JP2009201361A - Probe for catching hepatitis b virus dna and solid phase support on which probe is immobilized - Google Patents
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本発明は、Ｂ型肝炎ウイルス（本願明細書において「ＨＢＶ」ともいう。）ＤＮＡ捕捉用プローブ、および該プローブが固定化された固相担体に関する。 The present invention relates to a hepatitis B virus (also referred to herein as “HBV”) DNA capture probe and a solid phase carrier on which the probe is immobilized.
ＨＢＶの定量は、ＨＢＶ感染者の治療効果を観察するため、ならびに、輸血用血液あるいは移植用臓器を介したレシピエントのＨＢＶ感染を防ぐためのドナーのＨＢＶ感染の有無をチェックするための手段として重要である。 Quantification of HBV is a means for observing the therapeutic effect of HBV-infected persons, and as a means for checking the presence or absence of donor HBV infection to prevent recipient HBV infection via blood for transfusion or transplanted organs. is important.
血清等の試料中のＨＢＶを定量する方法としては、例えば、試料中のＨＢＶ−ＤＮＡを磁性粒子などの担体を用いて捕捉し、捕捉したＨＢＶ−ＤＮＡをＴａｑＭａｎ（商標）法等のリアルタイム検出ＰＣＲ法で定量する方法が挙げられる。この場合、ウイルス濃度が極めて少ない検体であっても、ＨＢＶ−ＤＮＡを確実に捕捉できることが好ましい。 As a method for quantifying HBV in a sample such as serum, for example, HBV-DNA in the sample is captured using a carrier such as magnetic particles, and the captured HBV-DNA is detected by real-time detection PCR such as TaqMan (trademark) method. The method of quantifying by a method is mentioned. In this case, it is preferable that HBV-DNA can be reliably captured even with a specimen having a very low virus concentration.
本発明は、ウイルス濃度が極めて少ない検体であってもＨＢＶ−ＤＮＡを確実に捕捉できるＢ型肝炎ウイルスＤＮＡ捕捉用プローブおよびプローブ固定化固相担体を提供する。 The present invention provides a probe for capturing hepatitis B virus DNA and a probe-immobilized solid phase carrier capable of reliably capturing HBV-DNA even in a specimen having a very low virus concentration.
本発明の一態様にかかるＢ型肝炎ウイルスＤＮＡ捕捉用プローブは、配列番号１〜１２で示されるいずれかの配列から選択される連続する１０塩基以上に対して８０％以上の配列相同性を有する塩基配列を含む塩基数１５〜８０のオリゴヌクレオチドである。 The probe for capturing hepatitis B virus DNA according to one embodiment of the present invention has a sequence homology of 80% or more with respect to 10 or more consecutive bases selected from any sequence shown in SEQ ID NOs: 1 to 12. It is an oligonucleotide having 15 to 80 bases containing a base sequence.
本発明の一態様にかかるプローブ固定化固相担体は、上記Ｂ型肝炎ウイルスＤＮＡ捕捉用プローブが固定化されている。 In the probe-immobilized solid phase carrier according to one embodiment of the present invention, the above-mentioned probe for capturing hepatitis B virus DNA is immobilized.
上記Ｂ型肝炎ウイルスＤＮＡ捕捉用プローブを用いることにより、ウイルス濃度が極めて少ない検体においても、ＨＢＶ−ＤＮＡを確実に捕捉することができる。 By using the above-mentioned probe for capturing hepatitis B virus DNA, HBV-DNA can be reliably captured even in a specimen having a very low virus concentration.
より具体的には、上記Ｂ型肝炎ウイルスＤＮＡ捕捉用プローブが固定化されたプローブ固定化固相担体を用いてＨＢＶ−ＤＮＡを捕捉することにより、ウイルス濃度が極めて少ない検体であっても、ＨＢＶ−ＤＮＡを確実に捕捉することができる。 More specifically, by capturing HBV-DNA using a probe-immobilized solid phase carrier on which the probe for capturing hepatitis B virus DNA is immobilized, even if the specimen has a very low virus concentration, HBV -The DNA can be captured reliably.
