JP2022119753A - Method for detecting gene mutation with high accuracy - Google Patents

Abstract

To provide methods for distinguishing between a specific gene mutation occurring at a cancer site and a gene mutation caused by a spontaneous or artificial chemical modification.SOLUTION: A method for detecting a mutated gene present in a nucleic acid sample comprises the steps of: detecting a first mutated gene (target); detecting a second mutated gene (for artifact determination) located in a region different from that of the first mutated gene; and comparing the detection result of the second mutated gene and the detection result of the first mutated gene to determine the presence or absence of the first mutated gene. The first and second mutated genes are genes in which a point mutation from cytosine to thymine or from guanine to adenine has generated.SELECTED DRAWING: None

Description

Applied for application of Article 30, Paragraph 2 of the Patent Act January 5, 2022, The Japan Society for Analytical Chemistry, Analytical Chemistry, Vol. 71, No. 1 and 2

TECHNICAL FIELD The present invention relates to a method for detecting genetic mutations with high sensitivity and accuracy.

In recent years, genetic testing that detects mutations in genes involved in cancer and utilizes them for treatment strategies has become widespread. In such cancer genetic testing, the quality of test materials (specimens) greatly affects the test results. In current clinical examinations using in-vitro diagnostic agents, the percentage of tumor cell content sites is determined for each examination. For example, the cobas EGFR test requires a tumor cell content of 10% or higher, and the cobas BRAF test requires a tumor cell content of 50% or higher. This is done for the purpose of increasing the amount of cancer cells contained in test materials and detecting mutations with high sensitivity from test materials in which normal cells coexist. In addition, in patients for whom surgical materials or biopsies cannot be obtained, free nucleic acids in the blood are used as test materials to detect gene mutations. is desired. In this way, it is important to use appropriate test materials and to detect cancer-related gene mutations with high sensitivity. On the other hand, there is also the possibility of generating false positives in cases where the test materials are not necessarily appropriate, such as stored specimens, or because high-sensitivity detection is pursued. A known cause of false positives is spontaneous or artificial chemical modification of nucleic acid bases derived from test materials. Nucleic acid base cytosine undergoes a deamination reaction and changes to uracil, and when the base sequence is examined by an amplification reaction such as PCR using the DNA that has undergone this change, it is detected as a test material having a mutation from cytosine to thymine. . This artifact appears when using test materials that have been stored for long periods of time. As a method for removing this false positive, the test material (DNA) is pretreated with uracil DNA glycosylase to cleave the DNA containing uracil, in which cytosine has undergone a deamination reaction, so that it can be used as a template DNA in an amplification reaction such as PCR. A method is known to prevent this from occurring (Non-Patent Document 1).

In addition, Japanese Patent Laid-Open No. 2019-191952 discloses a method for determining whether a base sequence is mutation-positive or mutation-negative, taking into account the probability of false positives due to reading errors in next-generation sequencers.

JP 2019-191952 A

Do H, Dobrovic A. Dramatic reduction of sequence artefacts from DNA isolated from formalin-fixed cancer biopsies by treatment with uracil- DNA glycosylase. Oncotarget. 2012 May;3(5):546-58.

However, since methylated cytosine (5-methylcytosine) becomes thymine by deamination reaction, it cannot be removed by uracil-DNA glycosylase treatment, and there was no effective method for judging false positives. Furthermore, many of the cytosines of CpGs in vivo are methylated, and the efficacy of uracil-DNA glycosylase treatment was limited. That is, in the case of cytosines other than CpGs in the base sequence, such as CT and CC, since they are not methylated, treatment with uracil DNA glycosylase is effective, but cytosines that are CpGs in the base sequence are subject to mutation detection. Treatment with uracil-DNA glycosylase was ineffective because in some cases CpG cytosines were methylated, and there was no effective method to determine false positives or true positives.
An object of the present invention is to provide a method for distinguishing between specific genetic mutations occurring at cancer sites and genetic mutations caused by spontaneous or artificial chemical modification. In other words, when detecting with high sensitivity the mutation from cytosine to thymine in the CpG sequence or the mutation from guanine to adenine, which is the complementary chain, the mutation derived from artifacts and the true mutation occurring in vivo can be distinguished. It is an object of the present invention to provide a simple and effective method by which this can be achieved.

As a result of intensive studies to solve the above problems, the present inventors found that when detecting a transition mutation from cytosine to thymine in the nucleotide sequence CpG, or from guanine to adenine, which is the complementary chain thereof, a mutation to be detected By examining mutations from cytosine to thymine, or from guanine to adenine, which is the complementary chain, and comparing the two, it is possible to distinguish whether the targeted mutation is due to spontaneous or artificial chemical modification. The inventors have found that the detection target can be detected with high precision and high sensitivity, and have arrived at the present invention.

The above problems can be solved by the following inventions:
[1] A method for detecting a mutated gene present in a nucleic acid sample, comprising:
detecting the first mutated gene (target);
A step of detecting a second mutated gene (for artifact determination) that is different from the first mutated gene;
Comparing the detection result of the second mutated gene and the detection result of the first mutated gene to determine the presence or absence of the first mutated gene;
The method, wherein the first and second mutated genes are genes with point mutations from cytosine to thymine or guanine to adenine.
[2] A method of detecting a mutated gene present in a nucleic acid sample, in which a mutation occurring in vivo and a mutation due to an artifact are distinguished, comprising:
detecting the first mutated gene (target);
A step of detecting a second mutated gene (for artifact determination) that is different from the first mutated gene;
Comparing the detection result of the second mutated gene and the detection result of the first mutated gene to determine the presence or absence of the first mutated gene;
The method, wherein the first and second mutated genes are genes with point mutations from cytosine to thymine or guanine to adenine.
[3] The method of [1] or [2], wherein the first and second mutations are mutations of 5-methylcytosine to thymine, or mutations of guanine to adenine at complementary positions of 5-methylcytosine. .
[4] The method of any one of [1] to [3], wherein the detection method is a nucleic acid amplification method, and the first and second mutation points are within the same amplification product.
[5] The method of [4], wherein the detection method is a nucleic acid amplification method using a probe.
[6] The method of [5], wherein the nucleic acid amplification method using a probe is a digital PCR method or a PNA-LNA-PCR clamp method.
[7] The method according to any one of [1] to [3], wherein the detection method is a next-generation sequencing method.
[8] The method of any one of [1] to [7], wherein the first mutation is the T790M mutation of the EGFR gene or the R132H mutation of the IDH1 gene.
[9] A primer capable of amplifying a region containing a first mutation point and a primer capable of amplifying a region containing a second mutation point, or a primer capable of amplifying a region containing a first mutation point and a second mutation point primers capable of amplifying a region;
a mutation probe that hybridizes to the first mutation point having the sequence of the mutated gene;
a mutation probe that hybridizes to a second mutation point having the sequence of the mutated gene;
A detection kit for the method of any one of [1] to [8], comprising:
[10] a wild-type probe that hybridizes to the first mutation point having the sequence of the wild-type gene;
The detection kit of [9], further comprising a wild-type probe that hybridizes to the second mutation point having the sequence of the wild-type gene.
[11] The mutation probe that hybridizes to the first mutation point and the mutation probe that hybridizes to the second mutation point are labeled with a fluorescent substance at one end and quenched at the other end to suppress the fluorescent substance. The detection kit of [9] or [10], which is labeled with a substance.
[12] the wild-type probe that hybridizes to the first mutation point and the wild-type probe that hybridizes to the second mutation point are labeled with a fluorescent substance having a wavelength different from that of the corresponding mutant probe at one end; The detection kit of [11], wherein the other terminus is labeled with a quencher that suppresses the fluorescent substance.

