JP2013070639A - Probe for predicting therapeutic effect on chronic hepatitis c - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a probe for detecting a single-nucleotide polymorphism (SNP) that affects the treatment rate of chronic hepatitis C, a method for detecting the SNP using the probe, and a kit for detecting the SNP.SOLUTION: A probe having a specific base sequence is used.

Description

The present invention relates to a probe for detecting a single nucleotide polymorphism (SNP) involved in the therapeutic rate of chronic hepatitis C, and a method for detecting SNP and a SNP using the probe. About the kit.

Hepatitis C virus (HCV) is said to cause 70-80% of infected people to transition to chronic hepatitis. In addition, many cases of transition from chronic hepatitis to cirrhosis and lung cancer have been reported.

A typical treatment for hepatitis C is pegylated interferon + ribavirin (PEG-IFN / RBV) combination therapy. However, it has been known that there are individual differences in the effectiveness of this treatment.

It has been reported that SNP around the IL28B gene is related as a reason why individual differences occur in the therapeutic effect of PEG-IFN / RBV (Non-patent Document 1, Non-patent Document 2, Patent Document 1).

Patent Document 1 describes an example of SNP inspection. In Patent Document 1, a method called sequence method or DigiTag2 method is used. The sequencing method is generally performed by nucleic acid amplification of a human gene as a specimen and identifying the sequence of the amplified product. However, there is a concern that this method may cause misdiagnosis due to carry-over contamination of nucleic acid amplification products. In addition, a period of several hours to one day is required from the start of the inspection to the determination of the result.

The DigiTag 2 method is a method of detection using two probes. In the method using a probe, contamination can be prevented by performing nucleic acid amplification and detection in the same closed system. However, the DigiTag2 method and the method using fluorescence resonance energy transfer (FRET) have the disadvantage that two or more probes are required for detection, which is costly.

JP2011-41526

Ge D. et al. Nature 461, 399-401, 2009 Tanaka Y. et al. et al. Nat Genet 41, 1105-1109, 2009

The present invention is intended to solve the problems in the conventional SNP test related to the treatment of hepatitis C, and an object thereof is to provide a method and a reagent for performing the SNP test quickly, accurately and inexpensively. is there.

  As a result of intensive studies in view of the above problems, the present inventors have made use of a single nucleic acid probe that specifically binds to a SNP that is said to be involved in the therapeutic rate of hepatitis C and its surrounding base sequences, thereby making it more conventional than the prior art. Has found that the SNP can be detected accurately, and has completed the present invention. That is, the present invention has the following configuration.

[Claim 1]
A probe for detecting a SNP that is said to be involved in the therapeutic rate of hepatitis C, the probe having a base sequence represented by any one of SEQ ID NOs: 1 to 3.
[Section 2]
Item 2. The probe according to Item 1, which is labeled with a detectable labeling substance.
[Section 3]
Item 3. The probe according to Item 2, wherein the detectable labeling substance is a fluorescent dye.
[Claim 4]
Item 4. The probe according to Item 2 or 3, wherein the terminal cytosine is labeled.
[Section 5]
A method for detecting an SNP that is said to be involved in the therapeutic rate of hepatitis C, comprising all of the following steps (1) to (3).
(1) A step of hybridizing the test nucleic acid with the probe according to any one of Items 1 to 4 to form a complex (2) After step (1), while continuously increasing the temperature, 260 nm A step of performing a melting curve analysis by measuring the absorbance of the probe or measuring the signal intensity of the label added to the probe.
(3) A step of determining the presence or absence of a mutation in the test nucleic acid from the fluctuation of the signal intensity accompanying the temperature change in the step (2) [Section 6]
Item 6. The method according to Item 5, comprising all of the following steps (1) to (3).
(1) The step of hybridizing the test nucleic acid and the probe according to any one of Items 1 to 4 to form a complex (2) After step (1), the temperature is increased continuously, A step of performing a melting curve analysis by measuring the fluorescence intensity of a fluorescent dye that labels the probe.
(3) A step of determining the presence or absence of a mutation in the test nucleic acid from the fluctuation of the fluorescence intensity accompanying the temperature change in the step (2) [Section 7]
Item 7. The method according to Item 5 or 6, wherein the test nucleic acid in step (1) is amplified from a template nucleic acid using a primer pair.
[Section 8]
Item 8. The method according to Item 7, wherein a DNA polymerase having a DNA synthesis rate of 100 bases / second or more and having no 5 ′ exonuclease activity is used for nucleic acid amplification.
[Claim 9]
Item 9. The method according to Item 7 or 8, wherein the primer pair is a pair of primers including a forward primer composed of an oligonucleotide (F) below and a reverse primer composed of an oligonucleotide (R) below.
(F) an oligonucleotide whose base sequence is represented by any one of SEQ ID NOs: 4 to 6 (R) an oligonucleotide whose base sequence is represented by any one of SEQ ID NOs: 7 to 9 [Item 10]
A primer pair for amplifying a SNP that is said to be involved in the treatment rate of hepatitis C by a nucleic acid amplification method, and comprising a primer pair configured by any of (1) to (3) below.
(1) Primer pair consisting of primers having the base sequences shown in SEQ ID NO: 4 and SEQ ID NO: 7 (2) Primer pair consisting of primers having the base sequences shown in SEQ ID NO: 5 and SEQ ID NO: 8 (3 ) Primer pair consisting of primers having the base sequences shown in SEQ ID NO: 6 and SEQ ID NO: 9 [Item 11]
Item 6. A mutation detection kit for use in a method for detecting an SNP that is said to be involved in the therapeutic rate of hepatitis C according to Item 5, comprising the probe according to any one of Items 1 to 4.