１．Ｂ型肝炎ウイルスＤＮＡ捕捉用プローブ
本発明の一実施形態にかかるＢ型肝炎ウイルスＤＮＡ捕捉用プローブ（以下「ＨＢＶ−ＤＮＡ捕捉用プローブ」ともいう。）は、配列番号１〜１２で示されるいずれかの配列から選択される連続する１０塩基以上に対して８０％以上（好ましくは９０％以上）の配列相同性を有する塩基配列を含む塩基数１５〜８０（好ましくは２５〜４５）のオリゴヌクレオチドである。ＨＢＶ−ＤＮＡ捕捉用プローブは、市販のものを用いても良いし、公知の合成方法により合成してもよい。
プローブＢＡ：５’−ＴＣＣＴＡＧＧＡＣＣＣＣＴＧＣＴＣＧＴＧＴＴＡＣＡＧＧＣＧＧＧＧＴＴＴＴＴＣＴ−３’（配列番号１）
プローブＢＢ：５’−ＣＣＣＣＴＧＣＴＣＧＴＧＴＴＡＣＡＧＧＣＧＧＧＧＴＴＴＴＴＣＴＴＧＴＴＧＡＣＡＡ−３’（配列番号２）
プローブＢＣ：５’−ＧＴＣＴＡＧＡＣＴＣＧＴＧＧＴＧＧＡＣＴＴＣＴＣＴＣＡＡＴＴＴＴＣＴＡＧＧＧＧ−３’（配列番号３）
プローブＢＤ：５’−ＴＡＴＣＧＣＴＧＧＡＴＧＴＧＴＣＴＧＣＧＧＣＧＴＴＴＴＡＴＣＡＴＡＴＴＣＣＴＣＴ−３’（配列番号４）
プローブＢＥ：５’−ＣＡＴＣＣＴＧＣＴＧＣＴＡＴＧＣＣＴＣＡＴＣＴＴＣＴＴＧＴＴＧＧＴＴＣＴＴＣＴＧＧＡ−３’（配列番号５）
プローブＢＦ：５’−ＣＣＴＡＴＧＧＧＡＧＴＧＧＧＣＣＴＣＡＧＴＣＣＧＴＴＴＣＴＣＴＴＧＧＣＴＣＡＧＴＴＴＡＣＴＡＧ−３’（配列番号６）
プローブＢＧ：５’−ＧＧＣＴＴＴＣＣＣＣＣＡＣＴＧＴＴＴＧＧＣＴＴＴＣＡＧＣＴＡＴＡＴＧＧＡＴＧＡＴ−３’（配列番号７）
プローブＢＨ：５’−ＴＧＣＣＡＡＧＴＧＴＴＴＧＣＴＧＡＣＧＣＡＡＣＣＣＣＣＡＣＴＧＧＣＴＧＧＧＧＣＴＴ−３’（配列番号８）
プローブＢＩ：５’−ＣＴＣＣＴＣＴＧＣＣＧＡＴＣＣＡＴＡＣＴＧＣＧＧＡＡＣＴＣＣＴＡＧＣＣＧＣＴＴＧ−３’（配列番号９）
プローブＢＪ：５’−ＣＣＧＴＧＴＧＣＡＣＴＴＣＧＣＴＴＣＡＣＣＴＣＴＧＣＡＣＧＴＴＧＣＡＴＧＧＡＧＡ−３’（配列番号１０）
プローブＢＫ：５’−ＴＧＴＴＣＡＡＧＣＣＴＣＣＡＡＧＣＴＧＴＧＣＣＴＴＧＧＧＴＧＧＣＴＴＴＧＧＧＧＣ−３’（配列番号１１）
プローブＢＬ：５’−ＧＣＡＧＧＴＣＣＣＣＴＡＧＡＡＧＡＡＧＡＡＣＴＣＣＣＴＣＧＣＣＴＣＧＣＡＧＡＣＧＡＡＧ−３’（配列番号１２）
ＨＢＶ−ＤＮＡ捕捉用プローブを例えばプローブ固定化固相担体（例えば磁性粒子）の表面に固定化して、検体を含む試料中で該捕捉用プローブとＨＢＶ−ＤＮＡとをハイブリダイズさせることにより、極めて低濃度にて検体中のＨＢＶ−ＤＮＡを捕捉することができる。検出対象となるＨＢＶ−ＤＮＡは、ＨＢＶを含有する疑いのある検体、例えば、血清、血漿等の体液中に含まれているものである。
1. Probe for capturing hepatitis B virus DNA The probe for capturing hepatitis B virus DNA according to one embodiment of the present invention (hereinafter also referred to as “probe for capturing HBV-DNA”) is any one of SEQ ID NOs: 1-12. An oligonucleotide having 15 to 80 bases (preferably 25 to 45 bases) containing a base sequence having a sequence homology of 80% or more (preferably 90% or more) with respect to 10 or more consecutive bases selected from is there. A commercially available probe may be used for the HBV-DNA capture probe, or it may be synthesized by a known synthesis method.
