JP4102087B2 - Method for detecting nucleic acid associated with disease - Google Patents

Description

【０１１０】
その結果、通常のＨＣＶ ＲＮＡ抽出法に比してＱＩＡａｍｐ ＤＮＡ Ｂｌｏｏｄ Ｍｉｄｉ Ｋｉｔを用いた方法では、ＨＣＶ ＲＮＡの抽出効率が約１０倍程度下がることがわかった。よって、ＱＩＡａｍｐ ＤＮＡ Ｂｌｏｏｄ Ｍｉｄｉ Ｋｉｔを用いた抽出法は、血中のウイルス量が極めて低い検体に対して行われる場合には、測定不能になる場合が生じたり、チップを用いたウイルスの定量等の用途には使用しにくいと考えられた。しかしながら、逆に通常法でのウイルス量が、全血中で１０ｃｏｐｙ／ｍｌ以上の濃度を占める検体では、理論的には、ＱＩＡａｍｐ ＤＮＡ Ｂｌｏｏｄ Ｍｉｄｉ Ｋｉｔを使用したＤＮＡおよびＲＮＡの同時抽出が可能であると示唆された。即ち、ＱＩＡａｍｐ ＤＮＡ Ｂｌｏｏｄ Ｍｉｄｉ Ｋｉｔを使用した抽出産物を用いて、ウイルスを定性的に測定できることが示唆された。
【０１１１】
上述の通りに逆転写反応を行って得たサンプルについて、ヒトゲノムとＨＣＶゲノムの同時増幅を行った。
【０１１２】
増幅のためのプライマーには、ヒトゲノムのためにはＭｘＡＦ０１とＭｘＡＲ０２を用いた。ＨＣＶゲノムのためにはｎｃ２（Ｋ．Ｃｈａｙａｍａ ｅｔ ａｌ．， Ｊ． Ｇａｓｔｒｏｅｎｔｅｒｏｌ．Ｈｅｐａｔｏｌ． ８， Ｓ４０−４４， １９９３， ｎｔ２７−４５）とｐｒｉｍｅｒ３３（Ｏｋａｍｏｔｏｅｔ ａｌ．， Ｊｐｎ Ｊ． Ｅｘｐ． Ｍｅｄ． ６０， ２１５−２２２， １９９０）を用いた。増幅は１本のチューブ内で行った。なお、ＰＣＲは９４℃で３０秒、５５℃で３０秒、７２℃で１分を５０ サイクル行い、その前に９４℃で４分、後に７２℃で７分の処理を行った。
【０１１３】

【０１１４】
その結果、ヒトゲノム由来の産物（ｓｉｚｅ ： ６１０ｂｐ）とＨＣＶゲノム由来の産物（ｓｉｚｅ ： ３００ｂｐ）は両者とも増幅された。よって、ゲノムの抽出から特定領域の増幅までの行程は、ヒトゲノムＤＮＡとＨＣＶゲノムＲＮＡの両者に対して同一の処理を行うことによって達成されることが明かとなった。
【０１１５】
このような本発明の態様によれば、個体から抽出した試料物質から、簡便に且つ短時間にその疾病に対する治療法を予測することが可能となる。また、このような態様によれば、個体から得た１試料から同時に複数の情報を得ることができるので、検査の途中で生じ得る試料の取り違いを防止できる。また、危険性の高い試料を扱う場合であっても、そのような試料による汚染の範囲の広がりを従来よりも狭くすることが可能であり、且つ試料による汚染事故の危険性も最小限に留めることが可能である。
【０１１６】
例２は、ＩＦＮ療法の効果を評価するためのＤＮＡチップを記載する
ＩＦＮ治療効果予測用ＤＮＡチップ
まず、患者（ヒト）の血液からヒトの染色体ＤＮＡとＨＣＶ−ＲＮＡを採取した。染色体ＤＮＡは、配列番号２０、配列番号２１のプライマーを用いて１３５ｂｐの断片を増幅した。また、ＨＣＶ−ＲＮＡは逆転写酵素を用いてｃＤＮＡを作製した。
【０１１７】
以上の工程により得られた核酸サンプルに対して、ファルマシア社製のキットを用いてＦＩＴＣ標識を行った。
【０１１８】
図１に本実施例のＤＮＡチップの概略図を示す。ポリリジンをコートしたスライドガラスからなる基体６上に第２プローブとして配列番号２２、配列番号２３、配列番号２４、配列番号２５、配列番号２６のＤＮＡプローブ及び第１プローブとして配列番号５、配列番号６、配列番号７のＤＮＡプローブ（図１中第１プローブ及び第２プローブは１〜５で示す）を２００ｎＬずつスポットして、乾燥した。その後、ＵＶ照射を行ってプローブを基体６上に固定化した。配列番号２２〜２５の塩基配列は、それぞれ配列番号１６〜１９の塩基配列の４５５位のＳＮＰ部位を含む断片である。配列番号５〜７の塩基配列はそれぞれＨＣＶウイルスの１型、２型、３型の遺伝子型を検出するプローブである。
【０１１９】
ＦＩＴＣ標識した核酸サンプルを２ｘＳＳＣ−１ｍｍｏｌ／Ｌ ＥＤＴＡ溶液に溶解した。これを、容量２０μＬのリアクションチャンバーにいれ、プローブ固定化スライドグラスでふたをした。５０℃、１２時間の反応を行った後、０．２ｘＳＳＣ−１ｍｍｏｌ／ＬＥＤＴＡ溶液で２回洗浄し、スライドガラス上の蛍光を測定した。
【０１２０】
その結果、配列番号２２のプローブを固定化したスポットからのみ有意な蛍光が観察された。この試料物質に含まれるヒト由来核酸におけるＭｘＡ−８８の遺伝子型はＴ／Ｔホモ型であると考えられた。また、この試料物質に含まれるウイルスは、２型である判定された。更に得られた蛍光強度から、ウイルス濃度は１０^６ｃｏｐｙ／ｍＬ以下であると見積もられた。従って、この試料物質が採取された患者には、ＩＦＮが有効に働くことが予測された。
【０１２１】
例３は、ＩＦＮ療法の効果を評価するためのＰＮＡチップを記載する
ＩＦＮ治療効果予測用ＰＮＡチップ
まず、患者（ヒト）の血液からヒトの染色体ＤＮＡとＨＣＶ−ＲＮＡを採取した。次に、染色体ＤＮＡは配列番号２０、２１のプライマーを用いて１３５ｂｐの断片を増幅した。また、ＨＣＶ−ＲＮＡは逆転写酵素を用いてｃＤＮＡを作製した。得られたｃＤＮＡを鋳型にして配列番号８（これはセンスである）、配列番号１０、配列番号１１、配列番号１２、配列番号１３、配列番号１４（これらはアンチセンスである）のプライマーを用いてＰＣＲ反応を行った。
【０１２２】
図２に本実施例のＰＮＡチップの概略図を示す。ガラス基板１２上に１０個の金電極１３をパターニングしたプローブ固定化チップに、Ｎ末端にシステインを修飾した第２プローブとして配列番号２７、配列番号２８、配列番号２９、配列番号３０、配列番号３１のＰＮＡプローブ、第１プローブとして配列番号３２、配列番号３３、配列番号３４、配列番号３５、配列番号３６のＰＮＡプローブ７〜１１を、それぞれ２００ｎＬずつスポットし、１時間放置した。
【０１２３】
配列番号２７〜３０の塩基配列は、それぞれ配列番号１６〜１９の塩基配列の４５５位のＳＮＰ部位を含む断片である。配列番号３２〜３６の塩基配列はそれぞれＨＣＶウイルスの１ａ型、１ｂ型、２ａ型、２ｂ型、３ａ型の遺伝子型を検出するプローブである。
【０１２４】
次に、サンプルを２ｘＳＳＣ−１ｍｍｏｌ／Ｌ ＥＤＴＡ溶液に溶解した。これを容量２０μＬのリアクションチャンバーにいれ、プローブ固定化チップでふたをした。５０℃、１時間の反応を行った後、０．２ｘＳＳＣ−１ｍｍｏｌ／ＬＥＤＴＡ溶液で２回洗浄した。次に、これに１０μｍｏｌ／Ｌのヘキスト３３２５８溶液を滴下した。別途対極と参照電極を設置し、金電極と対極間に電圧を印加したときのヘキスト３３２５８からの酸化電流を測定した。
【０１２５】
その結果、配列番号２７と配列番号２８と配列番号３４のプローブを固定化した金電極１３から有意な電流変化が観察され、この試料物質に含まれるヒト由来核酸におけるＭｘＡ−８８の遺伝子型はＧ／Ｔヘテロ型であると考えられた。また、この試料物質に含まれるウイルスは、２ａ型であると判定された。従って、この試料物質が採取された患者には、ＩＦＮが有効に働くことが予測された。
【０１２６】
例４
本発明に従うと、Ｃ型肝炎感染者の感染しているＨＣＶのウイルス型、感染者のＭｘＡ−８８、ＭｘＡ−１２３、ＭＢＬ−２２１およびＭＢＬのコラーゲン様ドメインのコドン５４の遺伝子型から、Ｃ型肝炎感染者におけるＩＦＮ療法の有効性を予測することが可能である。
【０１２７】
以下に図３を用いてウイルスの型、ＭＢＬおよびＭｘＡの遺伝子型よりＩＦＮ療法の効果を予測する方法の１例を説明する。
【０１２８】
ステップ３ａでは、実施者が、ＩＦＮを投与されるべき個体から血液サンプル等のサンプルを採取し、予測を開始する。採取されたサンプルは、必要に応じて精製および抽出などの処理がなされた後で、ウイルスの型、並びにＭＢＬおよびＭｘＡの遺伝子型、即ち、ＭｘＡ−８８とＭｘＡ−１２３の遺伝子型、ＭＢＬ−２２１の遺伝子型、ＭＢＬのコラーゲン様ドメインのコドン５４の遺伝子型を決定し、ステップ３ｂへ進む。
【０１２９】
ステップ３ｂでは、ステップ３ａで決定したウイルスの型にタイプ１が含まれている場合ステップ３ｃへ進むと判定し、タイプ２のみであれば（３ｄ）へ進むと判定する。
【０１３０】
ステップ３ｃでは、ステップ３ａで決定したＭＢＬおよびＭｘＡの遺伝子型を基に、遺伝子型とＩＦＮの効果を対応付けたテーブル２を検索して対応するＩＦＮの効果を抽出し、それによってＩＦＮ療法の有効性を予測し、全予測工程を終了する。
【０１３１】
ステップ３ｄでは、ステップ３ａで決定したＭＢＬおよびＭｘＡの遺伝子型を基に、遺伝子型とＩＦＮの効果を対応付けたテーブル３を検索し対応するＩＦＮの効果を抽出し、それによってＩＦＮ療法の有効性を予測し、全予測工程を終了する。
【０１３２】
ここで使用されるテーブルは遺伝子型とＩＦＮ感受性を対応付けた情報である。各テーブルを表として示したものがそれぞれのテーブル番号に対応する表である。上述の方法において使用した各表は例として示したものである。ここで使用されるテーブルおよび表は、各遺伝子型とＩＦＮの効果を対応付けたものであればよい。例えば、表２は、タイプ１のＨＣＶに感染している患者における遺伝子型とＩＦＮの効果の関係を示す表である。一方、表３は、タイプ２のＨＣＶに感染している患者における遺伝子型とＩＦＮの効果の関係を示す表である。また、使用される表は、そのうちの何れかの成分のみを含む表であってもよく、或いは他の組合せの成分からなる表であってもよい。上述の例ではステップ３ｃとステップ３ｄにて参照したテーブルとして、それぞれ表２と表３を使用し、夫々のステップ毎に必要な項目を予め選択して作製した２つの表を使用している。しかしながら、これらをあわせて作製した１つの表を用いてもよい。或いは他の組合せの成分からなる表であってもよい。成分の選択は、例えば、ステップ３ｃで使用されるテーブルには、タイプ１のＨＣＶ感染者における遺伝子型と著効または非著効の関連を示す情報を選択し、ステップ３ｄではタイプ２のＨＣＶ感染者における遺伝子型と著効または非著効の関連を示す情報を選択すればよい。しかしながらこれに限られるものではなく、成分の選択は実施者が任意に行ってよい。
【０１３３】
また、ここで使用される表２および３に記載される成分としての数値は、遺伝子型とＩＦＮ感受性またはＩＦＮの効果を対応付けた情報を示す表示の１例であり、当該表に記載の数値に限定されるものではない。即ち、遺伝子型と著効または非著効との相関関係を実質的に示すことが可能な表記であれば、例えば、「○」、「×」、「△」であっても、簡略化された数値によるスコア（例えば、１、２、３、４および５等）であってもよい。
【０１３４】
【表２−１】

