Method for testing canine cataracts, reagent for testing canine cataracts, and test kit for canine cataracts

Abstract

To provide canine cataract testing methods useful for canine cataract testing, to provide canine cataract testing reagents, and to provide canine cataract testing kit.SOLUTION: A canine cataract detecting method of the present invention comprises identifying the base of a polymorphism site, such as the base of the polymorphism site of the single nucleotide polymorphism international number rs23483462 or rs23507002. In addition, the canine cataract testing reagent of the present invention comprises a component, such as a primer capable of amplifying a region containing a base of a polymorphism site of the single nucleotide polymorphism international number rs23483462 or rs23507002. In addition, the testing kit of the present invention comprises the above-mentioned reagent.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for testing canine cataracts for identifying a base at a specific polymorphic site, a reagent for testing canine cataracts, and a test kit for canine cataracts. [Background technology]

[0002] Cataracts generally develop when the transparent lens loses its transparency due to denaturation of proteins in the nucleus and cortex of the lens, causing it to become cloudy. Cataracts are clinically classified into "initial stage," "immature stage," "mature stage," and "hypermature stage" depending on the degree of clouding. In the initial stage, vision is hardly impaired, but as the lens becomes cloudy and the condition progresses, vision gradually deteriorates. The dog then begins to bump into pillars and walls frequently, and becomes abnormally sensitive to even the slightest noise, causing problems in daily life. In addition, the degree of clouding of the lens becomes visible to the naked eye as the condition progresses. Therefore, owners often only notice cataracts in dogs when the condition progresses.

[0003] There is no effective treatment for cataracts, and if they are detected early, medical treatments, such as eye drops (N-acetylcarnosine, pirenoxine eye drops, etc.), can slow the progression of the disease. If the symptoms progress and vision is impaired, eye drops cannot restore vision, and surgical treatment is required. Early detection of cataracts cannot be achieved adequately without regular examinations, and there is a demand for a method that can diagnose the disease more reliably at an early stage.

[0004] Cataracts are classified according to etiology into "congenital cataracts" caused by genetic factors and "acquired cataracts" caused by aging, metabolic factors such as diabetes, retinopathy, etc. In the case of hereditary cataracts, they are often discovered at a young age. For canine cataracts, a search for disease susceptibility genes has been ongoing. For example, as disclosed in Non-Patent Document 1, of the 14 SNPs in the CRYAA gene and 6 SNPs in the CRYBA1 gene that show a significant correlation with the disease, one cataract-related polymorphic site has been found in the intron 3 region of the CRYBA1 gene. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Nobuyuki Inumaki et al., Azabu University Journal, Vol. 28 (2016), p. 78 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a method for examining canine cataracts, which is useful for examining canine cataracts, a reagent for examining canine cataracts, and a test kit for canine cataracts. [Means for solving the problem]

[0007] The present invention is based on the discovery of a disease susceptibility gene that is useful for the examination and diagnosis of canine cataract. In order to achieve the above object, one embodiment of the present invention provides a method for testing for canine cataracts, comprising identifying a base at at least one polymorphic site selected from the group consisting of a base at a polymorphic site of international single nucleotide polymorphism number rs23483462 (the 96,874th base of a nucleotide sequence of 289,560 bases in the MMP16 gene on dog chromosome 29), a base at a polymorphic site of international single nucleotide polymorphism number rs23507002 (the 109,224th base of a nucleotide sequence of 159,571 bases in the TMEM64 gene on dog chromosome 29), and bases at polymorphic sites in linkage disequilibrium with these polymorphisms.

[0008] A reagent for testing for canine cataracts in another embodiment of the present invention comprises at least one component selected from the group consisting of the following components (a) and (b): (a) Primers capable of amplifying a region including at least one polymorphic site base selected from the group consisting of the polymorphic site base of international single nucleotide polymorphism number rs23483462 (the 96,874th base of the 289,560 nucleotide sequence in the MMP16 gene on dog chromosome 29), the polymorphic site base of international single nucleotide polymorphism number rs23507002 (the 109,224th base of the 159,571 nucleotide sequence in the TMEM64 gene on dog chromosome 29), and polymorphic site bases in a state of linkage disequilibrium with these polymorphisms.

