertical axis shows the fluorescence intensity, and the horizontal axis shows the PCR cycle number. [Diagram 2] 1 shows PCR amplification curves for the detection method of the present invention using directly collected saliva samples. The right-hand figures show the amplification curves for Examples 1 to 4, and the left-hand figures show the amplification curves for the control. The vertical axis shows the fluorescence intensity, and the horizontal axis shows the PCR cycle number. [Diagram 3] 1 shows PCR amplification curves for the detection method of the present invention using saliva swab samples. The right-hand figures show the amplification curves for Examples 1 to 4, and the left-hand figures show the amplification curves for the control. The vertical axis shows the fluorescence intensity, and the horizontal axis shows the PCR cycle number. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The present invention will be described in detail below. As mentioned above, apolipoprotein E has three main isoforms, ApoE2, ApoE3, and ApoE4. These differ in two amino acid residues, 112th and 158th, with ApoE2 being Cys (cysteine ​​residue) / Cys (cysteine ​​residue), ApoE3 being Cys (cysteine ​​residue) / Arg (arginine residue), and ApoE4 being Arg (arginine residue) / Arg (arginine residue). Of these, ApoE3, which has Cys at 112th amino acid residue and Arg at 158th amino acid residue, is the wild type and is the most frequent. ApoE2 and ApoE4 are mutant types.

[0023] In addition, the codon encoding Cys at positions 112 and 158 of apolipoprotein E is "TGC," and the codon encoding Arg at positions 112 and 158 of apolipoprotein E is "CGC."

[0024] 1. Genetic polymorphism detection method according to the present invention The genetic polymorphism detection method of the present invention (hereinafter referred to as the "detection method of the present invention") is a method for detecting a genetic polymorphism of apolipoprotein E present in genomic DNA collected from a subject, and is characterized by comprising the following steps 1 to 3: 1. A step of isolating DNA from exocrine secretions or mucus containing epithelial cells collected from a subject to prepare a sample containing the DNA;

[0025] 2. The sample containing the DNA is (1) a PCR enzyme; (2) a PCR primer pair for amplifying a nucleic acid fragment of the apolipoprotein E gene, which contains a codon encoding the 112th or 158th amino acid residue (Cys or Arg) of apolipoprotein E; and (3) a fluorescently labeled probe having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene, which contains a codon encoding the wild-type Cys at the 112th amino acid residue of apolipoprotein E, and a fluorescently labeled probe having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene, which contains a codon encoding the mutant Arg at the 112th amino acid residue. a step of adding and mixing a set of optically labeled probes, the fluorescent labeled probe set being different from each other in fluorescent dyes used for labeling, or a set of fluorescent labeled probes having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene, the nucleic acid fragment containing a codon that codes for Arg, which is a wild type, at the 158th amino acid residue of apolipoprotein E, and a set of fluorescent labeled probes having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene, the nucleic acid fragment containing a codon that codes for Cys, which is a mutant type at the 158th amino acid residue of the apolipoprotein E, the fluorescent labeled probe sets being different from each other in fluorescent dyes used for labeling;

[0026] 3. A step of subjecting the mixture to PCR and measuring the fluorescence intensity of the PCR product corresponding to the 112th or 158th amino acid residue of the apolipoprotein E of the subject.

[0027] 1.1 Step 1 Step 1 is a process of isolating DNA from exocrine secretions or mucus containing epithelial cells collected from a subject, and preparing a sample containing the DNA. The external secretions or mucus are not particularly limited, but may include, for example, saliva and nasal mucous membrane fluid. In the present invention, saliva is preferred because it is easy to use. The method for collecting saliva from a subject is not particularly limited, but may include, for example, a method in which the subject directly spits out saliva or a method in which the inside of the cheek is rubbed with a tool such as a cotton swab.

[0028] The amount of the exocrine fluid or mucus collected may be small, specifically, for example, within the range of 0.1 to 10 μL, preferably within the range of 0.5 to 5 μL. When the subject is asked to directly spit out saliva as the exocrine fluid, an amount of saliva exceeding the above range may be collected and used as needed.

[0029] The release of DNA from the exocrine fluid or mucus can be carried out by a conventional method. For example, the collected exocrine fluid can be treated with a surfactant and protease K. Specifically, the collected exocrine fluid can be added to a solution containing a surfactant, sodium chloride, Tris-HCl (pH 8.0), and EDTA, or a solution containing these and protease K, and stirred using a vortex mixer or the like. Heating can also be carried out as necessary.

[0030] The surfactant may be selected from anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants. Among these, anionic surfactants are preferred, and sodium dodecyl sulfate is more preferred. The surfactant may be used within the range of 0.1 to 0.5% (w / v), for example. Protease K has the effect of inactivating DNA and RNA decomposition enzymes, and can be used, for example, within the range of 5 to 20 mg / mL.

[0031] 1.2 Step 2 Step 2 is a process in which a PCR enzyme, a PCR primer pair, a fluorescently labeled probe, etc. are added to and mixed with the sample to prepare the so-called PCR reaction solution necessary to initiate the polymerase chain reaction (PCR).

