Ophthalmic examination support methods and ophthalmic examination kits

Abstract

Rapid testing for marker genes of intraocular malignant lymphoma. [Solution] An ophthalmic examination support method comprising: a decomposition step to allow the reaction of the protease to proceed in a sample mixture obtained by mixing an ophthalmic specimen with a PCR buffer containing a protease; an inactivation step to inactivate the protease; and a detection step to add at least a portion of the sample mixture after the inactivation step to a PCR reaction composition and detect the presence or absence of mutations in the MYD88 gene and the CD79B gene by real-time PCR.

Description

Technical Field

[0001] The present invention relates to an ophthalmic examination assisting method and an ophthalmic examination kit. Background Art

[0002] Various techniques have been proposed for detecting disease marker genes, pathogen-derived genes, and the like. Among these, the PCR method utilizing polymerase chain reaction (PCR) is widely used because of its excellent detection sensitivity and detection accuracy. In particular, the real-time PCR method is extremely useful in clinical medical settings because it can provide results quickly.

[0003] In ophthalmic examination, since the amount of specimen obtained for examination is small, there is high demand for examinations that can be quickly performed using a small amount of specimen by means of real-time PCR. For example, Patent Document 1 discloses a method for efficiently extracting nucleic acid of a pathogen causing infectious uveitis and detecting the nucleic acid by a real-time PCR method.

[0004] Further, intraocular malignant lymphoma, which is a type of ophthalmic disease, is known as an intractable disease with a poor prognosis. Non-Patent Documents 1 to 3 mention the L265P mutation of MYD88 and the Y196 mutation of CD79B as markers for intraocular malignant lymphoma. Prior Art Documents Patent Documents

[0005] Patent Document 1 Japanese Unexamined Patent Publication No. 2020-198809 Non-Patent Documents

[0006] Non-Patent Document 1 Carbonell et al., Ocular Immunology and Inflammation, 29(3):507-520, 2021 Non-Patent Document 2 Genome Testing Guidelines 2023 Edition: Recommended Panel Testing Levels by Disease, Japanese Society of Hematology, December 2023 [Non-Patent Document 3] Yonese et. al., European Journal of Haematology, 102:191-196,2019 [Overview of the project] [Problems that the invention aims to solve]

[0007] However, there are currently no known examples of rapid testing using real-time PCR for marker genes of such intraocular malignant lymphoma.

[0008] One aspect of the present invention aims to realize a method for rapidly testing marker genes for intraocular malignant lymphoma. [Means for solving the problem]

[0009] To solve the aforementioned problems, an ophthalmic examination assistance method according to Embodiment 1 of the present invention includes: a degradation step of advancing the reaction of the protease in a sample mixture obtained by mixing an ophthalmic specimen containing at least a part of an eyeball or an appendage of the eyeball with a PCR buffer containing a protease; an inactivation step of inactivating the protease contained in the sample mixture; and a detection step of adding at least a part of the sample mixture after the inactivation step to a PCR reaction composition and amplifying and detecting a PCR product by real-time PCR, wherein the PCR reaction composition includes a first primer pair for amplifying the MYD88 gene and a first probe for detecting the presence or absence of mutation in the MYD88 gene, a second primer pair for amplifying the CD79B gene and a second probe for detecting the presence or absence of mutation in the CD79B gene.

[0010] In the ophthalmic examination assistance method according to aspect 2 of the present invention, the mutation in the MYD88 gene may be a mutation involving the L265P amino acid substitution of the MYD88 protein, as described in aspect 1.

[0011] The ophthalmic examination assistance method according to aspect 3 of the present invention may be such that, in aspect 1 or 2, the mutation in the CD79B gene is an amino acid substitution of the CD79B protein, and is a mutation involving at least one amino acid substitution selected from the group consisting of Y196N, Y196H, Y196S, Y196C, Y196F, and Y196D.

[0012] In any of the embodiments 1 to 3 of the present invention, the ophthalmic examination assistance method may include six types of probes, each detecting mutations involving amino acid substitutions of Y196N, Y196H, Y196S, Y196C, Y196F, and Y196D in the CD79B protein.

[0013] To solve the aforementioned problems, an ophthalmic examination kit according to aspect 5 of the present invention comprises a PCR buffer containing a protease and a PCR reaction composition, wherein the PCR reaction composition includes a first primer pair for amplifying the MYD88 gene and a first probe for detecting the presence or absence of mutations in the MYD88 gene, a second primer pair for amplifying the CD79B gene and a second probe for detecting the presence or absence of mutations in the CD79B gene. [Effects of the Invention]

[0014] According to one aspect of the present invention, a method for rapidly testing marker genes for intraocular malignant lymphoma can be realized. [Brief explanation of the drawing]

[0015] [Figure 1] This figure shows the amplification curve in a representative example where the vitreous humor was used as the ophthalmic specimen in Example 1. [Figure 2] This figure shows the amplification curve in a representative example where the vitreous humor was used as the ophthalmic specimen in Example 2. [Figure 3] This figure shows the amplification curve in a representative example where the vitreous humor was used as the ophthalmic specimen in Example 1. [Figure 4] It is a diagram showing an amplification curve in a representative example using anterior chamber aqueous humor as an ophthalmic specimen in Example 1. [MODE FOR CARRYING OUT THE INVENTION]

[0016] [1. Summary of the Present Invention] As a technique for amplifying and detecting nucleic acids, a PCR method utilizing the polymerase chain reaction (PCR) has been widely applied in research and clinical practice. Advantages of the PCR method include, for example, the ability to selectively amplify a specific DNA fragment, the ability to perform the method even from an extremely trace amount of specimen, the relatively short time required for amplification, and the simplicity of the process, such that amplification can be performed with a fully automatic benchtop apparatus, among others.

[0017] Among PCR methods, the real-time PCR method is useful in that it can detect the amount (copy number) of a nucleic acid that serves as a template for a PCR product. Furthermore, in the real-time PCR method, amplification results are obtained in real time, so no separate step is required for detecting the PCR product. Therefore, results can be obtained rapidly.

[0018] The real-time PCR method is utilized, for example, also in the field of ophthalmic testing. In order to detect abnormal cells such as intraocular malignant lymphoma or pathogens such as viruses and bacteria from an ophthalmic specimen where the amount of specimen obtained is limited, the real-time PCR method, which allows rapid detection of nucleic acids from a trace amount of specimen, can be suitably used.

