WO2016042830A1 - Method for analyzing fetal chromosome - Google Patents

Method for analyzing fetal chromosome Download PDF

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WO2016042830A1
WO2016042830A1 PCT/JP2015/062894 JP2015062894W WO2016042830A1 WO 2016042830 A1 WO2016042830 A1 WO 2016042830A1 JP 2015062894 W JP2015062894 W JP 2015062894W WO 2016042830 A1 WO2016042830 A1 WO 2016042830A1
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blood cells
red blood
nucleated red
amplification
cells
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Japanese (ja)
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靖幸 石井
雄喜 井上
彩 大内
雅也 長瀬
尭之 辻本
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富士フイルム株式会社
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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers

Definitions

  • fetal chromosome analysis method blood cells contained in blood collected from a pregnant mother are fixed on a substrate, and blood cells fixed on the substrate are analyzed with an optical instrument to identify nucleated red blood cells.
  • the method of the present disclosure is excellent in the ability to analyze genetic information. The reason is not limited by a specific mechanism, but is presumed to be due to the mechanism described below.
  • chromosome aneuploidy may not be detected with good reproducibility, or base sequence may not be determined with good reproducibility. It is thought that there is.
  • Fetal nucleated red blood cells are red blood cell precursors that pass through the placenta and are present in the maternal blood. When the mother is pregnant, fetal red blood cells can be nucleated. Since nucleated red blood cells have chromosomes, fetal chromosomes and fetal genes can be obtained by isolating fetal nucleated red blood cells. Fetal nucleated red blood cells are said to be present in a ratio of about 1 in 10 6 cells in maternal blood, and the probability of existence is very low in peripheral blood of the pregnant mother.
  • Nucleated red blood cells can be separated from plasma components and other blood cells present in the blood by density gradient centrifugation.
  • a known method may be applied to density gradient centrifugation for separating nucleated red blood cells.
  • nucleated red blood cells are fractionated by centrifuging and centrifuging blood diluted with physiological saline on a discontinuous density gradient in which two types of media with different densities (specific gravity) are layered on a centrifuge tube. It can be concentrated.
  • the density (specific gravity) of the medium to be stacked is set in order to separate fetal nucleated red blood cells with a density of about 1.065 to 1.095 g / mL from other blood cells. Since the density of the center of fetal nucleated red blood cells is about 1.080 g / mL, fetal nucleated red blood cells are present at the interface by layering two different density media adjacent to this density. It is possible to collect fractions.
  • the two-layer discontinuous density gradient is formed in the centrifuge tube as follows, for example.
  • the lower layer medium in a temperature state not lower than the freezing point and not higher than 14 ° C. preferably not higher than 8 ° C.
  • the lower layer medium is accommodated in the bottom of the centrifuge tube and then not higher than 14 ° C. ( It is preferably stored and cooled at a temperature of 8 ° C. or lower.
  • the upper layer medium is overlaid on the lower layer medium.
  • the blood cells fixed on the substrate are preferably stained in order to perform image analysis of the cells.
  • the method for staining blood cells is not particularly limited, and may be performed by a known method. Examples of the blood cell staining method include Giemsa staining and May Grünwald Giemsa staining.
  • the spectroscopic analysis includes, for example, analysis for measuring light absorption caused by hemoglobin.
  • Erythrocytes are characterized by having hemoglobin and differ from other blood cells in having hemoglobin. It is known that red blood cells having hemoglobin absorb blue and green light (especially light in the wavelength range of 400 nm to 500 nm and 525 nm to 580 nm) and appear red. Therefore, red blood cells can be distinguished from other blood cells by irradiating blood cells with single-wavelength light (monochromatic light) in the above wavelength region and measuring the absorbance. The absorbance of the cells can be measured with a microspectrophotometer.
  • the microspectrophotometer is a spectrophotometer that uses an optical system of a microscope, and may be a commercially available device.
  • the leukocyte extinction coefficient is preferably determined by measuring the cell absorbance of 2 to 20 leukocytes close to the nucleated erythrocyte candidate and obtaining the average of the measured extinction coefficients of 2 to 20 cells.
  • the DNA preparation step is a step of obtaining chromosomal DNA or its amplification product from nucleated red blood cells.
  • nucleated red blood cells are lysed using a surfactant and a proteolytic enzyme, and chromosomal DNA is extracted to obtain chromosomal DNA.
  • chromosomal DNA may be further amplified by DNA polymerase.
  • the DNA preparation step is preferably a step of obtaining an amplification product of chromosomal DNA by performing whole genome amplification (WGA).
  • WGA whole genome amplification
  • Whole genome amplification includes, for example, a step of lysing cells using a surfactant and a proteolytic enzyme, and a step of amplifying DNA by DNA polymerase using genomic DNA eluted from the cells as a template.
  • reagents based on PCR include PicoPLEXPLEWGA Kit (New England Biolabs), GenomePlex Single Cell Whole Genome Amplification Kit (Sigma-Aldrich), MALBAC method (Multiple Annealing and Looping disclosed in International Publication No. 2012/166425). -Based (Amplification (Cycles)).
  • reagents based on the strand displacement type DNA synthesis reaction include GenomiPhi DNA Amplification Kit (GE Healthcare, GenomiPhi is a registered trademark) and REPLI-g Single Cell Kit (QIAGEN, REPLI-g is a registered trademark).
  • it is preferable to use PicoPLEX WGA Kit (New England Biolabs).
  • the amplification step may be multiplex PCR (multiplex PCR) for performing multiplex amplification of a plurality of target regions using a plurality of primer pairs for the purpose of analyzing a plurality of regions on the chromosome.
  • the amplified target region is a mixture of a plurality of types of amplification products.
  • the primer pairs used in the first amplification step are one or more primer pairs designed according to the purpose of chromosome analysis.
  • the one or more primer pairs amplify the chromosomal position (target region) selected according to the purpose of chromosome analysis, using the chromosomal DNA obtained in the DNA preparation step or its amplification product as a template.
  • the base sequence complementary to the target region in the primer used in the first amplification step is preferably 15 to 25 bases, more preferably 20 bases.
  • the primer used in the first amplification step preferably has a base sequence at the 5 ′ end separately from the base sequence complementary to the target region.
  • oligonucleotides referred to as “first labels”.
  • the length of the first label is preferably 15 to 20 bases, more preferably 17 bases.
  • the first label added to one end of the target region and the first label added to the other end may have the same or different base sequences. In the present disclosure, the labels added to both ends of the target region regardless of the difference in both base sequences are collectively referred to as “first labels”.
  • the method of adding the first label to both ends of the target region may be ligation by DNA ligase instead of addition by PCR.
  • the addition of the first label by ligation is performed by the ligation reaction after the PCR reaction for amplification.
  • the primer used in the first amplification step may have a base sequence complementary to the target region.
  • PCR reagents applied to the first amplification step include, for example, Multiplex PCR-Assay Kit (Takara Bio Inc.), Multiplex PCR Asssay Kit Ver2 (Takara Bio Inc.), KAPA Library Amplification Kit (Nippon Genetics ( Co., Ltd.), Platinum® Multiplex® PCR® Master® Mix® Kit (Life Technologies, Platinum is a registered trademark).
  • the first amplification step is multiplex PCR, it is preferable to examine the reaction conditions because the optimal temperature for annealing may differ for each primer pair. It is preferable to set the number of PCR cycles based on the result of a real-time PCR study. PCR may change reaction temperature and / or reaction time in the middle.
  • the primer used in the second amplification step preferably has a base sequence at the 5 ′ end apart from the base sequence complementary to the amplification product of the first amplification step.
  • oligonucleotides referred to as “second labels”.
  • the second label added to one end of the amplification product and the second label added to the other end may have the same or different base sequences.
  • the labels added to both ends of the amplified product regardless of the difference in both base sequences are collectively referred to as “second labels”.
  • the second amplification step for adding the second label is sometimes referred to as “adapter-added PCR”, and the second label is sometimes referred to as “adapter”.
  • PCR reagents applied to the second amplification step include Multiplex PCR-Assay Kit (Takara Bio Inc.), Multiplex PCR Asssay Kit Ver2 (Takara Bio Inc.), KAPA Library Amplification Kit (Nippon Genetics Co., Ltd.) ), Platinum, Multiplex, PCR, Master, Mix, Kit (Life Technologies).
  • agarose gel electrophoresis After completion of the first amplification step and after the second amplification step, it is preferable to perform agarose gel electrophoresis to confirm the presence or absence of amplification products.
  • the sequence analysis step is a step of determining the base sequence and amount of the amplified target region. All or part of the final amplification product obtained in the amplification step is a substance that is applied to the sequence analysis apparatus in the sequence analysis step.
  • the sequence analysis step is preferably performed by a next-generation sequencer in terms of the accuracy and speed of analysis, the number of samples that can be processed at one time, and the like.
  • next-generation sequencer such as MiSeq, Burrows-Wheeler Aligner (BWA)
  • BWA Burrows-Wheeler Aligner
  • SAMtools and BEDtools it is preferable to analyze gene polymorphisms, gene mutations, and chromosome numbers using these analysis means.
  • Y chromosome detection in boys When the fetus is confirmed to be a boy by ultrasonography of the mother's body, it can be identified as a nucleated red blood cell derived from the fetus by confirming that the Y chromosome is present in the isolated nucleated red blood cell. .
  • a FISH (Fluorescence in situ hybridization) method using a fluorescent probe specific to the Y chromosome is known.
  • An example of the FISH method test kit is CEP X / Y DNA Probe Kit (Abbott, CEP is a registered trademark).
  • a primer pair having a base sequence specific to the Y chromosome is prepared, and PCR is used to confirm the presence or absence of the amplification, thereby identifying a nucleated red blood cell derived from a male fetus. Is preferred.
  • the amount of the amplification product derived from the mother and the amount of the amplification product derived from the fetus are approximately 1.0: 1.5 (or 2: 3). It is expected to be.
  • All primers were mixed at a final concentration of 25 nmol / L to prepare a primer mix solution.
  • Multiplex PCR was performed using Multiplex PCR Assay Kit (Takara Bio Inc.). 10 ng of the whole genome amplification product obtained from each cell as a template, 8 ⁇ L of primer mix solution, 0.125 ⁇ L of Multiplex PCR Mix1, 12.5 ⁇ L of Multiplex PCR Mix2, and water were prepared to prepare a reaction solution (final solution volume) 25 ⁇ L). The PCR reaction was denatured at 94 ° C./60 seconds, followed by 30 cycles of 94 ° C./30 seconds, 60 ° C./90 seconds, and 72 ° C./30 seconds.
  • the obtained PCR product was purified using AMPure® XP® Kit (BECKMAN® COULTER). The concentration of the PCR product after purification was measured using BioAnalyzer (Agilent). For more accurate PCR product quantification, KAPA Library Quantification Kits (Nippon Genetics Co., Ltd.) was used.
  • Examples 2 to 4 Peripheral blood was collected from 3 pregnant volunteers different from the pregnant volunteers collected in Example 1, and the chromosomes were analyzed in the same manner as in Example 1. In both cases, the same analysis results as in Example 1 were obtained, and it was estimated that none of the fetuses had any aneuploidy in the chromosome.
  • Example 1 the 16 primer pairs used in Example 1 were changed to another 16 primer pairs, and multiplex PCR was performed.
  • the 16 primer pairs used in Comparative Example 1 were designed so that the amplification region included the amplification region of Example 1 and was longer than the amplification region of Example 1.
  • Table 1 shows the names of the genes in which the 16 regions are located and the base lengths of the 16 regions.

Abstract

A method for analyzing a fetal chromosome, said method comprising: a collection step of immobilizing blood cells contained in blood collected from an impregnated mother body onto a substrate, analyzing the blood cells immobilized onto the substrate using an optical device to identify nucleated red blood cells, detaching the identified nucleated red blood cells from the substrate, and collecting the detached nucleated red blood cells; a DNA preparation step of obtaining chromosomal DNA or an amplification product thereof from the collected nucleated red blood cells; an amplification step of amplifying a region of interest, which has a length of 40 to 70 bp inclusive, by a polymerase chain reaction using the chromosomal DNA or a amplification product thereof as a template; and a sequence analysis step of determining the nucleotide sequence and the quantity of the amplified region.

Description

胎児染色体の解析方法Method for analyzing fetal chromosomes
　本発明は、胎児染色体の解析方法に関する。 The present invention relates to a method for analyzing a fetal chromosome.
　出生前遺伝学的検査として、従来、羊水穿刺により採取した羊水から胎児由来細胞を単離し、胎児由来細胞中の染色体を調べる羊水染色体検査が行われてきた。しかし、羊水穿刺には流産を引き起こす可能性がある。 As a prenatal genetic test, an amniotic fluid chromosome test has been performed in which fetal-derived cells are isolated from amniotic fluid collected by amniocentesis and the chromosomes in the fetal-derived cells are examined. However, amniocentesis can cause miscarriage.
　一方、妊娠母体の血液中に存在する胎児由来の無細胞ＤＮＡ、又は、妊娠母体の血液中に存在する胎児由来細胞の染色体ＤＮＡを解析して、胎児染色体の異常を検出する検査が、非侵襲な出生前遺伝学的検査として知られている。例えば、特表２０１４－５０７１４１号公報には、母親由来ＤＮＡと胎児由来ＤＮＡの混合試料から測定された遺伝子型データを利用して、胎児染色体の倍数性状態を決定する方法が開示されている。特許第５０３２３０４号公報には、胎児染色体の異常を単一のアッセイで検出する方法、及びそれに用いるキットが開示されている。特表２００４－５３１２７１号公報には、胎児由来細胞から得た核酸試料をポリメラーゼ連鎖反応で増幅し、シークエンサーで増幅産物の量を確定し、染色体不均衡を検出する方法が開示されている。 On the other hand, a test that detects fetal chromosomal abnormality by analyzing fetal cell-free DNA present in the blood of the pregnant mother or chromosomal DNA of fetal cell present in the blood of the pregnant mother is non-invasive. Known as prenatal genetic testing. For example, Japanese translations of PCT publication No. 2014-507141 discloses a method for determining the ploidy state of fetal chromosomes using genotype data measured from a mixed sample of maternal DNA and fetal DNA. Japanese Patent No. 5032304 discloses a method for detecting an abnormality of a fetal chromosome by a single assay, and a kit used therefor. Japanese translation of PCT publication No. 2004-53271 discloses a method of amplifying a nucleic acid sample obtained from fetus-derived cells by polymerase chain reaction, determining the amount of amplification product with a sequencer, and detecting a chromosomal imbalance.
