JPWO2006070667A1 - EGFR gene mutation detection method and detection kit - Google Patents

Abstract

A method and kit for rapidly, conveniently and sensitively detecting a deletion mutation in the EGFR gene associated with disease prediction, drug efficacy and side effects.

Description

  The present invention relates to a method and kit for rapidly, simply and sensitively detecting a deletion mutation of the EGFR gene related to disease prediction, drug efficacy and side effects.

In recent years, it has been revealed that various mutations (point mutations, deletions, insertions, duplications) at the gene level are closely related to lifestyle diseases as well as genetic diseases. Furthermore, pharmacogenomic analysis such as drug efficacy prediction and side effect prediction is being applied to clinical practice. Especially, the relationship between the EGFR inhibitor which is a lung cancer therapeutic agent, and the mutation of EGFR was clarified (refer nonpatent literature 1, 2, 3, 4).
As a method for detecting a point mutation among the above gene mutations, various methods have been reported (see Non-Patent Document 5). For example, a method for examining the presence or absence of nucleic acid amplification using a primer having a mutation-specific sequence at the 3 ′ end (see Patent Document 1), a method using a probe technique (TaqMan method, see Patent Documents 2 and 3), an Invader method (See Patent Document 4), Cycling probe technology (see Non-Patent Document 6), DataClave probe method (see Non-Patent Document 7), Cycle method (see Patent Document 5), Hybridization probe method (see Patent Document 6), Scorpion method (See Non-Patent Document 8), a sequencing method, a 1-base extension method, and the like. These methods are widely used for Ras mutation and various SNP typing, for example, and can also be applied to analysis of point mutations existing in EGFR (for example, L-> R mutation of codon 858 present in exon 21).

  Deletion mutation analysis has also been performed by sequencing and high performance electrophoresis. For example, in an example typified by a + / Δ32 deletion mutation of CC chemokine receptor 5 (CCR5) gene, since the deletion pattern of the gene is constant, nucleic acid amplification with normal primers for normal sequence is performed, and TaqMan The presence and intensity of signals are examined using two types of probes for normal sequences and defective junction regions, etc. (Non-patent Document 9).

Japanese Patent No. 2853864 US Pat. No. 5,210,015 US Pat. No. 5,487,972 US Pat. No. 5,846,717 International Publication No. 03/074696 Pamphlet International Publication No. 97/46714 Pamphlet Science, 304, p. 1497-1500 (2004) New England Journal of Medicine, 350, p. 2129-2139 (2004) British Journal of Cancer, 93, p. 355-363 (2005) Clinical Cancer Research, 11 (12), p. 4289-4294 (2005) Nature Reviews 3, p. 749-761 (2004) BioTechniques, 20, p. 240-248 (1996) Analytical Biochemistry, 333 (2), p. 246-255 (2004) Nucleic Acids Research, 28, p. 2752-3761 (2000) Clinical Chemistry, 46, p. 24-30 (2000)

  In this way, deletion mutation analysis with a constant deletion pattern has been studied, but in the detection of atypical deletion mutations with a non-fixation deletion pattern, high-performance electrophoresis after sequencing and nucleic acid amplification according to individual cases There is a problem that large and complicated processes such as analysis (for example, capillary electrophoresis) and mass spectrometry by TOF-MS have to be performed, and the process cannot be performed easily and quickly. Furthermore, when the specimen is a cancer tissue, it is not a mixture rate of 100% or 50% as in normal gene polymorphism analysis, but is a gene derived from cancer cells that are mixed in the presence of the majority of normal cells. Need to be detected. For example, it has been difficult to detect a mutated gene present at a mixing rate of 5%.

  An object of the present invention is to provide an anticancer agent (EGFR inhibitor) for somatic cell deletion mutations in exon 19 of the epidermal growth factor receptor (EGFR) gene with various mutation patterns and short deletion sequences, so-called deletion pattern is not constant. ) Is useful in terms of effect prediction, epidemiological studies, and disease prediction, and is intended to provide a simple, rapid and sensitive screening method.

  The present inventors diligently studied and developed a novel technique capable of detecting even a defective mutant gene mixed at a rate of 5% in a normal EGFR gene. Furthermore, a new technology has been developed to perform the above detection quickly in a closed / closed system in the same reaction system. In other words, the risk of cross-contamination is prevented with a single tube closed / homogeneous system, multiple samples can be measured simultaneously in a short time of 90 minutes, and detection is possible only by the presence of 5% defective mutant genes in normal genes. A highly sensitive detection method and reagent kit were developed to complete the present invention.

