WO2023013421A1 - 腫瘍マーカーｃａ１９－９によるがん診断の精度向上を目的とする血液型関連糖鎖遺伝子の迅速スクリーニングのための新規手法 - Google Patents

腫瘍マーカーｃａ１９－９によるがん診断の精度向上を目的とする血液型関連糖鎖遺伝子の迅速スクリーニングのための新規手法 Download PDF

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WO2023013421A1
WO2023013421A1 PCT/JP2022/028221 JP2022028221W WO2023013421A1 WO 2023013421 A1 WO2023013421 A1 WO 2023013421A1 JP 2022028221 W JP2022028221 W JP 2022028221W WO 2023013421 A1 WO2023013421 A1 WO 2023013421A1
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base
nucleotide sequence
represented
nucleotide
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利恵 佐野
伸 矢澤
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国立大学法人群馬大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material

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  • the present invention relates to genetic testing prior to serodiagnosis of malignant tumors, and specifically, synthesis of blood group-related sugar chain genes, namely Lewis blood group antigens, for improving the accuracy of cancer diagnosis using tumor marker CA19-9. It relates to a new rapid test method for single nucleotide polymorphism of FUT3 gene (also called Le gene) that produces ⁇ 1,3/4 fucosyltransferase involved in
  • CA19-9 which is a tumor marker for gastrointestinal cancer, especially pancreatic cancer, which is difficult to diagnose, and biliary tract cancer, has been widely clinically applied in Japan and overseas by serodiagnosis, and its usefulness has been recognized.
  • the antigenic determinant is Le c , Gal ⁇ 1,3GlcNAc (Le c ) , which is the backbone sugar chain of Lewis blood group antigens.
  • Sugar chain antigens are synthesized by glycosyltransferases produced by related sugar chain genes.
  • Non-Patent Document 1 Including analysis (Non-Patent Document 1), its synthetic mechanism (Non-Patent Document 2) and problems in serodiagnosis of malignant tumors (Non-Patent Document 3) have been elucidated.
  • Lewis blood group antigens include the Le a antigen and the Le b antigen. It is GlcNAc. Therefore, the presence of ⁇ 1,3/4 fucosyltransferase (Le enzyme) produced by the Le gene is essential for the expression of Lewis blood group antigen sugar chains for both Le a and Le b antigens.
  • nearly 20% of the Japanese population lacks this enzymatic activity, does not express Lewis blood group antigen sugar chains, and has a genotype of le/le and a blood type of Le(ab-).
  • the Lea antigen sugar chain which is a common sugar chain in the CA19-9 antigen sugar chain, is not synthesized, the expression of the CA19-9 antigen is not observed, and therefore it cannot be used as a diagnostic marker for malignant tumors. Nonetheless, CA19-9 testing is still being performed without discrimination of indications for CA19-9 testing based on Lewis blood group or genotype. Furthermore, in order to determine the Lewis blood group, it is usually performed from a serological test using anti-Le a antibody and anti-Le b antibody and the subject's red blood cells.
  • the phenotype may change, and even if it is a Le/- type (a genotype having a homozygous or heterozygous Le gene allele), the phenotype may become Le (ab-) (Non-Patent Documents 3 and 4).
  • Le/- type a genotype having a homozygous or heterozygous Le gene allele
  • the phenotype may become Le (ab-) (Non-Patent Documents 3 and 4).
  • determination using red blood cells in patients with malignant tumors is uncertain.
  • an extremely rare case of having Le enzymatic activity even if the erythrocyte type is Le(ab-) has been found (Non-Patent Document 5).
  • the present inventors investigated the presence or absence of Le enzyme, which is a key enzyme for CA19-9 sugar chain antigen synthesis, or deactivated Le enzyme activity in CA19-9 testing. It has already been clarified that it is necessary to investigate mutations in the associated Le gene by genetic analysis (Non-P
  • Lewis blood group antigens are synthesized by ⁇ 1,3/4 fucosyltransferase (Le enzyme) encoded by the FUT3 gene (Le gene).
  • Le enzyme fucosyltransferase
  • Le gene FUT3 gene
  • Le gene FUT3 gene
  • Le3 the dominant allele Le that produces the Le enzyme is recognized as the recessive allele le. I can't.
  • le3 which is extremely rare, is thought to have organ-specificity in the expression of the Le enzyme, and although the erythrocyte phenotype is Le(a-b-), the production of the Le enzyme is observed, and Le antigen is present in some tissues. detected. Therefore, the genotypes of individuals are Le/Le, Le/le, and le/le.
  • Non-Patent Document 5 The proportion of each allele in the Le gene is 68.9% Le allele, 24.8% le1 allele, 5.8% le2 allele, and 0.5% le3 allele (Non-Patent Document 5). Therefore, in order to identify individuals who have a homozygous le1 allele or le2 allele lacking the Le enzyme, or who have both alleles, which are not applicable for CA19-9 diagnosis in Japanese, detection of Le gene mutation, that is, on the Le gene Single-nucleotide polymorphism assays that include detection of three missense mutations c.59T>G, c.508G>A, and c.1067T>A are useful. However, due to the complexity of the operation and the use of special equipment, these factors have not been considered in practice. , CA19-9 has been repeatedly used for clinical diagnosis.
  • Single nucleotide polymorphism testing methods that have been used so far include the PCR-RFLP method, the direct sequence method, and the PCR-SSP method. These techniques require about one day from DNA extraction to amplification and detection, and require devices such as an electrophoresis device and a capillary sequencer. In the PCR-SSP method, determination is made by performing allele-specific PCR, but it is difficult to discriminate a single nucleotide difference using only sequence-specific primers. For these reasons, there has been no rapid and simple testing method for single nucleotide polymorphisms in the field of clinical testing.