[0009] (b) A probe capable of hybridizing to a region containing at least one polymorphic site base selected from the group consisting of a polymorphic site base of international single nucleotide polymorphism number rs23483462 (the 96,874th base of the 289,560 nucleotide sequence in the MMP16 gene on dog chromosome 29), a polymorphic site base of international single nucleotide polymorphism number rs23507002 (the 109,224th base of the 159,571 nucleotide sequence in the TMEM64 gene on dog chromosome 29), and polymorphic sites in linkage disequilibrium with these polymorphisms.

[0010] In the reagent, the probe may be immobilized on a solid phase. A test kit according to another embodiment of the present invention comprises the above-mentioned reagent. Effect of the Invention

[0011] According to the present invention, the usefulness of examination for canine cataracts can be improved. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] (First embodiment) Hereinafter, one embodiment of the canine cataract testing method of the present invention (hereinafter, simply referred to as the "testing method") will be described. The base identified by the testing method of this embodiment is at least one polymorphic site base selected from the group consisting of the polymorphic site base of the international single nucleotide polymorphism number (reference SNP ID number in dbSNP, the SNP database of NCBI, hereinafter, simply referred to as "rs") 23483462 (the 96,874th base of the 289,560 bases in the nucleotide sequence of the MMP16 gene of dog chromosome 29), the polymorphic site base of rs23507002 (the 109,224th base of the 159,571 bases in the nucleotide sequence of the TMEM64 gene of dog chromosome 29), and the polymorphic site base in linkage disequilibrium with these polymorphisms.

[0013] As described above, the base at the polymorphic site of rs23483462 is the 96,874th base of the 289,560 base nucleotide sequence in the MMP16 gene on dog chromosome 29. As described above, the base at the polymorphic site of rs23507002 is the 109,224th base of the 159,571-base nucleotide sequence in the TMEM64 gene on dog chromosome 29.

[0014] The polymorphism in linkage disequilibrium with the rs23483462 or rs23507002 polymorphism may be a polymorphism in the LD block of rs23483462 or rs23507002. The polymorphism in linkage disequilibrium with the rs23483462 or rs23507002 polymorphism may be another polymorphism with a p-value (an index of significant difference between cataract and healthy individuals) of less than 0.05 in a region including MMP16 and TMEM64 on dog chromosome 29.

[0015] If D' between SNPs is large, they are considered to be in linkage disequilibrium (Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics. 2005;21(2):263-265.; Gabriel SB, Schaffner SF, Nguyen H, et al. The structure of haplotype blocks in the human genome. Science. 2002;296(5576):2225-2229.). Thus, polymorphisms in linkage disequilibrium with the rs23483462 or rs23507002 polymorphism are, for example, polymorphisms with a larger D' between these SNPs.

[0016] LD blocks can be determined using the Haploview software (Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics. 2005;21(2):263-265.) according to the method of Gabriel et al. (Gabriel SB, Schaffner SF, Nguyen H, et al. The structure of haplotype blocks in the human genome. Science. 2002;296(5576):2225-2229.).

[0017] In this specification, single nucleotide polymorphisms (SNPs) are indicated by rs numbers, which are reference SNP ID numbers in dbSNP, an SNP database of NCBI, and base positions are based on the ENSEMBL database CanFam3.1 (GCA_000002285.2).

[0018] In this specification, the term "cataract examination" refers to a concept including an examination for determining whether a subject has a high or low probability of developing cataract, and an examination for definitively diagnosing cataract when the subject already has cataract. The cataract examination is preferably applied to examination of juvenile cataract caused by genetic factors.

[0019] rs23483462 is an adenine (A) / guanine (G) polymorphism at the 96,874th base of the 289,560-base nucleotide sequence in the MMP16 gene on dog chromosome 29. If the base at this site is G, it is determined that there is a high possibility of suffering from cataracts or that the patient is suffering from cataracts.

[0020] rs23507002 is an adenine (A) / guanine (G) polymorphism at the 109,224th base of the 159,571-base nucleotide sequence in the TMEM64 gene on dog chromosome 29. When the base at this site is A, it is determined that there is a high possibility of suffering from cataracts or that the individual is suffering from cataracts.

[0021] In addition, the NBN gene on dog chromosome 29, which is located near the MMP16 and TMEM64 genes, has also been suggested to be associated with cataracts. Therefore, testing for specific SNPs in the NBN gene may be as useful for testing for canine cataracts as rs23483462 or rs23507002.

[0022] The SNPs to be identified may be one type or a combination of multiple types. In addition, the sense strand or the antisense strand of the gene may be analyzed. In addition, homozygosity or heterozygosity of a polymorphism may serve as a diagnostic indicator.