That is, the first invention of the present invention relates to a method for detecting the presence or absence of a deletion mutation in exon 19 of the EGFR gene contained in a sample, which comprises the following steps:
Amplifying a target region in a sample using at least one primer for a defective sequence that can be annealed to a defective junction region of the EGFR gene and a primer opposite to the primer for the defective sequence, wherein 3 ′ of the primer for the defective sequence The terminal base sequence is a base sequence consisting of 2 to 5 bases derived from the non-deletion site on the 3 ′ side adjacent to the defective junction, and the primer opposite the defective sequence primer is a defective mutation or a normal gene. A primer capable of annealing; and detecting a deletion mutation based on the presence or absence of an amplification product.

  In the first invention of the present invention, a deletion mutation is further detected in the same reaction system as the amplification reaction using at least two probes including a probe that recognizes a defective site and a probe that recognizes a non-deficient site. Also good.

  In addition, although not particularly limited, in the first invention of the present invention, a deletion sequence primer selected from primers having a base sequence described in SEQ ID NO: 6 to 10 in the sequence listing and SEQ ID NO: 11 in the sequence listing. It is preferable to use a primer opposite to the deletion sequence primer having the described base sequence and / or a probe having the base sequence described in SEQ ID NOs: 16 to 17 in the sequence listing.

The second invention of the present invention relates to a composition for the method of the first invention of the present invention characterized in that it comprises:
(1) A primer for a defective sequence that can be annealed to a defective junction region of the EGFR gene, wherein the base sequence at the 3 ′ end of the primer for the defective sequence is derived from 2 derived from a non-defective site on the 3 ′ side adjacent to the defective junction It is a base sequence consisting of 5 bases.
(2) A primer opposite to the primer for the defective sequence of (1), wherein the primer is a primer capable of annealing to both a defective mutation and a normal gene, and (3) DNA polymerase.

  In the second invention of the present invention, (4) a probe that recognizes a defective site, (5) a probe that recognizes a non-defective site, and (6) for cleaving the probe when hybridized to a target sequence An enzyme may be contained.

  Further, although not particularly limited, in the second invention of the present invention, a deletion sequence primer selected from primers having a base sequence set forth in SEQ ID NO: 6 to 10 in the sequence listing and SEQ ID NO: 11 in the sequence listing. It is preferable to contain a primer facing the deletion sequence primer having the described base sequence and / or a probe having the base sequence described in SEQ ID NOs: 16 to 17 in the sequence listing.

A third invention of the present invention relates to a kit for the method of the first invention of the present invention characterized in that it comprises:
(1) A primer for a defective sequence that can be annealed to a defective junction region of the EGFR gene, wherein the base sequence at the 3 ′ end of the primer for the defective sequence is derived from 2 derived from a non-defective site on the 3 ′ side adjacent to the defective junction (2) A primer opposite to the deletion sequence primer of (1), wherein the primer is a primer capable of annealing to both a deletion mutation and a normal gene.
In the third invention of the present invention, it may further contain (3) a probe for recognizing a deficient site and (4) a probe for recognizing a non-deficient site.

  Further, although not particularly limited, in the third invention of the present invention, a deletion sequence primer selected from primers having a base sequence described in SEQ ID NO: 6 to 10 in the sequence listing and SEQ ID NO: 11 in the sequence listing. It is preferable to contain a primer facing the deletion sequence primer having the described base sequence and / or a probe having the base sequence described in SEQ ID NOs: 16 to 17 in the sequence listing.

  According to the present invention, it is possible to perform simple, rapid and highly sensitive screening of somatic cell-deficient mutations in exon 19 of the EGFR gene, which is useful from the viewpoint of anticancer drug (EGFR inhibitor) effect prediction, epidemiological investigation, and disease prediction. Become.

It is a figure which shows the pattern of the deletion mutation in the exon 19 of an EGFR gene. It is a figure which shows the pattern of the deletion mutation in the exon 19 of an EGFR gene. FIG. 6 is a diagram in which the fluorescence intensity of HEX and the HEX / FAM value per FAM fluorescence intensity in each sample of Example 5 are calculated and plotted.

  As used herein, a deletion mutation refers to a mutation in which a part of DNA is missing from DNA having a normal sequence.

  In the present specification, the term “deletion site” refers to a DNA site lacking due to the above-described deletion mutation.

  In this specification, the non-deficient site refers to a site other than the above-mentioned deficient site.

  In the present specification, a defective junction refers to a portion between bases adjacent to both sides of a defective site in a gene having a defective mutation. That is, it refers to the junction between non-deficient sites in a defective mutation.

  In the present specification, a defective junction region refers to a region containing bases adjacent to both sides of a defective site in a gene having a defective mutation. That is, it refers to a region containing bases adjacent to both sides of the junction between non-deficient sites in a defective mutation (that is, a defective junction).

  In the present specification, the primer opposite to the deletion sequence primer refers to a primer that constitutes a primer pair used in the nucleic acid amplification reaction together with the deletion sequence primer.