  • CA19-9 a tumor marker for cancer diagnosis widely used in Japan and overseas, detects sugar chain antigens with ⁇ 2,3 sialyl Le a structure from serum.
  • the FUT3 gene the gene that produces the Le enzyme involved in the synthesis of this antigen sugar chain, has a mutation that inactivates the enzyme activity, the CA19-9 antigen is not originally synthesized, resulting in a false negative result. Therefore, the accuracy of cancer diagnosis by CA19-9 was not necessarily high. Therefore, in order to improve the accuracy of cancer diagnosis, it has been desired to confirm the presence or absence of mutations that inactivate the enzyme activity in the gene that produces the Le enzyme involved in the synthesis of the antigen sugar chain.
  • the present invention provides a rapid and convenient method for confirming the presence or absence of gene mutations that result in inactivation of the enzyme activity in the gene that produces the Le enzyme involved in the synthesis of the antigen sugar chain at the time of CA19-9 testing in the field of clinical testing.
  • the task is to develop
  • the present inventors found that the recessive allele le of the FUT3 gene (Le gene) possessed by Japanese people is commonly present by a PCR method using a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • a single nucleotide polymorphism testing method comprising detecting a missense mutation in the FUT3 gene in a biological sample collected from a subject,
  • the missense mutation is a mutation from thymine to guanine at the base corresponding to the 59th base of the FUT3 gene nucleotide sequence represented by SEQ ID NO: 1
  • the inspection method is A step of amplifying a nucleic acid fragment of the FUT3 gene in which the nucleotide at the position corresponding to the 59th nucleotide of the nucleotide sequence of the FUT3 gene represented by SEQ ID NO: 1 is thymine
  • the amplification is (i) A peptide nucleic acid (PNA) that hybridizes to a region containing guanine in the complementary sequence of the FUT3 gene, in which the base at the position corresponding to the 59th base in the nucleotide sequence of the FUT3 gene represented by SEQ ID NO:
  • a forward primer that hybridizes to a region containing the thymine of the complementary sequence of the FUT3 gene, wherein the nucleotide at the position corresponding to the 59th nucleotide of the nucleotide sequence of the FUT3 gene represented by SEQ ID NO: 1 is thymine; iii) a reverse primer that hybridizes to a region on the 3′ end side of the region containing the base at the position corresponding to the 59th base in the nucleotide sequence of the FUT3 gene represented by SEQ ID NO: 1; and detecting an amplification product obtained by amplifying the nucleic acid fragment, which step is performed with a reagent comprising A method, including [2] The method of [1], wherein the forward primer has a tag sequence added.
  • the peptide nucleic acid is the nucleotide sequence represented by SEQ ID NO: 2, or 85% or more identical to the nucleotide sequence represented by SEQ ID NO: 2 and 7 of the nucleotide sequence represented by SEQ ID NO: 2 including a sequence in which the base at the position corresponding to the th base is guanine
  • the forward primer is the nucleotide sequence represented by SEQ ID NO: 3, or is 85% or more identical to the nucleotide sequence represented by SEQ ID NO: 3 and corresponds to the 18th base of the nucleotide sequence represented by SEQ ID NO: 3 contains a sequence in which the base at the position is thymine
  • the reverse primer contains the nucleotide sequence represented by SEQ ID NO: 4, or a sequence that is 90% or more identical to the nucleotide sequence represented by SEQ ID NO: 4, The method according to any one of [1] to [5].
  • a single nucleotide polymorphism test kit comprising detecting a missense mutation in the FUT3 gene, The missense mutation is a mutation from thymine to guanine at the base corresponding to the 59th base of the FUT3 gene nucleotide sequence represented by SEQ ID NO: 1,
  • the kit includes (i) A peptide nucleic acid (PNA) that hybridizes to a region containing guanine in the complementary sequence of the FUT3 gene, in which the base at the position corresponding to the 59th base in the nucleotide sequence of the FUT3 gene represented by SEQ ID NO: 1 is guanine.
  • PNA peptide nucleic acid
  • kits comprising a reagent for amplifying a nucleic acid fragment of the FUT3 gene in which the nucleotide at the position corresponding to the 59th nucleotide of the represented nucleotide sequence of the FUT3 gene is thymine, and a reagent for detecting an amplification product.
  • the peptide nucleic acid is the nucleotide sequence represented by SEQ ID NO: 2, or 85% or more identical to the nucleotide sequence represented by SEQ ID NO: 2 and 7 of the nucleotide sequence represented by SEQ ID NO: 2 including a sequence in which the base at the position corresponding to the th base is guanine
  • the forward primer is the nucleotide sequence represented by SEQ ID NO: 3, or is 85% or more identical to the nucleotide sequence represented by SEQ ID NO: 3 and corresponds to the 18th base of the nucleotide sequence represented by SEQ ID NO: 3 contains a sequence in which the base at the position is thymine
  • the reverse primer contains the nucleotide sequence represented by SEQ ID NO: 4, or a sequence that is 90% or more identical to the nucleotide sequence represented by SEQ ID NO: 4,
  • the detection reagent is a chromatographic membrane bound with a capture probe comprising a sequence complementary to the tag sequence when the amplification product is tagged with a tag sequence; A dye-containing solution for detecting amplification products, and a developing solution for chromatography,
  • the identification method which is identified as [13] Cases suitable for cancer diagnosis using tumor marker CA19-9, including the single nucleotide polymorphism test method according to any one of [1] to [8], and genetically synthesizing CA19-9 antigen sugar chains
  • a data acquisition method for improving accuracy by selecting unsuitable cases When a missense mutation from thymine to guanine is detected at the position corresponding to the 59th base in the nucleotide sequence of the FUT3 gene represented by SEQ ID NO: 1, it is determined that the test using the tumor marker CA19-9 is not applicable.