　非侵襲な出生前遺伝学的検査の中でも、胎児由来の無細胞ＤＮＡを解析する検査は、既に実用化されている。一方、胎児由来細胞の染色体ＤＮＡを解析する検査は、いまだ実用に至っていない。その理由として、胎児由来細胞（例えば、胎児由来の有核赤血球）が母体血数ｍｌ中に１個程度しか存在しない希少細胞であることが挙げられる。しかし、胎児由来細胞の染色体ＤＮＡを解析できれば、より検査精度に優れた非侵襲な出生前遺伝学的検査が期待できる。 Among non-invasive prenatal genetic tests, tests that analyze fetal cell-free DNA have already been put to practical use. On the other hand, the test for analyzing the chromosomal DNA of fetal cells has not yet been put into practical use. The reason for this is that fetal cells (for example, fetal nucleated red blood cells) are rare cells that are present in only about one in several milliliters of maternal blood. However, if the chromosomal DNA of fetal cells can be analyzed, a noninvasive prenatal genetic test with better test accuracy can be expected.
　血液細胞の中から有核赤血球または胎児由来細胞を識別する方法としては、光学機器によって行う方法があり、例えば、特表２００２－５１４３０４号公報、特開昭５８－１１５３４６号公報、特開２０１４－１４４８５号公報、及び特開２００４－２４８６１９号公報に開示されている。特表２００２－５１４３０４号公報には、細胞質を染色して透過可視光の吸収画像を生成し、励起光を照射して核の蛍光画像を形成し、細胞質と核のコントラスト画像を用いて有核赤血球を判別する方法が開示されている。特開昭５８－１１５３４６号公報には、ヘモグロビンの最大吸収波長付近の波長の光を用いて測光を行い、血球の種別を判別する血球種類識別装置が開示されている。特開２０１４－１４４８５号公報には、生体組織の光学的性質を利用した生体情報分析装置が開示されている。特開２００４－２４８６１９号公報には、血液をスライドガラスに塗布し、胎児由来細胞を形態により識別し胎児由来細胞のみを採取する標的細胞自動探索方法が開示されている。 As a method for discriminating nucleated red blood cells or fetal cells from blood cells, there are methods performed by an optical instrument. For example, JP 2002-514304, JP 58-115346, JP 2014 No. 14485 and Japanese Patent Application Laid-Open No. 2004-248619. In JP-T-2002-514304, the cytoplasm is stained to generate an absorption image of transmitted visible light, irradiated with excitation light to form a nuclear fluorescence image, and a cytoplasm-nucleus contrast image is used for nucleation. A method for discriminating red blood cells is disclosed. Japanese Patent Application Laid-Open No. 58-115346 discloses a blood cell type identification device that performs photometry using light having a wavelength near the maximum absorption wavelength of hemoglobin to determine the type of blood cell. Japanese Unexamined Patent Application Publication No. 2014-14485 discloses a biological information analysis apparatus that uses the optical properties of biological tissue. Japanese Patent Application Laid-Open No. 2004-248619 discloses an automatic target cell search method in which blood is applied to a slide glass, fetal-derived cells are identified by morphology, and only fetal-derived cells are collected.
　光学機器によって血液細胞の中から有核赤血球を識別する方法は、希少な有核赤血球を単離するために有用な技術である。しかし、この方法は例えば光学顕微鏡を用いるので、スライドガラス等の基板上に血液細胞を固定した標本を作製する必要があり、有核赤血球は基板上で乾燥に曝され、また、有核赤血球は基板上から剥離して回収されることになる。 A method for identifying nucleated red blood cells from blood cells using an optical instrument is a useful technique for isolating rare nucleated red blood cells. However, since this method uses, for example, an optical microscope, it is necessary to prepare a specimen in which blood cells are fixed on a substrate such as a glass slide. Nucleated erythrocytes are exposed to drying on the substrate. It is peeled off from the substrate and collected.
　一方で、細胞由来の染色体ＤＮＡを解析するためには、解析に十分な量のＤＮＡを得る目的で、微量の染色体ＤＮＡを増幅する必要がある。基板上で乾燥に曝され剥離により回収された有核赤血球からＤＮＡを増幅し解析を行った場合、染色体の異数性が再現性よく検出されなかったり、塩基配列が再現性よく決定されなかったりすることがある。 On the other hand, in order to analyze cell-derived chromosomal DNA, it is necessary to amplify a small amount of chromosomal DNA for the purpose of obtaining a sufficient amount of DNA for analysis. When DNA is amplified and analyzed from nucleated red blood cells that have been exposed to drying on the substrate and recovered by detachment, chromosomal aneuploidy cannot be detected with good reproducibility, or base sequences cannot be determined with good reproducibility. There are things to do.
　本発明の一実施形態は、上記状況のもとになされた。
　本発明の一実施形態は、遺伝情報の解析力に優れる、胎児染色体の解析方法を提供することを目的とする。 One embodiment of the present invention has been made under the above circumstances.
An object of one embodiment of the present invention is to provide a method for analyzing a fetal chromosome that is excellent in the ability to analyze genetic information.
　前記課題を解決するための具体的手段には、以下の態様が含まれる。
［１］　妊娠母体より採取された血液に含まれる血液細胞を基板上に固定し、基板上に固定された血液細胞を光学機器で解析して有核赤血球を識別し、識別された有核赤血球を基板から剥離して回収する回収工程と、回収された有核赤血球から染色体ＤＮＡ又はその増幅産物を得るＤＮＡ調製工程と、染色体ＤＮＡ又はその増幅産物を鋳型にして、４０ｂｐ以上７０ｂｐ以下の目的領域をポリメラーゼ連鎖反応により増幅する増幅工程と、増幅された目的領域の塩基配列及び量を決定する配列解析工程と、を含む、胎児染色体の解析方法。
［２］　染色体の異数性の有無の検出、遺伝子多型の検出、及び、遺伝子変異の検出の少なくともいずれかを行う、［１］に記載の胎児染色体の解析方法。
［３］　増幅工程が、複数のプライマー対を用いて複数の目的領域の多重増幅を行うことを含む、［１］又は［２］に記載の胎児染色体の解析方法。
［４］　ＤＮＡ調製工程が、回収工程で回収された有核赤血球から全ゲノム増幅を行って染色体ＤＮＡの増幅産物を得る工程である、［１］～［３］のいずれか１項に記載の胎児染色体の解析方法。
［５］　回収工程が、基板上に固定された血液細胞を染色し画像解析して有核赤血球を識別することを含む、［１］～［４］のいずれか１項に記載の胎児染色体の解析方法。
［６］　回収工程が、基板上に固定された血液細胞を分光学的に解析して有核赤血球を識別することを含む、［１］～［５］のいずれか１項に記載の胎児染色体の解析方法。
［７］　対立遺伝子の情報により、回収工程で回収された有核赤血球が胎児と母親のいずれの由来であるかを判別する工程をさらに含む、［１］～［６］のいずれか１項に記載の胎児染色体の解析方法。
［８］　配列解析工程が、次世代シークエンサーを用いて行われる、［１］～［７］のいずれか１項に記載の胎児染色体の解析方法。 Specific means for solving the problems include the following aspects.
[1] Blood cells contained in blood collected from a pregnant mother are fixed on a substrate, blood cells fixed on the substrate are analyzed with an optical instrument to identify nucleated red blood cells, and the identified nucleated red blood cells A recovery step for separating the substrate from the substrate, a DNA preparation step for obtaining chromosomal DNA or its amplification product from the recovered nucleated red blood cells, and a target region of 40 bp to 70 bp using the chromosomal DNA or its amplification product as a template A method for analyzing a fetal chromosome, comprising: an amplification step for amplifying the target region by polymerase chain reaction; and a sequence analysis step for determining the base sequence and amount of the amplified target region.
[2] The method for analyzing a fetal chromosome according to [1], wherein at least one of detection of presence / absence of chromosome aneuploidy, detection of gene polymorphism, and detection of gene mutation is performed.
[3] The method for analyzing a fetal chromosome according to [1] or [2], wherein the amplification step includes multiplex amplification of a plurality of target regions using a plurality of primer pairs.
[4] The DNA preparation step according to any one of [1] to [3], wherein the DNA preparation step is a step of obtaining a chromosomal DNA amplification product by performing whole genome amplification from the nucleated red blood cells recovered in the recovery step. Analysis method of fetal chromosome.
[5] The recovery step includes staining of blood cells fixed on the substrate and image analysis to identify nucleated erythrocytes. [1] The fetal chromosome according to any one of [1] to [4] analysis method.
[6] The fetal chromosome according to any one of [1] to [5], wherein the recovery step includes spectroscopic analysis of blood cells fixed on the substrate to identify nucleated red blood cells Analysis method.
[7] The method according to any one of [1] to [6], further including a step of discriminating whether the nucleated red blood cells recovered in the recovery step are derived from a fetus or a mother based on allele information The method for analyzing a fetal chromosome as described.
[8] The method for analyzing a fetal chromosome according to any one of [1] to [7], wherein the sequence analysis step is performed using a next-generation sequencer.
　本発明の一実施形態によれば、遺伝情報の解析力に優れる、胎児染色体の解析方法が提供される。 According to one embodiment of the present invention, there is provided a method for analyzing a fetal chromosome that is excellent in the ability to analyze genetic information.
　本明細書において「工程」との語は、独立した工程だけでなく、他の工程と明確に区別できない場合であってもその工程の所期の目的が達成されれば、本用語に含まれる。
　本明細書において「～」を用いて示された数値範囲は、「～」の前後に記載される数値をそれぞれ最小値および最大値として含む範囲を示す。
　本明細書において組成物中の各成分の量は、組成物中に各成分に該当する物質が複数種存在する場合、特に断らない限り、組成物中に存在する当該複数種の物質の合計量を意味する。 In this specification, the term “process” is not limited to an independent process, and is included in this term if the intended purpose of the process is achieved even when it cannot be clearly distinguished from other processes. .
In the present specification, a numerical range indicated by using “to” indicates a range including the numerical values described before and after “to” as the minimum value and the maximum value, respectively.
In the present specification, the amount of each component in the composition is the total amount of the plurality of substances present in the composition unless there is a specific indication when there are a plurality of substances corresponding to each component in the composition. Means.
　以下に、本発明の実施の形態について説明する。これらの説明及び実施例は本発明を例示するものであり、本発明の範囲を制限するものではない。 Hereinafter, embodiments of the present invention will be described. These descriptions and examples are illustrative of the invention and are not intended to limit the scope of the invention.
　本開示の方法は、胎児の遺伝情報の解析を目的に、胎児染色体を解析する方法である。本開示の方法は、妊娠母体より採取された血液に含まれる有核赤血球から染色体ＤＮＡを得て、染色体ＤＮＡ上の目的領域を増幅し、増幅された目的領域の塩基配列と量とを決定することにより行う、胎児染色体の解析方法である。本開示の方法は、例えば、染色体の異数性の有無の検出、遺伝子多型の検出、及び、遺伝子変異の検出の少なくともいずれかを行う出生前遺伝学的検査に適用できる。 The method of the present disclosure is a method for analyzing fetal chromosomes for the purpose of analyzing fetal genetic information. The method of the present disclosure obtains chromosomal DNA from nucleated red blood cells contained in blood collected from a maternal mother, amplifies a target region on the chromosomal DNA, and determines the base sequence and amount of the amplified target region This is a fetal chromosome analysis method. The method of the present disclosure can be applied to, for example, a prenatal genetic test that performs at least one of detection of the presence or absence of chromosome aneuploidy, detection of gene polymorphism, and detection of gene mutation.
　本開示の胎児染色体の解析方法は、妊娠母体より採取された血液に含まれる血液細胞を基板上に固定し、基板上に固定された血液細胞を光学機器で解析して有核赤血球を識別し、識別された有核赤血球を基板から剥離して回収する回収工程と；回収された有核赤血球から染色体ＤＮＡ又はその増幅産物を得るＤＮＡ調製工程と；染色体ＤＮＡ又はその増幅産物を鋳型にして、４０ｂｐ（base pair、塩基対）～７０ｂｐの目的領域をポリメラーゼ連鎖反応（polymerase chain reaction；ＰＣＲ）により増幅する増幅工程と；増幅された目的領域の塩基配列及び量を決定する配列解析工程と；を含む。
　かかる構成により本開示の方法は、遺伝情報の解析力に優れる。その理由は、特定のメカニズムに拘束されるものではないが、以下に説明するメカニズムによるものと推測される。 In the disclosed fetal chromosome analysis method, blood cells contained in blood collected from a pregnant mother are fixed on a substrate, and blood cells fixed on the substrate are analyzed with an optical instrument to identify nucleated red blood cells. A recovery step of separating and recovering the identified nucleated red blood cells from the substrate; a DNA preparation step of obtaining chromosomal DNA or its amplification product from the recovered nucleated red blood cells; and using the chromosomal DNA or its amplification product as a template, An amplification step of amplifying a 40 bp (base pair) to 70 bp target region by a polymerase chain reaction (PCR); a sequence analysis step for determining the base sequence and amount of the amplified target region; Including.
With this configuration, the method of the present disclosure is excellent in the ability to analyze genetic information. The reason is not limited by a specific mechanism, but is presumed to be due to the mechanism described below.
　基板上に血液細胞を固定し光学機器で解析を行って有核赤血球を識別する識別方法は、通常、血液を基板上に塗布し乾燥させる工程を含み、血液細胞が乾燥に曝されることになる。また、この識別方法は、血液細胞を染色したり、比較的強い光エネルギーを血液細胞に照射したりすることがある。したがって、基板上に固定されて解析を受けた有核赤血球は、染色体ＤＮＡが損傷を受けやすい状態になっていると考えられる。 An identification method for identifying nucleated red blood cells by immobilizing blood cells on a substrate and performing analysis with an optical instrument usually includes a step of applying blood on the substrate and drying it, and the blood cells are exposed to drying. Become. This identification method may stain blood cells or irradiate blood cells with relatively strong light energy. Therefore, it is considered that the nucleated red blood cells fixed on the substrate and analyzed are in a state in which the chromosomal DNA is easily damaged.
