Microarray kit, target detection method using microarray

Abstract

To accurately evaluate the success or failure of an amplification reaction even in the presence of a blocking nucleic acid.SOLUTION: A reaction solution is brought into contact with a microarray equipped with common probes, and the success or failure of the amplification reaction is evaluated on the basis of the signal intensity from the common probes. A microarray kit enables MYD88 gene mutation to be accurately evaluated.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a microarray kit including a microarray provided with detection probes for detecting a target nucleic acid to be detected and detection probes for detecting non-target nucleic acids, and also to a method for detecting a target nucleic acid using the microarray. [Background technology]

[0002] Microarrays are used to detect target nucleic acids in various fields, including clinical medicine, food, and the environment. In particular, single nucleotide polymorphisms (SNPs) are associated with individual differences in disease risk and drug efficacy, so there is a demand for microarrays to detect polymorphisms with high sensitivity and specificity.

[0003] For example, Patent Document 1 discloses a microarray having detection spots on which nucleic acid probes used for detecting target nucleic acids are immobilized and reference spots on which two or more types of nucleic acid probes are immobilized. The microarray and detection method disclosed in Patent Document 1 make it possible to detect deterioration of the microarray and defects in detection procedures such as nucleic acid amplification reactions based on the signal from the reference spots.

[0004] Furthermore, in the hybridization between target nucleic acids and nucleic acid probes, which is the detection principle of microarrays, accurate detection of the target nucleic acid of interest requires that the nucleic acid probe accurately recognize the target nucleic acid. Therefore, conventional hybridization methods involve adjusting the salt concentration and reaction temperature of the reaction solution appropriately, or using a blocking agent to suppress nonspecific hybridization between the nucleic acid probe and nucleic acid molecules other than those being measured. Examples of blocking agents include nucleic acid components that do not have a complementary base sequence to the nucleic acid molecules or nucleic acid probes being measured, such as salmon sperm DNA and yeast tRNA, surfactants such as SDS (sodium dodecyl sulfate) and N-lauroyl sarcosine (N-LS), and proteins such as bovine serum albumin (BSA) and casein.

[0005] Patent Document 2 discloses a method for detecting a target nucleic acid with high accuracy by mixing a buffer composition containing a target nucleic acid containing an allele to be detected in a genetic polymorphism and a non-target nucleic acid containing an allele other than the target allele with a blocking nucleic acid that specifically hybridizes to the non-target nucleic acid.

[0006] Patent Document 3 also discloses a method for detecting a target nucleic acid using a nucleic acid probe, in which a common probe that hybridizes to both the target nucleic acid and the non-target nucleic acid is used. This common probe is a nucleotide consisting of a base sequence complementary to a region commonly present in the target nucleic acid and the non-target nucleic acid. That is, when the target nucleic acid and the non-target nucleic acid are simultaneously amplified by an amplification nucleic acid reaction, the common probe specifically hybridizes to the amplified nucleic acid, regardless of whether the target nucleic acid is present in the reaction solution. In the method disclosed in Patent Document 3, the presence or absence of a mutation is determined using a value obtained by dividing the signal intensity from a nucleic acid probe detecting a non-target nucleic acid corresponding to the wild type by the signal intensity from the common probe. By using this value, the method disclosed in Patent Document 3 can accurately determine the presence or absence of a mutation when multiple mutations other than the wild type may exist. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 5607373 [Patent Document 2] Patent No. 6644297 [Patent Document 3] WO2019 / 004334 Summary of the Invention [Problem to be solved by the invention]

[0008] The use of a blocking nucleic acid in conventional techniques has the advantage that a target nucleic acid can be detected with high accuracy even when the amount of the target nucleic acid is small compared to non-target nucleic acids. However, the signal intensity from the nucleic acid probe detecting the non-target nucleic acid is lower than in a system that does not use a blocking nucleic acid. The signal from the nucleic acid probe detecting the non-target nucleic acid can be used, for example, to determine the presence of the target nucleic acid or to determine whether the amplification reaction of the target nucleic acid and non-target nucleic acid has been performed normally.

[0009] In view of the above circumstances, the present invention aims to provide a microarray kit including a microarray that can make a determination even when the signal intensity from a nucleic acid probe detecting a non-target nucleic acid is reduced by the presence of a blocking nucleic acid, and a method for detecting a target nucleic acid. [Means for solving the problem]

[0010] The present invention, which has achieved the above-mentioned objects, includes the following. (1) A microarray kit comprising: a probe for detecting a target nucleic acid for the MYD88 gene, the probe having a base sequence complementary to a region containing a target nucleic acid containing, as a target base, a genetic mutation in the MYD88 gene associated with lymphoplasmacytic lymphoma / Waldenström's macroglobulinemia; and a probe for detecting a non-target nucleic acid for the MYD88 gene, the probe having a sequence complementary to a region containing a non-target base in a non-target nucleic acid containing, as a non-target base, a wild-type corresponding to the genetic mutation in the MYD88 gene; and a buffer containing a blocking nucleic acid for the MYD88 gene, the blocking nucleic acid having a base sequence complementary to a region containing a non-target base in the non-target nucleic acid of the MYD88 gene.

[0011] (2) The microarray kit according to (1), further comprising a common probe for the MYD88 gene having a sequence complementary to a common region common to the target nucleic acid and non-target nucleic acid of the MYD88 gene, the common region not overlapping with the region containing the non-target base.

[0012] (3) The microarray kit according to (1), wherein the microarray further comprises: a probe for detecting a target nucleic acid for the CXCR4 gene, the probe having a base sequence complementary to a region containing a target base in a target nucleic acid containing, as a target base, a genetic mutation in the CXCR4 gene associated with lymphoplasmacytic lymphoma / Waldenström's macroglobulinemia; and a probe for detecting a non-target nucleic acid for the CXCR4 gene, the probe having a sequence complementary to a region containing a non-target base in a non-target nucleic acid containing, as a non-target base, a wild-type corresponding to the genetic mutation in the CXCR4 gene; and the buffer further comprises a blocking nucleic acid for the CXCR4 gene, the base sequence complementary to a region containing a non-target base in the non-target nucleic acid of the CXCR4 gene.