【０１３５】
【０１３６】
【表２−２】

【０１３７】
【表３】

【０１３８】
以上のような本発明の態様に従い、多くの項目を検討することにより、より正確な予測が可能になる。
【０１３９】
上述の本発明の態様に従う方法およびプローブ固相化チップを用いることにより、試料物質から、目的とする核酸情報を簡便に、短時間に、且つ正確に、回収することが可能である。
【０１４０】
また、上述の態様は本発明の１例であり本発明を制限することを目的として記載されるものではない。
【０１４１】
更なる利益および変更が当業者に容易に見出されるであろう。従って、その広範な側面における本発明は、ここに示し且つ記載した詳細および代表的な態様に制限するものではない。従って、種々の変更は、添付された請求の範囲およびそれらの均等物により明示されるような精神または一般的な本発明の概念の範囲から逸れることなく行われ得る。
【０１４２】
【発明の効果】
本発明により、試料物質から、目的とする核酸情報を簡便に、短時間に、且つ正確に、回収することが可能な方法が提供された。
【０１４３】
【配列表】

【図面の簡単な説明】
【図１】本実施例に係るＤＮＡチップの概略図。
【図２】本実施例に係るＰＮＡチップの概略図。
【図３】本実施例に係る方法を示すフローチャート。
【符号の説明】
１・・・ＤＮＡプローブ
２・・・ＤＮＡプローブ
３・・・ＤＮＡプローブ
４・・・ＤＮＡプローブ
５・・・ＤＮＡプローブ
６・・・基体
７・・・ＰＮＡプローブ
８・・・ＰＮＡプローブ
９・・・ＰＮＡプローブ
１０・・・ＰＮＡプローブ
１１・・・ＰＮＡプローブ
１２・・・基体
１３・・・金電極
１４・・・接続部[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for obtaining information about a nucleic acid associated with a disease obtained from an individual. The present invention relates to a method for obtaining information about an individual's nucleic acid, as well as information about a nucleic acid obtained about a disease, particularly about a nucleic acid of a pathogenic microorganism associated with a disease.
[0002]
The present invention also relates to a probe-immobilized substrate, particularly a probe-immobilized chip for use in the method.
[0003]
[Prior art]
DNA chip technology has attracted attention (Beattie et al., Clin Chem 39: 719-22, Fodor et al., Science, 251, 767 (1991), Khrapko et al., FEBS Lett, 256, 118 (1989 ), and Southern et al., Nucleic Acids Res., 22, 1368 (1994)). The DNA chip is a several cm square glass or silicon chip on which a plurality of types of DNA probes having different arrangements are immobilized. A sample nucleic acid labeled with a fluorescent dye or a radioisotope (RI) or a mixture of an unlabeled sample nucleic acid and a labeled oligonucleotide is reacted with a DNA probe on such a chip. When a sequence complementary to the DNA probe immobilized on the chip is present in the sample, a signal derived from the label used at a specific site on the chip is obtained. Therefore, if the sequence (sequence) and position of the immobilized DNA probe are known in advance, the base sequence (sequence) present in the sample nucleic acid can be examined (Pease et al., Proc. Natl. Acad). Sci. USA, 91, 5022 (1994), Parinov et al., Nucleic Acids Res., 24, 2998 (1996).
[0004]
By obtaining a sample substance such as blood obtained from a patient and extracting the nucleic acid from the sample substance, the patient-specific nucleic acid can be obtained. If the patient-specific nucleic acid is further subjected to genetic analysis, information related to the patient's constitution, such as the susceptibility to certain diseases, the efficacy of drugs, and / or the likelihood of side effects, can be obtained. Is possible. As a result, information specific to the patient can be obtained. A treatment designed specifically for an individual patient is called “tailor-made medicine”.
[0005]
When performing tailor-made medical care, it is required to develop a method for predicting a treatment method for a disease more accurately and simply from a sample material extracted from a patient.
[0006]
[Problems to be solved by the invention]
An object of the present invention is to provide a method capable of recovering target nucleic acid information from a sample substance simply, in a short time, and accurately.
[0007]
[Means for Solving the Problems]
Means for solving the above problem is a method for obtaining first information about nucleic acids of individuals exposed to a specific disease and second information about nucleic acids from pathogenic microorganisms present in the individuals. A method wherein the pathogenic microorganism is associated with the specific disease and comprises:
(A) reacting a nucleic acid extract from the individual with a probe-immobilized substrate comprising a first probe and a second probe, wherein the first probe is the pathogenic microorganism Detecting the presence of a specific nucleic acid sequence, the pathogenic microorganism is associated with the specific disease, and the second probe detects the presence of a specific nucleic acid in the individual; and
(B) obtaining the first information by detecting the presence of nucleic acid bound to the first probe as a result of the reaction of (a), and binding to the second probe; Obtaining second information by detecting the presence of the completed nucleic acid: and
A substrate;
A first probe immobilized on the substrate for detecting the presence of a specific nucleic acid sequence of a pathogenic microorganism, wherein the microorganism is associated with a specific disease;
A second probe immobilized on the substrate and having a base sequence different from the first probe for detecting the presence of a specific nucleic acid of the individual, wherein the nucleic acid of the individual is used to treat the disease Related to responsiveness to
Probe-immobilized substrate comprising:
It is.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method for obtaining information about nucleic acids from an individual exposed to a disease. The individual may be an individual at an early or late stage of the disease. The individual may be an individual suspected of suffering from the disease or an individual suspected of suffering in the future.
[0009]
The present invention relates to the correlation of nucleic acid information from both the individual and the disease. In the embodiments disclosed herein, the individual is exposed to a disease associated with a pathogenic microorganism, such as, for example, a virus, bacteria, yeast or mycoplasma. The present invention also relates to a correlation between nucleic acid information obtained from pathogenic microorganisms present in the individual and nucleic acids from the individual.
[0010]
Accordingly, in one aspect, the present invention provides:
Information about nucleic acids associated with a particular disease of an individual, in particular information about nucleic acids associated with susceptibility of said individual to treatment of the disease;
Information on nucleic acids related to the specific diseases derived from pathogenic microorganisms present in the individual
Provide a way to get.
[0011]
The invention also relates to specific nucleic acid probes used in the identification of nucleic acids from said individuals and nucleic acids from said pathogenic microorganisms.
[0012]
In some embodiments, the nucleic acid probe is immobilized on a substrate such as a chip. The present invention also relates to a probe-immobilized substrate, such as a probe-immobilized chip, for use in the method. Other substrates such as DNA capillary electrophoresis apparatus, beads, membrane base arrays, microtiter plates, electrodes, semiconductor-based devices, waveguide materials, etc., surface plasmon resonance (SPR) Quartz crystal microbalance (hereinafter referred to as QCM) and mass spectrometry (Mass-spectroscopy) can also be used in the present invention. Furthermore, allele-specific oligohybridization, restriction method (microtiterarray diagonal gel electrophoresis), ligation method (padlock probe, oligonucleotide ligation assay, dye-labeled oligonucleotide ligation) ), Nucleotide incorporation (mini-sequencing, template-directed dye-termination assay), and invader assay can also be used in the present invention. Other common solution hybridization techniques can also be used in the present invention, such as amplification methods (polymerase chain reaction; PCR), ligase chain reaction (LCR), nucleic acid sequence-based amplification (nucleic acid). sequence-based amplification (NASBA), strand displacement amplification (SDA), loop-mediated isothermal amplification (LAMP), isothermal and chimeric primer initiation of nucleic acid -Initiated amplification of nucleic acids (ICAN), branched DNA that can be used to detect nucleic acids of the solid and pathogenic microorganisms, and the like can be used.
[0013]
Whole blood, blood, serum, leukocytes, urine, stool, semen, saliva, biopsy tissue, cultured cells, sputum and the like may be used as the sample material containing nucleic acid collected from the individual. A preferred sample is blood. Moreover, it is preferable that sample material contains both the nucleic acid derived from the said individual relevant to the sensitivity with respect to the treatment of a disease, and the nucleic acid relevant to the pathogenic microorganism which exists in the said individual. Here, the pathogenic microorganism is associated with the disease. In addition, the sample may have been subjected to any necessary pretreatment such as homogenate and extraction as necessary. Such pretreatment could be selected by one skilled in the art depending on the biological sample of interest.
[0014]
  In the illustrative embodiment disclosed herein, a whole blood sample is first collected from a human. Next, from this collected whole blood sample, for example, QIAamp DNA Blood Midi Kit (registered trademark)Using a commercially available human genome extraction kit such as (QIAGEN, Tokyo, Japan), nucleic acids derived from the human genome and the virus genome derived from the human are extracted together. In some embodiments, a nucleic acid containing the sequence of interest is amplified. Then, the obtained amplification product is analyzed by hybridizing the obtained amplification product to the probe-immobilized chip and further detecting the nucleic acid bound to the probe-immobilized chip. In this way, information about nucleic acids related to a specific disease from the individual is obtained along with information about nucleic acids related to a specific disease obtained from a pathogenic microorganism present in the individual.
[0015]
In the above method, an example of a human individual has been described, but the individual is not limited to a human. In accordance with an embodiment of the present invention, the subject “individual” can be any mammal, including a human, dog, cat, cow, goat, pig, sheep, or monkey. However, humans are the most preferred individuals. According to the aspect of the present invention, the individual may be a healthy individual or an individual suffering from some kind of disease. However, in general, the method according to the present invention will be used more effectively by performing on an individual suffering from a disease caused by the pathogenic microorganism and the type of nucleic acid that the individual has. As used herein, the term “specific disease” includes diseases associated with pathogenic microorganisms such as viruses, bacteria, yeasts or mycoplasmas as well as oncological diseases, preferably diseases associated with pathogenic microorganisms. More preferably, depending on the type of nucleic acid contained in an individual suffering from the disease and / or the presence or absence of expression of a gene containing such a nucleus, its onset and therapeutic effect are influenced. Is a disease. However, it is not particularly limited to these diseases.
[0016]
As used herein, the term “target nucleic acid” refers to a nucleic acid containing a sequence that is to be detected and / or analyzed.
[0017]
According to the aspect of the present invention, the extracted nucleic acid derived from an individual may be any nucleic acid derived from an individual, and may be DNA, RNA, or genomic DNA. Further, the extracted nucleic acid derived from a pathogenic microorganism may be any nucleic acid derived from a target pathogenic microorganism, and may be DNA or RNA.
[0018]
The method for extracting the nucleic acid component from the sample substance is not particularly limited to the above means, and a liquid-liquid extraction method such as a phenol-chloroform method or a solid-liquid extraction method using a carrier can also be used. Moreover, although not limited to this, for example, a nucleic acid extraction method QIAamp (manufactured by QIAGEN), a genomic DNA purification kit (manufactured by Promega), Sumitest (manufactured by Sumitomo Metals) or the like is used. Is possible. Subsequently, an amplification operation such as PCR may or may not be performed.
[0019]
  When the extracted nucleic acid component is RNA, HCV-RNA may be used directly as a sample nucleic acid, or cDNA may be prepared from RNA using reverse transcriptase and then used as a sample nucleic acid. In this case as well, it may be performed without separating the nucleic acid derived from the individual and the nucleic acid derived from the pathogenic microorganism. For example, in the above-described method, QIAamp DNA Blood Midi KitAfter extraction using, reverse transcription may be performed followed by PCR amplification.
[0020]
The amplification performed according to the embodiment of the present invention may be any known publicly known means for generally amplifying nucleic acids, and is preferably performed by polymerase chain reaction (hereinafter referred to as PCR). Further, if the sample material contains a sufficient amount of nucleic acid, amplification may be omitted.
[0021]
A probe-immobilized substrate such as a probe-immobilized chip that can be used in accordance with an embodiment of the present invention is a probe-immobilized chip in which a first probe and a second probe having different base sequences are immobilized on a substrate. The first probe is a probe for detecting the presence of a specific nucleic acid sequence derived from a pathogenic microorganism associated with a specific disease. The second probe is a probe for detecting the presence of a specific nucleic acid sequence related to the specific disease and derived from an individual.
[0022]
Such a probe-immobilized substrate can be analyzed simultaneously by using the same substrate with respect to nucleic acids obtained from two different sources, that is, an individual and a pathogenic microorganism. Such a probe fixing chip is disclosed for the first time by the present applicant. According to the present invention, such a probe-immobilized substrate is also provided as one aspect of the present invention.
[0023]
In the probe-immobilized chip according to the embodiment of the present invention, the first probe detects the presence of a nucleic acid sequence derived from a pathogenic microorganism contained in a sample substance such as whole blood from a human patient. Such a probe may have a nucleotide sequence corresponding to a chromosomal DNA or gene (RNA) derived from a pathogenic microorganism that may be infected by an individual subject (for example, a human patient), Alternatively, it may have a base sequence corresponding to its cDNA or cRNA, chromosomal DNA, RNA, cDNA and cRNA fragment or a complementary sequence thereof.
[0024]
Further, the amount of pathogenic microorganisms in the sample material is also measured using the first probe, that is, a probe having a base sequence corresponding to the nucleic acid sequence derived from the pathogenic microorganism, a fragment thereof or a complementary sequence thereof. Is possible.
[0025]
Furthermore, the first probe is preferably a probe that detects not only the presence of the pathogenic microorganism in a sample material but also a genetic variation of the pathogenic microorganism.
[0026]
The nucleic acid (RNA) expressed by the pathogenic microorganism in the sample material is also measured using the first probe, that is, a probe having a base sequence corresponding to the nucleic acid sequence derived from the pathogenic microorganism, a fragment thereof or a complementary sequence thereof. Measurement is possible. In this case, the nucleic acid strand can detect or quantify RNA, cDNA or cRNA.
[0027]
In one embodiment where the pathogenic microorganism is a virus, a large amount of a specific gene is expressed during the growth phase of the virus, so that its efficacy can be predicted by measuring its expression pattern (quantity, quality). Become.
[0028]