　そして、上記の識別方法によれば、有核赤血球は、固定されている基板上から剥離されて回収されることになる。しかも、剥離および回収を効率よく行うには、先端の鋭利なガラス器具をマイクロマニピュレータで操作して、有核赤血球を基板上から１個ずつ剥離して回収することが好ましい。基板上の有核赤血球の染色体は損傷を受けやすい状態になっている上、ガラス器具の先端による機械的刺激も加わって、剥離の際に、染色体ＤＮＡの一本鎖あるいは二本鎖が切断されたり、染色体ＤＮＡが断片化したりすると考えられる。また、断片化した染色体ＤＮＡの一部が基板上に残留することもあると予想される。さらに、損傷を受けやすい状態になっている染色体ＤＮＡは、回収後の保管中にも、切断されたり分解されたりしていると考えられる。
　そして、染色体ＤＮＡの損傷は、細胞が曝された状態によって細胞ごとに発生頻度や発生箇所が異なる。そのために、染色体ＤＮＡを増幅した際に、細胞間で増幅倍率にばらつきが発生し、染色体の異数性の情報が失われてしまうものと推測される。また、染色体ＤＮＡが損傷しているため、目的領域が期待どおり増幅されず、塩基配列が安定して決定されないものと推測される。その結果、上記の識別方法で識別を行って回収した有核赤血球の染色体ＤＮＡを増幅すると、染色体の異数性が再現性よく検出されなかったり、塩基配列が再現性よく決定されなかったりすることがあると考えられる。 And according to said identification method, nucleated red blood cells will be peeled and collect | recovered from the fixed board | substrate. Moreover, in order to efficiently perform separation and recovery, it is preferable to operate a glass instrument having a sharp tip with a micromanipulator to separate and recover the nucleated red blood cells one by one from the substrate. The chromosomes of nucleated red blood cells on the substrate are in a state of being easily damaged, and the mechanical stimulation by the tip of the glass instrument is also applied, and when detaching, the single or double strand of the chromosomal DNA is broken. Or chromosomal DNA is considered to be fragmented. In addition, it is expected that a part of the fragmented chromosomal DNA may remain on the substrate. Furthermore, it is considered that chromosomal DNA that is easily damaged is cut or decomposed even during storage after collection.
The frequency of occurrence and location of chromosomal DNA damage varies from cell to cell depending on the state of the cell being exposed. For this reason, when chromosomal DNA is amplified, it is presumed that variation in amplification magnification occurs between cells, and information on chromosome aneuploidy is lost. Further, since the chromosomal DNA is damaged, it is presumed that the target region is not amplified as expected, and the base sequence is not stably determined. As a result, when chromosomal DNA of nucleated erythrocytes collected after identification by the above identification method is amplified, chromosome aneuploidy may not be detected with good reproducibility, or base sequence may not be determined with good reproducibility. It is thought that there is.
　上記事象に対して、本開示の方法においては、解析のために増幅される目的領域の長さを４０ｂｐ～７０ｂｐとする。本開示の方法の発明者が検討した結果、目的領域の長さが７０ｂｐ以下であると、細胞間において、染色体ＤＮＡの増幅倍率のばらつきが少ないことが分かった。この理由として、目的領域が長いほど配列中に損傷が少なくとも１箇所存在している確率が高いと予想されるところ、７０ｂｐ以下であると損傷の存在確率が十分に低く、目的領域が全長にわたって安定して増幅されるものと考えられる。したがって、目的領域の長さが７０ｂｐ以下であると、遺伝情報を安定して解析することができると考えられる。
　一方、プライマー長に関連する増幅適性、及びシークエンサー原理に関連する解析適性の点で、増幅され解析される領域はある程度の長さを要するため、目的領域の長さは４０ｂｐ以上である。
　上記の観点で、目的領域の塩基長は、４０ｂｐ～７０ｂｐであり、４０ｂｐ～６５ｂｐがより好ましく、４０ｂｐ～６０ｂｐが更に好ましい。 For the above event, in the method of the present disclosure, the length of the target region amplified for analysis is set to 40 bp to 70 bp. As a result of examination by the inventors of the method of the present disclosure, it has been found that when the length of the target region is 70 bp or less, there is little variation in the amplification factor of chromosomal DNA between cells. The reason is that the longer the target area, the higher the probability that at least one damage exists in the array. However, if it is 70 bp or less, the probability of damage is sufficiently low, and the target area is stable over the entire length. It is thought that it is amplified. Therefore, it is considered that genetic information can be stably analyzed when the length of the target region is 70 bp or less.
On the other hand, since the region to be amplified and analyzed requires a certain length in terms of the suitability for amplification related to the primer length and the suitability for analysis related to the sequencer principle, the length of the target region is 40 bp or more.
In view of the above, the base length of the target region is 40 bp to 70 bp, more preferably 40 bp to 65 bp, and still more preferably 40 bp to 60 bp.
　以下、本開示の方法が有する各工程、及び本開示の方法に用いられる生体試料について詳細に説明する。 Hereinafter, each step of the method of the present disclosure and a biological sample used in the method of the present disclosure will be described in detail.
［有核赤血球の回収工程］
　本開示の方法は、妊娠母体より採取された血液に含まれる有核赤血球を回収する回収工程を含む。回収工程は、妊娠母体より採取された血液に含まれる血液細胞を基板上に固定すること；基板上に固定された血液細胞を光学機器で解析して有核赤血球を識別すること；識別された有核赤血球を基板から剥離して回収すること；を含む。
　以下、血液、有核赤血球、及び回収工程に含まれる各工程について説明する。 [Recovery process of nucleated red blood cells]
The method of the present disclosure includes a recovery step of recovering nucleated red blood cells contained in blood collected from a pregnant mother. In the recovery process, blood cells contained in blood collected from the mother's body are fixed on the substrate; blood cells fixed on the substrate are analyzed with an optical instrument to identify nucleated red blood cells; Peeling and recovering nucleated red blood cells from the substrate.
Hereinafter, each step included in blood, nucleated red blood cells, and the recovery step will be described.
［妊娠母体の血液］
　本開示の方法は、妊娠母体より採取された血液から有核赤血球を得て、有核赤血球を胎児染色体の解析に供する。採取の対象となる妊娠母体の血液は、母体末梢血、臍帯血など、胎児由来細胞が存在することが知られている血液であればよく、妊娠母体への侵襲性を極力抑える観点で、母体末梢血が好ましい。本開示において血液には、血液そのもの、及び、生理食塩水で希釈した血液；血液にグルコースや抗血液凝固剤等の添加剤を加えた保存血液；これらの分画物；などの血液試料が含まれる。 [Pregnant mother's blood]
The method of the present disclosure obtains nucleated red blood cells from blood collected from a pregnant mother and uses the nucleated red blood cells for fetal chromosome analysis. The blood of the maternal body to be collected may be blood that is known to contain fetal cells, such as maternal peripheral blood and umbilical cord blood. Peripheral blood is preferred. In the present disclosure, blood includes blood samples such as blood itself and blood diluted with physiological saline; stored blood obtained by adding an additive such as glucose or an anticoagulant to blood; fractions thereof; It is.
　妊娠母体の末梢血には、母親由来の好酸球、好中球、好塩基球、単核球、リンパ球等の白血球；母親由来の、核のない成熟した赤血球；母親由来の有核赤血球；胎児由来の有核赤血球；などの血液細胞が含まれる。本開示の方法においては、これらの血液細胞から胎児由来の有核赤血球を単離し、胎児由来の有核赤血球から染色体ＤＮＡを得る。胎児由来の有核赤血球は、妊娠後６週程度から母体血中に存在するといわれている。したがって、本開示の方法における血液は、妊娠後６週程度以降の妊娠母体より採取した末梢血、又は、妊娠後６週程度以降の妊娠母体より採取した末梢血から調製した血液試料であることが好ましい。 Peripheral blood of the maternal body includes leukocytes such as eosinophils, neutrophils, basophils, mononuclear cells, and lymphocytes from the mother; mature red blood cells from the mother; nucleated red blood cells from the mother Blood cells such as fetal nucleated red blood cells; In the method of the present disclosure, fetal nucleated red blood cells are isolated from these blood cells, and chromosomal DNA is obtained from the fetal nucleated red blood cells. Fetal nucleated red blood cells are said to be present in maternal blood from about 6 weeks after pregnancy. Therefore, the blood in the method of the present disclosure may be a blood sample prepared from peripheral blood collected from a pregnant mother about 6 weeks after pregnancy or from peripheral blood collected from a pregnant mother about 6 weeks after pregnancy. preferable.
　胎児由来の有核赤血球は、胎盤を通過して母体の血液中に存在する、赤血球前駆体である。母体が妊娠中には、胎児の赤血球は有核であり得る。有核赤血球には染色体が存在するため、胎児由来の有核赤血球を単離することで、胎児染色体および胎児遺伝子の入手が可能となる。胎児由来の有核赤血球は、母体血中の細胞の約１０６個に１個の割合で存在しているといわれており、妊娠母体の抹消血中には非常に存在確率が低い。 Fetal nucleated red blood cells are red blood cell precursors that pass through the placenta and are present in the maternal blood. When the mother is pregnant, fetal red blood cells can be nucleated. Since nucleated red blood cells have chromosomes, fetal chromosomes and fetal genes can be obtained by isolating fetal nucleated red blood cells. Fetal nucleated red blood cells are said to be present in a ratio of about 1 in 10 6 cells in maternal blood, and the probability of existence is very low in peripheral blood of the pregnant mother.
　胎児由来の有核赤血球は、光学機器を用いた解析（「光学的解析」とも言う）により、血液中に存在するその他の血液細胞と区別され単離され得る。光学的解析は、好ましくは、画像解析及び／又は分光学的解析である。光学的解析を容易にするため、光学的解析に先だって、血液を密度勾配遠心分離に供し有核赤血球を濃縮することが好ましい。 Fetal nucleated red blood cells can be isolated from other blood cells present in blood by analysis using an optical instrument (also referred to as “optical analysis”). The optical analysis is preferably image analysis and / or spectroscopic analysis. In order to facilitate optical analysis, it is preferable to concentrate nucleated red blood cells by subjecting the blood to density gradient centrifugation prior to optical analysis.
［有核赤血球の密度勾配遠心分離（有核赤血球の濃縮）］
　有核赤血球は、密度勾配遠心分離により、血液中に存在する血漿成分やその他の血液細胞と分離され得る。有核赤血球を分離するための密度勾配遠心分離は、公知の方法を適用してよい。例えば、密度（比重）の異なる２種類の媒体を遠沈管に重層した不連続密度勾配の上に、生理食塩水で希釈した血液を重層して遠心を行うことにより、有核赤血球を分画し濃縮できる。 [Density gradient centrifugation of nucleated red blood cells (concentration of nucleated red blood cells)]
Nucleated red blood cells can be separated from plasma components and other blood cells present in the blood by density gradient centrifugation. A known method may be applied to density gradient centrifugation for separating nucleated red blood cells. For example, nucleated red blood cells are fractionated by centrifuging and centrifuging blood diluted with physiological saline on a discontinuous density gradient in which two types of media with different densities (specific gravity) are layered on a centrifuge tube. It can be concentrated.
　国際公開第２０１２／０２３２９８号に、胎児由来の有核赤血球を含めた母体の血球の密度が記載されている。その記載によると、想定される胎児由来の有核赤血球の密度は、1.065～1.095ｇ／ｍＬ程度、母親の血球の密度は、赤血球が1.070～1.120ｇ／ｍＬ程度、好酸球が1.090～1.110ｇ／ｍＬ程度、好中球が1.075～1.100ｇ／ｍＬ程度、好塩基球が1.070～1.080ｇ／ｍＬ程度、リンパ球が1.060～1.080ｇ／ｍＬ程度、単核球が1.060～1.070ｇ／ｍＬ程度である。 International Publication No. 2012/023298 describes the density of maternal blood cells including fetal nucleated red blood cells. According to the description, the expected density of fetal nucleated red blood cells is about 1.065 to 1.095 g / mL, the density of maternal blood cells is about 1.070 to 1.120 g / mL for red blood cells, and 1.090 to 1.110 for eosinophils. About g / mL, About 1.075 to 1.100 g / mL for neutrophils, About 1.070 to 1.080 g / mL for basophils, About 1.060 to 1.080 g / mL for lymphocytes, 1.060 to 1.070 g / mL for mononuclear cells Degree.
　密度1.065～1.095ｇ／ｍＬ程度の胎児由来の有核赤血球を、ほかの血液細胞と分離するために、積層する媒体の密度（比重）が設定される。胎児由来の有核赤血球の中心の密度は1.080ｇ／ｍＬ程度であるため、この密度をはさむ２つの異なる密度の媒体を隣接して重層することで、その界面に胎児由来の有核赤血球を有する画分を集めることが可能となる。好ましくは、下層の媒体の密度を1.08ｇ／ｍＬ～1.10ｇ／ｍＬ（より好ましくは1.08ｇ／ｍＬ～1.09ｇ／ｍＬ）、上層の媒体の密度を1.06ｇ／ｍＬ～1.08ｇ／ｍＬ（より好ましくは1.065ｇ／ｍＬ～1.08ｇ／ｍＬ）とする。本開示の方法においては、下層の媒体と上層の媒体は同じ種類でも異なる種類でもよく、同じ種類の媒体を用いることが好ましい。 The density (specific gravity) of the medium to be stacked is set in order to separate fetal nucleated red blood cells with a density of about 1.065 to 1.095 g / mL from other blood cells. Since the density of the center of fetal nucleated red blood cells is about 1.080 g / mL, fetal nucleated red blood cells are present at the interface by layering two different density media adjacent to this density. It is possible to collect fractions. Preferably, the density of the lower layer medium is 1.08 g / mL to 1.10 g / mL (more preferably 1.08 g / mL to 1.09 g / mL), and the density of the upper layer medium is 1.06 g / mL to 1.08 g / mL (more Preferably, it is 1.065 g / mL to 1.08 g / mL). In the method of the present disclosure, the lower layer medium and the upper layer medium may be the same type or different types, and the same type of medium is preferably used.
　媒体としては、ポリビニルピロリドンでコートされた直径１５ｎｍ～３０ｎｍのケイ酸コロイド粒子分散液であるPercoll（登録商標）、ショ糖から作られた側鎖に富んだ中性の親水性ポリマーであるFicoll-Paque（登録商標）、ポリスクロースとジアトリゾ酸ナトリウムを含むHistopaque（登録商標）等が挙げられる。本開示の方法においては、Percoll及び／又はHistopaqueを使用することが好ましい。Percollは、密度１．１３０の製品が市販されており、水で希釈することで密度勾配を調製することが可能である。Histopaqueは、市販されている密度１．０７７の媒体及び密度１．１１９の媒体と水とを用いて密度勾配を調製することが可能である。 As a medium, Percoll (registered trademark) which is a dispersion of 15 to 30 nm in diameter of silica silicate coated with polyvinylpyrrolidone, Ficoll- which is a neutral hydrophilic polymer rich in side chains made from sucrose. Examples include Paque (registered trademark), Histopaque (registered trademark) containing polysucrose and sodium ditrizoate. In the method of the present disclosure, it is preferable to use Percoll and / or Histopaque. Percoll is commercially available with a density of 1.130, and it is possible to prepare a density gradient by diluting with water. Histopaque can prepare density gradients using commercially available media of density 1.077 and media of density 1.119 and water.
　２層の不連続密度勾配は、例えば以下のようにして遠沈管に形成する。まず、凝固点以上かつ１４℃以下（好ましくは８℃以下）の温度状態にある下層の媒体を遠沈管の底部に収容する、又は、下層の媒体を遠沈管の底部に収容したのち１４℃以下（好ましくは８℃以下）の温度下で保存して冷却する。次に、下層の媒体の上に上層の媒体を重層する。 The two-layer discontinuous density gradient is formed in the centrifuge tube as follows, for example. First, the lower layer medium in a temperature state not lower than the freezing point and not higher than 14 ° C. (preferably not higher than 8 ° C.) is accommodated in the bottom of the centrifuge tube, or the lower layer medium is accommodated in the bottom of the centrifuge tube and then not higher than 14 ° C. ( It is preferably stored and cooled at a temperature of 8 ° C. or lower. Next, the upper layer medium is overlaid on the lower layer medium.