Probe BA: 5′-TCCTAGGACCCCTGCTCGGTGTTACAGGCGGGGTTTTTT-3 ′ (SEQ ID NO: 1)
Probe BB: 5'-CCCCCTGCTCTGGTTACAGGCGGGGTTTTCTTTGTCACA-3 '(SEQ ID NO: 2)
Probe BC: 5′-GTCTAGACTCGTGGGTGACTTCTCTCAATTTTCTAGGGGG-3 ′ (SEQ ID NO: 3)
Probe BD: 5′-TATCGCTGGATGTGTCTGCGGGCGTTTTATCATATTCCCTCT-3 ′ (SEQ ID NO: 4)
Probe BE: 5′-CATCCTGCTGCTATGCCCTCATCTTCTTGTTGGTTCTCTGGA-3 ′ (SEQ ID NO: 5)
Probe BF: 5′-CCTATGGGAGTGGGCCTCCAGCTCGTTTCTCTTGGCTCAGTTTACTAG-3 ′ (SEQ ID NO: 6)
Probe BG: 5′-GGCTTTCCCCCACTGTTTGGCTTTCAGCTATATGGATGAT-3 ′ (SEQ ID NO: 7)
Probe BH: 5′-TGCCAAGTGTTGCTGGACGCAACCCCCCACTGGCTGGGGCTT-3 ′ (SEQ ID NO: 8)
Probe BI: 5′-CTCCTCTGCGCATCATCATGCCGGAACTCCTAGCCGCTGTG-3 ′ (SEQ ID NO: 9)
Probe BJ: 5'-CCGTGGTCACTTCGCTTTCACCTCTGCACGTTGCATGAGAGA-3 '(SEQ ID NO: 10)
Probe BK: 5'-TGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGGGC-3 '(SEQ ID NO: 11)
Probe BL: 5′-GCAGGTCCCCTAGAAGAAGAACTCCCTCGCCCTGCAGACGAAG-3 ′ (SEQ ID NO: 12)
By immobilizing the HBV-DNA capture probe on, for example, the surface of a probe-immobilized solid phase carrier (for example, magnetic particles) and hybridizing the capture probe and HBV-DNA in a sample containing a specimen, extremely low HBV-DNA in the sample can be captured at a concentration. The HBV-DNA to be detected is contained in a sample suspected of containing HBV, for example, a body fluid such as serum or plasma.
また、ＨＢＶ−ＤＮＡ捕捉用プローブを用いて捕捉されたＨＢＶ−ＤＮＡは、例えば、リアルタイム検出ＰＣＲ法によって定量することができる。例えば、ＨＢＶ−ＤＮＡを捕捉した磁性粒子と、フォワード側プライマーと、リバース側プライマーと、蛍光プローブ（３’末端がドナー蛍光色素で標識化されかつ５’末端がアクセプター蛍光色素で標識化されたオリゴヌクレオチド（ＴａｑＭａｎ（商標）プローブ））とを含む系内でＰＣＲ反応を行ない、得られた蛍光強度に基づいて検体中のＢ型肝炎ウイルスＤＮＡ量を測定することができる。このリアルタイム検出ＰＣＲ法自体は公知であり、そのための装置及びキットも市販されているので、このような市販の装置及びキットを用いてリアルタイム検出ＰＣＲ法を行なうことができる。 In addition, HBV-DNA captured using a probe for capturing HBV-DNA can be quantified by, for example, a real-time detection PCR method. For example, magnetic particles capturing HBV-DNA, a forward primer, a reverse primer, and a fluorescent probe (an oligo having a 3 ′ end labeled with a donor fluorescent dye and a 5 ′ end labeled with an acceptor fluorescent dye) A PCR reaction is performed in a system containing nucleotides (TaqMan ™ probe), and the amount of hepatitis B virus DNA in the sample can be measured based on the obtained fluorescence intensity. This real-time detection PCR method itself is known, and devices and kits therefor are also commercially available. Therefore, the real-time detection PCR method can be performed using such commercially available devices and kits.
