JPWO2018199136A1 - Method for measuring expression level of ABL1 T315I mutation - Google Patents

Abstract

The present disclosure provides (1) a reverse binding to a region downstream of a T315I mutation position of ABL1 mRNA in the presence of a modified nucleic acid having a base sequence complementary to a region containing a T315I mutation position of wild type ABL1 mRNA. Reverse transcription of the RNA sample of interest using a primer and (b) a reverse primer that binds to a region upstream of the T315I mutation position of ABL1 mRNA in the same container, (2) reverse transcription with the primer of (b) And a kit therefor, which comprises the step of calculating the expression level of the ABL1 T315I mutation based on the ratio of the reverse transcription product by the primer of (a) to the product.

Description

This patent application claims priority to Japanese Patent Application No. 2017-0875578, which is hereby incorporated by reference in its entirety.
The present application relates to a method for measuring the expression level of ABL1 T315I mutation, or a kit therefor.

  The BCR-ABL1 fusion gene is a chromosomal abnormality found in chronic myelogenous leukemia (CML) and Philadelphia chromosome positive Acute Lymphocytic Leukemia (Ph positive ALL). The translocation t (9; 22) between chromosomes 9 and 22 fuses the ABL1 gene localized in the q34 band of chromosome 9 with the BCR gene localized in the q11 band of chromosome 22. It is formed. The chimeric protein BCR-ABL1 encoded by the BCR-ABL1 fusion gene has tyrosine kinase activity, constitutively stimulates cell proliferation signals, and acts to suppress apoptosis, thereby limiting hematopoietic stem cells indefinitely. Proliferate and cause abnormalities.

  As described above, since the chimeric protein BCR-ABL1 is one of the causative proteins of CML and Ph-positive ALL, a tyrosine kinase inhibitor (TKI) that acts with BCR-ABL1 as a target molecule is used as a therapeutic drug. Has been developed. Imatinib, a typical TKI, exerted a breakthrough therapeutic effect on CML and Ph-positive ALL cases. However, in the case where the BCR-ABL1 gene was mutated, its effect was reduced, which was a therapeutic problem. Nilotinib and dasatinib were subsequently developed as effective TKIs for many of the BCR-ABL1 gene mutations resistant to imatinib. However, in the case where the BCR-ABL1 gene was mutated so that threonine, which is the 315th amino acid in the ABL1 region of BCR-ABL1, was replaced by isoleucine (T315I mutation), the effect was significantly reduced (Non-patent document). 1-3). Conventionally, when T315I mutation occurs, the prognosis by TKI treatment is poor, and allogeneic stem cell transplantation has been the first choice, and early detection of T315I mutation before hematological recurrence has been desired.

  Ponatinib was developed as a drug having a tyrosine kinase inhibitory action also on BCR-ABL1 having this T315I mutation. Ponatinib was useful for existing TKI-resistant or intolerant CML and Ph-positive ALL, and showed a high effect, especially when having a T315I mutation (Non-patent Documents 4 and 5).

  Therefore, when a patient has a T315I mutation, it is necessary to quickly switch from the existing TKI to treatment with ponatinib. However, the direct sequencing method, pyrosequencing method, DHPLC method, high-resolution melting curve analysis method, PCR method using a specific primer, etc., which have been used for mutation analysis of BCR-ABL1 are not sufficiently sensitive, It was difficult to confirm the T315I mutation early (Non-patent documents 6 to 8). The late discovery of the T315I mutation and the progression of the stage increase the likelihood that remission cannot be achieved even with ponatinib effective for the T315I mutation. Therefore, early detection of the T315I mutation was considered to be an important medical problem.

  In the TKI treatment targeting BCR-ABL1, the therapeutic effect can be monitored by measuring the BCR-ABL1 mRNA expression level. Since the T315I mutation is resistant to many TKIs and has a poor prognosis, it is clinically useful to monitor the expression level of the BCR-ABL1 mRNA having the T315I mutation in the same manner as the BCR-ABL1 mRNA amount. Conceivable. It may also be useful for assessing the effects of ponatinib. However, the T315I mutation detection methods developed so far lack sufficient quantification and are insufficient for these purposes.

Druker BJ, Guilhot F, O'Brien SG, Gathmann I, Kantarjian H, Gattermann N, et al. Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia.N Engl J Med. 2006; 355: 2408-17 . Modugno M. New resistance mechanisms for small molecule kinase inhibitors of Abl kinase. Drug Discov Today Technol. 2014; 11: 5-10. Zabriskie MS, Eide CA, Tantravahi SK, Vellore NA, Estrada J, Nicolini FE, et al. BCR-ABL1 Compound Mutations Combining Key Kinase Domain Positions Confer Clinical Resistance to Ponatinib in Ph Chromosome-Positive Leukemia. Cancer Cell. 2014; 26: 428-42. Goldman JM. Ponatinib for chronic myeloid leukemia. N Engl J Med. 2012; 367: 2148-2149. Cortes JE, Kantarjian H, Shah NP, et al. Ponatinib in refractory Philadelphia chromosome-positive leukemias. N Engl J Med. 2012; 367: 2075-2088. Hofmann WK, Jones LC, Lemp NA, de Vos S, Gschaidmeier H, Hoelzer D, et al. Ph (+) acute lymphoblastic leukemia resistant to the tyrosine kinase inhibitor STI571 has a unique BCR-ABL gene mutation. Blood. 2002; 99 : 1860-2. Yamamoto M, Kakihana K, Ohashi K, Yamaguchi T, Tadokoro K, Akiyama H, et al. Serial monitoring of T315I BCR-ABL mutation by Invader assay combined with RT-PCR. Int J Hematol. 2009; 89: 482-8. Yin L, Dittman D, Chenn A. Rapid quantitative detection of the T315I mutation in patients with chronic myelogenous leukemia. Diagn Mol Pathol. 2012; 21: 34-39.

  An object of the present application is to provide a method for measuring the expression level of ABL1 T315I mutation in a subject, or a kit therefor.

  The present inventors used two reverse primers that bind an RNA sample of interest upstream and downstream of a T315I mutation site of ABL1 mRNA in the presence of a modified nucleic acid that specifically binds to a wild-type ABL1 mRNA gene. It was found that the expression level of the ABL1 T315I mutation can be measured by reverse transcribing with each other and comparing the amounts of the reverse transcription products with the two kinds of primers.

Accordingly, in one aspect, the present application provides a method of measuring the expression level of an ABL1 T315I mutation in a subject, comprising:
(1) In the presence of a modified nucleic acid having a base sequence complementary to a region containing a T315I mutation position of wild-type ABL1 mRNA, (a) a reverse primer that binds to a region downstream of the T315I mutation position of ABL1 mRNA, and ( b) reverse transcribing the RNA sample of interest using a reverse primer that binds to a region upstream of the T315I mutation position of ABL1 mRNA in the same container, and
(2) calculating the expression level of the ABL1 T315I mutation based on the ratio of the reverse transcript produced by the primer of (a) to the reverse transcript produced by the primer of (b),
A method including is provided.

In another aspect, the present application provides a kit for determining the expression level of ABL1 T315I mutation in a subject, comprising:
(A) a reverse primer that binds to a region downstream of the T315I mutation position of ABL1 mRNA;
(B) a reverse primer that binds to a region upstream of the T315I mutation position of ABL1 mRNA; and
(C) a modified nucleic acid having a base sequence complementary to the region containing the T315I mutation position of wild-type ABL1 mRNA;
A kit including is provided.

  According to the present invention, the expression level of ABL1 T315I mutation in a subject can be measured. It is expected that this will provide useful information for the treatment of patients with the T315I mutation.