［基板上への血液細胞の固定］
　光学機器で血液細胞を解析するために、妊娠母体より採取された血液に含まれる血液細胞を基板上へ固定する。血液細胞の基板上への固定は、例えば、血液を基板に塗布し乾燥することにより行われる。具体的には、密度勾配遠心分離により得た有核赤血球を含む画分を、透明基板（好ましくはスライドガラス）上に塗布し乾燥することで、血液細胞を基板上へ固定することが好ましい。 [Immobilization of blood cells on the substrate]
In order to analyze blood cells with an optical instrument, blood cells contained in blood collected from a pregnant mother are fixed on a substrate. The fixation of blood cells on the substrate is performed, for example, by applying blood to the substrate and drying. Specifically, it is preferable to fix blood cells on the substrate by applying a fraction containing nucleated red blood cells obtained by density gradient centrifugation onto a transparent substrate (preferably a slide glass) and drying.
　基板上に固定された血液細胞は、細胞の画像解析を行うため、染色されることが好ましい。血液細胞の染色法は、特に制限されず、公知の方法で行ってよい。血液細胞の染色法としては、例えば、ギムザ染色、メイ・グリュンワルド・ギムザ染色が挙げられる。 The blood cells fixed on the substrate are preferably stained in order to perform image analysis of the cells. The method for staining blood cells is not particularly limited, and may be performed by a known method. Examples of the blood cell staining method include Giemsa staining and May Grünwald Giemsa staining.
［基板上での有核赤血球の識別］
　基板上での有核赤血球の識別は、基板上に固定された血液細胞を光学機器で解析する、光学的解析により行われる。血液細胞を基板上で光学機器で解析することにより、有核赤血球を確度高く識別し得る。光学的解析は、好ましくは、画像解析及び／又は分光学的解析である。画像解析と分光学的解析は、いずれか一方を行ってもよく両方を行ってもよく、確実に有核赤血球を識別する観点で、両方を行うことが好ましい。 [Identification of nucleated red blood cells on a substrate]
Identification of nucleated red blood cells on a substrate is performed by optical analysis in which blood cells fixed on the substrate are analyzed with an optical instrument. By analyzing blood cells with an optical instrument on a substrate, nucleated red blood cells can be identified with high accuracy. The optical analysis is preferably image analysis and / or spectroscopic analysis. Either one or both of image analysis and spectroscopic analysis may be performed, and it is preferable to perform both from the viewpoint of reliably identifying nucleated red blood cells.
－血液細胞の画像解析－
　血液細胞の画像解析は、胎児由来の有核赤血球の候補を選ぶ目的で、基板（好ましくは透明基板）上の血液細胞の形態情報を取得して解析する。血液細胞の形態情報は、光学顕微鏡、デジタルカメラ、スライドガラス用のステージ、光学搬送系、画像処理用パーソナルコンピュータ（ＰＣ）、制御用ＰＣ、及びディスプレイを装備したシステムによって、標本から取得され画像解析されることが好ましい。光学搬送系は、例えば、対物レンズとＣＣＤカメラを備える。画像処理用ＰＣは、例えば、データ解析、データ記憶を行う処理系を備える。制御用ＰＣは、例えば、スライドガラス用のステージの位置制御や、全体の処理を制御する制御系を備える。 -Image analysis of blood cells-
The blood cell image analysis is performed by acquiring and analyzing blood cell morphology information on a substrate (preferably a transparent substrate) for the purpose of selecting fetal nucleated red blood cell candidates. Blood cell morphology information is acquired from specimens and analyzed by a system equipped with an optical microscope, digital camera, slide glass stage, optical transport system, image processing personal computer (PC), control PC, and display. It is preferred that The optical transport system includes, for example, an objective lens and a CCD camera. The image processing PC includes, for example, a processing system that performs data analysis and data storage. The control PC includes, for example, a control system for controlling the position of the slide glass stage and the entire process.
　胎児由来の有核赤血球の候補は、細胞質の面積に対する核の面積の割合、核の円形度、核の面積、等によって識別可能である。確実性の観点で、細胞質の面積に対する核の面積の割合、及び核の円形度、の少なくとも一方の条件（好ましくは両方の条件）を満たす細胞を、胎児由来の有核赤血球候補として識別することが好ましい。 Fetus-derived nucleated red blood cell candidates can be identified by the ratio of the nucleus area to the cytoplasm area, the circularity of the nucleus, the area of the nucleus, and the like. In terms of certainty, a cell satisfying at least one of the ratio of the nuclear area to the cytoplasmic area and the circularity of the nucleus (preferably both conditions) is identified as a fetus-derived nucleated red blood cell candidate. Is preferred.
　細胞質の面積に対する核の面積の割合については、下記の式（ａ）を満たす細胞を選択することが好ましい。核の円形度については、下記の式（ｂ）を満たす細胞を選択することが好ましい。
（ａ）０．２５＜（Ｎ／Ｃ）＜１．０
（ｂ）０．６５＜（Ｎ／（Ｌ×Ｌ））＜０．７８５
　式中、Ｃは、細胞質の面積、Ｎは、核の面積、Ｌは、核の長径の長さ、又は、複雑な形をした核に外接する楕円の長径の長さ、である。 Regarding the ratio of the area of the nucleus to the area of the cytoplasm, it is preferable to select cells that satisfy the following formula (a). Regarding the circularity of the nucleus, it is preferable to select a cell that satisfies the following formula (b).
(A) 0.25 <(N / C) <1.0
(B) 0.65 <(N / (L × L)) <0.785
In the formula, C is the area of the cytoplasm, N is the area of the nucleus, L is the length of the major axis of the nucleus, or the major axis of the ellipse circumscribing the complex-shaped nucleus.
　式（ａ）及び式（ｂ）を満たす細胞の中で、核の形状が真円あるいは楕円に近い順に順位をつけ、順位の高いものから優先して以降の工程に供してもよい。また、有核赤血球の由来を判別する工程まで行って、順位の高い複数個の中に胎児由来の細胞が含まれていなかった場合、次に順位の高い複数個の細胞を解析してもよい。 Among the cells satisfying the formulas (a) and (b), the nuclei may be ranked in the order close to a perfect circle or an ellipse, and may be used in the subsequent steps with priority from the highest ranking. In addition, when the origin of nucleated red blood cells is determined and fetal-derived cells are not included in the plurality of higher ranks, the plurality of cells having the next highest rank may be analyzed. .
－血液細胞の分光学的解析－
　分光学的解析としては、例えば、ヘモグロビンに起因する吸光を測定する解析が挙げられる。赤血球はヘモグロビンを有することを特徴とし、ヘモグロビンを有する点でほかの血液細胞と異なる。ヘモグロビンを有する赤血球は、青と緑の光（特には４００ｎｍ～５００ｎｍ及び５２５ｎｍ～５８０ｎｍの波長領域の光）を吸収し、赤色に見えることが知られている。したがって、上記波長領域の単波長の光（単色光）を血液細胞に照射し吸光度を測定することで、赤血球をほかの血液細胞と区別し得る。細胞の吸光度の測定は、顕微分光光度計によって可能である。顕微分光光度計は、顕微鏡の光学系を利用する分光光度計であり、市販の装置であってよい。 -Spectroscopic analysis of blood cells-
The spectroscopic analysis includes, for example, analysis for measuring light absorption caused by hemoglobin. Erythrocytes are characterized by having hemoglobin and differ from other blood cells in having hemoglobin. It is known that red blood cells having hemoglobin absorb blue and green light (especially light in the wavelength range of 400 nm to 500 nm and 525 nm to 580 nm) and appear red. Therefore, red blood cells can be distinguished from other blood cells by irradiating blood cells with single-wavelength light (monochromatic light) in the above wavelength region and measuring the absorbance. The absorbance of the cells can be measured with a microspectrophotometer. The microspectrophotometer is a spectrophotometer that uses an optical system of a microscope, and may be a commercially available device.
　血液細胞の分光学的解析は、画像解析によって有核赤血球候補を識別した後、この候補細胞について解析し、候補細胞が赤血球であることを確認する目的で、及び／又は、候補細胞から白血球を除く目的で、行われることが好ましい。
　具体的には、画像解析によって識別した有核赤血球候補と、その近傍に存在する白血球（形態情報により白血球と予想される細胞）とについて、細胞の吸光度を測定し、吸光係数を求めて、吸光係数を比べる。例えば４１５ｎｍ近傍の単色光を照射した場合、赤血球の吸光係数と白血球の吸光係数との比（赤血球／白血球）は１以上である。この赤血球の吸光係数と白血球の吸光係数との比が大きい順に優先順位をつけて、有核赤血球候補とすることが好ましい。白血球の吸光係数は、有核赤血球候補に近い２個～２０個の白血球について細胞の吸光度を測定し、測定した細胞２個～２０個の吸光係数の平均を求めることが好ましい。 In spectroscopic analysis of blood cells, nucleated red blood cell candidates are identified by image analysis, and the candidate cells are analyzed for the purpose of confirming that the candidate cells are red blood cells. It is preferable to be performed for the purpose of removing.
Specifically, for the nucleated red blood cell candidate identified by image analysis and the white blood cells (cells expected to be white blood cells based on morphology information) present in the vicinity, the absorbance of the cells is measured, the extinction coefficient is obtained, Compare the coefficients. For example, when the monochromatic light near 415 nm is irradiated, the ratio of the extinction coefficient of red blood cells to that of white blood cells (red blood cells / white blood cells) is 1 or more. It is preferable to give priority to the nucleated red blood cell candidates in descending order of the ratio of the red blood cell extinction coefficient to the white blood cell extinction coefficient. The leukocyte extinction coefficient is preferably determined by measuring the cell absorbance of 2 to 20 leukocytes close to the nucleated erythrocyte candidate and obtaining the average of the measured extinction coefficients of 2 to 20 cells.
［有核赤血球の基板からの剥離］
　識別された有核赤血球は、基板上から１個ずつ剥離され回収される。有核赤血球の剥離は、例えば、鋭利な先端を有するガラス器具をマイクロマニピュレータで操作して行われる。基板上から剥離された有核赤血球は、例えば、ガラス管内に回収される。回収された有核赤血球から、ＤＮＡ調製工程（好ましくは全ゲノム増幅）によって、染色体ＤＮＡ又はその増幅産物が調製される。 [Peeling of nucleated red blood cells from the substrate]
The identified nucleated red blood cells are peeled one by one from the substrate and collected. Separation of nucleated red blood cells is performed, for example, by operating a glass instrument having a sharp tip with a micromanipulator. Nucleated red blood cells separated from the substrate are collected in, for example, a glass tube. Chromosomal DNA or its amplification product is prepared from the collected nucleated red blood cells by a DNA preparation step (preferably whole genome amplification).
　以下、有核赤血球の染色体ＤＮＡを増幅して胎児染色体を解析するために行う、ＤＮＡ調製工程、増幅工程、及び配列解析工程について説明する。 Hereinafter, a DNA preparation process, an amplification process, and a sequence analysis process that are performed in order to amplify chromosomal DNA of nucleated red blood cells and analyze fetal chromosomes will be described.
［ＤＮＡ調製工程］
　ＤＮＡ調製工程は、有核赤血球から染色体ＤＮＡ又はその増幅産物を得る工程である。ＤＮＡ調製工程は、例えば、界面活性剤とタンパク質分解酵素を用いて有核赤血球を溶解させて染色体ＤＮＡを抽出し、染色体ＤＮＡを得る。ＤＮＡ調製工程は、さらに、ＤＮＡポリメラーゼによって染色体ＤＮＡを増幅させてもよい。 [DNA preparation step]
The DNA preparation step is a step of obtaining chromosomal DNA or its amplification product from nucleated red blood cells. In the DNA preparation step, for example, nucleated red blood cells are lysed using a surfactant and a proteolytic enzyme, and chromosomal DNA is extracted to obtain chromosomal DNA. In the DNA preparation step, chromosomal DNA may be further amplified by DNA polymerase.
　ＤＮＡ調製工程は、全ゲノム増幅（whole genome amplification；ＷＧＡ）を行って染色体ＤＮＡの増幅産物を得る工程であることが好ましい。全ゲノム増幅は、有核赤血球１個に存在する微量な染色体ＤＮＡを増幅することが可能であり、染色体ＤＮＡの解析を容易にする。 The DNA preparation step is preferably a step of obtaining an amplification product of chromosomal DNA by performing whole genome amplification (WGA). Whole genome amplification makes it possible to amplify a small amount of chromosomal DNA present in one nucleated red blood cell, facilitating analysis of chromosomal DNA.
　全ゲノム増幅の方法は、特に制限されず、公知の方法で行ってよい。全ゲノム増幅は、例えば、界面活性剤とタンパク質分解酵素を用いて細胞を溶解させる工程と、細胞から溶出したゲノムＤＮＡを鋳型にしてＤＮＡポリメラーゼによってＤＮＡを増幅する工程と、を含む。 The method of whole genome amplification is not particularly limited, and may be performed by a known method. Whole genome amplification includes, for example, a step of lysing cells using a surfactant and a proteolytic enzyme, and a step of amplifying DNA by DNA polymerase using genomic DNA eluted from the cells as a template.
　全ゲノム増幅は、市販の試薬を適用して行ってよい。ＰＣＲに基く試薬としては、例えば、PicoPLEX WGA Kit（New England Biolabs社）、GenomePlex Single Cell Whole Genome Amplification Kit（Sigma-Aldrich社）、国際公開第２０１２／１６６４２５号に開示のＭＡＬＢＡＣ法（Multiple Annealing and Looping-Based Amplification Cycles）に係る試薬が挙げられる。鎖置換型ＤＮＡ合成反応に基く試薬としては、例えば、GenomiPhi DNA Amplification Kit（ＧＥヘルスケア社、GenomiPhiは登録商標）、REPLI-g Single Cell Kit（QIAGEN社、REPLI-gは登録商標）が挙げられる。本開示の方法においては、PicoPLEX WGA Kit（New England Biolabs社）を用いることが好ましい。 Whole genome amplification may be performed using commercially available reagents. Examples of reagents based on PCR include PicoPLEXPLEWGA Kit (New England Biolabs), GenomePlex Single Cell Whole Genome Amplification Kit (Sigma-Aldrich), MALBAC method (Multiple Annealing and Looping disclosed in International Publication No. 2012/166425). -Based (Amplification (Cycles)). Examples of reagents based on the strand displacement type DNA synthesis reaction include GenomiPhi DNA Amplification Kit (GE Healthcare, GenomiPhi is a registered trademark) and REPLI-g Single Cell Kit (QIAGEN, REPLI-g is a registered trademark). . In the method of the present disclosure, it is preferable to use PicoPLEX WGA Kit (New England Biolabs).