The present invention also includes the following inventions:
When the detection target is mutation from cytosine to thymine (C>T) or guanine to adenine (G>A) (first mutation), mutation of cytosine or guanine (second mutation) different from the mutation point A method of detecting the mutation with high accuracy by examining the degree of spontaneous or artificial chemical modification (artifact) of the test material, examining the degree of false positive of the mutation to be tested, by also examining the presence or absence of the mutation.
A method for detecting a mutation point to be detected with high accuracy, characterized in that the cytosine or guanine (second mutation point) for which artifacts are examined is in the vicinity of the mutation point to be detected (first mutation point).
A high-precision mutation detection method characterized in that the cytosine or guanine (second mutation point) for which artifacts are to be examined is within the same amplification product as the mutation point (first mutation point) to be detected.
A highly accurate mutation detection method characterized by simultaneously detecting a mutation to be detected (first mutation) and a C>T or G>A mutation (second mutation) for examining artifacts.
A high-precision mutation detection method, wherein a highly sensitive detection method for examining a mutation (first mutation) and an artifact (second mutation) to be detected is a method using a probe or a next-generation sequencing method.

According to the present invention, genetic mutations can be distinguished from spontaneous or artificial chemical modifications (so-called artifacts) and detected with high accuracy. It is effective in detecting nucleobase cytosine to thymine and guanine to adenine mutations. In particular, it is an effective means for detecting a mutation from C (5-methylcytosine) to thymine in methylated 5'CpG on the nucleotide sequence while distinguishing it from artifacts.

1 is a graph showing the results of detection of human IDH1 gene R132H gene mutation by the PNA-LNA-PCR clamp method. 1 is a graph showing the results of detection of human EGFR gene T790M gene mutation by the PNA-LNA-PCR clamp method. 1 is a graph showing the results of detection of human EGFR gene F795F gene mutation by the PNA-LNA-PCR clamp method. 1 is a graph showing the results of detection of human IDH1 gene R119Q gene mutation by the PNA-LNA-PCR clamp method. 1 is a graph showing the results of detection of human IDH1 gene codon 132 antisense strand CpG mutation by the PNA-LNA-PCR clamp method.

In the present invention, terms such as DNA, RNA, nucleic acid, gene, gene expression, code, complement, template, promoter, probe, primer, hybridization, PCR, etc. are currently defined in terms of molecular biology, genetics, and genetic engineering. It has the same meaning as the term widely and commonly used in

As used herein, "cytosine" includes both unmethylated cytosine and methylated cytosine.

In the present invention, the "nucleic acid sample" is a sample containing nucleic acid, and is not particularly limited as long as it is a sample that may contain the first mutation point and the second mutation point. For example, DNA or RNA obtained from all cells in a "sample" such as blood or tissue, or cDNA prepared from RNA is applicable.

Although the origin of the "sample" that can be handled in the present invention is not particularly limited, it may be, for example, humans, animals such as mice, rats, and rabbits, biological tissues, Cultured cells established from cells may also be used.
In addition, the type of "sample" that can be handled in the present invention is not particularly limited, but biological samples such as blood, sputum, pleural effusion, bronchial washings such as brush washings, tissue specimens, or cell specimens can be mentioned. can be done. In particular, formalin-fixed paraffin-embedded tissue specimens or cell specimens (FFPE tissue specimens or cell specimens) are highly likely to have artifacts, and the present invention can be suitably used. A method for preparing genomic DNA or RNA from these samples may be a widely used preparation method for genomic DNA or RNA, such as an alkaline method. Alternatively, a commercially available DNA preparation kit or RNA preparation kit may be used. As a method for preparing cDNA from RNA, a widely used method using RT may be used, and a commercially available cDNA preparation kit may be used. Moreover, the method for preparing DNA from the FFPE sample may be performed by the method disclosed in WO2020/096031, for example.

In the present invention, a "wild-type gene" is a gene containing genetic information that is not mutated and has normal functions. The genetic information referred to here includes not only transcriptional regions encoding information such as mRNA, tRNA, rRNA and snRNA, but also regulatory regions necessary for gene expression such as promoters.

In the present invention, "mutation" means a change in a nucleic acid sequence such as DNA or RNA, and includes base substitution, insertion, deletion, inversion, duplication, translocation and the like used in genetics and the like. In the present invention, substitution of a single base (point mutation) is targeted for detection. In the present invention, the term "mutant gene" refers to a gene with a point mutation from cytosine to thymine or from guanine to adenine at its complementary position. In the mutated gene, the mutated region includes not only the transcriptional region but also regulatory regions necessary for gene expression, such as promoters. It should be noted that the mutation does not require the mutated gene to have a functional change. Mutations include both congenital and acquired mutations.

The "first mutation (target mutation)" in the present invention is a mutation to be detected, a transition mutation from cytosine to thymine, or from guanine to adenine at its complementary position, preferably CpG It is a transition mutation from cytosine to thymine in the sequence or from guanine to adenine at its complementary position. Mutations to be detected include, for example, gene mutations that are involved in the development, metastasis, and recurrence of cancer, and mutations that determine treatment strategies by examining the presence or absence of such mutations. Mutations that have a low mutation rate and are desired to be detected with high sensitivity are preferred, and examples thereof include tumor cell-specific somatic cell mutations and gene mutations associated with drug resistance. Such mutations include, for example, the R132H mutation in the IDH1 gene and the T790M mutation in the EGFR gene.

In the present invention, the first mutation may be in a strand having a sequence encoding genetic information (hereinafter referred to as a sense strand) or a strand having a sequence complementary to the sense strand (hereinafter referred to as an antisense strand). do). That is, the target mutation in the sense strand may be a base substitution from cytosine to thymine or a base substitution from guanine to adenine. Furthermore, the target mutation in the antisense strand may be a cytosine to thymine base substitution or a guanine to adenine base substitution.

In the present invention, the "first mutated gene" is the gene that has undergone the first mutation, and the "first mutation point" is the site where the base at which the substitution is observed in the first mutation exists, This is the site targeted for detection in the present invention.

In addition, when detecting a mutation of unmethylated cytosine, which is not a CpG sequence, to thymine via uracil, or a gene mutated from guanine to adenine at its complementary position, RNA containing uracil is used as a test material. The present invention can also be used as an alternative to the uracil-DNA glycosylase treatment method when detecting mutant genes.

In the present invention, the "second mutation (artifact)" is a transition mutation from cytosine to thymine at a site different from the first mutation point, or from guanine to adenine at its complementary position. The conversion of 5-methylcytosine to thymine is known to occur more readily than the conversion of cytosine to thymine via uracil, preferably from cytosine to thymine of CpG sequences that undergo in vivo methylation, or It is a guanine to adenine transition mutation at the complementary position.