[0019] In the real-time PCR method, pretreatment of the specimen is required. An ophthalmic specimen collected from a subject usually contains a large amount of substances that inhibit enzymatic reactions. Since these substances that inhibit enzymatic reactions inhibit PCR, it is necessary to remove the substances that inhibit enzymatic reactions in advance. As described above, in order to detect nucleic acids by the real-time PCR method, pretreatment such as removing substances that inhibit enzymatic reactions in the ophthalmic specimen or purifying the nucleic acids in the ophthalmic specimen has been necessary.

[0020] To reduce the complexity of such pretreatment and enable the detection of pathogens by real-time PCR without pretreatment of ophthalmic specimens, the Ampdirect® technology is known (Ann Clin Biochem 37, 674-680, 2000). Using such technology, nucleic acids extracted from ophthalmic specimens can be used directly in real-time PCR.

[0021] One method for extracting nucleic acids is to use a PCR buffer containing a surfactant, as described in Patent Document 1. However, this method is intended for liquid samples such as blood, and it is difficult to extract nucleic acids from tissue fragments. As a method for separating nucleic acids from tissue fragments, since the main component of tissue fragments is protein, a method is known in which the tissue fragment is added to a buffer containing a proteolytic enzyme to digest the protein in the tissue fragment. However, since proteolytic enzymes also digest DNA polymerase, it is not possible to have proteolytic enzymes and DNA polymerase coexist.

[0022] Therefore, proteolytic enzymes and DNA polymerases must be kept in separate reaction vessels, making it difficult to directly add tissue samples to PCR buffer containing DNA polymerase and detect nucleic acids using real-time PCR.

[0023] Furthermore, buffer solutions containing proteolytic enzymes and PCR buffer solutions containing DNA polymerase typically have different compositions. Therefore, adding a sample after the reaction with proteolytic enzymes can alter the composition of the PCR buffer solution containing DNA polymerase, potentially hindering subsequent PCR progress. For this reason, the amount of sample added to the PCR buffer solution containing DNA polymerase after the reaction with proteolytic enzymes was previously limited. To increase the amount of bacterial nucleic acid in the PCR buffer solution, for example, it was necessary to increase the amount of sample.

[0024] An ophthalmic examination support method according to one embodiment of the present invention (hereinafter sometimes referred to as "this examination method") can efficiently extract nucleic acids from ophthalmic specimens, regardless of whether the specimen is in liquid or solid form. Furthermore, in the examination of intraocular malignant lymphoma, even if intraocular malignant lymphoma cells are present in ophthalmic specimens, the amount is extremely small, and conventionally, rapid testing has been considered difficult.

[0025] The inventors have novelly discovered that this testing method can detect intraocular malignant lymphoma cells from ophthalmic specimens. Furthermore, this testing method enables testing for intraocular malignant lymphoma using the same principle as conventionally known real-time PCR-based testing kits for infectious uveitis. Therefore, it is possible to perform both intraocular malignant lymphoma testing and infectious uveitis testing simultaneously using a small amount of ophthalmic specimen.

[0026] Intraocular malignant lymphoma is known to be clinically difficult to differentiate from uveitis, including both infectious and non-infectious forms. For example, while the diagnosis of primary central nervous system lymphoma takes approximately 4.6 months, primary ocular lymphoma is often misdiagnosed as uveitis, and it has been reported that the diagnosis takes an average of 12.8 months. Therefore, differential testing between the two is strongly needed in clinical practice.

[0027] Furthermore, intraocular malignant lymphoma, a malignant disease presenting with uveitis-like findings (masked syndrome), is the seventh most common cause of uveitis according to the nationwide epidemiological survey of uveitis conducted by the Japanese Society for Ocular Inflammation, and its incidence has been increasing in recent years. While non-infectious uveitis is primarily treated with steroids, intraocular malignant lymphoma and infectious uveitis require treatment of the underlying cause, making differential diagnosis necessary for determining the treatment strategy. This diagnostic method can be suitably used for such differential diagnosis.

[0028] The following describes this testing method. For the sake of simplicity, unless otherwise specified, the auxiliary testing will simply be referred to as "testing." In this specification, "A~B" indicating a numerical range means "A or greater and B or less," including the last value.

[0029] [2. Methods to assist in ophthalmic examinations] This inspection method includes a decomposition step, a deactivation step, and a detection step.

[0030] <2-1. Decomposition process> The degradation process involves allowing the reaction of the proteolytic enzyme to proceed in a sample mixture, which is a mixture of an ophthalmic specimen and a PCR buffer containing a proteolytic enzyme. An ophthalmic specimen is a biological sample that includes at least a portion of the eyeball or its appendages.

[0031] Examples of parts of the eyeball include the cornea, vitreous humor, lens, sclera, uvea, and ciliary bodies. The aqueous humor present within the eyeball may also be included as part of the eyeball. The aqueous humor collected from the anterior chamber is preferably used.

[0032] Examples of ocular appendages include the ocular organs and secretions such as tears secreted from the eyeball or ocular organs. Examples of parts of the ocular organs include the conjunctiva, lacrimal apparatus, extraocular muscles, and parts of the eyelids.

[0033] Because it is difficult to collect large quantities of such ophthalmic specimens, the amount that can be used for testing is often limited. This testing method allows for real-time PCR without pretreatment to extract nucleic acids from ophthalmic specimens, making it possible to test for ophthalmic diseases such as lymphoma from minute amounts of ophthalmic specimens. Therefore, this testing method is particularly suitable for ophthalmic specimens, where the amount that can be collected tends to be small. Among ophthalmic specimens, aqueous humor is relatively easy to collect and is useful if it can be used for ophthalmic examinations. The inventors have novelly discovered that aqueous humor can be used in this testing method for testing for intraocular malignant lymphoma, or for testing for multiple diseases such as intraocular malignant lymphoma and infectious uveitis.

[0034] The collected ophthalmic specimen is mixed with a PCR buffer containing proteolytic enzymes (hereinafter sometimes referred to as the "pretreatment solution"). The pretreatment solution does not contain DNA polymerase. The method of mixing the ophthalmic specimen and the pretreatment solution is not particularly limited, but for example, the ophthalmic specimen may be added directly to the pretreatment solution, or a cotton swab or the like to which the ophthalmic specimen has been attached may be dipped into the pretreatment solution and then added. The pretreatment solution to which the ophthalmic specimen has been added is called the specimen mixture.