　全ゲノム増幅の終了後には、アガロースゲル電気泳動を行って、増幅産物の有無を確認することが好ましい。全ゲノム増幅の増幅産物は、精製することが好ましく、精製は、例えば、QIAquick PCR Purification Kit（QIAGEN社、QIAquickは登録商標）を用いて行う。全ゲノム増幅で得た増幅産物の量は、例えば、NanoDrop（登録商標、Thermo Fisher Scientific社）、BioAnalyzer（Agilent社）、Quantus Fluorometer（Promega社）を用いて濃度を測定することで確認し得る。 After completion of whole genome amplification, it is preferable to perform agarose gel electrophoresis to confirm the presence or absence of amplification products. The amplification product of whole genome amplification is preferably purified, and purification is performed using, for example, QIAquick PCR Purification Kit (QIAGEN, QIAquick is a registered trademark). The amount of amplification product obtained by whole genome amplification can be confirmed by measuring the concentration using, for example, NanoDrop (registered trademark, Thermo Fisher Fisher), BioAnalyzer (Agilent), or Quantus Fluorometer (Promega).
［増幅工程］
　増幅工程は、ＤＮＡ調製工程で得た染色体ＤＮＡ又はその増幅産物を鋳型にして、４０ｂｐ～７０ｂｐの目的領域をＰＣＲにより増幅する工程である。目的領域（増幅される領域）は、染色体解析の目的（例えば、染色体の異数性、遺伝子多型、及び遺伝子変異の少なくともいずれかを検出する）に応じて選択される、染色体上の領域である。本開示の方法における目的領域の長さは、４０ｂｐ～７０ｂｐであり、４０ｂｐ～６５ｂｐがより好ましく、４０ｂｐ～６０ｂｐが更に好ましい。 [Amplification process]
The amplification step is a step of amplifying a target region of 40 bp to 70 bp by PCR using the chromosomal DNA obtained in the DNA preparation step or its amplification product as a template. The target region (region to be amplified) is a region on the chromosome that is selected according to the purpose of chromosome analysis (for example, detecting at least one of chromosome aneuploidy, gene polymorphism, and gene mutation). is there. In the method of the present disclosure, the length of the target region is 40 bp to 70 bp, more preferably 40 bp to 65 bp, and still more preferably 40 bp to 60 bp.
　増幅工程は、染色体上の複数の領域を解析する目的で、複数のプライマー対を用いて複数の目的領域の多重増幅を行う多重ＰＣＲ（マルチプレックスＰＣＲ）としてもよい。この場合、増幅された目的領域は、複数種類の増幅産物の混合物である。 The amplification step may be multiplex PCR (multiplex PCR) for performing multiplex amplification of a plurality of target regions using a plurality of primer pairs for the purpose of analyzing a plurality of regions on the chromosome. In this case, the amplified target region is a mixture of a plurality of types of amplification products.
　本開示の方法において、増幅工程は、ＰＣＲを１回行ってもよく２回以上行ってもよい。配列解析工程を次世代シークエンサーで行う場合、増幅工程はＰＣＲを少なくとも２回行うことが好ましい。以下、増幅工程の好ましい態様として、次世代シークエンサーで配列解析を行うことを目的にＰＣＲを２回行う実施態様を説明する。この実施態様において、１回目のＰＣＲを第一増幅工程と言い、２回目のＰＣＲを第二増幅工程と言う。 In the method of the present disclosure, the amplification step may be performed once or twice or more. When the sequence analysis step is performed with a next-generation sequencer, the amplification step is preferably performed at least twice. Hereinafter, an embodiment in which PCR is performed twice for the purpose of performing sequence analysis with a next-generation sequencer will be described as a preferred embodiment of the amplification step. In this embodiment, the first PCR is referred to as a first amplification step, and the second PCR is referred to as a second amplification step.
－第一増幅工程－
　第一増幅工程は、ＤＮＡ調製工程で得た染色体ＤＮＡ又はその増幅産物を鋳型にしてＰＣＲを行い、増幅産物を得る工程である。 -First amplification step-
The first amplification step is a step of obtaining an amplification product by performing PCR using the chromosomal DNA obtained in the DNA preparation step or its amplification product as a template.
　第一増幅工程に用いるプライマー対は、染色体解析の目的に応じて設計される、１つ又は複数のプライマー対である。この１つ又は複数のプライマー対は、ＤＮＡ調製工程で得た染色体ＤＮＡ又はその増幅産物を鋳型にして、染色体解析の目的に応じて選ばれた染色***置（目的領域）を増幅する。第一増幅工程に用いるプライマーにおける目的領域に相補的な塩基配列は、１５～２５塩基が好ましく、２０塩基がより好ましい。 The primer pairs used in the first amplification step are one or more primer pairs designed according to the purpose of chromosome analysis. The one or more primer pairs amplify the chromosomal position (target region) selected according to the purpose of chromosome analysis, using the chromosomal DNA obtained in the DNA preparation step or its amplification product as a template. The base sequence complementary to the target region in the primer used in the first amplification step is preferably 15 to 25 bases, more preferably 20 bases.
　第一増幅工程に用いるプライマーは、目的領域に相補的な塩基配列とは別に、５’末端に塩基配列を有していることが好ましい。このようなプライマーを第一増幅工程に用いることによって、目的領域の両末端にオリゴヌクレオチド（「第一の標識」と言う）が付加される。第一増幅工程の増幅産物間で第一の標識を共通にすることにより、第二増幅工程において１種のプライマー対でＰＣＲを行うことが可能になる。第一の標識の長さは、好ましくは１５～２０塩基、より好ましくは１７塩基である。
　目的領域の一方の末端に付加される第一の標識と、もう一方の末端に付加される第一の標識とは、塩基配列が同じでもよく異なっていてもよい。本開示においては、両者の塩基配列の異同にかかわらず、目的領域の両末端に付加される標識は「第一の標識」と総称される。 The primer used in the first amplification step preferably has a base sequence at the 5 ′ end separately from the base sequence complementary to the target region. By using such a primer in the first amplification step, oligonucleotides (referred to as “first labels”) are added to both ends of the target region. By making the first label common among the amplification products of the first amplification step, it becomes possible to perform PCR with one primer pair in the second amplification step. The length of the first label is preferably 15 to 20 bases, more preferably 17 bases.
The first label added to one end of the target region and the first label added to the other end may have the same or different base sequences. In the present disclosure, the labels added to both ends of the target region regardless of the difference in both base sequences are collectively referred to as “first labels”.
　第一の標識を目的領域の両末端に付加する方法は、ＰＣＲによる付加に代えて、ＤＮＡリガーゼによるライゲーションでもよい。この場合、ライゲーションによる第一の標識の付加は、増幅目的のＰＣＲ反応後にライゲーション反応によって行う。ライゲーションによる付加を採用する場合、第一増幅工程に用いるプライマーは、目的領域に相補的な塩基配列を有していればよい。 The method of adding the first label to both ends of the target region may be ligation by DNA ligase instead of addition by PCR. In this case, the addition of the first label by ligation is performed by the ligation reaction after the PCR reaction for amplification. When employing addition by ligation, the primer used in the first amplification step may have a base sequence complementary to the target region.
　第一増幅工程に適用するＰＣＲ試薬としては、例えば、Multiplex PCR Assay Kit（タカラバイオ（株）社）、Multiplex PCR Assay Kit ver2（タカラバイオ（株）社）、KAPA Library Amplification Kit（日本ジェネティクス（株）社）、Platinum Multiplex PCR Master Mix Kit（ライフテクノロジーズ社、Platinumは登録商標）が挙げられる。本開示の方法においては、Multiplex PCR Assay Kit（タカラバイオ（株）社）を用いることが好ましい。第一増幅工程をマルチプレックスＰＣＲとする場合、プライマー対ごとにアニーリングの至適温度が異なることがあるため、反応条件の検討をすることが好ましい。ＰＣＲのサイクル数は、リアルタイムＰＣＲによる検討を予め行って、その結果に基き設定することが好ましい。ＰＣＲは、その途中で、反応温度及び／又は反応時間を変更してもよい。 PCR reagents applied to the first amplification step include, for example, Multiplex PCR-Assay Kit (Takara Bio Inc.), Multiplex PCR Asssay Kit Ver2 (Takara Bio Inc.), KAPA Library Amplification Kit (Nippon Genetics ( Co., Ltd.), Platinum® Multiplex® PCR® Master® Mix® Kit (Life Technologies, Platinum is a registered trademark). In the method of the present disclosure, it is preferable to use Multiplex PCR PCR Assay Kit (Takara Bio Inc.). When the first amplification step is multiplex PCR, it is preferable to examine the reaction conditions because the optimal temperature for annealing may differ for each primer pair. It is preferable to set the number of PCR cycles based on the result of a real-time PCR study. PCR may change reaction temperature and / or reaction time in the middle.
－第二増幅工程－
　第二増幅工程は、第一増幅工程の増幅産物を鋳型にしてＰＣＲを行い、増幅産物を得る工程である。 -Second amplification step-
The second amplification step is a step of obtaining an amplification product by performing PCR using the amplification product of the first amplification step as a template.
　第二増幅工程に用いるプライマーは、第一増幅工程の増幅産物に相補的な塩基配列を有していればよい。第一増幅工程において目的領域の両末端に第一の標識が付加された場合は、第二増幅工程に用いるプライマーは、第一の標識にアニールするプライマーであることが好ましい。このプライマーにおける、第一の標識にアニールする配列の塩基長は、第一の標識の塩基長と同じが好ましく、具体的には、１５～２０塩基が好ましく、１７塩基がより好ましい。 The primer used in the second amplification step only needs to have a base sequence complementary to the amplification product of the first amplification step. When the first label is added to both ends of the target region in the first amplification step, the primer used in the second amplification step is preferably a primer that anneals to the first label. In this primer, the base length of the sequence annealed to the first label is preferably the same as the base length of the first label, specifically, 15 to 20 bases are preferable, and 17 bases are more preferable.
　第二増幅工程に用いるプライマーは、第一増幅工程の増幅産物に相補的な塩基配列とは別に、５’末端に塩基配列を有していることが好ましい。このようなプライマーを第二増幅工程に用いることによって、増幅産物の両末端にオリゴヌクレオチド（「第二の標識」と言う）が付加される。
　増幅産物の一方の末端に付加される第二の標識と、もう一方の末端に付加される第二の標識とは、塩基配列が同じでもよく異なっていてもよい。本開示においては、両者の塩基配列の異同にかかわらず、増幅産物の両末端に付加される標識は「第二の標識」と総称される。 The primer used in the second amplification step preferably has a base sequence at the 5 ′ end apart from the base sequence complementary to the amplification product of the first amplification step. By using such a primer in the second amplification step, oligonucleotides (referred to as “second labels”) are added to both ends of the amplified product.
The second label added to one end of the amplification product and the second label added to the other end may have the same or different base sequences. In the present disclosure, the labels added to both ends of the amplified product regardless of the difference in both base sequences are collectively referred to as “second labels”.
　第二の標識は、増幅産物の解析を次世代シークエンサーで行うことを目的に増幅産物に付加されるオリゴヌクレオチドである。その塩基配列は、次世代シークエンサーの解析原理に従って設計される（次世代シークエンサーの詳細は後述する）。第二の標識の塩基長は、一例として５９～６４塩基が挙げられる。 The second label is an oligonucleotide added to the amplification product for the purpose of analyzing the amplification product with a next-generation sequencer. The base sequence is designed according to the analysis principle of the next generation sequencer (details of the next generation sequencer will be described later). An example of the base length of the second label is 59 to 64 bases.
　次世代シークエンサーとしてIllumina社のMiSeq又はHiSeq2000を使用する場合、第二の標識としては、例えば下記の一対のオリゴヌクレオチドが挙げられる。下記の２つのオリゴヌクレオチドの一方が、増幅産物の一方の末端に付加され、もう一方が、増幅産物のもう一方の末端に付加される。
・フローセル上に固定されたオリゴヌクレオチドにハイブリダイズするＰ５配列と、６～８塩基からなるサンプル識別配列（インデックス配列）と、シークエンスプライマーがアニールする配列（リード配列）と、を有するオリゴヌクレオチド。
・フローセル上に固定されたオリゴヌクレオチドにハイブリダイズするＰ７配列と、６～８塩基からなるサンプル識別配列（インデックス配列）と、シークエンスプライマーがアニールする配列（リード配列）と、を有するオリゴヌクレオチド。
　インデックス配列は、対となる２つのオリゴヌクレオチドの少なくとも一方にあればよく、解析精度を上げる観点で両方にあることが好ましい。リード配列には、第一の標識をあてることができ、その場合はリード配列を別途設けなくてよい。
　上記の例は一例であって、本開示の方法において、第二の標識が上記の例に限定されるものではない。 In the case where Illumina MiSeq or HiSeq2000 is used as the next-generation sequencer, examples of the second label include the following pair of oligonucleotides. One of the two oligonucleotides below is added to one end of the amplification product and the other is added to the other end of the amplification product.
An oligonucleotide having a P5 sequence that hybridizes to the oligonucleotide fixed on the flow cell, a sample identification sequence (index sequence) consisting of 6 to 8 bases, and a sequence (lead sequence) that the sequence primer anneals.
An oligonucleotide having a P7 sequence that hybridizes to an oligonucleotide fixed on the flow cell, a sample identification sequence (index sequence) consisting of 6 to 8 bases, and a sequence (lead sequence) that the sequence primer anneals.
The index sequence may be present in at least one of the two oligonucleotides to be paired, and is preferably present in both from the viewpoint of improving analysis accuracy. The lead array can be provided with a first label. In that case, the lead array need not be provided separately.
The above example is an example, and in the method of the present disclosure, the second label is not limited to the above example.
　以下、第二の標識を付加する第二増幅工程を「アダプター付加ＰＣＲ」、第二の標識を「アダプター」ということがある。 Hereinafter, the second amplification step for adding the second label is sometimes referred to as “adapter-added PCR”, and the second label is sometimes referred to as “adapter”.
　第二の標識を増幅産物の両末端に付加する方法は、ＰＣＲによる付加に代えて、ＤＮＡリガーゼによるライゲーションでもよい。この場合、ライゲーションによる第二の標識の付加は、増幅目的のＰＣＲ反応後にライゲーション反応によって行う。ライゲーションによる付加を採用する場合、第二増幅工程に用いるプライマーは、第一増幅工程の増幅産物に相補的な塩基配列を有していればよい。 The method of adding the second label to both ends of the amplified product may be ligation by DNA ligase instead of addition by PCR. In this case, the addition of the second label by ligation is performed by the ligation reaction after the PCR reaction for amplification. When employing addition by ligation, the primer used in the second amplification step only needs to have a base sequence complementary to the amplification product of the first amplification step.