[0023] rs23483462 and rs23507002 are present in the introns of genes, and other SNPs present in exons are also found. Polymorphisms in linkage disequilibrium with these polymorphisms may be present in the exons of genes, in regions that control gene expression (e.g., promoter regions, enhancer regions, etc.), in the introns of genes, or in regions before and after these genes that are in linkage disequilibrium with these genes. Types of polymorphisms include, but are not limited to, single nucleotide polymorphisms, and polymorphisms in which one to several tens of bases (sometimes several thousand bases) are substituted, deleted, inserted, transposed, or inverted.

[0024] In the test method of the present invention, the base at the polymorphic site (i.e., the base type) can be identified by a known single nucleotide polymorphism analysis method. Examples of single nucleotide polymorphism analysis methods include, but are not limited to, sequence analysis, PCR, PCR-SSCP, hybridization, RFLP method, Taqman-PCR method, Invader method, cycleave-PCR method, and HRM method.

[0025] In order to identify the base of the polymorphic site, genomic DNA may be extracted from a biological sample of a test individual. The biological sample may be, for example, the blood, skin, oral mucosa, tissue or cells collected or excised by surgery, or bodily fluids collected for the purpose of testing (saliva, lymph, respiratory mucosa, semen, sweat, urine, etc.) of the test individual. The biological sample is preferably whole blood collected from any of the limbs or a vein such as the neck. Genomic DNA can be extracted from the biological sample using a commercially available DNA extraction kit. Next, DNA containing the polymorphic site is isolated as necessary. The DNA can be isolated by PCR using a primer capable of hybridizing to DNA containing the polymorphic site and using genomic DNA or RNA as a template.

[0026] The dog breeds to be examined for cataracts are not particularly limited, but it is preferable to examine dog breeds that are susceptible to cataracts, such as Shiba Inu, American Cocker Spaniel, Dachshund, Beagle, Shetland Sheepdog, Shih Tzu, Lubrador Retriever, Maltese, Miniature Pinscher, Pug, Corgi, Chihuahua, etc.

[0027] Second embodiment Hereinafter, one embodiment of the canine cataract test kit (hereinafter simply referred to as the "test kit") of the present invention will be described. The test kit of this embodiment includes a reagent for testing canine cataract, which contains at least one component selected from the group consisting of the following components (a) and (b):

[0028] Component (a) is a primer capable of amplifying a region including at least one polymorphic site base selected from the group consisting of the rs23483462 polymorphic site base, the rs23507002 polymorphic site base, and polymorphic sites in a state of linkage disequilibrium with these polymorphisms, described in the first embodiment.

[0029] Component (b) is a probe capable of hybridizing to a region including at least one polymorphic site base selected from the group consisting of the polymorphic site base of rs23483462 described in the first embodiment, the polymorphic site base of rs23507002, and polymorphic sites in a state of linkage disequilibrium with these polymorphisms.

[0030] The above-mentioned primers and probes, which are components of the reagent of this embodiment, are preferably oligonucleotides having a chain length of at least 15 nucleotides. When the oligonucleotides are used as primers, their length is, for example, 15 bp to 100 bp, preferably 17 bp to 30 bp. The primers are not particularly limited as long as they can amplify at least a part of the DNA containing the polymorphic site. The length of the DNA that can be amplified by the primers is, for example, 15 to 1000 bp, preferably 20 to 500 bp, more preferably 20 to 200 bp.

[0031] When the oligonucleotide is used as a probe, its length is, for example, 5 bp to 200 bp, preferably 7 bp to 100 bp, and more preferably 7 bp to 50 bp. The probe is not particularly limited as long as it can hybridize with DNA containing the polymorphic site.

[0032] In this embodiment, the primer capable of amplifying a region containing a polymorphic site is preferably one that can initiate complementary strand synthesis toward the polymorphic site using DNA containing the polymorphic site as a template.

[0033] An example of the primer that is a component of the reagent of this embodiment is a primer pair consisting of a forward primer and a reverse primer.

[0034] An example of a primer capable of amplifying a region including the base of the polymorphic site of rs23483462 is a primer pair consisting of a forward primer and a reverse primer as shown below.

[0035] Forward Primer: 5'-TGAATAGAACTCTCACCAGTAA-3' (SEQ ID NO: 1) Reverse Primer: 5'-GTGCTTAATCCAATACTCAA-3' (SEQ ID NO: 2) Furthermore, examples of primers capable of amplifying a region containing the base of the rs23507002 polymorphic site include a primer pair consisting of a forward primer and a reverse primer as shown below.