According to the present invention, it is possible to quickly, accurately and easily perform an SNP test that is said to be involved in the treatment rate of hepatitis C.

It is a figure which shows the result of Example 1. It is a figure which shows the result of Example 2. It is a figure which shows the result of Example 3. It is a figure which shows the result of Example 4.

One embodiment of the present invention is a probe for detecting a SNP that is said to be involved in the treatment rate of hepatitis C, and has a base sequence represented by any one of SEQ ID NOs: 1 to 3. .

The “SNP that is said to be involved in the treatment rate of hepatitis C” of the present invention is a SNP that can predict the therapeutic effect of hepatitis C treatment, particularly interferon therapy, especially PEG-IFN / RBV combination therapy, Specifically, there are three SNPs indicated by rs8103142 and rs80999917rs129798860.

These three SNPs are all present in or around the IL28B gene. rs8103142 and the surrounding nucleotide sequence are represented by SEQ ID NO: 10. rs8103142 corresponds to the 389th base of SEQ ID NO: 10, and is represented by y (T / C) in SEQ ID NO: 10. rs8099917 and its surrounding nucleotide sequence are represented by SEQ ID NO: 11. rs8099917 corresponds to the 301st base of SEQ ID NO: 11, and is represented by k (T / G) in SEQ ID NO: 11. rs129798860 and its surrounding nucleotide sequence are represented by SEQ ID NO: 12. rs129798860 corresponds to the 301st base of SEQ ID NO: 12, and is represented by y (C / T) in SEQ ID NO: 12. The numbers representing the SNPs (rs numbers) and the sequences described in the sequence listing refer to human gene sequence information published in the NCBI database (Bulid 35).

The probe of the present invention is designed to specifically bind to each test nucleic acid one by one, and is suitable for melting curve analysis.
The method disclosed in Patent Document 1 requires two or more probes for detection, and is a method for detecting a test nucleic acid on a principle completely different from the present invention. It cannot be diverted directly to the invention, nor can it be used as a reference.

The probe of the present invention represented by SEQ ID NO: 1 is composed of a sequence complementary to a partial region containing rs8101032, and when rs810103142 is thymine which is a minor allyl, it becomes a complementary strand completely to the antisense strand. SNP in the antisense strand can be confirmed.

The probe of the present invention represented by SEQ ID NO: 2 is composed of a sequence complementary to a partial region containing rs8099917, and when rs8099917 is a minor allyl guanine, it becomes a complementary strand that is completely complementary to the antisense strand. SNP in the antisense strand can be confirmed.

The probe of the present invention represented by SEQ ID NO: 3 is composed of a sequence complementary to a partial region containing rs129798860, and when rs129798860 is a major allele of guanine, it becomes a complementary strand that is completely complementary to the antisense strand. SNP in the antisense strand can be confirmed.

  The probe of the present invention is preferably a nucleic acid sequence represented by any one of SEQ ID NOS: 1 to 3, but is complementary to a nucleic acid sequence of 10 to 30 bases including any one of rs810103142, rs80999917, and rs129798860 SNPs. The nucleic acid sequence is not particularly limited.

  The probe of the present invention may be a labeled probe labeled with a labeling substance. Preferably, a nucleic acid probe labeled with a fluorescent dye, more preferably a nucleic acid probe labeled with a fluorescent dye at the end can be used.

  The probe of the present invention is preferably a probe labeled with a labeling substance. The labeling substance is not limited, and examples thereof include fluorescent dyes (fluorophores). As a specific example of the labeled probe, for example, a probe that is labeled with the fluorescent dye, exhibits fluorescence alone, and fluorescence decreases (for example, quenches) by hybridization is preferable. Such a phenomenon is generally called a fluorescence quenching phenomenon. As a probe using this fluorescence quenching phenomenon, a probe generally called a guanine quenching probe is preferable. Such a probe is known as a so-called QProbe (registered trademark). A guanine quenching probe is, for example, a fluorescent dye designed so that the base at the 3 ′ end or 5 ′ end of an oligonucleotide becomes cytosine, and light emission becomes weaker when the base cytosine at the end approaches a complementary base guanine. It is a probe labeled at the end. In the probe of the present invention, for example, a fluorescent dye exhibiting a fluorescence quenching phenomenon may be bound to cytosine at the 3 ′ end of the oligonucleotide, or the 5 ′ end of the oligonucleotide is designed to be cytosine. It may be combined.

  Examples of the fluorescent dye include, but are not limited to, fluorescein, phosphor, rhodamine, polymethine dye derivatives, and the like. Examples of commercially available fluorescent dyes include BODIPY FL (trade name, manufactured by Molecular Probes), FluorePrime (trade name). Product name, Amersham Pharmacia), Fluoredite (trade name, manufactured by Millipore), FAM (ABI), Cy3 and Cy5 (Amersham Pharmacia), TAMRA (Molecular Probes), and the like. The probe detection conditions are not particularly limited and can be appropriately determined depending on the fluorescent dye to be used. For example, Pacific Blue has a detection wavelength of 450 to 480 nm, TAMRA has a detection wavelength of 585 to 700 nm, and BODIPY FL has a detection wavelength of 515 to 515. Detectable at 555 nm. If such a probe is used, hybridization and dissociation can be easily confirmed by signal fluctuation.