[0032] Examples of PCR enzymes include DNA polymerases. DNA polymerases are thermostable DNA polymerases derived from thermophilic bacteria, and include, but are not limited to, Taq, Tth, KOD, Pfu, and mutants thereof. In order to avoid non-specific amplification by DNA polymerases, hot-start DNA polymerases may be used. Examples of hot-start DNA polymerases include BIOTAQ (registered trademark) hot-start DNA polymerase. Hot-start DNA polymerases include DNA polymerases bound to anti-DNA polymerase antibodies and DNA polymerases whose enzyme active sites have been chemically modified to be heat-sensitive, and either of these can be used in the present invention.

[0033] The PCR primer pair used in the detection method of the present invention can use genomic DNA in exocrine fluids, etc. collected from a subject as a template to amplify, by PCR, a nucleic acid fragment containing the polymorphic site of the apolipoprotein E gene to be analyzed, i.e., the codon site encoding the 112th or 158th amino acid residue (Cys or Arg) of apolipoprotein E.

[0034] The length of the nucleic acid fragment to be amplified is suitably within the range of 20 to 1000 bases including the polymorphic site of the apolipoprotein E gene polymorphism, and is preferably within the range of 20 to 200 bases. As such a PCR primer pair, oligonucleotides (forward primer and reverse primer) that hybridize under stringent conditions to a region consisting of the base sequences shown in SEQ ID NOs: 13 and 14 are preferred. Here, stringent conditions refer to conditions under which binding between the template DNA and the primers is specific in annealing in PCR, which is the step in which the primers bind to the template DNA. The PCR primer pair according to the present invention preferably has a base length of 50 to 150 bases. Examples of more preferred base sequences of the PCR primer pair in the present invention include the following.

[0035] <Regarding the genetic polymorphism corresponding to the 112th amino acid residue> [ka]

[0036] <Regarding the genetic polymorphism corresponding to the 158th amino acid residue> [ka]

[0037] The following SEQ ID NOs: 13 and 14 are the nucleotide sequences of regions containing a polymorphism corresponding to the 112th amino acid residue and a polymorphism corresponding to the 158th amino acid residue, respectively, in the wild-type ApoE gene (ApoE3 / E3). In both cases, the portions in quotation marks are the codons corresponding to the 112th and 158th amino acid residues, respectively. Any nucleotide sequence in SEQ ID NOs: 13 and 14 can be amplified as long as it contains the polymorphic site.

[0038] [ka]

[0039] [ka]

[0040] The fluorescently labeled probe set used in the detection method of the present invention is a combination of a fluorescently labeled probe having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding wild-type Cys at the 112th amino acid residue of apolipoprotein E (hereinafter referred to as the "112th Cys probe") and a fluorescently labeled probe having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding mutant Arg at the 112th amino acid residue of apolipoprotein E (hereinafter referred to as the "112th Arg probe"), or a combination of a fluorescently labeled probe having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding wild-type Arg at the 158th amino acid residue of apolipoprotein E (hereinafter referred to as the "158th Arg probe") and a fluorescently labeled probe having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding mutant Cys at the 158th amino acid residue of apolipoprotein E (hereinafter referred to as the "158th Cys probe").

[0041] The fluorescent dyes in the 112th Cys probe and the 112th Arg probe, and the fluorescent dyes in the 158th Arg probe and the 158th Cys probe are different from each other, making it possible to distinguish whether the amino acid residue at the relevant site is Cys or Arg.

[0042] The Cys 112 probe and the Cys 158 probe hybridize under stringent conditions to a nucleic acid fragment amplified by PCR that encodes Cys at amino acid residue 112 or 158, but do not hybridize under stringent conditions to a nucleic acid fragment amplified by PCR that encodes Arg at amino acid residue 112 or 158. On the other hand, the Arg 112 probe and the Arg 158 probe hybridize under stringent conditions to a nucleic acid fragment amplified by PCR that encodes Arg at amino acid residue 112 or 158, but do not hybridize under stringent conditions to a nucleic acid fragment amplified by PCR that encodes Cys at amino acid residue 112 or 158.

[0043] Here, stringent conditions refer to conditions under which a specific hybrid is formed upon annealing between a nucleic acid fragment amplified by PCR and an oligonucleotide in a fluorescently labeled probe, and a non-specific hybrid is not formed.

[0044] The oligonucleotide in the fluorescently labeled probe used in the detection method of the present invention preferably has a base length of 10 to 25 bases, more preferably 15 to 20 bases. The base sequence of each oligonucleotide in the fluorescently labeled probe set, which is most preferred in the detection method of the present invention, is shown below. SEQ ID NO: 9 is a base sequence that binds to a nucleic acid fragment in which the 112th amino acid residue of apolipoprotein E is the wild type (Cys). Similarly, SEQ ID NOs: 10 to 12 are base sequences that bind when the 112th amino acid residue is mutant (Arg), and when the 158th amino acid residue is wild type (Arg) or mutant (Cys), respectively.

[0045] [ka]

[0046] The PCR reaction solution usually contains a PCR buffer. The PCR buffer is preferably Tris-HCl. The PCR buffer usually contains KCl, MgCl2, and dNTP mix (a mixture of deoxyribonucleotide 5'-triphosphate; dATP, dGTP, dCTP, and dTTP), but is not limited thereto. The concentrations of dNTP, MgCl2, KCl, and the buffer are appropriately set by those skilled in the art. For example, the MgCl2 may be 1.5 mM, the KCl may be 35 mM, the dNTP may be 200 μM, and the Tris-HCl may be 10 mM.

