JP5420256B2 - 精製方法及びキット - Google Patents
精製方法及びキット Download PDFInfo
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- JP5420256B2 JP5420256B2 JP2008558892A JP2008558892A JP5420256B2 JP 5420256 B2 JP5420256 B2 JP 5420256B2 JP 2008558892 A JP2008558892 A JP 2008558892A JP 2008558892 A JP2008558892 A JP 2008558892A JP 5420256 B2 JP5420256 B2 JP 5420256B2
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Description
メスの刃を用いて、フィトフソラ・ラモラムに感染したロドデンドロン種の葉の試料(1から2cm2)を小さな片に切断し、抽出緩衝液を含有するPocketDiagnosticsTM(CSL,York,UK)から得られたLFDキットのLFD抽出瓶の中においた。
各片に対するP.ラモラムとCOXのCt値が、図3に示されている(エラーバーは、反復反応(各片の半分2つ)に対する標準偏差を示している。)。PVYLFDを用いて、極めて類似したCt値が得られた(データは示さず。)。
PVYに感染した植物(ジャガイモ)抽出物に曝露され(従って、陽性結果を示した)PVY抗原に対して特異的な抗体を使用することを除き、実施例1に上述されているように調製されたジャガイモウイルスY(PVY)LFDを、6年間より長く、密封されたプラスチックバッグ中に、周囲温度で保存した。LFDから膜を取り出し、1から2mm幅の数個の片を膜から切断した。各片を半分に切断し、PVYに対するリアルタイムRT−PCR(Taqman)用のマスターミックス25μL(1×PCR緩衝液(50mMKCl、10mMTris−HCl、0.001%ゼラチン)、0.02U/μLM−MLV逆転写酵素、0.025U/μLHotTaq、0.2mMの各dNTP、5.5mMMgCl2、1×ROX受動参照色素、300nM各PVYプライマー及び100nMPVYローブ)又は植物チトクロムオキシダーゼ(COX)遺伝子に対するリアルタイムPCR(Taqman)用のマスターミックス25μL(1×PCR緩衝液、0.025U/μLHotTaq、0.2mMの各dNTP、5.5mMMgCl2、1×ROX受動参照色素、300nMの各COXプライマー及び100nMCOXプローブ)を含有する96ウェルプレートの別々のウェル中に、各半分の片を配置した。48℃で30分、次いで、95℃で10分、続いて、95℃で15秒と60℃で1分の2段階40サイクルのサイクル条件を用いて、ABI7900HT上でリアルタイムPCRを実行した。
PVY及びCOXは、それぞれ、RT−PCR及びPCRによって首尾よく検出された。PVY及びCOXのCt値が、表1に示されている。
以下のような市販の様々な膜として、P.ラモラムが接種されたロドデンドロンの抽出物を試料として用いてP.ラモラムを検出するために、実施例1に記載されている実験を反復した。
2Millipore Corp.HiFlow135
3Millipore Corp.HiFlow120
4Millipore Corp.HiFlow90
5Millipore Corp.HiFlow75
6Millipore Corp.HiFlow65
7Schleicher & Schuell Prima 40
LFD作製において使用された慣用のブロッキング溶液で、及び慣用のLFDのラテックス標識システムを適用するために使用された慣用の緩衝液を用いて又は用いずに、それぞれの膜を処理した。以降の実験では、処理されていない膜試料Millipore Corp.HiFlow75及びMillipore Corp.HiFlow180も使用した。
実施例1に記載されているようにP.ラモラムを検出するためのTaqManTMアッセイに、同じく実施例1に記載されているようにP.ラモラムに対する健康なロドデンドロンの未精製抽出物で処理されたLFD由来の膜と一緒に、純粋なP.ラモラムDNAの試料を供した。次いで、TaqManTMを実施する前に、P.ラモラムDNAの等量を試料に加えた。結果は表1に示されており、本明細書の以下の図4に示されている。
調製後に直接、実施例3に記載されている未精製抽出物を、実施例1に記載されているTaqManアッセイに直接供した。未精製抽出物は実施例1に記載されているようにLFDに対しても適用され、未精製抽出物が走行したら直ちに、本装置由来の膜を類似のアッセイに供した。結果(図5)は、調製後直ちに、両試料が陽性結果を与えたことを示している。
実施例1に記載されているLFD装置の膜に沿って、試料を走行させ、次いで、リアルタイムPCR、具体的にはTaqManアッセイ又は慣用のPCR及びLAMPアッセイを用いて、該試料中に存在する核酸が検出される多様な異なるアッセイを実施した。