  • the data acquisition method When a missense mutation from thymine to guanine is detected at the position corresponding to the 59th base in the nucleotide sequence of the FUT3 gene represented by SEQ ID NO: 1, it is determined that the test using the tumor marker CA19-9 is not applicable.
  • the present invention it is possible to test for single nucleotide polymorphisms including detection of the missense mutation c.59T>G that is commonly present in the recessive allele le of the FUT3 gene (Le gene) possessed by Japanese people.
  • Using a very small amount of blood we were able to provide a simple test method that can obtain rapid test results within 1 hour after blood collection.
  • CA19-9 by examining the presence or absence of gene mutations associated with inactivation of the enzyme activity in the gene that produces the Le enzyme involved in the synthesis of the CA19-9 antigen sugar chain, CA19-9 antigen By selecting CA19-9-specific cases limited to the Le/- type with production ability, it was possible to improve the diagnostic accuracy as a tumor marker.
  • FIG. 10 is a photograph of the results of examining conditions for the PNA-clamping PCR method.
  • Le / le type le is le1 or le2 blood samples from subjects (represented as Le (-) Blood in FIG. 1), two types of forward primers (Le-specific-F primer and FUT3-147 Primer), annealing temperature, and peptide nucleic acid concentration.
  • FIG. FIG. 10 is a photograph of the results of examining conditions for the PNA-clamping PCR method. Using blood samples from subjects of Le/- type (represented as Le(+) Blood in FIG. 2) and le/le (represented as le1 or le2) type (represented as le/le Blood in FIG. 2) FIG.
  • FIG. 2 is an electrophoresis photograph of the results of examining the concentration of peptide nucleic acid when Le-specific-F primer was used as a forward primer.
  • FIG. 10 is a photograph of the results of examining conditions for the PNA-clamping PCR method. Using blood samples from subjects of Le / - type (represented as Le (+) in FIG. 3) and le / le (le is le1 or le2, respectively) type (represented as Le (-) in FIG. 3) , the amount of blood sample added, and the results of examining the annealing temperature. As primers, Le-specific-F primer, control forward primer FUT3c222-F primer, and FUT3c373-R primer were used.
  • FIG. 10 is a photograph of the results of examining conditions for the PNA-clamping PCR method.
  • FIG. 10 is a photograph of the results of examining conditions for the PNA-clamping PCR method.
  • FIG. 5 is an electrophoresis photograph of the results of examination of the primer ratio using a blood sample derived from an Le/ ⁇ type (denoted as Le(+) sample in FIG. 4) subject.
  • As primers Le-specific-F primer and control forward primers FUT3c222-F primer and FUT3c373-R primer were used. It is a figure which compared this invention (lower figure) with a conventional method (upper figure). In the amplification in the middle diagram below, *primer 1 indicates the Le-specific-F primer with the added tag sequence, ⁇ primer 2 indicates the control forward primer with the added tag sequence, and primer 3 indicates the reverse primer.
  • FIG. 2 is a photographic diagram of the results of detection by the STH-PAS method after obtaining the target amplification product by the PNA-clamping PCR method.
  • Sample numbers 1-6 correspond to the sample numbers in Table 1.
  • a common line derived from the amplified product by primer 2 (tag sequence-added FUT3c200-F primer) and primer 3 (biotin-labeled FUT3c373-R primer) in each sample represented as control line in FIG. 6) and A band of the Le allele-specific line (indicated as test line in FIG.
  • a single nucleotide polymorphism testing method comprising detecting a missense mutation in the FUT3 gene in a biological sample collected from a subject,
  • the missense mutation is a mutation from thymine to guanine at the base corresponding to the 59th base of the FUT3 gene nucleotide sequence represented by SEQ ID NO: 1
  • the inspection method is A step of amplifying a nucleic acid fragment containing thymine at the position of the FUT3 gene in which the base at the position corresponding to the 59th base in the nucleotide sequence of the FUT3 gene represented by SEQ ID NO: 1 is thymine
  • the amplification is (i) A peptide nucleic acid (PNA) that hybridizes to a region containing guanine in the complementary sequence of the FUT3 gene, in which the base at the position corresponding to the 59th base in the nucleotide sequence of the FUT3 gene represented by SEQ ID NO: 1 is gu
  • a forward primer that hybridizes to a region containing the thymine of the complementary sequence of the FUT3 gene, wherein the nucleotide at the position corresponding to the 59th nucleotide of the nucleotide sequence of the FUT3 gene represented by SEQ ID NO: 1 is thymine; iii) a reverse primer that hybridizes to a region on the 3′ end side of the region containing the base at the position corresponding to the 59th base in the nucleotide sequence of the FUT3 gene represented by SEQ ID NO: 1; and detecting an amplification product obtained by amplifying the nucleic acid fragment, which step is performed with a reagent comprising
  • a method comprising:
  • the single nucleotide polymorphism in the FUT3 gene to be detected in the present invention is due to a missense mutation at the position corresponding to the 59th base in the nucleotide sequence of the FUT3 gene represented by SEQ ID NO:1.
  • This missense mutation is a mutation represented by NM_000149.3(FUT3):c.59T>G (p.Leu20Arg).