[0013] (4) The microarray kit according to (3), further comprising a common probe for the CXCR4 gene having a sequence complementary to a common region common to the target nucleic acid and non-target nucleic acid of the CXCR4 gene, the common region not overlapping with the region containing the non-target base.

[0014] (5) A method for detecting a target nucleic acid, comprising the steps of: preparing a reaction solution resulting from an amplification reaction between a target nucleic acid containing a target base and a non-target nucleic acid containing a non-target base corresponding to the target base; and preparing a hybridization buffer containing a blocking nucleic acid containing a base sequence complementary to a region containing the non-target base in the non-target nucleic acid; contacting the reaction solution and the hybridization buffer with a microarray comprising: a target nucleic acid detection probe having a sequence complementary to the region containing the target base in the target nucleic acid; a non-target nucleic acid detection probe having a sequence complementary to the region containing the non-target base in the non-target nucleic acid; and a common probe having a sequence complementary to a common region common to the target nucleic acid and the non-target nucleic acid, the common region not overlapping with the region containing the non-target base; and determining that the amplification reaction of the target nucleic acid and the non-target nucleic acid has been performed normally when the signal intensity from the common probe on the microarray exceeds a threshold value.

[0015] (6) The method for detecting a target nucleic acid according to (5), characterized in that after the determination step, the presence or absence of the target nucleic acid is determined based on the signal intensity from the target nucleic acid detection probe and the signal intensity from the non-target nucleic acid detection probe.

[0016] (7) A method for detecting a target nucleic acid according to (6), characterized in that the value of [signal intensity from the probe for detecting a target nucleic acid] / ([signal intensity from the probe for detecting a target nucleic acid]+[signal intensity from the probe for detecting a non-target nucleic acid]) is calculated, and if this value exceeds a threshold value, it is determined that the target nucleic acid is present.

[0017] (8) The amplification reaction of the target nucleic acid and the non-target nucleic acid was carried out with a template nucleic acid amount of 1.79 × 10 5 ~1.43×10 6 The method for detecting a target nucleic acid according to (5), wherein the target nucleic acid is a copy.

[0018] (9) The method for detecting a target nucleic acid according to (5), wherein the base to be detected contained in the target nucleic acid is a genetic mutation in the MYD88 gene associated with lymphoplasmacytic lymphoma / Waldenström's macroglobulinemia and / or a genetic mutation in the CXCR4 gene associated with lymphoplasmacytic lymphoma / Waldenström's macroglobulinemia. [Effects of the Invention]

[0019] The microarray kit according to the present invention can accurately determine gene mutations in the MYD88 gene associated with lymphoplasmacytic lymphoma / Waldenström's macroglobulinemia.

[0020] Furthermore, according to the target nucleic acid detection method of the present invention, even if the signal from the non-target nucleic acid detection probe is low in the presence of a blocking nucleic acid, it is possible to accurately determine whether the amplification reaction of the target nucleic acid and non-target nucleic acid has been carried out normally based on the signal intensity from the common probe. [Brief explanation of the drawings]

[0021] [Figure 1] FIG. 10 is a characteristic diagram showing the results of comparing the resolution when the length of the wild-type probe is changed. [Figure 2]FIG. 10 is a characteristic diagram showing the results of measuring the fluorescence intensity from the common probe, where (a) shows the results measured in the presence of a blocking nucleic acid, and (b) shows the results measured in the absence of a blocking nucleic acid. [Figure 3] FIG. 1 shows the results of measuring the fluorescence intensity from the wild-type probe, where (a) shows the results measured in the presence of a blocking nucleic acid, and (b) is a characteristic diagram showing the results measured in the absence of a blocking nucleic acid. [Figure 4] FIG. 10 is a characteristic diagram showing the results of calculating the resolution for each gene mutation in the presence of a blocking nucleic acid. [Figure 5] FIG. 10 is a characteristic diagram showing the results of calculating the resolution for each gene mutation in the absence of a blocking nucleic acid. DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention will be described in detail below. The method for detecting a target nucleic acid according to the present invention involves amplifying a target nucleic acid containing a target base and a non-target nucleic acid containing a non-target base corresponding to the target base by an amplification reaction, and detecting the amplified target nucleic acid and the non-target nucleic acid using a microarray comprising a target nucleic acid detection probe that hybridizes to the target nucleic acid contained in a reaction solution and a non-target nucleic acid detection probe that hybridizes to the non-target nucleic acid. The microarray according to the present invention also comprises a target nucleic acid detection probe for detecting the target nucleic acid and a non-target nucleic acid detection probe for detecting the non-target nucleic acid contained in a reaction solution obtained by amplifying a target nucleic acid containing a target base and a non-target nucleic acid containing a non-target base corresponding to the target base by an amplification reaction.

[0023] In particular, in this method, the reaction solution obtained by the nucleic acid amplification reaction and a hybridization buffer containing a blocking nucleic acid having a base sequence complementary to a region containing a non-target base in the non-target nucleic acid are contacted with the microarray. The reaction solution and the hybridization buffer may be premixed, and the resulting mixture may be contacted with the microarray. Alternatively, the reaction solution and the hybridization buffer may be contacted with the microarray separately. By using this hybridization buffer, a portion of the non-target nucleic acid is hybridized with the blocking nucleic acid, thereby preventing the non-target nucleic acid from nonspecifically hybridizing to the target nucleic acid detection probe.

[0024] In particular, the microarray used in this method is equipped with a common probe having a sequence complementary to a common region common to the target nucleic acid and the non-target nucleic acid. The common region is a region of base sequence common to the target nucleic acid and the non-target nucleic acid, which does not overlap with the region in the non-target nucleic acid to which the above-mentioned blocking nucleic acid hybridizes. In other words, the common region is a region that does not overlap with the region to which the target nucleic acid detection probe hybridizes or the region to which the non-target nucleic acid detection probe hybridizes. The common probe defined in this way can hybridize to the common region in the target nucleic acid and can also hybridize to the common region in the non-target nucleic acid even in the presence of the blocking nucleic acid.