This shows the base sequence of SEQ ID NO: 1. The 947th cytosine base is shown in bold and underlined. 1 shows the nucleotide sequence of SEQ ID NO: 1 (continuation). This shows the base sequence of SEQ ID NO: 3. Thymine base at 947 is shown in bold and underlined. The base sequence of SEQ ID NO: 3 (continuation) is shown. It is a schematic diagram of the reverse transcription reaction of wild type ABL1 mRNA using two reverse primers. It is a schematic diagram of the reverse transcription reaction of T315I mutant | variant ABL1 mRNA using two reverse primers. It is a schematic diagram of a reverse transcription reaction. It is a schematic diagram of quantitative real-time PCR of ABL1 mRNA. It is a schematic diagram of quantitative real-time PCR of T315I mutant type ABL1 mRNA. 8 shows amplification curves for amplification of cDNA derived from T315I reverse primer. 2 shows an amplification curve of amplification of cDNA derived from ABL reverse primer.

  Unless otherwise specifically defined, the terms used herein have the meanings as commonly understood by one of ordinary skill in the areas of organic chemistry, medicine, pharmacy, molecular biology, microbiology, and the like. Listed below are definitions for some of the terms used herein, but these definitions supersede the general understanding herein.

  As used herein, when a numerical value is accompanied by the term "about", it is intended to include a range of ± 10% of that value. For example, “about 20” includes “18 to 22”. The range of numerical values includes all numerical values between the endpoints and the numerical values of the endpoints. “About” with respect to a range applies to the endpoints of the range. Therefore, for example, “about 20 to 30” includes “18 to 33”.

  The ABL1 gene is a gene localized in the q34 band of chromosome 9. When exon 1, exons 1 to 13 or 1 to 14 of the BCR gene and exons 2 to 4 of the ABL1 gene are fused by translocation t (9; 22) between chromosomes 9 and 22, chronic bone marrow BCR-ABL1 gene found in adult leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia.

  The ABL1 gene typically has the nucleotide sequence of SEQ ID NO: 1, but many mutants are known. For purposes of this application, the ABL1 gene includes a polynucleotide that hybridizes under stringent conditions to a sequence complementary to the polynucleotide having the sequence of SEQ ID NO: 1. Regarding "hybridize under stringent conditions", the hybridization used herein is performed according to the usual method described in, for example, Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983). It can be carried out. The term “stringent conditions” means, for example, 6 × SSC (a solution containing 1.5M NaCl and 0.15M trisodium citrate is 10 × SSC) and a solution containing 50% formamide at 45 ° C. After forming a hybrid at 50 ° C. in 2 × SSC (Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6), and equivalent conditions. The conditions that bring about stringency can be mentioned.

  Furthermore, the ABL1 gene has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity with the polynucleotide having the sequence of SEQ ID NO: 1. Is included. Sequence identity refers to the degree of sequence similarity between two oligonucleotides, and refers to two sequences that are optimally aligned (states in which the sequences match) over the region of the base sequence to be compared. Determined by comparing. The sequence identity value (%) determines the number of matching sites by determining the same bases present in both sequences, and then dividing this number of matching sites by the total number of bases in the sequence region to be compared. It is calculated by multiplying the obtained numerical value by 100. Algorithms for obtaining optimal alignment and sequence identity include various algorithms commonly used by those skilled in the art (eg, BLAST algorithm, FASTA algorithm, etc.). The sequence identity of nucleotide sequences is determined using sequence analysis software such as BLAST and FASTA.

  In the present specification, of the ABL1 genes, the ABL1 gene in which the nucleotide at the T315I mutation position has cytosine is referred to as “wild-type ABL1 gene”, and its mRNA is referred to as “wild-type ABL1 mRNA”. Wild-type ABL1 mRNA typically has the nucleotide sequence of SEQ ID NO: 1 and the protein encoded by it has the amino acid sequence of SEQ ID NO: 2. For purposes of this application, "T315I mutation position" means the position corresponding to position 947 of SEQ ID NO: 1 when the nucleotide sequence of an ABL1 gene is optimally aligned with the nucleotide sequence of SEQ ID NO: 1.

  When the cytosine of the wild-type ABL1 gene is replaced with thymine, threonine corresponding to amino acid 315 of the ABL1 region of the BCR-ABL1 protein in the protein encoded by it is replaced with isoleucine. Such substitutions in the ABL1 gene or ABL1 protein are referred to herein as “T315I mutations”. The ABL1 gene having the T315I mutation is referred to as "T315I mutant ABL1 gene", and its mRNA is referred to as "T315I mutant ABL1 mRNA". The T315I mutant ABL1 mRNA typically has the nucleotide sequence of SEQ ID NO: 3 and the protein encoded by it has the amino acid sequence of SEQ ID NO: 4.

  In the context of the present application, “expression level of ABL1 T315I mutation” refers to the expression level of all genes having the T315I mutation, ie, the translocated ABL1 gene including the untranslocated ABL1 gene and the BCR-ABL1 gene having the T315I mutation. Means

  In the context of this application, the subject is typically a human. The subject may be one who has, or is suspected of having, chronic myelogenous leukemia (CML) or Philadelphia chromosome positive acute lymphoblastic leukemia (Ph + ALL). The subject may have the BCR-ABL1 gene.

  In the context of the present application, an "RNA sample" is RNA extracted from a sample containing hematopoietic stem cells, leukocytes or leukemia cells derived from a subject, which can be, for example, RNA extracted from blood, bone marrow fluid, lymph fluid. RNA extracted from isolated hematopoietic stem cells, blood cells, leukocytes or leukemia cells may be used. In one embodiment, the RNA sample is RNA extracted from peripheral blood leukocytes or bone marrow nucleated cells. In one embodiment, the RNA sample is RNA extracted from peripheral blood leukocytes.

  Any known method may be used to extract RNA from the sample. For example, a method of acidifying the solution in the PCI (phenol / chloroform / isoamyl alcohol extraction) method to extract from the aqueous layer, a method using a commercially available RNA extraction kit, and other known methods can be used. The RNA may be total RNA or purified mRNA.

  In step (1), a modified nucleic acid having a base sequence complementary to the region containing the T315I mutation position of wild-type ABL1 mRNA is used. In the present specification, the modified nucleic acid is referred to as "T315I clamp". The modified nucleic acid contains one or more artificial nucleotides and forms a complementary strand to mRNA having a certain sequence more strongly than DNA having the same base sequence as the modified nucleic acid, and is not decomposed by the exonuclease activity of reverse transcriptase. .. Reverse transcriptase stops the elongation reaction at the binding site when the modified nucleic acid is bound to the template mRNA.

  The T315I clamp binds to the wild-type ABL1 mRNA having a cytosine-containing nucleotide at the T315I mutation position and does not bind to the T315I-mutant ABL1 mRNA having a thymine at the nucleotide, thus suppressing the reverse transcription of the wild-type ABL1 mRNA. However, it is designed not to suppress reverse transcription of T315I mutant ABL1 mRNA. Specifically, the T315I clamp comprises about 10 to 22, about 12 to 20, about 14 to 18 or about 15 to 17, including about 16 nucleotides containing nucleotides at the T315I mutation position of wild-type ABL1 mRNA. It may be a modified nucleic acid complementary to a region consisting of nucleotides. In certain embodiments, the T315I clamp comprises the nucleotide sequence 5'-ATGAACTCAGTGATGA-3 '(SEQ ID NO: 5). In one embodiment, the T315I clamp consists of the base sequence of SEQ ID NO: 5.