  • This missense mutation is a common missense mutation in the recessive le allele of the FUT3 gene (Le gene) in Japanese, and the Le/- type without this missense mutation is capable of synthesizing the CA19-9 antigen sugar chain. Therefore, it is an applicable case for the tumor marker CA19-9 test.
  • the le/le type consisting of le1 and/or le2 does not synthesize the CA19-9 antigen sugar chain and is not applicable for the test.
  • the le3 allele is homozygous and when le3/- ((-) is either le1, le2, or le3), synthesis of the CA19-9 antigen sugar chain is considered. If the value is positive, it becomes a case where the test of the tumor marker is indicated.
  • the nucleotide sequence of the FUT3 gene is not particularly limited, but may have the nucleotide sequence represented by SEQ ID NO: 1, for example.
  • SEQ ID NO: 1 one or several nucleotides other than the nucleotide at the position corresponding to the 59th nucleotide, as long as an amplification product of the FUT3 gene can be obtained by PCR. may be a substituted, deleted, inserted and/or added base sequence.
  • One or several is not particularly limited, but includes, for example, 1 to 2, 1 to 3, 1 to 5, 1 to 10, and 1 to 20.
  • the step of amplifying the nucleic acid fragment of the present invention comprises a nucleic acid fragment containing thymine at the position corresponding to the 59th base of the FUT3 gene nucleotide sequence represented by SEQ ID NO: 1 of the FUT3 gene, wherein the base is thymine.
  • the PNA-clamping PCR method (Non-Patent Document 6), which is a PCR method that can efficiently amplify only nucleic acid fragments having a specific base sequence by using peptide nucleic acids, is performed. can be done.
  • peptide nucleic acids In peptide nucleic acids, the binding between nucleotides is not through phosphate bonds as in DNA, but the binding between portions corresponding to nucleotides in DNA is through peptide bonds. Therefore, unlike DNA, repulsion of negative charges due to the presence of phosphate groups does not occur, so that the melting temperature (Tm value) is high. Therefore, in annealing, the peptide nucleic acid with a high Tm value binds to the target DNA before the primer with a lower Tm value than the peptide nucleic acid, thereby inhibiting the binding of the primer to the DNA. That is, peptide nucleic acids are superior to DNA in base recognition ability and complementary strand binding ability, and have the characteristic of being able to accurately recognize a single base substitution in target DNA.
  • the peptide nucleic acid hybridizes to a region containing guanine in the complementary sequence of the FUT3 gene, in which the base at the position corresponding to the 59th base in the nucleotide sequence of the FUT3 gene represented by SEQ ID NO: 1 is guanine. It is not particularly limited as long as it is a substance.
  • the length of the peptide nucleic acid is not particularly limited as long as it can hybridize to the target sequence, and one with a higher melting temperature (Tm value) than the amplification primer is suitable. For example, it may be 10-30mer, 13-20mer, 14-16mer, or 15mer.
  • the peptide nucleic acid is specific without a mismatch to the region containing the guanine of the complementary sequence of the FUT3 gene in which the base at the position corresponding to the 59th base in the nucleotide sequence of the FUT3 gene represented by SEQ ID NO: 1 is guanine. It is not particularly limited as long as it hybridizes, for example, it may be set based on the nucleotide sequences within the range of 30mer on the 5'-terminal side and 3'-terminal side of the guanine at the position, and within the range of 20mer.
  • the peptide nucleic acid can contain a nucleotide sequence of 10-30mer, 13-20mer, 14-16mer, or 15mer including the 59th nucleotide in the nucleotide sequence of SEQ ID NO: 1, preferably , the nucleotide sequence represented by SEQ ID NO: 2, or 85% or more or 90% or more identical to the nucleotide sequence represented by SEQ ID NO: 2 and corresponding to the 7th base of the nucleotide sequence represented by SEQ ID NO: 2 It has a sequence in which the base at the position is guanine.
  • the amount of peptide nucleic acid used may be, for example, 15 to 30 pmol or 25 pmol in a PCR composition with a total volume of 25 ⁇ l.
  • Peptide nucleic acids may be commercially available products or may be produced by methods known to those skilled in the art.
  • the forward primer hybridizes to a region containing thymine in the complementary sequence of the FUT3 gene, wherein the nucleotide at the position corresponding to the 59th nucleotide in the nucleotide sequence of the FUT3 gene represented by SEQ ID NO: 1 is thymine.
  • the length of the forward primer is not particularly limited as long as it can hybridize to the target sequence and amplify the target nucleic acid by PCR. For example, it may be 18-30mer or 20-25mer.
  • the forward primer hybridizes to a region containing thymine in the complementary sequence of the FUT3 gene whose 3′ end side is thymine at the position corresponding to the 59th base in the nucleotide sequence of the FUT3 gene represented by SEQ ID NO: 1. Anything that soy is suitable.
  • the forward primer may contain an 18-30mer or 20-25mer nucleotide sequence containing the 59th nucleotide on the 3' end side in the nucleotide sequence of SEQ ID NO: 1, preferably the sequence The base sequence represented by number 3, or the position corresponding to the 18th base of the base sequence represented by SEQ ID NO: 3 that is 85% or more or 90% or more identical to the base sequence represented by SEQ ID NO: 3 It has a sequence in which the base is thymine.
  • the amount of forward primer used may be, for example, 1 to 5 pmol or 2.5 pmol in a PCR composition with a total volume of 25 ⁇ l.
  • the forward primer may be a commercially available product or may be produced by a method known to those skilled in the art.