In the probe-immobilized chip according to the embodiment of the present invention, the second probe corresponds to a chromosomal DNA sequence, gene (RNA) or cDNA or cRNA thereof, a fragment thereof or a complementary sequence thereof associated with a disease that an individual has. Examples include base sequences. The “nucleic acid sequence related to a disease” possessed by an individual is a nucleic acid sequence present in a chromosome in the cell of the individual, and is a nucleic acid sequence that can predict responsiveness to treatment. More specifically, the nucleic acid sequence is, for example, a nucleic acid sequence that predicts the responsiveness of an individual to a disease, determines the efficacy of a drug to be administered and / or the likelihood of occurrence of a side effect, and the like.
[0029]
The second probe can detect the presence of a specific nucleic acid sequence in an individual, the expression level of a specific gene, the expression pattern of a gene in the individual, and the like.
[0030]
The first probe or the second probe according to the present invention has at least 11 bases, preferably at least 12 bases, more preferably at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, It is desirable to have a base sequence with a length of 25, 26, 27, 28, 29, and 30 bases. If the length of the base sequence immobilized on the substrate is excessively long, it will be difficult to identify the difference between one base. On the other hand, if the length of the base sequence immobilized on the substrate is excessively short, it is difficult to determine the base sequence of the base sequence contained in the sample.
[0031]
As the first probe or the second probe, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), peptide nucleic acid (PNA), methyl phosphonate nucleic acid, oligonucleotide such as S-oligo, cDNA, cRNA, etc. Nucleic acids such as polynucleotides can be used.
[0032]
According to an embodiment of the present invention, the probe-immobilized substrate is a substrate, a porous body (Beattie et al., Clin. Chem., 41, 700 (1995)), a microtiter plate (Kawai et al., Anal. Biochem. , 209, 63 (1993)), beads (Mirkin et al., Nature, 382, 607 (1996), spherical materials, particulate materials, magnetic materials, magnetic beads (Miller et al., J. Magnet. Magn. Mater , 225, 138 (2001), DNA capillary electrophoresis chip (Chou et al., Proc. Natl. Acd. Sci., 96, 11 (1999), membrane base array (Lemieux et al., Molecular Breeding, 4, 277 (1998), devices based on semiconductors (Thewes et al., 2002 IEEE International Solid-State Circuits Conference, 350 (2002), waveguide materials (Piunno et al., Anal. Chim. Acta, 288, 205 (1994)), SPR (Nelson et al., Anal. Chem., 73, 1 (2001), QCM (Ito et al., Anal. Chim. Acta, 327, 29 (1996), mass spectrometry (Amexis et al., Proc. Natl. Acd. Sci., 98, 12097 (2001), etc. It can be prepared by immobilizing the probe and the second probe The material, size, shape, etc. of the substrate on which the nucleic acid is to be immobilized are not particularly limited, and any substrate capable of immobilizing the nucleic acid The detection of the nucleic acid sequence in the sample material using the probe-immobilized substrate may be performed by, for example, extracting the nucleic acid component from the sample material collected from the individual, and then the sample nucleic acid and the probe. A hybridization reaction between the probe on the probe-immobilized substrate and the sample nucleic acid may be detected by contacting a probe-immobilized substrate such as an immobilization chip.
[0033]
The present invention mainly includes (1) a method using a labeling substance and (2) electrochemical as means for detecting a hybridization reaction between a probe on the probe-immobilized chip and a nucleic acid component in a sample. Two types of methods may be included.
[0034]
In the case of the method using the labeling substance of (1), the sample nucleic acid is a fluorescent dye such as FITC, Cy3, Cy5 or rhodamine, or an enzyme such as biotin, hapten, oxidase or phosphatase, or ferrocene or quinones. Labeled with an electrochemically active substance. Alternatively, detection is performed by using a second probe labeled with the aforementioned substance. A plurality of labeling substances may be used simultaneously.
[0035]
In the case of the electrochemical method (2), a conductive substance is used as a substrate on which a probe is immobilized, and a probe-immobilized chip is used as an electrode. Detection of the presence of a hybridization reaction using this electrode uses a counter electrode or a reference electrode in addition to this electrode, as in other general electrochemical detection methods. When arranging the reference electrode, for example, a general reference electrode such as a silver / silver chloride electrode or a mercury / mercury chloride electrode can be used. Probes having different base sequences may be immobilized on different conductive substrates and constitute chips arranged on the same substrate or the like. This makes it possible to perform highly accurate measurement. In that case, it is detected to which probe the electrochemical signal obtained from each electrode corresponds.
[0036]
In some embodiments, a hybridization reaction between a nucleic acid component extracted from a sample substance and a probe immobilized on a probe-immobilized chip is performed, for example, as follows. That is, the hybridization reaction solution is performed in a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10. In this solution, dextran sulfate, which is a hybridization accelerator, salmon sperm DNA, bovine thymus DNA, EDTA, and a surfactant may be added. The nucleic acid component extracted here is added and heat-denatured at 90 ° C. or higher. The probe-immobilized chip may be inserted immediately after denaturation or after rapid cooling to 0 ° C. It is also possible to perform a hybridization reaction by dropping a solution on the substrate. During the reaction, the reaction rate may be increased by an operation such as stirring or shaking. The reaction temperature may be, for example, in the range of 10 ° C. to 90 ° C., and the reaction time may be about 1 minute to overnight. After the hybridization reaction, the electrode is removed and washed. For the washing, for example, a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10 is used.
[0037]
In the case of the method using the labeling substance in (1), the hybridization reaction is detected by detecting the labeled base sequence in the sample or the label in the secondary probe using an appropriate detection device according to the type of label. By doing. When the label is a fluorescent substance, for example, the label may be detected using a fluorescence detector.
[0038]
In the case of the electrochemical method (2), detection is carried out by the following procedure. After the substrate is washed, a double-strand recognition body that selectively binds to a double-strand portion formed on the electrode surface is allowed to act, and electrochemical measurement is performed. The double-stranded recognizer used here is not particularly limited. For example, bisintercalators such as Hoechst 33258, acridine orange, quinacrine, dounomycin, metallointercalator, bisacridine, trisintercalator and polyintercalator Etc. can be used. Furthermore, these intercalators can be modified with an electrochemically active metal complex such as ferrocene or viologen. The concentration of the DNA binding substance varies depending on the type, but is generally used in the range of 1 ng / mL to 1 mg / mL. In this case, a buffer solution having an ionic strength of 0.001 to 5 and a pH of 5 to 10 is used. After the electrode is reacted with the double-stranded recognizer, it is washed and subjected to electrochemical measurement.
[0039]
In the electrochemical measurement, a potential higher than the potential at which the double-stranded recognizer reacts electrochemically is applied, and the reaction current value derived from the double-stranded recognizer is measured. At this time, the potential can be swept at a constant speed, applied with a pulse, or a constant potential can be applied. For the measurement, for example, the current and voltage are controlled using a device such as a potentiostat, a digital multimeter, and a function generator. Based on the obtained current value, the concentration of the target nucleic acid can be calculated from the calibration curve.
[0040]
A base sequence detection apparatus using electrodes includes, for example, a nucleic acid extraction unit, a nucleic acid reaction unit, a double-stranded recognizer reaction unit, an electrochemical measurement unit, and a washing unit.
[0041]
Other examples of DNA chips based on electrochemical techniques are disclosed in, for example, the following documents (Hashimoto et al. 1994, Wang et al. 1998). Hashimoto et al. Reported sequence-specific gene detection using a gold electrode modified with a DNA probe and an electrochemically active dye. The anode current derived from the dye correlates with the concentration of the target DNA. Wang et al. Reported indicator-free electrochemical DNA hybridization. This biosensor configuration includes immobilization of an inosine displacement probe (without guanine) to a carbon paste electrode and chronopotentiometric detection of duplex formation due to the presence of the guanine oxidation peak of the label. These documents are incorporated herein by reference.
[0042]
The electrochemical method does not require labeling of the sample nucleic acid. Detection is performed by measuring an electrical signal. Therefore, a complicated system as required for fluorescence detection is not necessary. Therefore, such a method can be expected to reduce the size of the system.
[0043]
According to the embodiment of the present invention as described above, from a sample material such as blood collected from an individual such as a patient, a nucleic acid sequence related to the disease possessed by the patient itself, and information on the nucleic acid sequence of a pathogenic microorganism infected by the patient. It can be easily obtained.
[0044]
The probe-immobilized substrate according to an embodiment of the present invention is used to predict an appropriate treatment method in an individual exposed to a disease. The probe-immobilized base includes a second probe and a first probe fixed on the same base. The second probe is a probe (second probe) for obtaining information on a nucleic acid sequence linked to (linked to) the disease present in a chromosome in an individual cell, and the first probe is the disease It is a probe (first probe) for obtaining information related to the nucleic acid sequence of a pathogenic microorganism that induces the pathogen. Due to the advantages of the probe-immobilized substrate, information related to the prediction of the specific disease derived from the individual and the pathogenic microorganism and / or the responsiveness of the treatment of the specific disease can be easily and easily obtained from the sample material extracted from the individual. It is possible to obtain.
[0045]
In some embodiments, the method and probe-immobilized substrate can predict the therapeutic effect of hepatitis C. Another aspect is the method and probe-immobilized substrate used to evaluate AIDS treatment (eg, HIV quantification and mutation detection, etc.). Further embodiments are the methods and probe-immobilized substrates used to evaluate the treatment of hepatitis B (eg, HBV quantification, type and mutation detection, etc.).
[0046]
For example, it is known that infection with hepatitis C progresses to liver cancer via cirrhosis. One such treatment is IFN treatment in which interferon (hereinafter referred to as IFN) is administered. In many cases, IFNα and β are used for treatment. However, it is known that the effectiveness of IFN varies greatly. For example, IFN treatment is effective only in about 20 to 30% of Japanese human patients, and even if effective, very strong side effects have been reported. The difference in the therapeutic effect due to the difference in individual is that the hepatitis C virus (hereinafter referred to as HCV) genotype (J. Clin. Microbiol., 34, 2516 (1996)) and viral load that the individual has infected. This is considered to be caused by individual differences in the nucleic acid sequence possessed by the individual.
[0047]
At least six major genotypes have been reported (Forns et al., J. Clin. Microbiol., 34, 2516 (1996), based on a comparison of nucleotide and predicted amino acid sequences of different regions of HCV. Andonov et al., J. Clin. Microbiol., 33, 254 (1995).
[0048]
Thus, according to aspects of the present invention, it is possible to predict the effect of IFN treatment on hepatitis C. Here, in this specification, “interferon (IFN)” is used as a word indicating interferon α, β, γ and / or ω. For example, when the type of the infected virus is type 1b (common in Japanese), the effect of IFN treatment is small. This means that serum HCV-RNA, HCV antibodies, GOP and GPT are not reduced by IFN therapy. In the case of type 2a, the effect of treatment is higher. This means that serum HCV-RNA, HCV antibodies, GOP and GPT are reduced by IFN therapy. In addition, the viral load is 10⁶Even if it is more than copy / mL, the effect is small. Therefore, the first probe is used to examine the genotype and viral load of the infected virus at the nucleic acid level.
[0049]
For example, a probe used for detecting the presence of a nucleic acid sequence of hepatitis C virus (hereinafter also referred to as HCV) in a sample material includes a base sequence corresponding to HCV-RNA, a fragment thereof or a complementary sequence thereof. It is done. Probes according to aspects of the invention desirably have at least 11 bases, preferably at least 12 bases, more preferably at least 13, 14, 15, 16, 17, 18, for detecting HCV-RNA genotypes or mutations. It has a sequence of 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 bases. Alternatively, the probe is also preferred for detection of the HCV genome, even if it is longer than at least 30, preferably about 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000 bases. A length of 11 to 30 is suitable for chip formation, and probes longer than 30 bases are suitable for general hybridization formation. For example, it is possible to use those having a base sequence corresponding to SEQ ID NO: 1, 2, 3, 4 or their complementary sequences shown in the sequence listing. These sequences are most conserved in all HCV genotypes (Andonov et al., J. Clin. Microbiol., 33, 254 (1995).
[0050]
The first probe is preferably a probe that detects not only the presence of the pathogenic microorganism in a sample material but also a genetic mutation of the pathogenic microorganism. In the case of hepatitis C, it has been reported that the effect of IFN treatment varies depending on the mutation at a specific site in the viral nucleic acid sequence (Nagayama et al., Hepatology, 31, 745 (2000)). Examples of a first probe for detecting a mutation at a specific site in a nucleic acid sequence of a virus in a sample material are HCV polyprotein 434, 580, 938, 962, described in Hepatology, 31, 745 (2000). It is possible to use a probe having a base sequence that determines the difference between the 1176th and 2774th amino acids. The genomic sequence of HCV-1b from patients with hepatocellular carcinoma (hereinafter referred to as HCC) tends to have more mutated residues than that from asymptomatic carriers (hereinafter referred to as ASC). By using these, prediction with high accuracy becomes possible.
[0051]
As described above, it has been reported that the effect of IFN treatment varies depending on the genotype of HCV. Therefore, the first probe may be a base sequence corresponding to HCV-RNA, a fragment thereof or a complementary sequence thereof. For example, a probe that specifically detects genotypes of HCV type 1 (SEQ ID NO: 5), type 2 (SEQ ID NO: 6), and type 3 (SEQ ID NO: 7) is used as the first probe according to the genotype to be detected. It may be used. That is, a nucleic acid having a sequence corresponding to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or their complementary sequences shown in the sequence listing may be used as the first probe.
[0052]