  The T315I clamp contains one or more artificial nucleotides. An artificial nucleotide means a nucleotide having a modified nucleoside (base portion or sugar portion) and a structure different from that of a natural nucleotide. As the artificial nucleotide, one that enhances nuclease resistance and binding affinity with the target sequence can be selected. For example, artificial leaves described in Deleavey, GF, & Damha, MJ (2012). Designing chemically modified oligonucleotides for targeted gene silencing. Chemistry & biology, 19 (8), 937-954, which is incorporated herein by reference. Nucleotides may be used. Examples of artificial nucleotides include, for example, abasic nucleosides; arabinonucleosides, 2'-deoxyuridine, α-deoxyribonucleosides, β-L-deoxyribonucleosides, nucleosides having other sugar modifications; peptide nucleic acids (PNA). ), A peptide nucleic acid having a phosphate group bound thereto (PHONA), a cross-linked artificial nucleic acid (LNA), 2′-O, 4′-C-ethylene cross-linked nucleic acid (ENA), constrained ethyl (cEt), morpholino nucleic acid, and the like. .. Nucleosides with sugar modifications include substituted pentasaccharides such as 2'-O-methylribose, 2'-O-methoxyethylribose, 2'-deoxy-2'-fluororibose, 3'-O-methylribose; Included are 1 ', 2'-deoxyribose; arabinose; substituted arabinose sugars; hexosides and nucleosides with alpha-anomeric sugar modifications. Examples of modified bases include pyrimidines such as 5-hydroxycytosine, 5-methylcytosine, 5-fluorouracil, and 4-thiouracil; purines such as 6-methyladenine and 6-thioguanosine; and other heterocyclic bases. Is mentioned. The artificial nucleotides in the T315I clamp may all be of the same type or there may be two or more different artificial nucleotides.

  In some embodiments, the T315I clamp contains one or more PNAs as artificial nucleotides. PNA generally produces a structure in which N- (2-aminoethyl) glycine is peptide-bonded to replace the phosphate bond of DNA with a peptide bond (Nielsen et al. 1991 Science 254, 1457-1500). PNA is resistant to various nucleolytic enzymes and forms a complementary strand with DNA or RNA by the same molecular recognition as DNA or RNA. The affinity of PNA and DNA is higher than that of DNA and DNA or DNA and RNA. In one embodiment, the nucleotides in the T315I clamp are all PNAs.

  The amount of T315I clamp added may be any amount capable of suppressing reverse transcription of wild-type ABL1 mRNA. For example, the final volume molar concentration of the reverse transcription reaction solution is about 0.1 μM to 10 μM or about 0.5 μM to 5 μM. , For example, about 2 μM.

  In step (1), for reverse transcription, two types of reverse primers, namely (a) a reverse primer that binds to a region downstream of the T315I mutation position of ABL1 mRNA and (b) a T315I mutation position of ABL1 mRNA, A reverse primer is used that binds to the upstream region. The reverse primer in (a) reverse-transcribes a portion of ABL1 mRNA containing a region to which the T315I clamp binds. In the present specification, the reverse primer is referred to as “T315I reverse primer”. The reverse primer in (b) reverse-transcribes a part of ABL1 mRNA upstream of the region to which the T315I clamp binds. In the present specification, the reverse primer (b) is referred to as “ABL reverse primer”. Methods for designing suitable primers for reverse transcription are well known to those of skill in the art.

  In the step (1), reverse transcription with the T315I reverse primer and the ABL reverse primer is performed in one step, that is, in the same container, simultaneously or successively. In one embodiment, reverse transcription with the T315I reverse primer and the ABL reverse primer is performed simultaneously in the same container. Schematic diagrams of reverse transcription reactions of wild-type ABL1 mRNA and T315I mutant ABL1 mRNA using these reverse transcription primers and T315I clamp are shown in FIGS. 3 and 4, respectively.

  As the reagents such as reverse transcriptase, dNTP and buffer, those usually used in this field can be used, and the conditions such as the amount of various reagents, reaction time and temperature can be determined according to the instructions attached to the reverse transcriptase. It can be appropriately determined by a known method such as a description or a commonly used protocol. For example, the reverse transcriptase is any known reverse transcriptase that can be used in molecular biology experiments and the like, for example, Tth DNA polymerase, rTth DNA polymerase, AMV reverse transcriptase, MMLV reverse transcriptase, HIV reverse transcriptase and the like, or those thereof. Derivatives may be used.

  In the step (2), the expression level of the ABL1 T315I mutation is calculated based on the ratio of the reverse transcript produced by the T315I reverse primer in (a) to the reverse transcript produced by the ABL reverse primer in (b). For example, the value obtained by quantifying the reverse transcript by the T315I reverse primer in (a) is divided by the value obtained by quantifying the reverse transcript by the ABL reverse primer in (b), and the measured value of the expression level of the ABL1 T315I mutation is obtained. To do. Quantitation of reverse transcripts may be performed by any method known in the art. For example, quantitative PCR, electrophoresis of nucleic acid after amplification, hybridization method using a nucleic acid probe to which a detectable label is bound, double-stranded DNA staining with intercalating fluorescent dye, fluorescent probe method and the like can be mentioned. ..

In one embodiment, step (2) of the above method comprises the step of quantifying the reverse transcription product with the T315I reverse primer of step (2-1) (a) by quantitative PCR, and step (2-2) (b). Quantifying the reverse transcription product by the ABL reverse primer of 1. by quantitative PCR. The quantitative PCR may be, for example, quantitative real-time PCR. Various fluorescent PCR techniques can be used for quantitative real-time PCR. Examples of the fluorescent PCR technique include an intercalator method using a fluorescent nucleic acid labeling agent such as SYBR GREEN I (for example, LightCycler ^{(registered trademark)} (Roche), ABI Prizm 7700 Sequence Detection System ^{(registered trademark)} (Perkin Elmer, Applied Biosystems)), a TaqMan probe method for monitoring amplification in real time using the 5'exonuclease activity of DNA polymerase, a RNase activity of RNase H enzyme and a cycling probe using a dedicated chimeric RNA probe. However, the method is not limited to this.

  In one embodiment, quantitative real-time PCR is performed by the TaqMan probe method. Generally, in the TaqMan probe method, an oligonucleotide (TaqMan probe) having a 5'end modified with a fluorescent substance and a 3'end modified with a quencher substance is added to a PCR reaction system. The TaqMan probe hybridizes specifically to the template DNA in the annealing step, but since the quencher is present on the probe, the emission of fluorescence is suppressed even when irradiated with excitation light. When the TaqMan probe hybridized to the template is decomposed by the 5'exonuclease activity of Taq polymerase in the extension reaction step, the fluorescent dye is separated from the quencher and fluorescence is emitted. The TaqMan probe may be attached anywhere in the PCR product and designed by methods well known in the art. Also, any combination of fluorescent and quencher substances may be used.

  In quantitative real-time PCR, a standard solution containing a known concentration of cDNA and a cDNA of unknown concentration (sample) were simultaneously amplified, and the horizontal axis represents the number of amplification cycles and the vertical axis represents the fluorescence intensity (logarithmic conversion value) of the reporter dye. An amplification curve may be created. A line parallel to the horizontal axis is drawn near the median value of fluorescence intensity where the amplification curve becomes linear, and the number of amplification cycles when the line intersects each amplification curve can be obtained. In addition, a standard curve is created by plotting the concentration of each standard solution (logarithmic conversion value) on the horizontal axis and the amplification cycle number of the standard solution on the vertical axis, and applying the amplification cycle number of the sample on this standard curve, Can be calculated.