  In the probe of the present invention, for example, a phosphate group may be added to the 3 'end. As will be described later, a test nucleic acid (target nucleic acid) for detecting the presence or absence of a mutation can be prepared by a nucleic acid amplification method such as PCR. In this case, the probe of the present invention is allowed to coexist in the reaction system of the nucleic acid amplification reaction. be able to. In such a case, if a phosphate group is added to the 3 'end of the probe, the probe itself can be sufficiently prevented from extending due to the nucleic acid amplification reaction. The same effect can be obtained by adding a labeling substance as described above to the 3 'end.

  As described above, the probe of the present invention can be used for detection of SNPs related to the treatment of hepatitis C. The method for detecting this mutation is not limited at all, and any method using hybridization between a test nucleic acid and the probe may be used.

One of the embodiments of the present invention is a method for detecting SNPs that are said to be involved in the treatment rate of hepatitis C, and includes the following steps (1) to (3). is there.
A method for detecting an SNP that is said to be involved in the therapeutic rate of hepatitis C, comprising all of the following steps (1) to (3).
(1) A step of hybridizing the test nucleic acid with the probe according to any one of Items 1 to 4 to form a complex (2) After step (1), while continuously increasing the temperature, 260 nm A step of performing a melting curve analysis by measuring the absorbance of the probe or measuring the signal intensity of the label added to the probe.
(3) A step of determining the presence or absence of a mutation in the test nucleic acid from a change in signal intensity accompanying a temperature change in step (2).

  According to the SNP detection method of the present invention, the SNPs of rs810103142, rs80999917, and rs129798860 can be detected by using any of the probes of SEQ ID NOS: 1 to 3, respectively.

  The test nucleic acid in the present invention may be, for example, a single strand or a double strand. In the case of a double strand, for example, it is preferable to include a step of dissociating the double strand into a single strand by heating in order to hybridize a test nucleic acid and a probe to form a hybrid.

  The type of the test nucleic acid is not particularly limited, and examples thereof include DNA, RNA such as total RNA and mRNA, and the like. Examples of the test nucleic acid include nucleic acids contained in a sample such as a biological sample. The nucleic acid in the sample may be, for example, a nucleic acid originally contained in the biological sample. For example, since the accuracy of mutation detection can be improved, the nucleic acid in the biological sample is amplified by a nucleic acid amplification method as a template. Amplification products. Specific examples include, for example, a reverse transcription reaction (RT) from an amplification product amplified by a nucleic acid amplification method using DNA originally contained in the biological sample as a template, or RNA originally contained in the biological sample. An amplification product amplified by the nucleic acid amplification method using the cDNA generated by -PCR) as a template. These amplification products may be used as test nucleic acids in the present invention. The length of the amplification product is not particularly limited, and is, for example, 50 to 1000 bases, preferably 80 to 300 bases.

  Although it does not restrict | limit especially as said biological sample, For example, a blood cell sample, such as a white blood cell, a whole blood sample, a mouth wiping liquid, a pharyngeal wiping liquid, etc. are mention | raise | lifted. In the present invention, the method for collecting a sample and the method for preparing a nucleic acid such as DNA or RNA are not limited, and conventionally known methods can be employed.

  The melting curve analysis used in the method of the present invention will be described. For example, when a solution containing double-stranded DNA is heated, the absorbance at 260 nm increases. This is because hydrogen bonds between both strands in double-stranded DNA are unwound by heating and dissociated into single-stranded DNA (DNA melting). When all the double-stranded DNAs are dissociated into single-stranded DNA, the absorbance is about 1.5 times the absorbance at the start of heating (absorbance of only double-stranded DNA), thereby melting. Can be determined to be completed.

In the present invention, the measurement of the signal fluctuation accompanying the temperature change for performing the melting curve analysis can be performed by measuring the absorbance at 260 nm based on the principle as described above, but the signal of the label added to the probe of the present invention is measured. It is preferable to measure. For this reason, it is preferable to use the aforementioned labeled probe as the probe of the present invention.
Examples of the labeled probe include a labeled probe that shows a signal alone and does not show a signal by hybridization, or a labeled probe that does not show a signal alone and shows a signal by hybridization. In the former probe, no signal is shown when a hybrid (double strand) is formed with the detection target sequence, and a signal is shown when the probe is released by heating. In the case of the latter probe, a signal is shown by forming a hybrid (double strand) with the sequence to be detected, and the signal decreases (disappears) when the probe is released by heating. Therefore, by detecting the signal from the label under signal-specific conditions (absorbance and the like), the progress of melting can be grasped in the same manner as the absorbance measurement at 260 nm. The labeling substance in the labeling probe is, for example, as described above.
A preferred labeling substance is a fluorescent dye. When a fluorescent dye is used as the labeling substance, a melting curve analysis is performed by measuring the signal intensity of the fluorescent dye added to the probe in the above method. From this change in the signal intensity accompanying the temperature change, The presence or absence of a mutation in the test nucleic acid can be determined.