[0025] Here, the target nucleic acid refers to a nucleic acid molecule containing a base to be detected, i.e., a nucleic acid fragment. The target nucleic acid may be a nucleic acid molecule consisting of DNA, a nucleic acid molecule consisting of RNA, or a nucleic acid molecule containing DNA and RNA (DNA-RNA complex). The nucleic acid also includes adenine, cytosine, guanine, thymine, and uracil, as well as artificial nucleic acids such as peptide nucleic acid (PNA) and locked nucleic acid (LNA).

[0026] The target base refers to, for example, one or more nucleic acid residues at a specific position in a chromosome, and is not particularly limited, but refers to a specific type of base in a base sequence such as a single nucleotide polymorphism (SNP). For example, if a specific single nucleotide polymorphism can take A (adenine) or C (cytosine), either one of the bases, i.e., A (adenine) in the single nucleotide polymorphism, can be used as the target base. Here, the target base may be either a major allele or a minor allele in a genetic polymorphism, and may or may not be a risk allele.

[0027] A target nucleic acid containing a target base can be prepared by amplifying a predetermined region containing the target base by a nucleic acid amplification method. Alternatively, the target nucleic acid may be cDNA obtained by reverse transcription from a transcription product collected from an individual organism, tissue, or cell. The base length of the target nucleic acid is not particularly limited, but may be, for example, 60 to 1,000 bases, preferably 60 to 500 bases, and more preferably 60 to 200 bases.

[0028] In addition, for a target nucleic acid containing a target base, a nucleic acid molecule (nucleic acid fragment) containing a non-target base corresponding to the target base is referred to as a non-target nucleic acid. For example, when one of multiple bases that can be present at a predetermined position in a chromosome is the target base, bases other than the target base are the non-target bases. More specifically, when a single nucleotide polymorphism at a predetermined position can be A (adenine) or C (cytosine), if A (adenine) in the single nucleotide polymorphism is the target base, C (cytosine) in the single nucleotide polymorphism is the non-target base.

[0029] When the non-target base is present on a chromosome, the non-target nucleic acid containing the non-target base is simultaneously obtained when the target nucleic acid containing the non-target base is obtained as described above. For example, when the target nucleic acid is obtained by a nucleic acid amplification reaction such as a polymerase chain reaction, if one allele is a non-target base, the non-target nucleic acid will be amplified together with the target nucleic acid.

[0030] To obtain target nucleic acids and non-target nucleic acids by nucleic acid amplification, a series of nucleic acid amplification methods can be applied, including the steps of first extracting genomic DNA from a sample derived from a subject and then using the extracted genomic DNA as a template to amplify a region containing a target base or a non-target base.

[0031] The subject is typically a human, and examples thereof include patients suspected of having lymphoplasmacytic lymphoma / Waldenström's macroglobulinemia, as detailed in the Examples, and patients suffering from diseases such as cancer that can be diagnosed by testing for genetic polymorphisms. Healthy individuals may also be used as subjects. The sample derived from the subject is not particularly limited. Examples include blood-related samples (blood, serum, plasma, etc.), lymph, feces, cancer cells, tissue or organ fragments, and extracts.

[0032] The extraction method for extracting genomic DNA from a sample collected from a subject is not particularly limited, and for example, DNA extraction methods using phenol / chloroform, ethanol, sodium hydroxide, CTAB, etc. can be used.

[0033] Next, an amplification reaction is performed using the obtained genomic DNA as a template to amplify the target nucleic acid and non-target nucleic acid. Examples of applicable amplification reactions include polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN). In the amplification reaction, it is desirable to add a label so that the target nucleic acid and non-target nucleic acid can be distinguished after amplification. The method for labeling the amplified target nucleic acid and non-target nucleic acid is not particularly limited. For example, a method in which primers used in the amplification reaction are pre-labeled may be used, or a method in which labeled nucleotides are used as substrates in the amplification reaction may be used. Examples of labeling substances include, but are not limited to, radioisotopes, fluorescent dyes, and organic compounds such as digoxigenin (DIG) and biotin.

[0034] This reaction system also contains buffers necessary for nucleic acid amplification and labeling, a heat-stable DNA polymerase, a pair of primers designed based on the base sequences of the target nucleic acid and non-target nucleic acid, labeled nucleotide triphosphates (specifically, nucleotide triphosphates labeled with a fluorescent label, etc.), nucleotide triphosphates, magnesium chloride, etc.

[0035] On the other hand, the blocking nucleic acid contained in the hybridization buffer is designed to have a base sequence complementary to a region containing a non-target base in the non-target nucleic acid. Therefore, the blocking nucleic acid can hybridize to a region containing a non-target base in the non-target nucleic acid under conditions that allow hybridization of the target nucleic acid and the target nucleic acid detection probe. The blocking nucleic acid is not particularly limited, but preferably has a length that is 60% or more of the base length of the target nucleic acid detection probe. Furthermore, the blocking nucleic acid is preferably shorter than the base length of the target nucleic acid detection probe. For example, if the target nucleic acid detection probe is 25 bases long, the blocking nucleic acid is preferably 15 to 24 bases long.

[0036] In addition, in the blocking nucleic acid, the base complementary to the non-target base is preferably located at the center of the character string when the bases constituting the blocking nucleic acid are viewed as a character string. Note that the center of the character string includes the case where the base is shifted by one base toward the 5'-end or 3'-end in the case of a blocking nucleic acid consisting of an even number of bases.

[0037] Furthermore, the blocking nucleic acid may contain mismatched bases (non-complementary bases) at positions corresponding to bases other than the non-target bases to be detected contained in the non-target nucleic acid. When the blocking nucleic acid is 15 bases long, the number of mismatched bases can be 1 to 3, preferably 1 to 2. When the blocking nucleic acid is 24 bases long, the number of mismatched bases can be 1 to 3, preferably 1 to 2.