Hereinafter, the present invention will be described in detail.
(A) EGFR gene deletion mutation detection method of the present invention The detection method of the present invention comprises:
A method for detecting the presence or absence of a deletion mutation in exon 19 of an EGFR gene contained in a sample, wherein at least one primer for a deletion sequence that can be annealed to a deletion junction region of the EGFR gene, 'The base sequence of the terminal is a base sequence consisting of 2 to 5 bases derived from the non-deletion site adjacent to the deletion junction, and a primer opposite to the deletion sequence primer, wherein the primer is a deletion mutation and a normal gene Any of these primers can be annealed, and is characterized by amplifying a target region in a sample. The detection method of the amplification product is not particularly limited, and examples thereof include agarose gel electrophoresis and a detection method using a probe.
Further, the deletion mutation may be detected in the same reaction system as the amplification reaction by using at least two probes, ie, a probe for identifying the defective site and a probe for identifying the non-defective site.
Examples of the deletion mutation of exon 19 of the EGFR gene to be detected in the present invention include deletion mutations appearing at codons 746 to 752 of exon 19 of the EGFR gene. As a suitable example of such a detection target, the deletion mutation described in the previous non-patent documents 1 to 4 can be mentioned, and more preferably, the deletion mutation described in the non-patent documents 1 and 2 (FIG. 1) is exemplified. Is done. In FIG. 1, deletions Del-1a, Del-1b, Del-3, Del-4, Del-5, and Del-2 are the deletion of bases 2235 to 2249 (deletion of amino acids E746 to A750), base 2236, respectively. Deletion of ~ 2250 (deletion of amino acids E746-A750), deletion of bases 2239-2247 (deletion of amino acids L747-E749, substitution of A750P), deletion of bases 2240-2257 (deletion of amino acids L747-S752) Deletion, substitution of P753S), deletion of bases 2238 to 2255 (deletion of amino acids L747 to S752, substitution of E746V), deletion of bases 2254 to 2277 (deletion of amino acids S752 to I759). 1 and the above base numbers are numbers when the A of the start codon ATG of the human EGFR gene is 1, and the amino acid numbers are numbers when the methionine (Met) encoded by the start codon is 1. .
Moreover, one aspect of the method of the present invention is characterized in that at least one pair of primers for detecting the deletion mutation and at least two probes that recognize a non-deletion site and a non-deletion site are used. Another aspect of the method of the present invention is characterized in that a nucleic acid amplification reaction and signal detection are simultaneously performed in the same reaction system, and the presence or absence of a deletion mutation is detected rapidly and with high sensitivity.

  As the primer used in the method of the present invention, any primer can be suitably used as long as it does not substantially amplify the normal EGFR gene and can substantially amplify at least one deletion mutation. Although there is no particular limitation, for example, at least one of the primers used in the method of the present invention can be annealed to the deletion junction region of the EGFR gene, and the nucleotide sequence at the 3 ′ end of the primer is 3 ′ adjacent to the deletion junction. It is selected from base sequences derived from the non-deficient site. The base sequence at the 3 'end of the primer is preferably 2 to 5 bases from the 3' non-deletion site adjacent to the deletion junction, particularly preferably 2 to 3 bases. Two or more kinds of the primers can also be used for nucleic acid amplification reaction in the same reaction system. This is preferable in that two or more types of deletion mutations can be detected in the same reaction system. When two or more kinds of the primers are used, the positions where the 5 ′ ends of the primers are annealed coincide with each other in order to identify the chain length of the missing base sequence by determining the chain length by acarose gel electrophoresis or the like. Primers may be set in As said primer, what has the base sequence in any one of sequence number 6-10 of a sequence table can be used conveniently, for example.

Further, the other primer used in the method of the present invention is not particularly limited as long as it can be annealed to both a deletion mutation and a normal gene of the gene. For example, paraffin is difficult to amplify long-chain DNA. Primers set so as to be able to amplify DNA from the section are preferred. The primer is preferably a primer set such that the chain length of the amplification product obtained by the nucleic acid amplification reaction is 100 bp or less, and more preferably, the chain length of the amplification product is set to 87 bp or less. It is a primer. As said primer, what has a base sequence of sequence number 11 of a sequence table, for example can be used conveniently.
In the method of the present invention, the target defective gene can be detected with high sensitivity by the above primer pair.

  Moreover, when setting the primer used by the method of this invention, in addition to said matter, well-known literature, for example, the literature (Protein, a nucleic acid, an enzyme, Vol. 35, p3157-p3163, 1990) of Utsumi et al. You can refer to the guidelines for creating primers.

  In the present invention, the DNA fragment amplified by the method of the present invention may be detected using a probe.