When using a probe capable of detecting a genotype, a plurality of probes corresponding to different genotypes can be immobilized on the substrate at the same time and used for highly accurate detection.
[0053]
Further, in order to detect the genotype of a pathogenic microorganism in a sample material, in one embodiment, a PCR reaction is previously performed on the nucleic acid sequence of the pathogenic microorganism in the sample material using a primer characteristic of the genotype. Then, it is also possible to detect a universal probe capable of detecting all hepatitis C viruses without considering their genotypes with an immobilized probe (Andonov et al., J. Clin. Microbiol., 33, 254 (1995).
[0054]
In the case of hepatitis C, the sense strand having the nucleotide sequence shown in SEQ ID NO: 8 or 9 is used, and the antisense strand is SEQ ID NO: 10 for type 1a and SEQ ID NO: 11, 2a for type 1b. A PCR reaction is carried out using the nucleotide sequence of SEQ ID NO: 12 as the type, the sequence of SEQ ID NO: 13 as the type of 2b, and the nucleotide sequence of SEQ ID NO: 14 as the type of 3a. Good.
[0055]
The amount of pathogenic microorganisms in the sample material can also be measured using the first probe, that is, a probe having a base sequence corresponding to a nucleic acid sequence derived from the pathogenic microorganism, a fragment thereof or a complementary sequence thereof. It is.
[0056]
For example, in the case of hepatitis C, it has been reported that the effect of IFN therapy varies depending on the amount of HCV in the blood (J. Clin. Microbiol., 33, 254 (1995)). INF is 10 HCV in the serum of the patient⁶When the number of copies / mL is exceeded, it is difficult to obtain an effect (Yeh et al., J. Med. Biol., 66, 481 (2002). Therefore, HCV- is also used as a probe for quantitatively evaluating the viral load. For example, when the nucleic acid in the sample substance is fluorescently labeled, the probe on the chip is hybridized with the nucleic acid in the sample substance. After that, the concentration is calculated from the fluorescence intensity of the label on the substrate.When using an electrochemical method, the probe on the chip is hybridized with the nucleic acid in the sample substance, and then the concentration is determined from the current value obtained from the chip. Is calculated.
[0057]
In the probe-immobilized chip of the present invention, if the above-mentioned probe, SEQ ID NO: 5, 6 and / or 7 is used as the first probe, the virus genotype or virus amount of the pathogenic microorganism infected by the individual, the mutation at a specific site, Knowledge of the amount and quality of the expressed gene is obtained, and if the virus type is type 1b, which is more common in Japanese, the effect of IFN therapy is small, but if it is type 2a, it works effectively.⁶When it is more than copy / mL, it can be predicted that the effect of IFN therapy is small, and thereby the effectiveness of IFN therapy for the individual can be predicted.
[0058]
Another method for predicting the effect of IFN treatment on hepatitis C has been reported by the applicant in Japanese Patent Application Nos. 2001-62371 and 2001-62372. That is, a specific single nucleotide polymorphism (hereinafter referred to as SNP) present in the promoter region of a gene encoding the MxA protein of a human gene, that is, the -88 position of the MxA promoter region (hereinafter referred to as MxA-88). Is a method of predicting the magnitude of the IFN treatment effect by examining the SNP in order to determine the magnitude of the IFN treatment effect. That is, INF is effective in patients with a specific type of SNP in the MxA promoter region. Therefore, by checking the presence of the SNP, the effect of IFN therapy is evaluated. Accordingly, in one embodiment of the present invention, the second probe for detecting an individual's nucleic acid is one that detects SNP in MxA.
[0059]
Based on the above findings, when the -88 position of the promoter region of MxA (hereinafter referred to as MxA-88) is G / G type, the IFN treatment effect is low, and in the case of G / T and T / T types High therapeutic effect, low IFN therapeutic effect when position -123 (hereinafter referred to as MxA-123) in the same region is C / C type, and therapeutic effect for C / A and A / A types The therapeutic effect of IFN is predicted based on the fact that it is high. Here, the notations “−88” and “−123” are positions where the transcription start site of the MxA gene is +1. FIG. 1 of WO01 / 71001 shows the nucleotide sequence of the promoter region of the MxA gene.
[0060]
Therefore, when predicting the IFN treatment effect on hepatitis C according to the embodiment of the present invention, for example, the presence of the HCV gene, the first probe for detecting the HCV genotype or gene mutation, and the individual Diagnostic diagnosis of HCV in a sample substance collected from an individual using a probe-immobilized chip on which a second probe for detecting the type of base in the SNP of the promoter region of the gene encoding the MxA protein is immobilized, By performing genetic diagnosis of the promoter region of a gene encoding an MxA protein possessed by an individual, the effect of the IFN treatment can be easily predicted.
[0061]
Furthermore, as a method for predicting the effect of IFN treatment on hepatitis C, there is known a method for predicting the magnitude of IFN treatment effect depending on the two SNP types of the gene of mannose-binding lectin (hereinafter referred to as MBL). (M. Matsushita et al., J. Hepatology, 29; 695-700, 1998). MBL is an important factor of the innate immune system, and genetic polymorphism exists. The MBL genotype group “XB” does not respond to IFN. Rosen reports on the sequence of MBL (Immunology, 93, 421 (1998)). This document is incorporated herein by reference. Therefore, genetic diagnosis relating to the gene of mannose-binding lectin (hereinafter referred to as MBL) may be performed together with or in place of genetic diagnosis of the promoter region of the gene encoding MxA protein as described above.
[0062]
The polymorphic site of MBL that affects the effect of IFN exists at position -221 (hereinafter referred to as MBL-221) of the promoter region of the MBL gene and codons 52, 54 and 57 of exon 1 of the MBL gene. Here, the expression “−221” is a position when the transcription start site of the MBL gene is +1.
[0063]
The bases that can be taken by the genotype of MBL-221 are C or G. In the case of C, it is referred to as type X, and in the case of G, it is referred to as type Y (this nomenclature follows Madsen et al .; Madsen HO, Garred P, Thiel S, Kurtzhals JAL, Lamm LU, Ryder LP, et al. “The interaction between the promoter and the structural gene controls the lowest serum level of human mannan-binding protein” J Immunol 1995; 155: 3013-20 ). This document is incorporated herein by reference.
[0064]
Also, codons 52, 54 and 57 of exon 1 of the MBL gene are present in the collagen-like domain which is a structural gene. The genotype that codon 52 can take is CGT or TGT. According to the classification of Madsen et al., The former is type A and the latter is type D. Similarly, the genotype that can be taken by codon 54 is GGC or GAC. According to the same classification, the former is type A and the latter is type B. The genotype that can be taken by codon 57 is GGA or GAA. According to the same classification, the former is type A and the latter is type C. In all these codons, type A is a wild type allele and the other types B, C and D are mutant alleles. If alleles at codons 52, 54 and 57 are all type A, the effect of IFN is higher than in the other case, i.e. at least any allele is not type A.
[0065]
In the case of a YA-YA homozygous patient in which the above MBL-221 is type Y and the alleles of codons 52, 54 and 57 are type A, the effect of IFN is obtained compared to other types of patients. It is easy to be done. Also, in the case where the above-mentioned MBL-221 is type Y and any of the alleles of codons 52, 54 and 57 is not type A, that is, a patient who is YnonA-YA heterozygous or YnonA-YnonA homozygous Therefore, it is difficult to obtain the effect of IFN as compared with YA-YA homozygous patients.
[0066]
The second probe can be determined from the above knowledge.
[0067]
In one embodiment where the disease is hepatitis C and the drug is IFN, the second probe used in hybridization to the nucleic acid of the individual (including use on a probe-immobilized chip) is as described above. It is a promoter that detects the SNP of the MxA promoter. In other embodiments where the disease is hepatitis C and the drug is IFN, the second probe used in hybridization to the individual's nucleic acid (including use on a probe-immobilized chip) is as described above. Thus, it is a probe for detecting an MBL polymorphism associated with the effect of IFN.
[0068]
Each of these base sequences in the promoter region of the human MxA gene containing SNPs located at positions 455 and 420 is associated with the effect of IFN therapy. Therefore, the nucleotide sequence represented by SEQ ID NOs: 16, 17, 18, 19, 37, 38, 39 and 40
In a further embodiment, the second probe is selected from the group consisting of:
(at455) the base sequence shown in SEQ ID NO: 16 in the following sequence listing,
(bt455) Modified base sequence in which several bases except the base at position 455 in the base sequence shown in (at455) are deleted, substituted, or added with one or more bases; MBL polymorphism is detected The modified probes to do so maintain the ability to hybridize to individual nucleic acids encoding MBL. The hybridization conditions used for the modified probes are different from those for general probes. Desirably, the modified probe according to the present invention has at least 11 bases, preferably at least 12 bases, more preferably at least 13, 14, 15, 16, 17, 18 for detecting HCV-RNA genotypes or mutations. , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 bases. Alternatively, the probe is also preferred for detection of the HCV genome, even if it is longer than at least 30, preferably about 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000 bases. A length of 11 to 30 is suitable for chip formation, and probes longer than 30 bases are suitable for general hybridization formation.
[0069]
Here, a symbol in parentheses at the beginning of a line indicates a specific base sequence, and the content of the sequence is described on the right side of the symbol in the parentheses:
  (ct455) a base sequence comprising the sequence shown at positions 441 to 455 of SEQ ID NO: 16,
  (dt455) a nucleotide sequence comprising the sequence shown at positions 449 to 459 of SEQ ID NO: 16,
  (et455) a complementary sequence of a base sequence selected from (at455) to (dt455),
  (ag455)Sequence listingThe nucleotide sequence shown in SEQ ID NO:
  (bg455) a modified base sequence in which several bases except the base at position 455 in the base sequence shown in (ag455) are deleted, substituted, or added with one or more bases,
  (cg455) a base sequence comprising the sequence shown at positions 441 to 455 of SEQ ID NO: 17,
  (dg455) a nucleotide sequence comprising the sequence shown at positions 449 to 459 of SEQ ID NO: 17,
  (eg455) Complementary sequence of the base sequence selected from (ag455) to (dg455)
  (aa455)Sequence listingThe nucleotide sequence represented by SEQ ID NO: 18,
  (ba455) a modified base sequence in which several bases except the base at position 455 in the base sequence represented by (aa455) are deleted, substituted, or added with one or more bases,
  (ca455) a nucleotide sequence comprising the sequence shown at positions 441 to 455 of SEQ ID NO: 18,
  (da455) a nucleotide sequence comprising the sequence shown at positions 449 to 459 of SEQ ID NO: 18,
  (ea455) complementary sequence of the base sequence selected from (aa455) to (da455)
  (ac455)Sequence listingThe nucleotide sequence shown in SEQ ID NO: 19,
  (bc455) a modified base sequence in which several bases except the base at position 455 in the base sequence shown in (ac455) are deleted, substituted, or added with one or more bases,
  (cc455) a base sequence comprising the sequence shown at positions 441 to 455 of SEQ ID NO: 19,
  (dc455) a base sequence comprising the sequence shown at positions 449 to 459 of SEQ ID NO: 19,
  (ec455) a complementary sequence of a base sequence selected from (ac455) to (dc455),
A base sequence selected from the group consisting of:
[0070]
The base sequences of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40 are base sequences including the promoter region of the human MxA gene, SNPs at positions 455 and 420 of these nucleotide sequences are involved in responsiveness to the effects of IFN therapy
The base sequences of SEQ ID NOs: 16 to 19 have a common sequence except for the base at position 455. Position 455 of SEQ ID NO: 16 is thymine. Position 455 of SEQ ID NO: 17 is guanine. In addition, it is adenine at position 455 of SEQ ID NO: 18. Position 455 of SEQ ID NO: 19 is cytosine.
[0071]
Those HCV-infected persons having the nucleic acid sequence of SEQ ID NO: 16 whose base at position 455 is thymine have the nucleic acid sequence of SEQ ID NO: 16 whose base at position 455 is thymine, whereas IFN therapy is effective. For those with no HCV, IFN therapy is not effective. That is, HCV infection having a nucleic acid of SEQ ID NO: 16 whose base sequence at position 455 is thymine and a nucleic acid of SEQ ID NO: 17 whose base sequence at position 455 is guanine (hereinafter abbreviated as G / T hetero). Or a HCV-infected person having a nucleic acid of SEQ ID NO: 16 in which the nucleotide sequence at position 455 is thymine as a homozygote (hereinafter abbreviated as T / T homozygote), a sequence whose nucleotide sequence at position 455 is guanine IFN therapy is not effective for HCV-infected persons who have the nucleic acid of No. 17 in homozygosity (hereinafter abbreviated as G / G homo).
[0072]
Alternatively, compared to a T / non-T heterozygous or T / T homozygous HCV infected person, an HCV infected person (hereinafter referred to as non-T / non) having a promoter region of the MxA gene whose base sequence at position 455 is not thymine. (Abbreviated as -T homo) showed that IFN therapy was not effective. Non-T / non-T homo combinations include G / G, G / A, G / C, A / A, A / C, and C / C. T / non-T combinations include T / G, T / A, and T / C.
[0073]
The base sequences of SEQ ID NOs: 37 to 40 have a common sequence except for the base at position 425. Position 425 of SEQ ID NO: 37 is adenine. Position 425 of SEQ ID NO: 38 is cytosine. In addition, position 425 of SEQ ID NO: 39 is thymine. Position 425 of SEQ ID NO: 40 is guanine.
[0074]
In the probe-immobilized chip of the present invention, the base sequence of SEQ ID NO: 16, comprising the SNP site as a second probe for detecting the nucleic acid sequence of an individual and the base sequence of the SNP site is thymine, a fragment thereof, or a complement thereof Using the sequence ((at455) to (et455)), it is possible to examine whether or not the SNP site of the base sequence including the promoter region of the human MxA gene possessed by an individual is thymine before treatment. Thereby, the effectiveness of IFN therapy for the individual can be predicted.
[0075]
Therefore, prior to the implementation of IFN therapy, for example, by determining the base of the SNP site in the base sequence including the promoter region of the human MxA gene possessed by the HCV infected person, the IFN therapy is performed on the HCV infected person. Effectiveness can be detected.
[0076]
Further, in the probe-immobilized chip of the present invention, as a second probe for detecting the nucleic acid sequence of an individual, the base sequence of SEQ ID NO: 17, which contains the SNP site and the base sequence of the SNP site is guanine, and a fragment thereof Or a complementary sequence thereof ((ag455) to (eg455)), a base sequence of SEQ ID NO: 18, wherein the base sequence of the SNP site is adenine, a fragment thereof or a complementary sequence thereof ((aa455) to (ea455)), or Using the nucleotide sequence of SEQ ID NO: 19, wherein the nucleotide sequence of the SNP site is cytosine, a fragment thereof or a complementary sequence thereof ((ac455) to (ec455)), a nucleotide including the promoter region of the human MxA gene possessed by the individual The sequence of the SNP site of the sequence can be examined, whereby IFN therapy for the individual It can predict efficacy. (Japanese Patent Application Nos. 2001-62371 and 2001-62372).