  The primer pairs used in steps (2-1) and (2-2) are designed so that the reverse transcription product of step (1) can be amplified. Methods for designing primers suitable for PCR are well known to those skilled in the art. The primer pair is designed such that the amplified nucleic acid has a length suitable for quantification, eg, about 10-1000, about 20-500, about 50-300, about 100-200 nucleotides, for example about 150 nucleotides in length. obtain.

  The primer pair used in step (2-1) is designed so that the region containing the T315I mutation position of the reverse transcript can be amplified. That is, a forward primer having a base sequence of a region containing the T315I mutation position of the ABL gene or an upstream region thereof, and a base sequence complementary to the base sequence of a region containing the T315I mutation position of the ABL1 gene or a region downstream thereof. The reverse primer with is used. The forward primer used in the step (2-1) has a nucleotide sequence of a region containing the T315I mutation position of the ABL gene, and the nucleotide corresponding to the nucleotide at the T315I mutation position is replaced with uracil ribonucleotide. Good. In one embodiment, in step (2-1), the same T315I reverse primer as in step (1) (a) is used. In one embodiment, in step (2-2), the same ABL reverse primer as in step (1) (b) is used.

  In the step (2-1), the reverse transcription product by the T315I reverse primer may be quantified by RNaseH-dependent quantitative PCR specific for the T315I mutation. RNaseH-dependent PCR is a sequence-specific PCR method utilizing RNaseH (Boucard AA, et. Al. J Biol Chem, 289 (1): 387-402; Dobosy JR, et al., BMC Biotechnol, 11 ( 80): 1-18). RNase H recognizes an RNA / DNA heteroduplex and cleaves the phosphodiester bond with the 5'terminal DNA of RNA. In RNaseH-dependent PCR, RNA is used as at least one base of a primer to form a template DNA and an RNA / DNA heteroduplex, and a region (blocking region) to which a DNA polymerase cannot bind is provided on the 3 ′ side of RNA. This allows PCR to proceed only when RNase H recognizes the RNA / DNA heteroduplex and cleaves it. In order for RNase H to cleave the phosphodiester bond between RNA and DNA, the RNA in the primer must be complementary to the template DNA and not a mismatch.

  The RNase H-dependent quantitative PCR in step (2-1) has a nucleotide sequence of a region containing the T315I mutation position of the ABL1 gene, and the nucleotide corresponding to the nucleotide at the T315I mutation position is replaced with uracil ribonucleotide. A forward primer having a blocking region at the 3'end is used. In the present specification, the forward primer is referred to as "T315I forward primer". The uracil ribonucleotide in the T315I forward primer causes a mismatch with the nucleotide of the template in PCR of wild type ABL cDNA, so that the blocking region is not cleaved and PCR does not proceed. On the other hand, no mismatch occurs in PCR of T315I mutant ABL cDNA, so the blocking region is cleaved and PCR proceeds. Therefore, the T315I mutant ABL cDNA can be specifically amplified by RNaseH-dependent PCR using the T315I forward primer.

  The primer used for RNaseH-dependent PCR can be appropriately designed by those skilled in the art with reference to, for example, the protocol provided by Integrated DNA technologies. The T315I forward primer contains a sequence of a region upstream of the T315I mutation position of the ABL1 gene, and after the RNA and the blocking region are cut off, the remaining oligonucleotide is designed to function as a forward primer for PCR reaction. The T315I forward primer comprises, for example, a sequence of about 8-60, 10-30, 15-25 or 19-23 nucleotides upstream of the T315I mutation position of the ABL1 mRNA, for example about 20, 21 or 22 nucleotides. The nucleotide corresponding to the nucleotide at the T315I mutation position in the T315I forward primer is a uracil ribonucleotide. The blocking region on the 3'side contains a mismatching DNA and a blocking group such as a C3 spacer. For example, the blocking region consists of four deoxyribonucleotides having the same bases as the 945th to 948th nucleotides of the ABL1 gene, one deoxyribonucleotide having a different base from the 949th nucleotide, and one blocking group. In another example, the blocking region is one deoxyribonucleotide having the same base as the 945th nucleotide of the ABL1 gene, two blocking groups, one deoxyribonucleotide having the same base as the 946th nucleotide, and the 947th position. Consists of one deoxyribonucleotide having a base different from that of the nucleotide.

  In certain embodiments, the T315I forward primer is 5'-GAGCCCCCGTTCTATCATCATArUT / iSpC3 // iSpC3 / GC-3 '(SEQ ID NO: 6, where rU is uracil RNA and iSpC3 is spacer C3).

  Steps (2-1) and (2-2) may be performed in the presence of a T315I clamp. Therefore, the reaction can be carried out by adding the reagent necessary for step (2-1) or (2-2) to all or part of the product of step (1). For example, the steps (1) and (2-1) or the steps (1) and (2-2) can be performed in one step, that is, in the same container, simultaneously or successively.

  When RNaseH-dependent PCR is performed in step (2-1) and steps (1) and (2-1) are performed in one step, part of the T315I clamp and part of the T315I forward primer bind to each other complementarily. There are times when you can. Generally, the ability of an oligonucleotide to discriminate between single base mutations is thought to be maximal when the mutation site is in the center of the oligonucleotide, but to reduce this complementary binding, the T315I clamp is used to generate a T315I clamp. The nucleotide corresponding to the mutation position may be designed to be located 3 ′ to the center of the T315I clamp. That is, the T315I clamp can be designed such that the complementary regions of the T315I clamp and the T315I forward primer are less than 50% of the total length of the T315I clamp. For example, the T315I clamp consisting of the nucleotide sequence of SEQ ID NO: 5 has been optimized so that it has less complementary binding to the forward primer used for RNaseH-dependent PCR and has the ability to discriminate mutations. In addition, such adjustment of the modified nucleic acid sequence can be applied not only to the detection of the T315I mutation but also to other mutations. That is, when the reverse transcription reaction of wild-type mRNA is inhibited by the modified nucleic acid and the subsequent PCR reaction is inhibited by the RNaseH-dependent PCR, a portion of less than 50% of the total length of the modified nucleic acid is the forward primer of the RNaseH-dependent PCR. It is better to adjust so as to complementarily bind with.

  As the reagents such as DNA polymerase, RNaseH, dNTP, and buffer, those usually used in this field can be used, and the conditions such as the amount of various reagents, reaction time, temperature, etc. are described in the instructions attached to the enzyme. It can be appropriately determined by a known method such as or a commonly used protocol. For example, the DNA polymerase may be any known DNA polymerase that can be used in molecular biology experiments and the like, for example, rTth DNA polymerase, Taq polymerase and derivatives thereof. For example, as RNaseH, any known RNaseH, such as RNaseH2, that can be used in molecular biology experiments and the like can be used.

In one embodiment, the primers, clamps and probes in the table below are used. All of these may be used in combination, or at least one may be used.

In one aspect, the application provides a kit for performing the above method. At least the kit
(A) a reverse primer that binds to a region downstream of the T315I mutation position of ABL1 mRNA;
(B) a reverse primer that binds to a region upstream of the T315I mutation position of ABL1 mRNA; and
(C) a modified nucleic acid having a base sequence complementary to the region containing the T315I mutation position of wild-type ABL1 mRNA;
including.

  The kit may further comprise at least one of a forward primer for PCR amplification of the reverse transcript of ABL1 mRNA with the reverse primer of (a) and (b) and an additional reverse primer for PCR. The primer may be for RNase H dependent PCR. The kit may further include a probe for quantitative PCR. The kit may further comprise a standard, eg, a known amount of at least one of wild-type ABL1 mRNA and T315I mutant ABL1 mRNA.