The second mutation is a base substitution from cytosine to uracil or thymine that is caused as an artifact by spontaneous or artificial chemical modification, and furthermore, an artifact from cytosine to uracil or thymine is generated during PCR or cDNA synthesis. It is a base substitution to adenine formed in a nascent chain synthesized by elongation reaction with a polymerase or reverse transcriptase using the resulting mutant allele as a template. Artifacts caused by spontaneous chemical modification include, for example, when FFPE is stored at room temperature for a long period of time, hydrolytic deamination of cytosine bases in nucleic acids in tissues and cells proceeds, and bases to uracil and thymine are formed. Substitutions accumulate over time, and artificial chemical modification artifacts include those caused by excessive heat treatment when extracting nucleic acids from FFPE, or nucleic acids extracted from fresh tissue, blood, etc. Excessive heat treatment artificially promotes base substitution from cytosine to uracil or thymine.

That is, the present invention provides nucleic acid samples prepared from FFPE specimens stored for a long period of time, in which artifacts may pose a problem in the detection of disease-specific gene mutations occurring in cytosines of CpG sequences or guanines at their complementary positions, and preparation of FFPEs. It can be used for highly sensitive detection of gene mutations in nucleic acid samples for which nucleic acid preparation from FFPE was not appropriate, and nucleic acid samples for which storage after preparation was not appropriate.

In the present invention, the "second mutated gene" is a gene that has undergone the second mutation, and the "second mutation point" is the site where the base at which the substitution is observed in the second mutation exists, It is a site detected to examine artifacts in the present invention.

If the first mutation is a 5-methylcytosine to thymine base substitution or a guanine to adenine base substitution at its complementary site, then the second mutation is also any 5 at a site different from the first mutation point. It is preferably a base substitution from -methylcytosine to thymine, or a base substitution from guanine to adenine at its complementary site. The strand for detecting the first and second mutated genes can be selected from either the sense strand or the antisense strand, but preferably the second mutation point is on the same gene region as the first mutation point, Particularly preferably, it is selected from the vicinity of the first mutation point. By selecting the second mutation from the vicinity of the first mutation point, the region containing the first mutation point and the second mutation point can be simultaneously amplified with the same primer set.
Alternatively, the antisense strand CpG sequence of the first mutation may be selected as the second mutation. If the first mutation is a base substitution from cytosine to thymine in the CpG sequence or a base substitution from guanine to adenine at its complementary site, the second mutation is a base substitution from guanine to adenine in the CpG sequence of the first mutation. It is preferably a base substitution or a base substitution from cytosine to thymine at its complementary site.

As the "method for detecting a mutant gene" in the present invention, a highly sensitive detection method capable of detecting even when the content of the mutant gene is 1% or less is preferable. According to the present invention, mutations associated with 5-methylcytosine can be distinguished from gene mutations caused by artifacts even in a detection method with a detection sensitivity of less than 0.5%. Nucleic acids such as digital PCR method, PNA-LNA-PCR clamp method, Invader method, TaqMan-PCR method, Scorpion Arms method, etc. Amplification method, next-generation sequencing method, DNA chip method, PCR-HRM method, gFCS method and the like can be mentioned. Preferred are the nucleic acid amplification method and the next-generation sequencing method using probes, and particularly preferred are the digital PCR method, the PNA-LNA-PCR clamp method, and the next-generation sequencing method. In the present invention, "nucleic acid amplification method" refers to an amplification reaction of a template nucleic acid using a known polymerase reaction, and a method of amplifying a region containing a first mutation point and/or a second mutation point using primers. is.

The "nucleic acid amplification method using a probe" in the present invention is to selectively detect the region containing the first mutation point and the second mutation point of the mutated gene from the amplified product of the nucleic acid amplification reaction using the probe. The method. Digital PCR method, PNA-LNA-PCR clamp method, Invader method, TaqMan-PCR method, Scorpion Arms method and the like can be mentioned. The principle of the digital PCR method may be a droplet method (Bio-Rad, etc.) or a well-chip method (Thermo Fisher Scientific, etc.).

The step of detecting the first mutated gene (target) when using the next-generation sequencing method, and the step of detecting the second mutated gene (for artifact determination) are performed by detecting the first mutation point and the second mutation point. Preparing a library from the nucleic acid sample that may contain it, performing sequencing of the first mutation and the second mutation, and outputting the results. As long as the base sequences of the first mutation point and the second mutation point can be analyzed, commercially available reagents and devices may be used regardless of the analysis principle of the next-generation sequencing method.

An embodiment in which a nucleic acid amplification method using a probe is used for the step of detecting a mutated gene in the present invention will be described.

In this embodiment, the step of detecting the first mutated gene (target) is
a nucleic acid sample that may contain the first mutation point and the second mutation point;
a primer capable of amplifying a region containing the first mutation point;
a mutation probe that hybridizes to the first mutation point having the sequence of the mutated gene;
coexisting in the reaction solution, amplifying the region containing the first mutation point and selectively detecting the region containing the first mutation point of the mutated gene to detect the first mutated gene; include.

Preferably, a nucleic acid sample that may contain the first mutation point and the second mutation point;
a primer capable of amplifying a region containing the first mutation point;
a mutation probe that hybridizes to the first mutation point having the sequence of the mutated gene;
A wild-type probe that hybridizes to the first mutation point having the sequence of the wild-type gene may be coexisted in the reaction solution.

More preferably, the region containing the first mutation point of the wild-type gene is selectively detected together with the region containing the first mutation point of the mutant gene.

The “primers” used in this embodiment are primers capable of amplifying a region containing a mutation point, preferably two types of primers flanking the mutation point. For example, two types of primers may be used, consisting of a forward primer having a nucleotide sequence homologous to the upstream region of the mutation point and a reverse primer having a nucleotide sequence complementary to the downstream region of the mutation point. Alternatively, it may be an allele-specific primer containing a mutation point in the primer sequence. These primers can be designed and synthesized by conventional methods from sequence information of nucleotide sequences containing mutation sites, and are composed of nucleic acids such as DNA and RNA, or LNA, or a combination thereof. The number of bases of the primer can be appropriately determined in consideration of the Tm value of the primer, etc., and may be in the range of 10 to 40 bases, preferably 15 to 30 bases, and particularly preferably 18 bases. 25 bases from

A "mutation probe" used in this embodiment is a probe that specifically hybridizes to a region containing a mutation point having a sequence of a mutated gene. The bases in the sequence may be labeled with a fluorophore or a quencher, preferably one end labeled with a fluorophore and the other end labeled with a quencher that suppresses the fluorophore. The nucleotide sequence of the mutation probe is not particularly limited as long as it specifically hybridizes with part or all of the region containing the mutation point of the mutant gene, and it may hybridize to either the sense or antisense strand. can be designed. It can be designed and synthesized by a conventional method from the sequence information of the nucleotide sequence containing the mutation point, and may be composed of nucleic acids such as DNA and RNA, and part or all of the bases may be LNA, PNA, BNA, etc. It may be substituted with such an artificial nucleic acid. The length of the mutation probe is not particularly limited as long as it allows specific hybridization to the mutated allele or its complementary strand. to 20 bases is particularly preferred. In this embodiment, the term “fluorescent substance” refers to a substance that has the property of being excited by absorbing excitation light of a specific wavelength and emitting fluorescence when returning to the original ground state. The fluorescent substance used for labeling is not particularly limited as long as it can label the end of the mutation probe. For example, FAM, TET, HEX, Cy3, Cy5, Texas Red, TAMRA, FITC, or the like may be used.