[0035] The composition of the PCR buffer is not particularly limited, and conventionally known PCR buffers can be used. The PCR buffer may be, for example, a Tris buffer containing KCl, MgCl2, and a dNTP mix (a mixture of dATP, dGTP, dCTP, and dTTP).

[0036] The proteolytic enzymes included in the PCR buffer are not particularly limited, but examples include serine proteases, cysteine ​​proteases, threonine proteases, aspartate proteases, glutamate proteases, metalloproteases, and asparagine peptide lyases. Among these, proteinase K, a type of serine protease, is preferred from the viewpoint of proteolytic activity and availability.

[0037] The amount of ophthalmic specimen added to the pretreatment solution is 0.5 mg to 5 mg, more preferably 0.5 mg to 3 mg, and even more preferably 0.75 mg to 2 mg, when the ophthalmic specimen is in solid form such as a corneal and conjunctival scraping. When the ophthalmic specimen is in liquid form such as aqueous humor, the amount is preferably 12 μL to 20 μL, but may be less than 12 μL. In particular, when using aqueous humor, the amount may be 20 μL or less, 10 μL or less, or 5 μL or less. The ophthalmic specimen may also be used after dilution.

[0038] The resulting sample mixture is then subjected to a reaction with a proteolytic enzyme. From the viewpoint of efficiently degrading proteins with the proteolytic enzyme, it is preferable to heat the sample mixture at a temperature of 37°C to 60°C during this reaction. In this case, the heating time should be continued until almost all of the proteins in the sample mixture are degraded, for example, 30 minutes to 60 minutes.

[0039] <2-2. Deactivation process> The inactivation step is a process of inactivating the proteolytic enzyme contained in the sample mixture after the reaction with the proteolytic enzyme. The method of inactivating the proteolytic enzyme is not particularly limited, but examples include heating the sample mixture, adding a proteolytic enzyme inhibitor to the sample mixture, or allowing the proteolytic enzyme to autodigest. Among these, inactivation by heating is preferred from the viewpoint of simple and reliable inactivation of the proteolytic enzyme.

[0040] When inactivating proteolytic enzymes by heating, it is preferable to heat the sample mixture at a temperature of 90°C to 100°C. In this case, the heating time should be until the proteolytic enzymes are almost completely inactivated, for example, 5 to 10 minutes.

[0041] <2-3. Detection Process> The detection step involves adding at least a portion of the sample mixture after the inactivation step to the PCR reaction composition, and then amplifying and detecting the PCR product using real-time PCR.

[0042] The PCR reaction composition may include DNA polymerase, a PCR primer pair, and a probe. By adding a sample mixture to such a PCR reaction composition and performing real-time PCR, if the sample mixture contains the target nucleic acid corresponding to the PCR primer pair, the PCR product will be amplified, and this amplification can be detected in real time.

[0043] The DNA polymerase is not particularly limited as long as it is a heat-stable DNA polymerase, but may be, for example, Taq, Tth, KOD, Pfu, or their variants. From the viewpoint of avoiding nonspecific nucleic acid amplification by DNA polymerase, a hot-start DNA polymerase may be used. Examples of hot-start DNA polymerases include DNA polymerase to which an anti-DNA polymerase antibody is bound, and DNA polymerase in which the enzyme active site has been chemically modified to be heat-sensitive.

[0044] A PCR reaction composition includes a PCR primer pair and a probe corresponding to the amplification product amplified by the PCR primer pair. Details of the PCR primer pair and probe are described in section <2-4. Test Items> below.

[0045] From the viewpoint of simultaneously detecting multiple types of nucleic acids, two or more PCR reaction compositions containing at least one PCR primer pair may be used. In this case, by adding at least a portion of the sample mixture after the inactivation step to each of these two or more PCR reaction compositions and performing real-time PCR, multiple nucleic acids can be detected simultaneously. The nucleic acids to be detected simultaneously may be, for example, a combination of wild-type and mutant nucleic acids of the marker gene to be tested, or they may be the nucleic acids of each of multiple types of marker genes. They may also be the nucleic acids of the pathogen to be detected.

[0046] PCR primer pairs are preferably designed as primer pairs suitable for real-time PCR. Suitable for real-time PCR means, for example, a primer pair that achieves good amplification onset within a predetermined number of cycles in the presence of a fluorescent dye or fluorescently labeled probe for detecting amplification. The predetermined number of cycles is not particularly limited, but may be, for example, 35 cycles, 30 cycles, 25 cycles, or 20 cycles.

[0047] Furthermore, the multiplex method may be used for real-time PCR. The multiplex method is known as a method that saves the amount of sample mixture and amplifies multiple types of nucleic acids simultaneously (Sugita S, et al. Br J Ophthalmol. 2008;92:928-932, Sugita S, et al. Ophthalmology. 2013;120:1761-1768). The multiplex method is a method that simultaneously amplifies multiple nucleic acid regions by using multiple types of PCR primer pairs in a single PCR reaction system. In addition to saving the amount of sample mixture, this method has the advantage of being able to simultaneously detect multiple bacterial nucleic acids. When using the multiplex method, it is preferable to optimize the sequence of each primer and the reaction conditions so that the amplification of the target nucleic acid by each PCR primer pair proceeds well in a single PCR reaction system.

[0048] In this testing method, when it is stated that "the PCR reaction composition contains two or more PCR primer pairs," it may contain either or both of the following: (a) a single PCR reaction composition contains two or more PCR primer pairs; (b) the testing method is performed using multiple PCR reaction compositions, each containing one type of PCR primer pair.

[0049] The PCR reaction composition may be a solution or a solid prepared by freeze-drying or the like. If the PCR reaction composition is solid, PCR can be started simply by mixing the sample mixture after the inactivation step with the PCR reaction composition, making this test method easy to perform. In this case, storage of the PCR reaction composition is also easier.

[0050] Real-time PCR is performed by mixing the PCR reaction composition with the sample mixture after the inactivation step and performing thermal cycling using a thermal cycler compatible with real-time PCR. The PCR conditions (temperature, time, and number of cycles) may be set as appropriate depending on the expected nucleic acid sequence to be amplified. If the nucleic acid to be amplified is present in the sample mixture, the PCR product can be amplified.