[0038] Furthermore, the concentration of the blocking nucleic acid in the hybridization buffer is not particularly limited and can be appropriately set depending on, for example, the concentration of the non-target nucleic acid and / or the concentration of the target nucleic acid. Specifically, the concentration of the blocking nucleic acid in the composition can be 0.01 to 1 μM, preferably 0.05 to 0.75 μM, and more preferably 0.125 to 0.5 μM.

[0039] When there are multiple non-target nucleic acids for a given target nucleic acid, blocking nucleic acids may be prepared for all of the non-target nucleic acids, or blocking nucleic acids may be prepared for some of the non-target nucleic acids.

[0040] The blocking nucleic acid is more preferably single-stranded DNA, and can be chemically synthesized, for example, using a nucleic acid synthesizer, such as a DNA synthesizer, fully automated nucleic acid synthesizer, or automated nucleic acid synthesizer.

[0041] The hybridization buffer configured as described above contains a blocking nucleic acid, which can suppress nonspecific hybridization between non-target nucleic acids and target nucleic acid detection probes, thereby preventing inhibition of specific hybridization between the target nucleic acid and target nucleic acid detection probes. Therefore, by using a hybridization buffer, the target nucleic acid can be detected with high accuracy using the target nucleic acid detection probe, even when the target nucleic acid is present at a low concentration. Furthermore, by using a hybridization buffer containing a blocking nucleic acid, the target nucleic acid can be detected with high accuracy using the target nucleic acid detection probe, even when a non-target nucleic acid that differs from the target nucleic acid by only a single base is present.

[0042] The following describes a microarray used for detecting a target nucleic acid. A microarray is a device in which probes for detecting a target nucleic acid, probes for detecting non-target nucleic acids, and a common probe are immobilized on a carrier (including a substrate, hollow fiber, and microparticle), and the immobilized probes for detecting a target nucleic acid are used to detect (qualitatively and quantitatively) the target nucleic acid.

[0043] The material of the carrier in the microarray can be any known material in the art and is not particularly limited. For example, conductive materials such as noble metals such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten, and their compounds, and carbon such as graphite and carbon fiber; silicon materials such as single crystal silicon, amorphous silicon, silicon carbide, silicon oxide, and silicon nitride; composite materials of these silicon materials such as SOI (silicon-on-insulator); inorganic materials such as glass, quartz glass, alumina, sapphire, ceramics, forsterite, and photosensitive glass; polyethylene, ethylene, polypropylene, etc. Examples of suitable carriers include organic materials such as cyclic polyolefins, polyisobutylene, polyethylene terephthalate, unsaturated polyesters, fluorine-containing resins, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resins, polyacrylonitrile, polystyrene, acetal resins, polycarbonate, polyamides, phenolic resins, urea resins, epoxy resins, melamine resins, styrene-acrylonitrile copolymers, acrylonitrile-butadiene styrene copolymers, polyphenylene oxide, and polysulfone. The shape of the carrier is not particularly limited, but is preferably flat.

[0044] The carrier preferably has a carbon layer such as diamond-like carbon (DLC) on its surface and chemically modified groups such as amino groups, carboxyl groups, epoxy groups, formyl groups, hydroxyl groups, and active ester groups. Carriers having a carbon layer and chemically modified groups on their surface include those having a carbon layer and chemically modified groups on the surface of a substrate, and those having chemically modified groups on the surface of a substrate made of a carbon layer. Materials known in the art can be used for the substrate, and are not particularly limited, and the same materials as those listed above as carrier materials can be used.

[0045] The above-mentioned target nucleic acid detection probe, non-target nucleic acid detection probe and common probe may have their 5' ends immobilized directly to a support or may be immobilized to a support via a linker.

[0046] The target nucleic acid detection probe is designed to have a base sequence complementary to a region containing the target base in a target nucleic acid containing the target base. The target nucleic acid detection probe is not particularly limited, but may be, for example, 10 to 30 bases long, preferably 15 to 25 bases long. Furthermore, when the bases constituting the target nucleic acid detection probe are viewed as a character string, the base complementary to the target base is preferably located at the center of the character string. Note that, for target nucleic acid detection probes consisting of an even number of bases, the center of the character string includes cases where the base is shifted by one base toward the 5' end or the 3' end.

[0047] Similarly, a non-target nucleic acid detection probe is used to detect a non-target nucleic acid containing a non-target base and is designed to have a base sequence complementary to at least a region of the non-target nucleic acid containing the non-target base. The non-target nucleic acid detection probe is not particularly limited, but may be, for example, 10 to 30 bases long, preferably 15 to 25 bases long. Furthermore, when the bases constituting the non-target nucleic acid detection probe are viewed as a character string, the base complementary to the non-target base is preferably located at the center of the character string. Note that, for non-target nucleic acid detection probes consisting of an even number of bases, the center of the character string includes cases where the base is shifted by one base toward the 5' end or the 3' end.

[0048] In particular, the microarray used in this method is provided with a common probe having a sequence complementary to a common region common to the target nucleic acid and the non-target nucleic acid. The common probe is not particularly limited, but can be, for example, 10 to 30 bases long, and preferably 15 to 25 bases long.

[0049] In the method for detecting a target nucleic acid according to the present invention, a hybridization buffer containing the above-described blocking nucleic acid is brought into contact with the microarray. Here, first, it is determined whether the above-described series of nucleic acid amplification methods has been performed normally based on a signal from the common probe. Specifically, if the signal from the common probe exceeds a predetermined value (threshold), it is determined that the above-described series of nucleic acid amplification methods has been performed normally. After it is determined that the above-described series of nucleic acid amplification methods has been performed normally, it is possible to determine whether or not a target nucleic acid is present based on signals from the target nucleic acid detection probe and the non-target nucleic acid detection probe.