  • the forward primer is not particularly limited, but may have an arbitrary tag sequence added, and the tag sequence may be added to the 5' end of the forward primer.
  • the reverse primer hybridizes to a region on the 3′ end side of the region containing the base at the position corresponding to the 59th base in the nucleotide sequence of the FUT3 gene represented by SEQ ID NO: 1. It is not particularly limited.
  • the length of the reverse primer is not particularly limited as long as it can hybridize to the target sequence and amplify the target nucleic acid by PCR. For example, it may be 18-30mer or 20-25mer.
  • the reverse primer contains an 18-30 mer or 20-25 mer nucleotide sequence that is complementary to the nucleotide sequence of the region 100 nucleotides or more downstream of the 59th nucleotide in the nucleotide sequence of SEQ ID NO: 1.
  • FUT3-specific sequence that does not recognize the homologous gene FUT5,6, and is preferably 90% or more of the nucleotide sequence represented by SEQ ID NO: 4 or the nucleotide sequence represented by SEQ ID NO: 4, or Those having sequences that are 95% or more identical.
  • the amount of reverse primer used may be, for example, 1 to 5 pmol or 2.5 pmol in a PCR composition with a total volume of 25 ⁇ l.
  • the amount of the reverse primer used may be, for example, 2 to 20 pmol, or may be 5.0 pmol, in a PCR composition having a total volume of 25 ⁇ l.
  • a reverse primer may be a commercially available product, or may be produced by a method known to those skilled in the art.
  • the reverse primer may be one bound with a labeling element.
  • the labeling element may itself exhibit coloration, coloring, luminescence or fluorescence, or it may exhibit coloration, coloring, luminescence or fluorescence via a substance that binds to the labeling element.
  • any labeling element known to those skilled in the art can be used as long as it does not interfere with the amplification of the target nucleic acid fragment by the PCR method.
  • biotin can be used.
  • beads bound to avidin are formed by forming a complex between biotin in the amplification product and avidin previously bound to colored beads in the step of detecting the amplification product, which will be described later.
  • the coloring of the target amplification product can be detected.
  • a labeling element may be attached to the 5' end of the reverse primer.
  • the reagents in the step of amplifying the nucleic acid fragment of the present invention may further contain a control forward primer.
  • the control forward primer is located 3' to the region containing the base at the position corresponding to the 59th base in the nucleotide sequence of the FUT3 gene represented by SEQ ID NO: 1, and 5' to the region to which the reverse primer hybridizes. There is no particular limitation as long as it hybridizes with the complementary sequence of the FUT3 gene on the terminal side.
  • the length of the control forward primer is not particularly limited as long as it can hybridize to the target sequence and amplify the target nucleic acid by PCR. For example, it may be 18-30mer or 20-25mer.
  • the control forward primer has a nucleotide sequence of a region 50 bases or more downstream of the 59th base and 100 bases or more upstream of the region to which the reverse primer hybridizes. 18-30 mer or 20-25 mer nucleotide sequences, and the difference in the amplified length of these products is about 100 bp in order to be able to distinguish the control product and the Le-specific PCR product by electrophoresis It is desirable to Preferably, it has the nucleotide sequence represented by SEQ ID NO:5 or 7.
  • the amount of control forward primer used may be, for example, 1 to 10 pmol or 2.5 pmol in a PCR composition with a total volume of 25 ⁇ l.
  • the control forward primer may be a commercially available product or may be manufactured by a method known to those skilled in the art.
  • control forward primer is not particularly limited, but may have an arbitrary tag sequence different from the tag sequence to which the forward primer is added, and the tag sequence is added to the 5' end of the control forward primer. It's okay to be there.
  • the mixing ratio of the forward primer, the reverse primer, and the control forward primer in the PCR composition is such that the peptide nucleic acid corresponds to the 59th base of the nucleotide sequence of the FUT3 gene represented by SEQ ID NO: 1 before the forward primer.
  • FUT3 hybridizes with the FUT3 gene in which the base at the position is guanine, and the base at the position corresponding to the 59th base in the nucleotide sequence of the FUT3 gene represented by SEQ ID NO: 1 is thymine by the forward primer and the reverse primer
  • the mixing ratio of forward primer, reverse primer and control forward primer may be, for example, (1-1.5):(2-4):(1-1.5) or 1:2:1.
  • the conditions for nucleic acid amplification by the PCR method and the composition of the PCR reaction solution are such that the FUT3 gene in which the nucleotide at the position corresponding to the 59th nucleotide in the nucleotide sequence of the FUT3 gene represented by SEQ ID NO: 1 is guanine is amplified.
  • the FUT3 gene in which the nucleotide at the position corresponding to the 59th nucleotide in the nucleotide sequence of the FUT3 gene represented by SEQ ID NO: 1 is thymine by PCR using various primers. It is not particularly limited.
  • the PCR method may be performed by a method known to those skilled in the art, any DNA polymerase, dNTPs, MgCl 2 , buffers and other reagents may be used, and amplification may be performed in a 3-step cycle or a 2-step cycle. good. Annealing may be performed at 64°C to 69°C, 67°C to 68°C, or 68°C.
  • a peptide nucleic acid having a nucleotide sequence represented by SEQ ID NO: 2 a forward primer having a nucleotide sequence represented by SEQ ID NO: 3, a reverse primer having a nucleotide sequence represented by SEQ ID NO: 4, and KOD One (registered trademark) )
  • PCR Master Mix Toyobo
  • annealing is preferably carried out at 68°C. can be done most efficiently.