[0051] In real-time PCR, PCR products are generally detected by fluorescence detection. Methods for fluorescence detection in real-time PCR include, for example, the use of intercalator fluorescent dyes or fluorescently labeled probes. An example of an intercalator fluorescent dye is SYBR® Green. Intercalator fluorescent dyes bind to double-stranded DNA synthesized by PCR. By irradiating the fluorescent dye, which has accumulated due to binding to the double-stranded DNA, with excitation light and measuring the fluorescence intensity, the amount of PCR product produced can be measured.

[0052] Examples of fluorescently labeled probes used for real-time measurement include hydrolysis probes, molecular beacons, and cycling probes. Hydrolysis probes may be oligonucleotide probes in which the 5' end is modified with a fluorescent dye and the 3' end is modified with a quencher substance. The fluorescent dyes used in hydrolysis probes are not particularly limited, but may include the fluorescent dyes mentioned above. Examples of quencher substances include TAMRA®, Black Hole Quencher (BHQ®) 1, BHQ2, MGB-Eclipse®, and DABCYL. In order to distinguish and detect two or more different PCR products, it is preferable from the viewpoint of detection accuracy to perform PCR using two or more fluorescently labeled probes (e.g., hydrolysis probes) each labeled with a different fluorescent dye.

[0053] Examples of fluorescent dyes, though not particularly limited, include 6-caroxyfluorescein (FAM), 6-carboxy-X-rhodamine (ROX), Alexa Fluor® dyes (e.g., ALEXA594), cyanine dyes (e.g., Cy5), and 4,7,2',4',5',7'-hexachloro-6-carboxyfluorescein (HEX).

[0054] In real-time measurement of PCR products, the progress of PCR can be confirmed in real time by monitoring the amplification curve of the PCR product using a fluorescence filter corresponding to the fluorescent dye used. If the fluorescence intensity increases in accordance with the number of PCR cycles, it can be determined that the PCR product is being amplified.

[0055] In this testing method, it is preferable to perform the detection step under conditions in which a portion of the sample mixture after the inactivation step is added to the control PCR reaction composition. The control PCR reaction composition may, for example, include, in addition to the above-mentioned PCR reaction composition, a positive control nucleic acid and a PCR primer pair corresponding to the positive control nucleic acid. The positive control nucleic acid is used as an indicator that the PCR reaction was performed normally, and as an indicator that the nucleic acid derived from the ophthalmic sample was correctly added to the PCR reaction composition.

[0056] When used as an indicator to show that the PCR reaction was performed correctly, the positive control nucleic acid may be a nucleic acid that is thought to be present in the ophthalmic specimen, or an artificially synthesized artificial sequence.

[0057] When using nucleic acids that are thought to be present in ophthalmic specimens as positive control nucleic acids, it is more preferable to use housekeeping genes whose expression levels are less prone to fluctuation. Examples of housekeeping genes include the TATA-binding protein (TBP) gene, glyceraldehyde-3-phophate dehydrogenase (GAPDH) gene, β-actin gene, β2-microglobulin gene, hypoxanthine phosphoribosyl transferase 1 (HPRT1) gene, 18S rRNA gene, 5-aminolevulinate synthase (ALAS) gene, β-globin gene, glucose-6-phophate dehydrogenase (G6PD) gene, β-glucuronidase (GUSB) gene, importin 8 (IPO8) gene, porphobilinogen deaminase (PBGD) gene, phosphoglycerate kinase 1 (PGK1) gene, peptidylprolyl isomerase A (PPIA) gene, ribosomal protein L13a (RPL13A) gene, ribosomal protein large P0 (RPLP0) gene, and succinate dehydrogenase subunit Examples include the A(SDHA) gene, the transferrin receptor (TFRC) gene, the 3-monooxygenase / tryptophan 5-monooxygenase activation protein, and the zeta(YWHAZ) gene.

[0058] Positive control nucleic acids are also useful for quantifying the nucleic acid to be amplified by quantifying the nucleic acid copy number (absolute or relative quantification). The quantification result of the nucleic acid to be amplified can be used as an indicator of the number of pathogens in the ophthalmic sample, for example, if the nucleic acid to be amplified is a pathogen nucleic acid. When performing absolute quantification, for example, by creating a calibration curve based on the measurement results of a positive control nucleic acid of known concentration, the nucleic acid to be amplified, whose concentration is unknown, can be quantified with accuracy. In this case, it is preferable that the positive control nucleic acid is an artificially synthesized artificial sequence that is not present in the ophthalmic sample.

[0059] When performing relative quantification, for example, the number of cycles required for the PCR product to reach a certain level can be compared between the positive control nucleic acid and the target nucleic acid. Based on the characteristic of PCR that the PCR product is amplified twofold with each cycle, this comparison allows for the calculation of the relative concentration difference of the target nucleic acid relative to the positive control nucleic acid.

[0060] The positive control nucleic acid may be one of these or two or more. Using two or more positive control nucleic acids is preferable from the viewpoint of improving the reliability of nucleic acid quantification. For example, GAPDH and TBP may be used in combination as the positive control nucleic acid.

[0061] <2-4. Examination Items> (Intraocular malignant lymphoma, MYD88 and CD79B) Intraocular lymphoma is one of the target ophthalmic diseases for this diagnostic method. The L265P mutation in the MYD88 protein and the Y196 mutation in the CD79B protein are known markers for intraocular lymphoma. If gene mutations corresponding to these mutations are detected in an ophthalmic specimen, intraocular lymphoma should be suspected.

[0062] The PCR reaction composition includes a first primer pair for amplifying the MYD88 gene and a second primer pair for amplifying the CD79B gene. In this specification, gene amplification by PCR means amplifying at least a portion of the nucleic acid sequence as a PCR product, using the gene or the cDNA (complementary DNA) obtained from the transcript (mRNA) of the gene as a template. This testing method allows for rapid and highly accurate testing for the presence or absence of intraocular malignant lymphoma by detecting the presence or absence of mutations in the MYD88 gene and CD79B using real-time PCR.

[0063] The MYD88 gene (mRNA) has the nucleotide sequence shown in Sequence ID No. 1. The MYD88 gene encodes the MYD88 protein, which has the amino acid sequence shown in Sequence ID No. 2.

[0064] The first primer pair may be a pair of forward and reverse primers for amplifying at least a portion of the MYD88 gene by real-time PCR. The MYD88 gene may contain mutations such as SNPs in the nucleotide sequence shown in Sequence ID No. 1, insofar as it is amplified by the first primer pair.

[0065] The CD79B gene (mRNA) has the nucleotide sequence shown in Sequence ID No. 3. The CD79B gene encodes the CD79B protein, which has the amino acid sequence shown in Sequence ID No. 4.