　第二増幅工程に適用するＰＣＲ試薬としては、Multiplex PCR Assay Kit（タカラバイオ（株）社）、Multiplex PCR Assay Kit ver2（タカラバイオ（株）社）、KAPA Library Amplification Kit（日本ジェネティクス（株）社）、Platinum Multiplex PCR Master Mix Kit（ライフテクノロジーズ社）が挙げられる。本開示の方法においては、Multiplex PCR Assay Kit（タカラバイオ（株）社）を用いることが好ましい。ＰＣＲのサイクル数は、リアルタイムＰＣＲによる検討を予め行って、その結果に基き設定することが好ましい。ＰＣＲは、その途中で、反応温度及び／又は反応時間を変更してもよい。 PCR reagents applied to the second amplification step include Multiplex PCR-Assay Kit (Takara Bio Inc.), Multiplex PCR Asssay Kit Ver2 (Takara Bio Inc.), KAPA Library Amplification Kit (Nippon Genetics Co., Ltd.) ), Platinum, Multiplex, PCR, Master, Mix, Kit (Life Technologies). In the method of the present disclosure, it is preferable to use Multiplex PCR PCR Assay Kit (Takara Bio Inc.). It is preferable to set the number of PCR cycles based on the result of a real-time PCR study. PCR may change reaction temperature and / or reaction time in the middle.
　第一増幅工程の終了後および第二増幅工程の終了後には、それぞれ、アガロースゲル電気泳動を行って、増幅産物の有無を確認することが好ましい。 After completion of the first amplification step and after the second amplification step, it is preferable to perform agarose gel electrophoresis to confirm the presence or absence of amplification products.
　第一増幅工程の増幅産物、第二増幅工程の増幅産物はそれぞれ、精製することが好ましく、精製は、例えば、QIAquick PCR Purification Kit（QIAGEN社）、AMPure XP Kit（BECKMAN COULTER社）を用いて行う。各増幅産物の量は、例えば、NanoDrop（Thermo Fisher Scientific社）、BioAnalyzer（Agilent社）、Quantus Fluorometer（Promega社）、KAPA Library Quantification Kits（日本ジェネティクス（株）社）を用いて濃度を測定することで確認し得る。 It is preferable to purify the amplification product of the first amplification step and the amplification product of the second amplification step, respectively, and purification is performed using, for example, QIAquick PCR Purification Kit (QIAGEN), AMPure XP Kit (BECKMAN COULTER) . The amount of each amplification product is measured using NanoDrop (Thermo Fisher Fisher), BioAnalyzer (Agilent), Quantus Fluorometer (Promega), KAPA Library Quantification Kits (Nippon Genetics, Inc.), for example. It can be confirmed.
［配列解析工程］
　配列解析工程は、増幅された目的領域の塩基配列と量とを決定する工程である。増幅工程で得た最終の増幅産物の全部又は一部が、配列解析工程で配列解析用装置にかけられる物質である。配列解析工程は、解析の精度及び速さ、１度に処理可能な試料数の多さ等の点で、次世代シークエンサーによって行われることが好ましい。 [Sequence analysis process]
The sequence analysis step is a step of determining the base sequence and amount of the amplified target region. All or part of the final amplification product obtained in the amplification step is a substance that is applied to the sequence analysis apparatus in the sequence analysis step. The sequence analysis step is preferably performed by a next-generation sequencer in terms of the accuracy and speed of analysis, the number of samples that can be processed at one time, and the like.
　本開示において次世代シークエンサー（Next Generation Sequencer；ＮＧＳ）とは、サンガー法を利用したキャピラリーシークエンサー（第一世代シークエンサーと呼ばれる）に対比して分類されるシークエンサーを意味する。次世代シークエンサーは、第二世代、第三世代、第四世代、及び今後開発されるシークエンサーを含む。現時点で最も普及している次世代シークエンサーは、ＤＮＡポリメラーゼによる相補鎖合成又はＤＮＡリガーゼによる相補鎖結合に連動した蛍光又は発光をとらえ塩基配列を決定する原理のシークエンサーである。具体的には、MiSeq（Illumina社）、HiSeq2000（Illumina社、HiSeqは登録商標）、Roche454（Roche社）等が挙げられる。 In the present disclosure, the next generation sequencer (NGS) means a sequencer classified as compared with a capillary sequencer (called a first generation sequencer) using the Sanger method. Next generation sequencers include second generation, third generation, fourth generation, and sequencers that will be developed in the future. The most popular next-generation sequencer at present is a sequencer based on the principle of determining a base sequence by capturing fluorescence or light emission linked to complementary strand synthesis by DNA polymerase or complementary strand binding by DNA ligase. Specific examples include MiSeq (Illumina), HiSeq2000 (Illumina, HiSeq is a registered trademark), Roche454 (Roche).
　本開示の方法において、次世代シークエンサーとしては、Illumina社のMiSeq及びHiSeq2000が好適である。MiSeqは、最大９６種類のＰＣＲ産物を１回で測定することが可能である。９６種類のＰＣＲ産物は、１つのマルチプレックスＰＣＲ産物でもよく、複数のマルチプレックスＰＣＲ産物の混合物でもよい。複数のマルチプレックスＰＣＲ産物を混合してMiSeqで解析する場合には、それぞれのＰＣＲ産物を精度高く定量することが望ましい。定量は、例えば、BioAnalyzer（Agilent社）、Quantus Fluorometer（Promega社）又はKAPA Library Quantification Kits（日本ジェネティクス（株）社）を用いて行う。 In the method of the present disclosure, the next-generation sequencer is preferably Illumina's MiSeq or HiSeq2000. MiSeq can measure up to 96 PCR products at a time. The 96 kinds of PCR products may be one multiplex PCR product or a mixture of multiple multiplex PCR products. When mixing multiple multiplex PCR products and analyzing with MiSeq, it is desirable to quantify each PCR product with high accuracy. The quantification is performed using, for example, BioAnalyzer (Agilent), Quantus Fluorometer (Promega) or KAPA Library Quantification Kits (Japan Genetics).
　MiSeq等の次世代シークエンサーで得られた配列データをアライメントする手段としては、Burrows-Wheeler Aligner（ＢＷＡ）が挙げられ、ＢＷＡによって既知のヒトゲノム配列へ配列データをマッピングすることが好ましい。遺伝子を解析する手段としては、SAMtools及びBEDtoolsが挙げられ、これらの解析手段により遺伝子多型、遺伝子変異、及び染色体数を解析することが好ましい。 As a means for aligning sequence data obtained by a next-generation sequencer such as MiSeq, Burrows-Wheeler Aligner (BWA) can be mentioned, and it is preferable to map sequence data to a known human genome sequence by BWA. Examples of means for analyzing genes include SAMtools and BEDtools, and it is preferable to analyze gene polymorphisms, gene mutations, and chromosome numbers using these analysis means.
　配列解析工程で解析した、増幅された目的領域の塩基配列と量とから、例えば、染色体の異数性の有無の検出、遺伝子多型の検出、遺伝子変異の検出がなされる。染色体の異数性としては、例えば、１３番染色体、１８番染色体、及び２１番染色体のトリソミー；性染色体の過剰；等が挙げられる。染色体の異数性の検出方法の一例を、以下に説明する。 From the base sequence and amount of the amplified target region analyzed in the sequence analysis step, for example, detection of the presence or absence of chromosome aneuploidy, detection of gene polymorphism, and detection of gene mutation are performed. Examples of chromosome aneuploidy include trisomy of chromosomes 13, 18, and 21; excess of sex chromosomes; and the like. An example of a method for detecting chromosome aneuploidy will be described below.
［胎児由来の有核赤血球の同定］
　妊娠母体より採取された血液中の有核赤血球には、母親由来の有核赤血球と胎児由来の有核赤血球とが混在しており、胎児由来の有核赤血球であることの同定が必要となる。遺伝子配列により個人を識別する方法としては、対立遺伝子を調べてそこに存在する遺伝子多型を検出する方法が挙げられる。一例として、父子関係の判別には、遺伝子多型の一種であるＳＴＲ（short tandem repeat）を検出する方法が適用されており、個人の識別には、ＳＮＰ（Single Nucleotide Polymorphism、一塩基多型）を検出する方法が適用されている。本開示の方法においては、配列解析工程で得た配列情報に基き、例えば、対立遺伝子上のＳＴＲ及び／又はＳＮＰの差異により胎児由来細胞と母親由来細胞とを識別する。好ましくは、白血球（白血球は母親由来であることがほぼ確実である）を取得して、ＳＴＲ及び／又はＳＮＰを同様に解析することで、胎児由来細胞の同定の確実性を高めることがよい。 [Identification of fetal nucleated red blood cells]
Nucleated erythrocytes in blood collected from the mother's body contain both nucleated erythrocytes from the mother and nucleated red blood cells from the fetus, and it is necessary to identify the nucleated red blood cells from the fetus. . As a method for identifying an individual based on a gene sequence, there is a method of examining an allele and detecting a gene polymorphism present therein. For example, a method of detecting STR (short tandem repeat), which is a kind of gene polymorphism, is applied to discriminate between father and son, and SNP (Single Nucleotide Polymorphism) is used to identify individuals. A method to detect is applied. In the method of the present disclosure, based on the sequence information obtained in the sequence analysis step, for example, fetal-derived cells and maternal-derived cells are discriminated by differences in STR and / or SNP on alleles. Preferably, white blood cells (leukocytes are almost certain to be mother-derived) are obtained, and STR and / or SNP are analyzed in the same manner to improve the certainty of identification of fetal cells.
［男児の場合のＹ染色体検出］
　妊娠母体の超音波検査で胎児が男児であると確認されている場合、単離した有核赤血球内にＹ染色体が存在することを確認すれば、胎児由来の有核赤血球であると同定し得る。細胞内のＹ染色体の有無を検出する方法としては、Ｙ染色体特異的な蛍光プローブを用いるＦＩＳＨ（Fluorescence in situ hybridization）法が知られている。ＦＩＳＨ法の検査キットの一例として、CEP X/Y DNA Probe Kit（Abbott社、CEPは登録商標）が挙げられる。本開示の方法においては、Ｙ染色体に特異的な塩基配列を有するプライマー対を作製しＰＣＲを用い、その増幅の有無を確認することにより、男児の胎児由来の有核赤血球であることを同定するのが好ましい。 [Y chromosome detection in boys]
When the fetus is confirmed to be a boy by ultrasonography of the mother's body, it can be identified as a nucleated red blood cell derived from the fetus by confirming that the Y chromosome is present in the isolated nucleated red blood cell. . As a method for detecting the presence or absence of a Y chromosome in a cell, a FISH (Fluorescence in situ hybridization) method using a fluorescent probe specific to the Y chromosome is known. An example of the FISH method test kit is CEP X / Y DNA Probe Kit (Abbott, CEP is a registered trademark). In the method of the present disclosure, a primer pair having a base sequence specific to the Y chromosome is prepared, and PCR is used to confirm the presence or absence of the amplification, thereby identifying a nucleated red blood cell derived from a male fetus. Is preferred.
［染色体の異数性の有無の検出］
　胎児由来の有核赤血球と同定された細胞から得た染色体について、増幅産物量を例えば次世代シークエンサーで解析する。基準（あるいは参照）として、母親由来の有核赤血球と同定された細胞から得た染色体について、増幅産物量を例えば次世代シークエンサーで解析する。胎児が染色体の異数性を有しなければ、母親由来の増幅産物量と胎児由来の増幅産物量とは、ほぼ１：１の量比になると予想される。増幅領域が位置する染色体が胎児においてトリソミーである場合には、母親由来の増幅産物量と胎児由来の増幅産物量とは、ほぼ１．０：１．５（あるいは２：３）の量比になると予想される。 [Detection of chromosome aneuploidy]
For chromosomes obtained from cells identified as fetal nucleated red blood cells, the amount of amplified product is analyzed, for example, by a next-generation sequencer. As a standard (or reference), the amount of amplification product is analyzed with a next-generation sequencer, for example, for chromosomes obtained from cells identified as maternally derived nucleated red blood cells. If the fetus has no chromosomal aneuploidy, the amount of amplified product from the mother and the amount of amplified product from the fetus are expected to be approximately 1: 1. When the chromosome where the amplification region is located is trisomy in the fetus, the amount of the amplification product derived from the mother and the amount of the amplification product derived from the fetus are approximately 1.0: 1.5 (or 2: 3). It is expected to be.
　本開示の方法においては、下記の方法で、カットオフ値を予め決定しておき、このカットオフ値を解析結果の解釈に使用してもよい。
　染色体の異数性を有しない胎児を妊娠したことが判明している複数母体より採取された血液から、本開示の方法によって母親由来の増幅産物量に対する胎児由来の増幅産物量の比を解析し、その分布を求める。また、トリソミーの胎児を妊娠したことが判明している複数母体より採取された血液から、本開示の方法によって母親由来の増幅産物量に対する胎児由来の増幅産物量の比を解析し、その分布を求める。この２つの分布が重ならない領域をカットオフ値とする。このカットオフ値と、検査対象における母親由来の増幅産物量に対する胎児由来の増幅産物量の比とを比較して、その比がカットオフ値以下であれば胎児はトリソミーでなく、その比がカットオフ値以上であれば胎児はトリソミーである、との解釈をなし得る。 In the method of the present disclosure, a cutoff value may be determined in advance by the following method, and this cutoff value may be used for interpretation of the analysis result.
The ratio of the amount of amplified product derived from the fetus to the amount of amplified product derived from the mother was analyzed by using the method of the present disclosure from blood collected from multiple maternals who had been known to have conceived a fetus that did not have chromosome aneuploidy. Find its distribution. In addition, the ratio of the amount of amplification product derived from the fetus to the amount of amplification product derived from the mother was analyzed by using the method of the present disclosure from blood collected from a plurality of mothers known to have become pregnant with a trisomy fetus. Ask. A region where these two distributions do not overlap is defined as a cutoff value. Compare this cut-off value with the ratio of the amount of amplified product from the fetus to the amount of amplified product from the mother in the test. If the ratio is less than or equal to the cut-off value, the fetus is not trisomy and the ratio is cut If it is greater than or equal to the off value, it can be interpreted that the fetus is trisomy.
　以下に実施例を挙げて、本発明をさらに具体的に説明する。以下の実施例に示す材料、処理手順等は、本発明の趣旨を逸脱しない限り適宜変更することができる。したがって、本発明の範囲は以下に示す具体例により限定的に解釈されるべきものではない。 The present invention will be described more specifically with reference to the following examples. The materials, processing procedures, and the like shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples shown below.