  • PCR PCR Master Mix
  • Step of detecting amplified product Detection of the amplified product is not particularly limited as long as the desired amplified product can be detected, and detection methods known to those skilled in the art such as gel electrophoresis and various chromatographic methods may be used. For example, detection may be performed via a tag sequence carried by the amplification product by binding with a capture probe comprising a sequence complementary to the tag sequence. From the viewpoint of simplicity and rapidity, amplification products can be detected by, for example, the single-stranded tag hybridization chromatographic printed-array strip (STH-PAS) method (Non-Patent Document 7), which is one of the chromatography methods. is preferred.
  • STH-PAS single-stranded tag hybridization chromatographic printed-array strip
  • a capture probe is bound to a specific position on the membrane for chromatography, and by immersing one end of the membrane in a solution containing the amplification product, the amplification product can be easily and rapidly spread on the membrane.
  • the amplification product can be immobilized at a specific position on the membrane by binding the tag sequence of the amplification product to a capture probe having a sequence complementary to the tag sequence. Labels can be detected on the membrane by methods known to those skilled in the art.
  • the label When a labeling element is attached to the amplification product, the label can be detected at a specific position on the membrane by methods known to those skilled in the art. If the labeling element itself exhibits color development, coloring, luminescence or fluorescence, the color development, coloring, luminescence or fluorescence can be confirmed at a specific position on the membrane.
  • a labeling component when a labeling component is bound to the amplification product and the labeling component itself does not exhibit color development, coloring, luminescence or fluorescence, a substance that binds to the labeling component is added in advance to the developing solution used. It may be included and may exhibit coloration, coloration, luminescence or fluorescence via substances that bind to the labeling element.
  • the labeling element is biotin
  • the substance that binds to this labeling element is avidin, and by binding colored beads to this avidin in advance, coloration can be confirmed at a specific position on the membrane.
  • a position corresponding to the 59th base in the nucleotide sequence of the FUT3 gene represented by SEQ ID NO: 1 when color development, coloring, luminescence, or fluorescence due to the labeling element bound to the amplification product can be confirmed at a specific position on the membrane. It can be determined that there is a mutation from thymine to guanine at the base of , that is, it can be determined that it has a single base substitution.
  • the temperature at which the membrane is immersed in the developer for development is not particularly limited as long as it does not affect the development, and for example room temperature may be used.
  • the development time is not particularly limited as long as the amplified product can be developed, but it can be carried out in a short period of time, for example, 5 to 15 minutes.
  • the particle size and color of the colored beads are not particularly limited as long as the desired amplification product can be developed on the membrane by the STH-PAS method and the coloring can be confirmed.
  • control amplification product is amplified by the control forward primer and the reverse primer. Detection of the control amplification product is preferably carried out in the same manner as the detection of the desired amplification product described above.
  • control amplification product When the STH-PAS method is used to detect amplification products, the control amplification product has a tag sequence different from that of the target amplification product. Binding of the tag sequence of the control amplification product to a capture probe having a sequence complementary to the tag sequence results in binding at a specific position on the membrane different from the position where the capture probe for the target amplification product is bound. Control amplicons can be immobilized. The label can then be detected by methods known to those skilled in the art.
  • a labeling element is attached to the control amplification product, the label can be detected at a specific location on the membrane by methods known to those skilled in the art. If the labeling element itself exhibits color development, coloring, luminescence or fluorescence, the color development, coloring, luminescence or fluorescence can be confirmed at a specific position on the membrane.
  • a labeling element is bound to the control amplification product and the labeling element itself does not exhibit color development, coloring, luminescence or fluorescence
  • a substance that binds to the labeling element is preliminarily added to the developing solution to be used. and may exhibit color development, coloring, luminescence or fluorescence through the substance bound to the labeling element.
  • the labeling element is biotin
  • the substance that binds to this labeling element is avidin, and by binding colored beads to this avidin in advance, coloration can be confirmed at a specific position on the membrane.
  • the labeling elements of the control amplification product and the labeling elements of the desired amplification product are the same. It can be judged that the test method of the present invention was performed normally when color development, coloring, luminescence or fluorescence by the labeling element bound to the control amplification product was confirmed at a specific position on the membrane.
  • the biological sample is not particularly limited as long as it contains DNA, but is preferably a blood sample.
  • the biological sample may be diluted and used, and is not particularly limited as long as the nucleic acid of interest can be amplified by the PCR method. For example, it may be diluted 10- to 1000-fold, it may be diluted 50- to 200-fold, it may be diluted 100-fold, depending on what is used.
  • a blood sample dilution solvent is not particularly limited as long as it does not affect the PCR method.
  • the inspection method of the present invention may further include additional steps.
  • Another embodiment of the present invention is a single nucleotide polymorphism test kit comprising detecting a missense mutation in the FUT3 gene,
  • the missense mutation is a mutation from thymine to guanine at the base corresponding to the 59th base of the FUT3 gene nucleotide sequence represented by SEQ ID NO: 1,
  • the kit includes (i) A peptide nucleic acid (PNA) that hybridizes to a region containing guanine in the complementary sequence of the FUT3 gene, in which the base at the position corresponding to the 59th base in the nucleotide sequence of the FUT3 gene represented by SEQ ID NO: 1 is guanine.
  • PNA peptide nucleic acid
  • kits comprising a reagent for amplifying a nucleic acid fragment of the FUT3 gene in which the base at the position corresponding to the 59th base in the represented nucleotide sequence of the FUT3 gene is thymine, and a reagent for detecting an amplification product.
  • the detection of a missense mutation from thymine to guanine at the base corresponding to the 59th base in the nucleotide sequence of the FUT3 gene represented by SEQ ID NO: 1 was carried out according to the method described in the above single nucleotide polymorphism inspection method. can be used.