  As described above, when the test nucleic acid is an amplification product amplified from the template nucleic acid by the nucleic acid amplification method, the step (1) amplifies the nucleic acid from the template nucleic acid using the primer pair of the forward primer and the reverse primer. The process of carrying out may be included. In other words, the test nucleic acid in the step (1) may be a product amplified from a template nucleic acid using a primer pair of a forward primer and a reverse primer.

  The primer pair is not particularly limited as long as the region to which the probe of the present invention in the IL28B gene and / or the region surrounding the IL28B gene can hybridize is specifically amplified. The amplification region may be, for example, only a region where the probe hybridizes, or a region including the hybridization region.

Specifically, for example, a pair of primers including a forward primer composed of an oligonucleotide (F) below and a reverse primer composed of an oligonucleotide (R) below can be exemplified.
(F) an oligonucleotide whose base sequence is represented by any of SEQ ID NOs: 4 to 6 (R) an oligonucleotide whose base sequence is represented by any of SEQ ID NOs: 7 to 9

The specific nucleic acid amplification method used in the nucleic acid amplification step is not particularly limited, and a known method can be used as appropriate. For example, a PCR (Polymerase Chain Reaction) method, a NASBA (Nucleic acid sequence based amplification) method, a TMA (Transscription-mediated amplification) method, a SDA (Strand Displacement Amplification method), and a SDA (Strand Displacement Amplification method) are preferred. In each of these methods, the conditions for the amplification reaction are not particularly limited, and can be performed by a conventionally known method.

  When the PCR method is used for nucleic acid amplification, α-type DNA polymerase is preferably used as the DNA polymerase. The reason will be described below.

When the IL28B gene and / or IL28B gene peripheral region is amplified in a reaction system including the probe of the present invention, the nucleic acid probe binds to the IL28B gene and / or IL28B gene peripheral region of the sample or their amplification product during the nucleic acid amplification step. Yes. The nucleic acid probe bound to the IL28B gene and / or the region surrounding the IL28B gene during the nucleic acid amplification step inhibits the nucleic acid amplification reaction by the nucleic acid primer and the DNA polymerase.

PolI type DNA polymerases such as Taq DNA Polymerase are known to have 5 'to 3' exonuclease activity. Due to this activity, when there is a nucleic acid bound to the IL28B gene and / or the IL28B gene peripheral region as a template during the nucleic acid amplification reaction, the bound nucleic acid is degraded by the exonuclease activity. For this reason, the nucleic acid probe in the reaction system may be reduced, causing a problem in the nucleic acid detection process. Therefore, it is not preferable to carry out the present invention using PolI type DNA polymerase.

On the other hand, α-type DNA polymerases such as KOD DNA Polymerase (derived from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1) do not have 5 ′ → 3 ′ exonuclease activity but have 3 ′ → 5 ′ exonuclease activity. Therefore, the use of α-type DNA polymerase not only solves the above problem, but also exhibits high accuracy in the nucleic acid amplification step due to the 3 ′ → 5 ′ exonuclease activity.

Usually, α-type DNA polymerase has a 3 ′ → 5 ′ exonuclease activity, and therefore the nucleic acid amplification rate tends to be lower than that of PolI-type enzyme. However, although KOD DNA Polymerase is an α-type DNA polymerase, it has high DNA synthesis activity, has a DNA synthesis rate of 100 bases / second or more, and has excellent elongation efficiency. Therefore, in the practice of the present invention, it is preferable to use KOD DNA Polymerase (trademark, manufactured by Toyobo Co., Ltd.) among α-type DNA polymerases.

In addition, a mutant type in which α-type DNA polymerase is mutated to achieve a deoxyribonucleic acid synthesis rate of 100 bases / second or more, or a DNA polymerase composition in which the performance is achieved by a combination of wild type and / or mutant type Can be used as a DNA polymerase suitable for the practice of the present invention.
For example, as a DNA polymerase having a deoxyribonucleic acid synthesis rate of 100 bases / second or more in addition to the above KOD DNA Polymerase, “KOD FX (manufactured by Toyobo, registered trademark)”, “KOD-Plus- (trademark, manufactured by Toyobo)” “KOD Dash (manufactured by Toyobo, registered trademark)”, PrimeSTAR HS DNA polymerase (manufactured by Takara Bio, registered trademark), and the like can also be used.

  In the present invention, DNA synthesis activity refers to deoxyribonucleoside bound to deoxyribonucleic acid by covalently binding deoxyribonucleoside 5′-triphosphate α-phosphate to the 3′-hydroxyl group of the oligonucleotide or polynucleotide annealed to the template DNA. It refers to the activity of catalyzing the reaction of introducing 5′-monophosphate in a template-dependent manner.