  The kit may further comprise reagents necessary for performing the above method, such as reverse transcriptase, DNA polymerase, RNaseH, dNTPs, buffer, etc. for reverse transcription, PCR, quantitative PCR or RNaseH dependent PCR. .. The kit may further include other components desirable from a commercial and user standpoint, such as a package insert with instructions for use (eg, written or storage media, etc.).

  Each of the components included in the kit, individually or, if possible, in a mixture, dissolved in water or an appropriate buffer, or lyophilized, is stored in an appropriate container. Can be provided. Suitable containers include bottles, vials, test tubes, tubes, plates, multiwell plates and the like. The container may be formed from at least one material such as glass, plastic, metal and the like. The container may have a label.

  Hereinafter, the measurement principle of an example of the embodiment of the method and kit of the present application will be described using schematic diagrams, but the present invention is not limited to any theory.

1. Outline of measurement principle The expression level of T315I mutant ABL1 mRNA in which the 944th base of ABL1 mRNA in RNA extracted from peripheral blood leukocytes or bone marrow fluid nucleated cells is mutated from cytosine to thymine is measured. The measurement principle is a two-step two-step quantitative RT-PCR method consisting of a reverse transcription reaction (RT) for synthesizing complementary DNA (cDNA) using RNA as a template and real-time PCR for quantifying cDNA using a fluorescent labeled probe. Is. A region containing the T315I mutation position of T315I mutant ABL1 mRNA and a region not containing the T315I mutation position of all ABL1 mRNAs were respectively amplified, and the ratio obtained by dividing the former quantitative value by the latter quantitative value is taken as the reported value.

1.1 Reverse transcription reaction The outline of the reverse transcription reaction is shown in FIG. ABL1 mRNA in which the 944th base is cytosine is defined as wild-type ABL1 mRNA, and ABL1 mRNA in which thymine is thymine is defined as T315I mutant ABL1 mRNA. Two kinds of primers, an ABL reverse primer and a T315I reverse primer, are used for reverse transcribing a region for quantifying all ABL1 mRNA and a region for quantifying T315I mutant ABL1 mRNA, respectively.

  First, the ABL reverse primer and the T315I reverse primer bind to complementary sequences contained in the measurement sample (FIGS. 5 (1a) and (1b)). Starting from these reverse primers, the first-strand cDNA is synthesized by the reverse transcription activity of Tth DNA polymerase contained in the reaction solution (FIG. 5 (2a) or (2b)). The reaction solution contained a T315I clamp having a sequence complementary to the wild-type ABL1 mRNA, and when the template was the wild-type ABL1 mRNA during the synthesis of the first strand cDNA, the T315I clamp specifically bound, Since the reverse transcription reaction is inhibited (FIG. 5 (2a)), the amount of T315I reverse primer-derived cDNA synthesized is reduced (FIG. 5 (3a)). On the other hand, when the template is T315I mutant ABL1 mRNA, the T315I clamp does not bind and the reverse transcription reaction is not inhibited (FIGS. 5 (2b) and (3b)). Further, since the synthesis of cDNA derived from the ABL reverse primer is not affected by the T315I clamp, cDNA is similarly synthesized for the wild type ABL1 mRNA and the T315I mutant type ABL1 mRNA.

1.2 Quantitative real-time PCR of ABL1 mRNA
The outline of ABL1 mRNA quantitative real-time PCR is shown in FIG. ABL1 mRNA-derived cDNA synthesized by reverse transcription is amplified by real-time PCR (FIG. 6). In this amplification process, the fluorescence-labeled probe (ABL probe) bound to one side of the double-stranded cDNA is decomposed by the 5'-3 'exonuclease activity of Tth DNA polymerase in the reaction solution, and the reporter dye is released. By measuring the fluorescence intensity of the released dye for each cycle, the increase in the amount of cDNA derived from ABL1 mRNA is measured in real time (FIG. 6 (6)).

1.3 Quantitative real-time PCR of T315I mutant ABL1 mRNA
When amplifying the cDNA derived from the T315I mutant ABL1 mRNA, the principle of RNaseH-dependent PCR (rhPCR) is used to enhance the reaction specificity. RNase H2, which is a nucleic acid-degrading enzyme used in rhPCR, has the property of recognizing RNA / DNA heteroduplex and cleaving the phosphodiester bond with the 5'terminal DNA of RNA. In rhPCR, one deoxyribonucleotide of a primer is changed to a ribonucleotide to form a template DNA and an RNA / DNA heteroduplex, and a region (blocking region) to which a DNA polymerase cannot bind is provided on the 3 ′ side of RNA. PCR proceeds only when RNase H2 recognizes and cleaves the RNA / DNA heteroduplex. Using this principle, only the cDNA derived from the T315I mutant ABL1 mRNA in which the 944th base of ABL1 mRNA is thymine is amplified. The outline of quantitative real-time PCR of T315I mutant ABL1 mRNA is shown in FIG.

  In the T315I forward primer, the portion corresponding to the 944th base in the T315I mutant ABL1 mRNA was replaced with DNA (thymine) by RNA (uracil) (FIG. 7 (1a)). The RNA substitution base of the T315I forward primer does not form an RNA / DNA heteroduplex with respect to the wild-type ABL1 mRNA-derived cDNA, so that the blocking region is not cleaved by RNaseH2 and the PCR reaction does not occur (Fig. 7 ( 2a)).

  On the other hand, the RNA substitution base of the T315I forward primer forms an RNA / DNA heteroduplex with respect to the cDNA derived from the T315I mutant ABL1 mRNA, the blocking region is cleaved by RNaseH2, and the cDNA derived from the T315I mutant ABL1 mRNA is Specific amplification proceeds (Fig. 7 (1b), (2b)). The specific amplification of the cDNA derived from the T315I mutant ABL1 mRNA is repeated by PCR (FIGS. 7 (3) to (9)). In this amplification process, the fluorescence-labeled probe (ABLT315I probe) bound to one side of the double-stranded cDNA is decomposed by the 5'-3 'exonuclease activity of Tth DNA polymerase in the reaction solution, and the reporter dye is released. By measuring the fluorescence intensity of the released dye in each cycle, the increase in the amount of cDNA derived from the T315I mutant ABL1 mRNA is measured in real time (FIG. 7 (6)).

In another aspect, the present application provides a method of detecting an ABL1 T315I mutation in a subject, comprising:
(1) Using a reverse primer that binds to a region downstream of the T315I mutation position of ABL1 mRNA in the presence of a modified nucleic acid having a base sequence complementary to the region containing the T315I mutation position of wild-type ABL1 mRNA, Reverse transcribing the RNA sample,
(2) Using a forward primer having a nucleotide sequence of a region containing the T315I mutation position of ABL1 mRNA, the nucleotide corresponding to the nucleotide at the T315I mutation position is uracil ribonucleotide, and having a blocking region at the 3'end, ( Amplifying the reverse transcript of 1) by RNaseH-dependent PCR,
(3) A step of determining that the subject expresses the ABL1 T315I mutation when the nucleic acid is amplified in the step (2),
A method including is provided.

In another aspect, the present application is a kit for performing the above method, comprising:
(A) A forward primer having a nucleotide sequence of a region containing the T315I mutation position of ABL1 mRNA, in which the nucleotide corresponding to the nucleotide at the T315I mutation position is replaced with uracil ribonucleotide, and having a blocking region at the 3 ′ end;
(B) a reverse primer that binds to a region downstream of the T315I mutation position of ABL1 mRNA; and
(C) a modified nucleic acid having a base sequence complementary to the region containing the T315I mutation position of wild-type ABL1 mRNA;
A kit including is provided.

  The details of this method and kit follow the description of the method and kit for measuring the expression level of the ABL1 T315I mutation in the above-mentioned subject.