＜実施例１＞
［末梢血の採取］
　抗凝固剤としてＥＤＴＡ（エチレンジアミン四酢酸）のナトリウム塩１０．５ｍｇ入りの７ｍＬ採血管に、妊婦ボランティア１名から末梢血７ｍＬを採取した。その後、生理食塩水を用いて血液を希釈した。妊婦ボランティアからの採血は、インフォームドコンセントを得た上で行った。 <Example 1>
[Collecting peripheral blood]
As an anticoagulant, 7 mL of peripheral blood was collected from one pregnant volunteer in a 7 mL blood collection tube containing 10.5 mg of sodium salt of EDTA (ethylenediaminetetraacetic acid). Thereafter, the blood was diluted with physiological saline. Blood collection from pregnant volunteers was performed after obtaining informed consent.
［密度勾配遠心分離による有核赤血球の濃縮］
　Percoll液（シグマアルドリッチ社製）を使用して、密度１．０７０の液と密度１．０９５の液を調製し、遠沈管に密度１．０９５の液２ｍＬを入れ、４℃下に３０分間置き冷却した。その後、密度１．０９５の液の上に、密度１．０７０の液２ｍＬを、界面が乱れないようにゆっくり重層した。続けて、密度１．０７０の液の上に、希釈した血液１１ｍＬをゆっくり重層し、遠心分離を２０００ｒｐｍで２０分間行った。次いで、密度１．０７０の液と密度１．０９５の液の間に沈積した画分を、ピペットを用いて採取した。 [Concentration of nucleated red blood cells by density gradient centrifugation]
Using a Percoll solution (Sigma Aldrich), prepare a liquid with a density of 1.070 and a liquid with a density of 1.095. Place 2 mL of the liquid with a density of 1.095 in a centrifuge tube and place at 4 ° C. for 30 minutes. Cooled down. Thereafter, 2 mL of a liquid having a density of 1.070 was slowly layered on the liquid having a density of 1.095 so that the interface was not disturbed. Subsequently, 11 mL of diluted blood was slowly layered on the liquid having a density of 1.070, and centrifugation was performed at 2000 rpm for 20 minutes. Next, a fraction deposited between a liquid having a density of 1.070 and a liquid having a density of 1.095 was collected using a pipette.
［基板への血液の塗布］
　片手でスライドガラス１を保持し、その一端に、採取した画分を１滴点着した。もう一方の手で別のスライドガラス２を持ち、一端をスライドガラス１に３０度の角度で接触させ、スライドガラス２の接触下面を画分に触れさせ、毛管現象により２枚のスライドガラスに囲まれた空間に画分を広げた。次に上記角度を保ったまま、スライドガラス２を、スライドガラス１の画分を置いた領域と反対の領域の方向に滑らせて、スライドガラス１上に画分を均一に塗布した。塗布後、送風により１時間以上かけて十分に乾燥させた。 [Applying blood to the substrate]
The slide glass 1 was held with one hand, and one drop of the collected fraction was spotted on one end thereof. Hold another glass slide 2 with the other hand, one end is brought into contact with the glass slide 1 at an angle of 30 degrees, the lower surface of the glass slide 2 is touched to the fraction, and is surrounded by two glass slides by capillary action. Expanded the fraction in the space created. Next, the glass slide 2 was slid in the direction of the area opposite to the area where the fraction of the glass slide 1 was placed while keeping the above angle, and the fraction was uniformly applied onto the glass slide 1. After the application, it was sufficiently dried by blowing air over 1 hour.
［血液細胞の染色］
　画分を塗布したスライドガラス１をメイ・グリュンワルド染色液に３分間浸漬し、次いでリン酸緩衝液に浸漬して洗浄後、ギムザ染色液（リン酸緩衝液で希釈して濃度３％（v/v）とした）に１０分間浸漬した。次いで、純水で洗浄後、乾燥させた。こうして、画像解析用の標本スライドを複数枚作製した。 [Blood cell staining]
The slide glass 1 to which the fraction was applied was immersed in the May-Grünwald staining solution for 3 minutes, then immersed in a phosphate buffer solution, washed, and then Giemsa staining solution (diluted with a phosphate buffer solution to a concentration of 3% (v / v)) for 10 minutes. Then, after washing with pure water, it was dried. In this way, a plurality of specimen slides for image analysis were produced.
［画像解析による有核赤血球の識別］
　電動ＸＹステージ、対物レンズ及びＣＣＤカメラを備えた光学顕微鏡と、ＸＹステージ制御部及びＺ方向制御部を備えた制御部と、画像入力部、画像処理部、及びＸＹ位置記録部を備えた解析部と、を備えた画像解析システムを準備した。標本スライドをＸＹステージに乗せて、標本スライド上に焦点を合わせてスキャンし、光学顕微鏡から画像を解析部に取り込み、有核赤血球を探索した。 [Identification of nucleated red blood cells by image analysis]
An optical microscope including an electric XY stage, an objective lens and a CCD camera, a control unit including an XY stage control unit and a Z direction control unit, an analysis unit including an image input unit, an image processing unit, and an XY position recording unit And an image analysis system including The specimen slide was placed on an XY stage, focused and scanned on the specimen slide, an image was taken from the optical microscope into the analysis unit, and nucleated red blood cells were searched.
　画像解析によって、下記の式（ａ）及び式（ｂ）を満たす細胞を検出し、有核赤血球候補として識別し、ＸＹ位置を記録した。
（ａ）０．２５＜（Ｎ／Ｃ）＜１．０
（ｂ）０．６５＜（Ｎ／（Ｌ×Ｌ））＜０．７８５
　式中、Ｃは、細胞質の面積、Ｎは、核の面積、Ｌは、核の長径の長さ、又は、複雑な形をした核に外接する楕円の長径の長さ、である。 Cells satisfying the following formulas (a) and (b) were detected by image analysis, identified as nucleated red blood cell candidates, and XY positions were recorded.
(A) 0.25 <(N / C) <1.0
(B) 0.65 <(N / (L × L)) <0.785
In the formula, C is the area of the cytoplasm, N is the area of the nucleus, L is the length of the major axis of the nucleus, or the major axis of the ellipse circumscribing the complex-shaped nucleus.
［分光学的解析による有核赤血球の識別］
　画像解析を行い細胞の形態情報により識別した、標本スライド上の有核赤血球候補を１個ずつ、顕微分光光度計を用いて分析した。具体的には、下記の分析を行った。
　有核赤血球候補に４１５ｎｍ近傍の単色光を照射し吸光度を測定した。次に、その有核赤血球候補の近傍にある白血球と予想される細胞（式（ａ）及び式（ｂ）を満たさない細胞）３個について同様にして吸収度を測定した。有核赤血球候補の吸光係数と、白血球と予想される細胞の吸光係数（細胞３個の平均値）との比（赤血球／白血球）が１以上である場合、その有核赤血球候補を、より有力な有核赤血球候補と識別した。標本スライド上のすべての有核赤血球候補について上記の測定を行い、吸光係数の比が大きい順に、有核赤血球候補として１１個を識別した。 [Identification of nucleated red blood cells by spectroscopic analysis]
One nucleated red blood cell candidate on the specimen slide, which was identified by cell shape information through image analysis, was analyzed using a microspectrophotometer. Specifically, the following analysis was performed.
Absorbance was measured by irradiating mononuclear light near 415 nm to nucleated red blood cell candidates. Next, the absorbance was measured in the same manner for three cells (cells not satisfying the formulas (a) and (b)) expected to be white blood cells in the vicinity of the nucleated red blood cell candidate. If the ratio of the extinction coefficient of a nucleated red blood cell candidate to the expected white blood cell and the extinction coefficient of cells (average value of 3 cells) (red blood cell / white blood cell) is 1 or more, the nucleated red blood cell candidate is more effective. Were identified as potential nucleated red blood cell candidates. The above measurement was performed for all nucleated red blood cell candidates on the specimen slide, and 11 nucleated red blood cell candidates were identified in descending order of the extinction coefficient ratio.
［有核赤血球の回収］
　識別した有核赤血球候補１１個を、１個ずつ標本スライド上から剥離し、１個ずつ回収した。具体的には、マイクロマニピュレータでガラス針とガラス管（内径≦０．６ｍｍ）を操作し、ガラス針で細胞を標本スライド上から剥離し、ガラス管内に回収した。 [Recovery of nucleated red blood cells]
Eleven nucleated red blood cell candidates identified were peeled one by one from the specimen slide and collected one by one. Specifically, a glass needle and a glass tube (inner diameter ≦ 0.6 mm) were manipulated with a micromanipulator, and the cells were detached from the specimen slide with the glass needle and collected in the glass tube.
［全ゲノム増幅（ＤＮＡ調製工程）］
　回収した１１個の細胞それぞれから、PicoPLEX WGA Kit（New England Biolabs社）を用いて、本キットの添付文書に則り全ゲノム増幅を行い、ＤＮＡを約１００万倍に増幅した。 [Whole genome amplification (DNA preparation process)]
From each of the 11 cells collected, whole genome amplification was performed using the PicoPLEX WGA Kit (New England Biolabs) according to the package insert of this kit, and the DNA was amplified about 1 million times.
　得られた増幅産物は、QIAquick PCR Purification Kit（QIAGEN社）を用いて不純物を除去することで精製した。精製後の増幅産物の濃度を、Quantus Fluorometer dsDNA System（Promega社）を用いて測定した。 The obtained amplification product was purified by removing impurities using QIAquick® PCR® Purification® Kit (QIAGEN). The concentration of the amplified product after purification was measured using Quantus® Fluorometer® dsDNA® System (Promega).
［マルチプレックスＰＣＲ（第一増幅工程）］
　胎児由来細胞を同定する目的、並びに、染色体を解析する目的で、染色体上の１６箇所の領域を増幅する、１６対のプライマー対を設計した。１６箇所の領域は、内部にＳＮＰを有する領域とした。１６箇所の領域が位置する遺伝子名、及び１６箇所の領域の塩基長は、表１に示すとおりである。 [Multiplex PCR (first amplification step)]
For the purpose of identifying fetal cells and analyzing the chromosome, 16 primer pairs were designed to amplify 16 regions on the chromosome. The 16 regions were regions having SNPs inside. Table 1 shows the names of the genes in which the 16 regions are located and the base lengths of the 16 regions.
　各プライマーは、第一の標識の塩基配列１７塩基と、目的領域に相補的な塩基配列２０塩基と、からなる計３７塩基のオリゴヌクレオチドであり、５’末端に第一の標識の塩基配列が位置している。フォワードプライマー上の第一の標識の塩基配列は、CGCTCTTCCGATCTCTG（配列番号１）とし、リバースプライマー上の第一の標識の塩基配列は、CGCTCTTCCGATCTGAC（配列番号２）とした。 Each primer is an oligonucleotide with a total of 37 bases consisting of a base sequence of 17 bases of the first label and a base sequence of 20 bases complementary to the target region, and the base sequence of the first label is at the 5 ′ end. positioned. The base sequence of the first label on the forward primer was CGCTCTTCCGATCTCTG (SEQ ID NO: 1), and the base sequence of the first label on the reverse primer was CGCTCTTCCGATCTGAC (SEQ ID NO: 2).
　全プライマーを終濃度が各２５ｎｍｏｌ／Ｌとなるように混合し、プライマーミックス液を調製した。 All primers were mixed at a final concentration of 25 nmol / L to prepare a primer mix solution.
　マルチプレックスＰＣＲは、Multiplex PCR Assay Kit（タカラバイオ（株）社）を用いて反応を行った。各細胞から得た全ゲノム増幅産物を鋳型として１０ｎｇ、プライマーミックス液を８μＬ、Multiplex PCR Mix1を０．１２５μＬ、Multiplex PCR Mix2を１２．５μＬ、及び水を混合し反応液を調製した（最終液量２５μＬ）。ＰＣＲ反応は、９４℃／６０秒で変性した後、９４℃／３０秒、６０℃／９０秒、及び７２℃／３０秒を３０サイクル行った。 Multiplex PCR was performed using Multiplex PCR Assay Kit (Takara Bio Inc.). 10 ng of the whole genome amplification product obtained from each cell as a template, 8 μL of primer mix solution, 0.125 μL of Multiplex PCR Mix1, 12.5 μL of Multiplex PCR Mix2, and water were prepared to prepare a reaction solution (final solution volume) 25 μL). The PCR reaction was denatured at 94 ° C./60 seconds, followed by 30 cycles of 94 ° C./30 seconds, 60 ° C./90 seconds, and 72 ° C./30 seconds.
　得られたＰＣＲ産物は、QIAquick PCR Purification Kit（QIAGEN社）を用いて不純物を除去することで精製した。精製後のＰＣＲ産物の濃度を、Quantus Fluorometer dsDNA System（Promega社）を用いて測定した。 The obtained PCR product was purified by removing impurities using QIAquick® PCR® Purification® Kit (QIAGEN). The concentration of the PCR product after purification was measured using a Quantus® Fluorometer® dsDNA® System (Promega).
［アダプター付加ＰＣＲ（第二増幅工程）］
　Miseqを用いた配列解析を行うために、マルチプレックスＰＣＲ産物の両末端へアダプターを付加した。アダプターの付加は、下記のプライマー対を用いてＰＣＲ反応を行うことで実施した。
・５’末端から順に、フローセル結合用配列（Illumina社が提供しているＰ５配列）、サンプル識別用のインデックス配列、及び第一の標識にアニールする配列を有するオリゴヌクレオチド。インデックス配列としては、Illumina社が提供しているＤ５０１を用いた。
・５’末端から順に、フローセル結合用配列（Illumina社が提供しているＰ７配列）、サンプル識別用のインデックス配列、及び第一の標識にアニールする配列を有するオリゴヌクレオチド。インデックス配列としては、Illumina社が提供しているＤ７０１～Ｄ７１１のいずれかを１１個の細胞ごとに用いた。 [Adapter-added PCR (second amplification step)]
In order to perform sequence analysis using Miseq, adapters were added to both ends of the multiplex PCR product. The adapter was added by performing a PCR reaction using the following primer pair.
An oligonucleotide having a flow cell binding sequence (P5 sequence provided by Illumina), an index sequence for sample identification, and a sequence that anneals to the first label in order from the 5 ′ end. As the index array, D501 provided by Illumina was used.
An oligonucleotide having a flow cell binding sequence (P7 sequence provided by Illumina), an index sequence for sample identification, and a sequence that anneals to the first label in order from the 5 ′ end. As the index sequence, any one of D701 to D711 provided by Illumina was used for every 11 cells.