[0036] Forward Primer: 5'-AATTACACGGATGCTACAATA-3' (SEQ ID NO: 3) Reverse Primer: 5'-CAACTGCAGTGAAGACAGG-3' (SEQ ID NO: 4) The other primers have a nucleotide sequence capable of hybridizing to a region containing a polymorphic site, and may have a sequence identical to or complementary to a region shifted by several nucleotides from the region containing a polymorphic site identical to or complementary to the nucleotide sequences of SEQ ID NOs: 1 to 4.

[0037] In addition to the base sequence identical or complementary to the base sequence of the region containing the polymorphic site, any base sequence can be added to the primer. For example, in a primer for a method of analyzing polymorphism using a type IIs restriction enzyme, a primer to which a recognition sequence for the type IIs restriction enzyme is added is used. Furthermore, the primer may be modified. For example, a primer labeled with a fluorescent substance or a binding affinity substance such as biotin or digoxin may be used.

[0038] In this embodiment, the probe capable of hybridizing to the region containing the polymorphic site may be any probe capable of hybridizing to a polynucleotide having the base sequence of the region containing the polymorphic site, and is preferably a probe that specifically hybridizes to a DNA having the base sequence of the region containing the polymorphic site. Here, "specifically hybridize" means that under normal hybridization conditions, preferably stringent hybridization conditions (for example, the conditions described in Sambrook et al., Molecular Cloning, Cold Spring Harbour Laboratory Press, New York, USA, 2nd Edition 1989), cross-hybridization with DNA other than the DNA having the base sequence of the region containing the polymorphic site does not occur significantly. More specifically, a probe containing a polymorphic site in its base sequence is preferred. Alternatively, depending on the method of analyzing the base at the polymorphic site, the end of the probe may be designed to correspond to the base adjacent to the polymorphic site. Therefore, a probe that does not contain a polymorphic site in its base sequence but contains a base sequence complementary to the region adjacent to the polymorphic site can also be shown as a desirable probe in the present invention.

[0039] Examples of the probe capable of hybridizing to a region containing the base of the polymorphic site of rs23483462 include the following sequences. Probe 1: 5'-CCTTCAAATrAGT-3' (SEQ ID NO: 5) In addition, the third base from the 3' end in the sequence, adenine "rA," indicates RNA.

[0040] Probe 2: 5'-CTTCAAACrAGT-3' (SEQ ID NO: 6) In addition, the third base from the 3' end in the sequence, adenine "rA," indicates RNA. Furthermore, examples of the probe capable of hybridizing to a region containing the base of the polymorphic site of rs23507002 include the following sequences.

[0041] Probe 3: 5'-TGAAGrGGCT-3' (SEQ ID NO: 7) In addition, the fourth base from the 3' end in the sequence, guanine "rG," indicates RNA. Probe 4: 5'-TTGAAGrAGCT-3' (SEQ ID NO: 8) In addition, the fourth base from the 3' end in the sequence, adenine "rA," indicates RNA.

[0042] The probe may be modified, have a base sequence added, or be modified, like the primer. For example, a probe used in the Invader method is added with a base sequence unrelated to the genome constituting the flap. Such a probe is also included in the probe of this embodiment as long as it hybridizes to a region containing a polymorphic site. The base sequence constituting the probe of this embodiment can be designed according to the analysis method based on the base sequence of the DNA region surrounding the polymorphic site of this embodiment in the genome.

[0043] A person skilled in the art can design primers and probes according to the analytical method based on the base sequence information of the surrounding DNA region including the polymorphic site. The base sequences constituting the primers and probes are not limited to base sequences completely complementary to the base sequence of the genome, but can also be appropriately modified.

[0044] Primers and probes can be synthesized by any method based on the base sequence that constitutes them. Methods for synthesizing oligonucleotides having a given base sequence based on the base sequence are known. Furthermore, in the synthesis of oligonucleotides, any modification can be introduced into the oligonucleotide by using nucleotide derivatives modified with fluorescent dyes, biotin, etc. Alternatively, methods for binding fluorescent dyes, etc. to synthesized oligonucleotides are also known.