  • the detection reagent includes a chromatography membrane bound to a capture probe having a sequence complementary to the tag sequence, a solution containing a dye for detecting the amplification product, and A chromatographic developing solution may also be included.
  • the dye-containing solution for detection of the amplification product is a color development, coloring, luminescence or fluorescence via a substance that binds to the labeling component.
  • the detection dye-containing solution may be a solution containing avidin pre-bound with colored beads.
  • PCR reagents may include reagents such as DNA polymerase, dNTP, MgCl 2 , buffers, and distilled water, tools such as chips and tubes, and reagents such as NaOH for preparation of biological samples. .
  • Another embodiment of the present invention is a method for determining that the genotype of the Lewis blood group is le/le type, including the above-described single nucleotide polymorphism test method, represented by SEQ ID NO: 1 Lewis genotype is determined to be le/le genotype when a missense mutation from thymine to guanine is detected at the position corresponding to the 59th base in the nucleotide sequence of the FUT3 gene. is the above identification method.
  • the detection of a missense mutation from thymine to guanine at the base corresponding to the 59th base in the nucleotide sequence of the FUT3 gene represented by SEQ ID NO: 1 was carried out according to the method described in the above single nucleotide polymorphism inspection method. can be used.
  • STH-PAS method when used in the step of detecting the amplification product, when color development, coloring, luminescence or fluorescence by the labeling element bound to the amplification product can be confirmed at a specific position on the membrane, SEQ ID NO: 1 It can be judged that there is a mutation from thymine to guanine at the base at the position corresponding to the 59th base of the nucleotide sequence of the FUT3 gene represented by, that is, it can be judged to have a single base substitution. It can be determined that the genotype of the Lewis blood group is le/le type.
  • Another embodiment of the present invention includes the above-mentioned single nucleotide polymorphism test method for cancer diagnosis using the tumor marker CA19-9 and non-adaptive cases that are genetically incapable of synthesizing CA19-9 antigen sugar chains.
  • the detection of a missense mutation from thymine to guanine at the base corresponding to the 59th base in the nucleotide sequence of the FUT3 gene represented by SEQ ID NO: 1 was carried out according to the method described in the above single nucleotide polymorphism inspection method. can be used.
  • STH-PAS method when used in the step of detecting the amplification product, when color development, coloring, luminescence or fluorescence by the labeling element bound to the amplification product can be confirmed at a specific position on the membrane, SEQ ID NO: 1 It can be judged that there is a mutation from thymine to guanine at the base at the position corresponding to the 59th base of the nucleotide sequence of the FUT3 gene represented by, that is, it can be judged to have a single base substitution. It is possible to select suitable cases for examination by tumor marker CA19-9.
  • the missense mutation to be detected is present on the FUT3 gene. Since the FUT3 gene has high homology with the FUT5 gene and the FUT6 gene, primers capable of specifically amplifying the FUT3 gene should be set. is necessary.
  • 50 mM NaOH was added to 1 ⁇ l of a blood sample derived from an le/le type subject (denoted as Le( ⁇ ) Blood in FIG. 1) to prepare a blood sample diluent (100-fold dilution).
  • nucleic acid amplification (total volume: 25 ⁇ l) was performed by PCR in the presence of KOD One (registered trademark) PCR Master Mix (Toyobo), various primers, and peptide nucleic acid. Amplification of nucleic acids by PCR was performed by repeating 35 cycles of 98°C for 10 seconds and 68°C or 64°C for 5 seconds after heating at 94°C for 3 minutes.
  • Le-specific-F primer (5'-CCGCTGTCTGGCCGCACT-3' (SEQ ID NO: 3)) or FUT3-147F primer (5'-GTGAGTCACTTGGTCTCAGT-3' (SEQ ID NO: 6) was used as the forward primer, and FUT3c373- Using R primer (5'-GGGAAGGTGGGAGGCGTGACTTA-3' (SEQ ID NO: 4)), amplification efficiency, non-specific reaction, etc. by PCR were compared and examined.
  • the primers used in the PCR method used in the present invention were Le-specific-F primer (5'-CCGCTGTCTGGCCGCACT-3' (SEQ ID NO: 3)) and FUT3c373-R primer (5'-GGGAAGGTGGGAGGCGTGACTTA-3' (SEQ ID NO: 3). It was shown that the target amplification product can be efficiently obtained by using the primer set of 4)). In addition, as a result of comparing the annealing temperature, it was shown that non-specific reaction was less when annealing was performed at 68°C than at 64°C.
  • the addition amount of the specific peptide nucleic acid PNA-le-clamp was examined (Fig. 2). .
  • the blood samples used were blood samples from subjects of Le/ ⁇ type (denoted as Le(+) Blood in FIG. 2) and le/le type (denoted as le/le Blood in FIG. 2), respectively.
  • the PNA-le-clamp was 25 pmol, the amplification product of Le was confirmed in the blood sample derived from the Le/- type subject, while the amplified product of Le was confirmed in the blood sample derived from the le/le type subject.
  • Example 1 Sample preparation> When using conventional single nucleotide polymorphism detection, etc., a special device is required and it takes time due to the complexity of the operation. It is a specific, rapid and simple detection method for Le gene detection because it is completed within 1 hour including the standard (control). The features of the present invention are shown in comparison with the conventional method (Fig. 5).
  • a blood sample dilution (100-fold dilution) was prepared by adding 50 mM NaOH to 1 ⁇ l of the blood sample.