The present application provides the following embodiments, for example.
[1] A method for measuring the expression level of an ABL1 T315I mutation in a subject, comprising:
(1) In the presence of a modified nucleic acid having a base sequence complementary to a region containing a T315I mutation position of wild-type ABL1 mRNA, (a) a reverse primer that binds to a region downstream of the T315I mutation position of ABL1 mRNA, and ( b) reverse transcribing the RNA sample of interest using a reverse primer that binds to a region upstream of the T315I mutation position of ABL1 mRNA in the same container, and
(2) calculating the expression level of the ABL1 T315I mutation based on the ratio of the reverse transcript produced by the primer of (a) to the reverse transcript produced by the primer of (b),
Including the method.
[2] The step (2) is
Step (2-1) quantifying the reverse transcription product by the primer of (a) by quantitative PCR, and
Quantifying the reverse transcription product by the primer of step (2-2) (b) by quantitative PCR,
The method of paragraph 1, further comprising:
[3] The quantitative PCR of step (2-1)
(X) having a nucleotide sequence of a region containing the T315I mutation position of the ABL gene, wherein the nucleotide corresponding to the nucleotide at the T315I mutation position is replaced with uracil ribonucleotide, or
(Y) having a nucleotide sequence of a region upstream from the T315I mutation position of ABL mRNA,
The method according to item 2, wherein a forward primer is used.

[4] The method according to item 3, wherein the forward primer is the forward primer of (X).
[5] The method according to item 4, wherein the forward primer has a blocking region at the 3 ′ end.
[6] The method according to any one of items 2 to 5, wherein the quantitative PCR of step (2-1) is an RNaseH-dependent quantitative PCR specific for the T315I mutation.
[7] The method according to any one of items 2 to 6, wherein a forward primer containing the nucleotide sequence of SEQ ID NO: 6 is used in step (2-1).
[8] The method according to any one of items 2 to 7, wherein a forward primer consisting of the nucleotide sequence of SEQ ID NO: 6 is used in step (2-1).
[9] A part of the modified nucleic acid and a part of the forward primer of (X) are complementary, and the complementary region is less than 50% of the total length of the modified nucleic acid. The method described in either.
[10] The method according to any one of items 1 to 9, wherein the nucleotide corresponding to the T315I mutation position in the modified nucleic acid is located 3 ′ from the center of the modified nucleic acid.
[11] The method according to any one of items 1 to 10, wherein the modified nucleic acid comprises the base sequence of SEQ ID NO: 5.
[12] The method according to any one of items 1 to 11, wherein the modified nucleic acid comprises the base sequence of SEQ ID NO: 5.
[13] The method according to any one of items 1 to 12, wherein the modified nucleic acid contains PNA.

[14] The method according to any one of items 1 to 12, wherein the reverse primer in (a) of step (1) contains the sequence of SEQ ID NO: 7.
[15] The method according to any one of items 1 to 14, wherein the reverse primer in (b) of step (1) contains the sequence of SEQ ID NO: 10.
[16] The method according to any one of items 1 to 15, wherein the reverse primer in step (1) (a) comprises the sequence of SEQ ID NO: 7.
[17] The method according to any one of items 1 to 16, wherein the reverse primer in (b) of step (1) comprises the sequence of SEQ ID NO: 10.
[18] The method according to any one of items 2 to 17, wherein step (2-1) is performed in the presence of the modified nucleic acid used in step (1).
[19] The method according to any one of items 1 to 18, wherein the subject is a human.
[20] The method according to any one of Items 1 to 19, wherein the RNA sample is RNA extracted from peripheral blood leukocytes or bone marrow fluid nucleated cells.
[21] The method according to any one of items 1 to 19, wherein the RNA sample is RNA extracted from peripheral blood leukocytes.
[22] The method according to any one of items 1 to 21, which uses at least one of the primer, the clamp, and the probe described in Table 1.
[23] The method according to any one of items 1 to 22, which uses the primer, clamp, and probe described in Table 1.

[24] A kit for measuring the expression level of an ABL1 T315I mutation in a subject, comprising:
(A) a reverse primer that binds to a region downstream of the T315I mutation position of ABL1 mRNA;
(B) a reverse primer that binds to a region upstream of the T315I mutation position of ABL1 mRNA; and
(C) a modified nucleic acid having a base sequence complementary to the region containing the T315I mutation position of wild-type ABL1 mRNA;
A kit containing.
[25] The kit according to Item 24, further comprising a forward primer for amplifying a reverse transcription product of ABL mRNA by the reverse primer of (a) and (b) by PCR.
[26] The forward primer is
(X) having a nucleotide sequence of a region containing the T315I mutation position of ABL1 mRNA, wherein the nucleotide corresponding to the nucleotide of the T315I mutation is replaced with uracil ribonucleotide, or
(Y) has a nucleotide sequence of a region upstream from the T315I mutation position of ABL1 mRNA,
The kit according to Item 25, which is a forward primer.
[27] The kit according to Item 26, wherein the forward primer is the (X) forward primer.
[28] The kit according to Item 27, wherein the forward primer has a blocking region at the 3 ′ end.
[29] The kit according to any of paragraphs 25 to 28, wherein the forward primer comprises the sequence of SEQ ID NO: 6.
[30] The kit according to any of items 25 to 29, wherein the forward primer comprises the sequence of SEQ ID NO: 6.

[31] A part of the modified nucleic acid and a part of the forward primer of (X) are complementary, and the complementary region is less than 50% of the total length of the modified nucleic acid. The kit according to any one.
[32] The kit according to any one of items 24 to 31, wherein the nucleotide corresponding to the T315I mutation position in the modified nucleic acid is located 3 ′ from the center of the modified nucleic acid.
[33] The kit according to any of items 24 to 32, wherein the modified nucleic acid comprises the sequence of SEQ ID NO: 5.
[34] The kit according to any of items 24 to 33, wherein the modified nucleic acid comprises the sequence of SEQ ID NO: 5.
[35] The kit according to any of paragraphs 24 to 34, wherein the modified nucleic acid comprises PNA.
[35] The kit according to any of items 24 to 35, wherein the reverse primer of (a) contains the sequence of SEQ ID NO: 7.
[36] The kit according to any of items 24 to 35, wherein the reverse primer of (b) contains the sequence of SEQ ID NO: 10.
[37] The kit according to any of items 24 to 36, wherein the reverse primer of (a) comprises the sequence of SEQ ID NO: 7.
[38] The kit according to any of paragraphs 24 to 37, wherein the reverse primer of (b) comprises the sequence of SEQ ID NO: 10.
[39] The kit according to any of items 24 to 38, which further comprises a probe for quantitative PCR.
[40] The kit according to any of paragraphs 24 to 39, further comprising at least one of wild-type ABL1 mRNA and ABL1 mRNA having a T315I mutation.
[41] The kit according to any one of items 24 to 40, which comprises at least one of the primer, clamp and probe described in Table 1.
[42] The kit according to any one of items 24 to 41, which comprises the primer, clamp and probe described in Table 1.

All documents cited in this specification are incorporated herein by reference.
The above description is all non-limiting and can be modified without departing from the scope of the invention as defined in the appended claims. Furthermore, the following examples are all non-limiting examples, which are provided solely to illustrate the present invention.

Design of primer and probe The following primer set and probe were designed and synthesized.
Regarding the fluorescently labeled probe, the 5 ′ end of the probe was labeled with HEX (6-carboxyfluorescein), and the 3 ′ end of the probe was labeled with Iowa Black FQ (Integrated DNA technologies) as a quenching dye.
Table 2 shows the details of the primers and probes in this example.