[0045] The probes may be fixed to a solid phase (DNA array). In the DNA array, sample DNA (or RNA) is hybridized to a number of probes arranged on the same plane, and the plane is scanned to detect hybridization to each probe. Since the reaction to many probes can be observed simultaneously, the DNA array is useful, for example, for analyzing many polymorphic sites simultaneously. As an example of a method for fixing nucleotides (array), an array based on oligonucleotides developed by Affymetrix can be given. In the oligonucleotide array, the oligonucleotides are usually synthesized in situ. For example, in situ synthesis methods of oligonucleotides using the lithography method (Affymetrix), the inkjet method (Agilent), the bead array method (Illumina), etc. are known.

[0046] The oligonucleotide is composed of a base sequence complementary to a region containing a polymorphic site to be detected. When the oligonucleotide is immobilized on the substrate, the length of the nucleotide probe to be bound to the substrate is, for example, 10 to 100 bp, preferably 10 to 50 bp, and more preferably 15 to 25 bp.

[0047] A sample for SNP detection by the DNA array method can be prepared by a method well known to those skilled in the art based on a biological sample collected from a test individual. The biological sample is not particularly limited. For example, a DNA sample can be prepared from genomic DNA extracted from tissues or cells such as blood, skin, oral mucosa, tears, saliva, urine, feces, or hair of a test individual. A specific region of genomic DNA is amplified using primers for amplifying a region containing a polymorphic site to be determined. At this time, multiple regions can be amplified simultaneously by multiplex PCR. Multiplex PCR is a PCR method in which multiple primer sets are used in the same reaction solution. Multiplex PCR is useful when analyzing multiple polymorphic sites.

[0048] In general, in the DNA array method, a DNA sample is amplified by PCR and the amplified product is labeled. A labeled primer is used to label the amplified product. For example, genomic DNA is first amplified by PCR using a primer set specific to a region containing a polymorphic site. Next, biotin-labeled DNA is synthesized by labeling PCR using biotin-labeled primers. The biotin-labeled DNA thus synthesized is hybridized to an oligonucleotide probe on a chip. The hybridization reaction solution and reaction conditions can be appropriately adjusted depending on the length of the nucleotide probe immobilized on the solid phase, the reaction temperature, and other conditions. Those skilled in the art can design appropriate hybridization conditions. Avidin labeled with a fluorescent dye is added to detect the hybridized DNA. The array is analyzed by a scanner, and the presence or absence of hybridization is confirmed using fluorescence as an indicator.

[0049] An example of a procedure for carrying out the testing method of the first embodiment using the above-mentioned reagent and DNA array method is as follows: first, a solid phase to which DNA containing a polymorphic site prepared from a test individual and a nucleotide probe are immobilized is prepared, and then the DNA is brought into contact with the solid phase. Next, the base type of the polymorphic site is determined by detecting the DNA hybridized to the nucleotide probe immobilized on the solid phase.

[0050] In this specification, the term "solid phase" refers to a material capable of fixing nucleotides. The solid phase is not particularly limited as long as it is capable of fixing nucleotides, and specific examples include solid phases including microplate wells, plastic beads, magnetic particles, and substrates. As the solid phase, a substrate generally used in DNA array technology can be preferably used. In this specification, the term "substrate" refers to a plate-shaped material capable of fixing nucleotides. In addition, in the present invention, the term "nucleotide" includes oligonucleotides and polynucleotides.

[0051] In addition to the above methods, the allele specific oligonucleotide (ASO) hybridization method can be used to detect bases at specific sites. Allele specific oligonucleotides (ASOs) are composed of a base sequence that hybridizes to the region where the polymorphic site to be detected exists. When ASOs are hybridized to sample DNA, if a mismatch occurs at the polymorphic site due to polymorphism, the efficiency of hybridization decreases. Mismatches can be detected by the Southern blot method or a method that utilizes the property of a special fluorescent reagent that intercalates into the gap of the hybrid and quenches the light. Mismatches can also be detected by the ribonuclease A mismatch cleavage method.

[0052] The test kit of this embodiment may contain various enzymes, enzyme substrates, buffer solutions, etc. in addition to the above-mentioned reagents, depending on the base identification method. Examples of the enzymes include DNA polymerase, DNA ligase, IIs restriction enzyme, and other enzymes required for the various analysis methods exemplified as the above-mentioned base identification method. The buffer solution is appropriately selected from buffer solutions suitable for maintaining the activity of the enzymes used in these analyses. Furthermore, as the enzyme substrate, for example, a substrate for complementary strand synthesis is used.