When the enzyme activity is high, the activity is measured by diluting the sample with a storage buffer. In the present invention, 25 μl of the following solution A, 5 μl of each of solution B and C, and 10 μl of sterilized water are added to an Eppendorf tube and mixed with stirring. Thereafter, the mixture is ice-cooled, and 50 μl of E solution and 100 μl of D solution are added. This solution is filtered through a glass filter (Whatman GF / C filter), thoroughly washed with solution D and ethanol, and the radioactivity of the filter is measured with a liquid scintillation counter (manufactured by Packard) to incorporate nucleotides into the template DNA. taking measurement. One unit of enzyme activity is the amount of enzyme that incorporates 10 nmol nucleotides per 30 minutes into the acid-insoluble fraction under these conditions.
A: 40 mM Tris-HCl (pH 7.5)
16 mM magnesium chloride 15 mM dithiothreitol 100 μg / ml BSA
B: 2 μg / μl activated calf thymus DNA
C: 1.5 mM dNTP (250 cpm / pmol [3H] dTTP)
D: 20% trichloroacetic acid (2 mM sodium pyrophosphate)
E: 1 μg / μl carrier DNA

In the present invention, the 3′-5 ′ exonuclease activity refers to the activity of excising the 3 ′ terminal region of DNA and releasing 5′-mononucleotide.
The activity was measured by 50 μl of reaction solution (120 mM Tris-HCl (pH 8.8 at 25 ° C.), 10 mM KCl, 6 mM ammonium sulfate, 1 mM MgCl2, 0.1% Triton X-100, 0.001% BSA, 5 μg. Tritium-labeled E. coli DNA) is dispensed into 1.5 ml Eppendorf tubes and DNA polymerase is added. After reacting at 75 ° C. for 10 minutes, the reaction was stopped by cooling with ice, and then 50 μl of 0.1% BSA was added as a carrier, and then 100 μl of 10% trichloroacetic acid and 2% sodium pyrophosphate solution were added and mixed. To do. After leaving on ice for 15 minutes, the precipitate is separated by centrifugation at 12,000 rpm for 10 minutes. The radioactivity of 100 μl of the supernatant is measured with a liquid scintillation counter (manufactured by Packard), and the amount of nucleotide released in the acid-soluble fraction is measured.

In the present invention, the DNA synthesis rate of a DNA polymerase refers to the number of DNA synthesis per unit time. If the DNA synthesis rate is a commercially available product, the value described in the description of the commercially available product, or the value described in the literature or the like if not described in the description, etc. shall be indicated. If it cannot be specified by these methods, it can be determined by the following method.

Method for measuring DNA synthesis rate Reaction solution of DNA polymerase (20 mM Tris-HCl (pH 7.5), 8 mM magnesium chloride, 7.5 mM dithiothreitol, 100 μg / ml BSA, 0.1 mM dNTP, 0.2 μCi [α- 32P] dCTP) is reacted at 75 ° C. with M13mp181 single-stranded DNA with annealed primers. The reaction is stopped by adding an equal amount of a stop solution (50 mM sodium hydroxide, 10 mM EDTA, 5% Ficoll, 0.05% bromophenol blue). After the DNA synthesized by the above reaction is fractionated by alkaline agarose gel electrophoresis, the gel is dried and autoradiography is performed. Labeled λ / HindIII is used as a DNA size marker. The DNA synthesis rate is obtained by measuring the size of the synthesized DNA using the marker band as an index.

  Next, the mutation detection method of the present invention will be described with reference to an example in which a labeled probe labeled with a fluorescent dye is used as the probe of the present invention. The mutation detection method of the present invention is not limited by the following description.

First, for example, genomic DNA is isolated from whole blood. Isolation can be performed, for example, using a commercially available genomic DNA isolation kit.
Next, for example, QProbe is added as a labeled probe to the sample containing the isolated genomic DNA.

The timing of addition of the probe is not particularly limited. For example, the probe may be added to the amplification reaction reaction system before, during or after the amplification reaction described below.
Especially, since an amplification reaction and the said process (2) can be performed continuously, adding before an amplification reaction is preferable. Thus, when the probe is added before the nucleic acid amplification reaction, for example, as described above, it is preferable to add a fluorescent dye or a phosphate group to the 3 ′ end thereof.

The probe may be added to a liquid sample containing isolated genomic DNA or mixed with genomic DNA in a solvent. The solvent is not particularly limited, and examples thereof include conventionally known solvents such as a buffer solution such as Tris-HCl, a solvent containing KCl, MgCl ₂ , MgSO ₄ , glycerol and the like, and a PCR reaction solution.

  Subsequently, using the isolated genomic DNA as a template, a sequence containing a base site for detection is amplified by a nucleic acid amplification method such as PCR. In addition, conditions, such as PCR, are not specifically limited, It can carry out by a conventionally well-known method.

  The sequence of the PCR primer is not particularly limited as long as it can amplify a sequence including a base site for detection, and can be appropriately designed by a conventionally known method according to the target sequence as described above. The length of the primer is not particularly limited, and can be set to a general length (for example, 10 to 40 mer).

  Examples of the primer set used in the detection method of the present invention include the primer set of the present invention described above. The use of the primer set of the present invention is not limited to the detection method of the present invention, and the primer set used for the detection method of the present invention is not limited to the primer set of the present invention.

  Next, the PCR amplification product obtained is dissociated, and the single-stranded DNA obtained by the dissociation and the labeled probe are hybridized. This can be performed, for example, by changing the temperature of the reaction solution.

  The heating temperature in the dissociation step is not particularly limited as long as the amplification product can be dissociated, and is, for example, 85 to 98 ° C. The heating time is not particularly limited, but is usually 1 second to 10 minutes, preferably 1 second to 5 minutes.