　ＰＣＲは、Multiplex PCR Assay kit（タカラバイオ（株）社）を用いて反応を行った。マルチプレックスＰＣＲ産物を鋳型として５ｎｇ、プライマー（１．２５μｍｏｌ／Ｌ）を各１μＬ、Multiplex PCR Mix1を０．１２５μＬ、Multiplex PCR Mix2を１２．５μＬ、及び水を混合し反応液を調製した（最終液量２５μＬ）。ＰＣＲ反応は、９４℃／３分で変性した後、９４℃／４５秒、５０℃／６０秒、及び７２℃／３０秒を５サイクル行い、その後、９４℃／４５秒、５５℃／６０秒、及び７２℃／３０秒を１１サイクル行った。 PCR was performed using a Multiplex PCR Assay Kit (Takara Bio Inc.). 5 ng using the multiplex PCR product as a template, 1 μL each of the primer (1.25 μmol / L), 0.125 μL of Multiplex０．１PCR Mix1, 12.5 μL of Multiplex PCR Mix2, and water were prepared to prepare a reaction solution (final solution) Volume 25 μL). The PCR reaction was denatured at 94 ° C./3 minutes, followed by 5 cycles of 94 ° C./45 seconds, 50 ° C./60 seconds, and 72 ° C./30 seconds, and then 94 ° C./45 seconds, 55 ° C./60 seconds. And 72 ° C./30 seconds for 11 cycles.
　得られたＰＣＲ産物を、AMPure XP Kit（BECKMAN COULTER社）を用いて精製した。精製後のＰＣＲ産物の濃度を、BioAnalyzer（Agilent社）を用いて測定した。より正確なＰＣＲ産物の定量として、KAPA Library Quantification Kits（日本ジェネティクス（株）社）を用いて定量を行った。 The obtained PCR product was purified using AMPure® XP® Kit (BECKMAN® COULTER). The concentration of the PCR product after purification was measured using BioAnalyzer (Agilent). For more accurate PCR product quantification, KAPA Library Quantification Kits (Nippon Genetics Co., Ltd.) was used.
［配列解析工程］
　１１細胞由来のアダプター付加産物を混合し、MiSeq（Illumina社）及びMiSeq Reagent Kit v2 300 Cycle（Illumina社）を用いてシークエンスを行った。得られたFastQファイルを、ＢＷＡ（Burrows-Wheeler Aligner）を用いてヒトリファレンスゲノム（ｈｇ１９）へマッピングを行い、SAMtoolsにより遺伝子多型情報を抽出し、BEDtoolsにより各増幅領域のシークエンスリード数を算出することで解析を行った。 [Sequence analysis process]
The adapter addition product derived from 11 cells was mixed and sequenced using MiSeq (Illumina) and MiSeq Reagent Kit v2 300 Cycle (Illumina). The obtained FastQ file is mapped to the human reference genome (hg19) using BWA (Burrows-Wheeler Aligner), gene polymorphism information is extracted with SAMtools, and the number of sequence reads of each amplified region is calculated with BEDtools The analysis was performed.
［細胞の由来の同定］
　１３番染色体、１８番染色体、及び２１番染色体から増幅した領域のＳＮＰを解析したところ、１１個の細胞のうち１個の細胞が異なるＳＮＰ情報を有することが確認された。別途、核の形状から白血球と予想される細胞を、標本スライド上からマイクロマニピュレータを用いて回収し、１１個の細胞と同様にしてＤＮＡを増幅しＳＮＰを調べたところ、上記１個の細胞以外の１０個の細胞のＳＮＰと、白血球と予想される細胞のＳＮＰとが一致することが確認された。以上の解析により、１個の細胞が胎児由来の有核赤血球であり、１０個の細胞が母親由来の有核細胞であることが確認された。 [Identification of cell origin]
Analysis of SNPs in the regions amplified from chromosomes 13, 18, and 21 confirmed that one of 11 cells had different SNP information. Separately, cells expected to be white blood cells from the shape of the nucleus were collected from the specimen slide using a micromanipulator, and DNA was amplified and SNPs were examined in the same manner as 11 cells. It was confirmed that the SNPs of the 10 cells were consistent with the expected SNP of leukocytes. From the above analysis, it was confirmed that one cell was a nucleated red blood cell derived from a fetus, and 10 cells were a nucleated cell derived from a mother.
［異数性の有無の検出］
　胎児由来と同定された有核赤血球１個の２１番染色体の４領域の増幅産物量を、MiSeqを用いて決定した。また、母親由来と同定された有核細胞１０個の２１番染色体の４領域の増幅産物量を、MiSeqを用いて決定した。母親由来細胞１０個の間で４領域の各増幅産物量を比較したところ、ばらつきは少なく、量比を計算したところ、それぞれ１：１に近い値であった。また、母親由来細胞と胎児由来細胞との間で４領域の各増幅産物量を比較したところ、ばらつきは少なく、量比を計算したところ、それぞれ１：１に近い値であったので、胎児は２１番染色体に異数性を有しないことが推定された。
　同様にして、１３番染色体、１８番染色体、Ｘ染色体、及びＹ染色体についても、胎児は異数性を有しないことが推定された。 [Detection of aneuploidy]
The amount of amplification product of 4 regions of chromosome 21 of one nucleated erythrocyte identified as fetus was determined using MiSeq. In addition, the amount of amplification products of 4 regions of chromosome 21 of 10 nucleated cells identified as mother-derived was determined using MiSeq. When the amount of each amplified product in the four regions was compared among 10 maternal cells, the variation was small, and the amount ratio was calculated to be close to 1: 1. Moreover, when the amount of each amplification product in the four regions was compared between the mother-derived cell and the fetal-derived cell, there was little variation, and the amount ratio was calculated to be close to 1: 1. It was estimated that chromosome 21 has no aneuploidy.
Similarly, it was estimated that the fetus has no aneuploidy for chromosomes 13, 18, 18 and X.
＜実施例２～４＞
　実施例１で採血した妊婦ボランティアとは別の妊婦ボランティア３名から末梢血を採取し、実施例１と同様にして染色体を解析した。いずれも実施例１と同様の解析結果が得られ、いずれの胎児も染色体に異数性を有しないことが推定された。 <Examples 2 to 4>
Peripheral blood was collected from 3 pregnant volunteers different from the pregnant volunteers collected in Example 1, and the chromosomes were analyzed in the same manner as in Example 1. In both cases, the same analysis results as in Example 1 were obtained, and it was estimated that none of the fetuses had any aneuploidy in the chromosome.
＜比較例１＞
　実施例１で採血した妊婦ボランティアとは別の妊婦ボランティア１名から末梢血を採血し、実施例１と同様にして細胞の剥離工程まで行い９個の細胞を回収した。その後、実施例１と同様にして全ゲノム増幅を行った。 <Comparative Example 1>
Peripheral blood was collected from one pregnant volunteer different from the pregnant volunteers collected in Example 1, and 9 cells were collected in the same manner as in Example 1 until the cell peeling step. Thereafter, whole genome amplification was performed in the same manner as in Example 1.
　次いで、実施例１で使用した１６対のプライマー対を、別の１６対のプライマー対に変更してマルチプレックスＰＣＲを行った。比較例１で使用した１６対のプライマー対は、その増幅領域が実施例１の増幅領域を含み且つ実施例１の増幅領域よりも長くなるように設計した。１６箇所の領域が位置する遺伝子名、及び１６箇所の領域の塩基長は、表１に示すとおりである。 Subsequently, the 16 primer pairs used in Example 1 were changed to another 16 primer pairs, and multiplex PCR was performed. The 16 primer pairs used in Comparative Example 1 were designed so that the amplification region included the amplification region of Example 1 and was longer than the amplification region of Example 1. Table 1 shows the names of the genes in which the 16 regions are located and the base lengths of the 16 regions.
　マルチプレックスＰＣＲの後は、実施例１と同様にしてアダプター付加ＰＣＲ以降の各工程を行い、染色体の異数性の有無の検出を試みた。 After the multiplex PCR, each step after the adapter addition PCR was performed in the same manner as in Example 1 to try to detect the presence or absence of chromosome aneuploidy.
　９個の細胞のうち２個が胎児由来の有核赤血球であることが確認された。各増幅領域について、胎児由来細胞２個の間で、並びに、母親由来細胞７個の間で、各増幅産物量を比較したところ、ばらつきが大きく、異数性の有無を検出できるレベルから大きく外れた。 2 out of 9 cells were confirmed to be fetal nucleated red blood cells. For each amplification region, the amount of each amplification product was compared between two fetal-derived cells and seven maternal-derived cells. It was.
＜参考例１＞
　末梢血単核細胞（Peripheral blood mononuclear cell；ＰＢＭＣ）の凍結保存物（市販品）を用意し、解凍した。次いで、生理食塩水で３回洗浄し、死細胞および不純物を除去し、生理食塩水を入れた１０ｃｍ２培養ディッシュに１００細胞／１０ｍｌとなるように懸濁した。培養ディッシュ中のＰＢＭＣから、光学顕微鏡下でマイクロピペットを用いて細胞を５個回収した。５個のＰＢＭＣを用いて比較例１と同様にして全ゲノム増幅以降の各工程を行い、染色体の異数性の有無の検出を試みた。 <Reference Example 1>
A cryopreserved product (commercial product) of peripheral blood mononuclear cells (PBMC) was prepared and thawed. Next, the cells were washed three times with physiological saline to remove dead cells and impurities, and suspended in a 10 cm 2 culture dish containing physiological saline to a concentration of 100 cells / 10 ml. Five cells were collected from the PBMC in the culture dish using a micropipette under an optical microscope. Using the five PBMCs, each step after the whole genome amplification was performed in the same manner as in Comparative Example 1, and detection of the presence or absence of chromosome aneuploidy was attempted.
　各増幅領域について細胞５個の間で各増幅産物量を比較したところ、ばらつきは少なく、量比を計算したところ、それぞれ１：１に近い値であった。 When the amount of each amplification product was compared between 5 cells for each amplification region, there was little variation, and the amount ratio was calculated to be close to 1: 1.
　参考例１は、各増幅領域の長さが比較例１と同じであるが、染色体の異数性の有無の検出が可能であった。参考例１は、細胞が乾燥状態に曝されたり光エネルギー照射を受けたりしていないので、染色体ＤＮＡの損傷が抑制されており、５個の細胞すべてにおいて、増幅領域が安定して増幅されたと考えられる。 In Reference Example 1, the length of each amplification region is the same as that of Comparative Example 1, but it was possible to detect the presence or absence of chromosome aneuploidy. In Reference Example 1, since the cells were not exposed to a dry state or irradiated with light energy, damage to chromosomal DNA was suppressed, and the amplification region was stably amplified in all five cells. Conceivable.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　２０１４年９月１６日に出願された日本国特許出願２０１４－１８７９３５号の開示は、その全体が参照により本明細書に取り込まれる。
　本明細書に記載された全ての文献、特許出願、及び技術規格は、個々の文献、特許出願、及び技術規格が参照により取り込まれることが具体的かつ個々に記された場合と同程度に、本明細書中に参照により取り込まれる。 The disclosure of Japanese Patent Application No. 2014-187935 filed on September 16, 2014 is incorporated herein by reference in its entirety.
All documents, patent applications, and technical standards mentioned in this specification are to the same extent as if each individual document, patent application, and technical standard were specifically and individually stated to be incorporated by reference, Incorporated herein by reference.

Claims (8)

  1. 　妊娠母体より採取された血液に含まれる血液細胞を基板上に固定し、基板上に固定された前記血液細胞を光学機器で解析して有核赤血球を識別し、識別された前記有核赤血球を基板から剥離して回収する回収工程と、
    　回収された前記有核赤血球から染色体ＤＮＡ又はその増幅産物を得るＤＮＡ調製工程と、
    　前記染色体ＤＮＡ又はその増幅産物を鋳型にして、４０ｂｐ以上７０ｂｐ以下の目的領域をポリメラーゼ連鎖反応により増幅する増幅工程と、
    　増幅された前記目的領域の塩基配列及び量を決定する配列解析工程と、
    　を含む、胎児染色体の解析方法。     Blood cells contained in blood collected from a pregnant mother are fixed on a substrate, the blood cells fixed on the substrate are analyzed with an optical instrument to identify nucleated red blood cells, and the identified nucleated red blood cells are A recovery step for separating and recovering from the substrate;
    A DNA preparation step for obtaining chromosomal DNA or an amplification product thereof from the collected nucleated red blood cells;
    An amplification step of amplifying a target region of 40 bp to 70 bp by polymerase chain reaction using the chromosomal DNA or an amplification product thereof as a template;
    A sequence analysis step for determining the base sequence and amount of the amplified target region;
    A method for analyzing a fetal chromosome.
  2. 　染色体の異数性の有無の検出、遺伝子多型の検出、及び、遺伝子変異の検出の少なくともいずれかを行う、請求項１に記載の胎児染色体の解析方法。 The method for analyzing a fetal chromosome according to claim 1, wherein at least one of detection of the presence or absence of chromosome aneuploidy, detection of a gene polymorphism, and detection of a gene mutation is performed.
  3. 　前記増幅工程が、複数のプライマー対を用いて複数の前記目的領域の多重増幅を行うことを含む、請求項１又は請求項２に記載の胎児染色体の解析方法。 The method for analyzing a fetal chromosome according to claim 1 or 2, wherein the amplification step comprises performing multiplex amplification of a plurality of the target regions using a plurality of primer pairs.
  4. 　前記ＤＮＡ調製工程が、前記回収工程で回収された前記有核赤血球から全ゲノム増幅を行って染色体ＤＮＡの増幅産物を得る工程である、請求項１～請求項３のいずれか１項に記載の胎児染色体の解析方法。 The DNA preparation step according to any one of claims 1 to 3, wherein the DNA preparation step is a step of performing a whole genome amplification from the nucleated red blood cells recovered in the recovery step to obtain an amplification product of chromosomal DNA. Analysis method of fetal chromosome.
  5. 　前記回収工程が、基板上に固定された前記血液細胞を染色し画像解析して有核赤血球を識別することを含む、請求項１～請求項４のいずれか１項に記載の胎児染色体の解析方法。 The fetal chromosome analysis according to any one of claims 1 to 4, wherein the collecting step includes staining the blood cells fixed on the substrate and analyzing the image to identify nucleated red blood cells. Method.
  6. 　前記回収工程が、基板上に固定された前記血液細胞を分光学的に解析して有核赤血球を識別することを含む、請求項１～請求項５のいずれか１項に記載の胎児染色体の解析方法。 The fetal chromosome according to any one of claims 1 to 5, wherein the recovery step includes spectroscopic analysis of the blood cells immobilized on a substrate to identify nucleated red blood cells. analysis method.
  7. 　対立遺伝子の情報により、前記回収工程で回収された前記有核赤血球が胎児と母親のいずれの由来であるかを判別する工程をさらに含む、請求項１～請求項６のいずれか１項に記載の胎児染色体の解析方法。 The method according to any one of claims 1 to 6, further comprising a step of discriminating whether the nucleated red blood cells recovered in the recovery step originate from a fetus or a mother based on allele information. Method for analyzing fetal chromosomes.
  8. 　前記配列解析工程が、次世代シークエンサーを用いて行われる、請求項１～請求項７のいずれか１項に記載の胎児染色体の解析方法。 The method for analyzing a fetal chromosome according to any one of claims 1 to 7, wherein the sequence analysis step is performed using a next-generation sequencer.
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