The probe used in the method of the present invention is not particularly limited as long as it has a base sequence that recognizes a defective site and a base sequence that recognizes a non-defective site, but preferably has a sequence complementary to a target sequence. The probe is preferably, for example, a probe that is cleaved by a specific nucleolytic enzyme when hybridized to a target sequence. Such probes include TaqMan ^™ probes and DNA-RNA-DNA chimeric probes. A particularly preferred probe is a DNA-RNA-DNA chimeric probe. The chain length of the RNA portion of the chimeric probe is not particularly limited, but is preferably 1 to 5 bases, more preferably 1 base from the viewpoint of probe stability. The chain length of the entire probe is preferably 6 to 30 bases, more preferably 10 to 15 bases.
Of the above probes, a probe that recognizes a defective site preferably selects a region that is commonly deleted in various deletion mutations. A part of the sequence of the probe may include a non-deletion site, but it is preferable to avoid annealing the amplified defect DNA and the non-deletion site of the probe within the range of the amplification temperature condition. The non-deficient site of the probe is preferably 0 to 12 bases, more preferably 0 to 6 bases. Although it does not specifically limit, For example, what has the base sequence of sequence number 16 of a sequence table is preferable.

  In addition, as a probe for recognizing a non-deletion site, a probe in which a region that does not suffer from a defect in common among various defects can be suitably used. Although it does not specifically limit, For example, what has a base sequence of sequence number 17 of a sequence table is preferable.

In addition, if there is no region common to other deletion mutations in the deletion site, or there is a deletion mutation in which the chain length of the common deletion region is not sufficient to set the probe, the deletion mutation On the other hand, the above-described probe for recognizing a deficient site can be used as a probe for recognizing a non-deficient site, and the probe for recognizing a non-deficient site can be used as a probe for recognizing a deficient site.
Although not particularly limited, for example, the deletion mutation of exon 19 of the EGFR gene shown in FIG. 1 will be described as an example. The deletion sequences Del-1a, Del-1b, Del-3, Del-4, and Del-5 The probe DF (SEQ ID NO: 16) for recognizing a defective site can be used as a probe for recognizing a non-defective site with respect to the defective sequence Del-2. In addition, the probe NH (SEQ ID NO: 17) that recognizes non-deletion sites of the deletion sequences Del-1a, Del-1b, Del-3, Del-4, and Del-5 is a deletion site for the deletion sequence Del-2. Can be used as a probe for recognizing.

  Furthermore, in the method of the present invention, although not particularly limited, gene amplification methods often used in the art such as PCR method, ICAN method, LAMP method, SDA method and the like can be used. A particularly preferred method is, for example, the PCR method.

  Furthermore, examples of the real-time detection method that can be used in the present invention include TaqMan method, Scorpion method, Cycling probe method, hybridization probe method, and Cycle method. Although not particularly limited, the Cycle method is preferable. In the Cycle method, a hybrid is formed between the amplification product and the RNA-containing probe, and the RNA sequence of the probe is cleaved by RNase H in the reaction solution only when the amplification product and the base sequence of the probe are in perfect match. Is not disconnected. Taking advantage of this, when a probe that is labeled with two or more fluorescent dyes having different wavelengths and causes interference of fluorescence between the fluorescent dyes is used, the probe is cleaved. Since the fluorescence intensity of the fluorescent substance changes, the presence or absence of mutation can be determined. The fluorescent dye only needs to have a fluorescence wavelength of 20 nm or more. For example, 6-FAM (6-carbofluorescein) and HEX (Hexachloro-6-carboxyfluorescein), 6-FAM and ROX (6-carboxy-X-rhodamine), Can also be used.

  Further, the probe is suitable for both a fluorescent substance and a substance having an action of quenching the fluorescence emitted by the fluorescent substance, for example, Eclipse (manufactured by Epoch Biosciences) or DABCYL (4-dimethylaminoazobenzene-4'-sulfone). It may be labeled at intervals. In such a probe, a part of the fluorescence emitted from the fluorescent substance is converted into thermal energy by the quenching substance in an intact state, but when the distance between the fluorescent substance and the quenching substance is increased, the above action is achieved. It is eliminated and the intensity of the fluorescent signal detected increases.

  In the Cycle method, two or more types of probes can be used in the same reaction system. In such a case, each probe is labeled with a fluorescent dye having a different wavelength, and the presence or absence of mutation can be determined more clearly from the ratio of the change in fluorescence intensity. For example, when a probe for detecting a defective site in a target nucleic acid and a probe for detecting a normal site are labeled with fluorescent substances having different wavelengths and used in the Cycle method, if there is a defect, An increase in the fluorescence signal intensity due to the probe corresponding to the defect site is not detected, and only an increase in the fluorescence signal intensity due to the other probe corresponding to the normal site can be detected.

(B) Composition for the method of the present invention The composition of the present invention comprises:
A composition for the method described in (A) above,
(1) A primer for a defective sequence that can be annealed to a defective junction region of the EGFR gene, wherein the base sequence at the 3 ′ end of the primer for the defective sequence is derived from 2 derived from a non-defective site on the 3 ′ side adjacent to the defective junction It is a base sequence consisting of 5 bases,
(2) A primer opposite to the defective sequence primer of (1), wherein the primer is a primer capable of annealing to both a defective mutation and a normal gene, and (3) contains a DNA polymerase To do.
Furthermore,
(4) It contains a probe for identifying a defect site, and (5) a probe for recognizing a non-deletion site.