[0066] The second primer pair may be a pair of forward and reverse primers for amplifying at least a portion of the CD79B gene by real-time PCR. The CD79B gene may contain mutations such as SNPs in the nucleotide sequence shown in Sequence ID No. 3, insofar as it is amplified by the second primer pair.

[0067] The PCR reaction composition further comprises a first probe for detecting the presence or absence of a mutation in the MYD88 gene and a second probe for detecting the presence or absence of a mutation in the CD79B gene. The mutation in the MYD88 gene may be a mutation involving the L265P amino acid substitution of the MYD88 protein. The mutation in the CD79B gene may be an amino acid substitution of the CD79B protein, involving at least one amino acid substitution selected from the group consisting of Y196N, Y196H, Y196S, Y196C, Y196F, and Y196D.

[0068] The first probe may include a probe for detecting the presence or absence of mutations involving the L265P amino acid substitution in the MYD88 protein. The second probe may also include a probe for detecting the presence or absence of mutations involving amino acid substitutions in the CD79B protein, specifically at least one amino acid substitution selected from the group consisting of Y196N, Y196H, Y196S, Y196C, Y196F, and Y196D. The first and second probes may each be a single type of probe or a probe set containing multiple types of probes.

[0069] The first probe may be a set consisting of probe A, which detects the wild-type MYD88 gene in which the 265th amino acid of the MYD88 protein is L, and probe B, which detects the mutant MYD88 gene in which the 265th amino acid is P.

[0070] Specifically, the first probe may be a set consisting of probe A, which detects the wild-type MYD88 gene where the 794th base of the MYD88 gene is T, and probe B, which detects the mutant MYD88 gene where the 794th base of the MYD88 gene is C.

[0071] The second probe may include probe C, which detects the wild-type CD79B gene in which the 196th amino acid of the CD79B protein is Y. The second probe may further include at least one probe D, which detects the mutant CD79B gene in which the 196th amino acid is not Y. Probe D may be at least one of probes D1 to D6 shown below; Probe D1: A probe that detects a mutant CD79B gene in which the 196th amino acid of the CD79B protein is N. Probe D2: A probe that detects a mutant CD79B gene in which the 196th amino acid of the CD79B protein is H. Probe D3: A probe that detects a mutant CD79B gene in which the 196th amino acid of the CD79B protein is S. Probe D4: A probe that detects a mutant CD79B gene in which the 196th amino acid of the CD79B protein is C. Probe D5: A probe that detects a mutant CD79B gene in which the 196th amino acid of the CD79B protein is F. Probe D6: A probe that detects a mutant CD79B gene in which the 196th amino acid of the CD79B protein is D.

[0072] Specifically, the second probe may include probe C, which detects the wild-type CD79B gene where the 586th base of the CD79B gene is T and the 587th base is A. The second probe may also include probe D, which detects a mutant CD79B gene where the 586th base of the CD79B gene is not T, or the 587th base is not A. Probe D is at least one of probes D1 to D6 shown below; Probe D1: A probe that detects a mutant CD79B gene in which the 586th base of the CD79B gene is A. Probe D2: A probe that detects a mutant CD79B gene in which the 586th base of the CD79B gene is G. Probe D3: A probe that detects a mutant CD79B gene in which the 586th base of the CD79B gene is C. Probe D4: A probe that detects a mutant CD79B gene in which the 587th base of the CD79B gene is T. Probe D5: A probe that detects a mutant CD79B gene in which the 587th base of the CD79B gene is G. Probe D6: A probe that detects a mutant CD79B gene in which the 587th base of the CD79B gene is C.

[0073] The second probe may include at least one of probes D1 to D6 as probe D, preferably two or more, and more preferably all six.

[0074] In the first probe, probe A and probe B may each be fluorescently labeled probes. Examples of fluorescent dyes used for labeling are as described above. Preferably, probe A and probe B are labeled with fluorescent dyes having different excitation wavelengths. For example, probe A may be ROX-labeled and probe B may be FAM-labeled. In this case, both confirmation of whether or not the MYD88 gene is amplified and confirmation of whether the amplified MYD88 gene is wild-type or mutant can be clearly performed within the same tube.

[0075] Similarly, in the second probe, probe C and probe D may each be fluorescently labeled probes. Examples of fluorescent dyes are as described above. It is preferable that probe C and probe D are each labeled with fluorescent dyes having different excitation wavelengths. For example, probe C may be ROX-labeled and probe D may be FAM-labeled. In this case, both confirmation of the presence or absence of CD79B gene amplification and confirmation of whether the amplified CD79B gene is wild-type or mutant can be performed in the same tube.

[0076] If the second probe contains multiple types of probe D, each probe D may be labeled with the same type of fluorescent label. That is, it is sufficient that the fluorescence of probe C and the fluorescence of probe D can be distinguished, but it is not necessary to distinguish the type of mutation in the CD79B gene. Furthermore, multiple types of probe D may be labeled with fluorescent substances having different excitation wavelengths, as long as they can be distinguished from the fluorescence of probe C.

[0077] (Intraocular malignant lymphoma, IL-6 and IL-10) IL-6 and IL-10 are also known markers for intraocular malignant lymphoma. In ophthalmic specimens, if the amount of IL-10 mRNA is higher than that of IL-6 mRNA, intraocular malignant lymphoma is suspected. In real-time PCR, the amplification of the PCR product increases in proportion to the amount (copy number) of mRNA contained in the ophthalmic specimen. Therefore, the ratio of the amount of IL-6 PCR product amplification to the amount of IL-10 PCR product amplification from an ophthalmic specimen can be considered to be approximately proportional to the ratio of the amount of IL-6 mRNA to the amount of IL-10 mRNA in the ophthalmic specimen.

[0078] Furthermore, even if amplification of only the PCR product of IL-10 mRNA is detected and amplification of the PCR product of IL-6 mRNA is not detected after a predetermined number of cycles, it is still acceptable to conclude that "amount of IL-10 mRNA > amount of IL-6 mRNA".

[0079] The PCR reaction composition may include a third primer pair for amplifying the IL-6 gene and a fourth primer pair for amplifying the IL-10 gene. With this configuration, the presence or absence of intraocular malignant lymphoma can be rapidly and accurately tested by calculating the ratio of the amplification levels of the IL-6 gene to that of the IL-10 gene. Furthermore, by combining the test results for this item with the test results for the presence or absence of mutations in the MYD88 gene and CD79B gene, the presence or absence of intraocular malignant lymphoma can be tested with even greater accuracy and sensitivity.