[0077]
  In one embodiment where the disease is hepatitis C and the drug is IFN, the second probe (including use on a probe-immobilized chip) used in hybridization to an individual's nucleic acid is:
(aa420)Sequence listingThe nucleotide sequence shown in SEQ ID NO: 37,
(ba420) a modified base sequence in which several bases except the base at position 420 in the base sequence shown in (aa420) are deleted, substituted, or added with one or more bases,
(ca420) a base sequence comprising the sequence shown at positions 415 to 425 of SEQ ID NO: 37,
(da420) a complementary sequence of a base sequence selected from (aa420) to (ca420)
(ac420)Sequence listingThe nucleotide sequence shown in SEQ ID NO: 38,
(bc420) a modified base sequence in which several bases except the base at position 420 in the base sequence shown in (ac420) are deleted, substituted, or one or more bases are added,
(cc420) a nucleotide sequence comprising the sequence shown at positions 415 to 425 of SEQ ID NO: 38,
(dc420) Complementary sequence of the base sequence selected from (ac420) to (cc420)
(at420)Sequence listingThe nucleotide sequence shown in SEQ ID NO: 39,
(bt420) a modified base sequence in which several bases except the base at position 420 in the base sequence shown in (at420) are deleted, substituted, or added with one or more bases,
(ct420) a nucleotide sequence comprising the sequence shown at positions 415 to 425 of SEQ ID NO: 39,
(dt420) Complementary sequence of the base sequence selected from (at420) to (ct420)
(ag420)Sequence listingThe nucleotide sequence shown in SEQ ID NO: 40,
(bg420) a modified base sequence in which several bases except the base at position 420 in the base sequence shown in (at420) are deleted, substituted, or added with one or more bases,
(cg420) a nucleotide sequence comprising the sequence shown at positions 415 to 425 of SEQ ID NO: 40,
(dg420) complementary sequence of the base sequence selected from the above (ag420) ~ (cg420)
A base sequence selected from the group consisting of may be used.
Here, the symbol in parentheses at the beginning of the line indicates a specific base sequence, and the content of the sequence is described on the right side of the symbol.
[0078]
These HCV-infected persons having the nucleic acid sequence of SEQ ID NO: 37 whose base at position 425 is adenine have the nucleic acid sequence of SEQ ID NO: 37 whose base at position 425 is adenine, whereas IFN therapy is effective. For those with no HCV, IFN therapy is not effective. Details regarding the sequence and effectiveness may be understood as well as the description for the S455 at position 455 above.
[0079]
Furthermore, when the disease is a disease for which the efficacy of IFN therapy has been confirmed, for example, hepatitis C, the second probe has, for example, a sequence as described in (i) to (t) described below. Also good. These are suitable nucleic acids for use in determining the presence of the MBL-221 genotype and the polymorphisms of codons 52, 54 and 57.
[0080]
The MBL genes containing polymorphisms encoded by MBL-221 and codons 52, 54 and 57 are shown in SEQ ID NO: 41 to SEQ ID NO: 56. MBL-221 is present at position 425 of these sequences.
[0081]
Exon 1 starts from position 646 of these sequences, codon 52 is located from positions 868 to 870, codon 54 is located from positions 874 to 876, and codon 57 is located from positions 883 to 885. The SNP at codon 52 is at position 868, the SNP at codon 54 is at position 875, and the SNP at codon 57 is at position 884.
[0082]
(I) Nucleic acid fragments of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, including the SNP site of MBL-221, comprising SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44 A fragment comprising a nucleic acid having a sequence corresponding to positions 418 to 432.
[0083]
(J) Nucleic acid fragments of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, including the SNP site of MBL-221, comprising SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44 A fragment comprising a nucleic acid having a sequence corresponding to positions 421 to 430.
[0084]
(K) The complementary sequence according to (i) and (j) above.
[0085]
(L) SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53 including the SNP sites of the codons 52, 54 and 57 Fragments of the nucleic acids of SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56, comprising nucleic acids having sequences corresponding to positions 868 to 885 of these sequences. For example, a fragment comprising the nucleic acids shown in SEQ ID NO: 45 to SEQ ID NO: 56.
[0086]
(M) SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, sequence including the SNP site of the codons 52 and 54 A fragment of the nucleic acids of No. 54, SEQ ID No. 55 and SEQ ID No. 56, comprising nucleic acids having sequences corresponding to positions 868 to 876 of these sequences. For example, a fragment comprising the nucleic acids shown in SEQ ID NO: 45 to SEQ ID NO: 56.
[0087]
(N) SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 53, including the SNP sites of the codons 54 and 57 A fragment of the nucleic acid of No. 54, SEQ ID No. 55 and SEQ ID No. 56, comprising nucleic acids having sequences corresponding to positions 874 to 885 of these sequences. For example, a fragment comprising the nucleic acids shown in SEQ ID NO: 45 to SEQ ID NO: 56.
[0088]
(O) SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54 including the SNP site of the codon 54 Fragments of the nucleic acids of SEQ ID NO: 55 and SEQ ID NO: 56, comprising nucleic acids having sequences corresponding to positions 874 to 876 of these sequences. For example, a fragment comprising the nucleic acids shown in SEQ ID NO: 45 to SEQ ID NO: 56. In particular, a fragment containing positions 869 to 880 is desirable.
[0089]
(P) SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54 including the SNP site of the codon 52 Fragments of the nucleic acids of SEQ ID NO: 55 and SEQ ID NO: 56, comprising nucleic acids having sequences corresponding to positions 868 to 870 of these sequences. For example, a fragment comprising the nucleic acids shown in SEQ ID NO: 45 to SEQ ID NO: 56. In particular, a fragment containing positions 864 to 873 is desirable.
[0090]
(Q) SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54 including the SNP site of the codon 57 Fragments of the nucleic acids of SEQ ID NO: 55 and SEQ ID NO: 56, comprising nucleic acids having sequences corresponding to positions 883 to 885 of these sequences. For example, a fragment comprising the nucleic acids shown in SEQ ID NO: 45 to SEQ ID NO: 56. In particular, a fragment containing positions 880 to 890 is desirable.
[0091]
(R) SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54 including the SNP site of the codon 54 Fragments of the nucleic acids of SEQ ID NO: 55 and SEQ ID NO: 56, comprising a nucleic acid comprising position 875 of said sequence in which the SNP of the codon 54 is present. For example, a fragment comprising the nucleic acids shown in SEQ ID NO: 45 to SEQ ID NO: 56.
[0092]
(S) The nucleic acid fragment shown in the above (i) to (m) and having a length of 11 to 30. Preferred probe sequences include at least positions 420 to 430, positions 864 to 874, positions 870 to 880 and positions 879 to 889 for the MBL gene. The MxA gene preferably includes at least positions 450 to 460.
[0093]
(T) A nucleic acid fragment shown in (i) to (m) above, wherein one or several nucleotides excluding the polymorphic site are deleted, substituted, or added.
[0094]
In each sequence described in the sequence listing of the present specification, “N” and “n” represent any base of adenine, thymine, guanine or cytosine.
[0095]
In the probe-immobilized chip of the present invention, the nucleic acid (RNA) expressed by an individual is also measured by the second probe, that is, a probe having a base sequence corresponding to the nucleic acid sequence derived from the individual, a fragment thereof or a complementary sequence thereof. It is possible to measure using In this case, the nucleic acid strand is RNA, cDNA, or cRNA, and these can be detected or quantified. For example, since the efficacy of IFN varies depending on the individual constitution, it is possible to predict the drug efficacy by measuring the gene expression pattern (quantity, quality) upon administration.
[0096]
As described above, the target disease according to the embodiment of the present invention is not particularly limited as long as it is a disease induced by a pathogenic microorganism. The present invention includes diseases relating to pathogenic microorganisms. The present invention also includes oncological diseases. For example, when predicting the effectiveness of IFN treatment, even if it is a disease other than hepatitis C, the therapeutic effect can be similarly predicted as long as the disease has confirmed the effectiveness of IFN treatment. Such examples include hepatitis virus (A, B, C, D, E, F, G), HIV, influenza virus, herpes group virus, adenovirus, human polyoma virus, human papilloma virus, human parvovirus, mumps Viral, human rotavirus, enterovirus, Japanese encephalitis virus, dengue virus, rubella virus and HTLV virus infections, Staphylococcus aureus, hemolytic streptococci, pathogenic E. coli, Vibrio parahaemolyticus, Helicobacter pylori, Campylobacter, Vibrio cholerae Due to bacterial infections, parasites, and fungi such as Shigella, Salmonella, Yersinia, Neisseria gonorrhoeae, Listeria monocytogenes, Leptospira, Legionella, Spirocheta, Mycoplasma pneumonia, Rickettsia, Chlamydia, Malaria, Shigella amoeba and pathogenic fungi Disease and the like, but not limited thereto.
[0097]
Furthermore, hereditary diseases, retinoblastoma, Wilms tumor, familial colorectal polyposis, hereditary nonpolyposis colorectal cancer, neurofibromatosis, familial breast cancer, xeroderma pigmentosum, brain tumor, oral cancer, esophageal cancer Tumors such as gastric cancer, colon cancer, liver cancer, pancreatic cancer, lung cancer, thyroid tumor, breast tumor, urological tumor, male organ tumor, female organ tumor, skin tumor, bone / soft tissue tumor, leukemia, lymphoma and solid tumor It is possible to predict the effectiveness of IFN treatment for sex diseases.
[0098]
The pathogenic microorganism that can be detected according to the embodiment of the present invention is not particularly limited. For example, when predicting the effectiveness of IFN treatment, the pathogenic microorganism that induces a disease for which the efficacy of IFN treatment has been confirmed. Examples thereof include HCV, but are not particularly limited as long as they are pathogenic microorganisms related to diseases for which the efficacy of IFN treatment has been confirmed. A pathogenic microorganism is associated with the disease if the pathogenic microorganism is directly or indirectly correlated with the disease or symptoms of the disease.
[0099]
For example, hepatitis virus (A, B, C, D, E, F, G type), HIV, influenza virus, herpes virus, adenovirus, human polyoma virus, human papilloma virus, human parvovirus, mumps virus, human Viruses such as rotavirus, enterovirus, Japanese encephalitis virus, dengue virus, rubella virus and HTLV, Staphylococcus aureus, hemolytic streptococci, pathogenic E. coli, Vibrio parahaemolyticus, Helicobacter pylori, Campylobacter, Vibrio cholerae, Shigella, Salmonella, It can be used to detect bacteria, parasites and fungi such as Yersinia, Neisseria gonorrhoeae, Listeria monocytogenes, Leptospira, Legionella, Spirochete, Mycoplasma pneumonia, Rickettsia, Chlamydia, Malaria, Shigella amoeba and pathogenic fungi.
[0100]
In the above example, an example of analysis regarding IFN therapy and each disease and individual gene is shown. However, the present invention is not limited to this. In some embodiments, for example, the immunity of AIDS (acquired immunodeficiency syndrome; AIDS) is predicted by analyzing the human immunodeficiency virus (hereinafter referred to as HIV) and the individual gene. Is possible. Further, after analyzing the genotype and / or copy of HIV and a specific polymorphism of the individual gene, the following information is obtained from the information obtained, for example, ritonavir (hereinafter referred to as RTV) and saquinavir ( The therapeutic effect of protease inhibitors such as SQV (hereinafter referred to as SQV) and reverse transcription inhibitors such as azidothymidine (hereinafter referred to as AZT) and didanosine (hereinafter referred to as ddl) may be predicted. In some further embodiments, by predicting varicella or zoster susceptibility by analyzing varicella zoster virus (VZV) present in an individual and the individual gene Is possible. Moreover, you may analyze the effectiveness of antiviral drugs, such as acyclovir, from the information regarding the obtained nucleic acid. Alternatively, influenza susceptibility and the effectiveness of antiviral drugs such as Tamifulu may be analyzed by analyzing the influenza virus (influenza virus) genotype and copy number, and the genotype of the individual.
[0101]
When the extracted nucleic acid component is HCV-RNA, HCV-RNA may be used directly as sample nucleic acid, or cDNA may be prepared from HCV-RNA using reverse transcriptase and then used as sample nucleic acid.
[0102]
In addition, when using a probe capable of detecting a genotype, a plurality of probes corresponding to different genotypes can be immobilized on the substrate at the same time, thereby enabling highly accurate detection.
[0103]
The following examples are intended to illustrate the present invention and are not intended to limit the present invention.
[0104]
Example
Example 1 describes a method for extracting nucleic acids from a human subject
1. Extraction of human and viral genomes from patients
About 3 mL of blood from HCV-infected patients was collected in a vacuum blood collection tube containing EDTA2K. Thereafter, nucleic acid extraction was performed using QIAamp DNA Blood Midi Kit (QIAGEN). A reverse transcription reaction was performed on the HCV genomic RNA contained in the obtained extract. This reverse transcription reaction was performed on a part of the extract using Hexa-deoxyribonucleotide mixture (TAKARA) and M-MLV Reverse Transscriptase (LIFE TECHNOLOGIES) at 42 ° C. for 30 minutes.
[0105]
2. Examination of extraction efficiency when extracting HCV genomic RNA using human genome extraction kit
When extracting HCV genomic RNA, serum is usually used instead of whole blood. Further, after removing unnecessary impurities such as proteins as much as possible, HCV genomic RNA is extracted using a guanidine buffer or the like. However, the method according to embodiments of the present invention involves simultaneous extraction with human genomic DNA. Therefore, the extraction efficiency of the HCV genome was examined when human genomic DNA was extracted from whole blood using a human genome extraction kit.
[0106]
A method of extracting HCV genome from 100 μL of serum using SepeGeneRV-R (Sanko Junyaku) as a normal method and a method of extracting HCV genome from whole blood using QIAamp DNA Blood Midi Kit (QIAGEN) were compared. . Regarding the method of extraction from whole blood, reverse transcription reaction for the case where HCV genome is further extracted with QIAamp DNA Blood Midi Kit and then the resulting extract is purified by ethanol precipitation and when ethanol precipitation is not performed. A comparison was also made on the results obtained.
[0107]
Competitive PCR (K. Chayama et al., J. Gastroenterol. Hepatol. 8, S40-44, 1993) using the 5'UTR sequence of the HCV genome was used for evaluation of the extraction efficiency.
[0108]
The results are shown in Table 1.
[0109]
[Table 1]