[0050] If the normality of the above-described series of nucleic acid amplification methods is judged based on the signal from the non-target nucleic acid detection probe instead of the common probe, a sufficient signal may not be obtained, making it impossible to accurately judge whether the above-described series of nucleic acid amplification methods has been performed normally. For example, when the amount of genomic DNA used as a template is small or when a blocking nucleic acid is present, a sufficient signal from the non-target nucleic acid detection probe may not be obtained.

[0051] The method of the present invention utilizes a signal from a common probe that can hybridize with both the target nucleic acid and the non-target nucleic acid hybridized with a blocking nucleic acid, and therefore, even when the amount of template genomic DNA is small or when a blocking nucleic acid is present, it is possible to accurately determine whether the above-mentioned series of nucleic acid amplification methods have been performed normally.

[0052] In this way, after determining that the above-described series of nucleic acid amplification reactions have been carried out normally, it is possible to determine whether the target nucleic acid is present in the reaction solution after the amplification reaction based on the signals from the target nucleic acid detection probe and the non-target nucleic acid detection probe. In this case, by using the hybridization buffer according to the present invention described above, the blocking nucleic acid can suppress nonspecific hybridization between the non-target nucleic acid and the nucleic acid probe.

[0053] Here, hybridization reactions using a hybridization buffer are preferably carried out under stringent conditions. Stringent conditions refer to conditions under which specific hybrids are formed and nonspecific hybrids are not formed. For example, these conditions include a 16-hour hybridization reaction at 50°C, followed by washing in 2×SSC / 0.2% SDS at 25°C for 10 minutes and 2×SSC at 25°C for 5 minutes. Specifically, the hybridization buffer may contain salts necessary for the hybridization reaction, such as SSC, or known blocking agents, such as SDS. It is preferable to use a lower hybridization temperature for short probes, and a higher hybridization temperature for long probes. It goes without saying that a higher salt concentration increases the specific hybridization temperature, and a lower salt concentration decreases the specific hybridization temperature.

[0054] Alternatively, a reaction solution containing the target nucleic acid and non-target nucleic acid after the amplification reaction may be mixed with other compositions (such as a blocking nucleic acid) in advance to allow specific hybridization between the non-target nucleic acid and the blocking nucleic acid, and then the reaction solution may be contacted with a microarray to allow hybridization between the target nucleic acid and non-target nucleic acid and a common probe, hybridization between the target nucleic acid and the nucleic acid probe, and hybridization between the non-target nucleic acid and the probe for detecting the non-target nucleic acid to proceed. Alternatively, a reaction solution containing the target nucleic acid and non-target nucleic acid after the amplification reaction may be mixed with other compositions (such as a blocking nucleic acid) on a microarray to allow specific hybridization between the non-target nucleic acid and the blocking nucleic acid, hybridization between the target nucleic acid and non-target nucleic acid and the common probe, hybridization between the target nucleic acid and the nucleic acid probe, and hybridization between the non-target nucleic acid and the probe for detecting the non-target nucleic acid to proceed simultaneously.

[0055] For example, when a fluorescent label is used as the label, the fluorescent signal from each probe is detected using a fluorescent scanner, and the signal intensity can be quantified by analyzing the detected signal using image analysis software.

[0056] When determining whether or not a target nucleic acid is present based on signals from the target nucleic acid detection probe and the non-target nucleic acid detection probe, specifically, the signal intensities of the target nucleic acid detection probe and the non-target nucleic acid detection probe are measured, respectively, and a judgment value for evaluating the signal intensity from the target nucleic acid detection probe is calculated. An example of calculating the judgment value is a method using the formula: [signal intensity from the target nucleic acid detection probe] / ([signal intensity from the target nucleic acid detection probe]+[signal intensity from the non-target nucleic acid detection probe]).

[0057] Another known formula for calculating the judgment value is [signal intensity from the target nucleic acid detection probe] / [signal intensity from the common probe]. However, when the target nucleic acid is a specific gene mutation and the non-target nucleic acid is a wild type corresponding to that gene mutation, it is preferable to use the above-mentioned formula: [signal intensity from the target nucleic acid detection probe] / ([signal intensity from the target nucleic acid detection probe]+[signal intensity from the non-target nucleic acid detection probe]), as this has higher sensitivity for detecting mutations.

[0058] The determination value calculated by the above formula is then compared with a predetermined threshold (cutoff value), and if the determination value exceeds the threshold, it is determined that the amplified nucleic acid contains the target nucleic acid, and if the determination value is below the threshold, it is determined that the amplified nucleic acid does not contain the target nucleic acid. By using the determination value in this way, it is possible to determine the presence or absence of the target base such as the above-mentioned SNP in the subject (presence or absence of a gene mutation, presence or absence of a minor allele related to a disease, etc.).

[0059] Here, the threshold is not particularly limited, but can be determined, for example, based on a judgment value calculated by the above formula using a sample confirmed to be free of the target bases of the above-mentioned gene mutations, i.e., a sample in which the target nucleic acid is not amplified. More specifically, a plurality of judgment values ​​can be calculated using a plurality of samples confirmed to be free of the target bases of the above-mentioned gene mutations, and the value of the average value + 3σ (σ: standard deviation) can be used as the threshold. Note that the value of the average value + 2σ or the average value + σ can also be used as the threshold.

[0060] As described above, in the method for detecting a target nucleic acid using a blocking nucleic acid and a common probe, even if the amount of genomic DNA used as a template when amplifying a target nucleic acid and a non-target nucleic acid is small, it is possible to accurately determine whether the amplification reaction has proceeded normally and to accurately detect the presence of the target nucleic acid. Here, the amount of genomic DNA used as a template is not particularly limited, but even if it is, for example, 100 to 800 fg, it is possible to accurately determine the success or failure of the nucleic acid amplification reaction.