[0080] The IL-6 gene (mRNA) has the nucleotide sequence shown in Sequence ID No. 5. The third primer pair may be a pair of forward and reverse primers for amplifying at least a portion of the IL-6 gene by real-time PCR. The IL-6 gene may contain mutations such as SNPs from the nucleotide sequence shown in Sequence ID No. 5, insofar as it is amplified by the third primer pair.

[0081] The IL-10 gene (mRNA) has the nucleotide sequence shown in SEQ ID NO: 6. The fourth primer pair may be a pair of forward and reverse primers for amplifying at least a portion of the IL-10 gene by real-time PCR. The IL-10 gene may contain mutations such as SNPs from the nucleotide sequence shown in SEQ ID NO: 6, insofar as it is amplified by the fourth primer pair.

[0082] Amplification of the IL-6 and IL-10 genes may be detected by a fluorescently labeled probe, or by other labels such as intercalator fluorescent dyes. From the viewpoint of reducing the amount of ophthalmic sample required, it is preferable to use a fluorescently labeled probe capable of detecting multiple items in the same tube. Probes for detecting the amplification of PCR products of the IL-6 and IL-10 genes can be designed as appropriate.

[0083] (Infectious uveitis) This testing method may further include ophthalmic diseases other than intraocular malignant lymphoma as targets for testing. That is, the PCR reaction composition may include PCR primer pairs and probes other than those described above. These are not particularly limited, but examples include those that detect pathogens that cause infectious uveitis. The pathogens may be viruses, bacteria, or parasites (Toxoplasma). When bacteria are the target of detection, this testing method may further detect whether the bacteria possess drug resistance genes. Thus, this testing method may further include infectious uveitis as a target ophthalmic disease.

[0084] Thus, if this test method includes both intraocular malignant lymphoma and infectious uveitis as targets for examination, it becomes possible to quickly differentiate whether a patient's uveitis-like symptoms originate from intraocular malignant lymphoma or infectious uveitis.

[0085] Viruses that cause infectious uveitis include, for example, herpes simplex virus types 1 and 2, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus, Toxoplasma, HTLV-1, and human herpesvirus type 6. Bacteria that cause infectious uveitis include, for example, Treponema, Enterococcus, Klebsiella, Nocardia, Streptococcus, Staphylococcus, Pseudomonas aeruginosa, and Escherichia coli.

[0086] Examples of bacteria belonging to the genus Treponema include Treponema pallidum, which causes syphilis. Examples of bacteria belonging to the genus Enterococcus include Enterococcus faecalis and Enterococcus agalactie. Examples of bacteria belonging to the genus Klebsiella include Klebsiella pneumoniae. Examples of bacteria belonging to the genus Streptococcus include Group A Streptococcus, Group B Streptococcus, and Streptococcus pneumoniae. Examples of bacteria belonging to the genus Staphylococcus include Staphylococcus aureus or other Staphylococcus aureus. Examples of other Staphylococcus aureus include Staphylococcus epidermidis and other Staphylococcus epidermidis.

[0087] PCR primer pairs for detecting antibiotic resistance genes possessed by bacteria may be appropriately designed depending on the type of bacteria and antibiotic resistance gene present. The antibiotic resistance gene referred to here is a gene not originally present in bacteria, and may be, for example, a transferable antibiotic resistance gene such as a plasmid-borne one. It may also be an endogenous bacterial gene, but a mutant gene that exhibits antibiotic resistance due to gene mutation.

[0088] The PCR reaction composition may include a fifth primer pair for detecting pathogens that cause infectious uveitis. The fifth primer pair may be designed to amplify nucleic acids having sequences specific to the target pathogen. Such design can be carried out, for example, based on nucleotide sequence information obtained from a known sequence database (such as GenBank). Examples include known PCR primer pairs disclosed in Japanese Patent Application Publication No. 2020-198809 or Japanese Patent Application Publication No. 2023-093129.

[0089] By combining the test results for infectious uveitis with those for intraocular malignant lymphoma, the cause of ophthalmic diseases presenting with uveitis-like findings can be investigated in detail and rapidly. In this testing method, the tests for each item may be performed simultaneously or separately, as long as the same ophthalmic specimen is used. For example, the collected ophthalmic specimen may be divided, some of the tests may be performed first, and the remaining specimen may be stored and further tests may be performed as needed after the initial tests are completed.

[0090] This testing method allows for the extraction of nucleic acids from minute ophthalmic specimens without requiring complicated procedures, and enables their use in real-time PCR. Therefore, it enables efficient testing for ophthalmic diseases. This configuration, by simplifying and promoting the widespread use of bacterial infection testing, can contribute to achieving goals such as Sustainable Development Goal (SDG) 3, "Ensure healthy lives and promote well-being for all."

[0091] [3. Ophthalmic Examination Kit] An ophthalmic examination kit according to one embodiment of the present invention (hereinafter sometimes referred to as "this kit") comprises a PCR buffer containing a protease and a PCR reaction composition. The PCR reaction composition includes a first primer pair for amplifying the MYD88 gene and a first probe for detecting the presence or absence of mutations in the MYD88 gene. The PCR reaction composition also includes a second primer pair for amplifying the CD79B gene and a second probe for detecting the presence or absence of mutations in the CD79B gene.

[0092] Furthermore, this kit may include a third primer pair for detecting the IL-6 gene and a fourth primer pair for detecting the IL-10 gene. It may also include a fifth primer pair for detecting pathogens causing infectious uveitis. Additionally, this kit may include probes corresponding to these PCR primer pairs. Furthermore, it is preferable that this kit further includes the control PCR reaction composition described above. The descriptions of each component of this kit can be found in the above-described section [2. Methods for Assisting Ophthalmic Examinations].

[0093] This kit may comprise, for example, a sample collection container containing a PCR buffer solution including a proteolytic enzyme, and a PCR reaction container containing a PCR reaction composition. Furthermore, multiple PCR reaction containers may be provided, one for each type of PCR primer pair contained in the PCR reaction composition. In this case, the user of the kit can add the collected ophthalmic sample to the sample collection container and perform the degradation and inactivation steps directly within the sample collection container. The user can then add at least a portion of the sample mixture after the inactivation step to each PCR reaction container and perform the detection step directly within each PCR reaction container.