In the present embodiment, the term "quenching substance" refers to a substance that absorbs the excitation energy of a fluorescent substance, or the like. The type used may be any of BHQ1, BHQ2, Dabcyl, and the like. However, since the fluorescence suppression range differs depending on the type of the suppressing substance, it is necessary to select the type of quencher capable of suppressing the emission of the paired fluorescent substance on one mutant probe. There is no particular limitation as long as the selection range is such that light emission can be suppressed. For example, when the fluorescent substance is FAM or TET, the wavelength of the suppression range is slightly shorter (480 nm to 580 nm) BHQ1, and when the fluorescent substance is Cy3 or Texas red, the wavelength of the suppression range is slightly longer. A closer (550 nm to 650 nm) BHQ2 may be selected.

In this embodiment, the term “end” refers to the 5′ end or 3′ end of the base sequence of the mutation probe. In the present invention, "labeled" means that the fluorescent substance and/or quenching substance is attached to the end of the mutation probe by chemical bonding.

A "wild-type probe" in this embodiment is a probe that specifically hybridizes to a region containing a mutation point having a sequence of a wild-type gene. The bases in the sequence may be labeled with a fluorescent substance or a quencher, preferably one end is labeled with a different fluorescent substance that can be distinguished from the mutant probe, and the other end is quenched to suppress the fluorescent substance. labeled with a substance. As a result, the mutated gene containing the first mutation point can be detected, and the wild-type gene containing the first mutation point can also be detected separately. The wild-type probe is not particularly limited as long as it specifically hybridizes with part or all of the region containing the mutation point of the wild-type gene, and is designed to hybridize with either the sense strand or the antisense strand. You may Preferably, the wild-type and mutant probes are designed to hybridize to the same strand. Wild-type probes can also be designed and synthesized by conventional methods from sequence information of nucleotide sequences containing mutation sites, and may be composed of nucleic acids such as DNA and RNA, or may be composed of LNA or all of the bases. It may be substituted with an artificial nucleic acid such as PNA, BNA, or the like. The length of the wild-type probe is not particularly limited as long as it allows specific hybridization to the mutated allele or its complementary strand. Base to 20 bases is particularly preferred.

Mutant probes and wild-type probes may be labeled with fluorescent substances that can be distinguished from each other, and fluorescent substances with different excitation/fluorescence spectra may be used in combination. Examples include combinations of FAM and HEX, FAM and Cy5, and the like. Alternatively, one may be labeled and the other may be used unlabeled. As an example, the mutant probe has one end labeled with a fluorescent substance (e.g., FAM) and the other end labeled with a quencher that suppresses the fluorescent substance, and the wild-type probe has one end labeled with the mutant probe It is labeled with a fluorescent substance with a different wavelength (eg, HEX or Cy5), and the other end is labeled with a quenching substance that inhibits the fluorescent substance. As a result, for example, in the digital PCR method, it is possible to calculate the number of droplets such as "mutant type + wild type-" and the mutation rate. In another example, the mutant probe is labeled at one end with a fluorophore and at the other end with a quencher that suppresses the fluorophore, and the wild-type probe is used unlabeled. In addition, when detecting a mutated gene by the PNA-LNA-PCR clamp method, the mutated probe has at least one or more of the DNA sequences replaced with LNA, and one end is labeled with a fluorescent substance (eg, FAM). and the other end is labeled with a quencher that inhibits the fluorophore. A wild-type probe uses a probe whose entire sequence is composed of PNA. If necessary, a total probe that hybridizes to both the mutant gene and the wild-type gene is used together. The total probe may be labeled at one end with a different fluorophore (eg HEX or Cy5) distinguishable from the mutant probes, and labeled at the other end with a quencher that inhibits said fluorophore. This makes it possible to know the total amount of the mutant gene and the wild-type gene in the amplification product, and to determine the abundance ratio of the mutant gene even if the wild-type probe is not labeled with a fluorescent substance.

In the present embodiment, the "reaction solution" is a solution containing reagents and the like necessary for nucleic acid amplification and gene detection reaction in the nucleic acid amplification method. The composition of the reaction solution varies depending on the nucleic acid amplification method to be used, and one suitable for the nucleic acid amplification method may be selected.

In this embodiment, the nucleic acid amplification reaction and gene detection are not particularly limited as long as the regions containing the first mutation point and the second mutation point can be amplified and detected simultaneously or individually. Gene detection may be performed while the amplification reaction is being performed, or may be performed after the amplification. Reaction conditions and equipment suitable for the nucleic acid amplification method may be selected.

In this embodiment, the step of detecting the second mutated gene (for artifact determination) is
a nucleic acid sample that may contain a first mutation point and a second mutation point and primers capable of amplifying a region containing the second mutation point;
a mutation probe that hybridizes to a second mutation point having the sequence of the mutated gene;
coexisting in the reaction solution, amplifying the region containing the second mutation point and selectively detecting the region containing the second mutation point of the mutated gene to detect the second mutated gene; include.

Preferably, a nucleic acid sample that may contain the first mutation point and the second mutation point and primers capable of amplifying a region containing the second mutation point,
a mutation probe that hybridizes to a second mutation point having the sequence of the mutated gene;
A wild-type probe that hybridizes to the second mutation point having the sequence of the wild-type gene may be coexisted in the reaction solution.

More preferably, the region containing the second mutation point of the wild-type gene is selectively detected together with the region containing the second mutation point of the mutant gene.

Here, the primer capable of amplifying the region containing the second mutation point may be the same as or different from the primer capable of amplifying the region containing the first mutation point. Either forward primer or reverse primer may be used in common, or both may be used in common. Preferably, they can be amplified with the same primer set.

Further, the nucleic acid amplification reaction for the region containing the second mutation point may be carried out simultaneously with the nucleic acid amplification reaction for the region containing the first mutation point, or may be carried out separately. Moreover, the detection reaction of the second mutant gene may be performed simultaneously with the detection reaction of the first mutant gene, or may be performed separately.

When using a nucleic acid amplifier capable of detecting multiple types of fluorescent probes, it is possible to simultaneously amplify and detect the first and second mutant genes. When the first mutation point and the second mutation point are close to each other and PCR can be performed with the same primer set, the probes that detect the first and second mutant genes can be distinguished from each other. They are preferably labeled with different fluorescent substances, such as a combination of FAM and HEX. When the first mutation point and the second mutation point are in the same allele or their complementary strands that are very close to each other, PCR is performed in an independent reaction system to avoid mutual overlap or hybridization between the mutation probes. It is preferable to In that case, it is not necessary to distinguish between the fluorophores of the mutant probes.

Since the other requirements are the same as in the step of detecting the first mutant gene, the description thereof is omitted here.

The "comparison of detection results" of the first mutated gene and the second mutated gene in the present invention refers to the mutation ratio of both, or the number of mutant + wild type- droplets in digital PCR, the Ct value in real-time PCR, the fluorescence It is to compare the intensity, the number of cycles at which the signal rises, and the like. When detection is performed by a quantitative measurement method such as a digital PCR method, a numerical value obtained by adding an SD value or a CV value may be set as a threshold value, if necessary. If the value of the first mutant gene is greater than the threshold, the first mutant gene is determined to be positive.