  • the Lewis blood group genotype, erythrocyte phenotype and serum concentration of CA19-9 antigen of the blood samples used are shown in the following table.
  • Example 2 Amplification of nucleic acids by PCR> Using the PNA-clamping PCR method (Non-Patent Document 6), 1 ⁇ l of the diluted blood sample obtained in Example 1 was used as a template in the presence of KOD One (registered trademark) PCR Master Mix (Toyobo), various primers, and peptide nucleic acid. Amplification of the target nucleic acid fragment (total amount: 25 ⁇ l) was carried out by the PCR method.
  • KOD One registered trademark
  • Toyobo PCR Master Mix
  • primers used were primer 1, tag sequence-added Le-specific-F primer (5′-[F-1 (tag sequence)]-CCGCTGTCTGGCCGCACT-3′ (SEQ ID NO: 3), 0.1 pmol/ ⁇ l); Tag sequence-added FUT3c200-F primer (5'-[F-3 (tag sequence)]-TATGGACATGGCCTTTCCACATCCCT-3' (SEQ ID NO: 7), 0.1 pmol/ ⁇ l); primer 3, biotin-labeled FUT3c373-R primer (5'- Biotin-GGGAAGGTGGGAGGCGTGACTTA-3′ (SEQ ID NO: 4), 0.2 pmol/ ⁇ l).
  • a specific peptide nucleic acid PNA-le-clamp (N-CCGCACGGCTATTTC-C (SEQ ID NO: 2), 1 pmol/ ⁇ l) was also used.
  • This peptide nucleic acid was prepared to target and specifically hybridize to the c.59T>G mutation characteristic of the mutant allele (le).
  • Amplification of nucleic acids by the PCR method was performed by repeating 35 cycles of 94° C. for 3 minutes and then 98° C. for 10 seconds and 68° C. for 5 seconds. Two amplification products were obtained with primers 1 and 3 and primers 2 and 3 when an active allele (Le) was present in the sample DNA (Le/ ⁇ type).
  • the peptide nucleic acid PNA-le-clamp with a high Tm value hybridizes first to the sample DNA, thereby preventing the hybridization of Primer 1 to the sample DNA. Only amplification products with primers 2 and 3 were obtained because soybean was inhibited. Amplified products by primers 2 and 3 were used as internal standards for confirming that the nucleic acid amplification reaction by PCR was performed normally.
  • PCR amplification products After the nucleic acid amplification reaction by the PCR method in Example 2, a single-stranded tag hybridization chromatographic printed-array strip (STH-PAS ) method (Non-Patent Document 7), active allele (Le) was detected.
  • STH-PAS single-stranded tag hybridization chromatographic printed-array strip
  • Le active allele
  • primers 1 and 2 used in the PCR method of Example 1 each have a specific tag sequence (indicated by ⁇ and ⁇ in FIG. A sequence complementary to those tag sequences is printed on the PAS.
  • a PAS is inserted to develop the amplified product on a filter paper. bottom.
  • a common line derived from the products amplified by primers 2 and 3 appeared in all samples. Furthermore, in the samples 1 to 4 with the genotype Le/- type, a Le allele-specific line (represented as test line in FIG. 6) derived from the amplification product by the primers 1 and 3 appeared, but on the other hand, the le/le type This Le allele-specific line did not appear in samples 5 and 6 of . All cases with high serum CA19-9 levels were determined to be Le/- type, consistent with the conventional method.
  • Samples 3 and 4 are samples in which a discrepancy between the Lewis blood group genotype and the red blood cell phenotype was observed, and the Lewis blood group phenotype of patient red blood cells related to cancer-bearing This is a sample that changed to the Le(-) type.
  • Test lines appeared in both samples 3 and 4, demonstrating that the present invention enables genotype determination regardless of erythrocyte phenotype. That is, according to the present invention, the appearance of the control line enables confirmation of normal PCR amplification, and the appearance of the test line clearly distinguishes between the genotypes Le/ ⁇ and le/le. It was shown that it is possible to determine
  • the present invention does not require any special equipment, and can be sufficiently performed in hospital inspection departments, making it possible to determine in advance whether the CA19-9 test is applicable or not.
  • the rapid and simple test method of the present invention when diagnosing cancer using the tumor marker CA19-9, which is widely used clinically, genetic testing of the subject's active allele (Le) is performed at the same time, and Le / -
  • Le active allele

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004173623A (ja) * 2002-11-28 2004-06-24 Nippon Flour Mills Co Ltd Ｐｃｒクランピング法
US20150176065A1 (en) * 2013-06-28 2015-06-25 Michael J. Powell Detection of pna clamping
JP2015518727A (ja) * 2012-05-29 2015-07-06 アボツト・モレキユラー・インコーポレイテツド ヒトｂｒａｆのコドン６００における一塩基多型（ｓｎｐ）を検出する方法
CN107630080A (zh) * 2016-11-16 2018-01-26 复旦大学附属肿瘤医院 一种Lewis血型抗原检测方法

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JP2004173623A (ja) * 2002-11-28 2004-06-24 Nippon Flour Mills Co Ltd Ｐｃｒクランピング法
JP2015518727A (ja) * 2012-05-29 2015-07-06 アボツト・モレキユラー・インコーポレイテツド ヒトｂｒａｆのコドン６００における一塩基多型（ｓｎｐ）を検出する方法
US20150176065A1 (en) * 2013-06-28 2015-06-25 Michael J. Powell Detection of pna clamping
CN107630080A (zh) * 2016-11-16 2018-01-26 复旦大学附属肿瘤医院 一种Lewis血型抗原检测方法

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