[0047] Furthermore, in the detection method of the present invention, the PCR buffer solution can contain negatively charged substances of biological origin (e.g., certain sugars and pigments, etc.) that adsorb to DNA polymerase and positively charged substances of biological origin (e.g., certain proteins, etc.) that adsorb to DNA and bind to substances that inhibit PCR, thereby neutralizing the PCR-inhibiting action of the negatively charged and positively charged substances.

[0048] Specific examples of the PCR buffer include gene amplification reagents Ampdirect (registered trademark) and Ampdirect (registered trademark) Plus (both manufactured by Shimadzu Corporation).

[0049] 1.3 Step 3 Step 3 is a process in which PCR is performed on the prepared sample and the fluorescence intensity of the PCR product corresponding to the 112th or 158th amino acid residue of apolipoprotein E is measured.

[0050] In the detection method of the present invention, PCR is used to detect apolipoprotein E gene polymorphism. Those skilled in the art can easily set PCR conditions (temperature, time, and number of cycles) as appropriate. In the detection method of the present invention, the detection of PCR products can be carried out by real-time measurement. Real-time measurement of PCR products is also called real-time PCR.

[0051] In the detection method of the present invention, a fluorescently labeled probe having an oligonucleotide can be used to detect the PCR product by fluorescence. Examples of the fluorescently labeled probe include, but are not limited to, hydrolysis probes (such as TaqMan (registered trademark) probes), Molecular Beacons, and cycling probes. Among these, from the viewpoint of detection specificity and multiplex analysis possibility, it is preferable to use a hydrolysis probe. Furthermore, from the viewpoint of high sensitivity detection, it is preferable to use a Molecular Beacon. Furthermore, from the viewpoint of high accuracy detection, it is preferable to use a cycling probe.

[0052] A hydrolysis probe is an oligonucleotide modified with a fluorescent dye at the 5' end and a quencher at the 3' end. The hydrolysis probe hybridizes specifically to the template DNA during PCR annealing, but due to the presence of a quencher on the probe, the generation of fluorescence is suppressed (quenched) by fluorescence resonance energy transfer (FRET) even when irradiated with excitation light. In the subsequent extension reaction step, when the hydrolysis probe hybridized to the template DNA is decomposed by the 5'→3' exonuclease activity of Taq DNA polymerase, the fluorescent dye is released from the probe, and the quenching by the quencher is released, causing the probe to emit fluorescence. The amount of amplified product produced can be measured by measuring the intensity of this fluorescence.

[0053] From the viewpoint of detecting gene polymorphisms with higher accuracy, the above-mentioned fluorescently labeled probe may be an embodiment containing a flap endonuclease (FEN). Examples of such fluorescently labeled probes include a molecular set containing FEN, a detection probe, and a detection FRET cassette corresponding to the probe. The detection probes may be for detecting wild-type DNA and for detecting mutant-type DNA, and each may have a different flap sequence at its end. The wild-type detection probe may have a structure containing an oligonucleotide having a base sequence that specifically hybridizes with wild-type DNA, and the mutant detection probe may have a structure containing an oligonucleotide that similarly specifically hybridizes with mutant-type DNA. The detection FRET cassette is a molecule that recognizes each detection probe, and may have a structure in which a fluorescent dye is bound to the end of the recognition site. The fluorescent dye may be in a state of being quenched by a quencher in the FRET cassette.

[0054] When PCR is performed using such a fluorescently labeled probe, and the oligonucleotide and the detection probe hybridize to the template DNA in the annealing and extension reaction steps, a triple-stranded structure (flap structure) consisting of the oligonucleotide, the detection probe, and the template DNA can be formed. In this case, FEN breaks the bond and releases the flap sequence at the end of the detection probe (note that the oligonucleotide becomes a PCR amplification product after the extension reaction step). The released flap sequence hybridizes to the corresponding FRET cassette, forming a triple structure (flap structure) consisting of the flap sequence, the FRET cassette, and the fluorescent dye bound to it. In this case, FEN breaks the bond and releases the fluorescent dye. After release, the fluorescent dye is released from FRET quenching and emits fluorescence. The amount of PCR amplified product produced can be measured by measuring the intensity of this fluorescence.

[0055] Examples of the fluorescent dye include, but are not limited to, FAM (6-carboxyfluorescein), ROX (6-carboxy-X-rhodamine), Cy3 and Cy5 (cyanine dyes), and HEX (4,7,2',4',5',7'-hexachloro-6-carboxyfluorescein). Examples of the quencher include, but are not limited to, TAMRA (registered trademark), BHQ (Black Hole Quencher, registered trademark) 1, BHQ2, MGB-Eclipse (registered trademark), DABCYL, BHQ3, and the like.

[0056] In the detection method of the present invention, two types of fluorescently labeled probes, a 112th Cys probe and a 112th Arg probe, or a 158th Arg probe and a 158th Cys probe, are used to distinguish and detect two types of DNA target base sequences. For this reason, the two types of fluorescently labeled probes are labeled with different fluorescent dyes. The combination of different fluorescent dyes is not particularly limited as long as they have different fluorescent properties and do not interfere with each other in fluorescence measurement. Specifically, for example, FAM can be used for the 112th Cys probe, ROX can be used for the 112th Arg probe, FAM can be used for the 158th Arg probe, and ROX can be used for the 158th Cys probe. That is, FAM can be used for the wild type, and ROX can be used for the mutant type.