最近、東アフリカ全体にわたってバラ穀物が、根頭癌腫病として知られる病気によって深刻な影響を受けている。本疾患は、Ti−プラスミドとして知られるDNAの小さな環状片を有する細菌アグロバクテリウム・ツメファシエンスの株によって引き起こされる。これらのプラスインドは、細菌の内部に保有されているので、また、多くの非病原性アグロバクテリウム株が天然に存在するので、病原体を検出する唯一の方法は、細菌DNAを単離し、次いで、Ti−プラスミドの存在に関して、本DNAを検査することによる方法である。
(a)LFD装置の加工。HarrisUnicoreHolePunchを用いて、接種された各LFD膜から2つの直径1.25mmの円板を採取した。96ウェルPCRぷラートの1つのウェル中にこれらの円板を直接配置した。全ての膜を試料として採取した後、円板を含有する各ウェルに、リアルタイムPCRマスターミックス25μLを添加した。ディスク及びマスターミックスの両者が各ウェルの底に位置するようにするため、PCRプレートを短時間遠心した。次いで、リアルタイムPCR機の中に、プレートを直接配置し、サイクリング反応を開始した。(b)WhatmanFTAカード。HarrisUnicoreHolePunchを用いて、接種された各FTAカードから2つの直径1.25mmの円板を採取した。96ウェルPCRプレートの1つのウェル中にこれらの円板を直接配置した。全ての膜を試料として採取した後、Whatman精製緩衝液200μLを各ウェルに添加し、室温で5分間、プレートを温置した。5分後、円板がウェル中に残存するように注意を払いながら、各ウェルから緩衝液を除去した。この精製工程を、さらに2回繰り返した。第三の精製工程後、1×TE緩衝液200μLをを各ウェルに添加し、室温で5分間、プレートを温置した。5分後、円板がウェル中に残存するように注意を払いながら、各ウェルから緩衝液を除去した。この工程を、さらに1回繰り返した。ウェルから、1×TE緩衝液の最後の分取試料を取り出した後、56℃で20分間、PCR機ヒートブロックを用いて、プレートを乾燥させた。円板が乾燥したら、円板を含有する各ウェルに、リアルタイムPCRマスターミックス25μLを添加した。ディスク及びマスターミックスの両者が各ウェルの底に位置するようにするため、PCRプレートを短時間遠心した。次いで、リアルタイムPCR機の中に、プレートを直接配置し、サイクリング反応を開始した。
バラ植物の様々な異なる点から試料が採取された。接木部によって創出された創が病原性アグロバクテリウム属種を惹きつけ、従って、病原体がこの点に集合するので、茎部分、特に、開花変種が台木変種に融合されている接木部が、根頭癌腫病の病原体を検出するための最も可能性が高い場所であることが見出された。以下に示されている結果は、液体に浸して柔らかくされ、2つの小さなDNAキット瓶の中に供給された緩衝液を用いて両装置に適用された10個の異なる1から2cmの幹切片から得られたものである。これらの試料のうち4つが、接木部を横切って採取された。
これは、本発明の方法から得ることができる結果が、WhatmanFTAカードを用いて得られる結果より優れていないとしても、少なくとも同等であることを示している。しかしながら、記載されているプロトコールから明らかであるように、本方法は操作がずっと簡便であった。
さらに、LFDの使用は、予備的タンパク質診断アッセイを同時に実施し得るというさらなる利点を提供する。
Claims (45)
- 液体試料から核酸を分離するための方法であって、前記核酸を含有する液体試料又は前記核酸を含有することが疑われる液体試料を、核酸に対する特異的な結合剤を含まないセルロースをベースとした吸水性膜の試料受容部分に添加する工程と、核酸が吸水性膜の長手方向に分布されるように、前記液体試料を前記吸水性膜に沿って流す工程と、その後、試料受容部分から離れた前記膜の領域における核酸を検出する又は試料受容部分から離れた前記膜の領域から核酸を回収する工程とを含む、方法。
- 液体が膜に沿って流れた後、核酸を検出又は回収する前に、膜が乾燥され、又は膜を乾燥させる、請求項1に記載の方法。
- 乾燥された膜が保存され、及び、その後の工程において、膜上の核酸の存在が検出され、又は核酸が前記膜から回収される、請求項2に記載の方法。
- 分離された核酸がさらなる分析に供せられる、請求項1から3の何れか一項に記載の方法。
- さらなる分析が膜の試料を用いて実施される、請求項4に記載の方法。
- 前記さらなる分析の前に、核酸が膜から溶出される、請求項4に記載の方法。
- さらなる分析がSNPの種類決定、DNAフィンガープリント法又は配列決定を含む、請求項4から6の何れか一項に記載の方法。
- 特異的な標的核酸が検出される、請求項4から7の何れか一項に記載の方法。
- セルロースをベースとした吸水性膜がニトロセルロース膜である、請求項1から8の何れか一項に記載の方法。
- セルロースをベースとした吸水性膜が液体流動装置の要素である、請求項1から9の何れか一項に記載の方法。
- 液体流動装置が試料受容部分及び膜上の前記試料受容部分から離隔して配置された吸収性部分を含む、請求項10に記載の方法。
- 膜が固形収納体の中に収容されており、又は膜が積層化されている、請求項1から11の何れか一項に記載の方法。