In the composition of the present invention, although not particularly limited, for example, a primer for a deletion sequence selected from primers having a base sequence described in SEQ ID NO: 6 to 10 in the sequence listing and a base described in SEQ ID NO: 11 in the sequence listing You may contain the primer which opposes the said primer for deletion sequences which has a sequence | arrangement.
Furthermore, it may contain a probe having the base sequence described in any one of SEQ ID NOS: 16 and 17 in the sequence listing.
The probe may be labeled with the label described in (A) above.

  The DNA polymerase contained in the composition of the present invention is not particularly limited, but a DNA polymerase generally used for DNA amplification is preferable. For example, a DNA polymerase derived from Taq (Thermus aquaticus), Pfu (Pyrococcus furiosus-derived DNA polymerase, Tli (Thermococcus literalis) -derived DNA polymerase, KOD (Thermococcus kodakaraensis) -derived DNA polymerase, Bca (Bacillus caldotenax) -derived DNA polymerase, Bst (Geobacilus-derived DNA polymerase, Bst (Geobacilus) Mix more DNA polymerase, and more preferably Taq-derived DNA polymerase.

  In addition, the composition of the present invention may contain an enzyme for cleaving the probe when the composition can be used for the cycling probe method or the cycle method, and is not particularly limited. Is, for example, RNase H (ribonuclease H). RNaseH is more preferably derived from archaea, and in particular, RNaseH (Tli RNaseH), which can be prepared by the method described in WO 02/22831, can be suitably used. Further, the composition may contain a nucleic acid amplification reaction reagent and the like.

(C) Kit for the method of the present invention
A kit for the method described in (A) above,
(1) A primer for a defective sequence that can be annealed to a defective junction region of the EGFR gene, wherein the base sequence at the 3 ′ end of the primer for the defective sequence is derived from 2 derived from a non-defective site on the 3 ′ side adjacent to the defective junction (2) a primer opposite to the deletion sequence primer of (1), wherein the primer is a primer that can be annealed to both a deletion mutation and a normal gene. It is characterized by things.
Furthermore,
(3) It contains a probe for identifying a defect site, and (4) a probe for recognizing a non-deletion site.

In the kit of the present invention, although not particularly limited, for example, a primer for a deletion sequence selected from primers having a base sequence described in SEQ ID NO: 6 to 10 in the sequence listing and a base sequence described in SEQ ID NO: 11 in the sequence listing It may contain a primer opposite to the defective sequence primer.
Furthermore, it may contain a probe having the base sequence described in any one of SEQ ID NOS: 16 and 17 in the sequence listing. The probe may be labeled with the label described in (A) above.

  Further, as the DNA polymerase contained in the kit of the present invention, those exemplified in the above (2) can be suitably used.

  The kit of the present invention may contain the enzyme exemplified in the above (2). Further, it may contain a nucleic acid amplification reaction reagent or the like.

  By using the composition of the present invention and / or the kit of the present invention, the presence or absence of a deletion mutation in the EGFR gene can be easily detected.

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

  First, control template DNA and a primer for detecting a defective sequence were synthesized. That is, a normal sequence control template DNA (SEQ ID NO: 1 in the sequence listing) was synthesized from the 164856th to 164948th nucleic acid sequence of the human EGFR gene sequence (GenBank Accession No. AF2888738). This sequence includes a part of human EGFR gene exon 19 (SEQ ID NO: 18 in the sequence listing). In addition, the amino acid sequence information of the deletion sequence Del-1a, Del-1b, Del-3, Del-4 of EGFR exon 19 reported in Paez et al., Science, 304, 1497-1500 (2004) and the above human From the EGFR gene sequence information, CTDEL-1a (SEQ ID NO: 2 in the sequence listing), CTDEL-1b (SEQ ID NO: 3 in the sequence listing), CTDEL-3 (SEQ ID NO: 4 in the sequence listing), and CTDEL-4 (in the sequence listing) Each of SEQ ID NO: 5) was synthesized by a DNA synthesizer and used as a defective sequence control template DNA. The obtained normal sequence control template DNA and defective sequence control template DNA were each prepared to be 100 fg / μl with sterile distilled water.