[Test 1]
(1) Preparation of standard product (ABL1 T315I mutant RNA) As a standard product, a synthetic RNA containing a sequence of ABL1 mRNA having a T315I mutation was used. In order to prepare a DNA fragment that serves as a template for synthetic RNA, a plasmid vector containing the PCR amplification region of the above-mentioned primers in wild-type ABL1 mRNA and the T7 promoter sequence serving as the origin of RNA synthesis was prepared. A single nucleotide mutation was introduced into the portion corresponding to the T315I mutation of the ABL1 mRNA-derived sequence contained in this plasmid vector by the site-directed mutagenesis method. The plasmid vector introduced with this mutation was transformed into Escherichia coli. Escherichia coli was cultured to prepare a large amount of plasmid vector, which was cleaved at one site with a restriction enzyme to prepare a linear DNA fragment having a sequence encoding ABL1 mRNA having a T315I mutation.

Using T7 RNA polymerase, RNA containing the sequence of ABL1 mRNA having T315I mutation (ABL1 T315I mutant RNA) was synthesized using the above DNA fragment as a template. The synthesized RNA was diluted with TE buffer containing 100 ng / μL of E. coli transfer RNA to prepare an RNA standard product. The thus prepared ABL1 T315I mutant RNA standard was adjusted to a concentration of 1 × 10 ² , 1 × 10 ³ , 1 × 10 ⁴ , 1 × 10 ⁵ , 1.0 × 10 ⁶ , 1 × 10 ⁷ copies / test. .. As a negative control product, TE buffer containing 100 ng / μL of E. coli transfer RNA was used.

(2) Reverse transcription reaction (2-1) Preparation of reaction solution The volume of the reaction solution was 50 μL, the T315I reverse primer and the ABL reverse primer (Integrated DNA technologies) were each at a final concentration of 0.2 μM, and dNTPs (Toyobo Co., Ltd.) were used. Final concentration 0.1 mM, MnOAc (Toyobo) final concentration 2.4 mM, PNA clamp (Panagene) 2 μM final concentration, rTth DNA polymerase (Toyobo) 2.5 U per reaction, test RNA per reaction It was adjusted to 1 μg. One sample standard for each concentration was used in the reaction. The PNA clamp had a base sequence of ATGAACTCAGTGATGA (SEQ ID NO: 5) and had all nucleotides peptide-bonded.
(2-2) Reaction conditions :
The reverse transcription reaction was performed at 60 ° C. for 60 minutes.

(3) PCR
In the PCR reaction, cDNA of T315I reverse primer-derived cDNA and ABL reverse primer-derived cDNA (control) were amplified in separate tubes.
(3-1) Measurement of cDNA derived from T315I reverse primer (a) Preparation of reaction solution The volume of the reaction solution was 30 μL, and the final concentration of T315I forward primer and T315I reverse primer (Integrated DNA technologies) were 0.3 μM and T315I fluorescence, respectively. Labeled probe (Integrated DNA technologies) at a final concentration of 0.15 μM, dNTPs (Toyobo) at a final concentration of 0.2 mM, MnOAc (Toyobo) at a final concentration of 2.4 mM, RNase H2 (Integrated DNA technologies) per reaction. 100 mU, rTth DNA polymerase (Toyobo Co., Ltd.) was prepared so that 1.25 U per reaction and the reverse transcription reaction product would be 25 μL. The reverse transcription reaction product of (2) was used without purification. The reverse transcription reaction product at each concentration was simply measured.
(B) PCR reaction
Using Applied Biosystems 7500 Fast Realtime PCR system (Life Technologies), after reacting at 95 ° C. for 10 seconds, reaction at 95 ° C. for 10 seconds → 60 ° C. for 60 seconds was repeated 40 times.

(3-2) Measurement of cDNA derived from ABL reverse primer (a) Preparation of reaction solution The reaction solution volume was set to 30 μL, ABL forward primer and ABL reverse primer (Integrated DNA technologies) were each at a final concentration of 0.3 μM, and ABL fluorescence was used. Labeled probe (Integrated DNA technologies) at a final concentration of 0.2 μM, dNTPs (Toyobo) at a final concentration of 0.4 mM, MnOAc (Toyobo) at a final concentration of 2.0 mM, Tth DNA polymerase at 1.125 U per reaction, The reverse transcription reaction product was prepared at 5 μL per reaction. The reverse transcription reaction product at each concentration was simply measured.
(B) PCR reaction
Using Applied Biosystems 7500 Fast Realtime PCR system (Life Technologies), after reacting at 95 ° C. for 10 seconds, reaction at 95 ° C. for 10 seconds → 60 ° C. for 60 seconds was repeated 40 times.

(4) Results (a) When the T315I reverse primer-derived cDNA was amplified, and (b) when the ABL reverse primer-derived cDNA was amplified, the fluorescence amplification curve and the amplification cycle number of the standard product and the negative control product, respectively. It was confirmed.
FIG. 8 shows an amplification curve when (a) T315I reverse primer-derived cDNA was amplified, and FIG. 9 (b) shows an amplification curve when ABL reverse primer-derived cDNA was amplified. Table 3 shows the amplification cycle when the T315I reverse primer-derived cDNA and the ABL reverse primer-derived cDNA were amplified. Both T315I reverse primer-derived cDNA and ABL reverse primer-derived cDNA were measurable in a wide range of 1 × 10 ² to 1 × 10 ⁷ copies / test.

[Test 2] Examination of detection limit In this test, in order to determine the lower limit of detection of the ABL1 T315I mutation assay system, RNA extracted from human leukemia-derived cells HL60 was subjected to various ABL1 T315I mutant RNAs used as standard products in Test 1. The T315I reverse primer-derived cDNA and the ABL reverse primer-derived cDNA were measured using the sample added at the above concentration, and the ratio of the former measured value divided by the latter measured value was determined.

(1) Preparation of standard product (ABL1 T315I mutant RNA) The standard product was prepared in the same manner as in Test 1.

(2) Test sample As a test sample, a sample prepared by adding ABL1 T315I mutant RNA used as a standard product at various concentrations to RNA extracted from a human leukemia cell line HL60 that was BCR-ABL1-negative was used. Specifically, ABL1 T315I mutant RNA used as a standard product was added to Total RNA extracted from HL60 at a concentration of 25, 50, or 100 copies / test, and a final concentration of RNA was 100 ng / μL using TE buffer. Was prepared. As a control product, Total RNA extracted from HL60 and adjusted to 100 ng / μL with TE buffer was used.

(3) Reverse transcription reaction The reverse transcription reaction was performed in the same manner as in Test 1. The standard product was used for one sample reaction at each concentration. As a test sample, 12 samples for each concentration were used in the reaction.
(4) PCR
PCR was performed in the same manner as in Test 1. For the standard product, the reverse transcription reaction product was simply measured. As test samples, reverse transcripts of 12 samples at each concentration were individually measured.

(5) Results Table 4 shows the measured values of T315I reverse primer-derived cDNA, Table 5 shows the measured values of ABL reverse primer-derived cDNA, and the ratio of the measured value of T315I reverse primer-derived cDNA divided by the measured value of reverse primer-derived cDNA (ABL1 The T315I mutation / ABL1 ratio) is shown in Table 6. The measured values of T315I reverse primer-derived cDNA in the samples to which 100, 50, and 25 copies / test of ABL1 T315I mutant RNA were added were 109.4, 47.4, and 27.3, respectively, on average, which were in agreement with the theoretical values. .. On the other hand, no fluorescence amplification was detected in the measurement of the T315I reverse primer-derived cDNA of HL60 (control) containing no ABL1 T315I mutant RNA.