[0057] 2. Determination of apolipoprotein E gene polymorphism In the detection method of the present invention, the amplification curve of the PCR product is monitored using a fluorescence filter corresponding to the fluorescent dye used in real-time measurement of the PCR product. If the fluorescence intensity increases substantially according to the number of PCR cycles, the presence of the target DNA in the sample is determined to be positive, whereas if the fluorescence intensity does not increase substantially during PCR, the result is determined to be negative.

[0058] In the present invention, the detection method of the present invention can obtain amplification curves based on the 112th amino acid residue and the 158th amino acid residue, for example, as shown in FIG. 1, for each data based on the 112th amino acid residue, the solid line represents the fluorescence intensity derived from Cys, and the dashed line represents the fluorescence intensity derived from Arg. Meanwhile, for each data based on the 158th amino acid residue, the solid line represents the fluorescence intensity derived from Arg, and the dashed line represents the fluorescence intensity derived from Cys. Thus, the solid line corresponds to the amino acid residues encoded by the wild-type apolipoprotein E gene, and the dashed line corresponds to the amino acid residues encoded by the mutant apolipoprotein E gene.

[0059] Then, from the obtained amplification curve, the apolipoprotein E gene polymorphism can be determined as follows. In the amplification curve based on the 112th amino acid residue, if a substantial increase in the fluorescence intensity derived from the 112th Cys probe (solid line in Figure 1; same below) is observed only while a substantial increase in the fluorescence intensity derived from the 112th Arg probe (dashed line in Figure 1; same below) is not observed, and in the amplification curve based on the 158th amino acid residue, if a substantial increase in the fluorescence intensity derived from the 158th Arg probe (solid line in Figure 1; same below) is observed only while a substantial increase in the fluorescence intensity derived from the 158th Cys probe (dashed line in Figure 1; same below) is not observed, the apolipoprotein E gene polymorphism can be determined to be E3 / E3 (homozygous type).

[0060] If, in the amplification curve based on the 112th amino acid residue, a substantial increase in the fluorescence intensity derived from the 112th Cys probe is observed only, and no substantial increase in the fluorescence intensity derived from the 112th Arg probe is observed, and, in the amplification curve based on the 158th amino acid residue, no substantial increase in the fluorescence intensity derived from the 158th Arg probe is observed, and a substantial increase in the fluorescence intensity derived from the 158th Cys probe only is observed, the apolipoprotein E gene polymorphism can be determined to be E2 / E2 (homozygous type).

[0061] If, in the amplification curve based on the 112th amino acid residue, a substantial increase in the fluorescence intensity derived from the 112th Cys probe is observed only, and no substantial increase in the fluorescence intensity derived from the 112th Arg probe is observed, and, in the amplification curve based on the 158th amino acid residue, a substantial increase in the fluorescence intensity derived from the 158th Arg probe is observed, and also a substantial increase in the fluorescence intensity derived from the 158th Cys probe is observed, the apolipoprotein E gene polymorphism can be determined to be E2 / E3 (heterozygous type).

[0062] If, in the obtained amplification curve based on the 112th amino acid residue, no substantial increase in the fluorescence intensity derived from the 112th Cys probe is observed, and only a significant increase in the fluorescence intensity derived from the 112th Arg probe is observed, and, in the obtained amplification curve based on the 158th amino acid residue, only a substantial increase in the fluorescence intensity derived from the 158th Arg probe is observed, and no substantial increase in the fluorescence intensity derived from the 158th Cys probe is observed, the apolipoprotein E genetic polymorphism can be determined to be E4 / E4 (homozygous type).

[0063] If a substantial increase in the fluorescence intensity derived from both the Cys probe at position 112 and the Arg probe at position 112 is observed in the amplification curve obtained based on the 112th amino acid residue, and if a substantial increase in the fluorescence intensity derived from only the Arg probe at position 158 is observed in the amplification curve obtained based on the 158th amino acid residue, but no substantial increase in the fluorescence intensity derived from the Cys probe at position 158 is observed, the apolipoprotein E gene polymorphism can be determined to be E3 / E4 (heterozygous type).

[0064] If a substantial increase in the fluorescence intensity derived from both the Cys probe at position 112 and the Arg probe at position 112 is observed in the amplification curve based on the obtained amino acid residue at position 112, and if a substantial increase in the fluorescence intensity derived from both the Arg probe at position 158 and the Cys probe at position 158 is observed in the amplification curve based on the obtained amino acid residue at position 158, the apolipoprotein E gene polymorphism can be determined to be E2 / E4 (heterozygous type).

[0065] Here, a "substantial increase" in fluorescence intensity does not refer to an increase in a false signal due to, for example, artifacts or other factors (e.g., the probe is broken down by temperature, causing it to dissociate from the quencher, resulting in a gradual increase in fluorescence), but rather refers to an increase that a person skilled in the art can determine is an increase in fluorescence intensity that is actually derived from the probe bound to the base sequence corresponding to the amino acid residue in question.

[0066] In other words, in each data item in Figure 1, if the fluorescence intensity only increases along the solid line, this is indicated as judgment indication 1; if the fluorescence intensity only increases along the dashed line, this is indicated as judgment indication 2; and if both lines increase substantially, this is indicated as judgment indication 12, then a judgment table for apolipoprotein E gene polymorphisms as shown in Table 1 below can be obtained.