For example, in the case of the droplet digital PCR method, a value twice the number of droplets of "mutant + wild type-" obtained with the second mutation is set as the threshold, and the same value obtained with the first mutation is If it is equal to or greater than the threshold, it is determined that there is a mutation. A sample in which only the first mutation is detected is considered to be an in vivo mutation rather than an artifact, and is determined to be a mutation detection. Samples in which both the first and second mutations are detected are considered mutations induced by spontaneous or artificial chemical modification. Samples that have undergone mutations that occur in vivo may undergo chemical modification during the storage period, so the relative If the number of droplets of the first mutation “mutant + wild type-” is less than twice that of the second mutation, chemical modification occurred during the storage period. , and it is determined that it is not detected. If the number of first mutations is at least two times greater than that of the second mutations, the sample with mutations is considered to have undergone chemical modification during storage and is determined to be detected. In addition, if necessary, the CV value in this measurement method is calculated in advance, and the value added to the number of droplets of the second mutation "mutant type + wild type-" can be set as a threshold value. good.

For example, in the case of the PNA-LNA-PCR clamp method, etc., the first mutated gene and the second mutated gene, for example, 0.1% positive control are simultaneously measured in advance, and the positive control is used as an indicator to determine positive or negative. Then, the mutation rate can be examined by the difference in Ct value between the nucleic acid sample and the corresponding positive control (positive control Ct value - sample Ct value). The difference (positive control Ct value - sample Ct value) of the first mutated gene and the second mutated gene with respect to each positive control is compared, and the Ct value difference of the first mutated gene and the Ct of the second mutated gene If the value difference is 1 or more, it can be determined to be detected, and if it is less than 1, it can be determined not to be detected. In the case of the next-generation sequencing method, after eliminating reading errors, the mutation ratio of the first mutation and the second mutation is compared, and the first mutation ratio is sufficiently large relative to the second mutation ratio, for example If the first mutation is greater than 2-fold, it can be determined to be detected, and if it is less than 2-fold, it can be determined that it is not detected. In the case of the next-generation sequencing method, it is preferable to use this method after using a molecular barcode to eliminate reading errors.

The "detection kit" of the present invention is configured to detect the first mutated gene and the second mutated gene.
The detection kit of the present invention is a reaction component for detecting the first mutation and the second mutation, a primer for amplifying the region containing the first or second mutation, an allele-specific primer containing the mutation point , containing probes that detect mutation points. Probes can also include artificial nucleic acids suitable for detection of mutation sites.

A detection kit of the present invention comprises at least primers for amplifying a region containing a first mutation and a second mutation point, and a mutation probe that hybridizes to the first mutation point having the sequence of the mutant gene; a mutation probe that hybridizes to a second mutation point having the sequence of the mutated gene. Preferably, it further comprises a wild-type probe that hybridizes to the first mutation point having the sequence of the wild-type gene and a wild-type probe that hybridizes to the second mutation point having the sequence of the wild-type gene. A mutant probe that hybridizes to the first mutation point and a mutation probe that hybridizes to the second mutation point are labeled with a fluorescent substance at one end and labeled with a quenching substance that suppresses the fluorescent substance at the other end. ing. Each fluorescent substance may be labeled with the same fluorescent substance, or may be labeled with fluorescent substances having different excitation/emission spectra.

The wild-type probe that hybridizes to the first mutation point and the wild-type probe that hybridizes to the second mutation point may be labeled with a fluorescent substance or left unlabeled. In the case of labeling with a fluorescent substance, one end is labeled with a fluorescent substance having an excitation/fluorescence spectrum different from that of the mutant probe that hybridizes to the same mutation point, and the other end is labeled with a quenching substance that suppresses the fluorescent substance. be.

The detection kit of the present invention can also contain a substrate, a reaction solution, a polymerase for nucleic acid amplification, etc., depending on the gene mutation detection method to be used. A person skilled in the art can appropriately select and design according to the configuration of a known kit.

EXAMPLES The present invention will be specifically described below with reference to Examples, but these are not intended to limit the scope of the present invention.

<<Example 1: Confirmation of artifacts in DNA derived from healthy human blood cells>>
(1-1: Preparation of DNA)
DNA was extracted from the peripheral blood of healthy individuals who gave informed consent using a Blood & Cell Culture DNA mini Kit (manufactured by Qiagen). 50 μL of the DNA solution (15 ng/μL) was overlaid with light mineral oil, and heat-treated at 70° C. for 24 hours, 70° C. for 96 hours, and 70° C. for 7 days. After the heating time was over, it was stored in a refrigerator (4°C).

(1-2: Droplet digital PCR (ddPCR))
EGFR gene exon20 T790M (c.2369C>T) mutation and EGFR gene exon20 F795F (c. 2385C>T) Reaction solutions for mutation detection were prepared. Reactions were performed in duplicate.

Table 2 shows the human EGFR gene exon 20 nucleotide sequence (SEQ ID NO: 1). Here, C of ACG shown in bold and underlined is the T790M mutation point, and CG shown in bold and underlined is the CpG sequence constituting the F795F mutation point.
The sequences of primers and probes are shown in Tables 3 and 4. Here, lower case letters in the sequence indicate DNA, upper case letters indicate LNA (Locked Nucleic Acid), HEX and FAM indicate fluorescent dyes, and BHQ1 indicates a fluorescence suppressing substance, respectively. Bold and underlined g or A are mutation points.

The 96-well plate into which each reaction solution was dispensed was set in the Bio-Rad Automated Droplet Generator (AutoDG ^™ ) to prepare droplets, and the C1000 Touch thermal cycler (Bio-Rad) was used under the temperature conditions shown in Table 5. Nucleic acid amplification was performed using After that, analysis was performed by QuantaSoft ^™ Software using QX200 ^™ Droplet Reader (Bio-Rad).

(1-3: Droplet digital PCR (ddPCR) analysis results)
When performing a similar experiment using only the wild type as a template, 1 piece may be detected in Ch1 + Ch2- (mutant type + wild type-), so 1 piece is detected in Ch1 + Ch2- (mutant type + wild type-). In the case of , it was determined as "not detected", and when two or more of Ch1+Ch2- (mutant type + wild type-) were detected, it was determined as "detected".

Neither the T790M mutation nor the F795F mutation was detected from the untreated healthy human DNA sample, but both mutations were detected from the sample heated at 70°C, and the rate of mutation increased with heating time. It turned out that the proportions were also approximately the same. Both T790M and F795F 5'CpG of blood cell-derived DNA are methylated, and the storage state of the sample causes artifact-induced mutation of 5-methylcytosine to thymine, and 5-methylcytosine to thymine. was confirmed to occur equally at both mutation points.
As a result, by examining the mutation point to be detected and the rate of mutation from cytosine to thymine at a site different from the detection target, or from guanine to adenine, which is the complementary chain, it is possible to determine whether the mutation point to be detected is spontaneous or artificial chemical. It was found that it is possible to distinguish between mutations due to modifications (artifacts) and in vivo mutations that occur in cancer cells.

<<Example 2: Confirmation of artifacts in PCR products>>
A pGEM T Easy Vector containing the gene fragment of SEQ ID NO: 1, which is the EGFR gene exon 20 wild type, was constructed and transformed into E. coli JM109. This E. coli was cultured, and plasmid DNA was extracted using Plasmid Mini Kit (manufactured by Qiagen). A reaction solution was prepared according to the composition shown in Table 7 using a plasmid containing the extracted EGFR gene exon20 wild-type gene sequence as a template. As primers, primers for the SP6 region and the T7 region located at both ends of the inserted gene fragment were used. PCR was performed under the temperature conditions shown in Table 8.