Next, as upstream primers for detection of deletion sequences Del-1a, Del-1b, Del-3, Del-4 of EGFR exon 19, F1D1a (SEQ ID NO: 6 in the sequence listing), F2D1b (SEQ ID NO: 7 in the sequence listing), F3D3 (SEQ ID NO: 8 in the sequence listing), F4D4 (SEQ ID NO: 9 in the sequence listing), and F5D5 (SEQ ID NO: 10 in the sequence listing) were each synthesized by a DNA synthesizer. Further, RV (SEQ ID NO: 11 in the sequence listing) was synthesized by a DNA synthesizer as a downstream primer for detection common to the deletion sequence and normal sequence of EGFR exon 19. Furthermore, as an upstream primer for detection of the deletion sequences Del-1a, Del-1b, Del-3, and Del-4 of EGFR exon 19, F1-2D1a (SEQ ID NO: in the sequence listing) is different from the normal sequence only at the 3 ′ end. 12), F2-2D1b (SEQ ID NO: 13 in the Sequence Listing), F3-2D3 (SEQ ID NO: 14 in the Sequence Listing), and F4-2D4 (SEQ ID NO: 15 in the Sequence Listing) were also synthesized by a DNA synthesizer. FIG. 1 shows the position of the downstream primer RV. In FIG. 1, the position of each upstream primer is shown with an underline.

  High-sensitivity detection of EGFR exon 19-deficient gene by multiplex PCR using the defective sequence primer prepared in Example 1 was examined. First, a template DNA was prepared as follows. That is, the 100 fg / μl CTdel-1a deficient sequence control template DNA solution prepared in Example 1 was adjusted to 100 fg / μl normal sequence control template DNA solution so that the deficient sequence CTDEL-1a was 5 mol% with respect to the total control template. To obtain a 5 mol% CTDela-1a-deficient sequence control template DNA solution. Similarly, the CTDEL-1b solution, the CTDEL-3 solution, and the CTDEL-4 solution were each diluted with a 100 fg / μl normal sequence control template DNA solution to obtain a 5 mol% defective sequence control template DNA solution.

Detection of defective sequence DNA in each 5 mol% defective sequence control template DNA solution prepared above was performed by multiplex PCR. The composition of the reaction solution was 10 × PCR buffer (manufactured by Takara Bio Inc.), 100 fg of control template DNA containing 5 mol% of the defective sequence or 100 fg of normal sequence control template DNA, and 1.25 U of TaKaRa Taq DNA polymerase ( The final concentration is 0.2 mM dNTP, 2 mM MgCl ₂ , 0.2 μM F1D1a primer, 0.2 μM F2D1b primer, 0.2 μM F3D3 primer, 0.2 μM F4D4 primer, 0.2 μM F5D5 Primers and 0.2 μM RV primer were prepared. The reaction solution volume is 25 μl. The PCR amplification apparatus used was a thermal cycler MP (Takara Bio), and the reaction conditions were a 35-cycle reaction with 95 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 30 seconds. After completion of the reaction, the amplified product was electrophoresed with 3% NuSieve 3: 1 agarose (manufactured by Takara Bio Inc.), and ethidium bromide staining was performed to detect the amplified product.

  As a result, in all samples using four types of 5 mol% deletion sequence control templates as templates, only amplification bands corresponding to the amplification chain length specific to each deletion sequence control DNA were detected. In addition, in the sample using the normal sequence control template DNA as a template, an 87 bp amplification band corresponding to the normal amplified chain length could not be confirmed. From this, it was confirmed that the defective sequence in the sample can be accurately amplified by the method of the present invention.

The detection of EGFR exon 19-deficient gene by the multiplex PCR method using a deletion sequence primer that differs from the normal sequence only at the 3 'terminal 1 base reported as the ARMS method and the method of the present invention were compared and examined.
That is, the upstream primers F1-2D1a, F2-2D1b, F3-2D3, F4-2D4, and F5- for the EGFR exon 19-deficient sequence, which differ from the normal sequence of EGFR exon 19 only in the 1 ′ base at the 3 ′ end prepared in Example 1. Detection of defective sequence DNA in a 5 mol% defective sequence control template DNA solution was performed using 2D5. PCR conditions and subsequent detection of amplification products were performed under the same conditions as in Example 2 except for the upstream primer used for detecting the defective sequence.

As a result, when a 5 mol% deletion sequence control template DNA of Del-1 and Del-1b was used as a template, only an 88 bp amplification product corresponding to the amplified chain length using the normal sequence DNA as a template was detected. In Del-4, the amplification product of 88 bp corresponding to the amplification chain length using normal sequence DNA as a template and the amplification product of 70 bp corresponding to the amplification chain length specific to Del-4 deletion sequence DNA are 1: 1. Detected in percentage.
Based on the above results and the results of Example 2, the highly sensitive detection of the defective sequence DNA in the presence of the majority of normal sequence DNAs requires that the 3 ′ end of the primer is specific to the defective sequence as used in Example 2 It was confirmed that a primer having a sequence different from the normal sequence by 2 to 3 bases was effective.

The detection limit of detection of the EGFR exon 19 deficient gene in the detection method of the present invention was examined.
That is, the 100 fg / μl 5 mol% defective sequence control template DNA solution prepared in Example 2 was further diluted with 100 fg / μl normal sequence control template DNA solution to obtain 1, 0.1, 0.01 mol% deficient. A sequence control template DNA solution was prepared. Using 100 fg of each of these DNAs, PCR amplification was performed under the same conditions as in Example 2, and the detection sensitivity for defective DNA in the presence of normal sequence DNA was examined. As a result, the deletion sequence control template DNA could be detected up to 1 mol% for Del-1a and Del-1b, and up to 0.1 mol% for Del-3 and Del-4.