[0067] [Table 1]

[0068] As shown in Table 1, the combinations of 112 and 158 with 1 and 1, 1 and 2, and 2 and 1, respectively, are homozygous, of which 1 and 1 are wild type and the rest are E2 or E4 mutant. The combinations of 112 and 158 with 12 and 12 are E2 / E4 heterozygous, and the rest are wild type and mutant type heterozygous, which can be determined as E2 / E3 or E3 / E4.

[0069] 3. Kit according to the present invention The kit of the present invention (hereinafter referred to as the "kit of the present invention") is a kit for detecting or determining a genetic polymorphism of apolipoprotein E present in genomic DNA collected from a subject, and is characterized by comprising the following (1) to (3): (1) PCR enzyme, (2) a PCR primer pair for amplifying a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding the 112th or 158th amino acid residue (Cys or Arg) of apolipoprotein E; and (3) A fluorescently labeled probe set including a 112th Cys probe and a 112th Arg probe, the fluorescent dyes used for labeling being different from each other, or a fluorescently labeled probe set including a 158th Arg probe and a 158th Cys probe, the fluorescent dyes used for labeling being different from each other.

[0070] In the kit of the present invention, the terms PCR primer pair, fluorescently labeled probe set, etc. are the same as those defined above. The kit of the present invention can be used in a rapid genotyping system for performing a simple and rapid genetic test in a clinical setting. An example of the rapid genotyping system is GTS-7000 manufactured by Shimadzu Corporation.

[0071] In one embodiment of the present invention, the kit of the present invention may include a PCR buffer solution containing a surfactant and proteinase K used in sample pretreatment to facilitate application to a genotype rapid determination system. The surfactant is preferably an anionic surfactant, more preferably sodium dodecyl sulfate. In one embodiment, the PCR buffer solution may be a Tris buffer solution containing KCl, MgCl2, and a dNTP mix (a mixture of dATP, dGTP, dCTP, and dTTP). In one embodiment, the PCR buffer solution may include a substance that binds to a biologically derived negatively charged substance that adsorbs to DNA polymerase and a biologically derived positively charged substance that adsorbs to DNA and inhibits PCR, and neutralizes the PCR inhibitory effect of the negatively charged substance and the positively charged substance. In one embodiment, the kit of the present invention may include a gene amplification reagent Ampdirect (registered trademark) or Ampdirect (registered trademark) Plus (both manufactured by Shimadzu Corporation).

[0072] When applying the kit of the present invention to a rapid genotype determination system, by incorporating, as positive controls, an artificially synthesized wild-type gene containing a codon encoding the wild-type Cys at the 112th amino acid residue of apolipoprotein E and a mutant gene containing a codon encoding the mutant Arg at the 112th amino acid residue, and / or a wild-type gene containing a codon encoding the wild-type Arg at the 158th amino acid residue of apolipoprotein E and a mutant gene containing a codon encoding the mutant Cys at the 158th amino acid residue, automatic genotype determination is possible.

[0073] The present invention can also provide a dementia diagnosis system including the kit of the present invention and MRI and / or CT. The accuracy of dementia diagnosis can be improved by combining the kit of the present invention or the detection method of the present invention using the kit with MRI and / or CT. The dementia can be, for example, Alzheimer's dementia. EXAMPLES

[0074] The present invention will be described below with reference to examples, but the present invention is not limited to these examples in any way.

[0075] [Measurement of ApoE gene polymorphism using saliva samples] Using human saliva samples (four individuals), the accuracy of determining ApoE gene polymorphisms was confirmed based on the kit and detection method of the present invention. The reagents used are as follows: Cell lysis solution: Prepared by mixing Tris-HCl (pH 8.0, 20 mM), EDTA (5 mM), NaCl (400 mM), SDS (0.3%), and distilled water (each concentration indicates the final concentration of each component). PCR buffer: Ampdirect Plus Reagent (Shimadzu Corporation) Enzyme: BIOTAQ® DNA polymerase (Bioline) 112-PT: primer (sequence numbers 1 and 2) / fluorescently labeled probe (containing oligonucleotides of sequence numbers 9 and 10) mixture 158-PT: primer (sequence numbers 5 and 6) / fluorescently labeled probe (containing oligonucleotides of sequence numbers 11 and 12) mixture E2 / E2: ApoE2 / ApoE2 control DNA E3 / E3: ApoE3 / ApoE3 control DNA E4 / E4: ApoE4 / ApoE4 control DNA E2 / E3: ApoE2 / ApoE3 control DNA E2 / E4: ApoE2 / ApoE4 control DNA E3 / E4: ApoE3 / ApoE4 control DNA In addition, hydrolysis probes (TaqMan (registered trademark) probes) were used as the above-mentioned fluorescently labeled probes. As for the fluorescent dye, FAM was used for the probes having the oligonucleotides of SEQ ID NOs: 9 and 11, and ROX was used for the probes having the oligonucleotides of SEQ ID NOs: 10 and 12, respectively.

[0076] [Examples 1 to 4] Measurement using directly collected samples (Collection Method 1) The samples used were 2 μL of human saliva (four individuals) spat out directly into microtubes (1.5 mL). The PCR reaction solution for detecting the polymorphism corresponding to the 112th amino acid residue of the protein encoded by ApoE was prepared by mixing PCR buffer, Enzyme, 112-PT, and purified water. 24 μL of the PCR reaction solution was dispensed into PCR reaction tubes, and 1 μL of each saliva sample was added thereto and mixed. The PCR reaction solution for detecting the polymorphism corresponding to the 158th amino acid residue was prepared by mixing PCR buffer, Enzyme, 158-PT, and purified water. 24 μL of the reaction solution was also dispensed into PCR reaction tubes, and 1 μL of each saliva sample was added thereto and mixed.