[0061] In particular, the method of the present invention can be applied to obtaining data used for a definitive diagnosis of lymphoplasmacytic lymphoma / Waldenstrom's macroglobulinemia. For example, it can be applied to detecting a specific single nucleotide polymorphism in the MYD88 gene when a B-cell lymphoma patient with IgM M-proteinemia shows a worsening condition (such as a tendency for M-protein levels to increase), or to detecting a specific single nucleotide polymorphism in the CXCR4 gene to predict the effectiveness of ibrutinib as a therapeutic agent. Lymphoplasmacytic lymphoma / Waldenstrom's macroglobulinemia is described in detail in the reference (NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines), Waldenstrom Macroglobulinemia / Lymphoplasmacytic Lymphoma, Version 1.2021-September 1, 2020).

[0062] More specifically, genetic mutations in the MYD88 gene associated with lymphoplasmacytic lymphoma / Waldenström's macroglobulinemia include those encoding amino acid substitution mutations such as L265P, and genetic mutations in the CXCR4 gene associated with lymphoplasmacytic lymphoma / Waldenström's macroglobulinemia include those encoding amino acid substitution mutations such as S338X and R334X. [Example]

[0063] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0064] Example 1 In this example, when a B-cell lymphoma patient with IgM M-proteinemia showed a worsening condition (e.g., an increase in M-protein), we analyzed the MYD88 and CXCR4 genes, which are useful for definitively diagnosing and assisting in the treatment of WM / LPL patients. Table 1 shows the target mutations in MYD88 / CXCR4. Table 2 also shows the primer sets used to amplify the target regions.

[0065] [Table 1]

[0066] [Table 2]

[0067] The base sequences of primers MYD_F, IC5-MYD_R, CXCR_F and IC5-CXCR_R shown in Table 2 are SEQ ID NOs: 1 to 4, respectively.

[0068] Using DNA samples prepared as follows, the two target regions of MYD88 and CXCR4 were amplified by PCR.

[0069] [Table 3]

[0070] The temperature conditions for PCR were set as shown in Table 4.

[0071] [Table 4]

[0072] Hybridization reactions were then performed using a previously prepared MYD88 / CXCR4 analysis chip (WM chip). The prepared hybridization buffer (2.25x SSC, 0.23% SDS) and PCR product were removed from the freezer and returned to room temperature. The hybridization buffer (2.25x SSC, 0.23% SDS) was supplemented with the blocking nucleic acids listed in Table 5. The blocking nucleic acids were added to suppress nonspecific hybridization of the mutation detection probe, ensuring sufficient detection sensitivity even when the mutation rate of the target gene is low. They were designed to specifically hybridize with the wild-type amplification product.

[0073] [Table 5]

[0074] Here, ICHIhyb is an oligonucleotide that hybridizes with the positional probe (ICHI) shown in Table 8 below. Based on the hybridization between ICHIhyb and the positional probe (ICHI), the presence or absence of microarray degradation and the position of the probe on the microarray can be indicated. ICHIhyb consists of a sequence unrelated to the target mutation and is added to the hybridization buffer solution together with a blocking nucleic acid. The base sequences of the blocking nucleic acids MYD_B, CXCR_1012B, CXCR_1000B, CXCR_952B, and ICHIhyb shown in Table 5 are designated SEQ ID NOS: 5 to 9. Thirty microliters of PCR product was mixed with 15 μl of hybridization buffer and placed in an automated detection device (HySHOT HT-32 or BIOSHOT HT-32, manufactured by Toyo Kohan Co., Ltd.). The cleaning solution (0.1x SSC / 0.1% SDS solution), rinse solution, and detection solution (1x SSC) were prepared, and the mixed solution, cleaning solution, and rinse solution were loaded into the BIOHSHOT according to the instrument's instruction manual. Measurements were then performed using the fluorescence intensity estimation method. The instrument test conditions are shown in Table 6.

[0075] [Table 6]

[0076] [Experiment 1] In this experiment 1, the judgement value and resolution for the wild-type probe length were evaluated. Test conditions The mutant probes corresponding to the MYD88 mutations and the corresponding wild-type probes are shown in Table 7.

[0077] [Table 7]

[0078] The base sequences of probes MYD_W1, MYD_W2 and MYD_M01 shown in Table 7 are SEQ ID NOs: 10 to 12, respectively.

[0079] The judged value, which is the intensity ratio of the fluorescence intensity of each probe, was calculated using the following formula. Judgment value = mutant probe intensity / (wild-type probe intensity + mutant probe intensity)

[0080] ·result As shown in Figure 1, the W2 / M1 probe combination, which has a shorter MYD88 base length and a lower Tm, had a higher resolution (5% mutation threshold minus wild-type threshold) compared to the W1 / M1 combination. This is thought to be due to the shortening of the wild-type probe, which increased the specificity of the PCR product binding to the probe. This suggests that shortening the probe length is effective in improving detection performance.

[0081] [Experiment 2] In Experiment 2, we investigated the minimum amount of DNA required when using a common probe. The target specimens for the genetic testing kit (WM chip) are assumed to be bone marrow and formalin-fixed, paraffin-embedded (FFPE) specimens. FFPE specimens are generally known to be prone to DNA fragmentation due to the effects of chemicals used during the fixation process, reducing the amount of DNA that can be amplified. Therefore, when using the WM chip, it is extremely important to improve the amount of DNA that can be determined, i.e., to enable determination with a smaller amount of DNA.

[0082] On the other hand, when using the WM chip, a blocking nucleic acid with a sequence complementary to the wild-type target is used. The blocking nucleic acid hybridizes to the wild-type target, which hybridizes nonspecifically to the mutant probe, thereby preventing the wild-type target from hybridizing to the mutant probe. This increases the detection sensitivity for even smaller amounts of mutant target.

[0083] However, the use of blocking nucleic acids has the problem of reducing the amount of wild-type target that specifically reacts with the wild-type probe. When determining whether the amplification reaction for amplifying the wild-type target and mutant target has proceeded normally based on the fluorescence intensity of the wild-type probe, the blocking nucleic acid reduces the amount of wild-type target that can hybridize to the wild-type probe, resulting in a problem of insufficient fluorescence and making it impossible to determine (amplification failure).