  The dissociated single-stranded DNA and the labeled probe can be hybridized, for example, by lowering the heating temperature in the dissociation step after the dissociation step. As temperature conditions, it is 35-50 degreeC, for example.

  The volume and concentration of each composition in the reaction system (reaction system) of the hybridization process are not particularly limited. As a specific example, in the reaction system, the concentration of DNA is, for example, 0.01 to 1 μmol / L, preferably 0.1 to 0.5 μmol / L, and the concentration of the labeled probe is, for example, A range satisfying the addition ratio with respect to DNA is preferable, for example, 0.001 to 10 μmol / L, and preferably 0.001 to 1 μmol / L.

  Then, the temperature of the reaction solution is changed, and a signal value indicating the melting state of the hybrid formed of the amplification product and the labeled probe is measured. Specifically, for example, the reaction solution (hybridized body of the single-stranded DNA and the labeled probe) is heated, and a change in signal value accompanying a temperature rise is measured. As described above, when a probe labeled with a terminal C base (guanine quenching probe) is used, fluorescence is reduced (or quenched) in a hybridized state with single-stranded DNA, and in a dissociated state, Fluoresce. Therefore, for example, the hybrid formed body in which the fluorescence is decreased (or quenched) may be gradually heated, and the increase in the fluorescence intensity accompanying the temperature increase may be measured.

  The temperature range for measuring the fluctuation of the fluorescence intensity is not particularly limited. For example, the start temperature is room temperature to 85 ° C, preferably 25 to 70 ° C, and the end temperature is 40 to 105 ° C, for example. is there. Moreover, the rate of temperature rise is not particularly limited, but is, for example, 0.05 to 20 ° C./second, and preferably 0.08 to 5 ° C./second.

  Determination of the genotype (wild type, mutant type, etc.) at the target base site can be performed, for example, by measuring signal fluctuations during hybridization. That is, when a hybrid is formed by lowering the temperature of the reaction solution containing the probe, the signal fluctuation accompanying the temperature drop is measured.

  As a specific example, when a labeled probe (for example, a guanine quenching probe) that shows a signal alone and does not show a signal by hybridization, it emits fluorescence when the single-stranded DNA and the probe are dissociated. However, when a hybrid is formed due to a decrease in temperature, the fluorescence is reduced (or quenched). Therefore, for example, the temperature of the reaction solution may be gradually decreased to measure the decrease in fluorescence intensity accompanying the temperature decrease. On the other hand, when a labeled probe that does not show a signal alone and shows a signal by hybridization, it does not emit fluorescence when the single-stranded DNA and the probe are dissociated, but the hybrid is not released due to a decrease in temperature. Once formed, it will fluoresce. Therefore, for example, the temperature of the reaction solution may be gradually lowered and the increase in fluorescence intensity accompanying the temperature drop may be measured.

  In the present invention, in order to determine the genotype at the target base site, the signal variation may be analyzed and determined as a Tm (melting temperature) value.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples.

Example 1: Detection of rs8103142 from purified human genome
(1) Preparation of sample Purified human genome (hereinafter referred to as HM) in which rs810103142 has already been found to be major homo (T / T), and purification in which rs810103142 has been found to be hetero (T / C) Sampled human genome (hereinafter referred to as HH), and purified human genome (hereinafter referred to as Hm) in which rs810103142 is known to be a minor homozygote (C / C), and 10 mM Tris-HCl (NC) as a negative control (NC). The sample was pH 7.5).
(2) Nucleic acid amplification and melting curve analysis The following reagents were added to the human genome sample and the negative control, respectively, and rs8103142 was detected under the following conditions. GENECUBE (registered trademark) manufactured by Toyobo Co., Ltd. was used for nucleic acid amplification and melting curve analysis.

Reagents A 13.2 μl solution containing the following reagents was prepared:
Nucleic acid primer (SEQ ID NO: 4) 300 nM
Nucleic acid primer (SEQ ID NO: 7) 1500 nM
Nucleic acid probe (SEQ ID NO: 1, 3 ′ end labeled with BODIPY-FL) 300 nM
× 10 buffer 1.3 μl
dNTP 0.2 mM
MgSO ₄ 4 mM
BSA 1.3μg
DMSO 0.98μL
KOD plus DNA polymerase (manufactured by Toyobo) 0.4U
Sample 4μl

Nucleic acid amplification and melting curve analysis 94 ° C, 2 minutes (above one cycle)
97 ° C · 1 second 60 ° C · 3 seconds 63 ° C · 5 seconds (over 50 cycles)
94 ° C, 30 seconds 39 ° C, 30 seconds 39 ° C to 75 ° C (temperature rise at 0.09 ° C / second)

Results FIG. 1 is a graph showing the results of analyzing the change in fluorescence intensity with increasing temperature in the melting curve, with the horizontal axis of the graph being the temperature and the vertical axis being the differential value of the fluorescence signal. HM has a peak at 57 to 58 ° C, and Hm has a peak at around 67 ° C. HH has peaks at both temperatures. The end of the probe of SEQ ID NO: 1 used in this example is labeled with BODIPY-FL. This probe is characterized in that the fluorescence is quenched while bound to a nucleic acid having a complementary sequence, and conversely, the fluorescence is emitted while the probe is released alone. The probe of SEQ ID NO: 1 binds most strongly to the nucleic acid containing the minor homo rs8103142, and binds weaker to the nucleic acid containing the major homo rs81010314 due to a single base mismatch. Accordingly, the peak at 57-58 ° C. in FIG. 1 indicates that rs8101032 in the test nucleic acid is major allyl, and the peak near 67 ° C. indicates that rs810103142 in the test nucleic acid is minor allyl. ing. The test nucleic acid emitting both peaks indicates that the nucleic acid has both major and minor alleles.
From the above, it was shown that the allele of rs8103142 can be easily identified by using the probe of the present invention.