The resulting PCR product was purified using MiniElute PCR Purification Kit (manufactured by Qiagen), the absorbance OD260 was measured, and the copy number was calculated from the value and the molecular weight. Light mineral oil was added to 50 μL of the EGFR gene exon20 wild-type PCR product solution of about 3000 copies/μL, and the mixture was heated at 70° C. for 24 hours and 70° C. for 96 hours. After the heating was completed, it was stored at refrigerated 4°C. Using 5 μL of the DNA before heating and the DNA after heating as a template, ddPCR was performed in the same manner and under the same conditions as in Example 1-2. The ddPCR analysis conditions were the same as in Example 1-3.

In the EGFR gene exon 20 wild-type PCR product, T790M and F795F mutations appeared with the passage of heating time, similarly to the result of heating the genomic DNA, and the mutation ratios of both were substantially the same.
Similar results were obtained not only with genomic DNA but also with artificial PCR products, so not only mutations at 5-methylcytosine of methylated CpG but also mutations from cytosine to uracil due to artifacts It was found that the detection method of the present invention can be an effective means for this mutation as well.

<<Example 3: Confirmation of artifacts in DNA derived from cultured lung cancer cells>>
DNA was extracted from lung cancer-derived cultured cell line H1975 using a Blood & Cell Culture DNA mini Kit (manufactured by Qiagen). A 15 ng/μL DNA solution was prepared, overlaid with light mineral oil, and heated at 75° C. for 3 days and 75° C. for 6 days. After the heating time was over, it was stored in a refrigerator (4°C).
A gene amplification reaction was carried out under the same conditions as in Example 1-2 using the heat-treated DNA and the DNA before heat treatment (stored in a refrigerator at 4° C.) as templates. Reactions were performed in duplicate. The ddPCR analysis conditions were the same as in Example 1-3.

Lung cancer-derived cultured cell line H1975 has EGFR gene L858R mutation and T790M mutation. At the untreated time point, the T790M mutation had approximately 78%, while the F795F mutation had none. The F795F mutation appeared as the heating time progressed, and the rate of the F795F mutation increased to 2.4% after the heat treatment at 75°C for 6 days.
The usefulness of the method of the present invention lies in comparing the mutation point to be detected and the rate of mutation due to spontaneous or artificial chemical modification. Mutation detection by the nucleic acid amplification method may be carried out with high sensitivity from samples that are not suitable test materials, for example, samples with a small amount of starting template nucleic acid. In this case, it is necessary to detect the mutation from a small amount of the mutant allele by an amplification reaction such as the PCR method. In this case, it is a more useful means to examine mutations from cytosine to thymine or from guanine to adenine at sites different from the detection target mutation point occurring at the cancer site and compare the mutation rates.

<<Example 4: PNA-LNA-PCR clamp method>>
The isocitrate dehydrogenase 1 gene (IDH1 gene) R132H gene mutation was detected by the PNA-LNA-PCR clamp method, which is a highly sensitive detection method for mutation sites. Using the unheated DNA derived from healthy subjects prepared in Example 1 and the DNA heat-treated at 70° C. for 7 days as templates, a reaction solution was prepared according to the composition shown in Table 11, and nucleic acid amplification was carried out under the temperature conditions shown in Table 12. gone. As positive controls, DNA containing 1.0% R132 mutation and DNA containing 0.1% R132 mutation were measured simultaneously.

Table 13 shows the human IDH1 gene base sequence (SEQ ID NO: 8). Here, G in CGT shown in bold and underlined is the R132H mutation point.
The primer and probe sequences are shown in Table 14. An LNA probe is a probe containing LNA (Locked Nucleic acid) in part of its sequence, and a PNA probe is a probe composed of PNA (Peptide Nucleic acid). Here, lower case letters indicate DNA, upper case letters indicate LNA or PNA, FAM and Cy5 indicate fluorescent dyes, and BHQ1 and BHQ2 indicate fluorescent inhibitors.

The results are shown in FIG. The vertical axis of the graph indicates the fluorescence intensity, and the horizontal axis indicates the cycle number of PCR. "PC 1.0%" is 1.0% R132H mutation-containing positive control, "PC 0.1%" is 0.1% R132H mutation-containing positive control, "circled number 1" is healthy Human DNA was heat-treated at 70° C. for 7 days, and “circled number 2” is non-heat-treated healthy human DNA (n=2).
Here, the 1.0% R132H mutation-containing positive control is DNA containing 100 copies of the R132H mutation PCR product for 10,000 copies of healthy human genomic DNA (1.0% R132H mutation-containing, 2 × 1000 copies Genomic DNA / μL), and 0.1% R132H mutation-containing positive control is DNA containing 10 copies of R132H mutation PCR product for 10,000 copies of healthy human genomic DNA (0.1% R132H mutation-containing, 2×1000 copies of genomic DNA/μL). Healthy human DNA is 15 ng/μL genomic DNA.

P. C. 1.0%, P.I. C. For 0.1% and DNA heat-treated at 70° C. for 7 days, the Cp value was calculated, exponential amplification was observed, and the R132H mutation was detected. On the other hand, unheated DNA from healthy subjects was measured in duplicate, but no Cp value was calculated, no exponential amplification was observed, and no R132H mutation was detected. The H132H mutation detected in heat-treated DNA is the result of hydrolytic deamination of 5'-CGT guanine to adenine (complementary strand 5'-ACG) and is an artificially induced mutation. . This artifact-induced mutation showed a higher fluorescence intensity than the control containing 0.1% of the R132H mutation, confirming that false positives due to artifacts pose a problem in highly sensitive mutation detection methods.
From this heat-treated DNA, the F795F mutation was confirmed as shown in Example 1, and the mutation caused by artificial or spontaneous chemical modification is induced in cytosine on the genome without specificity. Investigating and comparing a mutation to be detected (R132H) with a second mutation that is different from the detection target is an effective means for highly sensitive and accurate determination of the target mutation point.

<<Example 5: Confirmation of artifacts in clinical sample DNA>>
Artifacts were confirmed using actual clinical specimens (FFPE tissue specimens). A QIAamp DNA FFPE Tissue kit (manufactured by Qiagen) was used to extract DNA from the specimen. ddPCR was performed in the same manner and under the same conditions as in Example 1-2, and the reaction was performed in a single assay. The ddPCR analysis conditions were the same as in Example 1-3. Determination of T790M mutation positive using the mutation rate of T790M and F795F, if the ratio of the mutation rate of T790M and F795F is 2 times or more, it is assumed that there is an amount of mutation that exceeds the artifact due to deamination, positive determination and We have set the criteria for
For comparison, cobas EGFR mutation test kit v2.0 (manufactured by Roche Diagnostics), Oncomain Dx Target Multi CDx System (manufactured by Life Technologies Japan), PNA-LNA-PCR clamp method Measured by either method. did. The PNA-LNA-PCR clamp method was measured by the method described in Canadian Respiratory Journal 2016; Volume 2016 Article ID 5297329.