[0084] In this example, we designed a common probe that can hybridize to both wild-type and mutant targets, even in the presence of a blocking nucleic acid. This common probe detects fluorescence intensity that depends on the amount of product (the total amount of wild-type and mutant targets), regardless of whether the target mutation is present or not. Therefore, by using the signal from the common probe as an amplification indicator, it is possible to determine whether samples that do not meet the fluorescence intensity criteria with the wild-type probe meet the minimum DNA detection standard, and we believe this will improve the minimum DNA detection amount.

[0085] Test conditions The sequences of the mutant probes corresponding to the mutant targets, the wild-type probes corresponding to the wild-type targets, and the common probes are shown in Table 8.

[0086] [Table 8]

[0087] The base sequences of probes MYD88_W, MYD88_M, CXCR4_952_W, CXCR4_952_M, CXCR4_1000_W, CXCR4_1000_M, CXCR4_1012_W, CXCR4_1012_M1, CXCR4_1012_M2, CXCR4_1012_M3, MYD_C, CXCR4_C and ICHI shown in Table 8 are set as SEQ ID NOs: 13 to 25, respectively.

[0088] 3200 fg of wild-type and mutant artificial genes containing the target mutation site were used as templates and amplified by PCR. The PCR amplification products were then diluted with TE to prepare samples with template amounts of 10 to 3200 fg. The test compared fluorescence intensity and resolution. As a criterion for evaluation, the fluorescence intensity required for stable analysis in this test system was set to 1000. Furthermore, a resolution of 0.25 or greater was set as an indicator of whether the wild-type target and mutant target (5%) could be sufficiently distinguished. The test conditions are shown in Table 9. As shown in Table 9, test condition 1 was a condition in which a blocking nucleic acid was present, and the wild-type, mutant, and common probes were evaluated.

[0089] [Table 9]

[0090] Test results The results of evaluating the fluorescence intensity from each probe in the presence of blocking nucleic acid are shown in Table 10, and the results of evaluating the fluorescence intensity from each probe in the absence of blocking nucleic acid are shown in Table 11. Conditions under which the fluorescence intensity was 1000 or higher were marked with an ◯, and all other conditions were marked with an ×. The results of measuring the fluorescence intensity from the common probe are shown in Figure 2. Figure 2(a) shows the results measured in the presence of blocking nucleic acid, and Figure 2(b) shows the results measured in the absence of blocking nucleic acid. The results of measuring the fluorescence intensity from the wild-type probe are shown in Figure 3. Figure 3(a) shows the results measured in the presence of blocking nucleic acid, and Figure 3(b) shows the results measured in the absence of blocking nucleic acid.

[0091] [Table 10]

[0092] [Table 11]

[0093] As shown in Table 10, in the presence of blocking nucleic acid, the common probe detected fluorescence intensity at 100 fg or more of template DNA, and the wild-type probe detected fluorescence intensity for all probes at 800 fg or more of template DNA. On the other hand, as shown in Table 11, in the absence of blocking nucleic acid, both the common probe and the wild-type probe detected fluorescence intensity at 200 fg or more of template DNA.

[0094] The results of evaluating the resolution in the presence of blocking nucleic acid are shown in Table 12, and the results of evaluating the resolution in the absence of blocking nucleic acid are shown in Table 13. Conditions under which the resolution was 0.25 or higher were marked with an ◯, and all other conditions were marked with an ×. The results of calculating the resolution for each gene mutation in the presence of blocking nucleic acid are shown in Figure 4, and the results of calculating the resolution for each gene mutation in the absence of blocking nucleic acid are shown in Figure 5.

[0095] [Table 12]

[0096] [Table 13]

[0097] As shown in Table 12, in the presence of blocking nucleic acid, the resolution was 0.25 or more when the template was 100 fg or more. On the other hand, as shown in Table 13, in the absence of blocking nucleic acid, the resolution was less than 0.25 under all conditions. These results demonstrate that blocking nucleic acid is essential for highly sensitive detection of mutations.

[0098] The above results were summarized for test conditions 1 to 4 in Table 9, and the test results evaluating each fluorescence intensity and resolution are shown in Table 14. In Table 14, conditions under which the fluorescence intensity was 1000 or more and the resolution was 0.25 or more were marked with a circle, and other conditions were marked with an ×.

[0099] [Table 14]

[0100] The results of this example confirmed that, in the presence of blocking nucleic acid, sufficient resolution was achieved for DNA template amounts of 100 fg or more. However, the fluorescence intensity detection limit for the wild-type probe under test condition 2 was 800 fg of DNA template. This suggests that conventional methods for determining the success or failure of an amplification reaction based on the signal from the wild-type probe were unable to determine the success or failure of a sample containing 100 to 800 fg of DNA due to insufficient fluorescence intensity. In contrast, by determining the success or failure of an amplification reaction based on the signal from the common probe, as under test condition 1, it was confirmed that amplification and mutation detection were possible even at low DNA concentrations.

[0101] Here, based on the molecular weight of the artificial gene used in the test, the unit of DNA template amount is converted from weight to copy number, 100 fg = 1.79 × 10 5 copies, 800fg = 1.43 × 10 6 Therefore, under test condition 1, the specimen is at least 1.79 × 10 copies. 5 It can be said that detection is possible if there are 1.43 × 10 copies. 6 The usefulness of the common probe can be demonstrated because the wild-type probe lacks fluorescence intensity when the number of copies is less than 1.