[0110]
As a result, it was found that the extraction efficiency of HCV RNA was reduced by about 10 times in the method using QIAamp DNA Blood Midi Kit as compared with the normal HCV RNA extraction method. Therefore, when the extraction method using the QIAamp DNA Blood Midi Kit is performed on a sample with a very low amount of virus in the blood, it may become impossible to measure, or the quantification of the virus using a chip, etc. It was thought that it was difficult to use for the purpose. However, in contrast, in a specimen in which the amount of virus in the normal method occupies a concentration of 10 copies / ml or more in whole blood, it is theoretically possible to simultaneously extract DNA and RNA using the QIAamp DNA Blood Midi Kit. It was suggested. That is, it was suggested that viruses can be qualitatively measured using an extract using QIAamp DNA Blood Midi Kit.
[0111]
About the sample obtained by performing reverse transcription reaction as mentioned above, the human genome and the HCV genome were simultaneously amplified.
[0112]
MxAF01 and MxAR02 were used as primers for amplification for the human genome. For the HCV genome, nc2 (K. Chayama et al., J. Gastroenterol. Hepatol. 8, S40-44, 1993, nt27-45) and primer33 (Okamoto et al., Jpn J. Exp. 2 Med. 60. Med. -222, 1990). Amplification was performed in a single tube. The PCR was performed at 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 1 minute for 50 cycles, followed by treatment at 94 ° C. for 4 minutes and then at 72 ° C. for 7 minutes.
[0113]