２．プローブ固定化固相担体
本発明の一実施形態にかかるプローブ固定化固相担体は、上記ＨＢＶ−ＤＮＡ捕捉用プローブが固定化されている。
2. Probe-immobilized solid phase carrier The probe-immobilized solid phase carrier according to one embodiment of the present invention has the HBV-DNA capture probe immobilized thereon.
固相担体としては、例えばクロマトグラフィ担体や診断薬として使用可能な担体粒子、例えばＤＮＡチップやＤＮＡマイクロアレイとして使用可能なチップ（基板）などが挙げられる。 Examples of the solid phase carrier include carrier particles that can be used as a chromatography carrier or a diagnostic agent, such as a chip (substrate) that can be used as a DNA chip or a DNA microarray.
本実施形態にかかるプローブ固定化固相担体は、ＨＢＶを検出するための固相担体（より具体的には、ＨＢＶ−ＤＮＡ捕捉用プローブが表面に固定化された磁性粒子）であることがより好ましい。ＨＢＶ−ＤＮＡ捕捉用プローブを磁性粒子に固定化する方法としては、例えば、３’末端がアミノ基で修飾されたＨＢＶ−ＤＮＡ捕捉用プローブをカルボキシル基含有磁性粒子と反応させる方法や、３’末端がビオチン化されたＨＢＶ−ＤＮＡ捕捉用プローブをアビジン化磁性粒子と反応させる方法が挙げられる。 The probe-immobilized solid phase carrier according to the present embodiment is more preferably a solid phase carrier for detecting HBV (more specifically, a magnetic particle having a probe for capturing HBV-DNA immobilized on the surface). preferable. Examples of the method for immobilizing the HBV-DNA capture probe on the magnetic particle include a method of reacting the HBV-DNA capture probe whose 3 ′ end is modified with an amino group with a carboxyl group-containing magnetic particle, or the 3 ′ end. And a biotinylated HBV-DNA capture probe is reacted with avidinized magnetic particles.
３．実施例
以下、本発明を実施例に基づきより具体的に説明する。もっとも、本発明は下記実施例に限定されるものではない。
3. EXAMPLES Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
３．１．ＨＢＶ−ＤＮＡ捕捉用プローブを有する磁性粒子の作製
３．１．１．ＨＢＶ−ＤＮＡ捕捉用プローブ
ＨＢＶ−ＤＮＡ捕捉用プローブとして、上記配列番号１〜１２で示される配列ＢＡ〜ＢＬを用いた。
3.1. Production of magnetic particles having a probe for capturing HBV-DNA 3.1.1. HBV-DNA capture probes The sequences BA to BL shown in SEQ ID NOs: 1 to 12 were used as the HBV-DNA capture probes.
３．１．２．ＨＢＶ−ＤＮＡ捕捉用プローブの磁性粒子への固定化
３’末端がアミノ基で修飾されたＨＢＶ−ＤＮＡ捕捉用プローブをカルボキシル基含有磁性粒子と反応させる方法および３’末端がビオチン化されたＨＢＶ−ＤＮＡ捕捉用プローブをアビジン化磁性粒子と反応させる方法の２通りの方法により、ＨＢＶ−ＤＮＡ捕捉用プローブが固定化された磁性粒子を作製した。
3.1.2. Immobilization of HBV-DNA capture probe to magnetic particles Method of reacting HBV-DNA capture probe modified with an amino group at the 3 'end with a carboxyl group-containing magnetic particle, and HBV- 3' end biotinylated Magnetic particles on which the HBV-DNA capture probe was immobilized were prepared by two methods of reacting the DNA capture probe with avidinized magnetic particles.