[0053] Furthermore, the test kit of this embodiment can be provided with a control in which the bases at the polymorphic site are clear. As the control, genomic DNA or a fragment of genomic DNA in which the base type at the polymorphic site is clear in advance can be used. Genomic DNA extracted from cells can be provided as a control, or cells or a cell fraction can be provided as a control from which the user can extract genomic DNA. If cells are used as a control, the results of the control can prove that the genomic DNA extraction operation was performed correctly. Alternatively, DNA consisting of a base sequence including a polymorphic site can be used as a control. Specifically, a YAC vector or a BAC vector containing DNA derived from a genome in which the base type at the polymorphic site is clear can be used as a control. Alternatively, a vector in which only tens to hundreds of bp corresponding to the polymorphic site is cut out and inserted can be used as a control.

[0054] The functions and effects of the method for testing for canine cataract, the reagent for testing for canine cataract, and the test kit for canine cataract according to the above embodiment will be described below. (1) In the above embodiment, the device includes a configuration for identifying or a reagent for identifying a base at a polymorphic site such as a base at a polymorphic site of rs23483462 or rs23507002. Therefore, according to the above embodiment, the usefulness of canine cataract testing can be improved. More specifically, canine cataract can be tested and diagnosed earlier and more accurately. In individuals that have already developed cataract, a definitive diagnosis can be made, and active treatment can be performed. In addition, in individuals that have not yet developed cataract, it is possible to predict the onset of the disease, and frequent testing can be recommended, leading to early detection. Therefore, the present invention can be widely used in the fields of veterinary medicine, diagnosis, and the like.

[0055] The above embodiment can be modified as follows: The above embodiment and the following modifications can be combined with each other as long as they are not technically inconsistent. The above embodiment may be applied to dog mating. EXAMPLES

[0056] Next, the above embodiment will be described in more detail with reference to examples and comparative examples. Note that the present invention is not limited to the configurations described in the examples section. <Example 1 group> (A) Diagnosis of canine cataract and DNA purification 1) Both eyes of young (under 6 years old) pure-bred Shiba Inu dogs that visited Azabu University Animal Hospital were examined. Animals that showed cataract symptoms in either the left or right eye after the "initial stage" were judged to have cataracts, while others were judged to be healthy. A total of 55 dogs were examined, with 9 having cataracts and 46 healthy.

[0057] 2) 1 cc of blood was collected from the jugular vein of each individual, and DNA was purified using a DNA whole blood kit spin method (FUJI FILM). 3) DNA concentration and purity were measured using GeneQuant Pro (GE Health Sciences).

[0058] (B) SNP analysis Primers (SEQ ID NOs: 1 and 2) and probes (SEQ ID NOs: 5 and 6) capable of amplifying a region including the base at the polymorphic site of rs23483462 were used as reagents.

[0059] 4) CycleavePCR was performed using CycleavePCR Reaction Mix and CycleavePCR 2 Probe Set. 5) The conditions for Cycleave PCR are as follows:

[0060] <Reagents> Cycleave PCR Reaction Mix 12.5μL Primer (SEQ ID NO: 1) (5 μM) 1 μL Primer (SEQ ID NO:2) (5 μM) 1 μL Probe (SEQ ID NO: 5) (5 μM) 1 μL Probe (SEQ ID NO: 6) (5 μM) 1 μL Template 1μL 7.5μL dH2O Total volume: 25 μL <Cycleave PCR conditions> 95°C for 10 seconds, 1 cycle 95°C for 5 seconds 55°C for 10 seconds, 45 cycles 72°C for 20 seconds 6) SNPs were analyzed using the analysis software (Thermal Cycler Dice Real Time System Ver 3.00) attached to the real-time PCR device.

[0061] 7) For the SNP (rs23483462) of the canine MMP16 gene, when the fluorescence intensity of FAM was strong, it was determined as the non-risk type; when the fluorescence intensity of ROX was strong, it was determined as the risk type; when the fluorescence intensities of both were strong, it was determined as the hetero type.

[0062] 8) For the SNP (rs23507002) of the canine TMEM64 gene described later, when the fluorescence intensity of FAM was strong, it was determined as the non-risk type; when the fluorescence intensity of ROX was strong, it was determined as the risk type; when the fluorescence intensities of both were strong, it was determined as the hetero type.

[0063] 9) When each SNP was of the risk type, hetero type, or non-risk type, the number of risk alleles was set to 2, 1, and 0 respectively, and the risk allele frequency was calculated. 10) The risk allele frequencies of the cataract group (analyzed separately for juvenile and senile cataracts) and the healthy group were compared, and odds ratio test and Fisher's exact test were performed.