[0077] For each of the resulting mixtures, the PCR reaction was immediately measured using a real-time PCR device (GTS-7000, manufactured by Shimadzu Corporation) (Examples 1 to 4). In the measurement, the enzyme was first activated under conditions of 95°C / 10 minutes, and then 50 cycles of PCR were performed under conditions of 95°C / 10 seconds to 60°C / 30 seconds. As positive and negative controls, mixtures were prepared by adding each of the above E2 / E2 to E3 / E4 DNAs or distilled water instead of the saliva samples, and the PCR reaction was measured in the same manner.

[0078] The measurement results are shown in Figure 2. In all of Examples 1 to 4, substantial amplification was observed only in the fluorescence intensity (solid line) derived from the 112th Cys probe, and no amplification was observed in the fluorescence intensity (dashed line) derived from the 112th Arg probe. In addition, for the 158th, substantial amplification was observed only in the fluorescence intensity (solid line) derived from the 158th Arg probe, and no substantial amplification was observed in the fluorescence intensity (dashed line) derived from the 158th Cys probe. From these results, each Example could be determined to be E3 / E3 (homozygous type).

[0079] [Examples 5 to 8] Measurement using swab samples (Collection method 2) The samples used were saliva and oral epithelial cells collected from humans (the same four individuals as in Examples 1 to 4) using a cotton swab. Specifically, the inside of each individual's cheek was rubbed with a cotton swab, immersed in 100 μL of cell dissolving solution in a microtube (1.5 mL), and stirred for about 20 seconds. 2 μL of the solution after stirring was used as the sample. A mixture was prepared in the same manner as in the above Examples 1 to 4, and the PCR reaction was measured under the same conditions. The same was true for the positive and negative controls.

[0080] The measurement results are shown in Figure 3. In all of Examples 5 to 8, substantial amplification was observed only in the fluorescence intensity (solid line) derived from the 112th Cys probe, and no amplification was observed in the fluorescence intensity (dashed line) derived from the 112th Arg probe. In addition, for the 158th, substantial amplification was observed only in the fluorescence intensity (solid line) derived from the 158th Arg probe, and no substantial amplification was observed in the fluorescence intensity (dashed line) derived from the 158th Cys probe. From these results, each Example could be determined to be E3 / E3 (homozygous type).

[0081] From the above, it was shown that the ApoE polymorphism can be accurately identified by using the kit and the detection method of the present invention. In each of the above examples, the amplification curve begins to increase about 30 minutes after the start of the reaction, and the rise in fluorescence intensity is faster than when measuring a normal blood sample. It can be said that the detection method of the present invention can determine gene polymorphism more quickly than the conventional detection method using blood. [Sequence List Free Text]

[0082] SEQ ID NO: 1: PCR forward primer for amplifying a nucleic acid fragment of the apolipoprotein E gene containing the codon encoding the 112th amino acid residue of apolipoprotein E. SEQ ID NO: 2: PCR reverse primer for amplifying a nucleic acid fragment of the apolipoprotein E gene containing the codon encoding the 112th amino acid residue of apolipoprotein E. SEQ ID NO: 3: PCR forward primer for amplifying a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding the 112th amino acid residue of apolipoprotein E. SEQ ID NO: 4: PCR reverse primer for amplifying a nucleic acid fragment of the apolipoprotein E gene containing the codon encoding the 112th amino acid residue of apolipoprotein E. SEQ ID NO: 5: PCR forward primer for amplifying a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding the 158th amino acid residue of apolipoprotein E. SEQ ID NO: 6: PCR reverse primer for amplifying a nucleic acid fragment of the apolipoprotein E gene containing the codon encoding the 158th amino acid residue of apolipoprotein E. SEQ ID NO: 7: PCR forward primer for amplifying a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding the 158th amino acid residue of apolipoprotein E. SEQ ID NO: 8: PCR reverse primer for amplifying a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding the 158th amino acid residue of apolipoprotein E. SEQ ID NO: 9: Probe for detecting an amplified nucleic acid fragment of the apolipoprotein E gene containing a wild-type codon for the 112th amino acid residue of apolipoprotein E. SEQ ID NO: 10: Probe for detecting an amplified nucleic acid fragment of the apolipoprotein E gene containing a mutant codon for the 112th amino acid residue of apolipoprotein E. SEQ ID NO: 11: Probe for detecting an amplified nucleic acid fragment of the apolipoprotein E gene containing a wild-type codon for the 158th amino acid residue of apolipoprotein E. SEQ ID NO: 12: Probe for detecting an amplified nucleic acid fragment of the apolipoprotein E gene containing a mutant codon for the 158th amino acid residue of apolipoprotein E. SEQ ID NO: 13: Nucleotide sequence of a region containing a genetic polymorphism corresponding to the 112th amino acid residue in the ApoE gene. SEQ ID NO: 13: The positions from 122 to 124 in the sequence are a wild-type codon corresponding to the 112th amino acid residue of apolipoprotein E. SEQ ID NO: 14: Nucleotide sequence of a region containing a genetic polymorphism corresponding to the 158th amino acid residue in the ApoE gene. SEQ ID NO: 14: The positions from 135 to 137 in the sequence are a wild-type codon corresponding to the 158th amino acid residue of apolipoprotein E.