（ａ）３’末端がアミノ基で修飾されたＨＢＶ−ＤＮＡ捕捉用プローブをカルボキシル基含有磁性粒子と反応させることによる、ＨＢＶ−ＤＮＡ捕捉用プローブの磁性粒子への固定化
カルボキシル基含有磁性粒子（Ｍａｇｎｏｓｐｈｅｒｅ（商標） Ｍ３００／Ｃａｒｂｏｘｙｌ（ＪＳＲ株式会社製））１ｗｔ％分散液５００μｌをチューブに取り、磁気スタンドにて磁気分離し、上澄みを除去した。０．０００１Ｎ塩酸により３回洗浄した後、５００μｌの０．０００１Ｎ塩酸に分散し、３’末端がアミノ化されたＨＢＶ−ＤＮＡ捕捉用プローブ（プローブＢＡ）１００μＭ溶液を５μｌおよびＥＤＣ（１−エチル−３−（３−ジメチルアミノプロピル）カルボジイミド塩酸塩）０．２５ｍｇを加え、１０℃に設定した恒温槽で１５時間攪拌した。その後、磁気スタンドにて磁気分離し、上澄みを除去した。０．５ｍＭＥＤＴＡおよび１．０ＭＮａＣｌを含む５ｍＭｔｒｉｓ−ＨＣｌ（ｐＨ７．４）にて３回洗浄後５０μｌの５ｍＭｔｒｉｓ−ＨＣｌバッファーに分散して、アミド結合を介してＨＢＶ−ＤＮＡ捕捉用プローブ（プローブＢＡ）が結合されたプローブ固定化磁性粒子（ａｍ−ＢＡ）の分散液（１０重量％）を得た。
(A) Immobilization of an HBV-DNA capture probe to a magnetic particle by reacting an HBV-DNA capture probe whose 3 ′ end is modified with an amino group with the carboxyl group-containing magnetic particle Magnosphere (trademark) M300 / Carboxyl (manufactured by JSR Corporation)) 1 wt% dispersion 500 μl was taken in a tube, magnetically separated by a magnetic stand, and the supernatant was removed. After washing three times with 0.0001N hydrochloric acid, 5 μl of an HBV-DNA capture probe (probe BA) 100 μM solution dispersed in 500 μl of 0.0001N hydrochloric acid and aminated at the 3 ′ end and EDC (1-ethyl- 0.25 mg of 3- (3-dimethylaminopropyl) carbodiimide hydrochloride) was added, and the mixture was stirred for 15 hours in a thermostatic bath set at 10 ° C. Thereafter, magnetic separation was performed with a magnetic stand, and the supernatant was removed. After washing three times with 5 mM tris-HCl (pH 7.4) containing 0.5 mM EDTA and 1.0 M NaCl, the mixture was dispersed in 50 μl of 5 mM tris-HCl buffer, and a probe for capturing HBV-DNA (probe BA) was obtained via an amide bond. A dispersion (10% by weight) of bonded probe-immobilized magnetic particles (am-BA) was obtained.
また、アミド結合を介してＨＢＶ−ＤＮＡ捕捉用プローブ（プローブＢＢ、ＢＣ、ＢＤ、ＢＥ、ＢＦ、ＢＧ、ＢＨ、ＢＩ、ＢＪ、ＢＫ、ＢＬ）が結合されたプローブ固定化磁性粒子（ａｍ−ＢＢ、ａｍ−ＢＣ、ａｍ−ＢＤ、ａｍ−ＢＥ、ａｍ−ＢＦ、ａｍ−ＢＧ、ａｍ−ＢＨ、ａｍ−ＢＩ、ａｍ−ＢＪ、ａｍ−ＢＫ、ａｍ−ＢＬ）を同様の方法にて得た。 In addition, probe-immobilized magnetic particles (am-BB) to which probes for capturing HBV-DNA (probes BB, BC, BD, BE, BF, BG, BH, BI, BJ, BK, BL) are bonded via an amide bond. , Am-BC, am-BD, am-BE, am-BF, am-BG, am-BH, am-BI, am-BJ, am-BK, am-BL) were obtained in the same manner.