Claims

1. a microarray comprising: a target nucleic acid detection probe for the MYD88 gene, which has a base sequence complementary to a region containing a target nucleic acid containing a genetic mutation in the MYD88 gene associated with lymphoplasmacytic lymphoma / Waldenstrom's macroglobulinemia as a detection target base; a non-target nucleic acid detection probe for the MYD88 gene, which has a sequence complementary to a region containing a non-target base containing a wild-type corresponding to the genetic mutation in the MYD88 gene as a non-target base; and a common probe for the MYD88 gene, which has a sequence complementary to a common region common to the target nucleic acid and non-target nucleic acid of the MYD88 gene, which does not overlap with the region containing the non-target base; a buffer containing a blocking nucleic acid for the MYD88 gene, the blocking nucleic acid having a base sequence complementary to a region containing a non-target base in the non-target nucleic acid of the MYD88 gene; A microarray kit comprising: the probe for detecting a target nucleic acid for the MYD88 gene comprises the base sequence shown in SEQ ID NO: 14, the non-target nucleic acid detection probe for the MYD88 gene comprises the base sequence represented by SEQ ID NO: 11 and / or 13, the common probe for the MYD88 gene comprises the base sequence shown in SEQ ID NO: 23, The blocking nucleic acid for the MYD88 gene comprises the base sequence shown in SEQ ID NO:

5. The microarray kit.

2. The microarray further comprises a target nucleic acid detection probe for the CXCR4 gene, which has a base sequence complementary to a region containing a target nucleic acid containing a genetic mutation in the CXCR4 gene associated with lymphoplasmacytic lymphoma / Waldenström's macroglobulinemia as a target base to be detected, a non-target nucleic acid detection probe for the CXCR4 gene, which has a sequence complementary to a region containing a non-target base to be detected in a non-target nucleic acid containing a wild type corresponding to the genetic mutation in the CXCR4 gene as a non-target base to be detected, and a common probe for the CXCR4 gene, which has a sequence complementary to a common region common to the target nucleic acid and non-target nucleic acid of the CXCR4 gene, which does not overlap with the region containing the non-target base to be detected, the buffer further comprises a blocking nucleic acid for the CXCR4 gene, the blocking nucleic acid having a base sequence complementary to a region including a non-target base in the non-target nucleic acid of the CXCR4 gene; the probe for detecting a target nucleic acid for the CXCR4 gene comprises at least one base sequence selected from the group consisting of SEQ ID NOs: 16, 18, and 20 to 22; the non-target nucleic acid detection probe for the CXCR4 gene comprises at least one base sequence selected from the group consisting of SEQ ID NOs: 15, 17, and 19; the common probe for the CXCR4 gene comprises the base sequence shown in SEQ ID NO: 24, The blocking nucleic acid for the CXCR4 gene comprises at least one base sequence selected from the group consisting of SEQ ID NOs: 6 to 8. The microarray kit according to claim 1 .

3. A process of preparing a reaction solution after an amplification reaction between a target nucleic acid containing a target base that is a genetic mutation in the MYD88 gene and / or CXCR4 gene associated with lymphoplasmacytic lymphoma / Waldenström's macroglobulinemia and a non-target nucleic acid containing a wild-type non-target base that corresponds to the target base; and a hybridization buffer containing a blocking nucleic acid that contains a base sequence complementary to a region containing the non-target base in the non-target nucleic acid; contacting the reaction solution and the hybridization buffer with a microarray comprising: a target nucleic acid detection probe having a sequence complementary to a region containing a base to be detected in the target nucleic acid; a non-target nucleic acid detection probe having a sequence complementary to a region containing a base to be detected in the non-target nucleic acid; and a common probe having a sequence complementary to a common region common to the target nucleic acid and the non-target nucleic acid, the common region not overlapping with the region containing the base to be detected; determining that the amplification reaction of the target nucleic acid and the non-target nucleic acid has been performed normally when the signal intensity from the common probe in the microarray exceeds a threshold; determining the presence or absence of a target nucleic acid based on the signal intensity from the target nucleic acid detection probe and the signal intensity from the non-target nucleic acid detection probe; A method for detecting a target nucleic acid, comprising: the target nucleic acid detection probe is a probe for detecting a target nucleic acid for the MYD88 gene or a probe for detecting a target nucleic acid for the CXCR4 gene, the non-target nucleic acid detection probe is a non-target nucleic acid detection probe for the MYD88 gene or a non-target nucleic acid detection probe for the CXCR4 gene, the common probe is a common probe for the MYD88 gene or a common probe for the CXCR4 gene, the blocking nucleic acid is a blocking nucleic acid for the MYD88 gene or a blocking nucleic acid for the CXCR4 gene; the probe for detecting a target nucleic acid for the MYD88 gene comprises the base sequence shown in SEQ ID NO: 14, the non-target nucleic acid detection probe for the MYD88 gene comprises the base sequence represented by SEQ ID NO: 11 and / or 13, the common probe for the MYD88 gene comprises the base sequence shown in SEQ ID NO: 23, The blocking nucleic acid for the MYD88 gene comprises the base sequence shown in SEQ ID NO: 5, the probe for detecting a target nucleic acid for the CXCR4 gene comprises at least one base sequence selected from the group consisting of SEQ ID NOs: 16, 18, and 20 to 22; the non-target nucleic acid detection probe for the CXCR4 gene comprises at least one base sequence selected from the group consisting of SEQ ID NOs: 15, 17, and 19; the common probe for the CXCR4 gene comprises the base sequence shown in SEQ ID NO: 24, The blocking nucleic acid for the CXCR4 gene comprises at least one base sequence selected from the group consisting of SEQ ID NOs: 6 to 8. The method for detecting a target nucleic acid.

4. The method for detecting a target nucleic acid according to claim 3, characterized in that the value of [signal intensity from the probe for detecting a target nucleic acid] / ([signal intensity from the probe for detecting a target nucleic acid]+[signal intensity from the probe for detecting a non-target nucleic acid]) is calculated, and if this value exceeds a threshold value, it is determined that the target nucleic acid is present.

5. The amplification reaction of the target nucleic acid and the non-target nucleic acid is carried out such that the amount of template nucleic acid is 1.79 × 10 5 ~1.43×10 6 5. The method for detecting a target nucleic acid according to claim 3, wherein the target nucleic acid is a copy.

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