The measured value of ABL reverse primer-derived cDNA was 2.08 × 10 ⁵ on average because HL60 used for dilution expressed a certain amount of ABL1 mRNA, and all measured samples showed almost the same value. Thus, even when RNA extracted from HL60 cells expressing 10 ⁵ copies or more of wild-type ABL was measured, T315I reverse primer-derived cDNA of HL60 containing no ABL1 T315I mutant RNA was not detected. It is considered that this assay system has very high specificity for ABL1 T315I mutant RNA.

  The ABL1 T315I mutation / ABL1 ratios of the samples to which 100, 50 and 25 copies / test of ABL1 T315I mutant RNA were added were 0.052%, 0.023% and 0.013%, respectively, on average. From this, the mutation detection rate of this measurement system was about 0.01%, which was extremely high sensitivity.

[Test 3] Reaction inhibitory effect by PNA clamp This test was conducted for the purpose of confirming the effect of PNA clamp in the reverse transcription reaction.
(1) Preparation of standard product (ABL1 T315I mutant RNA) The standard product was prepared in the same manner as in Test 1.
(2) Test sample The test sample was prepared in the same manner as in Test 2.
(3) Reverse transcription reaction The reverse transcription reaction was performed in the same manner as in Test 2 except that the PNA clamp was not added.
(4) PCR
PCR was performed in the same manner as in Test 2.

(5) Results Table 7 shows the measured values of the T315I reverse primer-derived cDNA, Table 8 shows the measured values of the ABL reverse primer-derived cDNA, and the ratio of the measured value of the T315I reverse primer-derived cDNA divided by the measured value of the reverse primer-derived cDNA (ABL1 The T315I mutation / ABL1 ratio) is shown in Table 9. The measured values of T315I reverse primer-derived cDNA in the samples to which 100, 50 and 25 copies / test of ABL1 T315I mutant RNA were added were 333.7, 235.3 and 202.3 copies / test, respectively, on average. The measured value of T315I reverse primer-derived cDNA of RNA (control) extracted from HL60 was 186.9 copies / test.

The measured value of cDNA derived from ABL reverse primer was 2.29 × 10 ⁵ on average because HL60 used for dilution expressed a certain amount of ABL1 mRNA, and all measured samples showed almost the same value. The ABL1 T315I mutation / ABL1 ratios of the samples to which 100, 50 and 25 copies / test of ABL1 T315I mutant RNA were added were 0.147%, 0.107% and 0.090%, respectively, on average. The ABL1 T315I mutation / ABL1 ratio of HL60 (control) containing no ABL1 T315I mutant RNA was 0.077% on average.

  Unlike Test 2, in the absence of the PNA clamp, the T315I reverse primer-derived cDNA of the sample without addition of ABL1 T315I mutant RNA was measured as 186.9 copies / test, and the difference from the sample with addition of ABL1 T315I mutant RNA was observed. I couldn't get enough. Therefore, using the PNA clamp in the reverse transcription reaction is considered to be important for highly sensitively detecting the ABL1 T315I mutation.

  The present application can provide a highly sensitive method for detecting or quantifying the ABL1 T315I mutation as compared with conventional methods. That is, by using the above method or kit, the expression level of the ABL1 T315I mutation can be quantified with high sensitivity. The expression level of the ABL1 T315I mutation thus quantified is expected to be a useful index in the diagnosis of leukemia onset and recurrence, prognosis, timing of bone marrow transplantation, and the like.

Claims (21)

A method of measuring the expression level of an ABL1 T315I mutation in a subject, comprising:
(1) In the presence of a modified nucleic acid having a base sequence complementary to a region containing a T315I mutation position of wild-type ABL1 mRNA, (a) a reverse primer that binds to a region downstream of the T315I mutation position of ABL1 mRNA, and ( b) reverse transcribing the RNA sample of interest using a reverse primer that binds to a region upstream of the T315I mutation position of ABL1 mRNA in the same container, and
(2) calculating the expression level of the ABL1 T315I mutation based on the ratio of the reverse transcript produced by the primer of (a) to the reverse transcript produced by the primer of (b),
Including the method. Step (2) is
Step (2-1) quantifying the reverse transcription product by the primer of (a) by quantitative PCR, and
Quantifying the reverse transcription product by the primer of step (2-2) (b) by quantitative PCR,
The method of claim 1, further comprising: The quantitative PCR of step (2-1)
(X) has a nucleotide sequence of a region containing the T315I mutation position of ABL mRNA, wherein the nucleotide corresponding to the nucleotide at the T315I mutation position is replaced with uracil ribonucleotide, or
(Y) having a nucleotide sequence of a region upstream from the T315I mutation position of ABL mRNA,
The method according to claim 2, wherein a forward primer is used.   The method according to claim 3, wherein the forward primer is (X) having a blocking region at the 3'end.   The method according to any one of claims 2 to 4, wherein the quantitative PCR in step (2-1) is an RNaseH-dependent quantitative PCR specific for the T315I mutation.   The method according to claim 2, wherein a forward primer containing the nucleotide sequence of SEQ ID NO: 6 is used in step (2-1).   The method according to any one of claims 2 to 6, wherein a forward primer having the nucleotide sequence of SEQ ID NO: 6 is used in step (2-1).   The part of the modified nucleic acid and the part of the forward primer of (X) are complementary, and the complementary region is less than 50% of the total length of the modified nucleic acid. Method.   The method according to any one of claims 1 to 8, wherein the nucleotide corresponding to the T315I mutation position in the modified nucleic acid is located 3'to the center of the modified nucleic acid.   The method according to any one of claims 1 to 9, wherein the modified nucleic acid comprises the base sequence of SEQ ID NO: 5.   The method according to any one of claims 1 to 10, wherein the modified nucleic acid comprises the base sequence of SEQ ID NO: 5.   12. The method of any of claims 1-11, wherein the modified nucleic acid comprises PNA.   13. The method according to any one of claims 1 to 12, wherein the reverse primer in (a) of step (1) comprises the sequence of SEQ ID NO: 7 and the reverse primer of (b) comprises the sequence of SEQ ID NO: 10. A kit for determining the expression level of ABL1 T315I mutation in a subject, comprising:
(A) a reverse primer that binds to a region downstream of the T315I mutation position of ABL1 mRNA;
(B) a reverse primer that binds to a region upstream of the T315I mutation position of ABL1 mRNA; and
(C) a modified nucleic acid having a base sequence complementary to the region containing the T315I mutation position of wild-type ABL1 mRNA;
A kit containing.   The kit according to claim 14, further comprising a forward primer for amplifying a reverse transcription product of ABL mRNA by the reverse primer of (a) and (b) by PCR. Forward primer
(X) having a nucleotide sequence of a region containing the T315I mutation position of ABL1 mRNA, wherein the nucleotide corresponding to the nucleotide of the T315I mutation is replaced with uracil ribonucleotide, or
(Y) has a nucleotide sequence of a region upstream from the T315I mutation position of ABL1 mRNA,
The kit according to claim 15, which is a forward primer.   The kit according to claim 16, wherein the forward primer is (X) having a blocking region at the 3'end.   The kit according to claim 16 or 17, wherein a part of the modified nucleic acid and a part of the forward primer of (X) are complementary, and the complementary region is less than 50% of the entire length of the modified nucleic acid.   The kit according to any one of claims 14 to 18, wherein the nucleotide corresponding to the T315I mutation position in the modified nucleic acid is located 3'to the center of the modified nucleic acid.   The kit according to any one of claims 14 to 19, further comprising a probe for quantitative PCR.   The kit according to any one of claims 14 to 20, further comprising at least one of wild-type ABL mRNA and ABL mRNA having a T315I mutation.

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