（ｂ）３’末端がビオチン化されたＨＢＶ−ＤＮＡ捕捉用プローブをアビジン化磁性粒子と反応させることによる、ＨＢＶ−ＤＮＡ捕捉用プローブの磁性粒子への固定化
アビジン化磁性粒子（Ｍａｇｎｏｓｐｈｅｒｅ（商標） Ｍ３００／Ｓｔｒｅｐｔａｖｉｄｉｎ（ＪＳＲ株式会社製））１ｗｔ％分散液５００μｌをチューブに取り、磁気スタンドにて磁気分離し、上澄みを除去した。０．５ｍＭＥＤＴＡおよび１．０ＭＮａＣｌを含む５ｍＭｔｒｉｓ−ＨＣｌ（ｐＨ７．４）にて３回洗浄した後、５００μｌの０．５ｍＭＥＤＴＡおよび１．０ＭＮａＣｌを含む５ｍＭｔｒｉｓ−ＨＣｌ（ｐＨ７．４）に分散し、３’末端がビオチン化されたＨＢＶ−ＤＮＡ捕捉用プローブ（プローブＢＡ）１００μＭ溶液を５μｌ加え、室温下３０分間攪拌した。その後、磁気スタンドにて磁気分離し、上澄みを除去した。０．５ｍＭＥＤＴＡおよび１．０ＭＮａＣｌを含む５ｍＭｔｒｉｓ−ＨＣｌ（ｐＨ７．４）にて３回洗浄した後、５０μｌの５ｍＭｔｒｉｓ−ＨＣｌバッファーに分散し、ＨＢＶ−ＤＮＡ捕捉用プローブ（プローブＢＡ）がビオチン−アビジン結合により結合した磁性粒子（ｂｉ−ＢＡ）分散液（１０重量％）を得た。
(B) Immobilization of HBV-DNA capture probe to magnetic particles by reacting HBV-DNA capture probe with biotinylated 3 ′ end with avidinized magnetic particles (Magnosphere ™) 500 μl of 1 wt% dispersion of M300 / Streptavidin (manufactured by JSR Corporation) was placed in a tube and magnetically separated by a magnetic stand, and the supernatant was removed. After washing 3 times with 5 mM tris-HCl (pH 7.4) containing 0.5 mM EDTA and 1.0 M NaCl, it was dispersed in 500 μl of 5 mM tris-HCl (pH 7.4) containing 0.5 mM EDTA and 1.0 M NaCl. 5 μl of 100 μM HBV-DNA capture probe (probe BA) with biotinylated ends was added and stirred at room temperature for 30 minutes. Thereafter, magnetic separation was performed with a magnetic stand, and the supernatant was removed. After washing three times with 5 mM tris-HCl (pH 7.4) containing 0.5 mM EDTA and 1.0 M NaCl, the mixture was dispersed in 50 μl of 5 mM tris-HCl buffer, and the HBV-DNA capture probe (probe BA) was bound to biotin-avidin. To obtain a magnetic particle (bi-BA) dispersion (10 wt%) bonded thereto.
また、プローブＢＢ、ＢＣ、ＢＤ、ＢＥ、ＢＦ、ＢＧ、ＢＨ、ＢＩ、ＢＪ、ＢＫ、ＢＬがそれぞれビオチン−アビジン結合により結合したプローブ固定化磁性粒子（ｂｉ−ＢＢ、ｂｉ−ＢＣ、ｂｉ−ＢＤ、ｂｉ−ＢＥ、ｂｉ−ＢＦ、ｂｉ−ＢＧ、ｂｉ−ＢＨ、ｂｉ−ＢＩ、ｂｉ−ＢＪ、ｂｉ−ＢＫ、ｂｉ−ＢＬ）を同様の方法で得た。 Probe-immobilized magnetic particles (bi-BB, bi-BC, bi-BD) in which probes BB, BC, BD, BE, BF, BG, BH, BI, BJ, BK, and BL are bonded by biotin-avidin bonds, respectively. , Bi-BE, bi-BF, bi-BG, bi-BH, bi-BI, bi-BJ, bi-BK, bi-BL) were obtained in the same manner.
３．２．ＨＢＶ−ＤＮＡ捕捉用プローブ固定化磁性粒子によるＨＢＶ−ＤＮＡの抽出および検出
３．２．１．ＨＢＶ−ＤＮＡの抽出
上記工程で調製されたＨＢＶ−ＤＮＡ捕捉用プローブ固定化磁性粒子を用いて、ヒト血漿中からＨＢＶ−ＤＮＡを抽出した。濃度既知のＨＢＶを含有するヒト血漿の検体を、正常なヒト血漿を用いて希釈して、１０倍ずつの希釈列を調製した。これらの希釈された血漿０．１ｍｌずつからＨＢＶ−ＤＮＡを抽出した。
3.2. Extraction and detection of HBV-DNA with probe-immobilized magnetic particles for HBV-DNA capture 3.2.1. Extraction of HBV-DNA HBV-DNA was extracted from human plasma using probe-immobilized magnetic particles for capturing HBV-DNA prepared in the above step. A specimen of human plasma containing HBV with a known concentration was diluted with normal human plasma to prepare a 10-fold dilution series. HBV-DNA was extracted from 0.1 ml of each diluted plasma.