[0114]
As a result, a product derived from the human genome (size: 610 bp) and a product derived from the HCV genome (size: 300 bp) were both amplified. Therefore, it has been clarified that the process from genome extraction to amplification of a specific region can be achieved by performing the same treatment on both human genomic DNA and HCV genomic RNA.
[0115]
According to such an aspect of the present invention, it is possible to predict a treatment method for a disease easily and in a short time from a sample material extracted from an individual. Further, according to such an aspect, a plurality of pieces of information can be obtained from one sample obtained from an individual at the same time, so that it is possible to prevent the sample from being mixed during inspection. In addition, even when dealing with highly dangerous samples, it is possible to narrow the extent of contamination by such samples compared to conventional methods, and to minimize the risk of contamination accidents due to samples. It is possible.
[0116]
Example 2 describes a DNA chip for assessing the effect of IFN therapy
DNA chip for IFN treatment effect prediction
First, human chromosomal DNA and HCV-RNA were collected from the blood of a patient (human). The chromosomal DNA was obtained by amplifying a 135 bp fragment using the primers of SEQ ID NO: 20 and SEQ ID NO: 21. As HCV-RNA, cDNA was prepared using reverse transcriptase.
[0117]
The nucleic acid sample obtained by the above steps was subjected to FITC labeling using a Pharmacia kit.
[0118]
FIG. 1 shows a schematic diagram of the DNA chip of this example. On a substrate 6 made of a slide glass coated with polylysine, the second probe is a DNA probe of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 and the first probe is SEQ ID NO: 5, SEQ ID NO: 6 The DNA probe of SEQ ID NO: 7 (the first probe and the second probe in FIG. 1 are indicated by 1 to 5) was spotted 200 nL at a time and dried. Thereafter, UV irradiation was performed to fix the probe on the substrate 6. The base sequences of SEQ ID NOs: 22 to 25 are fragments containing a SNP site at position 455 of the base sequences of SEQ ID NOs: 16 to 19, respectively. The nucleotide sequences of SEQ ID NOs: 5 to 7 are probes for detecting HCV virus type 1, 2, and 3 genotypes, respectively.
[0119]
The FITC-labeled nucleic acid sample was dissolved in 2 × SSC-1 mmol / L EDTA solution. This was put in a reaction chamber with a capacity of 20 μL and covered with a probe-immobilized slide glass. After the reaction at 50 ° C. for 12 hours, the plate was washed twice with 0.2 × SSC-1 mmol / LEDTA solution, and the fluorescence on the slide glass was measured.
[0120]
As a result, significant fluorescence was observed only from the spot where the probe of SEQ ID NO: 22 was immobilized. It was considered that the genotype of MxA-88 in the human-derived nucleic acid contained in this sample material was T / T homotype. Moreover, the virus contained in this sample substance was determined to be type 2. Furthermore, from the fluorescence intensity obtained, the virus concentration was 10⁶It was estimated to be less than copy / mL. Therefore, IFN was expected to work effectively for patients from whom this sample material was collected.
[0121]
Example 3 describes a PNA chip for evaluating the effects of IFN therapy
PNA chip for IFN treatment effect prediction
First, human chromosomal DNA and HCV-RNA were collected from the blood of a patient (human). Next, a 135 bp fragment was amplified from the chromosomal DNA using the primers of SEQ ID NOs: 20 and 21. As HCV-RNA, cDNA was prepared using reverse transcriptase. Using the obtained cDNA as a template, primers of SEQ ID NO: 8 (this is sense), SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 (these are antisense) were used. PCR reaction was performed.
[0122]
FIG. 2 shows a schematic diagram of the PNA chip of this embodiment. SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 as a second probe in which cysteine is modified at the N-terminus on a probe-immobilized chip in which ten gold electrodes 13 are patterned on a glass substrate 12 Each PNA probe of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36 was spotted 200 nL each and left for 1 hour.
[0123]
The nucleotide sequences of SEQ ID NOs: 27 to 30 are fragments containing a SNP site at position 455 of the nucleotide sequences of SEQ ID NOs: 16 to 19, respectively. The nucleotide sequences of SEQ ID NOs: 32 to 36 are probes for detecting HCV virus type 1a, 1b, 2a, 2b, 3a genotypes, respectively.
[0124]
Next, the sample was dissolved in 2 × SSC-1 mmol / L EDTA solution. This was placed in a reaction chamber with a capacity of 20 μL and covered with a probe-immobilized tip. After reacting at 50 ° C. for 1 hour, it was washed twice with 0.2 × SSC-1 mmol / LEDTA solution. Next, 10 μmol / L Hoechst 33258 solution was added dropwise thereto. Separately, a counter electrode and a reference electrode were installed, and the oxidation current from Hoechst 33258 when a voltage was applied between the gold electrode and the counter electrode was measured.
[0125]
As a result, a significant current change was observed from the gold electrode 13 on which the probes of SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 34 were immobilized. The genotype of MxA-88 in the human-derived nucleic acid contained in this sample material was G / T heterozygous. Moreover, the virus contained in this sample substance was determined to be type 2a. Therefore, IFN was expected to work effectively for patients from whom this sample material was collected.
[0126]
Example 4
In accordance with the present invention, from the genotype of codon 54 of the infected HCV virus type, MxA-88, MxA-123, MBL-221 and MBL collagen-like domain of the infected person, hepatitis C infection type C It is possible to predict the effectiveness of IFN therapy in people with hepatitis infection.
[0127]
An example of a method for predicting the effect of IFN therapy from the virus type, MBL and MxA genotype will be described below with reference to FIG.
[0128]
In step 3a, the practitioner collects a sample, such as a blood sample, from the individual to be administered IFN and starts prediction. The collected sample is subjected to treatment such as purification and extraction as necessary, and then the virus type, and the MBL and MxA genotypes, that is, the MxA-88 and MxA-123 genotypes, MBL-221. Genotype, the genotype of codon 54 of the collagen-like domain of MBL, and go to step 3b.
[0129]
In step 3b, if type 1 is included in the virus type determined in step 3a, it is determined to proceed to step 3c, and if only type 2 is determined, it is determined to proceed to (3d).
[0130]
In step 3c, based on the genotypes of MBL and MxA determined in step 3a, the table 2 in which the genotype and the effect of IFN are associated is searched to extract the corresponding IFN effect, thereby the effectiveness of the IFN therapy Predict the sex and finish the whole prediction process.
[0131]
In step 3d, based on the genotypes of MBL and MxA determined in step 3a, the table 3 in which the genotype and the effect of IFN are associated is searched, and the corresponding IFN effect is extracted, thereby the effectiveness of the IFN therapy Is predicted, and the entire prediction process is completed.
[0132]
The table used here is information that associates genotypes with IFN susceptibility. Each table is a table corresponding to each table number. Each table used in the above method is given as an example. The table and table used here may be any table in which each genotype is associated with the effect of IFN. For example, Table 2 is a table showing the relationship between genotype and IFN effects in patients infected with type 1 HCV. On the other hand, Table 3 is a table showing the relationship between genotype and IFN effect in patients infected with type 2 HCV. Further, the table used may be a table including only any one of the components, or may be a table composed of other combinations of components. In the above example, Tables 2 and 3 are used as the tables referred to in Step 3c and Step 3d, respectively, and two tables prepared by selecting necessary items in advance for each step are used. However, one table prepared by combining these may be used. Or the table | surface which consists of a component of another combination may be sufficient. For the selection of components, for example, in the table used in step 3c, information indicating a relationship between genotype and effective or ineffective in a person infected with type 1 HCV is selected, and in step 3d, type 2 HCV infection is selected. What is necessary is just to select the information which shows the relationship between a genotype in a person and a remarkable or ineffective. However, the present invention is not limited to this, and selection of components may be arbitrarily performed by a practitioner.
[0133]
Moreover, the numerical value as a component described in Tables 2 and 3 used here is an example of a display indicating information in which a genotype and IFN sensitivity or IFN effect are associated with each other. It is not limited to. In other words, if it is a notation that can substantially indicate the correlation between genotype and effective or ineffective, for example, “○”, “×”, and “△” are simplified. A numerical score (for example, 1, 2, 3, 4, 5, etc.) may be used.
[0134]
[Table 2-1]