- 液体の膜に沿ったウィッキングを増強するように膜が加工されている、請求項1から12の何れか一項に記載の方法。
- 試料が生理的若しくは臨床的試料又は組織抽出物である、請求項1から13の何れか一項に記載の方法。
- 試料が植物又は動物組織の抽出物である、請求項14に記載の方法。
- 試料が血液、血清、尿、乳又は唾液又は糞便抽出物である、請求項14に記載の方法。
- 試料が環境試料である、請求項1から13の何れか一項に記載の方法。
- 試料が水試料又は固体抽出物である、請求項17に記載の方法。
- 試料中の標的核酸の存在を検出するためのアッセイにおいて使用される請求項1に記載の方法であって、前記核酸を含有すると疑われる液体試料を液体流動装置の膜に沿って流す工程と、及び試料受容部分から離れた前記膜の領域上の特異的な核酸の存在を検出する工程とを含む、方法。
- 膜に沿って試料を走行させた後に膜の一部が採取され、及び膜上の核酸の存在が検出される、請求項1から19の何れか一項に記載の方法。
- 核酸が核酸増幅反応を用いて検出される、請求項20に記載の方法。
- 試料が膜に沿って流れた後に、前記核酸増幅反応において有用な試薬が膜上に存在する、請求項21に記載の方法。
- 試料を添加する前に、前記試薬が膜上に存在する、請求項22に記載の方法。
- 前記試薬が増幅プライマーである、請求項22又は23に記載の方法。
- 増幅反応がポリメラーゼ連鎖反応(PCR)である、請求項21から24の何れか一項に記載の方法。
- PCRの進行がリアルタイムにモニターされる、請求項25に記載の方法。
- 膜に添加された試料の量が知られており、及び存在する核酸の量を定量するためにモニタリングの結果が使用される、請求項26に記載の方法。
- 膜上の2以上の核酸の存在が検出される、請求項1から27の何れか一項に記載の方法。
- 膜の同じ試料を用いて複数のPCR反応が実施される、請求項28に記載の方法。
- 膜の異なる試料を用いて複数のPCR反応が実施される、請求項28に記載の方法。
- 単一のPCR中で2以上の核酸を検出できるようにするために、多重PCR反応が実施される、請求項29又は30に記載の方法。
- 2以上の核酸が膜から回収される、請求項1から27の何れか一項に記載の方法。
- 膜が、さらなる分析物のためのアッセイを実施するように配置された液体流動装置の一部である、請求項1から32の何れか一項に記載の方法。
- アッセイがイムノアッセイである、請求項33に記載の方法。
- 分析物の存在を検出し、液体試料中の核酸も場合によって検出するための二段階アッセイであって、(i)さらなる分析物の存在を検出するためのアッセイを実施することができる液体流動装置のセルロースをベースとした吸水性膜の試料受容部分に、前記分析物及び核酸を含有すると疑われる液体試料を添加する工程と、ここで、膜は核酸に対する特異的な結合剤を含まず、液体試料は前記膜の長手方向に沿って流れる、(ii)工程(i)の結果に応じて、試料受容部分から離れた前記膜の領域における核酸を検出する又は試料受容部分から離れた前記膜の領域から回収する工程とを含む、アッセイ。
- 工程(i)のアッセイがイムノアッセイである、請求項35に記載のアッセイ。
- 前記分析物及び核酸が同一の生物又は該生物の特定の種若しくは遺伝子型に由来する、請求項35又は請求項36に記載のアッセイ。
- 分析物が生物の種類に特徴的であり、及び核酸が前記生物の特定の種に特徴的である、請求項37に記載のアッセイ。
- 分析物又は核酸の一方が特定の感染性生物に特徴的であり、及び他方が前記生物の宿主に特徴的である、請求項35又は請求項36に記載のアッセイ。
- 分析物が感染性生物に特徴的であり、及び前記生物に対する宿主の感受性又は耐性に対して影響を与える宿主の遺伝子中に核酸が見出される、請求項39に記載のアッセイ。
- 分析物が植物に適用可能な殺虫剤であり、及び核酸が植物の遺伝子中に見出される、請求項35又は請求項36に記載のアッセイ。
- 請求項1から34の何れか一項に記載の方法又は請求項35から請求項41の何れか一項に記載のアッセイを実施するためのキットであって、核酸に対する特異的な結合剤を含まないセルロースをベースとした吸水性膜と、核酸の検出において使用される、膜に組み込まれた少なくとも1つの試薬とを含み、吸水性膜は液体流動装置の要素であり、液体流動装置はポリペプチドを検出するためのイムノアッセイを実施するように配置されている、キット。
- 試料が膜に沿って流れた後に、前記試薬が膜上に存在する、請求項42に記載のキット。
- 試料を付与する前に、前記試薬が膜上に存在する、請求項43に記載のキット。
- 前記少なくとも1つの試薬が標的核酸配列の増幅に適したプライマーを含む、請求項42から44の何れか一項に記載のキット。
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PCT/GB2007/000865 WO2007104962A1 (en) | 2006-03-11 | 2007-03-12 | Purification method and kits |
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