まず、血漿０．１ｍｌをチューブにとり、検体希釈液（１％Ｎ−ラウロイルサルコシン酸ナトリウム、１０ｍＭＥＤＴＡ、２００ｍＭＮａＣｌ、２％ ２−メルカプトエタノール、２００ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５））３８０μｌ、および１０％デキストランＴ２０００（アマシャム社製）５μｌを加えて攪拌した後、５５℃で３０分間静置した。次に、８Ｍグアニジン塩酸塩２５０μｌを加え攪拌した後５５℃で１５分間静置した。その後、９５℃で１０分間静置した後室温まで冷却した。次に上記工程で調製されたＨＢＶ−ＤＮＡ捕捉用プローブを有する磁性粒子分散液（１０重量％）１０μｌを加え、室温で１０分間攪拌した後、磁気スタンドにて磁気分離し液体を除去した。その後、０．０１％ｔｒｉｔｏｎＸ−１００および０．１ＭＮａＣｌを含む１０ｍＭｔｒｉｓ−ＨＣｌ（ｐＨ７．５）溶液にて粒子を１回洗浄した後、４０μｌのＤＮＡｓｅ−ｆｒｅｅ水に分散し、ＨＢＶ−ＤＮＡを含む磁性粒子分散液を得た。この磁性粒子分散液をテンプレートとして、そのままリアルタイム検出ＰＣＲ増幅反応処理にかけた。 First, 0.1 ml of plasma was placed in a tube, and 380 μl of a sample diluent (1% N-lauroyl sarcosyl sodium salt, 10 mM EDTA, 200 mM NaCl, 2% 2-mercaptoethanol, 200 mM Tris-HCl (pH 7.5)), and 10% dextran. After adding 5 μl of T2000 (Amersham) and stirring, the mixture was allowed to stand at 55 ° C. for 30 minutes. Next, 250 μl of 8M guanidine hydrochloride was added and stirred, and then allowed to stand at 55 ° C. for 15 minutes. Then, after leaving still at 95 degreeC for 10 minutes, it cooled to room temperature. Next, 10 μl of a magnetic particle dispersion (10 wt%) having a probe for capturing HBV-DNA prepared in the above step was added and stirred for 10 minutes at room temperature, followed by magnetic separation on a magnetic stand to remove the liquid. Thereafter, the particles were washed once with a 10 mM tris-HCl (pH 7.5) solution containing 0.01% triton X-100 and 0.1 M NaCl, dispersed in 40 μl of DNAse-free water, and magnetic particles containing HBV-DNA. A particle dispersion was obtained. Using this magnetic particle dispersion as a template, it was directly subjected to a real-time detection PCR amplification reaction treatment.
３．２．２．リアルタイム検出ＰＣＲによる増幅および検出
反応チューブ一本当り下記表１に示す反応液を調製し、上記で抽出されたＨＢＶ−ＤＮＡを含む磁性粒子分散液４０μｌを加えてリアルタイム検出ＰＣＲを行った。
3.2.2. Amplification and detection by real-time detection PCR A reaction solution shown in Table 1 below was prepared per reaction tube, and 40 μl of the magnetic particle dispersion containing HBV-DNA extracted above was added to perform real-time detection PCR.
［反応液の組成］
ＰＣＲグレード蒸留水 ３２．２μｌ
１０×ｂｕｆｆｅｒ １０μｌ
ＭｇＣｌ２（２５ｍＭ） １０μｌ
ｄＡＴＰ（１０ｍＭ） １μｌ
ｄＧＴＰ（１０ｍＭ） １μｌ
ｄＣＴＰ（１０ｍＭ） １μｌ
ｄＴＴＰ（１０ｍＭ） １μｌ
フォワード側プライマー（１００μＭ） １μｌ
リバース側プライマー（１００μＭ） １μｌ
ＴａｑＭａｎ（商標）プローブ （２５μＭ） ０．４μｌ
ＦａｓｔＳｔａｒｔ Ｔａｑ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ（５Ｕ／μｌ）０．４μｌ
ＲＯＸレファレンスダイ（インビトロジェン（株）社製） １μｌ
合計 ６０μｌ
フォワード側プライマー、リバース側プライマー、およびＴａｑＭａｎ（商標）プローブは特表２００５−５０５２８９記載の下記の配列を有するものを使用した。
フォワード側プライマーの配列：５’−ＡＴＣＴＴＡＴＣＡＡＣＡＣＴＴＣＣＧＧＡ−３’（配列番号１３）
リバース側プライマーの配列：５’−ＡＧＡＴＴＧＡＧＡＴＣＴＴＣＴＧＣＧＡＣ−３’（配列番号１４）
ＴａｑＭａｎ（商標）プローブの配列：５’−［ＦＡＭ］ＡＧＧＴＣＣＣＣＴＡＧＡＡＧＡＡＧＡＡＣＴＣＣＣＴ［ＴＡＭＲＡ］−３’（配列番号１５）
ＦａｓｔＳｔａｒｔ Ｔａｑ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅおよび１０×ｂｕｆｆｅｒは、ＦａｓｔＳｔａｒｔ Ｔａｑ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ（ロッシュ・ダイアグノスティック株式会社製）付属の試薬を用いた。
[Composition of reaction solution]
PCR grade distilled water 32.2 μl
10 × buffer 10 μl
MgCl 2 (25 mM) 10 μl
dATP (10 mM) 1 μl
dGTP (10 mM) 1 μl
dCTP (10 mM) 1 μl
dTTP (10 mM) 1 μl
Forward primer (100 μM) 1 μl
Reverse side primer (100 μM) 1 μl
TaqMan ™ probe (25 μM) 0.4 μl
FastStart Taq DNA Polymerase (5 U / μl) 0.4 μl
ROX reference dye (manufactured by Invitrogen Corporation) 1 μl
60μl total
The forward side primer, reverse side primer, and TaqMan (trademark) probe having the following sequences described in JP-T-2005-505289 were used.