[0094] Both the sample collection container and the PCR reaction container are preferably shaped to fit a thermal cycler used for real-time PCR. Examples of such containers include tube strips with multiple tubes connected together or PCR well plates with multiple wells.

[0095] Furthermore, this kit may contain reagents other than those described above, such as protease inhibitors. The term "contains" used in this kit description may refer to a state where such reagents are contained within one of the individual containers that make up the kit. This kit may also include instructions for performing this test method.

[0096] This kit allows for efficient assistance in testing ophthalmic diseases by collecting small amounts of ophthalmic specimens and following the prescribed testing method.

[0097] The present invention is not limited to the embodiments described above, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. [Examples]

[0098] This test method was performed on ophthalmic specimens, specifically vitreous humor or aqueous humor, collected from patients who tested positive for intraocular malignant lymphoma or infectious uveitis.

[0099] (method) Vitreous humor or aqueous humor was added to the pretreatment solution (PCR buffer containing proteolytic enzymes) to prepare the sample mixture. The composition of the pretreatment solution before addition was 200 μg / mL proteinase K, 0.05% (w / v) nonionic surfactant, 1.5 mM MgCl2, 35 mM KCl, and dNTP Mix (200 μM dATP, dGTP, dCTP, and dTTP, respectively).

[0100] After the decomposition and inactivation processes by heating, 10 μL of the sample mixture was added to a microtube containing 90 μL of PCR reaction composition (Ampdirect® RT, Shimadzu Corporation), and mixed by pipetting. 20 μL of the mixed sample mixture containing the PCR reaction composition was dispensed into each tube of an 8-tube strip. The PCR reaction composition in the strip tubes contained reverse transcriptase, DNA polymerase, and PCR primer pairs and fluorescently labeled probes corresponding to each test item.

[0101] In Example 1, the markers for intraocular malignant lymphoma, MYD88, CD79B, IL-6, and IL-10, were used as test items, and each tube contained the following PCR primer pairs and fluorescently labeled probes: Tube A: Internal control (IC, artificial arrangement) and TBP, Tube B: IL-6 and IL-10, Tube C: MYD88 wild type and mutant, Tube D: Wild-type and variant forms of CD79B.

[0102] The nucleic acid used for IC, the PCR primer pair for detecting IC, and the fluorescently labeled probe were designed with nucleic acid sequences suitable for this testing method. A ROX-labeled probe was used as the fluorescently labeled probe for IC.

[0103] In Example 2, among the markers for intraocular malignant lymphoma, only IL-6 and IL-10 were selected as test items, and each tube contained the following PCR primer pairs and fluorescently labeled probes; Tube E: TBP, Tube F: IL-6 and IL-10.

[0104] In Example 3, the following viruses, which cause infectious uveitis, were tested: human herpes simplex virus type 1 (HSV-1), human herpes simplex virus type 2 (HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV-6), human T-cell leukemia virus type 1 (HTLV-1), Treponema pallidum (TP), and Toxoplasma (TXO). Each tube contained the following PCR primer pairs and fluorescently labeled probes; Tube G: GAPDH and TBP, Tube H: HSV-1 and VZV, Tube I: HSV-2 and HHV-6, Tube J: EBV and CMV, Tube K:HTLV-1 and TP, Tube L: TXO.

[0105] The PCR primer pairs and fluorescently labeled probes corresponding to each test item in Examples 1-3 are shown in Table 1 below. In Table 1, forward primers are denoted as "Fwd" and reverse primers as "Rev". Fluorescently labeled probes using ROX or FAM were used. In the base sequence, "R" indicates adenine (A) or guanine (G), and "M" indicates adenine (A) or cytosine (C).

[0106] [Table 1]

[0107] In this way, we experimented to enable the detection of two types of PCR products in each tube using two types of fluorescent dyes (FAM and ROX).

[0108] The sample mixture after the inactivation step was monitored for PCR reaction using a real-time PCR instrument and the hydrolysis probe method. For PCR conditions, in Examples 1 and 2, which included IL-6 and IL-10 as test items, initial denaturation was performed at 90°C / 30 seconds, reverse transcription at 61°C / 10 minutes, denaturation at 95°C / 30 seconds, followed by 45 cycles of PCR at 95°C / 5 seconds - 62.5°C / 20-30 seconds. In Example 3, initial denaturation was performed at 95°C / 10 seconds, followed by 45 cycles of PCR at 98°C / 5 seconds - 60°C / 20-30 seconds. The presence or absence of amplification of the PCR product was determined based on the Cq value (the number of cycles at which the amplification curve of the PCR product crosses the threshold line).

[0109] In Examples 1 and 3 described above, the tests were performed using different PCR conditions. This is because Example 1 used PCR conditions that included a reverse transcription reaction, while Example 3 used PCR conditions that did not include a reverse transcription reaction. In this case, if one wishes to test for both intraocular malignant lymphoma and infectious uveitis, it is necessary to perform one test first and then the other test, or to use multiple real-time PCR devices.

[0110] Therefore, as Example 4, we attempted to see if it was possible to simultaneously perform tests for intraocular malignant lymphoma and infectious uveitis. Specifically, we removed the IL-6 and IL-10 test items (tube B) from Example 1 and attempted the tests using the aforementioned tubes A, C, D, G-L, including the test items from Example 3.

[0111] Furthermore, the primer and probe sequences used in Example 4 were the same as those used in Examples 1 and 3, with a few exceptions. The "exceptions" made in Example 4 involved changing probe C, used to detect the wild-type CD79B, to probe C', which has the sequence of sequence number 62 shown in Table 3. Additionally, probe D5, used to detect the mutant form of CD79B, was changed to probe D5', which has the sequence of sequence number 63 shown in Table 3. The PCR conditions were the same as those used in Example 3.

[0112] [Table 2]

[0113] Examples 1 and 4 combined involved 53 ophthalmic specimens (47 vitreous humor specimens, 6 aqueous humor specimens) from patients diagnosed with intraocular malignant lymphoma and 18 ophthalmic specimens (6 vitreous humor specimens, 12 aqueous humor specimens) from patients diagnosed with infectious uveitis. The study was also conducted on 3 ophthalmic specimens (1 vitreous humor specimen, 2 aqueous humor specimens) from patients diagnosed with non-intraocular malignant lymphoma and non-infectious uveitis, as well as other ophthalmic diseases. Example 2 involved 6 ophthalmic specimens (all vitreous humor) from patients diagnosed with intraocular malignant lymphoma and 1 ophthalmic specimen (aqueous humor) from a patient diagnosed with infectious uveitis.