  The simple detection using the cycling probe method of EGFR exon 19-deficient gene amplification product by deletion-specific primer multiplex PCR was examined. As a probe for detecting an EGFR exon 19 deletion sequence used for detection, a probe DF (SEQ ID NO: in the sequence listing) is labeled with FAM (Applied Biosystems) at the 5 ′ end and Eclipse quencher (Epoch) at the 3 ′ end. 16) and a probe NH (SEQ ID NO: 17 in the sequence listing) each labeled with HEX (Applied Biosystems) at the 5 ′ end and Eclipse quencher at the 3 ′ end were synthesized by a DNA synthesizer.

Using the above probe, simple detection using the cycling probe method of the EGFR exon 19-deficient gene amplification product by the deletion-specific primer multiplex PCR method was performed. The composition of the reaction solution was 10 × PCR buffer (Takara Bio Inc.), 100 fg of control template DNA containing 5 mol% of defective sequence as a template, 100 fg of normal sequence control template DNA or 100 ng of normal genomic DNA, 1.25 U TaKaRa Taq DNA polymerase (manufactured by Takara Bio Inc.) and 50U Tli RNase H II (Takara Bio International Publication No. 02/22831 pamphlet) are used, and final concentrations are 0.2 mM dNTP, 2 mM MgCl ₂ , 0.2 μM F1D1a primer, 0 .2 μM F2D1b primer, 0.2 μM F3D3 primer, 0.2 μM F4D4 primer, 0.2 μM F5D5 primer, 0.2 μM RV primer, 0.2 μM probe NH, 0.2 μM probe It was prepared in such a way that DF. The reaction solution volume is 25 μl. ABI7500 system (Applied Biosystems) was used for PCR amplification and fluorescence intensity detection. PCR conditions were carried out in a 40-cycle reaction with 95 ° C for 15 seconds, 60 ° C for 40 seconds, and 72 ° C for 30 seconds as one cycle. Simultaneously with PCR, the fluorescence intensity of FAM and HEX was measured by ABI7500 system.

  As a result, the increase in the fluorescence intensity of HEX caused by the cleavage of the probe NH in the region not subjected to the defect by RNase H was remarkably observed in all the reactions using the 5 mol% defective sequence control template DNA. On the other hand, the increase in the fluorescence intensity of FAM caused by the cleavage of the probe DF in the region commonly subjected to the defect by RNase H was hardly confirmed in all the reactions using the 5 mol% defective control template DNA as a template. On the other hand, in the reaction using the normal sequence control template DNA and the normal genomic DNA as templates, almost no increase in fluorescence intensity caused by cleavage of the probe DF with RNase H and cleavage of the probe NH with RNase H was confirmed. From this, it was confirmed that real-time detection was possible using the probe.

Further, in order to clarify the difference in fluorescence intensity between the case where the normal sequence DNA is used as a template and the case where the mutant sequence DNA is used as a template, the HEX fluorescence intensity and the HEX / FAM value per FAM fluorescence intensity in each sample are calculated. Calculated and plotted. The result is shown in FIG. Here, N is the HEX / FAM value when the normal sequence control template DNA is used as a template, N1-N5 is the HEX / FAM value when the normal genomic DNA is used as a template, Del1-1a, Del1-1b, Del -3 and Del-4 show HEX / FAM values when the defective sequence control template DNA is used as a template. As shown in FIG. 2, in the reactions using the normal sequence control template DNA and 5 types of normal genomic DNAs as templates, all showed extremely low HEX / FAM values of 0.5 or less, whereas 5 mol% In the defective sequence control template DNA, the HEX / FAM value was 5.8 to 52.5, and the sample containing the defective sequence control template DNA showed a value 10 times higher than the sample containing only the normal sequence DNA. . Thus, it was shown that the presence of the EGFR exon 19 deletion sequence can be clearly determined by using the HEX / FAM value as an index.
As described above, by combining the cycling probe method and the deletion sequence-specific primer multiplex PCR method, 5 mol% of EGFR exon 19-deficient gene can be detected even in the presence of a normal gene. It was confirmed that the detection method using a single tube was effective.

  The present invention provides a simple, rapid and highly sensitive method for detecting somatic deletion mutations in exon 19 of the EGFR gene with a non-constant deletion pattern. The detection method is useful in predicting the effects of anticancer agents, epidemiological studies, and disease prediction.