[0064] 11) The genotype (non-risk homozygous, hetero, risk homozygous) of the sample was determined based on the nucleotide sequence of one SNP (rs23483462) of the canine MMP16 gene. The results of the gene polymorphism analysis of 9 cataract Shiba Inus and 46 healthy Shiba Inus are shown in Table 1.

[0065] At the polymorphic site of rs23483462 in the group of Example 1, the risk allele frequency in the cataract group was about 27.8%, and the risk allele frequency in the healthy group was about 2.2%. When comparing the two groups, the risk of developing cataracts in the case of carrying the risk allele was 17.31 times, and a significant difference (P<0.01) was observed by chi-square test.

[0066] <Example Group 2> A bilateral eye examination was carried out on an old pure-bred Shiba Inu different from the group in Example 1. Individuals with cataract symptoms in either the left or right eye that had developed symptoms after the "initial stage" were judged to have cataracts, and others were judged to be healthy. A total of 67 dogs were examined, and 21 had cataracts and 46 were healthy.

[0067] Genetic polymorphism analysis was carried out in the same manner as in Example 1. The results are shown in Table 1. At the polymorphic site of rs23483462 in Example 2, the risk allele frequency of the cataract group was about 23.8%, and the risk allele frequency of the healthy group was about 2.2%. When comparing the two groups, the risk of developing cataracts in the case of carrying the risk allele was 14.06 times, and a significant difference (P<0.01) was observed by chi-square test.

[0068] <Example Group 3> A bilateral eye examination was conducted on young (under 6 years old) purebred beagles. Those that showed cataract symptoms in either the left or right eye after the "initial stage" were judged to have cataracts, while others were judged to be healthy. A total of 18 dogs were examined, with 3 having cataracts and 15 healthy.

[0069] Genetic polymorphism analysis was carried out in the same manner as in Example 1. The results are shown in Table 1. At the polymorphic site of rs23483462 in the Example 3 group, the risk allele frequency in the cataract group was 0%, and the risk allele frequency in the healthy group was 0%.

[0070] [Table 1] <Example Group 4> DNA from a young Shiba Inu (pure line) from Example 1 was used as a sample. Primers (SEQ ID NOs: 3 and 4) and probes (SEQ ID NOs: 7 and 8) capable of amplifying a region containing the base of the polymorphic site of rs23507002 were used as reagents. Gene polymorphism analysis was otherwise performed in the same manner as in Example 1. The results are shown in Table 2.

[0071] At the rs23507002 polymorphism site in Example 4, the risk allele frequency in the cataract group was about 27.8%, and the risk allele frequency in the healthy group was about 1.1%. When comparing the two groups, the risk of developing cataracts in those who carried the risk allele was 35 times higher, and a significant difference (P<0.01) was observed by chi-square test.

[0072] <Example Group 5> DNA from an old Shiba Inu (pure line) from Example 2 was used as a sample. Primers (SEQ ID NOs: 3 and 4) and probes (SEQ ID NOs: 7 and 8) capable of amplifying a region containing the base of the polymorphic site of rs23507002 were used as reagents. Gene polymorphism analysis was otherwise performed in the same manner as in Example 1. The results are shown in Table 2.

[0073] At the rs23507002 polymorphism site in Example 5, the risk allele frequency in the cataract group was about 23.8%, and the risk allele frequency in the healthy group was about 1.1%. When comparing the two groups, the risk of developing cataracts in the case of carrying the risk allele was 28.44 times, and a significant difference (P<0.01) was observed by chi-square test.

[0074] <Example Group 6> DNA from young beagles (pure line) in Example 3 was used as a sample. Primers (SEQ ID NOs: 3 and 4) and probes (SEQ ID NOs: 7 and 8) capable of amplifying a region containing the base of the polymorphic site of rs23507002 were used as reagents. Gene polymorphism analysis was otherwise performed in the same manner as in Example 1. The results are shown in Table 2.

[0075] At the rs23507002 polymorphism site in Example 6, the risk allele frequency in the cataract group was 100%, and the risk allele frequency in the healthy group was about 43.3%. When comparing the two groups, the risk of developing cataracts in the case of carrying the risk allele was 16.85 times, and a significant difference (P<0.05) was observed by chi-square test.