[0114] In Examples 1 and 2, the specimens that underwent testing for intraocular malignant lymphoma were also tested for infectious uveitis as described in Example 3. In Example 4, testing for intraocular malignant lymphoma and infectious uveitis were performed simultaneously.

[0115] (result) The results for Examples 1, 3, and 4 are summarized in Table 3 below, and the results for Example 2 are summarized in Table 4 below.

[0116] [Table 3]

[0117] [Table 4]

[0118] Furthermore, a representative example of the amplification curve obtained in Example 1 is shown in Figure 1, and a representative example of the amplification curve obtained in Example 2 is shown in Figure 2. Both Figures 1 and 2 are representative examples using vitreous humor as a specimen. In addition, a representative example using vitreous humor as an ophthalmic specimen in Example 1 is shown in Figure 3, and a representative example using aqueous humor is shown in Figure 4.

[0119] As shown in Tables 3 and 4 and Figures 1-4, it was demonstrated that nucleic acids of intraocular malignant lymphoma cells, even when present in trace amounts in ophthalmic specimens such as aqueous humor and vitreous humor obtained from patients, can be detected. Furthermore, it was shown that the detection sensitivity was higher when MYD88 and CD79B were used as test items than when IL-6 and IL-10 were used. It should be noted that using all four items—MYD88, CD79B, IL-6, and IL-10—as test items may prevent missed detections that may occur when using only MYD88 and CD79B.

[0120] Furthermore, the results of this embodiment demonstrate that even when all six types of probes D1 to D6 are included in a single tube as a second probe for detecting the presence or absence of CD79B mutations and real-time PCR is performed, mutant CD79B can be detected without any problems. This demonstrates that many mutant types of CD79B can be easily detected using this test method.

[0121] Furthermore, in samples that tested positive for intraocular malignant lymphoma (positive for at least one of the MYD88 variant, CD79B variant, and IL-10), all tests for infectious uveitis were negative. Conversely, in samples that tested negative for intraocular malignant lymphoma (negative for all of the MYD88 variant, CD79B variant, and IL-10), one of the tests for infectious uveitis was positive. In other words, the testing method shown in Examples 1, 3, and 4 demonstrated that it can accurately detect either intraocular malignant lymphoma or infectious uveitis, respectively.

[0122] Thus, this testing method allows for convenient ophthalmic examinations for malignant lymphoma, infectious uveitis, and other conditions from ophthalmic specimens that are difficult to collect in large quantities.

[0123] Furthermore, in Example 4, it was demonstrated that intraocular malignant lymphoma and infectious uveitis could be detected with no inferiority to Examples 1 and 3. These results from Example 4 demonstrate that this testing method can simultaneously test for intraocular malignant lymphoma and infectious uveitis. In other words, the results from Example 4 indicate that this testing method is suitably applicable for differentiating between intraocular malignant lymphoma and infectious uveitis.

[0124] In intraocular lymphoma, the positivity rate for the MYD88 mutation is known to be high, at 70-80%. Furthermore, the positivity rate for CD79B, a poor prognostic factor associated with central nervous system recurrence, in intraocular lymphoma is reported to be approximately 35%. MYD88 and CD79B mutations are not observed in healthy individuals or in infectious and non-infectious uveitis other than intraocular lymphoma. Therefore, MYD88 and CD79B mutations are important markers for useful diagnosis, exclusion diagnosis, and prognosis in intraocular lymphoma.

[0125] As can be seen from the positive rates of MYD88 and CD79B mutations in intraocular lymphoma, not all ophthalmic specimens from patients diagnosed with intraocular lymphoma used in each example contain cells with MYD88 and / or CD79B mutations. In other words, it should be noted that the above examples do not represent the positive predictive value of this test method. On the other hand, the results of the above examples demonstrate that if an ophthalmic specimen contains cells with at least MYD88 and / or CD79B mutations, it can be detected by this test method.

[0126] Furthermore, a cell composition consisting solely of MYD88 wild-type and / or CD79B wild-type cells suggests a disease other than intraocular malignant lymphoma. Therefore, the infective uveitis test, which can be performed concurrently with the intraocular malignant lymphoma test, can assist in the diagnosis of major infectious uveitis conditions or in excluding them (mainly non-infectious uveitis). [Industrial applicability]

[0127] The present invention can be used, for example, in the examination of ophthalmic diseases.

Claims

1. A sample mixture obtained by mixing an ophthalmic specimen containing at least a portion of the eyeball or its appendages with a PCR buffer containing a proteolytic enzyme is subjected to a decomposition step to allow the reaction of the proteolytic enzyme to proceed, An inactivation step in which the proteolytic enzyme contained in the sample mixture is deactivated, The detection step includes adding at least a portion of the sample mixture after the inactivation step to the PCR reaction composition and amplifying and detecting the PCR product by real-time PCR, The PCR reaction composition, A first primer pair for amplifying the MYD88 gene, and a first probe for detecting the presence or absence of mutations in the MYD88 gene, An ophthalmic examination assistance method comprising a second primer pair for amplifying the CD79B gene, and a second probe for detecting the presence or absence of mutations in the CD79B gene.

2. The ophthalmic examination assistance method according to claim 1, wherein the mutation in the MYD88 gene is a mutation involving the L265P amino acid substitution of the MYD88 protein.

3. The ophthalmic examination assistance method according to claim 1 or 2, wherein the mutation in the CD79B gene is an amino acid substitution of the CD79B protein, and the mutation involves at least one amino acid substitution selected from the group consisting of Y196N, Y196H, Y196S, Y196C, Y196F, and Y196D.

4. The ophthalmic examination assistance method according to claim 3, wherein the second probe includes six types of probes for detecting mutations involving amino acid substitutions of Y196N, Y196H, Y196S, Y196C, Y196F, and Y196D in the CD79B protein, respectively.

5. The PCR buffer comprises a proteolytic enzyme and a PCR reaction composition. The PCR reaction composition, A first primer pair for amplifying the MYD88 gene, and a first probe for detecting the presence or absence of mutations in the MYD88 gene, An ophthalmic examination kit comprising a second primer pair for amplifying the CD79B gene, and a second probe for detecting the presence or absence of mutations in the CD79B gene.

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