Sequence of forward primer: 5′-ATCTTATCAACACTTCCGGA-3 ′ (SEQ ID NO: 13)
Reverse primer sequence: 5′-AGATTGAGATCTTCTGCGAC-3 ′ (SEQ ID NO: 14)
TaqMan ™ probe sequence: 5 ′-[FAM] AGGTCCCCTAGAAGAAGAACTCCCT [TAMRA] -3 ′ (SEQ ID NO: 15)
For FastStart Taq DNA Polymerase and 10 × buffer, reagents attached to FastStart Taq DNA Polymerase (manufactured by Roche Diagnostics) were used.
反応チューブをリアルタイム検出ＰＣＲ装置であるＰｒｉｓｍ ７７００（商品名、アプライドバイオシステムズ社）にセットし、以下の条件で定量ＰＣＲ反応を行なった。
初期活性化ステップ ９５℃ １５分間
ＰＣＲ 変性 ９５℃ １５秒間
ＰＣＲ エクステンション ６０℃ ６０秒間
このＰＣＲサイクルを５０回繰り返した。
The reaction tube was set in Prism 7700 (trade name, Applied Biosystems), which is a real-time detection PCR apparatus, and a quantitative PCR reaction was performed under the following conditions.
Initial activation step 95 ° C. for 15 minutes PCR denaturation 95 ° C. for 15 seconds PCR extension 60 ° C. for 60 seconds This PCR cycle was repeated 50 times.
５０サイクルまでに蛍光強度が指数関数的に増加したものを陽性、増加しなかったものを陰性と判断した。各磁性粒子を用いて得られた実験結果を以下の表１に示す。表１によれば、上記ＨＢＶ−ＤＮＡ捕捉用プローブを有する磁性粒子は、少なくとも１０個／０．１ｍｌ血漿のウイルスを含むサンプルからＢ型肝炎ウイルスを捕捉できることが理解できる。 Those in which the fluorescence intensity increased exponentially by 50 cycles were judged positive, and those that did not increase were judged negative. Table 1 below shows the experimental results obtained using each magnetic particle. According to Table 1, it can be understood that the magnetic particles having the HBV-DNA capture probe can capture hepatitis B virus from a sample containing at least 10 viruses / 0.1 ml of plasma.
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| CN1184330C (en) * | 2000-06-30 | 2005-01-12 | 中国科学院上海冶金研究所 | Process for preparing polymorphic test chip for hepatitis B virus and its application |
| CN1405323A (en) * | 2001-08-09 | 2003-03-26 | 上海博华基因芯片技术有限公司 | Gene chip for detecting drug-resistance of hepatitis, its preparation and detection method |
| CN1200115C (en) * | 2002-10-14 | 2005-05-04 | 山东省医药生物技术研究中心 | Oligonucleotide chip and its application of detecting muatatonal site of hepatitis B virus |
| CN201083740Y (en) * | 2007-06-29 | 2008-07-09 | 上海裕隆生物科技有限公司 | Hepatitis B e antigen immune escape detection gene chip and its reagent kit |
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| JPH07502653A (en) * | 1991-12-23 | 1995-03-23 | カイロン コーポレイション | HBV amplification probe for use in solution phase sandwich hybridization assays |
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