[0076] [Table 2] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

[0077] Next, the technical ideas that can be understood from the above-described embodiment and other examples will be described below together with their effects. (i) A reagent for testing for canine cataracts, comprising at least one component selected from the group consisting of the following components (a) and (b):

[0078] (a) a primer of any one of SEQ ID NOs: 1 to 4; (b) any one of the probes of SEQ ID NOs: 5 to 8. Therefore, according to the invention described in (A), the usefulness of testing for canine cataracts can be further improved. [Sequence List Free Text]

[0079] <Sequence number 1> SEQ ID NO: 1 shows the nucleotide sequence of the forward primer used for detecting the polymorphic site of rs23483462. <Sequence number 2> SEQ ID NO: 2 shows the base sequence of the reverse primer used for detecting the polymorphic site of rs23483462.

[0080] <Sequence number 3> SEQ ID NO: 3 shows the nucleotide sequence of the forward primer used for detecting the polymorphic site of rs23507002. <Sequence number 4> SEQ ID NO: 4 shows the base sequence of the reverse primer used for detecting the polymorphic site of rs23507002.

[0081] <Sequence number 5> SEQ ID NO:5 shows a probe capable of hybridizing to a region containing the base of the polymorphic site of rs23483462.

[0082] <Sequence number 6> SEQ ID NO: 6 shows a probe capable of hybridizing to a region containing the base of the polymorphic site of rs23483462.

[0083] <Sequence number 7> SEQ ID NO: 7 shows a probe capable of hybridizing to a region containing the base of the polymorphic site of rs23507002.

[0084] <Sequence number 8> SEQ ID NO: 8 shows a probe capable of hybridizing to a region containing the base of the polymorphic site of rs23507002.

Claims

1. The base of the polymorphic site of the international single nucleotide polymorphism number rs23483462 (the 96,874th base of the 289,560 bases in the nucleotide sequence of the MMP16 gene on dog chromosome 29), The base of the polymorphic site of the international single nucleotide polymorphism number rs23507002 (the 109,224th base of the 159,571 bases in the nucleotide sequence of the TMEM64 gene on dog chromosome 29), and identifying at least one polymorphic site base selected from the group consisting of polymorphic site bases in linkage disequilibrium with said polymorphisms, The polymorphisms in linkage disequilibrium with these polymorphisms are polymorphisms within the LD block of rs23483462 or rs23507002, other polymorphisms with a p-value of less than 0.05, which is an indicator of a significant difference between cataractous and healthy individuals, within a region including MMP16 and TMEM64 on dog chromosome 29, or polymorphisms with a larger D' between the SNP of rs23483462 or rs23507002.

2. A reagent for testing for canine cataracts, comprising at least one component selected from the group consisting of the following components (a) and (b): (a) the base at the polymorphic site of the international single nucleotide polymorphism number rs23483462 (the 96,874th base of the 289,560 base nucleotide sequence in the MMP16 gene on dog chromosome 29); The base of the polymorphic site of the international single nucleotide polymorphism number rs23507002 (the 109,224th base of the 159,571 bases in the nucleotide sequence of the TMEM64 gene on dog chromosome 29), and bases of polymorphic sites that are in a state of linkage disequilibrium with these polymorphisms; The polymorphisms in linkage disequilibrium with these polymorphisms are primers for a polymorphism in the LD block of rs23483462 or rs23507002, another polymorphism in the region including MMP16 and TMEM64 on dog chromosome 29 with a p-value of less than 0.05, which is an indicator of a significant difference between cataractous and healthy individuals, or a polymorphism with a larger D' between the SNP of rs23483462 or rs23507002; (b) the base at the polymorphic site of the international single nucleotide polymorphism number rs23483462 (the 96,874th base of the 289,560 base nucleotide sequence in the MMP16 gene on dog chromosome 29); The base of the polymorphic site of the international single nucleotide polymorphism number rs23507002 (the 109,224th base of the 159,571 bases in the nucleotide sequence of the TMEM64 gene on dog chromosome 29), and a region including at least one base of a polymorphic site selected from the group consisting of bases of polymorphic sites that are in linkage disequilibrium with these polymorphisms; The polymorphisms in linkage disequilibrium with these polymorphisms are polymorphisms within the LD block of rs23483462 or rs23507002, other polymorphisms within the region containing MMP16 and TMEM64 on dog chromosome 29 with a p-value of less than 0.05, which is an indication of a significant difference between cataractous and healthy individuals, or probes that have a larger D' between the SNP of rs23483462 or rs23507002.

3. The reagent of claim 2 , wherein the probe is immobilized on a solid phase.

4. A test kit for canine cataract, comprising the reagent according to claim 2 or 3.

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