[Example 2: Detection of rs8099917 from purified human genome]
(1) Sample preparation A purified human genome (hereinafter referred to as HM) in which rs8099917 has already been found to be a major homo (T / T), and purification in which rs8099917 has been found to be hetero (T / G). Sampled human genome (hereinafter referred to as HH), and purified human genome (hereinafter referred to as Hm) in which rs8099917 is known to be a minor homozygote (G / G), and 10 mM Tris-HCl (NC) as a negative control (NC). The sample was pH 7.5).
(2) Nucleic acid amplification and melting curve analysis The following reagents were added to the human genome sample and the negative control, respectively, and rs8099917 was detected under the following conditions. GENECUBE (registered trademark) manufactured by Toyobo Co., Ltd. was used for nucleic acid amplification and melting curve analysis.

Reagents A 13.2 μl solution containing the following reagents was prepared:
Nucleic acid primer (SEQ ID NO: 5) 500 nM
Nucleic acid primer (SEQ ID NO: 8) 2500 nM
Nucleic acid probe (SEQ ID NO: 2, 3 ′ end labeled BODIPY-FL) 250 nM
× 10 buffer 1.3 μl
dNTP 0.2 mM
MgSO ₄ 4 mM
BSA 1.3μg
DMSO 0.98μL
KOD plus DNA polymerase (manufactured by Toyobo) 0.4U
Sample 4μl

Nucleic acid amplification and melting curve analysis Same as Example 1.

Results FIG. 2 is a graph showing the results of analyzing the change in fluorescence intensity with increasing temperature in the melting curve, with the horizontal axis of the graph being the temperature and the vertical axis being the differential value of the fluorescence signal. HM has a peak around 54 ° C., and Hm has a peak around 61 ° C. HH has peaks at both temperatures. The end of the probe of SEQ ID NO: 2 used in this example is labeled with BODIPY-FL. This probe is characterized in that the fluorescence is quenched while bound to a nucleic acid having a complementary sequence, and conversely, the fluorescence is emitted while the probe is released alone. The probe of SEQ ID NO: 2 binds most strongly to the nucleic acid containing the minor homo rs80999917 and binds weaker to the nucleic acid containing the major homo rs81031142 due to a single base mismatch. Therefore, the peak near 54 ° C. in FIG. 2 indicates that rs8099917 in the test nucleic acid is a major allyl, and the peak near 61 ° C. indicates that rs8099917 in the test nucleic acid is a minor allyl. Yes. The test nucleic acid emitting both peaks indicates that the nucleic acid has both major and minor alleles.
From the above, it was shown that the allele of rs8099917 can be easily identified by using the probe of the present invention.

[Example 3: Detection of rs12979860 from purified human genome]
(1) Preparation of sample Purified human genome (hereinafter referred to as HM) in which rs129798860 is already known to be a major homo (C / C), and purification in which rs129798860 is heterozygous (C / T) Sampled human genome (hereinafter referred to as HH) and purified human genome (hereinafter referred to as Hm) in which rs129798860 is known to be a minor homozygote (T / T), and 10 mM Tris-HCl (NC) as a negative control (NC). The sample was pH 7.5).
(2) Nucleic acid amplification and melting curve analysis The following reagents were added to the human genome sample and the negative control, respectively, and rs129798860 was detected under the following conditions. GENECUBE (registered trademark) manufactured by Toyobo Co., Ltd. was used for nucleic acid amplification and melting curve analysis.

Reagents A 13.2 μl solution containing the following reagents was prepared:
Nucleic acid primer (SEQ ID NO: 6) 300 nM
Nucleic acid primer (SEQ ID NO: 9) 1500 nM
Nucleic acid probe (SEQ ID NO: 3, 3 ′ end labeled with BODIPY-FL) 300 nM
× 10 buffer 1.3 μl
dNTP 0.2 mM
MgSO ₄ 4 mM
BSA 1.3μg
DMSO 0.98μL
KOD plus DNA polymerase (manufactured by Toyobo) 0.4U
Sample 4μl

Nucleic acid amplification and melting curve analysis Same as Example 1.

Results FIG. 3 is a graph showing the results of analyzing the change in fluorescence intensity with increasing temperature in the melting curve, with the horizontal axis of the graph being the temperature and the vertical axis being the differential value of the fluorescence signal. HM has a peak around 62 ° C., and Hm has a peak around 48 ° C. HH has peaks at both temperatures. The end of the probe of SEQ ID NO: 3 used in this example is labeled with BODIPY-FL. This probe is characterized in that the fluorescence is quenched while bound to a nucleic acid having a complementary sequence, and conversely, the fluorescence is emitted while the probe is released alone. The probe of SEQ ID NO: 3 binds most strongly to the nucleic acid containing the major homo rs129798860, and binds weaker to the nucleic acid containing the minor homo rs129798860 due to a single base mismatch. Accordingly, the peak at around 48 ° C. in FIG. 3 indicates that rs12979860 in the test nucleic acid is a minor allyl, and the peak near 62 ° C. indicates that rs12979860 in the test nucleic acid is a major allyl. Yes. The test nucleic acid emitting both peaks indicates that the nucleic acid has both major and minor alleles.
From the above, it was shown that the allele of rs129798860 can be easily identified by using the probe of the present invention.