SEQ ID NO: 1: Control template DNA
SEQ ID NO: 2: Control template DNA CTDel-1a
SEQ ID NO: 3: Control template DNA CTDel-1b
SEQ ID NO: 4: Control template DNA CTDel-3
SEQ ID NO: 5: Control template DNA CTDel-4
SEQ ID NO: 6: PCR primer F1D1a to amplify a deletion mutant "Del-1a"
SEQ ID NO: 7: PCR primer F2D1b to amplify a deletion mutant "Del-1b"
SEQ ID NO: 8: PCR primer F3D3 to amplify a deletion mutant "Del-3"
SEQ ID NO: 9: PCR primer F4D4 to amplify a deletion mutant "Del-4"
SEQ ID NO: 10: PCR primer F5D5 to amplify a deletion mutant "Del-5"
SEQ ID NO: 11: PCR primer RV to amplify a deletion mutant of EGFR exon 19
SEQ ID NO: 12: PCR primer F1-2D1a to amplify a deletion mutant "Del-1a"
SEQ ID NO: 13: PCR primer F2-2D1b to amplify a deletion mutant "Del-1b"
SEQ ID NO: 14: PCR primer F3-2D3 to amplify a deletion mutant "Del-3"
SEQ ID NO: 15: PCR primer F4-2D4 to amplify a deletion mutant "Del-4"
SEQ ID NO: 16: Chimeric oligonucleotide probe DF to detect the DNA fragment of human mutant type EGFR exon 19. "nucleotides 4 is a ribonucleotides-other nucleotides are deoxyribonucleotides"
SEQ ID NO: 17: Chimeric oligonucleotide probe NH to detect the DNA fragment of human mutant type EGFR exon 19. "nucleotides 5 is a ribonucleotides-other nucleotides are deoxyribonucleotides"

Claims (12)

A method for detecting the presence or absence of a deletion mutation in exon 19 of the EGFR gene contained in a sample, which comprises the following steps:
Amplifying a target region in a sample using at least one primer for a defective sequence that can be annealed to a defective junction region of the EGFR gene and a primer opposite to the primer for the defective sequence, wherein 3 ′ of the primer for the defective sequence The terminal base sequence is a base sequence consisting of 2 to 5 bases derived from the non-deletion site on the 3 ′ side adjacent to the defective junction, and the primer opposite the defective sequence primer is a defective mutation or a normal gene. A primer capable of annealing; and detecting a deletion mutation based on the presence or absence of an amplification product.   The method according to claim 1, further comprising detecting a defective mutation in the same reaction system as the amplification reaction using at least two probes including a probe that recognizes a defective site and a probe that recognizes a non-defective site.   A primer for a deletion sequence selected from primers having a base sequence described in SEQ ID NO: 6 to 10 in the sequence listing and a primer opposite to the primer for a deletion sequence having a base sequence described in SEQ ID NO: 11 in the sequence listing are used. The method of claim 1 wherein:   The method according to claim 2, wherein a probe having the base sequence of any one of SEQ ID Nos. 16 and 17 in the sequence listing is used. A composition for a process according to claim 1, characterized in that it comprises:
(1) A primer for a defective sequence that can be annealed to a defective junction region of the EGFR gene, wherein the base sequence at the 3 ′ end of the primer for the defective sequence is derived from 2 derived from a non-defective site on the 3 ′ side adjacent to the defective junction It is a base sequence consisting of 5 bases.
(2) A primer opposite to the primer for the defective sequence of (1), wherein the primer is a primer capable of annealing to both a defective mutation and a normal gene, and (3) DNA polymerase. further,
(4) a probe for recognizing a defect site,
The composition according to claim 5, comprising (5) a probe for recognizing a non-deficient site, and (6) an enzyme for cleaving the probe when hybridized to a target sequence.   A deletion sequence primer selected from a primer having a base sequence described in SEQ ID NO: 6 to 10 in the sequence listing and a primer opposite to the deletion sequence primer having a base sequence described in SEQ ID NO: 11 in the sequence listing The composition according to claim 5.   The composition according to claim 6, comprising a probe having the base sequence set forth in SEQ ID NO: 16 or 17 in the sequence listing. A kit for the method according to claim 1, characterized in that it comprises:
(1) A primer for a defective sequence that can be annealed to a defective junction region of the EGFR gene, wherein the base sequence at the 3 ′ end of the primer for the defective sequence is derived from 2 derived from a non-defective site on the 3 ′ side adjacent to the defective junction (2) A primer opposite to the deletion sequence primer of (1), wherein the primer is a primer capable of annealing to both a deletion mutation and a normal gene. further,
The kit according to claim 9, comprising: (3) a probe that recognizes a defective site; and (4) a probe that recognizes a non-defective site.   A deletion sequence primer selected from a primer having a base sequence described in SEQ ID NO: 6 to 10 in the sequence listing and a primer opposite to the deletion sequence primer having a base sequence described in SEQ ID NO: 11 in the sequence listing The kit according to claim 9. The kit according to claim 10, comprising a probe having the base sequence of any one of SEQ ID NOS: 16 and 17 in the sequence listing.

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