Claims

1. A method for detecting a polymorphism in apolipoprotein E gene present in genomic DNA collected from a subject, comprising the following steps 1 to 3:

1. A step of isolating DNA from exocrine secretions or mucus containing epithelial cells collected from a subject to prepare a sample containing the DNA; 2. A sample containing the DNA is subjected to (1) PCR enzyme, (2) a PCR primer pair for amplifying a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding the 112th or 158th amino acid residue (Cys or Arg) of apolipoprotein E; and (3) A set of a fluorescently labeled probe having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene, the nucleic acid fragment containing a codon that codes for the wild-type Cys at the 112th amino acid residue of apolipoprotein E, and a set of a fluorescently labeled probe having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene, the nucleic acid fragment containing a codon that codes for the mutant Arg at the 112th amino acid residue, the fluorescently labeled probe sets being different in fluorescent dyes used for labeling. or a step of adding and mixing a set of a fluorescently labeled probe having an oligonucleotide capable of binding to a nucleic acid fragment of the apolipoprotein E gene, the nucleic acid fragment containing a codon encoding the wild-type Arg at the 158th amino acid residue of apolipoprotein E, and a set of a fluorescently labeled probe having an oligonucleotide capable of binding to a nucleic acid fragment of the apolipoprotein E gene, the nucleic acid fragment containing a codon encoding the mutant Cys at the 158th amino acid residue, the fluorescently labeled probe sets being different from each other in fluorescent dyes used for labeling; 3. A step of carrying out PCR on the mixture and measuring the fluorescence intensity from the PCR product corresponding to the 112th or 158th amino acid residue of the apolipoprotein E of the subject.

2. 2. The method for detecting a genetic polymorphism according to claim 1, wherein in step 1, the DNA is released using a surfactant and protease K.

3. The method for detecting a genetic polymorphism according to claim 2 , wherein the surfactant is sodium dodecyl sulfate.

4. 4. The method for detecting a genetic polymorphism according to claim 1, further comprising adding a Tris-HCl buffer solution containing potassium chloride, magnesium chloride, and a dNTP mix to the sample and mixing the sample.

5. The method for detecting a genetic polymorphism according to any one of claims 1 to 4, further comprising adding to and mixing with the specimen a negatively charged substance derived from a living organism that adsorbs to a PCR enzyme and a positively charged substance derived from a living organism that adsorbs to DNA, the substance binding to a substance that inhibits PCR and neutralizing the PCR inhibitory action of the negatively charged substance and the positively charged substance.

6. The method for detecting a genetic polymorphism according to any one of claims 1 to 5, wherein the exocrine fluid or mucus is saliva.

7. The method for detecting a genetic polymorphism according to claim 6, wherein the saliva is collected by a cotton swab, absorbent cotton, spit, or a DNA collection kit.

8. The method for detecting a genetic polymorphism according to any one of claims 1 to 7, wherein the PCR primer pair is a pair of base sequences represented by the following SEQ ID NO:1 and SEQ ID NO:2, or SEQ ID NO:3 and SEQ ID NO:4, or SEQ ID NO:5 and SEQ ID NO:6, or SEQ ID NO:7 and SEQ ID NO:

8. 【Chemistry 1】

9. The method for detecting a genetic polymorphism according to any one of claims 1 to 8, wherein in the fluorescently labeled probe, the base sequence of the oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding wild-type Cys for the 112th amino acid residue of apolipoprotein E, and the base sequence of the oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding mutant Arg for the 112th amino acid residue, are the base sequences represented by the following SEQ ID NOs: 9 and 10, respectively; and the base sequence of the oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding wild-type Arg for the 158th amino acid residue of apolipoprotein E, and the base sequence of the oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding mutant Cys for the 158th amino acid residue, are the base sequences represented by the following SEQ ID NOs: 11 and 12, respectively. 【Chemistry 2】

10. A method for determining a genetic polymorphism of apolipoprotein E present in genomic DNA collected from a subject, comprising the following steps 1 and 2:

1. A step of obtaining an amplification curve based on the 112th amino acid residue and the 158th amino acid residue by the method for detecting a genetic polymorphism according to any one of claims 1 to 9; 2. (a) In the amplification curve based on the 112th amino acid residue obtained in the above step, A substantial increase in fluorescence intensity was observed only in a fluorescently labeled probe (112th Cys probe) having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding a wild-type Cys, and no substantial increase in fluorescence intensity was observed in a fluorescently labeled probe (112th Arg probe) having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding a mutant Arg, and In the amplification curve based on the 158th amino acid residue obtained in the above step, determining that the sample is apolipoprotein E3 / E3 when a substantial increase in fluorescence intensity is observed only in the fluorescent-labeled probe (158th Arg probe) having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon that codes for wild-type Arg, and no substantial increase in fluorescence intensity is observed in the fluorescent-labeled probe (158th Cys probe) having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon that codes for mutant Cys; (b) In the amplification curve based on the 112th amino acid residue obtained in the above step, A substantial increase in the fluorescence intensity derived from the 112th Cys probe was observed, and no substantial increase in the fluorescence intensity derived from the 112th Arg probe was observed; and In the amplification curve based on the 158th amino acid residue obtained in the above step, determining that the sample is alipoprotein E2 / E2 when no substantial increase in the fluorescence intensity derived from the 158th Arg probe is observed and only a substantial increase in the fluorescence intensity derived from the 158th Cys probe is observed; (c) In the amplification curve based on the 112th amino acid residue obtained in the above step, A substantial increase in the fluorescence intensity derived from the 112th Cys probe was observed, and no substantial increase in the fluorescence intensity derived from the 112th Arg probe was observed; and In the amplification curve based on the 158th amino acid residue obtained in the above step, determining that the sample is alipoprotein E2 / E3 when a substantial increase in the fluorescence intensity derived from the 158th Arg probe and a substantial increase in the fluorescence intensity derived from the 158th Cys probe are both observed; (d) In the amplification curve based on the 112th amino acid residue obtained in the above step, No substantial increase in the fluorescence intensity derived from the 112th Cys probe was observed, and only a significant increase in the fluorescence intensity derived from the 112th Arg probe was observed; and In the amplification curve based on the 158th amino acid residue obtained in the above step, determining that the sample is alipoprotein E4 / E4 when a substantial increase in the fluorescence intensity derived from the 158th Arg probe is observed but no substantial increase in the fluorescence intensity derived from the 158th Cys probe is observed; (e) In the amplification curve based on the 112th amino acid residue obtained in the above step, A substantial increase in the fluorescence intensity derived from the 112th Cys probe and the 112th Arg probe was observed, and In the amplification curve based on the 158th amino acid residue obtained in the above step, determining that the sample is alipoprotein E3 / E4 when a substantial increase in the fluorescence intensity derived from the 158th Arg probe is observed and no substantial increase in the fluorescence intensity derived from the 158th Cys probe is observed; or (f) In the amplification curve based on the 112th amino acid residue obtained in the above step, A substantial increase in the fluorescence intensity derived from the 112th Cys probe and the 112th Arg probe was observed, and In the amplification curve based on the 158th amino acid residue obtained in the above step, a step of determining that the sample is alipoprotein E2 / E4 when a substantial increase in the fluorescence intensity derived from both the 158th Arg probe and the 158th Cys probe is observed;

11. A kit for detecting or determining a genetic polymorphism of apolipoprotein E present in genomic DNA collected from a subject, the kit comprising the following items (1) to (3): (1) PCR enzyme, (2) a PCR primer pair for amplifying a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding the 112th or 158th amino acid residue (Cys or Arg) of apolipoprotein E; and (3) A set of a fluorescently labeled probe having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene, the nucleic acid fragment containing a codon that codes for the wild-type Cys at the 112th amino acid residue of apolipoprotein E, and a set of a fluorescently labeled probe having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene, the nucleic acid fragment containing a codon that codes for the mutant Arg at the 112th amino acid residue, the fluorescently labeled probe sets being different in fluorescent dyes used for labeling. or a set of a fluorescently labeled probe having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene, the nucleic acid fragment containing a codon encoding the wild-type Arg at the 158th amino acid residue of apolipoprotein E, and a fluorescently labeled probe having an oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene, the nucleic acid fragment containing a codon encoding the mutant Cys at the 158th amino acid residue, the fluorescently labeled probe set using different fluorescent dyes for labeling.

12. The kit of claim 11 , further comprising a surfactant and protease K.

13. The kit of claim 12, wherein the surfactant is sodium dodecyl sulfate.

14. The kit according to any one of claims 11 to 13, further comprising a Tris-HCl buffer containing potassium chloride, magnesium chloride, and a dNTP mix.

15. The kit according to any one of claims 11 to 14, further comprising a negatively charged substance of biological origin that adsorbs to a PCR enzyme and a positively charged substance of biological origin that adsorbs to DNA, which binds to a substance that inhibits PCR and neutralizes the PCR inhibitory action of the negatively charged substance and the positively charged substance.

16. The kit according to any one of claims 11 to 14, further comprising a cotton swab, absorbent cotton, a saliva collection tool, and a DNA collection kit.

17. The kit according to any one of claims 11 to 16, wherein the PCR primer pair is a pair of base sequences represented by the following SEQ ID NO:1 and SEQ ID NO:2, or SEQ ID NO:3 and SEQ ID NO:4, or SEQ ID NO:5 and SEQ ID NO:6, or SEQ ID NO:7 and SEQ ID NO:

8. 【Chemistry 3】

18. The kit according to any one of claims 11 to 17, wherein in the fluorescently labeled probe, the base sequence of the oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding wild-type Cys for the 112th amino acid residue of apolipoprotein E, and the base sequence of the oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding mutant Arg for the 112th amino acid residue, are the base sequences represented by the following SEQ ID NOs: 9 and 10, respectively, and the base sequence of the oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding wild-type Arg for the 158th amino acid residue of apolipoprotein E, and the base sequence of the oligonucleotide that binds to a nucleic acid fragment of the apolipoprotein E gene containing a codon encoding mutant Cys for the 158th amino acid residue, are the base sequences represented by the following SEQ ID NOs: 11 and 12, respectively. 【Chemistry 4】

19. A dementia diagnosis system comprising the kit according to any one of claims 11 to 18 and MRI or CT.

20. The dementia diagnostic system according to claim 19, wherein the dementia is Alzheimer's disease.

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