[0135]
[0136]
[Table 2-2]

[0137]
[Table 3]

[0138]
By examining many items according to the above-described aspect of the present invention, more accurate prediction is possible.
[0139]
By using the method according to the above-described embodiment of the present invention and the probe-immobilized chip, it is possible to easily collect target nucleic acid information from a sample substance in a short time and accurately.
[0140]
Further, the above-described embodiment is an example of the present invention, and is not described for the purpose of limiting the present invention.
[0141]
Additional benefits and modifications will be readily apparent to those skilled in the art. Accordingly, the invention in its broader aspects is not limited to the details and representative embodiments shown and described herein. Accordingly, various modifications may be made without departing from the spirit or general inventive concept as defined by the appended claims and their equivalents.
[0142]
【The invention's effect】
According to the present invention, there has been provided a method capable of easily recovering target nucleic acid information from a sample substance in a short time and accurately.
[0143]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a schematic view of a DNA chip according to the present example.
FIG. 2 is a schematic diagram of a PNA chip according to the present embodiment.
FIG. 3 is a flowchart showing a method according to the embodiment.
[Explanation of symbols]
1 ... DNA probe
2 ... DNA probe
3 ... DNA probe
4 ... DNA probe
5 ... DNA probe
6 ... Substrate
7 PNA probe
8 PNA probe
9 ... PNA probe
10 ... PNA probe
11 ... PNA probe
12 ... Substrate
13 ... Gold electrode
14 ... Connection part

Claims (5)

In a method for detecting a first nucleic acid which is a specific nucleotide sequence of a nucleic acid of the hepatitis C virus present in an individual exposed to a particular disease, the second nucleic acid is a specific sequence of nucleic acid of said individual there are, wherein said specific disease is associated with the hepatitis C virus, without nucleic acid nucleic acid of the individual and the hepatitis C virus is also separated either nucleic acids, QIAamp from blood collected from the individual (Registered trademark) A method that is extracted using DNA Blood Kit and further comprises:
(A) amplifying the extract of nucleic acid from the individual, and here, the steps from extraction to amplification of the nucleic acid are performed simultaneously in the same process without separating any nucleic acid;
(B) reacting the amplified product obtained by amplification in (a) with a probe-immobilized substrate comprising a first probe and a second probe, wherein the first probe detects the presence of the first nucleic acid of the hepatitis C virus, and the second probe detects the presence of a second nucleic acid of said individual;
(C) As a result of the reaction of (b), detecting the presence of the first nucleic acid by detecting the presence of the nucleic acid bound to the first probe, and binding to the second probe Detecting the presence of a second nucleic acid by detecting the presence of the nucleic acid; Second nucleic acids associated with responsiveness to treatment of the particular disease, and the presence of the second nucleic acid both of the first nucleic acid and the individual of the hepatitis C virus of the individual, for the treatment of the disease The method of claim 1, which correlates with reactivity.   The method of claim 1, wherein the individual is a human. The method according to claim 1, wherein the blood is whole blood, the nucleic acid of said individual is derived from a nucleic acid contained in the blood, the hepatitis C virus are derived from serum of the blood A method comprising simultaneously detecting these nucleic acids without isolation of these nucleic acids. Wherein the nucleic acid of the individual is human genomic nucleic acid, and the method of claim 4 wherein the nucleic acid of the hepatitis C virus is a hepatitis C virus genomic nucleic acid.

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