The T790M mutation was detected in all 11 cases, but by confirming the F795F mutation, 3 cases (specimens 7, 10, and 11) could be determined to be artifacts. The 6 specimens that differed from the results of the clinical test were all the results of the Cobas EGFR Mutation Test Kit v2.0, and the minimum detection sensitivity of T790M is said to be 2 to 3%. can. In addition, the Oncomine Dx Target multi-CDx system is a diagnostic agent using a next-generation sequencer. Although there is no clear description of the minimum detectable sensitivity for the T790M mutation, the published minimum detectable sensitivity for EGFR L858R is 5.3%. Therefore, the determination of specimen 7 was also considered to be negative because it was below the sensitivity, and in this method, the determination was consistent because it was negative due to the artifact determination. Since the minimum detection sensitivity of T790M in the PNA-LNA-PCR clamp method is set at 0.5%, it is considered that there was no discrepancy in the determination of specimens 8 to 11. In other methods, the cut-off value of the mutation rate for T790M mutation positive determination may be set slightly higher in consideration of artifacts, and the detection method of the present invention can be an effective means when detecting a trace amount of T790M mutation. . In particular, it has been found to be an effective technique when detecting mutations involving less than 0.5% 5-methylcytosine.

<<Example 6: PNA-LNA-PCR clamp method EGFR gene mutation detection>>
EGFR T790M gene mutation and F795F gene mutation were detected by the PNA-LNA-PCR clamp method, which is a highly sensitive detection method for mutation sites. In the same manner as in Example 4, reaction solutions for detecting the T790M mutation and the F795F mutation were prepared according to the compositions shown in Tables 16 and 17, using unheated DNA from a healthy subject and DNA heat-treated at 70°C for 7 days as templates. , Tables 18 and 19, nucleic acid amplification was performed.

As positive controls, DNA containing 1.0% T790M or F795 mutation and DNA containing 0.1% T790M or F795 mutation were measured simultaneously.
The sequences of primers and probes are shown in Tables 20 and 21. An LNA probe is a probe containing LNA (Locked Nucleic acid) in part of its sequence, and a PNA probe is a probe composed of PNA (Peptide Nucleic acid). Here, lower case letters indicate DNA, upper case letters indicate LNA or PNA, FAM and Cy5 indicate fluorescent dyes, and BHQ1 and BHQ2 indicate fluorescent inhibitors.

FIG. 2 shows the measurement results of the T790M mutation, and FIG. 3 shows the measurement results of the F795F mutation. The notations in the figure are the same as those in the fourth embodiment. P. C. 1.0%, P.I. C. Cp values were calculated for 0.1% and DNA heat-treated at 70° C. for 7 days, exponential amplification was observed, and both the T790M and F795F mutations were detected. On the other hand, unheated DNA from healthy subjects was measured in duplicate, but no Cp value was calculated, no exponential amplification was observed, and neither mutation was detected. In the T790M mutation, the difference between the Ct value (38.85) of the 0.1% positive control and the Ct value (36.05) of DNA heat-treated at 70 ° C. for 7 days is 2.8, and in the F795F mutation, The difference between the Ct value (35.95) of the 0.1% positive control and the Ct value (33.52) of DNA heat-treated at 70°C for 7 days was 2.43. Since the difference (0.37) between the Ct value difference of the T790M mutation and the Ct value difference of the F795F mutation was less than 1, the detected T790M mutation was determined to be false positive. From this result, it was shown that the PNA-LNA-PCR clamp method can also determine artifacts by confirming the F795F mutation together with the T790M mutation.

<<Example 7: PNA-LNA-PCR clamp method IDH1 R119Q gene mutation detection (artifact detection)>>
From the results of Example 4, it was confirmed that false positives due to artifacts pose a problem in the detection of IDH1 R132H (c.395G>A) gene mutations by the PNA-LNA-PCR clamp method, which is a highly sensitive detection method for mutation sites. rice field. Therefore, the IDH1 R119Q (c.356G>A) gene mutation was detected as a second mutation different from the R132H mutation to be detected. In the same manner as in Example 4, using unheated DNA from a healthy individual and DNA heat-treated at 70°C for 7 days as templates, a reaction solution was prepared according to the composition shown in Table 22, and nucleic acid amplification was carried out under the temperature conditions shown in Table 12. gone. As positive controls, DNA containing 1.0% R119 mutation and DNA containing 0.1% R119 mutation were measured simultaneously.

Table 23 shows the human IDH1 gene base sequence (SEQ ID NO: 8). Here, the CG shown in bold and underlined is the CpG sequence that constitutes the R119Q mutation point.
The primer and probe sequences are shown in Table 24. An LNA probe is a probe containing LNA (Locked Nucleic acid) in part of its sequence, and a PNA probe is a probe composed of PNA (Peptide Nucleic acid). Here, lower case letters indicate DNA, upper case letters indicate LNA or PNA, FAM and Cy5 indicate fluorescent dyes, and BHQ1 and BHQ2 indicate fluorescent inhibitors.

The results are shown in FIG. The notations in the figure are the same as those in the fourth embodiment. P. C. 1.0%, P.I. C. For 0.1% and DNA heat-treated at 70° C. for 7 days, the Cp value was calculated, exponential amplification was observed, and the R119Q mutation was detected. On the other hand, unheated DNA from healthy subjects was measured in duplicate, but no Cp value was calculated, no exponential amplification was observed, and no R119Q mutation was detected. The R119Q mutation detected in heat-treated DNA is the result of hydrolytic deamination of CpG guanine of 5'-CGG to adenine, and is an artificially induced mutation. The difference between the Ct value (36.6) of the 0.1% positive control in Example 4 (first mutation) and the Ct value (35.12) of DNA heat-treated at 70°C for 7 days was 1.48. In this example (second mutation), the difference between the Ct value (36.94) of the 0.1% positive control and the Ct value (34.94) of DNA heat-treated at 70°C for 7 days was 2 Met. Since the difference (0.52) of each Ct value difference was less than 1, it was shown that the R132H mutation detected in Example 4 can be determined as false positive.

<<Example 8: PNA-LNA-PCR clamp method Confirmation of IDH1 gene codon 132 antisense strand CpG artifact>>
CpG artifact detection on the codon 132 antisense strand of the IDH1 gene was performed by the PNA-LNA-PCR clamp method. When the guanine to adenine mutation of the R132H mutation 5'-CGT was regarded as the first mutation, the guanine to adenine mutation of the complementary strand 5'-ACG was considered to be the second mutation and used for artifact determination.
In the same manner as in Example 4, using unheated DNA from a healthy individual and DNA heat-treated at 70°C for 7 days as templates, a reaction solution was prepared according to the composition shown in Table 22, and nucleic acid amplification was carried out under the temperature conditions shown in Table 12. gone. As a positive control, DNA containing 1.0% R132C (c.394C>T: guanine of antisense strand 5'-ACG to adenine) mutation and DNA containing 0.1% R132C mutation were measured simultaneously. .

Table 25 shows the human IDH1 gene base sequence (SEQ ID NO: 8). Here, the CG shown in bold and underlined is a CpG sequence in which the CG of the complementary strand can be an artifact of the R132H gene mutation.
The primer and probe sequences are shown in Table 26. An LNA probe is a probe containing LNA (Locked Nucleic acid) in part of its sequence, and a PNA probe is a probe composed of PNA (Peptide Nucleic acid). Here, lower case letters indicate DNA, upper case letters indicate LNA or PNA, FAM and Cy5 indicate fluorescent dyes, and BHQ1 and BHQ2 indicate fluorescent inhibitors.