[Example 4: Detection of rs8099917 from whole blood diluted solution]
(1) Preparation of sample 90 μl of 10 mM Tris-HCl (pH 7.5) was added to 10 μl of whole blood to prepare a diluted whole blood solution having a dilution ratio of 10 times. This whole blood dilution was used as a sample. Since the detection system is 10 μl and the sample is 1 μl, the final concentration of whole blood in the detection system is 1%.
(2) Nucleic acid amplification and melting curve analysis The following reagents were added to the whole blood dilution, and rs8099917 was detected under the following conditions. GENECUBE (registered trademark) manufactured by Toyobo Co., Ltd. was used for nucleic acid amplification and melting curve analysis.

Reagents A 10 μl solution containing the following reagents was prepared.
Nucleic acid primer (SEQ ID NO: 5) 500 nM
Nucleic acid primer (SEQ ID NO: 8) 2500 nM
Nucleic acid probe (SEQ ID NO: 2, 3 ′ end labeled BODIPY-FL) 250 nM
× 10 buffer 1 μl
dNTP 0.2 mM
MgSO ₄ 4 mM
1 μg of BSA
DMSO 0.75μL
KOD plus DNA polymerase (manufactured by Toyobo) 0.3U
Sample 1μl

Nucleic acid amplification and melting curve analysis Same as Example 1.

Results FIG. 5 is a graph showing the results of analyzing the change in fluorescence intensity with increasing temperature in the melting curve, with the horizontal axis of the graph as the temperature and the vertical axis as the differential value of the fluorescence signal. As is apparent from FIG. 5, rs8099917 can be detected even from a whole blood dilution by using the probe of the present invention.

By applying the present invention to the development of a SNP test relating to the treatment of hepatitis C and the development of a SNP test reagent relating to the treatment of hepatitis C, it is possible to rapidly, reliably and simply detect mutations in genes associated with chronic myeloproliferative diseases.

Claims (11)

A probe for detecting a SNP that is said to be involved in the therapeutic rate of hepatitis C, the probe having a base sequence represented by any one of SEQ ID NOs: 1 to 3. The probe according to claim 1, which is labeled with a detectable labeling substance. The probe according to claim 2, wherein the detectable labeling substance is a fluorescent dye. The probe according to claim 2 or 3, wherein the terminal cytosine is labeled. A method for detecting an SNP that is said to be involved in the therapeutic rate of hepatitis C, comprising all of the following steps (1) to (3).
(1) A step of hybridizing the test nucleic acid with the probe according to any one of Items 1 to 4 to form a complex (2) After step (1), while continuously increasing the temperature, 260 nm A step of performing a melting curve analysis by measuring the absorbance of the probe or measuring the signal intensity of the label added to the probe.
(3) A step of determining the presence or absence of a mutation in the test nucleic acid from a change in signal intensity accompanying a temperature change in step (2). It is a method of Claim 5, Comprising: The method of including all the processes of (1)-(3) below.
(1) The step of hybridizing the test nucleic acid and the probe according to any one of Items 1 to 4 to form a complex (2) After step (1), the temperature is increased continuously, A step of performing a melting curve analysis by measuring the fluorescence intensity of a fluorescent dye that labels the probe.
(3) A step of determining the presence or absence of a mutation in the test nucleic acid from the change in fluorescence intensity associated with the temperature change in step (2). The method according to claim 5 or 6, wherein the test nucleic acid in step (1) is amplified from a template nucleic acid using a primer pair. The method according to claim 7, wherein a DNA polymerase having a DNA synthesis rate of 100 bases / second or more and having no 5 'exonuclease activity is used for nucleic acid amplification. The method according to claim 7 or 8, wherein the primer pair is a pair of primers including a forward primer composed of the following oligonucleotide (F) and a reverse primer composed of the following (R) oligonucleotide.
(F) an oligonucleotide whose base sequence is represented by any of SEQ ID NOs: 4 to 6 (R) an oligonucleotide whose base sequence is represented by any of SEQ ID NOs: 7 to 9 A primer pair for amplifying a SNP that is said to be involved in the treatment rate of hepatitis C by a nucleic acid amplification method, and comprising a primer pair configured by any of (1) to (3) below.
(1) Primer pair consisting of primers having the base sequences shown in SEQ ID NO: 4 and SEQ ID NO: 7 (2) Primer pair consisting of primers having the base sequences shown in SEQ ID NO: 5 and SEQ ID NO: 8 (3 ) Primer pair consisting of primers having the base sequences shown in SEQ ID NO: 6 and SEQ ID NO: 9, respectively A mutation detection kit for use in the method for detecting a SNP that is said to be involved in the therapeutic rate of hepatitis C according to claim 5, comprising the probe according to any one of claims 1 to 4.