The results are shown in FIG. The notations in the figure are the same as those in the fourth embodiment. P. C. 1.0%, P.I. C. For 0.1% DNA and DNA heat-treated for 7 days at 70° C., the Cp value was calculated, exponential amplification was observed, and the R132C mutation was detected. On the other hand, unheated DNA from healthy subjects was measured in duplicate, but no Cp value was calculated, no exponential amplification was observed, and no R132C mutation was detected.
This indicated that an artifact due to hydrolytic deamination of methylated cytosine was also detected in the complementary strand CpG of the first mutation. The difference between the Ct value (36.6) of the 0.1% positive control in Example 4 (first mutation) and the Ct value (35.12) of DNA heat-treated at 70°C for 7 days was 1.48. In this example (second mutation), the difference between the Ct value (37.31) of the 0.1% positive control and the Ct value (35.31) of DNA heat-treated at 70°C for 7 days was 2. Met. Since the difference (0.52) of each Ct value difference was less than 1, it was shown that the R132H mutation detected in Example 4 can be determined as false positive.

<<Example 9: Confirmation of droplet digital PCR EGFR gene codon 790 antisense strand CpG artifact>>
When the mutation from cytosine to thymine in the T790M mutation 5'-ACG was regarded as the first mutation, the mutation from cytosine to thymine in the complementary strand 5'-CGT was regarded as the second mutation and used for artifact determination.
Droplet digital PCR was performed according to the composition shown in Table 1 using as templates unheated DNA derived from healthy subjects and DNA heat-treated at 70°C for 24 hours, 70°C for 96 hours, and 70°C for 7 days in the same manner as in Example 1. was measured by

The sequences of primers and probes are shown in Tables 3 and 27. Here, lower case letters in the sequence indicate DNA, upper case letters indicate LNA (Locked Nucleic Acid), HEX and FAM indicate fluorescent dyes, and BHQ1 indicates a fluorescence suppressing substance, respectively.

The 96-well plate into which each reaction solution was dispensed was set in the Bio-Rad Automated Droplet Generator (AutoDG ^™ ) to prepare droplets, and the C1000 Touch thermal cycler (Bio-Rad) was used under the temperature conditions shown in Table 5. Nucleic acid amplification was performed using After that, analysis was performed by QuantaSoft ^™ Software using QX200 ^™ Droplet Reader (Bio-Rad).
When performing a similar experiment using only the wild type as a template, 1 piece may be detected in Ch1 + Ch2- (mutant type + wild type-), so 1 piece is detected in Ch1 + Ch2- (mutant type + wild type-). In the case of , it was determined as "not detected", and when two or more of Ch1+Ch2- (mutant type + wild type-) were detected, it was determined as "detected".

Neither the T790M mutation nor the CpG artifact in the codon 790 antisense strand was detected from the untreated healthy human DNA sample, and the rate of mutation increased with increasing heating time. It was found that the mutation rate increased with heating time and both mutation rates were almost the same.
From this, it was shown that, similarly to the results of Example 1, by examining the artifact of methylated cytosine in the antisense strand as the second mutation, it is possible to determine the false positive of the first mutation.

<<Example 10: Confirmation of artifacts by next-generation sequencing>>
The sequence data of the clinical sample (DNA derived from FFPE tissue sample: case 7) measured with the Oncomine Dx Target multi-CDx system (manufactured by Life Technologies Japan) in Example 5 was analyzed with the Integrative Genomics Viewer after reading errors were eliminated. (manufactured by Broad Institute). By analyzing using Integrative Genomics Viewer, it is possible to count the number of reads with mutations in the total number of reads in the amplification target region read by the next-generation sequencer, and to calculate the approximate mutation rate. . For example, when the first mutation to be detected is the EGFR gene T790M mutation and the second mutation is the F795F mutation, the ratio of each mutation was calculated to be 0.22% for the T790M mutation and 0.44% for the F795F mutation. Since the first mutation rate was lower than the second mutation rate, the T790M mutation could be determined as a false positive. It was also confirmed that by using a next-generation sequencer, any other CpG sequence can be used as a second mutation for artifact determination as long as it is within the region to be amplified.

INDUSTRIAL APPLICABILITY The present invention can be used in the field of genetic testing.

Each base sequence represented by SEQ ID NOs: 13, 16, 19 and 21 in the sequence listing is a PNA probe.

Claims (12)

A method of detecting a mutated gene present in a nucleic acid sample, comprising:
detecting the first mutated gene;
detecting a second mutated gene that is different from the first mutated gene;
Comparing the detection result of the second mutated gene and the detection result of the first mutated gene to determine the presence or absence of the first mutated gene;
The method, wherein the first and second mutated genes are genes with point mutations from cytosine to thymine or guanine to adenine. A method for detecting a mutated gene present in a nucleic acid sample, in which a mutation occurring in vivo and a mutation due to an artifact are distinguished, comprising:
detecting the first mutated gene;
detecting a second mutated gene that is different from the first mutated gene;
Comparing the detection result of the second mutated gene and the detection result of the first mutated gene to determine the presence or absence of the first mutated gene;
The method, wherein the first and second mutated genes are genes with point mutations from cytosine to thymine or guanine to adenine. 3. The method of claim 1 or 2, wherein the first and second mutations are mutations of 5-methylcytosine to thymine or guanine to adenine at the complementary position of 5-methylcytosine. The method according to any one of claims 1 to 3, wherein the detection method is a nucleic acid amplification method and the first and second mutation points are within the same amplification product. 5. The method according to claim 4, wherein the detection method is a nucleic acid amplification method using probes. The method according to claim 5, wherein the nucleic acid amplification method using probes is digital PCR method or PNA-LNA-PCR clamp method. The method according to any one of claims 1 to 3, wherein the detection method is a next-generation sequencing method. The method of any one of claims 1-7, wherein the first mutation is the T790M mutation of the EGFR gene or the R132H mutation of the IDH1 gene. Primer capable of amplifying the region containing the first mutation point and the primer capable of amplifying the region containing the second mutation point, or amplifying the region containing the first mutation point and the second mutation point a primer capable of
a mutation probe that hybridizes to the first mutation point having the sequence of the mutated gene;
a mutation probe that hybridizes to a second mutation point having the sequence of the mutated gene;
A detection kit for the method according to any one of claims 1 to 8, comprising a wild-type probe that hybridizes to the first mutation point having the sequence of the wild-type gene;
10. The detection kit of claim 9, further comprising a wild-type probe that hybridizes to the second mutation point having the sequence of the wild-type gene. The mutant probe that hybridizes to the first mutation point and the mutation probe that hybridizes to the second mutation point are labeled with a fluorescent substance at one end and labeled with a quenching substance that suppresses the fluorescent substance at the other end. The detection kit according to claim 9 or 10, wherein The wild-type probe that hybridizes to the first mutation point and the wild-type probe that hybridizes to the second mutation point are labeled with a fluorescent substance having a wavelength different from that of the corresponding mutant probe at one end, and the other end is is labeled with a quencher that inhibits said fluorescent substance.

Images